Jury: Prof. dr. ir. Walter Steurbaut (chairman) Prof. dr. ir. Erick Vandamme (promotor) Prof. dr. ir. Wim Soetaert (promotor) Prof. dr. ir. Nico Boon Prof. dr. ir. Monica Höfte Prof. dr. Els Van Damme Dr. Roel Bovenberg Dr. Thierry Dauvrin
Promotors: Prof. dr. ir. Erick VANDAMME Prof. dr. ir. Wim SOETAERT Laboratory of Industrial Microbiology and Biocatalysis Department of Biochemical and Microbial Technology Ghent University
Dean: Prof. dr. ir. Herman VAN LANGENHOVE
Rector: Prof. dr. Paul VAN CAUWENBERGE
S. De Maeseneire was supported by a fellowship of the Flemish Institute for the Promotion of Scientific and Technological Research in the Industry (IWT-Vlaanderen).
The research was conducted at the Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University.
ir. Sofie De Maeseneire
MYROTHECIUM GRAMINEUM AS A NOVEL FUNGAL EXPRESSION HOST
Thesis submitted in fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Cell and Gene Biotechnology
Titel van het doctoraatsproefschrift in het Nederlands: Myrothecium gramineum als een nieuwe fungale expressie-gastheer
Cover illustration: ‘ Myrothecium gramineum’ by Sofie De Maeseneire (June 2007)
To refer to this thesis: De Maeseneire, S. L. (2007) Myrothecium gramineum as a novel fungal expression host. PhD-thesis, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium, 308 p.
ISBN-number: 978-90-5989-176-0
The author and the promotors give the authorisation to consult and to copy parts of this work for personal use only. Every other use is subject to copy right laws. Permission to reproduce any material contained in this work should be obtained from the author.
Woord vooraf
Vier jaren, een eeuwigheid, doch véél te snel voorbij gegleden… Hoog tijd voor een woordje van dank!
Graag wil ik mijn promotoren, prof. dr. ir. Erick Vandamme en prof. dr. ir. Wim Soetaert bedanken om mij toe te laten mijn doctoraatsonderzoek uit te voeren in hun labo, en voor de vrijheid en het vertrouwen waarvan ik steeds heb mogen genieten.
Ik wil ook de leden van de leescommissie bedanken, prof. dr. ir. Nico Boon, prof. dr. ir. Monica Höfte, prof. dr. ir. Els Vandamme en dr. Roel Bovenberg voor de kritische beoordeling van dit proefschrift. Een speciaal woord van dank ook voor dr. Thierry Dauvrin, voor de hulp tijdens het schrijven van dit proefschrift en voor de vele nuttige suggesties. Ik mag ook zeker de ‘leescommissie van het thuisfront’ niet vergeten! Liefste tante, dank je wel voor de vele uren die je doorgebracht hebt met het lezen en verbeteren van deze voor jou ongetwijfeld moeilijke en vermoedelijk ook saaie tekst ☺!! En Wouter, bedankt voor de last- minute correcties!
Dit proefschrift zou ook nooit af geraakt zijn moest de sfeer op het labo niet zo goed zijn als ze is! Ik wil graag iedereen bedanken die bijdraagt tot het ‘Limab-gevoel’. Ik weet het, het is een beetje sentimenteel, maar ik geloof dat iedereen die op ons labo werkt of gewerkt heeft, weet wat wij hiermee bedoelen. De vriendschap en de steun die wij van elkaar krijgen helpen ons door te gaan, zelfs tot in de late uurtjes en tijdens de weekends. Het risico lopend hier iemand te vergeten, wil ik toch een paar mensen speciaal bedanken. Marjan in het bijzonder, voor alles; Cassandra, voor het delen van de whisky in de Jan Van Gent; An, Bart en Katja, voor de gezellige en lekkere etentjes; Joeri, voor je steun tijdens de overuren en Inge, voor het luisteren naar mijn schimmelgeklaag. Bedankt voor jullie vriendschap en steun, voor de vele onvergetelijke momenten, voor het aanhoren van mijn gezaag en om er mij steeds weer in te laten geloven.
Een oprecht woord van dank ook voor mijn thesisstudenten, voor hun onvermoeibare inzet tijdens de vele uren die ze op het labo doorbrachtten en om er ondanks hun vele tegenslagen toch in te blijven geloven. In het bijzonder dank ik Manu, Veerle en Eline voor hun bijdrage aan dit doctoraat.
En, last but not least, wil ik ook mijn familie bedanken. Liefste mama en papa, bedankt voor jullie liefde en onvoorwaardelijke steun, bedankt om steeds het beste voor ons te willen en uit ons te halen, bedankt om er steeds voor ons te zijn! Ook Zussie en Wesley, dank jullie wel en veel succes als jullie binnenkort in jullie eigen stekje gaan wonen. Liefste nichteke, bedankt voor de peptalks tijdens onze ‘vrouwenonderonsjes’! Ook voor Do en Jan: merci voor alles en dat we nog lang samen van alles mogen genieten!!! Lieve schat, woorden schieten te kort om jou te bedanken… Dank je wel voor je liefde, voor je eeuwige optimisme, voor je ongelooflijk geduld, voor het aanhoren van mijn razernijen en teleurstellingen, om er mij steeds weer bovenop te brengen en zo veel meer! Nu dit hoofdstuk is afgerond zal ik mijn best doen wat minder te werken, zodat er wat meer tijd is voor ons tweetjes. The best has yet to come!
Sofie, Zaterdag 9 juni 2007
‘Success is the ability to go from one failure to another with no loss of enthousiasm’ Sir Winston Churchill, British politician of, vrij vertaald:
‘Een echte winnaar is een verliezer die nooit opgeeft!’ Prof. Erick Vandamme, Professor at the University of Ghent Table of contents
Table of contents Abbreviations i Literature review 1. Introduction 1 2. Introduction to the fungi 7 2.1. Organisation of the thallus 7 2.2. Reproduction and life cycle 8 2.3. Regulatory circuits in filamentous fungi 11 2.3.1. Glucose repression in fungi 12 2.3.2. Nitrogen catabolite repression 12 2.3.3. pH regulation 13 2.3.4. Phosphorous regulation 13 2.3.5. Sulphur regulation 14 3. The fungus Myrothecium gramineum 14 4. Transformation methods of filamentous fungi 18 4.1. Electroporation 19 4.2. Lithium acetate treatment 19 4.3. Biolistical transformation 19 4.4. Agrobacterium tumefaciens -mediated transformation 20 4.5. Protoplast transformation 21 4.5.1. Preparation of the protoplasts 21 4.5.2. DNA-uptake 23 4.5.3. Protoplast regeneration 24 5. Selection markers 25 6. Fate of the transforming DNA 29 7. Applications of transformation technology 32 7.1. Using transformation for cloning genes (heterologous and homologous) 32 7.2. Gene inactivation and gene targeting 34 7.3. Manipulation of gene expression using transformation 39 8. (Heterologous) protein production by fungi 40 8.1. Structure of fungal expression systems 40 8.1.1. Promoters of filamentous fungi 41 8.1.2. Introns 48 8.1.3. Transcription termination 49 8.1.4. Initiation of translation 49 8.1.5. Secretion of heterologous proteins 50 Table of contents
8.2. The fungal secretion pathway 51 8.3. Post-translational modifications 54 8.4. Reporter genes and expression studies 56 8.5. Strategies for the improvement of (heterologous) protein production 58 9. Research objectives 64 Part I Chapter I 69 Transformation of M. gramineum with the hygromycin B resistance marker gene 69 1. Introduction 69 2. Materials and methods 70 2.1. Strains and plasmids 70 2.2. Culture conditions 71 2.2.1. Standard culture conditions 71 2.2.2. Test of the hygromycin selection medium 71 2.2.3. Test of the acetamidase selection medium 71 2.2.4. Amylase production conditions 71 2.3. Plasmid preparation 71 2.4. Protoplast preparation and transformation of M. gramineum 72 2.5. Amylase enzyme test 73 2.5.1. Cell dry weight measurement 73 2.6. PCR control of the transformants 73 2.7. Southern analysis of the transformants 74 2.7.1. Construction and labelling of the probe 74 2.7.2. Genomic DNA preparation and Southern analysis 74 3. Results and discussion 74 3.1. Development of the selective media 74 3.1.1. Hygomycin selective medium 74 3.1.2. Acetamide selective medium 75 3.2. Transformation of M. gramineum 75 3.3. Development of an amylase enzyme test 76 3.4. Amylase enzyme tests on the transformant cultures 78 3.5. Control of the stability of the amylase production 79 3.6. PCR control of the transformants 80 3.7. Southern analysis 81 4. Conclusion 83
Table of contents
Chapter II 84 Rapid sample preparation for long distance PCR on genomic DNA of M. gramineum 84 1. Introduction 84 2. Materials and methods 86 2.1. Strains and culture conditions 86 2.2. Collection of material 86 2.3. Extraction methods 86 2.4. PCR amplifications 86 2.4.1. Amplification of ‘small’ fragments 86 2.4.2. Amplification of ‘longer’ fragments 87 3. Results and discussion 90 3.1. DNA extractions 90 3.2. PCR 1 and 2 91 3.3. PCR 3 93 4. Conclusion 95 Part II Chapter III 99 Cloning and sequence analysis of the gpd -gene of M. gramineum 99 1. Introduction 99 2. Materials and methods 102 2.1. Strains and growth conditions 102 2.2. Standard DNA manipulation 102 2.3. Cloning of the Myrothecium gramineum gpd -gene 103 2.3.1. Genomic DNA isolation 103 2.3.2. Genome walking 103 2.3.3. Cloning and sequence analysis 103 3. Results and discussion 105 3.1. Isolation of the full length gpd -gene 105 3.1.1. Construction of the BD GenomeWalker banks 106 3.1.2. Genomic walking 106 3.1.3. Cloning and sequencing of the complete gpd -gene of M. gramineum 109 3.2. Nucleotide sequence analysis 109 3.2.1. Analysis of the coding sequence 109 3.2.2. Analysis of the 5’ and 3’ flanking sequences 111 3.3. Determination of the gpd copy number 116 3.4. Protein sequence analysis 117 3.5. Phylogenetic analysis 119 Table of contents
3.6. Construction of expression vectors with the M. gramineum gpd -promoter 120 4. Conclusions 123 Chapter IV 124 Expression of an Aspergillus oryzae amylase gene ( amy3 ) in Myrothecium gramineum 124 1. Introduction 124 2. Materials and methods 125 2.1. Strains, plasmids and growth conditions 125 2.2. Standard DNA manipulation 126 2.3. Transformation of M. gramineum and control of transformants 126 3. Results and discussion 127 3.1. Cloning of the A. oryzae amy3 gene into pGEM-T® 127 3.2. Construction of pGPDkpAmyAO and pGPDlpAmyAO 127 3.3. Transformation of Myrothecium gramineum and analysis of the transformants 130 4. Conclusion 138 Chapter V 140 Expression of a Penicillium griseofulvum endo-1,4-βββ-xylanase gene in M. gramineum 140 1. Introduction 140 2. Materials and methods 142 2.1. Strains, plasmids and growth conditions 142 2.2. Standard DNA manipulation 143 2.3. Transformation of M. gramineum and control of transformants 143 3. Results and discussion 144 3.1. Cloning of the P. griseofulvum xylanase gene into pGEM-T® 144 3.2. Construction of pGPDkpXylPG and pGPDlpXylPG 145 3.3. Transformation of Myrothecium gramineum and analysis of the transformants 148 4. Conclusion 153 Chapter VI 155 Expression of a Bacillus subtilis xylanase gene ( xynA ) in M. gramineum 155 1. Introduction 155 2. Materials and methods 158 2.1. Strains, plasmids and growth conditions 158 2.2. Standard DNA manipulation 158 2.3. Transformation of M. gramineum and control of transformants 159 3. Results and discussion 159 Table of contents
3.1. Construction of the plasmids pGPDnocarXylBS and pGPDcarBS 159 3.2. Transformation of Myrothecium gramineum and analysis of the transformants 162 4. Conclusion 165 Part III Chapter VII 169 Cloning, sequence analysis and expression of the M. gramineum ompd-gene 169 1. Introduction 169 2. Material and methods 171 2.1. Strains, plasmids and growth conditions 171 2.2. Standard DNA manipulation 171 2.3. Cloning of the Myrothecium gramineum ompd -gene 171 2.3.1. Genomic DNA isolation 171 2.3.2. Degenerate PCR 171 2.3.3. Genome walking 171 2.3.4. Cloning and sequence analysis 172 2.4. Complementation of the uracil auxotrophic Aspergillus nidulans A722 strain 173 2.4.1. Transformation conditions 173 2.4.2. Verification of the transformants 173 3. Results and discussion 174 3.1. Cloning of the Myrothecium gramineum ompd -gene 174 3.1.1. Development of a degenerate primer pair 174 3.1.2. Degenerate PCR on genomic DNA of M. gramineum 176 3.1.3. Genome walking 177 3.2. Nucleotide sequence analysis 181 3.2.1. Analysis of the coding sequence 181 3.2.2. Analysis of the promoter region and the 3’ flanking sequences 182 3.3. Determination of the copy number of the ompd -gene 183 3.4. Protein sequence and comparison to other OMPD sequences 184 3.5. Phylogenetic analysis 187 3.6. Functional complementation of a defined OMPD-deficient Aspergillus strain 189 4. Conclusion 191 Chapter VIII 192 Development of a new selection system based on the ompd -gene of M. gramineum 192 1. Introduction 192 2. Materials and methods 200 2.1. Strains and standard cultivation techniques 200 2.2. Standard DNA manipulation 200 Table of contents
2.3. Selective media 200 2.3.1. Selection against OMPD deficient strains 200 2.3.2. Selection against OMPD positive strains 200 2.4. Creation of OMPD-negative M. gramineum strains 201 2.4.1. Transformation and homologous recombination 201 2.4.2. UV mutagenesis 201 3. Results and discussion 202 3.1. Development of selective media 202 3.1.1. Selection against OMPD deficient strains 202 3.1.2. Selection against OMPD positive strains 203 3.2. Construction of OMPD knock-out vectors 207 3.2.1. Principle 207 3.2.2. Knock-out vector with mutation at 3’ end of the ompd -gene 208 3.2.3. Knock-out vector with mutation at 5’ end of the ompd -gene 209 3.3. Creation of OMPD-negative M. gramineum strains 210 3.3.1. Via transformation and homologous recombination 210 3.3.2. UV mutagenesis 216 4. Conclusion 219 Part IV General discussion, conclusion and research perspectives 223
References 241 Summary 267 Samenvatting 275 Curriculum Vitae 283
Abbreviations i
Abbreviations 5-FdUMP 5-Fluoro-2’-deoxyUridine MonoPhosphate 5-FUMP 5-Fluoro-Uridine MonoPhosphate 5-FUMP 5-Fluoro-Uridine TriPhosphate AA Amino Acid Acetyl-CoA Acetyl-Coenzyme A AMM Aspergillus Minimal Medium AMT Agrobacterium Mediated Transformation AP Adaptor Primer ARS Autonomously Replicating Sequences ATCA AurinTriCarboxylic Acid ATP Adenosine Triphosphate BCCM TM Belgian Co-ordinated Collections of Micro-organismsTM BRENDA BRaunschweig ENzyme DAtabase cAMP cyclic Adenosine MonoPhosphate CDART-tool Conserved Domain Architecture Retrieval Tool CDD Conserved Domain Database cDNA copy DeoxyriboNucleic Acid CDW Cell Dry Weight CHIPS Codon Heterozygosity (Inverse of) in a Protein-coding Sequence DEO 1,2,7,8-DiEpoxyOctane DIG DIGoxigenin DNA DeoxyriboNucleic Acid DNS DiNitroSalicylic acid dNTP deoxyriboNucleotide TriPhosphate EDTA EthyleneDiamineTetraacetic Acid ELSD Evaporative LightScattering Detector EMBOSS European Molecular Biology Open Software Suite EMS Ethyl Methane Sulfonate ER Endoplasmatic Reticulum ERAD Endoplasmatic Reticulum Associated protein Degradation ERE Estrogene Responsive Elements FGSC Fungal Genetics Stock Center FOA 5-Fluoro-Orotic Acid gDNA genomic DeoxyriboNucleic Acid (E)GFP (Enhanced) Green Fluorescent Protein GH Glucoside Hydrolase GPD Glyceraldehyde-3-Phosphate Dehydrogenase GRAS Generally Recognised As Safe GSP Gene Specific Primer GT Glucosyl Transferase Abbreviations ii
HPLC High Pressure Liquid Chromatography HR Homologous Recombination HSE Heat Shock Elements HSP Heat Shock Protein ITS Internal Transcribed Spacer LMW Low Molecular Weight MAR Matrix Attachment Regions MMLV Moloney Murine Leukemia Virus MNNG N-Methyl-N’-Nitro-N-NitrosoGuanidine MOPS 3-(N-Morpholino)-PropaneSulfonic acid mQ milliQ water mRNA messenger RiboNucleic Acid MUCL Mycothèque de l'Université Catholique de Louvain MW Molecular Weight NAD Nicotinamide Adenine Dinucleotide NADP Nicotinamide Adenine Dinucleotide Phosphate Nc Effective Number of codons NCBI National Center for Biotechnology Information NHEJ Non-Homologous End Joining nmr nitrogen metabolic regulation NMR Nuclear Magnetic Resonance OMP Orotidine-5-MonoPhosphate OMPD Orotidine MonoPhosphate Dehydrogenase OPRT Orotic acid PhosphoRibosylTransferase PAGE PolyAcrylamide Gel Electrophoresis PCB’s PolyChlorinated Biphenyls PCR Polymerase Chain Reaction PD Potato Dextrose PDA Potato Dextrose Agar PDI Protein Disulfide Isomerase PEG Poly Ethylene Glycol PNP P-NitroPhenol PPI Peptidyl Prolyl Isomerase RACE Rapid Amplification of cDNA Ends RBB-xylan 4-O-methyl-D-glucurono-D-xylan-Remazol Brilliant Blue R rDNA ribosomal DeoxyriboNucleic Acid REMI Restriction Enzyme Mediated Integration RNA RiboNucleic Acid RNAi RiboNucleic Acid Interference rRNA ribosomal RiboNucleic Acid RT Reverse Transcriptase SDS Sodium Dodecyl Sulphate Abbreviations iii
SRP Signal Recognition Particle SRPR Signal Recognition Particle Receptor ss Secretion signal TAGKO Transposon Arrayed Gene Knock-Out TBP TATA Binding Protein T-DNA Transferred-DeoxyriboNucleic Acid Tr Transformants tRNA transfer RiboNucleic Acid tsp transcription start point UDP Uridine DiPhosphate UMP Uridine MonoPhosphate UPR Unfolded Protein Response UTR UnTranslated Region VIB Vlaams Instituut voor Biotechnologie WT Wild Type Xgal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside YNB Yeast Nitrogen Base (Broth) YPD Yeast extract/Peptone/Dextrose
Literature review
Literature review 1
Literature review
1. INTRODUCTION For centuries, humans have unwittingly used microorganisms for their own benefit. Filamentous fungi, yeasts and their enzymes are indeed instrumental since thousands of years in the making of beer, wine, bread and cheese and in several other fermented foods . Nowadays, fungi and their enzymes are still widely used in the food industry, but they are also increasingly applied in other industrial fields.
Brewing and baking have been practiced for ages and both are dependent on the conversion of sugar into alcohol and carbon dioxide by yeast enzymes. In bread making, carbon dioxide causes the dough to rise whilst the alcohol is driven off during baking. Early processes were dependent on contamination by ‘wild’ yeasts. Today pure strains are usually employed and over 1.5 million tons of baker’s yeast ( Saccharomyces cerevisiae ) are produced worldwide every year. Modern baking technologies also use enzymes, produced by filamentous fungi, for example to improve the taste, the digestibility or the preservation of bread. Cheese production also has ancient origins. The growth of the mould Penicillium roqueforti in the body of blue-veined cheeses and the surface growth of the moulds Penicillium candidum, Penicillium caseicolum or Penicillium camembertii on Camembert, Brie and related types of cheeses play an important role in the development of the characteristic flavours of these cheeses. Oriental food fermentations such as the ones used to produce soy sauce could originally take several months, but this time span has been considerably reduced in modern processes. The fungus involved, Aspergillus oryzae , is now also used to produce a range of commercially important enzymes. A recent innovation in food technology has been the development of Quorn myco-protein from a strain of Fusarium venenatum . Quorn is produced from mycelia grown in 155,000 litre airlift fermenters: the biomass is responsible for the meat-like texture and appearance of the final product (1).
In addition to being important in food applications, fungi and their enzymes also play a significant role in a lot of other industrial processes. A number of examples are given in Table 1-1 and Table 1-2. Again, these applications have a long history. At the end of the 19th century, in 1894, the commercial production of enzymes started in the United States with diastase (Taka-amylase) from Aspergillus oryzae (2). Nowadays, this enzyme is still used in the baking industry, for example to make foods more digestible. Also in the United States, Pfizer started the commercial production of citric acid using Aspergillus niger in 1923. Prior to 1923, citric acid was produced from lemons mainly in Italy.
Industrial microbiology really started to develop after the discovery of penicillin in 1929. It began when Alexander Fleming published his observation about the inhibition of growth of Staphylococcus aureus on an agar plate contaminated with Penicillium notatum (3). Three years later, it was shown that the growth inhibition was due to penicillin. During the late Literature review 2
1940s, the fungus Cephalosporium acremonium (now renamed Acremonium chrysogenum ) was isolated from the sea at Cagliari, Italy. This fungus was first found to produce penicillin N; later, another antibiotic was discovered in the culture broth, which was found to consist of different derivatives of a β-lactam compound designated cephalosporin. The discovery of cephalosporin C generated a whole new group of clinically significant β-lactams. The success of β-lactams in the treatment of infectious diseases is due to their high specificity and their low toxicity. Despite a growing number of antibiotics and the incidence of penicillin-resistant isolates, β-lactams are still by far the most frequently used antibiotics. Industrial production of penicillin and cephalosporin is achieved with P. chrysogenum and A. chrysogenum , respectively, up till today. Cyclosporin A, produced by the Tolypocladium inflatum , is currently a widely used drug for preventing rejection of human organ transplants (1, 4). Nowadays, fungi are well known as a source of antibiotics and new therapeutic compounds with novel pharmacological activities (5). Table 1-1. Use of fungi in biotechnology (1) Food Applications Useful Products Other Processes Baking Alkaloids Biobleaching/biopulping Beverages and Brewing Antibiotics Biological control agents Cheese-making Ethanol Bioremediation of soils Chocolate industry Enzymes Coal solubilisation Mushroom cultivation Gibberellins Dyes/dye intermediates Oriental food fermentations Immunomodulators Micro encapsulation Quorn myco-protein production Organic acids Mycorrhizal inoculants Flavour production Polysaccharides Steroid bioconversions Vitamins Waste treatment
Many people cannot control their cholesterol at a healthy level by diet alone but must depend on hypocholosterolemic drugs. One huge success has been based on the fungal statins, including lovastatin, pravastatin and others. They inhibit the rate-limiting enzyme of cholesterol biosynthesis in the liver (4). They are mainly produced by Penicillium sp ., Monascus ruber and Aspergillus terreus .
Most natural anticancer agents are made by Actinomycetes (Streptomyces sp.), but a recent non-actinomycete molecule is Taxol , which was originally discovered in plants but is also produced by the fungus Taxomyces andreanae . It is approved for breast and ovarian cancer and it is the only commercial antitumor drug known to act by blocking depolymerisation of microtubules (4).
The biopolymer chitin and its derivative chitosan have a wide range of industrial applications. Chitosans are non-toxic, biocompatible, and biodegradable and have been widely used for pharmaceutical purposes and for other purposes such as clarification of waste water, in food products, in feed ingredients, and in the paper industry. Hydrogels, controlled release dosage Literature review 3 forms, mucoadhesive dosage forms, microcapsules, microparticles, and nanoparticles have been developed for pharmaceutical use (6). These polysaccharides are currently obtained commercially by treatment of prawn- and crab-shell wastes. However, problems with product variability and processing difficulties have prompted research into alternative supplies. Filamentous fungi grown under controlled conditions are an attractive source of chitin and chitosan where a high-quality product is required (e.g. for cosmetic, medical and pharmaceutical applications) (7).
Fungi can also be used in new production processes which are less polluting than traditional chemical processes. For example, textile dyes could be produced by fermentation in the future. Many fungi produce pigments during their growth and have potential as agents for the direct production of textile dyes. This production could replace chemical synthesis which has inherent waste disposal problems (8). Another way to resolve the ecological problems associated with colour production is to use fungal enzymes to detoxify industrial waste waters. The dye effluent from the textile industry contains a mixture of various dyes that are usually aromatic and difficult to degrade. For example, the white-rot fungus Irpex lateus is able to degrade dyes in the contaminated soil as well as to decolour various types of synthetic dyes in aqueous cultures and packed bed bioreactors (9). Recent studies have suggested that lignin-degrading or white-rot fungi such as Phanerochaete chrysosporium and Trametes versicolor could replace some of the chemical steps used in paper making. An industrial biopulping/biobleaching process would eliminate the pollution problems associated with the use of chemicals (10). Lignin-degrading fungi or their enzymes also have the ability to degrade highly toxic organic compounds such as dioxins and PCB’s (polychlorinated biphenyls), and could play an important role in the remediation of contaminated soils or the disposal of chemical wastes (11, 12). Biological treatment technologies for the remediation of soils and groundwater contaminated with organopollutants are widely used for their environmentally friendly impact combined with low cost compared to other treatment alternatives. Some species of white rot fungi are being employed to degrade toxic wastes. Fungi display a high ability to immobilize toxic metals. Moreover, due to the low substrate specificity of their degradative enzyme machinery (e.g. laccase, lignin peroxidase, and Mn-peroxidase), fungi are able to perform the breakdown of a wide range of organopollutants in contaminated soils. Many of these applications are still under development (13).
Literature review 4
Table 1-2. Fungal products of commercial importance (non exhaustive list, adapted from 14, 1, www.AMFEP.org , 2004 1) Fermentation product Source 2 Enzymes Aminoacylase A. melleus Asparaginase Aspergillus spp., Penicillium sp. α-Amylase A. niger, A. oryzae Catalase A. niger, A. oryzae, Penicillium sp. Cellulase A. niger, A. oryzae, Humicola insolens, P. funiculosum, H. jecorina, Dextranase P. lilacinum Esterase Rhizomucor miehei α- & β-Galactosidase A. niger, A. oryzae ß-Glucanase A. aculeatus, A. niger, A. oryzae, P. emersonii, P. funiculosum, P. ticolor, H. jecorina, T. viride Gluco-amylase A. niger, A. oryzae , A. phoenicis, R. delemar, R. niveus, R. oryzae Glucose oxidase A. niger, A. oryzae, P. chrysogenum Glucosidases A. niger, P. decumbens, P. funiculosum, T. harzarium Glucosyltransferase A. foetidus, A. niger Hemicellulase A. foetidus, A. niger, A. oryzae, P. emersonii, H. jecorina, T. viride Inulinase A. niger, A. oryzae Invertase A. niger, A. oryzae Laccase A. oryzae, Coriolus (Trametes) versicolor, Pyricularia oryzae Lipases & phospholipases A. niger, A. oryzae, M. javanicus, P. camenbertii, P. roquefortii, Rhizomucor miehei, R. delemar, R. niveus, R. oryzae, H. jecorina Mannanase A. niger, P. funiculosum, Hypocrea jecorina Pectinase A. aculeatus, A. japonicus, A. niger, A. oryzae, A. pulverulentus, A. sojae, Humicola insolens, P. funiculosum, R. oryzae, H. jecorina Phytase A. niger, A. oryzae, H. jecorina Phosphatase A. niger Proteases & peptidases A. melleus, A. niger, A. oryzae, A. sojae, Mucor sp. , P. citinum, Rhizomucor meihei, R. delemar, R. niveus, R. oryzae, Trichoderma sp. Rennine (microbial) Endothia parasitica, M. miehei, M. pusillus Ribonuclease A. oryzae, Penicillium sp. Tannase A. niger, A. oryzae Xylanase A. foetidus, A. niger, A. oryzae, P. funiculosum, H. jecorina, T. viride Antibiotics and other pharmaceutically active compounds Penicillin P. chrysogenum Cephalosporine Acremonium chrysogenum Ergot alkaloids Claviceps purpurea Griseofulvin P. griseofulvum Mevalonin A. terreus Cyclosporin Tolypocladium inflatum Organic acids Citric acid A. niger Itaconic acid A. terreus 1 AMFEP or ‘The Association of Manufacturers and Formulators of Enzyme Products’ is a European industry association founded in 1977. The members of AMFEP produce and sell enzymes for use in food, feed, detergents and other non-food industries, excluding enzymes for pharmaceutical and diagnostic use. 2 Abbreviations: A., Aspergillus ; H., Hypocrea ; M., Mucor ; P., Penicillium ; R., Rhizopus
Literature review 5
The use of fungi as biocontrol agents to kill insects, nematodes and weeds in the control of plant diseases has the potential to replace many of the toxic chemicals currently in use (4). Several species of fungi have now been commercially formulated as mycopesticides. Trichoderma atroviride , for example, is a filamentous soil fungus that functions as a biocontrol agent for a wide range of economically important aerial and soil borne plant pathogens. The mycoparasitic activity of this organism is attributed to a combination of successful nutrient competition, the production of cell wall-degrading enzymes and antibiosis. Several strains of the genus Trichoderma are being tested as alternatives to chemical fungicides (15), such as strobilurins. Strobilurins are fungal metabolites which have been used to develop several commercially useful antifungal products. They are produced by many species of the Basidiomycetes , such as Strobilurus and others (4).
In their natural environment, filamentous fungi such as Aspergillus and Trichoderma are capable of metabolising a large number of carbon and nitrogen sources by the secretion of a huge diversity of enzymes under a wide variety of conditions (2). Together with the capacity of secreting these enzymes in large amounts, this makes fungi attractive hosts for the large scale production of enzymes. For commercial processes, yields of more than 20 to 30 g/L of a specific protein are not uncommon (14, 16), while only a few yeast genera ( Pichia , Hansenula ) reach the g/L production level (17, 18). Fungi are also capable of excreting high amounts of metabolites and organic acids in the growth medium (19). Compared to intracellular production, the secretion of products into the medium does not disturb the metabolism of the fungus, it protects the protein from exposure to intracellular proteases, it facilitates purification of the (recombinant) protein and it simplifies the downstream processing a lot, which keeps production costs low. In contrast to animal and plant tissue, fungi can be grown relatively easy and cheap; another characteristic which keeps costs in fungal production systems low.
Fungi are eukaryotic, which enables them to carry out post-transcriptional modifications, such as intron excision (in contrast to bacteria), and post-translational modifications, such as the formation of disulfide bridges, proteolysis and glycosylation during the passage of the secretion pathway (20). Together with the protein folding processes, these modifications play an important role in the determination of the structure of the enzyme and thus, in the activity of it. The structure and function of secreted proteins can be strongly influenced by glycosylation. Glycosylations in filamentous fungi, as far as known, are similar to those in other (higher) eukaryotes. This is in contrast to yeast ( Saccharomyces cerevisiae ), where hypermannosylation frequently occurs (21). This makes fungi promising hosts for the production of enzymes of higher eukaryotes. Some filamentous fungi are used since decades in the food industry and have gained the ‘GRAS-status’ ( Generally Recognised As Safe ).
Taking all these points into consideration, together with the knowledge drawn from the long tradition of using fungi in industrial processes, fungi can be considered as attractive hosts for the synthesis of new products. Literature review 6
The high production yields usually are only obtained for homologous enzymes. In early approaches towards the high-level production of proteins of non-fungal origin, initial optimistic promises were not completely met. Although in several cases, production of the protein of interest could be detected at levels comparable to those obtained in various other expression systems (such as bacteria and yeast), the levels obtained in filamentous fungi were always much lower than those for fungal proteins (16, 22). The commercial uses of the fungal secretion system and the limitations concerning the production of heterologous proteins have stimulated researchers to investigate the genetics of filamentous fungi (19).
Despite the fact that fungi already have a broad spectrum of industrial applications, until now, only a limited number of fungal host species has been explored for recombinant protein production. The field of modern fungal biotechnology is competitive and is attracting considerable interest from industrial companies outside of the traditional fermentation industry, interested in the applications of enzymes and other proteins. Therefore, it is not surprising that several parties started to explore the alternatives to those covered by several patent applications. Patents and intellectual property rights have necessitated searching for expression hosts other than the fungal species traditionally used. Recent research in several groups has focused on developing Fusarium venenatum (16), Aspergillus sojae (7), A. japonicus (14), Neurospora crassa (20), Mortierella alpinis (21) and Chrysosporium lucknowense (22) as hosts for protein and metabolite production. In most cases, the evaluation of the true potential of these species is still subject to further research (16).
Till today, estimations claim that only 1 % of all microorganisms is known and even less are used in industrial applications. This is a limiting factor for companies, which wish to exploit a broad market and thus need a range of applications as varied as possible. The fact that many currently used fungal expression hosts are protected by patent applications, taken together with the consideration that, undoubtedly, micro-organisms and, more specific, filamentous fungi, exist which have new characteristics and produce enzymes with new application possibilities, was an inspiration to explore the BCCM/MUCL agro-industrial fungi- and yeast collection for a potential new expression host. Study of this new fungal host could also contribute to some more fundamental areas of fungal research.
The aim of our research was to further develop a transformation system and to develop an expression system to enable us to use the filamentous fungus Myrothecium gramineum as a new, universal expression host. Myrothecium gramineum was selected in a preliminary study in which the BCCM/MUCL agro-industrial fungi- and yeast collection of the “Université Catholique de Louvain” (UCL) was screened for potential, new fungal hosts. This preliminary study was carried out by the company Beldem in collaboration with the Laboratory of Microbiology of the UCL.
Literature review 7
In the next section a general introduction to the fungi will be given, followed by a section describing the fungus M. gramineum in more detail. In order to use this fungus as an expression host, a transformation system based on an appropriate selection marker is needed. General ways to transform fungi and to select the transformed cells will be discussed. The current state of the art of the expression systems used to enable fungi to produce proteins and the optimisation of this production will be illustrated in the final paragraphs of this chapter. In the last section, a more detailed description of the objectives of our research is given.
2. INTRODUCTION TO THE FUNGI
2.1. Organisation of the thallus The fungal kingdom is a diverse assemblage of organisms with a great variety of structural types (23). All organisms we recognise as fungi have the basic characteristics of eukaryotic cells. The cellular basis of the thallus organisation ranges from unicellular forms to coenocytic, filamentous hyphae and structures with a considerable degree of differentiation.
The vast majority of fungi is composed of hyphae - unique threadlike, tubular structures, bounded by walls and forming extensive, branched systems called mycelia. The hyphae of most fungi are segmented by septae that divide the mycelium into units similar to cells, but the presence of pores in the septae allows mass movement of cytoplasm and organelles and the migration of nuclei through the mycelium to distant parts. This communication has important consequences for the physiology of the fungus. Of the variety of septal pores found among the fungi, two are very common: the simple pore, typical for the mycelial Ascomycota , and the more complex dolipore septum, typical for many Basidiomycota (Figure 2-1). Although there is cytoplasmatic continuity through these pores, the parenthesome and septal swelling at the pore of the dolipore septum and the Woronin granules or other inclusions in fungi with simple pores may act to occlude the pores, preventing free movement of materials between the segments. The numbers of nuclei per segment are variable: in some fungi the nuclei are strictly regulated to one per segment, but in many the numbers are variable. Often there is a large number in the apical segment and progressively fewer nuclei in the subapical segments, so that only a few segments from the tip there is one nucleus per segment. The dikaryotic mycelium of the Basidiomycota typically has two nuclei per segment which divide in a co-ordinate way.
Figure 2-1. Hyphal segments are delimited by septae with pores. A. Simple pore B. Dolipore. Literature review 8
The occurrence of anastomosis (fusion between two hyphae, resulting in the exchange of nuclei and cytoplasm) and the subsequent nuclear migration create heterokaryons, mycelia with two or more genetically different nuclei, adding to the genetic capabilities of fungi and permitting the asexual recombination of genetic material between nuclei in the hyphae.
This phenomenon, known as parasexuality, occurs in place of or in addition to the normal sexual cycle of the fungus. It is genetic recombination without meiosis, explaining the term parasexuality. Mycelial fungi are able to maintain genetic variability within a single thallus with an extraordinary flexibility, which is not found anywhere else among living things.
Hyphae forming the somatic body of the fungus normally are undifferentiated and unorganised. Differentiation occurs during reproduction, although some fungi also produce differentiated somatic structures, such as root like mycelial strands, rhizomorphs and more compact hyphal masses (sclerotia).
2.2. Reproduction and life cycle Fungi reproduce both sexually and asexually. The sexual life cycle consists of mating and meiosis, while the asexual cycle is mitotic. Fungi produce spores as means of reproduction, dispersion, and/or survival of harsh conditions. Sexual spores are usually associated with meiosis (meiospores). Between and during the cardial stages of syngamy (mating) and meiosis occur periods of mitotic growth and development, characteristic for the several taxonomic groups of fungi. Most fungi are haploid during the main assimilative phase of their existence (mitotic growth, asexual reproduction, mitospores), although there are several noticeable exceptions to this (e.g. Pythium sp. and Phytophthora sp. ( Oomycota), Saccharomyces sp. (Ascomycota)). The mitospores serve to clonally disperse the individual. Mating of the fungi may require two genetically different individuals to achieve sexual combination (heterothallism); however, with many fungi a single haploid strain completes the sexual cycle by itself (homothallism).
There exist 12 phyla of fungi (CABI bioscience Databases). Three of these are well studied and are most generally discussed. They can be classified according to their sexual reproduction structures (23): - The Ascomycota : the phylum of the Ascomycota is the largest of the fungal groups, which can contain more than 75 % of all fungal species. Some well known members of this phylum are Saccharomyces cerevisiae , Candida albicans, Penicillium sp., Aspergillus sp., and the fungus used in this research, Myrothecium gramineum . The Ascomycota are characterised by the formation of ascospores within specialised hyphal segments of the cell, the asci. The ascus is commonly the site of nuclear fusion and meiosis, and the zygotic nucleus formed within it, is the only diploid stage of the life cycle, except for some yeast, such as Saccharomyces . The Ascomycota can be classified in subgroups, according to the structures in which the asci are born (Figure 2-2). Some Ascomycota have naked asci, formed by the differentiation of hyphal segments (or yeast cells), with no Literature review 9
protective sterile tissue associated; e.g. Saccharomyces . Other Ascomycota form asci within mycelial structures (the ascomata) which differentiate as part of the sexual cycle into perithecia (small opening), cleistothecia (closed) or apothecia (wide open, like a cup). Fungi belonging to this subgroup are Neurospora , Sordaria , Gibberella ( Fusarium ), Emericella ( Aspergillus ), Claviceps and Sclerotinia . The third and last subgroup differs from the second group in forming asci within cavities, pseudothecia, formed within stroma, a dense, usually black-pigmented tissue.
Figure 2-2. Structures of ascomata.
- The Basidiomycota : the Basidiomycota constitute nearly 25 % of the described species of fungi. They are characterised by the formation of spores on differentiated hyphal tips (basidia), which are usually the site of nuclear fusion and meiosis. Basidia are analogous to asci and differ in that the basidiospores are born externally to the basidium, rather than internally, as with the asci. Basidiomycota are classified according to the form of the basidia and their supporting structures: the basidia can be fleshy, as with the well known mushrooms, or they lack the fleshy structures and produce basidia from thick-walled spores, the teliospores, as with the fungi which cause rusts and smuts on plants (e.g. Ustilago maydis and Puccinia graminis f. sp. tritici ). - The Zygomycota : the Zygomycota comprise 1 % of all known fungi. They are fast growing fungi and are commonly found in the soil or on sugar containing fruits. They produce zygospores in zygosporangia, which originate after the fusion of special hyphae, from which 1 zygospore is produced per zygosporangium (24). The Zygomycota include Rhizopus , Mucor and Phycomyces species.
Since the fungus Myrothecium gramineum studied here belongs to the Ascomycota , a more detailed description of the life cycle of these fungi will be given (Figure 2-3). Ascospores germinate to form septate haploid mycelium. Asexual reproduction (arrow 1 in Figure 2-3) is accomplished by macroconidia and microconidia produced on specialised aerial hyphae, called conidiophores. The macroconidia are multinucleate and germinate rapidly, while the microconidia are predominantly uninucleate and germinate poorly. The sexual cycle is initiated by the differentiation of an ascogonium consisting of a swollen, coiled hypha with an apical branched trichogyne. The ascogonium is quickly enclosed by nearby hyphae that grow closely around it, forming a protoperi-, cleisto, apo- or pseudothecium with the trichogyne projecting out. The trichogyne fuses with a spermatial element (spermatocyst) of the opposite mating type or of the same mating type. Spermatia Literature review 10 may be microconidia, macroconidia or hyphae. The nuclei of the spermatium (spermatocyst) enter and migrate through the trichogyne to the ascogonium, forming the ‘dikaryotic’ stage (arrow 2) (plasmogamy). Ascogenous hyphae grow from the fertilised ascogonium, forming a basal hymenial layer within the developing ascomata (arrow 3). Most of the tissue of the ascomata arises from the monokaryotic mycelium that produced the ascogonium. The spermatial and ascogonial nuclei migrate into the growing ascogenous hyphae, dividing as they go. As development proceeds, nuclei in the tips of the ascogenous hyphae pair together and divide simultaneously, while the tip of the hyphae grows back on itself to form a hook, or crosier (Figure 2-4).
Figure 2-3. Life cycle of the Ascomycota .
Figure 2-4. Hook development during dikaryotic growth of Ascomycota . Two septae form in the crosier making the cell tip uninucleate, the second cell binucleate and the third cell uninucleate. The first and the third cells fuse, restoring the binucleate condition and a short branch grows from the third cell to repeat crosier formation (arrow 4). The nuclei in the second cell fuse (karyogamy), forming a zygote and immediately proceed to meiosis. This cell is called the ascus (arrow 5). After meiosis, a single mitosis produces an eight- nucleate ascus and each nucleus is enclosed within a cell wall within the ascus, forming the Literature review 11 ascospores (haploid). When the ascus is mature, the ascospores are released, upon which germination and the start of a new cycle follows.
When the hyphae which mate are of the same mating type, the resulting ascogenous hyphae will be homothallic. On the other hand, when they are of the opposite mating type (as in Figure 2-3) they will be heterothallic. Aspergillus sp. are homothallic, while Neurospora sp. are heterothallic.
The germ tube of a spore grows with an accelerating growth speed until a maximum linear rate is achieved. Some distance back form the tip, branches form at characteristic intervals and grow at rates equal to or less than that of the main axis. A variety of factors, including the activity of the axis hyphal tip, the ingredients of the medium and the temperature affect the timing and density of branch formation in ways that can be best described as apical dormancy. The acceleration phase of the growth of the germ-tube is exponential, that is, the logarithm of length is proportional to time. As the maximum linear growth rate of the tip is established, branching begins behind the tip, such that the sum of the lengths of the germ tube and its branches increases exponentially with time. An extended portion of the mycelium contributes to the synthesis of the protoplasm required for continued growth at the hyphal apex. Microscopic examination of hyphae showed vigorous forward streaming of protoplasm in young hyphae and vacuolation in older hyphae. Thus two events, synthesis of new material and forward migration of the protoplast, are occurring as the mycelium expands. The apical pattern of growth is correlated with the presence of a vesicular aggregation at the growing tip. Some of these vesicles apparently fuse with the plasmalemma and deliver several ‘products’, such as membrane to the plasmalemma, prefabricated polysaccharides to the periplasmic space, enzymes and activated monomers, digestive enzymes and other secretions for release to the external medium. A variety of constraints may be applied to this tendency in a growing thallus.
2.3. Regulatory circuits in filamentous fungi In fungi - as in other organisms - growth requires the presence of certain chemical substances, in appropriate concentrations. The nutritional requirements may be divided into two concentration classes (23): macronutrients required at about 10 -3 M and micronutrients, required at 10 -6 M or less. Of the macronutrient elements (carbon, hydrogen, oxygen, phosphorous, potassium, nitrogen, sulphur and magnesium), carbon is required as the major structural element of the organism, primarily in combination with hydrogen, oxygen and nitrogen. Examples of essential micronutrients are iron, copper, manganese, zinc and molybdenum. Since fungi are capable of growing on many different substrates, are degrading many compounds, are recycling carbon, nitrogen and sulphur compounds as nutrients for their own growth and that of other organisms, and are breaking down and detoxifying wastes and other pollutants, they are expected to have a huge set of genes and enzymes required for such capacities and to possess regulatory circuits which enable them to adapt to and to grow in these variable and sometimes extreme conditions. Thus, filamentous fungi are excellent Literature review 12 experimental subjects for the study of gene regulation in eukaryotic organisms (25). The ability to regulate gene expression and to adapt to environmental changes helps living organisms to survive. Study of regulation phenomena by genetic and molecular biological methods is therefore of growing interest, not only because of biotechnological importance but also from an evolutionary point of view.
2.3.1. Glucose repression in fungi Microorganisms usually turn off a range of genes required for the utilisation of less favoured carbon sources in the presence of glucose or other more readily used carbon sources. In yeast and filamentous fungi, the mechanism of glucose repression is completely different from that in bacteria, where the so called carbon catabolite repression is triggered by glucose phosphorylation causing a drop in the level of cAMP. The mechanism in fungi includes a global regulatory gene coding for a negative acting repressor protein, for example, the CreA repressor in A. nidulans . In A. nidulans , a large number of mutations which altered response to carbon catabolite repression have been isolated. They are located in the creA , creB and creC loci. The creA mutations lead to derepression of many genes which are normally under control of carbon catabolite repression. The gene creA codes for activator proteins of specific pathways which bind to the 5’ untranslated region of the genes at the consensus sequence 5’-SYGGRG-3’. Not all possible sites derived from this consensus do in fact bind. This binding site was found under the form of 5’-CTGGGG-3’ and 5’-GCGGAG-3’ in the proline gene cluster and as 5’-SYGGGG-3’ in the alcR promoter of A. nidulans (25). The structural gene alcA (encoding alcohol dehydrogenase) is both directly repressed by CreA and indirectly repressed via CreA- mediated repression of transcription of the specific activator gene alcR (2). Moreover, the binding sites of AlcR and CreA partially overlap each other. This system is referred to as the ‘double-lock mechanism’. An enzyme with a function similar to CreA in A. nidulans was identified in Hypocrea jecorina (Trichoderma reesei ) and is referred to as Cre1. Cre1-binding sites were for example found in the cellobiohydrolase I promoter around positions -700 and - 1000 upstream of the protein coding region, two of which are arranged as a tandem repeat (26).
2.3.2. Nitrogen catabolite repression Utilisation of various nitrogen-containing compounds in fungi requires the regulated expression of a set of unlinked structural genes which code for various nitrogen catabolic enzymes. Those enzymes required for assimilation of other than ammonium nitrogen sources are expressed only when concentrations of ammonium, glutamine or glutamate become growth limiting. The major regulatory genes, nit-2 ( N. crassa ), areA ( A. nidulans ), nre ( P. chrysogenum ) or nmc ( P. roquefortii (27)) positively regulate the expression of the structural genes for assimilation of secondary nitrogen sources, including nitrate, nitrite, urea, proteins and amino acids. Knocking out the genes coding for these regulatory factors results in the inability to use the secondary nitrogen sources. All these genes code for regulatory products which bind to Literature review 13 specific promoter elements upstream of the functional genes by a single zinc-finger DNA- binding domain. There is a high degree of conservation between the major regulatory proteins. The specific binding sites for the nitrogen regulatory proteins all appear to possess at least two GATA (or TATC) sequence elements. The binding sites differ in number, orientation, distance between them and their location with respect to the transcription initiation sites. Nearly all substitutions for bases within the GATA elements result in complete loss of binding. It was recently found that AreA not only acts as a transcriptional activator in response to nitrogen limitation, but that it also regulates the expression of ammonium transporter proteins in A. nidulans . GATA sequences were found in the transporter encoding genes meaA , mepA , mepB and mepC (28).
Another major nitrogen regulatory gene, named nmr (nitrogen metabolic regulation), is unlinked to any of the nitrogen-related structural genes and codes for a negative acting regulatory protein. Mutations in this locus led to a loss of nitrogen catabolite repression (25).
Besides the global-acting nitrogen regulatory proteins, the expression of structural genes is dependent upon pathway-specific regulatory proteins (25).
2.3.3. pH regulation Besides carbon and nitrogen repression, changes in environmental pH are a major determinant of gene expression. The response to pH changes in the medium of A. nidulans is mediated by the zinc-finger pacC transcription factor, an activator of alkaline-expressed genes and a repressor for acidic-expressed genes. Six other genes, palA , palB , palC , palF , palH and palI , are involved in pH-mediated control by transduction of the extracellular pH signals, which is important for conversion of pacC into a functional form. Under alkaline conditions, the pal gene products induce processes which allow the proteolysis of the 73 kDa pacC translation product to a shorter zinc finger protein by removal of the C-terminus. This activated regulatory protein is then able to prevent transcription of acidic-expressed genes and to activate transcription of alkaline-expressed genes by binding to the DNA consensus sequence 5’-GCCARG-3’ (2, 25).
2.3.4. Phosphorous regulation When fungi are grown in media with limiting amounts of inorganic phosphate or rate-limiting phosphorous sources, a number of enzymes are expressed which make phosphate more available to the cell. Such enzymes are alkaline or acidic phosphatases, phosphate permeases and a number of nucleases. At least 4 genes are involved in N. crassa : nuc-2, preg , pgov , and nuc-1. The nuc-1 gene product is necessary for the expression of the structural genes which are involved in phosphorous acquisition. The NUC-1 is part of a signal transduction pathway involved in supplying the cell with phosphorous under conditions of phosphate starvation. The protein binds to the motif CACGTG included in promoters of the NUC-1 target genes. The products of the preg- and pgov -genes act together and prevent the action of the nuc -1 product. The nuc-2 product inhibits the function of the preg- and pgov -products. Phosphate or Literature review 14 its derivates inhibits the action of the nuc-2 product. In this model of the signal transduction pathway, the amount of NUC-1 is constant and the preg and pgov negative regulatory factors act together and inactivate NUC-1, unless they are inactivated by the gene product of nuc-2 (25).
2.3.5. Sulphur regulation The sulphur containing amino acids methionine and cysteine play a critical role in the de novo synthesis of nearly all proteins. Therefore, efficient regulation is important to ensure a steady supply of sulphur. Fungi can use various inorganic and organic sulphur sources, but the favoured ones are methionine and cysteine. Utilisation of secondary sources requires the transcription of corresponding sulphur catabolic genes. In N. crassa , a major positively acting regulatory gene, cys-3, was found. This gene encodes a sequence-specific DNA binding protein which recognises elements in the promoter regions of sulphur-regulated structural genes, such as aryl sulphatase, sulphate permease, extracellular alkaline proteases and others. The gene products of scon-1 and scon-2, two other regulatory genes, repress the synthesis of cys-3 mRNA when primary sulphur sources are available. Furthermore, the expression of cys-3 after sulphur exhaustion is the subject of strong positive autogenous regulation leading to an amplified level of CYS-3 protein (25).
3. THE FUNGUS MYROTHECIUM GRAMINEUM In a preliminary study, the BCCM/MUCL agro-industrial fungi- and yeast collection was screened for potentially interesting, new fungal hosts (29). Initially, 3000 different fungal strains and species of the MUCL collection were selected, based on their non-pathogenetic characteristics and the fact that a transformation system was not yet described, nor in the scientific literature, nor in patents. These strains were cultivated in liquid culture and on solid medium. To begin with, strains were selected based on their growth rate, biomass production, protein production, enzyme production and on their morphology. One tenth of the strains were picked out. These strains were submitted to a second selection round, this time carried out in liquid cultures. Different media were used, each with a specific purpose: maximal protein production, maximal biomass production or enzyme induction. The screening criteria retained were the same as for the first step. Based on statistical analysis, 50 strains were selected which had the highest scores for one or more criteria in the different media. In a next round of selection, all strains producing anti-bacterial or anti-fungal compounds were eliminated. The 30 remaining strains were tested as to their ability to take up foreign DNA and express the TAKA-amylase of Aspergillus oryzae as a reporter gene.
Finally, Myrothecium gramineum MUCL 39210 (syn. Xepiculopsis graminea ) seemed to possess all desired qualities necessary to perform as an interesting expression host. These qualities are, amongst others, a good expression of homologous and heterologous enzymes, poor protease production, a good transformability and good fermentation characteristics. The systematics of the Myrothecium genus and some biotechnologically relevant fungi is presented in Table 3-1. Literature review 15
The strain used in this research, Myrothecium gramineum MUCL 39210, was isolated from a dead branch from the Royal Chitwan National Park beneath the Himalaya in Nepal and is available in the Belgian Co-ordinated Collection of Micro-organisms (BCCM /MUCL). A taxonomical reidentification of the strain Myrothecium gramineum MUCL 39210 confirmed that it presents all morphological characteristics of the species Myrothecium gramineum , as described by Tulloch (30) and Nag (31). Myrothecium gramineum MUCL 39210 has also been characterised on molecular level, together with 4 other strains currently assigned as Myrothecium gramineum . The DNA coding for the RNA ITS (Internal Transcribed Spacer) and the 28S regions was amplified and sequenced. The sequences were aligned using the software ClustalX 1.5b and then a bootstrap tree was constructed using the software PAUP 4.0b10. The phylogenetic trees revealed that some of the investigated strains have been erroneously assigned to the species ‘ gramineum’ . However, all the strains studied, with the exception of strain MUCL 11831, belong to the Myrothecium genus (29).
The genus Myrothecium was first described in 1790 by Tode who depicted it as a cup-shaped fungus with spores that become slowly viscous. At first only three species were described: M. inundatum , M. roridum and M. verrucaria . In 1972, Tulloch revised the genus and described 8 known species. Nowadays, the genus includes 65 different species (Cabi Bioscience Databases, Dictionary of the Fungi). Their conidiophores are united together to form fruiting structures (sporodachia) which are usually flat, but may be on a stalk. The spores (conidia) are born from the tips of the long philiades which are in turn born on densely branched and brush- like conidiophores. The mass of dark green conidia collect in large green to blackish wet drops (Figure 3-1) (30).
Many Myrothecium species, for example M. cinctum , M. roridum and M. verrucaria are able to degrade cellulose. Because of their strong cellulolytic activities, M. verrucaria species are used as standard test organisms for the degradation of textiles by fungi (32). M. verrucaria was once described as ‘one of the best cellulose degrading organisms known so far’ (33).
Some Myrothecium species produce antibiotic compounds, such as M. roridum and M. verrucaria . Examples are the myrothecins, roridins and verrucarians (34). The possibility to use myrothecins as a tyrosinase inhibitor for the treatment of local hyperpigmentation diseases is currently investigated (35). Roridin A, a cytostatic compound, was isolated from cultures of Myrothecium verrucaria and Myrothecium roridum . The accumulation of roridins can cause livestock poisoning and other veterinary problems. These compounds may even be a dangerous threat to human health (36). The toxins are believed to have been the causative agents of several mycotoxicoses in both humans and animals, in Japan, in the U.S.A., in the former U.S.S.R. and in Northern Europe. Various toxicoses have been ascribed to the intake of food contaminated by Myrothecium verrucaria (37). The production of dermatitic or skin- irritating compounds by Myrothecium verrucaria has also been described (38). Literature review 16
Table 3-1. Systematics of Myrothecium gramineum and some biotechnologically relevant fungi (modified after 16)1
Phylum Class Subclass Order Family Genus
Asco - Eurotiomycetes Eurotio- Eurotiales Trichocomaceae Aspergillus * (G,P) mycota Mycetidae Penicillium * (G,P) Talaromyces * (P) Incertae sedis Monascaceae Monascus * (F) Sordariomycetes Hypocreo- Hypocreale s Hypocreaceae Hypocrea /Trichoderma * (R) mycetidae Incertae sedi s Acremonium * (P) Myrotheciu m Tolypocladium * (P) Nectriaceae Fusarium /Gibberella * (G,P) Sordario- Diaporthales Cryphonectriaceae Cryphonectria /Endothia (G) * Mycetida Sordariales Sordariaceae Neurospora * (F) Saccharo- Saccharo- Saccharo- Eremotheciaceae Eremothecium /Ashbya * (G) mycetes mycetidae Mycetidae mycetales Incertae sedis Candida* (G) Geotrichum (F) Yarrowia * (F) Saccharo- Hansenula /Pichia * (R) mycetaceae Kluyveromyces *(G) Saccharomyces * (G) Schizosaccharo- Schizosaccharo- Schizosaccharo- Schizosaccharo- Schizosaccharomyces * (F) mycetes mycetidae mycetales mycetaceae Basidio- Basidiomycetes Agarico- Agaricales Agaricaceae Agaricus * (F) mycota Mycetidae Marasmiaceae Flammulina *(F) Lentinula * (F) Pleurotaceae Pleurotus * (F) Pluteaceae Volvariella * (F) Polyporales Ganodermataceae Ganoderma * (F) Incertae sedis Auriculariales Auriculariaceae Auricularia (F) Zygo- Zygomycetes Incertae sedis Mortierellales Mortierellaceae Mortierella * (G) mycota Mucorales Mucoraceae (Rhizo)mucor * (G) Rhizopus * (G) * Abbreviations: F = food; G = GRAS-status; P = production of pharmaceutical compounds; R = recombinant protein production. Genera for which genetic transformation procedures are already described are indicated with an asterix (recently developed for Flamminula (39), Ganoderma (40), Monascus (41) and Rhizopus (42 and 43). The systematics of M. gramineum are underlined.
1 Classification based on the Ainsworth and Bisby's Dictionary of the Fungi, 9 th edition, available at www.indexfungorum.org/Names/fundic.asp (June, 7 th , 2007) Literature review 17
Figure 3-1. The genus Myrothecium . Left: conidiophores and conidia. Middle: green spores of Myrothecium formed like an American football (Copyright 2006, Envirox, Inc). Right: Pure colony of Myrothecium roridum (Copyright 2006, The American Phytopathological Society). Most Myrothecium species are cosmopolitic and are associated to plant waste or found in the soil, in moderate climates, as well as in tropical climates (44). Despite their ecological success, no sexual form of a Myrothecium species is known (45). Thus, M. gramineum has an asexual reproduction cycle, which is mitotic. Most fungi are haploid during their mitotic growth.
Most species are saprophytes, but only M. roridum and M. verrucaria can be considered as serious plant pathogens as they often cause leaf spot disease (46, 47). Myrothecium leaf spot appears in severe form causing even defoliation. The fungus causes small, dark brown circular lesions, which most frequently appear on wounded areas of leaves. It appears first on young plant leaves only, but later it may cause damping of the seedlings. The disease is favoured by wet weather conditions and has been observed mostly in the Lower Rio Grande Valley. It can appear for Figure 3-2. Myrothecium leaf example on begonia’s, on New Guinea Impatiens plants, spot on New Guinea Impatiens on watermelon, on soybean and on peanuts (Figure 3-2). (by Jan Byne).
The genus Myrothecium is actually an artificial genus and is liable to criticism. Not only the results of the molecular characterisation (ITS and 28S rDNA sequences) of 5 Myrothecium gramineum strains by Jonniaux et al. (29) indicated problems with the (incorrect) classification of certain strains. Tulloch (30) already recognised difficulties to delimit Myrothecium morphologically from other genera. The genus has been controversial since it was created (48). For example, the decision to select the colour of the conidial mass as the main characteristic was criticised by Nag Raj (48, 49). Ahrazem and co-workers (46) were able to conclude that Myrothecium is a heterogeneous genus, based on a study of the composition of the cell wall polysaccharides of different Myrothecium species.
Literature review 18
Next to morphological and molecular characteristics, cell wall mono- and polysaccharides of fungi can be useful to classify fungi and study their evolution (50). Because the cell wall polysaccharides of M. gramineum , M. atrum and M. cincutum are so different of those of other Myrothecium species, they concluded these three species probably do not belong to this genus. To which other genus they do belong was not investigated. In contrast to the results of Ahrazem et al. , Seifert and co-workers (48) decide that the species Myrothecium gramineum and M. cinctum are correctly included in the Myrothecium genus. Their study was based on the analysis of the sequence of the 5’ end of the large subunit rDNA. In short, one can state that the classification of certain species within the Myrothecium genus remains uncertain, as will be discussed further in this work.
The fungus Myrothecium gramineum was chosen during a preliminary study because no transformation system was described for this species, or for other species of the genus at that time (29). In the patent resulting from the study, a basic transformation system using an antibiotic resistance marker as selection system is described. In the following paragraphs, the general methodology of transforming filamentous fungi are explained, as well as the selection markers most regularly used and the ways fungi integrate foreign DNA in their genome.
4. TRANSFORMATION METHODS OF FILAMENTOUS FUNGI Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material.
The ability to transform Saccharomyces cerevisiae using auxotrophic markers and the development of Escherichia coli shuttle vectors for this organism represented the beginning of fungal molecular genetics (51). In 1973, Mishra and Tatum reported the first successful transformation of a filamentous fungus, Neurospora crassa (14, 52). The results of these early investigations were not generally accepted until, in 1979, Case et al. (53) were able to prove via Southern analysis that exogenous DNA had integrated in the genome of N. crassa . Since the development of a protoplast transformation system for N. crassa , many fungi were transformed with success. Amongst them are some industrially important ones, such as Aspergillus niger (54), A. oryzae (55) and Hypocrea jecorina (Trichoderma reesei ) (56). By now, all major groups of fungi, including the Ascomycota , Basidiomycota and Zygomycota , can be transformed.
Presently, five different techniques for the transformation of filamentous fungi are described in the literature (51): protoplast transformation, electroporation, lithium acetate mediated transformation, biolistical (or shot-gun) transformation and Agrobacterium tumefaciens mediated transformation. For shot-gun transformation (i.e. biolistical transformation) and for electroporation, expensive special equipment is necessary and the method with A. tumefaciens has been patented. The lithium acetate method is not generally applied, except for Saccharomyces cerevisiae . In our research, the protoplast transformation system was chosen, because no special equipment is required and because it is one of the most commonly used Literature review 19 techniques to transform fungi and the method has already been used with success to transform M. gramineum (29).
4.1. Electroporation This technique is based on the reversible permeabilisation of bio-membranes by the application of short electronic pulses with high amplitudes. The changes in the membranes during the electric pulse allow the uptake of (recombinant) DNA, which, in turn, can result in molecular transformation. Electroporation seems to be based on the general ability of biomembranes to produce channels when exposed to an electric field (57). The technique is rapid, simple to perform, and avoids the use of chemicals such as polyethylene glycol (PEG). The electric field strength and the time constant of the electric pulse are important factors in electroporation, although the high amplitude of the electric pulse is of greater importance than its duration. Electroporation is used for the transformation of animal cells, plant protoplasts, bacteria, yeasts and filamentous fungi. For fungi, three procedures for electroporation are applied (51). In the first procedure, the conidia of the fungi are pre-treated with a cell wall weakening agent. An alternative procedure is the electroporation of protoplasts. In the third method the use of protoplasts is avoided by using germinating spores to electroporate. This method is for example used with A. nidulans (58). Electroporation is frequently used for filamentous fungi and has already been successfully employed with A. oryzae (59), A. nidulans (58), A. niger (60), A. fumigatus (61), Leptosphaeria maculans (59), N. crassa (59), Penicillium urticae (59), Scedosporium prolificans (62) and Wangiella dermatidis (61).
4.2. Lithium acetate treatment The lithium acetate treatment, which avoids protoplast formation, is often used with S. cerevisiae and has already been used with success for N. crassa (63), Coprinus cinerus (64) and Ustilago violacea (65). In all procedures, germinating spores are exposed to the transforming DNA in the presence of 0.1 M lithium acetate. Exactly how alkali metal cations assist the passage of DNA into cells does not seem to be well understood (51). The addition of high concentrations of PEG after an initial period of exposure to the transforming DNA is a step used in the lithium acetate method as well as in transformation procedures using protoplasts (51). Usually, about ten volumes of 40 % PEG 4000 are used. The PEG is added together with CaCl 2 and buffer. It causes the coagulation of the cells and the DNA and may enhance in this way the uptake of DNA.
4.3. Biolistical transformation Not all filamentous fungi can be transformed by the above described methods. Moreover, fungi exist of which protoplast production is not possible so far. Foreign DNA can be introduced in these fungi by biolistic (biological-ballistic) transformation procedures. In these methods, thick walled, intact cells are shot with tungsten particles. The particles are coated with the DNA of interest, which enters the cells at high speed. One can use fungal hyphae or even spores. Single penetrations underlie the vast majority of biolistic transformation events. Literature review 20
A disadvantage of the method is that expensive, specialised equipment is required. Advantages over other existing transformation methods are the fact that the simplicity of the technique allows one to target hundreds of millions of cells simultaneously and without preparatory treatments, and the fact that it is not limited to any particular species and has been successfully employed both in systems already transformable by other means as in less tractable systems (66). Biolistical transformation, also called ‘shot gun transformation’ has been fruitfully applied on filamentous fungi like A. nidulans (67), Magnaporthe grisea (68), N. crassa (68), Trichoderma harzianum (68) and Hypocrea jecorina (69).
4.4. Agrobacterium tumefaciens -mediated transformation The Agrobacterium tumefaciens mediated transformation procedure is a method frequently used for plant cells (4, 70). This method is based on the fact that A. tumefaciens is able to transport a part of the extrachromosomal tumour inducing Ti plasmid, more precisely the T-DNA which is flanked by 25 bp imperfect direct repeats, to an eukaryotic target cell. The T-DNA then integrates into the host nuclear genome at a random position. The process of T-DNA transfer depends on the induction of a set of virulence ( vir ) genes, which are also located on the Ti-plasmid (71). The DNA is carried over as a single stranded molecule, assisted by the DNA-binding proteins VirD2 and VirE2 (reviewed in reference 72). As observed in plants, an intact virulence system of A. tumefaciens is necessary for T-DNA transfer in yeasts and filamentous fungi.
The transformation of filamentous fungi with the aid of Agrobacterium tumefaciens was successfully performed for the first time by De Groot et al. (71). They showed that protoplasts, as well as conidia of Aspergillus awamori could be efficiently transformed via A. tumefaciens . Transformation efficiencies which were 600 times higher as compared to conventional transformation with protoplasts were achieved. The majority of the transformants had integrated one T-DNA copy. The T-DNA is integrated at random in the genomic DNA, similarly as known for plants. Different filamentous strains have been transformed by this method, including Agaricus bisporus , A. niger, Colletotrichum gloeosporioides, Fusarium venenatum, Hypocrea jecorina and N. crassa (71). Unfortunately, this method is protected by different patent applications.
Recently, several transformation methods were compared for Aspergillus giganteus (73). A. giganteus was transformed using four methods: protoplast transformation, electroporation, biolistical transformation and Agrobacterium mediated transformation. Electroporation and biolistical transformation appeared to be inefficient, while the conventional method via protoplasts produced 55 transformants out of 10 8 protoplasts per µg DNA. This number of transformants was multiplied 140 times with A. tumefaciens transformation. Furthermore, it could be noticed that these A. giganteus transformants integrated only one copy of the T- DNA, randomly, while several copies were found with protoplast transformation.
Literature review 21
Gouka et al. (74) proved when the Ti DNA is homologous to the A. awamori genome homologous recombination can occur. In this way, transformants which have one or more copies of a certain gene integrated at a defined place in the genome can be achieved. Michielse and co-workers (70) compared the efficiency of homologous recombination with
Agrobacterium mediated transformation in A. awamori to its efficiency with CaCl 2/PEG mediated transformation. They found that the frequency of homologous recombination with
Agrobacterium mediated transformation is 3 to 6 times higher as with CaCl 2/PEG transformation. Additionally, for efficient homologous recombination, shorter homologous flanks could be used as compared to the CaCl 2/PEG protoplast transformation.
4.5. Protoplast transformation Protoplast transformation basically involves three steps: - the preparation of the protoplasts - the DNA- uptake - the regeneration of the protoplasts and the selection of the transformed cells.
4.5.1. Preparation of the protoplasts Protoplasts are osmotically stabilised ‘buds’ of cytoplasm surrounded by a cytoplasmatic membrane (the plasmalemma), which have been liberated from hyphae treated with cell wall degrading enzymes. The fungal cell wall is a multilayered structure (Figure 4-1, after 23). Exocellular and capsular material, the outermost layers, have not been demonstrated in all fungi, but they are very common. More inside, the walls of most fungi have two or more discernable layers. The outer most layer is amorphous and is composed of hot water or alkali- extractable material. This layer merges with, and perhaps extends into an inner layer that contains the most chemically resistant, fibrillar component of the wall. Intermediate layers of various kinds also have been described. The alkali soluble fraction layer contains glycans, heteroglycans and glycoprotein. The alkali insoluble fraction contains chitin and/or cellulose and insoluble glucan. The chitin, cellulose and β-glucan form fibrillar networks which give the wall its strength and form. The composition of the fungal cell wall not only varies between different fungal strains, but also between different developmental stages. The form of the fungi depends on the composition of the wall as well as on the materials within it (23).
Figure 4-1. Model of the layered structure of the hyphal wall. Literature review 22
The intimate contact between the fungus and its environment and the critical roles of the cell surface in the structure and development of fungi make a thorough understanding of the cell wall and the plasmalemma critical to the understanding of fungal physiology. The wall contains recognition factors on its surface which are involved in agglutination, mating and other interactions. Several enzymes, including the synthases and hydrolases involved in wall metabolism and the hydrolases involved in the digestion of difficult nutrients are located within the wall. Digestive enzymes secreted into the medium must pass through the wall, and some wall components serve as storage reserves that are accumulated at certain times and hydrolysed at certain others. Clearly, the wall is a vital, living component of the fungus, as important as any cytoplasmatic organelle (23).
The first report describing the liberation of protoplasts from the cell wall of the filamentous fungus N. crassa appeared in 1958 and since then protoplasts have been obtained from a variety of fungi (75).
As starting material for the preparation of protoplasts one can use germinating spores, young mycelium or basidiospores. Germinating macroconidia are mostly used for N. crassa . They are mainly multinuclear. Mononuclear microconidia can also be used, but they are difficult to obtain (76). For Basidiomycota , one can use basidiospores, dikaryotic mycelium or asexually produced conidia. The choice of the cell type which will be used to start with is a matter of preference and ease (51). In our research, young mycelium, grown for 24 hours in liquid cultures will be used.
The choice of the cell wall degrading enzymes is crucial for the preparation of viable protoplasts (77). Relating factors required to be optimised are the enzyme digestion time and the incubation temperature (78). Formerly used mixtures are for example Helicase and Glusulase mixtures from snail stomachs or an enzyme concentrate produced by the bacteria Arthrobacter luteus (Zymolase 100T). The first successful transformations of N. crassa were performed with Glusulase, but until recently the most commonly used enzyme mixture was that produced by Trichoderma viride , commercially known as ‘Novozyme 234’ (68). Today, this mix is not on the market anymore, but plenty of other enzymes and mixture can be used. All of them contain complex mixtures of hydrolytic enzymes, such as 1,3-glucanases, chitinases (79), cellulases (51), β-glucuronidase (80) and proteases (77). In our research, a mixture of enzymes consisting of driselase, α-1,4-glucanase and yeast lytic enzymes will be used (29). The use of proteases or their presence as contaminants in the mixtures has to be carefully considered because they can damage the plasmamembrane, which hampers correct regeneration of the protoplasts. On the other hand, more protoplasts can be formed when proteases are used (77).
Protoplast preparation is always carried out in the presence of osmotically stabilising agents to prevent the protoplasts from bursting. One can use NaCl (0.6-0.7 M), KCl, MgSO 4 (1.2 M),
(NH 4)2SO 4, mannitol (0.8 M), sorbitol or sucrose (77). Sorbitol in concentrations between 0.8 Literature review 23 and 1.2 mol/L is most often used (51). Neurospora protoplasts stabilised in sorbitol are viable for a long time at -70°C (79). Generally speaking, inorganic salts have proved more effective with filamentous fungi, and sugars or sugar alcohols are more effective with yeast. The virtues of particular stabilisers are understood only in an empirical sense, and the differences in effectiveness must relate to unknown factors in the uptake and utilisation of the particular compounds (81).
Osmotic stabilisation is also depending on the pH of the solution wherein the protoplasts are suspended during the breakdown of the cell wall. Buffers are used in order to keep the pH stabilised. In the literature, sodium phosphate buffers, potassium phosphate and succinate buffers at pH 5.6 or 5.8 are described most.
When large scale production of protoplasts is necessary, the use of enzymes to break down the cell wall can become expensive, either in cost of the purchase of the commercial enzyme or in time for the laboratory production of enzymes. Some laboratories have therefore developed procedures to create protoplasts without lytic enzymes. For example, a method for Schizosaccharomyces pombe involves the culture of cells in the presence of 2-deoxy-D- glucose and high concentrations of magnesium sulphate. Another highly specialised process of protoplast release has been reported for an Entomophthora. Conidia of this entomogenous fungus inoculated into insect tissue culture medium produced germ tubes that later released their cytoplasmic contents as protoplasts (81). Costanzo and Fox (79) have reported successful transformation of S. cerevisiae cells by suspending them in 1 M sorbitol and DNA, adding glass beads and agitating the mixture at the highest rate of a vortex mixer for 30 seconds. This violent treatment killed 80 to 90 % of the cells, but transformed colonies were produced. The efficiency of transformation was rather low, but the simplicity of the method is attractive. After the cell wall disruption, the protoplasts have to be separated from the remainders of the mycelium and from the enzymes. This is done by filtration, followed by centrifugation and several wash steps. Optimalisation of the centrifugation parameters and the number of wash steps is critical.
4.5.2. DNA-uptake The quality of the transforming DNA, usually either linear or circular double stranded, can be of crucial importance for the efficiency of a transformation procedure. Plasmids which are purified twice by CsCl-centrifugation steps can perform 5 to 10 times better than those which have been centrifuged only once (82). At the present time, CsCl-centrifugation is not done any more because it is a very laborious and because of the fact that toxic compounds are involved. Many plasmid purification kits are currently for sale, which provide large amounts of high quality DNA.
In some cases, linearization of the transforming plasmids can enlarge the efficiency of transformation (80). The length of the transforming DNA also has an effect on the amount of Literature review 24 transformants obtained. In certain experiments it was shown that the efficiency is higher when the DNA is shorter. The elimination of bacterial sequences present on the plasmids used for transformation could not only improve the transformation efficiency, but is also recommended because in that way, the incorporation of exogenous DNA in the host genome is avoided. This can be useful when the transformants will be used in industrial applications, for example in the food industry or for pharmaceutical purposes.
The uptake of DNA by protoplast takes place in the presence of PEG and CaCl 2. The main variables in a transformation protocol are the concentration of the DNA, of the protoplasts, of
CaCl 2 and PEG, the incubation time and the buffer in which the DNA is dissolved (77). In 7 8 most protocols, between 1-10 µg DNA is mixed with 10 to 10 protoplasts. The CaCl 2- concentration can be about 10 to 50 mM. The buffers can vary, but 10 mM Tris-Cl (pH 7.5 or 8.0) or MOPS (pH 6) are commonly used. The amount of PEG can rise to 10 volumes of PEG 4000. The time of incubation with the transforming DNA mostly lasts 10 to 30 minutes.
In some procedures, a heat shock is used to increase the capability of the protoplasts to take up the DNA. The use of a heat shock increased that competence of protoplasts from Podospora anserina 5 to 10 times (82). The protoplasts were incubated for 5 minutes at 48°C and then transferred immediately to ice. The effect only occurred when the heat shock was given before the addition of the DNA. The increase was observed immediately after the shock and remained till ten minutes later.
4.5.3. Protoplast regeneration Protoplast regeneration is the process of de novo synthesis of the cell wall and the associated return to the normal cell form when the protoplasts are incubated in osmotically stabilised (selective) medium. Unfortunately, not all protoplasts are able to regenerate a wall and/or to reverse. In fact, the reversion frequency can be quite variable for any single species and in some cases it can be very low. The reasons for this are unknown. The absence of a nucleus is one obvious factor, and this can be a feature of a large fraction of a protoplast preparation. In filamentous fungi, another significant factor can be the origin of protoplasts in relation to hyphal organisation: protoplasts from distal regions of hyphae may be lacking the capacity for reversion (81).
Broadly speaking, two patterns of morphological development are found in protoplast reversion in the mycelial fungi. In one pattern, protoplasts give rise to abnormally shaped germ tubes, which resemble chains of budding cells and ultimately change to normal hyphae at their tips. An apparent variation to this pattern was described for Trichoderma viride , where the protoplasts having produced the abnormal budlike structure proceeded to produce a normal cell tube from itself. The second and totally different form of development was described in Rhizopus nigricans and later in Schizosaccharomyces commune. In the latter the protoplasts first develop a wall, maintaining a spherical shape, to form a primary cell that later produces a normal germ tube. This distinction in patterns of development is not always as Literature review 25 clear-cut and sometimes both types of development have been described for the same species. This variability may be yet another expression of the heterogeneity of protoplasts isolated from the highly differentiated hyphal structure (81).
The regeneration of the protoplasts is also influenced by external factors, such as the lytic enzymes which were used to break down the cell wall during the protoplast preparation. When developing a protoplast based transformation system for the first time, it is strongly advised to spend a little time in studying the effect of different osmotic stabilisators since they also have an important influence on the regeneration capacity of the protoplasts (77). Another factor which is important for the regeneration is the composition of the (selective) regeneration medium (75). Studies with Fusarium culmorum showed that reversion frequency was influenced by the carbon source in the regeneration medium and values of 5 to 82 % were obtained. Contradictory, in Aspergillus nidulans , no difference was found with complex and defined media. Observations made with protoplasts cultured in liquid media suggest that sometimes higher levels of reversion can be obtained in liquid, but protoplasts of S. cerevisiae and a few other yeasts stand out in comparison to all other fungal protoplasts in that reversion to normal cells only occurs in solid media. In liquid medium, these protoplasts form an incomplete wall that prevents complete reversion (81). In some cases, a lower concentration of osmotic stabiliser is utilised in the medium on which the transformants are plated, for example 0.2 mol L -1 sucrose or sorbitol in the plates, and 1 mol L -1 sucrose or sorbitol in the top agar (see below). This lower concentration of stabiliser has been found to give increased numbers of transformants, and permits a more rapid growth of the colonies than if 1 mol L -1 concentration is used in both the plates and the top agar (83). This is presumably because the high concentration is inhibitory to the regeneration and growth of the protoplasts.
The protoplasts can be plated on selective medium immediately after their exposure to DNA or they can be incubated for a certain time in liquid, osmotically stabilised complete medium. In some cases this approach can lead to a higher number of protoplasts (84). Another method used to increase the number of transformants is the use of a top layer: the protoplasts are incubated on solid, non-selective medium until their cell wall is recovered, after which a thin layer of stabilised, selective medium is poured over the plates. In some strategies, the layer below the top layer already contains a selective agent, but in a lower concentration.
5. SELECTION MARKERS In order to distinguish between the cells (protoplasts) which are transformed and the ones which are not, one needs a selectable marker present on the transforming DNA. Selection of transformants from the background of non-transformed cells depends on the expression of genes conferring readily selectable dominant phenotypes. The first selection marker employed for filamentous fungi was the qa-2 gene, used to select transformants of an inositol-dependent N. crassa mutant (53). The gene encodes catabolic dehydroquinase and is part of the qa gene cluster. The recipient strain carried a stable qa-2- mutation and an arom-9- mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were Literature review 26 selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were qa-2+ and had normal levels of catabolic dehydroquinase.
Since then, different selectable markers have become available for fungal transformation systems. Generally, these can be divided into two major groups. On the one hand, there are dominant selectable markers, such as antibiotic resistance markers. On the other hand, one has wild type genes which complement an auxotrophic mutant (14). For filamentous fungi, as opposed to yeasts, dominant selection markers are preferred, because they do not require a specific mutant genotype (25). Generally, with protoplast transformation, the efficiencies of transformation range between 1 and 10 3 transformants/ g DNA when dominant selection markers are used, and between 10 2 and 10 5 transformants/ g DNA when auxotrophic markers are used (85).
The antibiotic resistance markers commonly used are: - the bacterial hygromycin B resistance marker gene: the E. coli HygB r gene codes for a hygromycin phosphotransferase which inactivates the antibiotic by phosphorylation - the bleomycin/phleomycin resistance marker gene: the Streptoalloteichus hindustanus ble gene encodes a small acidic protein which binds with strong affinity to the phleomycin family of antibiotics. When these antibiotics are bound by the Sh protein, phleomycins can no longer be activated by ferrous ions and oxygen to break down DNA (86) - the neomycin/kanamycin/G418 resistance gene: the neomycin resistance gene (Neo r) of transposon Tn5 codes for a neomycin phosphotransferase which confers kanamycin resistance in E. coli and G418 resistance in yeast. The gene encodes a phosphotransferase which inactivates the antibiotics by catalysing the transfer of the terminal phosphate of ATP to the drug (87) - the fungal benomyl (benlate) resistance gene: the N. crassa bml gene or the A. nidulans benA gene code for a benomyl resistant β-tubulin gene which confers an altered β-tubulin structure. The benomyl resistance is caused by a phenylalanine-to-tyrosine amino acid change at a highly conserved position (88) - the oligomycin resistance gene: certain alleles of the A. niger or A. nidulans oliC gene code for an oligomycin-resistant form of subunit 9 of the mitochondrial ATP synthase complex (51, 79, 89). The use of plasmids bearing the genes coding for the resistance markers allows the transformation of a large variety of fungal species without the need for auxotrophic mutants (90). Preliminary research with Myrothecium gramineum MUCL 39210 showed that the minimal required antibiotic concentrations are 500 µg/mL hygromycin B and >100 µg/mL phleomycin (29).
Another, non-antibiotic, dominant selection marker which can be used for filamentous fungi is the amdS -gene. The acetamidase gene ( amdS ) was used as a selection marker for the first time by Tilburn and co-workers (91) for the transformation of Aspergillus nidulans (51) and has since then been widely used for the transformation of filamentous fungi of industrial Literature review 27 importance, including A. niger, P. chrysogenum and Hypocrea jecorina (92). Because acetamide is a poor nitrogen source for wild type A. nidulans and most other fungi, the acetamidase gene can be used as a selectable marker for many fungi. Transformants which incorporated the amdS gene and which express it are able to grow on media with acetamide as the only nitrogen (and carbon) source. Moreover, the degree of acetamide metabolisation reflects the gene copy number and thus can be used for the selection of transformants with a high gene copy number (Ruiz-Díez, 2002). Rahim and co-workers developed a rapid and qualitative plate assay based on pH change for screening acetamidase producers (93). The method is based on the principle that amidase production is accompanied by the release of ammonia and thus by an increase in the pH of the culture filtrate. Phenol red was added to the selective medium containing acetamide as sole carbon and nitrogen source and amidase production caused colourisation of the medium (the colour of phenol red exhibits a gradual transition from yellow to red over the pH range 6.6 to 8.0). Counter selection for amdS positive strains can be performed using their sensitivity for fluoroacetamide (94). Debets et al. and Wernars et al. used the amdS selection marker for genetic mapping, for example of non- selectable recessive markers (94, 95). Because Myrothecium gramineum MUCL 39210 can not grow on minimal medium with acetamide as the sole nitrogen source, the amdS gene could theoretically be used as a selection marker for M. gramineum (29).
These dominant selection markers have as big advantage in that they can be used for fungal species which have not been studied a lot. In addition, the amdS system avoids the use of antibiotics, which is required in certain industrial applications. Next to the dominant selection markers, auxotrophic markers can be used. Unfortunately, these have the disadvantage that the desired genotypes have to be created. For example, arginine auxotrophic mutants can be transformed with the A. nidulans argB gene, coding for orithine transcarbamylase. This system was used to transform Aspergillus niger for the first time (54). Tryptophan requiring mutants can be complemented with the trpC gene of A. nidulans or A. niger , a trifunctional gene coding for glutamine-amino transferase, indol-glycerol-phosphate synthase and phosphoribosyl-anthranilate isomerase (96, 97) or the trp-1 gene of N. crassa , coding for the latter two reactions (98). Glutamate auxotrophic mutants can be transformed with the Am gene of N. crassa , which codes for the NADP-specific glutamate dehydrogenase (GDH) (99). Because these systems require the use of defined (and thus expensive) minimal growth media for continuous selection during large scale fermentation, Elrod et al. (100) developed a system based on the 5-aminolevunilate synthase gene ( hemA ) of Aspergillus oryzae . This gene codes for the first enzyme in heme biosynthesis, a cofactor required for functional respiratory cytochromes. Deletion of the gene is lethal, since aerobic growth is no longer possible. The phenotype can only be rescued by adding 5-aminolevulinic acid to the medium or by transformation with the wild type hemA gene, and not by growth on rich media, which thus can be used for large scale fermentation. Oxygen limitation helped to maintain plasmid selection.
Literature review 28
An extremely useful characteristic of some selection systems is that the marker is both selectable and counter selectable. These systems allow the use of positive screening methods for auxotrophic mutants of a defined gene and thus can be used for genetically poorly characterised fungi. The system which is used most commonly is the chlorate resistance system. Mutants which are resistant to chlorate are nitrate reductase negative and can be transformed with the niaD gene of A. nidulans as heterologous selection marker (101). This method was already used for industrially important fungi such as A. niger and A. oryzae , and in plant pathogenic strains like Colletotrichum lindemuthianum and Nectria haematococca . Next to the possibility of positive selection, this system offers other advantages. The nitrate reductase negative strains can be obtained by spontaneous mutation, which reduces the possibility of secondary mutations arising in genes of commercial interest or in genes encoding essential catalytical steps. The desired mutants have a simple growth type, i.e. inability to utilise nitrate as sole nitrogen source. The pathway is dispensable and therefore mutations in the nitrate pathway should not alter growth or metabolic fluxes through important pathways. And last, most filamentous fungi will utilise nitrate as a sole nitrogen source and heterologous hybridisation signals afford the opportunity to isolate the corresponding niaD from various fungi in order to develop a homologous transformation system (102).
Another method is the use of selenate resistant mutants which are ATP sulphurylase negative and can be complemented with the sC gene of A. nidulans (103). Also fluoroacetate selection to obtain acetyl-CoA synthetase mutants proved to be useful (104). Another procedure which is used a lot is the selection of orotidine monophosphate decarboxylase (OMPD) negative mutants which are resistant to 5-fluoro-orotic acid (5-FOA) and are uracil and/or uridine auxotrophic. These mutants can be complemented with the homologous OMPD gene or with the heterologous pyrG ( A. nidulans ), pyr4 ( Neurospora crassa ) or ura3 ( S. cerevisiae , for yeasts) genes which code for orotidine monophosphate decarboxylase (105).
In some special cases, the expression of the transforming gene can be seen visually on an indicator plate. For example, Van Gorcom et al . (106) provided a valuable technique for the study of Aspergillus transformation by fusing the E. coli lacZ ( β-galactosidase) gene in frame into the coding region of the Aspergillus trpC gene. Aspergillus colonies transformed with this construction turned blue on medium containing the chromogenic β-galactosidase substrate X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside).
In cases in which a transforming gene cannot easily be selected for directly, one option is to look for its assimilation along with a more readily selectable marker. This phenomenon, referred to as co-transformation, is discussed in section 6.
In view of high expression of a gene of interest, special consideration should be made when choosing a particular auxotrophic marker. Lubertozzi et al. (107) very recently found significant differences in mean expression levels when comparing single-copy transformants Literature review 29 with the same promoter but a different marker. Transformants with the argB marker had the highest average expression, approximately threefold over the trpC or niaD clones.
6. FATE OF THE TRANSFORMING DNA In S. cerevisiae and other yeast, plasmids with selection markers nearly all integrate in the genome by homologous recombination to give relatively low frequency transformation (108). The construction of plasmids with autonomously replicating sequences (ARS) allows replication of the plasmids, which results in unstable transformants at a high frequency. The addition of centromere sequences to ARS vectors can result in stable transformants which have 1 to 3 copies of the vector. These features of S. cerevisiae have contributed greatly to the utility of this organism, because they allow, for example, predicting the fate of the transforming DNA (i.e. the site where it will integrate in the genome).
In general, filamentous fungi differ from S. cerevisiae in that high frequency transformation often results from non-homologous (ectopic) integration of DNA into the genome, as well as homologous integration (25). Since the integration of vector molecules at different places in the genome or in tandem repeats at a specific site occurs frequently, it is possible to obtain transformants with high copy numbers (up to 100 copies). In contrast to transformants with replicating vectors, they are mitotically stable. For example, a tandem duplication of the A. nidulans argB locus separated by plasmid sequences was not detectably lost during growth for more than 50 generations (90). This high degree of mitotic stability is desirable for genetic engineering of industrial strains, because introduced gene copies will be retained during large- scale fermentations. By contrast, introduced DNA sequences are meiotically unstable. Most economically important filamentous fungi lack a sexual phase though, thus with many species the behaviour of inserted DNA sequences in meiosis is irrelevant.
Genomic integration of circular plasmids occurs in several ways (90). Some transformants appear to arise from simple gene conversion or double crossover events; no integrated plasmid sequences are detected (Figure 6-1 A, after 90). Another name for this type of integration is ‘type III integration’. In other transformants, however, plasmid molecules/sequences are incorporated in the genome. As stated above, integration sometimes occurs at homologous sites, leading to formation of (tandem) reiterations of the target region separated by plasmid sequences (Figure 6-1 B). This type of integration is also referred to as ‘type I integration’ or ‘homologous additive integration’ (79): the transformants contain the wild type allele, as well as the mutant allele, separated by plasmid sequences. Integration also occurs at heterologous sites, with insertion of single or multiple plasmid copies (Figure 6-1 C). This integration is also called ‘type II integration’ or ‘ectopic integration’. Rearrangements in integrated plasmids may occur at readily detectable frequencies. The frequency with which the various types of integration events occur varies according to the plasmids and recipient strains or species used. Ectopic integration, with different integration sites from one transformant to another, appears generally to be the commonest mode of transformation. Genomic integration of linear molecules also occurs, often by a process Literature review 30 formally equivalent to a double crossover event. However, circularisation of linear molecules prior to integration can produce tandem duplications.
Figure 6-1. Transformation with circular plasmid DNA. Thin lines represent plasmid and thick lines chromosomal DNA. Shaded rectangles represent genes. Vertical lines within genes represent mutations. A. Type III integration or gene conversion or double crossover events. B. Type I integration or homologous additive integration. C. Type II integration or ectopic integration.
An important feature of fungal transformations is that high frequencies of co-transformation (30-90 %) of non-selected plasmids occur and that high copy numbers can be obtained. When cells are exposed to two different kinds of DNA, there is a great chance that, if one molecule is taken in, the other is absorbed too (79). With high ratios of selectable plasmids as compared to non-selectable plasmids, co-transformation efficiencies of more than 50 % and even up to 95 % with Aspergillus nidulans can be achieved (109, 110). The high frequency of co- transformation observed has led to the assumption that there is a subpopulation of cells or nuclei especially competent for DNA integration. It is supposed that not all protoplasts are equally prone to take up DNA and those most competent to do so will tend to take up several molecules simultaneously (79).
Research has shown that the competence of taking in multiple copies of the same or different molecules is not determined at the cell level, but by the status of the nucleus, possibly by the occurrence of chromosomal breaks during replication (108). It is likely that this competence phenomenon contributes to the variation in transformation events seen with different protocols with the same and with different organisms. Recombination between free plasmids Literature review 31 to generate multimers before integration (as in the right part of Figure 6-1), thereby generating the tandem clusters of integrated sequences often observed, is a possible explanation for this phenomenon (51), but it cannot account for the high frequency of scattered ectopic sites of integration. The high copy number can also be caused by combinations of homologous and heterologous integration, under the form of tandem repeats or at different places. A higher copy number, integrated in the genome or present on an autonomously replicating vector, does not always lead to a higher production of a (heterologous) protein (14). The correlation between copy number and production can be checked by combining Southern analysis (estimation of the copy number) with activity tests on the enzyme of interest.
No useful centromeric sequences have been obtained for filamentous fungi. However, a few ARS sequences have been used. The construction of autonomously replicating vectors has been a major goal for the transformation of filamentous fungi because they give high transformation frequencies and allow the construction of shuttle vectors (108). A given DNA molecule will replicate independently of integration into the host genome only if it contains an initiation site recognised by the essential replication enzymes and factors (111). Propagation of such extrachromosomal DNA molecules can be ensured by selecting for the expression of a linked marker, e.g. a gene encoding a drug resistance or a gene capable of complementing a host lesion. The isolation of a 383 bp element of Ustilago maydis with sequences similar to the S. cerevisiae ARS sequences made this possible for this basidiomycete (112). Gems et al. (113) discovered in A. nidulans a 6 kb long origin of replication , AMA-1, which found to give extremely high frequencies of unstable transformants. The frequencies of co-transformation using an AMA-1-containing plasmid without a selectable marker are high, and result from the formation of autonomously replicating co-integrates. These transformants contain 10-30 free plasmids per genome. The AMA-1 sequence appeared to be functional in A. niger and A. oryzae , in Gaeumannomyces graminis and Penicillium chrysogenum (108). Other ARS systems were found for filamentous fungi, but they seem to be rare.
Many fungi are able to add telomere sequences to exogenous DNA (51). For example, the yeast Cryptococcus neoformans , the dimorphic ascomycete Histoplasma capsulatum , the filamentous ascomycete Fusarium oxysporum and the taxol-producing filamentous fungus Pestalotiopsis microspora can add telomeric repeats to the transforming DNA. The reasons for this are unknown. Linear transformation vectors containing telomeric sequences were created in Fusarium oxysporum and functioned efficiently as autonomously replicating vectors in the plant pathogens Nectria haematococca and Cryphonectria parasitica, and in F. oxysporum . In A. nidulans , plasmids with human telomeric DNA sequences can replicate autonomously. The isolation of telomeric sequences of other fungi and their characterisation could lead to the development of linear autonomously replicating vectors, useful for fungal transformation.
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In some cases, linearization of the vector DNA strongly enlarges the transformation efficiency and the efficiency of homologous recombination. Linearization also enhances double crossovers, which results in gene replacement and/or conversion (14). Gene replacement is often used for the construction of defined deletion mutants to study the function of the deleted gene (paragraph 7.2). Commonly, a linear gene replacement cassette which consists of a selection marker gene flanked by DNA fragments homologous to the target gene is used (70). After this construct is introduced in the target cell, it can integrate via homologous as well as non-homologous recombination. The way exogenous DNA sequences integrate in the genome depend on the dominant pathway of the host cell to repair double stranded DNA breaks (114). In S. cerevisiae , double stranded DNA breaks are mainly repaired by homologous recombination, while in plants this generally happens non-homologously. With these higher eukaryotes, the frequency of integration via illegitimate recombination is thus much higher than targeted integration via homologous recombination. In yeasts, such as S. cerevisiae and Schizosaccharomyces pombe , short homologous regions of 50-100 basepairs are already enough for the integration of the gene replacement cassette in the homologous locus with an efficiency of 50-100 % (115, 116). In filamentous fungi, longer sequences of at least 1000 bp are necessary to obtain efficiencies of 10-30 % (108). The possibility to integrate the DNA at a specific site in the genome is of crucial importance for many researches, as is further discussed in 7.2.
7. APPLICATIONS OF TRANSFORMATION TECHNOLOGY
7.1. Using transformation for cloning genes (heterologous and homologous) The ability to isolate genes by function (complementation) is one of the greatest rewards of developing a transformation system (108). The opportunity to clone any gene that can provide a recognizable phenotype means that there is absolutely no requirement to know anything about the product of the gene or its pattern of expression. Genes defined by recessive mutants can be cloned using a standard and defined wild-type library and transforming a mutant strain. Genes defined by dominant mutations can be obtained by the construction of a library from a mutant strain and transforming a wild-type recipient. Where known genes have been cloned by molecular methods such as by hybridisation, the identity of functional sequences can be determined by complementation. Heterologous expression of genes is very useful for cloning genes from species where the genetics is poorly developed because it enables the use of model organisms with many mutants available as recipients for complementation by DNA from gene banks from the species under study.
Genes have been cloned by using gene banks constructed in plasmid vectors or in cosmid vectors. The plasmid or cosmid libraries can be maintained as individual clones in microtitre dishes. The method of ‘sib selection’ (selection in broth selection) uses successively smaller pools of clones in transformation experiments, selecting for the desired gene until a single complementing cosmid (plasmid) is identified.
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Another method to clone genes by complementation is the ‘instant gene bank method’. The method is based on the complementation of mutants defective in the gene of interest and employs co-transformation of fragmented chromosomal DNA together with a plasmid capable of autonomous replication. This plasmid (pHELP1) contains the AMA -1 sequence which confers replicative potential if included in fungal vectors and which catalyzes recombination in the host cell and enhances transformation frequency considerably. When pHELP1 and genomic DNA of the donor strain are co-transformed into the mutant strain, replicating co- integrate plasmids are generated which may be recovered by transformation of E. coli with these plasmids (25). The instant gene bank method has been applied for A. nidulans and for other fungal species by heterologous complementation in A. nidulans and avoids the need for gene-library construction (108).
‘Tagging’ of genes by known sequences is another potential way of cloning genes. T-DNA tagging is a form of insertional mutagenesis relying on Agrobacterium mediated transformation (AMT) to mutate the recipient genome at random sites by integration of T- DNA carrying a selectable marker. T-DNA tagging has been used successfully to find new genes and T-DNA tagging projects on fungi have recently been initiated in many laboratories around the world. AMT is very suitable for insertional mutagenesis as it can cause a relatively high frequency of transformation and often creates single-copy integrations. Also, T-DNA appears to integrate approximately at random, although integration may be targeted towards transcribed regions and promoters in particular. The majority of AMT transformants may contain small (~100 bp) genomic deletions and even with single-copy T-DNA integrations, small or large genomic rearrangements are frequently observed. For this reason it is important that putatively T-DNA tagged mutants are tested to see if the T-DNA insertion site is linked to the mutant phenotype (117). The technique of ‘restriction enzyme mediated integration’ (REMI) also is a potential method for insertional mutagenesis and tagging of genes. It is a transformation-based technology which leads to mutants tagged by an integrated plasmid. As outlined above, ectopic integration is frequent in hyphal fungi. REMI involves the use of a transforming vector without major homology to the host’s genome. The transformation assay includes significant amounts of a restriction enzyme, which randomly cuts the genomic DNA, leading to a random integration of vector DNA at these sites. To a certain extent, differing between the fungal systems, the plasmid can be recovered from genomic DNA of the transformant by digestion with the REMI enzyme. If a restriction enzyme is used which does not cut within the plasmid, the plasmid plus parts of the tagged gene are excised and can be recovered from E. coli . By increasing the target sites for recombination, REMI usually improves the overall transformation rate and frequency of single copy integration events although this is not always the case. The high frequency with which the mutant phenotype is not linked to the integrated DNA greatly complicates the use of this system for gene discovery (117).
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As sequences accumulate in the databases, a common approach is to clone genes on the basis of homologous (conserved) sequences (the ‘candidate gene’ approach). The function of such cloned genes can be investigated by complementation using transformation.
Yet another way to clone (inducible) genes is by ‘differential cDNA screening’ (25). In the typical set up, a cDNA library prepared from mRNA of a culture, in which the desired gene is active, is screened with labelled cDNA from induced and non-induced (or repressed) cultures.
In addition to vectors used to clone genes, more specialised shuttle vectors were designed to search in the genome for effective promoters and upstream activators by including a potentially selectable but promoter-less gene in the cloning vector. When random sequences are cloned into such a plasmid just upstream of the promoter-less gene and selection is made for transformants exhibiting the gene activity, the clones selected should be a rich source of transcription-promoting sequences (79). For enhancer traps, a reporter gene, such as the green fluorescent protein, downstream from a minimal promoter which is insufficient for its expression, is positioned near the end of the transferred DNA. Insertion of the DNA near an enhancer element will allow expression of the reporter gene from the minimal promoter (117).
7.2. Gene inactivation and gene targeting The use of transformation to inactivate the function of a gene (gene knockout) is a valuable tool, since such a mutant can provide essential information for understanding the role of a gene. For example, this technique has been especially valuable in the study of phytopathogenic fungi for functional analyses of putative pathogenicity or virulence genes (25). The deletion should be as precise as possible (full coding region), since insertional inactivation or partial deletion leaves the possibility of a partial gene function being retained, while deletion of flanking sequences has the potential to cause additional phenotypes. Complete deletion of the coding region can be achieved by a one step disruption technique (Figure 7-1 A): a (usually) linear fragment, in which gene sequences are replaced with a selectable marker, allows the selection of disruptants generated by homologous recombination (double crossover). In filamentous fungi, the flanking homologous regions usually need to be at least 1 kb long to yield the desired replacement at a reasonable frequency. The efficiency of gene targeting in fungi is dependent on the length of the homologous sequence, the GC- content of the flanks, the extent of homology, the transformation method, the homologous recombination efficiency of the host, and the genomic position of the target gene, i.e. the transcriptional state of the target gene and the chromatin structure (70, 117). For efficient gene targeting, the level of homology between the target sequence and the homologous region of the transferred DNA needs to be very high, almost 100 %.
The ability to make strains containing multiple disruptions is of great value, particularly in fungi for which there is little or no classical genetics and fungi with few transformation markers available. Therefore, selection systems with markers which can be both positively and negatively selected are important (see also paragraph 5). One possible procedure to create Literature review 35 multiple disruptions is the use of the ‘ura-blaster’ or the ‘pyr-blaster’ system. A functional ura3 , pyr4 or pyrG gene flanked by direct repeats is used for gene disruption, selecting for uracil prototrophy in an URA3/OMPD negative strain. Counter selection using 5-fluoro-orotic acid selects for excision of the functional ura3 , pyr4 or pyrG gene by recombination between the repeats, which leaves the gene disrupted. Further rounds of disruptions in additional genes can then follow.
Figure 7-1. Procedures for gene targeting. A. One step gene replacement B. Targeted integration C. Targeted gene replacement.
There are basically three ways to increase the percentage of transformants resulting from a homologous recombination event among the total number of transformants picked (118): turn off the non-homologous recombination system, increase the efficiency of the homologous recombination system and select against non-homologous recombination events. Most methods described aim to improve the efficiency of the homologous recombination, but some techniques also aim to achieve the goal using the first and the third way.
Next to being important for the creation of gene knockouts, the ability to target introduced DNA to specific locations in the genome is also crucial for many other studies. For example, quantitative measurements of gene expression are best done by introducing single gene copies at defined loci (see also paragraph 8.4). The one step replacement method is one way to obtain targeted integration, but another widely applied method is to use two different mutations within one gene so that a selectable phenotype is only generated by a single crossover at the site of interest or by gene conversion (Figure 7-1 B and C). One approach is Literature review 36 to use 5’ and 3’ deletions of a selectable gene so that only a homologous crossover will result in an intact gene. The argB gene of A. nidulans , the his3 -gene of N. crassa and the pyrG gene of A. awamori have been used for such strategies (108, 119). As a result, wild type and doubly mutant genes can be separated by integrated (plasmid) sequences, which for example can be expression cassettes used to study a promoter of interest.
Very recently, Nielsen et al. (120) used the pyr-4 gene of N. crassa to develop an efficient PCR-based gene targeting system for Aspergillus nidulans . A. nidulans is a good model organism to develop gene targeting systems, because the gene-targeting success rates are typically quite high in this organism, ranging between 10 % and 50 %, which is much higher than for most other multicellular eukaryotes. For this reason, most types of genome manipulations have been reported for A. nidulans . Unfortunately, the methods often employ several E. coli based cloning steps and the development of a versatile cloning-free method is desirable. Therefore, PCR-based methods have recently been successfully used to perform gene deletions and promoter replacements in e.g. A. nidulans , using a one-step replacement strategy. The use of fragments generated by PCR can drastically increase the frequency of homologous recombination when compared to linearised plasmids. The method of Nielsen et al. (120) employs PCR to generate two composite DNA fragments, called a “bipartite gene- targeting substrate”, which are fused in vivo by homologous recombination (HR) to form the active gene-targeting substrate. The bipartite gene targeting substrate exists of two fragments, which each carry a targeting sequence as well as a part of a selectable marker gene. None of the two individual fragments contains a functional marker, but if the fragments fuse by HR via overlapping marker sequences, a complete and functional marker sequence is generated after co-transformation. The selectable marker fragments ( pyr-4) are designed so that a Direct Repeat (DR) is formed when they integrate into the genome, which allows multiple rounds of gene targeting to be performed in the same strain as the marker can be excised via direct- repeat recombination.
Nielsen and co-workers (120) showed that the use of this bipartite substrate results in a higher frequency of correct targeting events compared to that obtained with the corresponding continuous substrates.
The method can be used for gene deletion and for the construction of point mutations (Figure 7-2, reproduced after 120), and since one of the direct-repeat sequences remains in the genome, the method can be easily adjusted to allow the construction of fusion genes. The only requirement is a vector where the pyr-4 marker is surrounded by the relevant sequence as a direct repeat.
Literature review 37
Figure 7-2. Integration of bipartite gene targeting substrates and subsequent direct-repeat recombination events to delete a gene of interest (left part) or to create a point mutation in the endogenous copy of your gene of interest.
Another PCR-based gene replacement system was developed for Ustilago maydis (117, 121). The 5’ and 3’ regions of the target gene are first amplified by PCR using PCR primers which include restriction sites compatible with those in the selection cassette and subsequently ligated directionally to a marker cassette via two distinct SfiI sites, providing the flanking homologies needed for homologous recombination in U. maydis . Then the ligation product is gel purified and used as a template for the amplification of the deletion construct, which can be used directly to transform U. maydis . In this way, different genes of U. maydis were disrupted. The efficiency of this ligation-mediated PCR system strongly depends upon the chromosomal context of the targeted gene.
An alternative technique to that of Kämper et al . (121) has been developed for the generation of deletion constructs in Cryptococcus neoformans and Dictyostellium discoideum . In these systems, fusion of the flanking regions to the central cassette is facilitated by the use of a modified PCR overlap technique. The central cassette and the flanking regions are amplified with primers which produce overlaps of approximately 40 bp. Subsequently, the three PCR products are combined and used as the template for PCR amplification of the entire disruption cassette. Since the central cassette (containing a marker and sometimes a reporter gene) is PCR-amplified twice, the risk of accumulating mutations that might interfere with the functionality of the marker or the reporter increases. The ligation-mediated PCR method has an advantage over overlap PCR in that the selection cassette is not reamplified (117).
Jadoun and collaborators (122) used a transposon-based mutagenesis technique to disrupt the Aspergillus fumigatus argB gene. This approach uses a modified transposon containing the pyr-4 gene of N. crassa , which is randomly inserted in vitro into a target sequence of interest, for example, the argB gene. Randomly transposon-integrated vectors were cloned in E. coli and clones in which the gene of interest was disrupted were identified by PCR. Their plasmids Literature review 38 were linearised and used to transform a pyrG -deficient strain of A. fumigatus . Integration of this cassette into the argB -locus could be selected based on the ability of the transformants to grow on selective medium without uracil and their inability to grow on medium without arginine. However, a transposition tagging system usually will consist of an ectopically integrated construct containing a marker gene inactivated by a transposon carrying a second selectable marker gene. Excision of the transposon can be selected through restoration of the first marker gene and stable reintegration can be selected through the presence of the second marker gene (117). The transposon-based insertional strategy circumvents the need to identify suitable restriction sites inside the target gene into which a selectable marker can be inserted (122). In vitro transposition was recently used to initiate genome-wide mutagenesis studies in filamentous fungi. The main advantage of a transposon mutagenesis strategy is that a high efficiency transformation system is not required (117). The transposon Impala has been highly studied for use as a gene tagging element in fungi. Impala transposes by a cut and paste method. It encodes a single transposase enzyme that recognizes inverted terminal repeat sequences on the transposon and mediates the excision and reintegration of the transposon without the involvement of host proteins. Consequently, transposons like Impala can typically be used in heterologous hosts.
Other approaches take advantage of an E. coli strain expressing the phage λ Red functions ( α, β and γ) to increase the length of the homologous DNA flanking the transformation markers (123, 124, 125): cosmids are produced in which the gene of interest has been replaced by a selectable marker. The λ Red functions drastically increase homologous recombination in E. coli , requiring flanking regions of only 50 basepairs. Recombination in E. coli occurs between a PCR-amplified bi-functional transformation marker and a selected cosmid carrying an A. nidulans genomic region after transformation with the marker of an E. coli strain carrying the cosmid of interest. The bifunctional marker carries a marker for E. coli (Zeocin R) and for the fungus (e.g. the A. nidulans pyrG ) flanked by the 50 bp homologies. Recombinant cosmids are selected by the resistance to Zeocin of their host. These cosmids are then used to transform A. nidulans , linearised or circular, yielding transformants with the appropriate gene replacement at frequencies up to 60 %. The efficiencies of gene replacement are so high because the cosmids have large flanking regions surrounding the selectable marker. Cosmids are also used in transposon arrayed gene knockout (TAGKO). In TAGKO, a cosmid library is created from the whole fungal genome. Transposon Tn7 carrying a selectable marker gene integrates, by a transposase-catalysed in vitro reaction, into a pool of cosmids or a single cosmid, randomly disrupting genes. Sequencing primed by transposon sequences identifies disrupted genes, allowing selection of cosmids carrying disruptions in genes of interest, for gene disruption by homologous recombination. Restriction digestion of selected cosmids releases a linear insert suitable for transformation methods, and phenotypic characterisation of disruption mutants can then follow (117).
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In addition to the development of methods facilitating the construction of gene replacement vectors with long homologous DNA flanks, methods to identify non-homologous integration events have been developed. To discriminate between homologous and non-homologous recombination, a second dominant selectable marker, which flanks the gene replacement cassette, was added. Upon homologous recombination the second selection marker will be lost, whereas upon non-homologous recombination transformants carrying both selectable markers will be obtained (70). An alternative method which employs selection against non- homologous integration events to increase the yield of desired gene-replacement mutants is described by Pratt et al. (118) for N. crassa . They use a replacement cassette consisting of a lethal gene followed by a left flank, a marker and a right flank, in that order. Upon homologous recombination, the lethal gene is not integrated into the genome and the transformants can survive. Upon ectopic integration, however, the lethal gene co-integrates and causes the death of the transformants.
Another possible strategy for increasing the efficiency of gene targeting in fungi with a very low frequency of homologous recombination is to increase the expression of genes involved in homologous recombination or decrease the expression of genes involved in non- homologous end-joining (NHEJ) (117). Homologous recombination in S. cerevisiae is dependent on the RAD51 protein. In A. nidulans , overexpression of the rad51 homologue uvsC resulted in an improvement in targeting efficiency through homologous recombination. However, elevated expression of uvsC also suppressed colony growth rate and leads to increased genomic instability. Knockout of mus-51 and mus-52 genes required for NHEJ in N. crassa , allowed a frequency of homologous recombination of 100 %. N. crassa mus-51 and mus-52 mutants did not show defects in growth or morphology, suggesting that this might be a viable strategy.
A method which circumvents the problem of lethal mutants disrupts gene expression at a post-transcription level by targeting the mRNA rather than the gene. RNA interference (RNAi) is a sequence-specific post-transcriptional gene-silencing phenomenon. However, RNAi is still a relatively undeveloped technique which does not always reduce expression to a point where a phenotype change is seen. For a more detailed description of this technique, see Weld et al. (117).
7.3. Manipulation of gene expression using transformation One of the major uses of transformation is to bring about overexpression or inappropriate expression of cloned genes. In pathway design, for example, the optimisation of biosynthetic pathways of industrially relevant products can be performed by amplification of a gene coding for a ‘bottleneck’ enzyme or by cutting of undesirable side pathways by inactivation of the corresponding genes. A good example for the successful application of such techniques is the production of β-lactam antibiotics in Penicillium chrysogenum and Acremonium chrysogenum (25). Production of heterologous proteins is especially important for proteins of Literature review 40 other eukaryotes which are not readily available on a larger scale and cannot be efficiently produced in bacterial hosts.
The ability to generate transformants containing multiple copies of integrated molecules allows uncontrolled high-level gene expression. Uncontrolled gene overexpression can also be obtained by the use of strong constitutive promoters, such as the gpdA promoter of A. nidulans . Controlled gene expression can be achieved by the construction of transcriptional fusions with inducible promoters, like the alcA promoter of the gene coding for alcohol dehydrogenase in A. nidulans . These systems allow switching on high level expression in a controlled way (at a specific time) (108).
Further comment on the manipulation of gene expression in fungi will be given in section 8.
8. (HETEROLOGOUS) PROTEIN PRODUCTION BY FUNGI Fungi are generally employed as ‘cell factories’ for the production of recombinant proteins, using strong and regulated promoters (126). One of the most common used promoters is the promoter of the Aspergillus oryzae Taka-amylase, which is used for a large number of enzymes (127). Other fungal promoters which are used a lot are the Aspergillus gpdA , alcR and glaA promoters (128, 129, 130). Aspergillus promoters have already been successfully used with other Ascomycota for genetic studies or for the development of transformation systems.
8.1. Structure of fungal expression systems Heterologous gene expression systems in filamentous fungi exist of an expression vector which contains the gene of interest flanked by non-translated sequences, and an expression host which can optimally synthesise the heterologous protein (20). Fungal expression vectors usually contain expression cassettes consisting of several components. As shown in Figure 8-1, the (heterologous) gene is flanked upstream by DNA sequences responsible for the transcriptional control and the initiation of translation. When the gene product needs to be secreted, a signal sequence is fused to the coding region of the (heterologous) gene. Downstream, the heterologous gene is flanked by sequences which control the transcription termination.
binding sites of specific ATG translational TAA/TAG/TGA translational transcription factors start codon stop codon
transcriptional control signal sequence heterologous gene transcription + initiation of translation for secretion terminaton Figure 8-1. Schematic representation of an expression cassette for the synthesis and secretion of (heterologous) proteins in filamentous fungi .
Literature review 41
8.1.1. Promoters of filamentous fungi Efficient synthesis of proteins can be achieved by putting a gene coding for the protein under the control of a strong fungal promoter. Generally, fungal promoters for (heterologous) gene expression can be divided into two groups, namely constitutive promoters originating from household genes and inducible promoters (131). Filamentous fungal promoters are often large and ill-defined fragments of DNA. Very few have been functionally characterised in any detail, but, interestingly, there appear to be significantly different sequence requirements for promoter activity from those reported for either lower eukaryotes, such as Saccharomyces cerevisiae , or mammalian systems (14). In most cases homologous promoters give more efficient expression than heterologous (14).
Transcriptional control in fungal promoters When discussing promoters, it is useful to differentiate between the upstream elements which determine the transcription start site (tsp) and initiate a minimal level of transcription, and other sequences which are called ‘transcriptional activation elements’ (14). Although this is a somewhat artificial distinction, the term ‘core promoter’ is used to define the minimum sequence requirement to initiate transcription from the normal major transcription start point. This may not always result in mRNA levels as high as those ‘basal’ levels defined for wild type genes.
In most eukaryotic systems, the TATA-box (consensus TATAAA) appears to be essential for a functional core promoter. In higher eukaryotes, this sequence almost always lays between 25 and 30 bp (20-40 bp) upstream the tsp and mutations within this sequence usually lead to aberrant initiation of transcription. In S. cerevisiae , however, both the presence and/or the siting of the TATA-element appear to be far less critical. Some genes, even highly expressed, do not have a TATA-box, while other genes have one or more copies of this element up to 120 bp upstream of the tsp (132). In this sense, filamentous fungal genes resemble those of yeast more closely: consensus TATA-boxes have been found in few fungal promoters (133). Although AT-rich sequences were observed upstream of the transcription initiation sites, their location is variable and their functional significance unknown. Deletion analysis of the A. nidulans oliC promoter (134) indicated that the TATA-box is important for the determination of the tsp , but does not affect the level of expression. Similar findings have been reported for the A. nidulans trpC promoter. However, with so few fungal genes functionally characterised, it is possible that, rather like yeast, there is more than one functional class of TATA-element in filamentous fungal promoters: if so, some of the AT-rich sequences may still be important for core promoter functions.
The element which is most important in determining the tsp in filamentous genes is the CT- box. This is a pyrimidine-rich sequence of 8 to 12 bp which is found directly upstream a considerable number of fungal genes (135). The length of this pyrimidine stretch can vary, as particularly marked in the A. nidulans oliC gene, where it extends to 96/100 bases (133). Functional analysis of the gpdA promoter of A. nidulans has shown that deletion of this Literature review 42 sequence results in aberrant initiation of transcription and suggests that this region alone is sufficient for the determination of the tsp (136). The fact that, in cases where the CT-box has been deleted, the alternative transcription start points used are often found downstream of other CT-rich sequences supports the involvement of this motif in determining the tsp .
In many higher eukaryotic systems, the CAAT-box is another motif which shows core promoter activity and which is usually situated between 70/80 and 90 bp upstream the tsp . In fungal genes, the CAAT box often is absent, or when present, is situated at a variable distance of the tsp (60-120 bp).
Lack of sufficient experimental data makes it difficult to draw firm conclusions as to what sequences represent the core promoter in filamentous fungi. Although the multi-competent nature of the RNA-polymerase II mediated transcriptional initiation makes it likely that no simple generalisations can be made, it does appear that in several cases remarkably short (circa 50 bp) upstream sequences can initiate transcription precisely in filamentous fungi. Such relatively simple sequence requirements could be responsible for the multiplicity of initiation sites observed in fungal genes, and might help to explain why transcriptional signals appear to transfer so readily between filamentous fungi.
Despite the little research effort already performed in the field, a number of sequences which are important in controlling the level of transcription (transcription activation or repression elements) have already been identified for some fungal genes. The majority appear to involve binding sites for positively acting regulatory proteins (14): - in the qa -gene cluster of N. crassa , each gene can be induced by the binding of the qa-1F activator protein to a 16 bp motif found upstream of the genes (consensus GGRTAARYRYTTAYCC). The qa-2 gene encodes a catabolic dehydroquinase which catabolises dehydroquinic acid to dehydroshikimic acid (137). - both the amdS gene and the gatA gene of A. nidulans are positively regulated by the amdR gene product, but the presence of multiple copies of a 60-80 bp upstream fragment where the amdR activator protein binds on, titres the activator out, resulting in reduced growth (YGAAGCYR motif). The amdS gene of A. nidulans is one of the best studied systems and genetic analysis has revealed that the gene is regulated by at least six regulatory factors (AmdA, AmdR, AmdX, FacB, AreA, CreA) acting via binding on short sequences in the promoter region of the gene (138). Other promoters of which regulatory sequences are already known and extensively studied involve the promoters of genes of which expression is controlled by the carbon source, the nitrogen source, the pH and the sulphur and phosphor availability (2, 25). Examples are the Hypocrea jecorina cellobiohydrolase I and II promoters, the Hypocrea jecorina endoxylanase I and II promoters (26), the xylanase xlnA and B promoters of A. nidulans (2), the xylanase xyn1 of Hypocrea jecorina (139) and an arabinofuranosidase of P. purpurogenum (140). Regulatory sequences of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter have also been identified in several fungi, as is discussed in paragraph 8.1.1 and Literature review 43 chapter III. Several of these elements show inverted repeat homologies which are often associated with the binding sites of regulatory proteins or transcription factors. An interesting program to search for regulatory sites in a new promoter is the ‘Motif Finder’ (141), based on the TRANSFAC ® database. TRANSFAC ® is the database on eukaryotic transcription factors, their genomic binding sites and DNA-binding profiles (142).
Constitutive promoters The use of strong promoters for the expression of (heterologous) proteins in suitable host organisms is of great importance for biotechnological applications. The most frequently used constitutive promoter is the glyceraldehyde-3-phosphate dehydrogenase (gpdA ) promoter of A. nidulans . This promoter is functional in different species, including industrially important Penicillium and Aspergillus species.
The glyceraldehyde-3-phosphate dehydrogenase (GPD; EC 1.2.1.12) promoter is a promising candidate (39) because in many eukaryotic microorganisms, the GDP genes are expressed constitutively and in large amounts. GPD is one of the key enzymes in the glycolytical and gluconeogenesis pathways. It catalyses the conversion of D-glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate and vice versa. The soluble cellular proteins of S. cerevisiae and other higher eukaryotes can contain up to 5 % GPD (143). Moreover, the GPD mRNA counts for 2- 5 % of the total poly (A) + RNA present in yeasts (144). The fact that GPD is present in large amounts indicates that it is regulated by a very active promoter (39).
Punt et al. (145) compared the promoter region of the gpdA gene of A. nidulans with promoter sequences of other glycolytical Aspergillus genes. They found two regions of similar sequences, more precisely, a pgk box 600 basepairs upstream of the transcription start point (tsp ) and a CT-rich region immediately before the transcription start site. By comparing the promoter region of the gpdA gene of A. nidulans with the gpdA promoter region of A. niger , Punt et al. (136) found a region with 96 % homology, which was called the gpd box. This gpd box is situated 250 bp upstream of the major tsp in A. nidulans and at approximately the same distance in A. niger . Both the gpd box and the pgk box contain a considerable amount of inverted repeats. Since inverted repeats are considered as regulatory sites in promoter sequences by the binding of transcription factors and/or regulatory proteins, this could mean that the pgk and the gpd box are part of such a regulatory site (146).
Gpd promoters have been successfully used for the expression of heterologous genes in filamentous fungi (147, 148). A few examples are given in Table 8-1.
Literature review 44
Table 8-1. Use of the gpd promoter in filamentous fungi for the heterologous expression of fungal and other eukaryotic genes ( A. = Aspergillus , E. = Escherichia , P. = Penicillium ) Host Promoter Heterologous protein Reference A. awamori gpd (Aspergillus sp. ) glucoamylase (A. niger) 149 A. awamori gpdA (A. nidulans) thaumatin (plant) 150 A. nidulans gpdA (A. nidulans) aspartase ( E. coli ) 151 A. nidulans gpdA (A. nidulans) elastomer (synthetic) 152 A. niger gpd (A. niger) xylanase ( Hypocrea jecorina ) 153 A. niger gpd (A. niger) laccase ( Pycnoporus cinnabarinus ) 154 A. niger gpdA (A. nidulans) α-amylase (barley) 147 A. niger gpdA (A. nidulans) lysozyme (chicken) 155 P. chrysogenum gpdA (A. nidulans) β-galactosidase ( E. coli ) 156
Other constitutive promoters which have been used for example in Aspergillus sp. are the trpC promoter from A. nidulans and the TEF1-alpha promoter of A. oryzae (4). These promoters control the expression of the phosphoribosyl anthranilate isomerase (tryptophan biosynthesis) and the translation elongation factor 1-alpha , respectively. The steady-state levels of trpC mRNA are regulated by both nutritional regimen and developmental state - (157). They are highest in cultures grown in minimal medium with NO 3 as the sole nitrogen + source, are 3- to 4-fold lower in cultures grown in minimal medium with NH 4 as the sole nitrogen source, and are about ten-fold lower in complex medium with yeast extract as the nitrogen source. The promoter does not contain CCAAT or TATAA like sequences, nor is it GC rich. Another recently developed expression system is based on the constitutive promoter of the histone H4 of P. funiculosum (158). The promoter of the histone 4.1 gene reached high steady-state expression in the early stages of batch cultures and was successfully used to express the bacterial uidA gene and a fungal xylanase.
A class of promoters distinct of the constitutive promoters are the so called ‘culture-specific’ expression promoters, which are constitutively expressed, but only in certain culture conditions. Recently, Ishida et al. (159) reported that the tyrosinase gene promoter (melO ) was four times stronger for the heterologous expression of the β-glucoronidase reporter gene in a submerged culture as compared with other gene promoters, such as amyB , glaA and gpdA , previously used for heterologous gene expression in A. oryzae . The melO promoter was used to express a glucoamylase in A. oryzae and reached a maximum yield of 3.3 g/L and 99 % purity, establishing the melO promoter for high-level protein expression and high-purity production in A. oryzae . They also isolated the glucoamylase promoter ( glaB ) for solid state cultures. Since enzyme production with the melO promoter causes problems due to the time- consuming culture period, this research group searched for another promoter, useful in submerged cultures and reported in 2004 (160) the isolation of the superoxide dismutase encoding gene ( sodM ) promoter. Extracellular glucoamylase production under the sodM promoter reached 1 g/L in a shorter production period. Literature review 45
Inducible promoters Inducible promoters are particularly useful when the expressed gene product is unstable or toxic to the host (20). Moreover, the use of strong promoters can overload the fungal secretion system which results in incorrect folding and glycosylation of the produced proteins (161). This can induce the protein degradation pathway resulting in lower protein yields.
The glucoamylase ( glaA ) promoter of A. niger is the most commonly used inducible promoter. Other inducible promoters used in fungal expression systems are: - the acid phosphatase ( phoA ) promoter of Penicillium chrysogenum (162) - the alcohol dehydrogenase ( alcA ) and the aldehyde dehydrogenase ( aldA ) promoter of A. nidulans (163) - the amylase ( amyA, amyB ) promoter of A. niger and of A. oryzae (amyA ) - the cellobiohydrolase promoter of Hypocrea jecorina ( cbhI ) or of A. fumigatus ( cbhB ) (164) - the copper inducible metallothionein promoter of N. crassa (108) - the endoxylanase ( xylP ) promoter of P. chrysogenum (165) - the endoxylanase II ( exlA ) promoter of A. awamori (22, 166), inducible by xylose but not by sucrose or maltodextrin - the enolase ( enoA ) promoter of A. oryzae (167) - the glutamate dehydrogenase ( gdhA ) promoter of A. awamori (150) or P. chrysogenum (168) - the isopenicillin N-synthase ( pcbC ) promoter of P. chrysogenum (150) - the phosphoglycerate kinase ( pgk ) promoter of Rhizopus niveus and Penicillium citrinum (169, 170, 171, 172)
The expression signals of glaA are frequently used for the overexpression of homologous and heterologous genes in various filamentous fungi, e.g. the human interleukin 6 gene in A. nidulans (173) and the phytase genes of A. awamori and A. fumigatus in A. awamori (174). The regulation of the glaA promoter of A. niger was studied by Verdoes et al. (175) as a fusion to the β-glucoronidase encoding reporter gene. Gene expression controlled by these signals is highly induced by growth on starch or maltose/maltodextrin, whereas no expression is seen in the presence of xylose as carbon source. Verdoes et al. studied single copy transformants with the cassette of interest integrated at the pyrG locus. Expression in cells grown on glucose was roughly 50-80 % of the maximum level, reached with maltose/maltodextrin. The glucoamylase promoters of A. oryzae and A. nidulans also are starch and maltose inducible (4). They are repressed by glucose. The effect of nitrogen sources and pH on the expression of glaA in chemostat cultures was investigated by Swift et al. (176). It was proven that trans-acting regulatory proteins are involved in the expression of the A. niger glaA , since the introduction of multiple copies of the glaA promoter leads to a decreased expression (titration of one or more activator protein) (175). Apparently the available amount of regulatory protein is sufficient to activate only a limited number of glaA copies per nucleus. According to Fowler et al. (177), a region between -562 and -318 appears Literature review 46 to direct high-level expression, whereas only 214 bp of the 5’ flanking region is required to initiate the start to transcription. Verdoes et al. found that deletion of the fragment between -815 and -517 decreased expression to about 5 %. Three putative CreA-binding sites were found in the promoter. Analysis of the cis-regulatory regions revealed the presence of a CCAAT motif between position -464 and -426. This motif is considered as a functional element essential for high-level expression of a large number of genes. Introduction of multiple copies of this CCAAT motif in the promoter of glaA significantly improved the transcriptional activity of the promoter (178).
A. nidulans is able to grow on ethanol as the sole carbon source via the combined action of alcohol dehydrogenase (encoded by aclA , ethanol to acetaldehyde) and aldehyde dehydrogenase (encoded by aldA , aldehyde to acetate). The alcA promoter is tightly regulated by glucose repression via the major carbon catabolite repressor CreA and pathway specific induction by a positive regulatory protein encoded by the alcR gene product. AlcR also induces aldA , but aldA is not regulated by CreA. AlcA and aldA are among the most highly transcribed, inducible genes known to date (179). AlcA is inducible by threonine, ethanol or cyclopentanone in the absence of glucose. It is possible to generate very high levels of inducible expression with transformants containing multiple copies (25, 108). This promoter was, for example, used for the expression of human lactoferrin in A. nidulans (180).
The alpha-amylase promoters are inducible by starch. The Taka-amylase promoter of A. oryzae (amyB) is inducible by starch, but not by glucose (181). The Taka-amylase promoter of A. oryzae was used for the expression in A. oryzae of, for example, the manganese peroxidase gene of Phanerochaete chrysosporium (182), a dye decolourising peroxidase of Geotrichum candidum (183), a human lysozyme (172) and Mortierella alpina desaturase genes (180, 184).
The cellobiohydrolase ( cbhI ) promoter from Hypocrea jecorina , next to other promoters of cellulase genes, is best induced by sophorose (2 β-1,2-linked glucose units), which is considered to be the natural inducer of cellulase formation. Sophorose is formed from cellobiose, the major soluble end-product formed from cellulose by the concerted action of cellulases (26). The cbhI promoter also is strongly repressed by glucose. The cellobiohydrolase I promoter has been studied considerably and its regulation was recently revised by Mach et al. (26). Examples of cbhI -promoter driven expression in Hypocrea jecorina are the expression of calf chymosin (185), the endochitinase of T. harzarium (186), of the human N-acetylglucosaminyltransferase I (187), of a xylanase of Humicoli grisea (188) and of the Melanocarpus albomyces laccase (189). Very recently Bromley et al . (164) investigated the possibility to use the cellobiohydrolase promoter ( cbhB ) of A. fumigatus for controlled protein expression. They concluded this promoter can be exploited for these purposes because it is tightly regulated: CbhB message and reporter message was present at high levels in the presence of carboxymethylcellulose and undetected in the presence of glucose. The reporter construct gave lower message levels than the A. nidulans alcA promoter Literature review 47 under repressing conditions in A. fumigatus . This alcA promoter was previously described by Romero et al. (190) as part of the first conditional expression system for A. fumigatus .
The metallothionein promoter of N. crassa is copper inducible and was used for the expression of tyrosinase (191) and laccase (192) in N. crassa . The promoter of the cytoplasmatic filament protein P59Nc ( cfp ) gene of N. crassa was recently investigated as to its capacities to drive the expression of transgenes in filamentous fungi (193). mRNA levels of CFP modify rapidly in response to either inducing or repressing culture conditions. Constructs wherein a minimal cfp promoter drives the expression of reporter genes were highly expressed in N. crassa in media containing glucose or sucrose and repressed in media containing ethanol or ethanol plus glucose. Analogous results were obtained in A. nidulans . They propose that the cfp promoter of N. crassa can be used to control the expression of transgenes in filamentous fungi in a carbon-source dependent fashion.
The endoxylanase promoters are transcriptionally regulated: xylan and xylose are efficient inducers, whereas glucose strongly represses the promoter activity. Zadra et al. (165) developed an expression system based on the xylP promoter of P. chrysogenum which appears to be 65-fold more efficient than the pcbC promoter in Penicillium and 23-fold more efficient than the niaD promoter (nitrate reductase) in Aspergillus under induced conditions. Upon xylose induction, the exlA promoter of A. oryzae was threefold more efficient than the frequently used glaA promoter of A. niger under maltodextrin induction (194). MacCabe et al. (2) suggest that the differential control of the xlnA and B (coding for endo-β-1,4-xylanases) expression in response to ambient pH in A. nidulans provides the possibility to produce enzymes under pH conditions which may be more favourable for their stability. These promoters may be used as starting points for the rational design of more efficient pH- regulated promoters.
The enolase ( enoA ) is one of the most highly expressed genes of A. oryzae and its mRNA levels can reach approximately 3 % of the total mRNA in glucose-induced conditions. The enzyme catalyses the interconversion of 2-phosphoglycerate into phosphoenolpyruvate. Reporter activity is highest when a 512 bp fragment upstream from the start codon is used as promoter. Insertion of conservative regions of the promoters of the amylolytic genes of A. oryzae into this promoter further increased its activity (195).
The glutamate dehydrogenase ( gdhA ) promoter of A. awamori can be used when expression of the gene of interest is wanted in young cultures, since the gene is known for its early expression in A. awamori (150). Moraleyo et al. (150) compared this promoter to the B2 esterase promoter of Acremonium chrysogenum , the pcbC promoter of P. chrysogenum and the gpdA promoter of A. nidulans for the production of plant thaumatin. While maximum production with the latter three promoters was reached after 72h, the transformants with the gdhA promoter reached maximum production after 48h. The B2 promoter is largely expressed when the growth phase has been completed. A combination of early and late promoters may Literature review 48 provide a valuable tool with which to prolong gene expression during fermentation. Transformants of A. awamori with the B2 esterase promoter and the pcbC promoter produced between 0.25 and 2 mg/L thaumatin, while better results (1-11 mg/L) were obtained with the gpdA and the gdhA promoter, both originating from Aspergillus .
The isopenicillin N-synthase ( pcbC ) promoter of P. chrysogenum and the transcription termination signals of the gene were, for example, used for the successful expression of the Streptomyces clavuligerus penicillin N expandase in P. chrysogenum (196).
Recently, the A. oryzae thiA promoter was developed as an inducible promoter (197). The thiA gene encodes an enzyme involved in the biosynthesis of thiamine and is transcriptionally regulated by thiamine. The expression levels can be controlled by the concentration of extern thiamine. Pachlinger et al. (161) developed a metabolically independent and strictly controllable expression system for Aspergillus species. They use estrogene-responsive elements (ERE’s) of the human estrogene receptor in such way that Aspergillus species become sensitive for estrogenous compounds. In this way, the ERE’s could regulate the transcription of a reporter gene.
In order to solve the metabolic problems caused by the regulated use of the alcA promoter of A. nidulans in A. fumigatus , Vogt et al. (198) recently developed a tetracycline/doxycycline regulated gene expression system for this fungus. In the absence of tetracycline, the repressor protein TetR binds the tetracyclin resistance operon of E. coli and prevents transcription. When minute concentrations of tetracyline are present, a resistance response is induced since in those conditions TetR is no longer able to bind the operon. Different adaptations to this system were made in order to be able to use it in eukaryotes. Since this system uses prokaryotic regulatory elements, it has no effect on eukaryotic physiology. A limitation of the system is that only 10 to 15 % of the transformants could be regulated by doxycycline.
Glucose-repressed promoters are not useful in continuous cultures for a long period of time: depletion of glucose very often leads to the induction of proteases which decrease the yield of the product of interest (25). Deletion of the binding sites for the glucose repressor proteins in the promoters can offer a solution to this problem This strategy was followed by Hintz and Lagosky for the alcA -promoter of A. nidulans (198).
8.1.2. Introns Many protein encoding genes in eukaryotes are interrupted by non-translated intervening sequences, or introns (133). In higher eukaryotes, these introns can be several kilobases in size, in sharp contrast with those of yeast, which are rare, relatively short (a few hundred bases or less) and tend to be located at the 5’ end of the coding sequence. Higher eukaryotic genes often contain several introns, whereas in yeast genes only one or two introns are found. Filamentous fungal genes appear to be somewhere in between: they are present in approximately two-thirds of the genes, but they are very short (50 - 250 bp). Literature review 49
Several sequences are important for the correct splicing of fungal genes: - the 5’ splice site can be represented by the conserved sequence g\GTAYGTT - the splicing signal (lariat formation) usually has a sequence as WRCTRAC - the 3’ splice site is represented by MYAG\g There are sufficient differences between the conserved sequences in yeast and in filamentous fungi for yeasts to fail to slice fungal introns.
8.1.3. Transcription termination Till today, little is known about the mechanisms involved in transcription termination in filamentous fungi. Information is still limited on the effects of both the nature and location of mRNA processing signals on parameters such as the stability, the transport or the translational competence of the messenger RNA. Most expression cassettes have the 3’ untranslated region of a known homologous gene downstream the coding region of the heterologous gene. In this way, efficient termination of transcription is achieved.
The most important sequences which are associated with transcription termination and polyadenylation of the mRNA in eukaryotes are the YGTGTTYY and the AAUAAA motif (14). These sequences were already found in fungal genes, but mostly they are present in a shortened version (AUAA) or missing altogether in many others, so that their function is unclear. Filamentous fungal genes can also have several apparent polyadenylation sites. Evidence has shown that at least some of them are real rather than artefactual since 3’ heterogeneity has been observed in the sequences of cDNA clones (133).
From studies with higher eukaryotes, it became clear that the 3’ untranslated regions not only control the transcription termination, but also that they have important functions related to the mRNA stability (199). Various studies have shown that the terminator sequence with recombinant constructs plays an important role in the production and/or the stability of the mRNA. Construction of gpdA-lacZ fusions in A. nidulans reduced the mRNA levels to one- third of the wild type gpdA gene (136). A possible cause of this phenomenon is the use of a heterologous terminator ( A. nidulans trpC ) of the lacZ sequences. If these reduced mRNA levels were caused by instability of the mRNA or by reduced transcription was not investigated. By Northern blot analysis, Punt et al. (200) showed that mRNA derived from a recombinant glaA gene ( A. niger ) under the control of the gpdA promoter ( A. nidulans ) was less stable than the wild type glaA transcript.
8.1.4. Initiation of translation The translational initiation site in filamentous fungal genes usually occurs at the first ATG in the sequence and almost always has a purine (usually A) at the -3 position. Translation in eukaryotes often can be modulated by the sequences flanking the start codon. For example, in A. nidulans it has been shown that restoration of the 8 bp’s immediately upstream the AUG in the sequence of the native alcA promoter doubled the expression of human α-interferon (14). Kozak (201) worked out a consensus sequence for the AUG environment, based on 31 fungal Literature review 50 genes: TCAMM AUG KC. These sequences are presumably involved in recognition of the correct AUG by the ribosome.
The length of non-translated mRNA prior to the presumed initiation codon varies from approximately 25 to over 400 bases in filamentous fungi, though this tends to be less than 100 bases in Aspergillus (133).
8.1.5. Secretion of heterologous proteins The secretion of heterologous proteins by fungi offers several advantages as compared to intracellular production (20). Examples are, amongst others: - the purification of the protein is easier because the mycelium does not need to be broken - the protein is not exposed to intracellular proteases - the metabolism of the fungus is not disturbed by the foreign protein
Proteins can be translocated to the secretion system by the insertion of a secretion signal sequence between the promoter and the coding sequence of the heterologous gene. This sequence codes for a signal peptide of 13 to 50 amino acids which directs the protein to the endoplasmatic reticulum and subsequently to the secretory pathway, which is discussed in paragraph 8.2. The signal can be derived from well-secreted homologous proteins or, when the heterologous gene is secreted per se , the endogenous secretion signal can be used.
Not only signal peptides, but also prosequences may play a role in secretion efficiency. Prosequences are normally removed in the late stages of secretion and perform functions as organelle targeting and protein folding. Fusions of prosequences to heterologous proteins to improve secretion may have opposite effects, depending on the strain, the prosequence and the enzyme to be produced. Care must be taken in choosing a particular signal, especially if the target protein already contains a prosequence (14). Studies in eukaryotic and prokaryotic systems have shown that signal sequences do not contain certain recognition amino acid sequences necessary for secretion, rather the physical characteristics of the leader peptide is important (110). As a result, signal sequences are usually versatile and can be used to target heterologous proteins into the extracellular space with usually only little gene- or species- specific limitations. On the other hand, the signal (pre) and also the pro-sequence following in many enzymes can largely influence the quality and yield of the secreted product.
Besides fusing the heterologous gene to a secretion signal or prosequence, an alternative strategy for the production of heterologous proteins in filamentous fungi is the fusion of the heterologous gene to the complete coding region of a highly expressed homologous gene. In this way, the secreted homologous gene serves as a carrier for the heterologous protein, which in some cases leads to improved protein yields. To date, this approach is probably the most successful modification to increase heterologous protein production (19). It has resulted in 5 to 100 fold increases in the secretion of heterologous proteins (202). For example, the yield of calf chymosin in A. awamori was significantly improved by the fusion of the prochymosin Literature review 51 gene to the complete coding region of the fungal glucoamylase, in stead of only to the glucoamylase leader sequence (203). A similar strategy in A. awamori where Korman et al. (204) used α-amylase-prochymosin fusions also resulted in higher yields. Gouka et al. (205) showed that the limitation of the production of non-fused human interleukin-6 ( hil6 ) and guar galactosidase ( aglA ) at the transcriptional and (post)transcriptional level could be resolved by fusing the genes to the A. niger glucoamylase gene ( glaA ). A glaA fusion to the to the 3’ end of the hil6 gene produced almost no protein, but a fusion to the 5’ end resulted in a large increase in the hIL6 yield, while mRNA levels of both constructs were very similar and not clearly different from the non-fused hIL6. In contrast, a fusion of glaA to the aglA gene resulted in a 25-fold increase in the mRNA level and in a similar increase in the protein level. This study was carried out in defined single-copy strains.
These observations suggest that the coding sequence of extracellular proteins contains information which promotes the secretion by improving the translocation of the protein into the endoplasmatic reticulum, aiding folding and protecting the protein from degradation. The problem of splicing the heterologous protein and the homologous carrier can mostly be solved by inserting a recognition site for a KEX2 endopeptidase (20). This endopeptidase splices proteins endoproteolyticly between two adjacent basic amino acids, preferably lysine and arginine.
The glucoamylase of A. niger and A. awamori and the cellobiohydrolase of Hypocrea jecorina are the most commonly used fusion partners (22). Interestingly, both enzymes can be divided into three domains: an N-terminal catalytic domain, a C-terminal starch or cellulose binding domain and a flexible O-glycosylated linker region. The C-terminal domain can be efficiently replaced by the heterologous protein, although full-length fusions are also successful. It has been suggested that the positive effect of the fusion is caused by the fact that the linker region permits the catalytic domain and the rest of the fusion protein to fold independently. Paloheimo et al. (206) studied the effect of various carrier polypeptides on the expression of a bacterial xylanase in the Hypocrea jecorina , controlled by the cbhI promoter. They conclude that high-yield production requires a carrier with an intact domain structure and that a flexible hinge region (connection) between the carrier and the xylanase had a positive effect on both the production of the xylanase and the efficiency of cleavage of the fusion polypeptide. It was also shown that sequences of some recombinant proteins are incompatible with the secretory pathway of filamentous fungi, resulting in stalling of the proteins, targeting to other cell compartments or proteolytic attack by intracellular proteases associated specifically with the secretory pathway (14).
8.2. The fungal secretion pathway The high capacity of the secretion machinery of filamentous fungi has been widely exploited for the production of homologous and heterologous proteins; however, our knowledge of the fungal secretion pathway is still at an early stage (19). Most of the knowledge comes from Literature review 52 models developed in yeast and higher eukaryotes, which have served as a reference for the studies on fungal species.
A schematic view of the fungal secretion pathway and the cellular compartments important for post-transcriptional modifications is given in Figure 8-2. The translation of the proteins starts in the cytosol by binding of the ribosomes to the mRNA. As soon as the secretion signal is translated, the signal recognition particle (SRP) binds on it. As a result, the translation process is paused, and the whole moves to the endoplasmatic reticulum (ER). Secretory proteins begin their journey to the extracellular medium by entering the ER via an interaction between the SRP and an SRP-receptor (docking protein) present in the membrane of the ER. The SRP is released and the translation resumes, resulting in a growing peptide chain which is translocated through the ER membrane. In the ER lumen, the N-terminus of the peptide chain is processed by membrane bound signal peptidases and the maturation of the protein starts.
Figure 8-2. The secretion pathway in filamentous fungi.
Next to the signal peptidase, at least tree other proteases have been identified in S. cerevisiae : KEX1 is a carboxypeptidase B-like serine protease, KEX2 is a trypsin-like serine protease with dibasic endoprotease activity (cuts between lysine-arginine, arginine-arginine or arginine-lysine, with lysine-arginine being the most efficient) and STE13 is a dipeptidyl- aminopeptidase involved in amino-terminal maturation. Similar processing activities have been postulated for filamentous fungi.
Literature review 53
In S. cerevisiae , another pathway (the SRP independent pathway) has been described in which proteins are targeted to the ER posttranslationally thereby assisted by the BiP-protein (an ER chaperone) (19). Both routes are depicted in Figure 8-3 (after 19). The targeting route of each protein is determined by the hydrophobicity of the signal sequence: proteins with a less hydrophobic signal sequence are targeted through the SRP-independent route, whereas both routes can be followed by proteins with a more hydrophobic signal. Although both targeting routes may be universal, the specificity of the system and the proteins which follow either route may not be interchangeable, which could be of importance when heterologous expression is studied. It is not yet clear if both routes exist in filamentous fungi, but the identification of an SRP-homologue in A. niger and of KAR2/BiP homologues in numerous fungal species suggests that also in fungi both routes are present.
Figure 8-3. Two pathways of protein targeting and translocation to the ER. ss = secretion signal, SRP = signal recognition particle, SRPR = SRP receptor.
In the ER lumen, proteins are folded and can undergo distinct modifications such as glycosylation, disulfide bridge formation, phosphorylation, and subunit assembly (see 8.3).
Subsequently, the proteins leave the ER packed in transport vesicles and head to the Golgi compartment, where additional modifications can take place, such as further glycosylation and peptide processing. Finally, again packed in secretory vesicles, proteins are directed to the plasma membrane from where they are secreted. In some cases, proteins will not reach the extracellular space, but are targeted to intracellular compartments such as the vacuole, either to become resident proteins, or to undergo proteolytic degradation.
Literature review 54
Although there has been some controversy, it can be stated that a special quality of protein secretion in filamentous fungi is that the proteins predominantly are secreted into the medium at the end of growing hyphae (207).
8.3. Post-translational modifications During the passage through the secretion system, proteins are submitted to series of post- transcriptional modifications such as glycosylation, disulfide bridge formation, phosphorylation, proteolysis, and subunit assembly (20). Together with the protein-folding processes, these modifications play an important role in determining the structure of many proteins.
In the endoplasmatic reticulum, immature proteins are associated with the Binding Protein BiP-protein (also known as the glucose-regulated protein GRP78). This protein is a member of the heat shock 70 protein family (Hsp70) and has three functions. It participates in the ER- translocation of nascent proteins; it functions as a chaperone by keeping the proteins unfolded in order to prevent aggregation and it binds to incorrectly folded proteins to keep them in the ER lumen till they are broken down. In yeast, this protein is encoded by KAR2 .
During translocation through the ER membrane, N-glycanes (Glc 3Man 9GlcNac2) are added to the side chain of asparagine residues in the consensus sequence N-X-S/T by an oligosaccharyltransferase (Figure 8-4). These glycanes are removed again by the sequential action of glucosidase I and II (GI and GII). Glycoproteins containing high-mannose-type oligosaccharides are transiently reglycosylates in the ER lumen by the luminal UDP-glucose- glycoprotein glucosyltransferase (GT) to generate monoglucosylated structures. Monoglucosylated polypeptides which arise from the stepwise removal of glucoses and GT reglucasylation are recognised by and bind to calnexin (or is soluble homologue calreticulin), allowing the chaperone to provide folding assistance. Upon GII action, the last glucose is trimmed off and glycoproteins are released from calnexin. Correctly and completely folded proteins will continue their journey to the Golgi, whereas incompletely folded proteins are recognised by GT and reglucosylated to regenerate the monoglucosylated glycoprotein, which will start a new cycle of folding and deglucosylation (19).
The folding of secreted proteins takes place in the lumen of the (ER) and is an energy- consuming process, similar to that of many other eukaryotes. The proteins are assisted by helper proteins named chaperones and foldases, more precisely protein disulfide isomerases (PDIs) and peptidyl prolyl isomerases (PPIs), the latter of which catalyses the isomerisation of cis and trans peptide bonds on the N-terminal side of proline residues. PDIs catalyse the oxidation, reduction and isomerisation of protein disulfide bonds. The tertiary structure of many extracellular proteins is stabilised by disulfide bonds. The first fungal protein-disulfide isomerase was cloned from A. niger (208). Chaperones and foldases are ubiquitous: they are not only present in the ER, but also in the cytosol, mitochondria, chloroplasts and periplasm. They are conserved between organisms. The process of protein folding is a complex network Literature review 55 of interactions which is dependent on the characteristics of the folding proteins, the environment in the ER, and the availability of specific cofactors. Chaperones may cobind or act sequentially in protein folding.
Proteolytic digestion is another important type of post-translational modification in the ER. The most occurring proteolytic modification is the removing of signal sequences by signal peptidases (see also 8.2) after translocation through the ER membrane (209) and the endoproteolytic cutting by endoproteases such as KEX2.
When the proteins went through all modifications which take place in the ER, they are submitted to a quality check (Figure 8-4, after 19): calnexine or calreticulin recognises the correctly folded proteins after which the proteins leave the ER. If the proteins are folded in a good way, they move to the Golgi apparatus, where glycosylation takes place and where the propeptides are split off. In contrast, UDP-glucose-glycoprotein glucosyltransferases recognise incorrectly folded proteins and keep them in the ER. As a result, the ‘unfolded proteins response’ (UPR) and the ‘ER-associated protein degradation’ (ERAD) are initiated. The UPR detects the presence of unfolded proteins and induces the synthesis of folding enzymes, and the ERAD degrades those proteins which fail to reach the correct conformation. The ERAD system eliminates misfolded proteins via degradation in the cytosol.
Figure 8-4. Calnexin/calreticulin cycle.
The structure and the function of secreted proteins can be strongly influenced by glycosylation, which takes place in the Golgi-apparatus. Glycosylations in filamentous fungi are, as far as known, very similar to those in other eukaryotes, especially S. cerevisiae (20). In contrast to S. cerevisiae , where hypermannosylation frequently occurs, the glycosylation patterns in filamentous fungi resemble those of higher eukaryotes more (21): oligomannose N- and O-glycans are predominant. O-Glycosylation mainly attaches glycosyl structures to serine or to threonine residues (14). The presence of glucose, galactose, phosphate, sulphate Literature review 56 and simple N-acetylglucosamine on the linked glycans has also been reported. Other more complex glycan structures typical of mammalian glycoproteins are not found in fungal proteins, probably because fungi lack some of the glycosyltransferases of higher eukaryotes. This difference between the mammalian and fungal glycosylation machinery limits the applicability of the fungal cell for the synthesis of mammalian proteins when correct glycosylation is essential for the protein activity (19). Overglycosylation is generally believed to occur in filamentous fungi when the protein secretion pathway is saturated and the export is slowed down as a result of proteins overloading the system (110). Directed modification of the glycosylation can be used to improve the heterologous gene expression. In this way, the insertion of an additional N-glycosylation site in calf chymosin could improve the yield more than three times in A. awamori (131) and also resulted in higher yields after addition in preproinsuline in A. niger . Other approaches introduced the genes of the missing mammalian glycosyltransferases into the fungal hosts (19).
After passage through the Golgi proteins are directed either to the plasma membrane for secretion or to the vacuole.
8.4. Reporter genes and expression studies The use of reporter genes (and their encoded proteins) for the analysis of various biological mechanisms is well established for many organisms. The first studies about heterologous gene expression with filamentous fungi have been done with model genes, such as the lacZ and gusA reporter genes from E. coli . These studies have been very fruitful for the study of gene expression and protein secretion. The lacZ gene coding for β-galactosidase and the gusA gene and uidA gene coding for β-glucuronidase are frequently used (200). They allow, for example, the study of 5’ sequences which are necessary for controlled gene expression (20, 108): determination of promoter strength, identification of regulatory elements, induction or repression analysis, etc.. Mini-promoters fused to reporter genes can be used to examine the regulatory effects of inserted sequences (128, 200).
Bacterial reporter genes are a powerful tool in different studies and they have been used with success in fungal systems. On the other hand, eukaryotic reporter genes are recently used more frequently. Shoji et al. (197) for example used the ‘enhanced green fluorescent protein’ (EGFP) for expression studies with the inducible A. oryzae thiA promoter. Lopes et al. (210) used the GFP-gene to study multicopy integration in Penicillium . GFP has been used as a reporter for gene expression, for tagging of proteins to monitor their localisation within living cells and for monitoring enzyme secretion. The success of GFP as a reporter protein can be attributed to its unique characteristics, such as a non-requirement of co-factors or substrates for its activity, high stability under different temperature and pH regimes, the ease with which it is detected in vivo and the possibility of being fused to other proteins without losing its fluorescence property. In this view, Tavoularis et al. (211) notify that the number of linker amino acids is crucial for the correct expression and/or translocation of the fusion construct to their target location and for the fluorescence of the GFP. Mutations of the gfp gene have led to Literature review 57 the design of improved GFP varieties, which are not only brighter than the wild type, but also are available in different colours (212). Mikkelsen and collaborators recently used the red fluorescent protein DsRed from the reef coral Discoma sp. as a new marker in Penicillium and Trichoderma species (212). It was fused to the A. nidulans gpd -promoter and trpC -terminator. Dual marked transformants expressed GFP and DsRed in the same mycelium. Mostly, reporter genes are used for which convenient and established activity assays exist. Obviously, also other secreted proteins, for which no activity assay exists, may be used as secretion reporter proteins, but in those cases analysis will mainly have to be carried out by immunological methods. In this respect, the A. niger glucoamylase can be mentioned, for which numerous poly- and monoclonal antibodies are available (207).
In order to be able to compare the production levels of different proteins in a reliable manner, it is essential that the production strains take in the expression vectors in single-copy and at a specific chromosomal site since the copy number and the chromosomal environment can influence gene expression (119) (see also paragraphs 6 and 7.2). For different Aspergillus species, expression and/or analysis systems have already been developed which simplify the selection of single-copy transformants containing the expression vector at a specific locus. These systems are based on a mutant pyrG of A. niger (106) or mutant argB genes of A. nidulans (157) as selection marker and the corresponding pyrG or argB mutants as recipients. Gouka and co-workers (119) developed for A. awamori a site specific integration system based on mutant pyrG genes. After transformation of a defined OMPD-negative mutant (containing a mutation at the 3’ site of the pyrG gene) with vectors having a mutation at the 5’ site of pyrG , mutants with a single copy of the vector at the pyrG locus are obtained with high frequency. In this way, an expression vector can be integrated in single copy into the genome (Figure 8-5, after 119).
Figure 8-5. Strategy for the creation of single-copy transformants.
Literature review 58
The use of site specific integration vectors enables the study of parameters which have an influence on the expression of recombinant genes. A few examples are given: - Van Hartingsveldt et al. (213) used such vectors in A. niger . By the use of glucoamylase leader peptide-chymosine fusions they were able to observe two important things. First of all, it appeared that depending on the leader peptide which was used, the mRNA levels could vary with a factor 10. This shows that the sequences which code for the leader peptides of highly expressed genes are co-responsible for the initiation of transcription or for the stability of the mRNA. Further it was observed that there is no direct correlation between the mRNA levels and the final protein yield. A construct which yielded 90 % less mRNA as compared to another produced only 50 % less protein. - Gouka and co-workers (214) compared in A. awamori the production of a homologous protein to that of different fungal and non-fungal proteins. They observed strong differences in the steady-state mRNA levels, whereby fungal genes had the highest levels and non-fungal genes reached much lower levels. In all cases the protein levels were correlated with the mRNA levels, except for the human interleukin 6. In this case, relatively high levels of mRNA were observed, while only a very low protein yield was attained, probably due to intracellular protein degradation. - Van Gemeren et al. (166) studied the effect of pre- and pro-sequences and multicopy integration on the expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori . This gene was used as reporter gene and expression could be followed by the formation of clearing zones around the colonies on plates containing an olive oil/arabic gum emulsion or quantitatively in an assay (215). The analysis was carried out in single copy transformants containing an expression cassette with the cutinase gene between the A. awamori exlA promoter and terminator integrated at the pyrG locus. Transformants containing a construct encoding a direct, in frame fusion of the xylanase pre-peptide to the mature cutinase showed a two-fold higher cutinase production compared to strains containing constructs with an additional cutinase pro-peptide. Strains with more than one copy of the cutinase construct showed a 6- to 12-fold increased production of extracellular cutinase compared to the single copy transformants. No linear dose response relation to the number of expression cassettes present in the strains was observed, but the amount of active enzyme correlated with the amount of mRNA.
8.5. Strategies for the improvement of (heterologous) protein production Different strategies have been developed to improve the (heterologous) protein yield (214): - use of strong promoters and secretion signals (as discussed above: 8.1.1 and 8.1.5) - use of gene fusion with highly secreted homologous proteins (8.1.5) - introduction of a high copy number of the expression cassette in the genome - modification of the protein, random mutagenesis and screening for higher production levels - use of protease-deficient cloning and expression hosts - development of a suitable fermentation medium.
Literature review 59
As mentioned above, transformation of filamentous fungi results in the non-specific integration of the recombinant DNA in the chromosome. A frequently observed feature of this process is the duplication of the integrated DNA, resulting in a high copy number of the DNA of interest. Although it can generally be said that a higher copy number results in a higher protein yield, these two phenomena are not strictly correlated (14). There is a more or less linear dose/response relation with strains with a small number of gene copies, whereas no dose/response relation is observed with strains with higher copy numbers. With higher copy numbers, the production level is lower than expected from the copy number (216). The fact that DNA can integrate in chromosome regions with different transcription efficiencies can be an explanation for this. Another explanation could be that rearrangements occur during integration and some copies may be rendered non-functional (110). To prevent the recombinant DNA from integrating at a site in the chromosome with low transcription efficiency, one can use site specific integration vectors. These vectors contain the expression cassette flanked by sequences of a non-essential gene which is known to be highly expressed. Via homologous recombination, the expression cassette is integrated in transcriptionally active regions. Such gene replacement vectors based on, for example, the A. niger glaA (213) and the Hypocrea jecorina cbh1 (217) flanking sequences have been used with success. An alternative possibility for ameliorating the effects caused by the site of integration is the inclusion of matrix attachment regions (MARs) at either end of the expression cassette. These sequences bind the DNA to the nuclear scaffold and appear to define a transcriptional ‘domain’ (14). It was suggested that the pronounced damping of position effects observed is due to the MAR maintaining the DNA in a transcriptionally active form. Interestingly, these elements appear to function in heterologous systems. It is also possible to engineer artificially high copy number strains. Using a cosmid vector containing up to 10 copies of an A. niger glaA expression cassette, transformants with up to an estimated 160 copies of the glaA gene were obtained, with a concomitant rise (five- to tenfold) in glucoamylase production.
Transformants with high copy numbers of DNA, carrying sequences where positively acting regulatory proteins bind on, appear to have a lower expression of genes which are activated by these regulators (108). This phenomenon is attributed to the titration of the transcription factors involved in the activation process. This vision is supported by the fact that more copies of the regulatory proteins are able to compensate the loss. In A. nidulans the yield of α- interferon under the control of the alcA promoter could be increased by the overproduction of the positively acting transcription factor AlcR which regulates the alcA promoter (218).
In filamentous fungi, codon bias is regularly observed, especially in genes which are highly expressed (219). This correlates strongly with the number of copies of tRNA for each codon, which in turn determines cellular tRNA levels (220). In many cases, this codon bias is seen as the preferred occurrence of a C or G at the third position. In Aspergillus and Neurospora genes there is a tendency to avoid usage of codon ending with A (133). Generally, the bias is less pronounced in Aspergillus than in Neurospora and in fungi less evident than in yeast. The Literature review 60 introduction of rare codons (NNA) in the am gene of N. crassa resulted in a decrease in protein production to 35 % of the wild type level, while the mRNA levels remained the same (221). Optimalisation of the codon usage of the Dictyoglomus xynB to suit expression in Trichoderma resulted in a dramatic increase in the production of the enzyme (220).
The often low production yields obtained for heterologous proteins using the fungal production system present an intriguing problem. Analysis of limiting factors which could be responsible for this phenomenon has shown that transcription of heterologous proteins and mRNA steady state levels are mostly satisfactory, suggesting that the bottlenecks are present mainly at the posttranscriptional stage, possibly along the secretion pathway (19). Different genetic approaches have been used to analyse and alleviate these putative blockages. Two factors already discussed (see 8.1.5) are the choice of the secretion signal and the use of translational fusions. Other means to intervene at the posttranscriptional stage are the introduction of changes in the glycosylation machinery (8.3) or to modify the levels of chaperones. It has been argued that in a heterologous system proteins may encounter folding restrictions limiting their production efficiency and that this problem can probably be overcome by provision of the cell with an increased level of the helper proteins. Bearing this idea in mind, the effect of chaperone levels on heterologous protein production has been assessed for a number of systems. However, results are far from conclusive and frequently even seem contradictory (for an overview of the strategies already tested, see 3).
The apical localisation of protein secretion has led to the suggestion of employing morphological mutants displaying an increased apical surface, i.e. hyperbranching mutants, as ‘supersecretion’ strains (19). Moreover, these strains often grow as compact pellets, which results in low-viscosity cultures having additional technical advantages in the fermentation process. However, as different, often pleiotropic, mutations can alter hyphal morphology, an unequivocal correlation between hyperbranching and secretion seems unlikely. Hyperbranching mutants of T. viride have an increased production of extracellular cellulase and a N. crassa mutant displaying an enlarged growth surface area produces more protein than the wild type strain, but a similar mutant of A. nidulans secretes less acid phosphatase.
Even when heterologous proteins are successfully excreted into the medium, a substantial part of the production can be lost via degradation by extracellular proteases. Moreover, heterologous proteins are more sensitive to proteolysis than homologous proteins (222). Mattern et al. (223) were able to show that the use of protease-deficient mutants of A. niger results in higher yields of heterologous proteins. Proteolytic degradation by fungal proteases is considered to be one of the major problems with heterologous protein production. Thus, the reduction or the elimination of protease activity has been a major area of interest in host strain improvement. Some approaches have met with success. The gene encoding the major secreted aspartyl protease ( pepA ) of A. awamori and A. niger has been successfully deleted from the strains, resulting in improved production hosts: 2-fold higher yields were obtained (110). Zheng et al . Literature review 61
(224) were able to reach a more stable and higher expression of lysozyme in Aspergillus oryzae by expressing antisense RNA of the carboxypeptidase O gene in the production host. As a result, carboxypeptidase activity dropped to 30 % of that of the parent strain and peptidase degradation of lysozyme reduced. A more speculative approach to control protease activity is to clone and express genes coding for protease inhibitors. Simple addition of protease inhibitors to the medium may have limited appeal in large-scale production (14). Attempting to make use of gene regulatory phenomena to control protease action offers the possibility of finer tuning when certain protease activities can be down-regulated in the growth phase when expression and secretion of the heterologous proteins is to be achieved (110). Such a strategy can only be based on the detailed knowledge of the regulation of different protease encoding genes. Jarai et al . (1997) studied three extracellular proteases, PepA, PepB and PepF and two intracellular proteases, PepC and PepE under different conditions. All three extracellular proteases appeared to be under complex transcriptional regulation as carbon catabolite repression, nitrogen repression and external pH affected the expression of all three enzymes. Overall similarities as well as significant differences in their regulation were found. The two intracellular proteases did not show any noticeable variation in expression levels and probably are not governed by the above mentioned regulatory circuits. These complex regulations make it difficult to down-regulate certain protease activities precisely. Besides the extracellular proteases, the large amounts of organic acids produced by many fungi under particular growth conditions can be a problem once the proteins are secreted. As a result of the secretion, the external pH drops to as low as pH 1.5 to 2.0 in some cases, which can alter the structure of certain proteins or lead to increased sensitivity to acid proteases active in the culture broth. Altering the culture conditions or genetic engineering to avoid the secretion of certain organic acids thus can be another objective in improving the protein production yield (14).
Classical methods such as random mutagenesis followed by a screening of the resulting mutants were already successfully applied to improve protein production, for example for the improvement of the chymosin production in A. awamori (225). Random mutagenesis approaches result in mutants with defects in unknown functions, possibly in genes important for industrial production of the target enzyme, and thus these strategies should be avoided.
Next to the above mentioned strategies to improve the heterologous gene expression in filamentous fungi, one needs to consider physiological factors such as the role of the fermentation medium and the cultivation techniques. In the wild, fungi grow on the surface of soil particles, plant material, animals and other substances. On the other hand, in fermentation industry, submerged cultures are normally used because they are easier to handle and because it is easier to control the fermentation. In solid-state fermentations, as used in the Japanese traditional food and beverage industry (‘koji’-type), fungi also grow on the surface of medium, which is one of their common characteristics. The amounts of enzymes secreted by filamentous fungi in solid state culture are large and frequently exceed the amounts secreted Literature review 62 in submerged culture. This high productivity of solid state culture is not only observed for the production of homologous proteins, but also for the production of heterologous proteins. The different culture conditions between solid-state and submerged culture can alter the expression of various genes, which can affect various phenotypes, such as growth, development and mycotoxin and enzyme production. Solid-state specific gene expression appears to be responsible for this increased production (202) and some regulatory elements in the 5’ untranslated regions of fungal genes responsible for the specific expression have been discovered. Proteins can be differently targeted, depending on the culture condition (solid- state versus submerged). In submerged culture, some enzyme activities are mainly found in the cell wall of mycelia, while in solid-state culture these enzyme activities are observed in the medium and little activity is observed in the cell wall. This suggests that the destination of some genes is affected by culture conditions.
From the classical study of solid state culture, it is known that several environmental factors, such as water activity and cultivation temperature significantly affect growth and enzyme production. For example, at low water activity, the production of glucoamylase is optimal at 35-40°C, while the production of proteases is optimal at 30-35°C and significantly decreases at 42°C. Other culture conditions which can affect the protein yield are the inoculum size, the culture temperature, the pH of the medium, the dissolved oxygen and the medium composition (14).
Fungal cultures may be inoculated by using either conidial suspensions or vegetative mycelia. Levels of secreted hen egg-white lysosyme in A. niger have been shown to be maximal at a specific conidial inoculum size in shake flasks. This may be due to the presence of an inhibitor at high spore concentrations, as washing of the spores can relieve some inhibitory effects on germination. Vegetative mycelial inocula can also be used to circumvent this problem and have proved effective in achieving more rapid increases in fungal biomass in bioreactors.
Evidence from various systems has indicated that protein secretion is favoured at temperatures lower than that for optimal growth. This is probably due to a decrease in protease activity, although other factors have not been ruled out. Processes have been developed for fungal systems, such as for Hypocrea jecorina , whereby fungal biomass production is promoted at the growth optimum temperature, followed by a period of protein secretion at a lower temperature.
Related to the pH of the medium, strains can be used which acidify the medium to a lesser extent or components of the medium can be altered to reduce the problem. Buffering the pH of the medium can also be a solution to the problem.
In large scale bioreactors, the critical growth-limiting substrate is most often dissolved oxygen, due to its low solubility in water. Much effort has therefore been applied to achieve Literature review 63 sufficient oxygen transfer to satisfy demand. Oxygen demand in filamentous fungi, as in other microorganisms, varies with the growth phase, and the supply of oxygen is critical, especially when considering the changes in culture rheology from Newtonian to non-Newtonian as the degree of hyphal branching increases. Oxygen transfer into ‘pellet’ fungi will also differ when compared to that into the true mycelial form. Some enzymes, such as the glucoamylase, are maximally produced at low levels of dissolved oxygen and this should be taken into account when using the gla A promoter to express heterologous proteins. Dissolved oxygen levels in other systems are normally maintained between 25 and 50 %, either by varying the agitation rate or altering the gas composition.
Variations in the medium composition can produce dramatic increases in protein productivity. Genes controlled by catabolite repression tend to be better expressed when the supply of inducer is gradually added in a fed-batch matter. Good knowledge of the inducers and repressors of the expression system in use is therefore required to optimise protein production. Soybeans can be added to the medium because they are known to contain protease inhibitors, and are rich sources of manganese ions, which are known to increase hyphal branching. Literature review 64
9. RESEARCH OBJECTIVES Humans have used microorganisms without knowing for their own benefit since ages. Filamentous fungi, yeasts and their enzymes are indeed instrumental since thousands of years in the making of beer, wine, bread and cheese. Nowadays, fungi and their enzymes are still widely used in the food industry, but they are also applied in other industrial fields. Despite the fact that fungi already have a broad spectrum of industrial applications, until now, only a limited number of fungal host species has been explored for recombinant protein production. Therefore, it is not surprising that several parties started to explore the alternatives to those covered by several patent applications. Patents and intellectual property rights have necessitated searching for expression hosts other than the fungal species traditionally used.
The fact that many currently used fungal expression hosts are protected by patent applications, taken together with the consideration that, undoubtedly, micro-organisms and, more specific, filamentous fungi, exist which have new characteristics and produce enzymes with new application possibilities, was a stimulation to explore the BCCM/MUCL agro- industrial fungi- and yeast collection for a potential new expression host. The aim of our research was to further develop a transformation system and to develop an expression system to enable us to use the filamentous fungus Myrothecium gramineum MUCL 39210 (syn. Xepiculopsis graminea ) as a new, universal expression host.
In chapter I , further research was performed on the development of a transformation system which would enable us to use the filamentous fungus Myrothecium gramineum as a new, universal expression host. Transformants were selected based on their resistance to hygromycin B and their ability to overproduce the Taka amylase of Aspergillus oryzae , which was used as a reporter gene.
A quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host or to study the molecular systematics of an organism. While rapid and high-throughput PCR-methods or other molecular techniques are amply available for bacteria and even for yeast, this is not the case for filamentous fungi. In chapter II , we compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR.
The use of strong promoters for the expression of proteins in suitable host organisms is of great importance for biotechnological applications. However, it has been shown that there is not much homology between fungal promoters, not even in related fungi. Because little is known about the critical parts of fungal promoters till today, a strong homologous promoter is usually searched for when developing a new expression system. It is assumed that this strong promoter will function ideally in the new expression host. In chapter III , the cloning of a homologous promoter of M. gramineum is described. Literature review 65
In order to explore its capabilities to produce (heterologous) enzymes, Myrothecium gramineum , was tested as to its production of an α-amylase of A. oryzae . This aspect of the research is discussed in chapter IV . α-Amylases constitute an important class of enzymes which find many biotechnological applications in processes which involve, for example, the degradation of starch and the determination of soluble and insoluble dietary fiber in rice and wheat bran. Such applications include baking, brewing, detergents and desizing (in textile industries). In chapter V , Myrothecium gramineum was tested as to its production of an endo −1,4−β−xylanase of Penicillium griseofulvum MUCL 41920. Recently, there has been much industrial interest in xylan and its hydrolytic complex. For example, increasing concern over preserving the environment from industrial wastes has initiated a growing interest in applying microbial enzyme systems in the paper and pulp industry. Besides in the paper and pulp industry, xylanases find their applications as supplements in animal feed, for the manufacturing of bread, food, juice and wine, in the textile industry, and for the production of ethanol and xylitol.
Although production of homologous fungal proteins is usually quite efficient, equally successful production of heterologous proteins from filamentous fungi is not always achieved. Unfortunately, the published yields of non-fungal enzymes from filamentous fungi often are even lower. On the basis of limitations observed for the production of non-fungal enzymes, several strategies have been developed to improve protein yields. The application of a gene fusion strategy has been especially successful. In chapter VI , Myrothecium gramineum was tested as to its production of a bacterial enzyme, more precisely an endo −1,4−β−xylanase of Bacillus subtilis .
To be able to use this fungus for the industrial production of enzymes, an efficient expression system is required, as well as an easy selection method for clones of this fungus carrying the introduced expression cassette. The literature describes many different methods to select transformants of fungi. One can use dominant selection markers, such as antibiotic resistance markers, or auxotrophic markers. Since antibiotic resistance markers cannot be used for some industrial applications and homologous transformation systems are more efficient, the orotidine monophosphate decarboxylase gene ( ompd -gene) was chosen as selection marker and isolated from the genome of Myrothecium gramineum . In chapter VII , the cloning of the ompd -gene of Myrothecium gramineum is discussed. In chapter VIII , the development of a homologous transformation system based on the orotidine monophosphate decarboxylase gene is described. In this research, attempts were made to develop an OMPD-negative strain which can be used in expression studies. In such expression experiments, it is important that the expression cassette integrates at a defined site in the genome, in single copy and preferably at a high frequency.
Part I: Method Development
Chapter I: Transformation of M. gramineum 69
Chapter I
Transformation of Myrothecium gramineum with the hygromycin B resistance marker gene
1. INTRODUCTION Since the advent of genetic engineering, a number of living cells have been transformed with DNA in order to enable them to produce homologous or heterologous proteins or to modify their metabolism by, for example, introducing new metabolic pathways. The variety of transformed organisms spans from bacteria to human cells and comprises organisms as diverse as yeast, fungi, plant tissue and insect cells. Among these organisms fungi have been the subject of many studies because of their interesting features, as described in § 1 of the Introduction.
In 1973, Mishra and Tatum reported the first successful transformation of a filamentous fungus, Neurospora crassa (14, 52). Since then, many fungi were transformed with success. Presently, five different techniques to transform filamentous fungi are described in the literature (51): protoplast transformation, electroporation, lithium acetate mediated transformation, biolistical transformation and Agrobacterium tumefaciens mediated transformation. In our research, the protoplast transformation system was chosen, because no special machinery is required, because it is one of the most frequently used techniques to transform fungi and because the method has already been used with success to transform Myrothecium gramineum (29).
In order to distinguish between the cells (protoplasts) which are transformed and the ones which are not, one needs a selectable marker present on the transforming DNA. Different selectable markers have become available for fungal transformation systems, as also discussed in § 5 of the Introduction. Generally, the selection markers can be divided into two major groups. Firstly, there are dominant selectable markers, such as antibiotic resistance markers and secondly, one has wild type genes which complement auxotrophic mutants (14). For filamentous fungi, as opposed to yeasts, dominant selection markers are preferred, because they do not require a specific mutant genotype (25). The E. coli hygromycin B resistance marker gene, the Streptoalloteichus hindustanus bleomycin/phleomycin resistance marker gene, the neomycin/kanamycin/G418 resistance gene, the fungal benomyl resistance gene and the oligomycin resistance gene are antibiotic resistance markers which are commonly used in fungal transformation systems. Another, non-antibiotic, dominant selection marker which can be used for filamentous fungi is the acetamidase gene ( amdS ). Because acetamide is a poor nitrogen source for wild type A. nidulans and most other fungi, the acetamidase gene can be used as a selectable marker for many fungi. Transformants which incorporated and express the amdS gene are able to grow on media with acetamide as the only nitrogen (and carbon) source (51). The dominant selection markers have as big advantage that
Chapter I: Transformation of M. gramineum 70 they can be used for fungal species which have not been studied a lot. In addition, the amdS system avoids the use of antibiotics, which is required in certain industrial applications.
The aim of our research was to further develop a transformation system to enable us to use the filamentous fungus Myrothecium gramineum as a new, universal expression host. Myrothecium gramineum is a fungus which was selected in a preliminary study carried out by the company Beldem in collaboration with the Laboratory of Microbiology of the UCL. Myrothecium gramineum MUCL 39210 (syn. Xepiculopsis graminea ) seemed to possess all desired qualities for an interesting expression host. The basic transformation protocol used to transform M. gramineum is the one described by Punt and Van den Hondel, which uses protoplast generation for the transformation of filamentous fungi (226). This protocol was used by Jonniaux et al. for the transformation of Myrothecium (29). Transformants were selected based on their resistance to hygromycin B and their ability to overproduce the Taka amylase of Aspergillus oryzae , which was used as a reporter gene. This yielded 0.6 hygromycin transformants per µg DNA and per 10 6 protoplasts. During this research, the transformation protocol was modified in order to increase the transformation efficiency, which would allow screening for higher production strains. The preculture was carried out in
Aspergillus minimal medium (AMM, see below) + V 8 (a blend of 8 fruit and vegetable juices) instead of in medium containing 2% malt extract, 1% peptone and 3% glucose. In this medium, the strain grew under the form of filamentous mycelium instead of in pellets, which are less accessible for the cell wall digesting enzymes and thus less suitable for protoplast production. A heat shock was introduced after DNA was added to the protoplasts.
2. MATERIALS AND METHODS
2.1. Strains and plasmids Myrothecium gramineum BCCM TM /MUCL 39210 was used as the wild type strain. Escherichia coli JM109 was used for plasmid amplification. Three plasmids were used for the transformation: - pCSN43: this plasmid contains the 2.4 kb Sal I fragment of pDH25 in the Sal I site of pBSSK + (Stratagene). The fragment encloses the Escherichia coli hygromycin B resistance gene ( hph ) surrounded by the trpC -promoter and -terminator of Aspergillus nidulans (227, 228) - p3SR2: this plasmid contains the amdS -gene of Aspergillus nidulans as a selectable marker (229) - p2G-S: this plasmid has 2 copies of the Aspergillus oryzae Taka-amylase gene ( amy3 , Genbank accession number X12727 , under the transcriptional control the promoter of the gpd -gene of A. nidulans and its own 3’ flanking sequences) and the amdS -gene of A. nidulans (29).
Chapter I: Transformation of M. gramineum 71
2.2. Culture conditions
2.2.1. Standard culture conditions E. coli was grown on Luria Broth medium (230) supplemented 0.1 g/L ampicillin (Acros) and 0.04 g/L X-gal (Acros) when necessary. Liquid cultures were grown at 37°C and 200 rpm. M. gramineum was grown on Potato Dextrose Agar (PDA, Oxoid) for spore production. The spores of Myrothecium were inoculated into 350 mL Aspergillus minimal medium (AMM) + 9 V8 to a final concentration of 2.10 spores per litre in order to produce mycelium for the protoplast preparation. Cultures were grown at 25°C (200 rpm) for 24 hour. AMM was used as described by Barrat et al. (231), supplemented with 5 g/L yeast extract (Difco). AMM + V 8 consisted of the same components, with the addition of 4.1 g/L Na-acetate, 2 g/L CaCO 3 and
200 mL/L V 8. For the isolation of genomic DNA the strains were grown in 100 mL Potato dextrose broth (PD, Oxoid) during 3 days (25°C, 200 rpm).
2.2.2. Test of the hygromycin selection medium M. gramineum was grown on PDA supplemented with 100 µg/L, 250 µg/L, 500 µg/L and 1000 µg/L hygromycin B. To test the influence of the osmotic stabilisation on the toxicity of the antibiotic, 1 M glucose was added to the medium. To test the possibility to use a top layer, spores were inoculated on PDA and grown for 2 hours, after which 8 mL of a selective top layer (PD with 0.8 % agar and 500 µg/L hygromycin B) was poured over the plates.
2.2.3. Test of the acetamidase selection medium In order to test the possibility to use the amdS gene as a selectable marker, the strains were tested on medium with acetamide as the sole nitrogen source (AMMA). AMM without
NaNO 3 was supplemented with 10 mM acetamide and 10 mM CsCl 2. Agar Noble (12 g/L, Difco) was added to solidify the medium.
2.2.4. Amylase production conditions Spores were inoculated in 5 mL or 100 mL AMM supplemented with 3 % sucrose (final concentration of 22.10 6 spores/mL). The cultures were grown at 25°C (155 rpm and 175 rpm, respectively). The 5 mL cultures were incubated for 4 days.
2.3. Plasmid preparation The plasmids used for transformation were isolated from E. coli with the HiSpeed Plasmid Maxi kit (Qiagen). They were concentrated according to the following procedure in order to obtain concentrations between 1 and 10 µg plasmids/µL: - add 1/10 volume of sodium acetate (3 M) - add 2 volumes of 100 % ethanol - keep at -20°C for 10 to 20 minutes - centrifuge at 12000 rpm during 20 minutes - remove supernatant - wash the pellet with 500 µL ice cold 70 % ethanol
Chapter I: Transformation of M. gramineum 72
- centrifuge at 12000 rpm during 20 minutes - remove supernatant and air dry the pellet - resolve the pellet in 30 µL mQ-water
2.4. Protoplast preparation and transformation of M. gramineum The procedure was carried out as follows: - harvest the mycelium by filtration through a Miracloth-filter (Calbiochem, 22-25 µM)
- wash with 250 mL water and 250mL CaCl 2 (0.27 M CaCl 2.2H 2O; 0.6 M NaCl) - gather the mycelium in a Falcon tube and determine the wet weight
- fill the Falcon tube with CaCl 2 up to 50 mL - mix (per 5 g mycelium) 600 mg β-D-glucanase (Interspex), 375 mg driselase (Interspex) and 848 mg Yeast Lytic Enzymes (ICN) in a Falcon tube, add water up to 50 mL, mix well and centrifuge during 4 minutes at 4000 rpm and 4°C - mix the mycelium and the supernatant of the enzyme mix in an Erlenmeyer - incubate at 25°C ( ± 55 rpm) during 0.5 to 1.5 hours - harvest the protoplasts in an Erlenmeyer placed on ice by filtration through a Miracloth- filter
- wash with 100 mL cold GTC (1 M Glucose, 10 mM Tris pH 8, 50 mM CaCl 2.2H 2O) - divide over 4 Falcon tubes, centrifuge at 3500 rpm during 10 minutes at 4°C and remove the supernatant - suspend the pellet of 1 Falcon tube in 25 mL GTC and pour this in a second Falcon tube - rinse the content of the first Falcon tube with 25 mL GTC and add it to the second Falcon tube, repeat these steps for the remaining two Falcon tubes - centrifuge at 3500 rpm during 10 minutes at 4°C and remove the supernatant - suspend the pellets as described above - centrifuge at 3500 rpm during 10 minutes at 4°C - remove the supernatant keeping a little bit of it to suspend the pellet - count the density of the protoplasts with a Thoma counting chamber (the protoplast concentration should be about 1.10 8 protoplasts per mL) - test the viability of the protoplasts before transformation by plating an equivalent of 100 and 500 protoplasts on PDAG (PDA supplemented with 1 M glucose) - add 1 µL 15 mM aurintricarboxylic acid (ATCA) per 100 µL protoplasts - add the DNA: maximum 10 µg DNA per 100 µL protoplasts in maximum 10 µL water per 100 µL protoplasts - keep on ice for 10 minutes - keep at room temperature for 10 minutes - add stepwise (per 100 µL protoplasts): 250 µL, 250 µL and 850 µL PEG (60 % PEG
4000, 10 mM Tris pH 7.5, 50 mM CaCl 2.2H 2O) - keep at room temperature for 20 minutes - add GTC (at room temperature) up to 50 mL - centrifuge at 3500 rpm during 10 minutes at 4°C - remove the supernatant keeping a little bit of it to suspend the pellet
Chapter I: Transformation of M. gramineum 73
- test the viability of the protoplasts after transformation by plating an equivalent of 100 and 500 protoplasts on PDAG - plate on selective medium (150 µL per plate).
2.5. Amylase enzyme test Samples of 500 µL were taken from the AMM + 3 % sucrose cultures. To test the amylase production of the transformants, the amylase test kit AMYL (Roche Diagnostics) was used. The following procedure was used:
- 5 µL sample + 125 µL R 1 + 25 µL R 2 + 595 µL mQ water are mixed in a cuvet and this mixture is incubated in the spectrophotometer at 37°C. The absorbance is measured every minute during 20 minutes.
One unit amylase is defined as the amount of enzyme able to raise the OD 405 nm by 1 during 15 minutes in a reaction mixture consisting of 5 µL sample + 125 µL R 1 + 25 µL R 2 + 595 µL mQ water incubated at 37°C.
2.5.1. Cell dry weight measurement The cell dry weight of the cultures was determined after filtration of the broth over Millipore filters (0.22 µm). The cell dry weight was determined with the Moisture Analyzer XM60 (Led Techno, boost program).
2.6. PCR control of the transformants The mycelium was frozen with liquid nitrogen and grinded in a mortar. The DNA was isolated with DNeasy Plant Mini kit (Qiagen). The PCR was carried out in a Progene thermocycler (Techne). The reaction mix consisted of 5 µL sample, 5 µL Taq -polymerase buffer (Roche Diagnostics), 2 µL primers (10 pmol/µL, Eurogentec), 1 µL nucleotides (10 mM, Sigma), 0.2 µL Taq -polymerase (5 u/µL, Roche Diagnostics) and 34.8 µL mQ-water. One primer pair is based on the hph gene of the plasmid pCSN43 (not present in the wild type). The sequence of the primers is 5’- ATGCCTGAACTCACCGCGACG -3’ for the forward primer and 5’- CTATTCCTTTGCCCTCGG -3’ for the reverse primer. The second primer pair is based on the A. oryzae amy3 gene preceded by the A. nidulans gpd -promoter. One primer anneals to the gene, the other to the promoter, forming a combination not present in the wild type genome. The sequence of the forward primer (GPD-primer) is 5’-TCTGGCATGCGGAGAG -3’ and the one of the reverse primer (AMY-primer) is 5’-GTCGATGATGCCCTGCC -3’. A first denaturation step of 4 minutes at 94°C was followed by 25 cycles of 30 s denaturation at 94°C, 30 s annealing at 55 °C and 1.5 minutes elongation at 72°C. The PCR was completed with a final extension of 7 minutes at 72°C.
Chapter I: Transformation of M. gramineum 74
2.7. Southern analysis of the transformants
2.7.1. Construction and labelling of the probe The probe to check the presence of the amylase gene in the genome of the transformants was constructed by restriction of the plasmid p2G-S (13.2 µg) with Nco I and Xho I (20 units, Roche Diagnostics). The restriction was carried out overnight at 37°C. The desired fragment of 1.6 kb (spanning part of the gpd -promoter and the amylase coding sequence) was gel purified with the QiaexII Gel Extraction kit (Qiagen). The purified fragment had a concentration of 21.6 ng/µL (30 µL). The fragment was labelled according to the procedure described in the DIG High Prime DNA Labelling and Detection Starter kit I (Roche Diagnostics). The labelling of the probe was controlled as described in the kit.
2.7.2. Genomic DNA preparation and Southern analysis The mycelium was frozen with liquid nitrogen and homogenised with a mortar. The DNA was isolated with DNeasy Plant Mini kit (Qiagen). The samples were concentrated with 3 M sodium acetate and ethanol, as described in 2.3. The DNA was cut with Nru I: 24 µL sample, 1X buffer, 20 units Nru I, 19 µL mQ-water (overnight, 37°C). This enzyme cuts once in the probe. Gel electrophoresis, blotting, hybridisation and detection were carried out according to a combined protocol described by Sambrook & Russell (2001) (230) and the DIG High Prime DNA Labelling and Detection Starter kit I (Roche Diagnostics). Routine recombinant DNA methodology was performed according to Sambrook & Russell (2001) (230). DNA concentrations were measured with the NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies). The Smartladder TM of Eurogentec was used to control the length of the amplified products.
3. RESULTS AND DISCUSSION
3.1. Development of the selective media
3.1.1. Hygomycin selective medium Myrothecium gramineum was grown on PDA supplemented with 100 µg/L, 250 µg/L, 500 µg/L and 1000 µg/L hygromycin B. To test the influence of the osmotic stabilisation on the selectivity of the medium, 1 M glucose was added to the medium. The possibility to use a top layer was examined. A top layer is used when the fungus is resistant to high concentrations of the antibiotic (226). The wild type strain was unable to grow on PDA with 500 or 1000 µg/L hygromycin B, even after 15 days of incubation. M. gramineum was able to grow on the plates with lower concentrations of the antibiotic (100 µg/mL and 250 µg/µL). The wild type was not able to grow through the top layer, and the addition of 1 M glucose to the medium did not have an effect on the selectivity of the medium. Other causes for the fact that there was no growth through the top layer (such as the creation of anaerobic conditions) were excluded, because transformants were able to grow through the top (3.2).
Chapter I: Transformation of M. gramineum 75
3.1.2. Acetamide selective medium In order to test the possibility to use the amdS gene as a selectable marker, the strains were tested on AMMA (medium with acetamide as the sole nitrogen source). The wild type Myrothecium did not grow during 10 days, but on the 11 th day, clear mycelium formation was visible and after 15 days the plates were fully covered with mycelium. After 25 days, a slight sporulation was detected.
3.2. Transformation of M. gramineum M. gramineum protoplasts were transformed with a plasmid containing mixture and with water as a control. The concentration of the protoplasts before transformation was 2.8.10 8/mL for the protoplasts transformed with plasmids and 3.43.10 8/mL for the control. Since the concentration optimally is 1.10 8/mL, the protoplasts were diluted. After the dilution 2850 µL protoplasts were obtained for the control and 4000 µL protoplasts were gathered for the transformation with the plasmids. The amount of DNA or water which was added is given in Table I. 3-1. Table I. 3-1. Amount of DNA/water added to transform the protoplasts of M. gramineum Transformation Control µg DNA µL DNA µg DNA µL water pCSN43 (2.5 µg/µL) 75 30 - 30 p2G-S (4.4 µg/µL) 132 30 - 30
The protoplast density after transformation decreased to 4.10 7/mL for the transformed protoplasts and to 8.6.10 7 for the control. The viability of the protoplasts was checked before and after transformation for both protoplast mixtures. Equivalents of 100 and 500 protoplasts were inoculated on PDAG and on AMMA, supplemented with 6 g/L NaNO 3 and 1 M glucose, and without CsCl 2.
After the transformation, half of both protoplast mixtures were inoculated on PDAG and the other half on AMMA stabilised with 1 M glucose. The PDAG plates were incubated for 2 hours at 25°C and then overlaid with 8 mL top layer containing 500 µg/L hygromycin. None of the plates inoculated with the protoplasts which were transformed only with water produced a colony, which indicated that growth of false positives was not possible.
After 6 days, 172 colonies arose on the PDAG top layer plates and this number increased to 320 colonies the next day. No colonies grew on the plates where the protoplasts were directly selected for their ability to grow on medium with acetamide as the sole nitrogen source.
The viability of the protoplasts before and after transformation was rather low: - for the PDAG plates a viability of 6.3 % and 2.1 % before and after transformation, respectively, was calculated for the protoplasts which were transformed with the plasmids, and of 7.1 and 0.5, respectively, for the control. The decrease of the protoplast viability after
Chapter I: Transformation of M. gramineum 76
transformation could be due to the extra centrifugation steps and handling of the protoplasts during transformation - the viability of the protoplasts calculated from the AMMA plates supplemented with
NaNO 3 and glucose was zero.
A transformation efficiency of 5.6 transformants per µg DNA and per 10 6 viable protoplasts was achieved, which is 9.6 times higher than the efficiency reported by Jonniaux et al . (29).
A possible explanation for the fact that there was no growth on the amdS selective medium could be that expression of this gene is repressed by the high amounts of glucose. The amdS gene on plasmid p2G-S is preceded by its own promoter, which is repressed by CreA, the major carbon catabolite repressor in many fungi (91, 232). Acetamide is added as the sole nitrogen source, but it can also be used as a carbon source and therefore is subjected by repression by glucose. Another possibility is that the medium is not rich enough for the regeneration of the protoplasts. This explanation is supported by the fact that there is also no growth on the plates used to test the viability of the protoplasts. These plates contain glucose and acetamide, but also nitrate as nitrogen source. Expression of the amdS gene in this case is not necessary for growth, since both a carbon and a nitrogen source different from acetamide are available.
The 320 transformants were inoculated on PDA + 500 µL/L Triton ® X-100 + 500 µg/L hygromycin B for a second round of selection. After 5 days 149 of the 320 colonies (46.5 %) grew back, which means they had stably integrated the hygromycin resistance gene. All of them were inoculated on AMMA + 500 µL/L Triton ® X-100. After 3 days mycelium formation was visible for 23 colonies (15.43 %). This number increased to 54 colonies (36.24 %) after 7 days and to 69 colonies (46.31 %) after 9 days. The fact that some of the transformants were able to grow on medium with acetamide as the sole nitrogen source (with only 10 g/L glucose) proves that they did incorporate the p2G-S plasmid and supports the fact that the high glucose concentrations in the osmotically stabilised medium inhibits the expression of the amdS gene. Nevertheless, it is not excluded that the medium is not rich enough for regeneration of the protoplasts.
3.3. Development of an amylase enzyme test α-Amylases (or 1,4-α,D-glucanohydrolases, EC 3.2.1.1) are hydrolases which hydrolyse the α-1,4 glucosidic bounds of oligosaccharides and polysaccharides, such as amylose, amylopectin and glycogen. The method which is used in the enzyme test is a colorimetric method based on the hydrolysis of a substrate ‘protected’ by an ethylidene group, more specific 4,6-ethylidene-(G 7)-1,4-nitrophenyl-(G 1)-α,D-maltoheptaoside. This hydrolysis is followed by a hydrolysis of all the degradation products catalysed by the α-glucosidase helper enzyme, which releases p-nitrophenol (100 % chromophore release). The intensity of the yellow colour is related to the α-amylase activity and is spectrophotometrically determined at 405 nm (Figure I. 3-1). The aim of this experiment was to determine to which extend the
Chapter I: Transformation of M. gramineum 77 colour formation in the enzyme test increases linearly with the amount of enzyme added. The test was done with a reference amylase sample provided by the company Beldem.
Figure I. 3-1. Principle of the Amyl enzyme test.
The result of the experiment is presented in Figure I. 3-2 and Figure I. 3-3. The sample was diluted 0 to 100 000 times. The absorbance decreases linearly with the dilution of the sample when the slope is lower than 0.12. The correlation coefficient R² is high enough (0.99). Repetition of the measurement of the 1.10 4 times diluted sample proves the method is reproducible. It was decided to use this procedure for future amylase tests.
One unit amylase was defined as the amount of enzyme able to raise the OD 405nm by 1 in 15 minutes in a reaction mixture consisting of 5 µL sample + 125 µL R 1 + 25 µL R 2 + 595 µL mQ water incubated at 37°C.
5 Sample 4,5 10x 4 100x 1000x 3,5 5000x 3 10000x 2,5 10000xbis 13333x 2 20000x
nm) (405 Absorbance 1,5 40000x
1 50000x 80000x 0,5 100000x 0 0 5 10 15 20
Time
Figure I. 3-2. Plot of the absorbance of the diluted samples increasing in time.
Chapter I: Transformation of M. gramineum 78
1,4 0,14
y = 166210x + 0,1625 1,2 0,12 R2 = 0,9908 1 0,1 0,8 0,08
Slope 0,6 Slope 0,06
0,4 0,04
0,2 0,02
0 0 0 0,2 0,4 0,6 0,8 1 0 0,00002 0,00004 0,00006 0,00008 0,0001 Dilution Dilution Figure I. 3-3. Plot of the slopes of the graphs of Figure I. 3-2 versus the dilution factor.
3.4. Amylase enzyme tests on the transformant cultures Spores of 80 transformants which grew the fastest on PDA + Triton ® X-100 + hygromycin were inoculated in 5mL AMM + 3 % sucrose to test their amylase production (co- transformation with p2G-S). The result of the enzyme tests is given in Figure I. 3-4. Eighty colonies were tested, but only the ones producing the recombinant amylase are pictured. The numbers accord to the number of the colony.
60
50
40
30
20 Units amylase/mL
10
0 16 26 27 28 53 91 93 94 96 55 59 72 73 83 84 85 114 117 297
Transformant number
Figure I. 3-4. Amylase production of the Myrothecium transformants in 5 mL cultures (units recombinant amylase = units transformant- units wild type).
Chapter I: Transformation of M. gramineum 79
Table I. 3-2. Units amylase per mL of the 7 best transformants and the wild type strain (5 mL cultures) Units A. oryzae Strain amylase/mL Transformant 59 54.90 Transformant 55 47.40 Transformant 16 47.31 Transformant 72 22.14 Transformant 73 13.77 Transformant 83 8.27 Transformant 114 5.73 Wild type 0.47
Four of the best transformants were also able to grow on acetamide medium after three days, more precisely transformants 16, 55, 72 and 73.
From Figure I. 3-4 it can be deduced that 19 of the 80 selected transformants produce the amylase of Aspergillus oryzae . The values in Figure I. 3-4 correspond to the units of amylase produced by the transformants reduced with the background amylase production of the wild type strain (0.47 ± 0.047 units). The seven best transformants produce between 54.90 and 5.73 units/mL in 5 mL cultures (Table I. 3-2).
An important feature of fungal transformation systems is that high frequencies of co- transformation with a non selected plasmid can occur (30-90 %) (110). In this case, a co- transformation efficiency of 23.75 % and 36.24 % was achieved for genes present on different plasmids (selection for hygromycin resistance and co-transformation with the amylase gene or selection for hygromycin resistance and co-transformation with the amdS gene, respectively). 7.5 % of the transformants which were hygromycin resistant were able to grow on acetamide as the sole carbon source and produced more amylase than the wild type strain.
3.5. Control of the stability of the amylase production The stability of the amylase production was checked by inoculating new 5 mL cultures of AMM + 3 % sucrose. The results were not reproducible, since growth in the 5 mL cultures was very heterogeneous. It was decided to increase the volume to 20 mL and finally to 100 mL. The cultures were inoculated with a standardised number of spores. Only in the 100 mL cultures a more or less stable production could be achieved. The results for transformants 16, 55 and 114 are given in Figure I. 3-5. Amylase production was followed during 320 hours. The production is reproducible for the wild type (background), transformant 16 and transformant 55, but for transformant 114 it starts fluctuating significantly after 170 hours. Remarkably, transformant 114, which had the lowest amylase production in the 5 mL cultures, now seems the best producer. The highest production in the cultures was achieved after 263h for all cultures except that of transformant 55 (after 239 h), reaching 56.24 ± 25.56
Chapter I: Transformation of M. gramineum 80 units for transformant 114; 27.37 ± 3.18 units for transformant 16; and 5.87 ± 1.26 units for transformant 55. The maximum background amylase production was 2.51 ± 0.52 units.
90 80
70
60 50 40 30 units/mL Mean 20 10 0 0 100 200 300 400 Time (h) WT 16 55 114
Figure I. 3-5. Mean amylase production of the wild type and 3 transformants (in 3 100 mL cultures, for transformants: units of amylase produced by the transformants reduced with the background amylase production of the wild type str ain) .
3.6. PCR control of the transformants In order to prove that the transformants had incorporated the foreign DNA, four of them were tested by PCR. Two of them are high amylase producers (transformants 55 and 59) and two of them did not produce more amylase than the wild type (transformants 1 and 2). The PCR was carried out as described in 2.6. With the hph primers a fragment of 1.1 kb is expected and with the GPD-primer and the AMY-primer a fragment of 1 kb is amplified. The results of the PCRs are given in Figure I. 3-6. In Figure I. 3-6 it can be seen that the genomic DNA is present as one band and that no fragmentation of the DNA has occurred.
WTg WTA WTH Tr1g Tr1A Tr1H Marker Tr2g Tr2A Tr2H Tr55g Tr55A Tr55H Marker Tr59g Tr59A Tr59H Control A Control H Figure I. 3-6. Result DNA isolation and PCR on the wild type and four transformants (WT, wild type; Tr, transformant; g, genomic DNA; H, PCR with hph gene primers; A, PCR with second primer pair,
5 µL sample per lane).
Gel electrophoresis of the PCRs shows that all four transformants have integrated the hph gene: in all lanes a fragment of 1 kb is detected. No fragment is visible in the lane of the wild
Chapter I: Transformation of M. gramineum 81 type or the control. All four transformants also have the gpd -promoter-amylase construct (1.1 kb). The enzyme tests showed that transformants 1 and 2 do not produce more amylase than the wild type, but here it is proven they do have at least one copy of the amylase gene in their genome. It is possible that recombination has occurred which made the enzyme inactive, but also that the construct has not completely integrated or that it integrated at a non-active site in the genome or that a small number of copies have integrated so that expression is not higher than in the wild type.
3.7. Southern analysis A higher copy number does not necessarily lead to a higher production of the (heterologous) protein (14). This can be checked by a combination of Southern analysis (copy number determination) and enzyme tests. Five transformants were checked by Southern analysis. The DIG High Prime DNA Labelling and Detection Starter kit I (Roche Diagnostics) was used to label the probe. Control of the labelling proved the reaction was successful (results not shown). The probe consists of a 1.6 kb fragment which anneals to a part of the gpd -promoter of A. nidulans and a part of the amylase encoding sequence of A. oryzae (Figure I. 3-7). Genomic DNA was extracted from the wild type and transformants 16, 55, 59, 72 and 114. The result of the DNA extractions and of their restrictions with Nru I is shown in Figure I. 3-8. The concentration of the genomic DNA was high enough (bands higher than 10 kb in the left part of the figure) and the DNA was successfully cut with Nru I (right part of the figure: smear of restricted genomic DNA).
XhoI XhoI NruI NcoI XhoI XhoI NruI NcoI
amy amdS gene Prom gpd amy Prom gpd NcoI, XhoI
Probe 1,6 kb Figure I. 3-7. Schematic representation of the plasmid p2G-S and the probe based on part of the gpd promoter and part of the amylase gene (thin lines represent coding sequences, grey boxes promoter sequences and thick lines plasmid sequences).
WT 16 55 72 59 Marker 114
Marker WT 16 59 114
Figure I. 3-8. Gel electrophoresis of the isolated genomic DNA of the Myrothecium wild type and the transformants (left) and of the restriction of this DNA with Nru I (right).
Chapter I: Transformation of M. gramineum 82
The result of the blot of the restricted genomic DNA of the wild type and the transformants, and of the restricted plasmid p2G-S and the detection with the anti-DIG antibody coupled alkaline phosphatase is given in Figure I. 3-9. Unfortunately, the marker is not visible. The probe is specific for the recombinant amylase construct since there is no hybridisation to the wild type genome (lane ‘WT’ in Figure I. 3-9). The two fragments in the lane of p2G-S can be explained by the fact that there are two copies of the ‘ gpd -promoter-amylase gene’ construct present on this plasmid, and that Nru I cuts one time in each of them, resulting in two fragments which are of different size and to which the probe hybridises (see also Figure I. 3-7). This means that also for the transformants at least two fragments are expected, when the plasmid has entirely integrated at a certain place in the genome. Two fragments (and even more) are clearly visible for transformants 59 and 114. Nevertheless, the fact that the bands are positioned lower than for the plasmid indicates that rearrangements must have occurred. For transformants 16 and 55 only one clear band is visible, but it is possible that the concentration of the genomic DNA was not high enough for the second band to be easily detected. For transformant 72 there are no bands, but this strain did not produce more amylase than the wild type. Nevertheless it was able to grow on AMMA and thus it should have incorporated the amdS gene. Possibly, a rearrangement of the plasmid p2G-S occurred, but this was not confirmed. It has to be noticed that the concentration of the genomic DNA sample of this strain was clearly lower than that of the other samples (Figure I. 3-8).
Tr 16 Tr 55 Tr 72 Marker p2G-S WT Tr 59 Tr 114
Figure I. 3-9. Southern analysis of the wild type and the transformants . This Southern analysis proofs that the amylase overproducers have incorporated at least one and probably more copies of the ‘ gpd -promoter-amylase gene’ construct. It was not possible to draw any conclusions about the correlation between the copy number and the amylase activity from this experiment. Transformant 16 was the best in the 5 mL cultures, but the hybridisations to the probe are weaker than with transformant 59 and 114, which were the second best and the worst producers in the 5 mL cultures. Transformant 114 was the best producer in the 100 mL cultures, followed by transformant 16 and 55. Again, transformant 55 has a (slightly) more intense hybridisation than transformant 16. Transformant 59 was not tested in the 100 mL cultures. More probably, the differences in intensity can be explained by different concentrations of DNA loaded on the gel.
Chapter I: Transformation of M. gramineum 83
4. CONCLUSION M. gramineum was successfully transformed using a protoplast based transformation method and selection for the hygromycin B resistance marker. A protoplast solution of a density high enough for successful transformation was obtained. Although the viability of the protoplasts was rather low, 320 hygromycin resistant colonies grew on the selective plates. A transformation efficiency of 5.6 transformants per µg DNA and per 10 6 viable protoplasts was achieved.
Direct selection on a medium with acetamide as the sole nitrogen source was not possible, but 36.24 % of the transformants selected on the antibiotic containing plates were able to grow on the acetamide medium. Since the wild type is not able to grow on these plates (within 10 days) the transformants must have integrated a copy of the amdS gene. Clear mycelium formation was visible after 3 to 7 days.
An amylase enzyme test and culture conditions were developed to reproducibly test the amylase production of the wild type and M. gramineum transformants. From a selection of 80 transformants, 19 produce significantly more amylase than the wild type strain. The seven best transformants produce between 54.90 and 5.73 units/mL units amylase in 5 mL cultures. The stability of the amylase production was checked by inoculating 100 mL cultures of AMM + 3 % sucrose. Unfortunately, the results of the 5 mL cultures could not be reproduced in this way. Only in the 100 mL cultures a more or less stable production could be achieved, but the production levels were not as high as the ones obtained in the 5 mL cultures. The production is reproducible for the wild type, transformant 16 and transformant 55, but for transformant 114 it starts fluctuating after 170 hours. The mean highest production in the three cultures was 56.24 ± 25.56 units for transformant 114; 27.37 ± 3.18 units for transformant 16; and 5.87 ± 1.26 units for transformant 55. The maximum background amylase production was 2.51 ± 0.52 units.
A feature of many fungal transformation systems is that high frequencies of co-transformation can occur. Here, a co-transformation efficiency of 23.75 % and 36.24 % was achieved for genes present on different plasmids (selection for hygromycin resistance and co- transformation with the amylase gene or selection for hygromycin resistance and co- transformation with the amdS gene, respectively). 7.5 % of the transformants which were hygromycin resistant were able to grow on acetamide as the sole carbon source and produced more amylase than the wild type strain.
The presence of recombinant DNA in the genome of the transformants was confirmed by PCR and Southern analysis. It was not possible to draw any conclusions about the correlation between the copy number and the amylase activity from the Southern analysis.
Chapter II: PCR-based analysis of transformants 84
Chapter II
Rapid sample preparation for long distance PCR on genomic DNA of Myrothecium gramineum
1. INTRODUCTION A quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host or to study the molecular systematics of an organism. While rapid and high-throughput PCR-methods or other molecular techniques are amply available for bacteria and even for yeast, this is not the case for filamentous fungi.
Because Southern blot analysis is laborious and time consuming, several colony hybridisation methods have been developed for the analysis of a large number of transformants. Unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (233). Recently methods for PCR-based analysis of fungal transformants have been described, but most of these methods require high quality DNA.
Many methods for DNA extraction from fungi have been described in the past few years. These methods often are tedious, time consuming, costly or limited to a small number of samples per run. Most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (234). Other methods require the use of specific equipment, such as grinders or filters (235, 236, 237). The methods need excessive amounts of starting material. Many methods involve the use of toxic chemicals such as phenol or chloroform (238), which is not only hazardous, but furthermore, in these steps loss of DNA can occur, which is particularly undesirable when attempting to isolate DNA from a small number of fungal cells (239). In addition to being lengthy, the current methods also involve the handling of large amounts of glassware, which is inconvenient when handling a large number of samples (234).
Lately, a number of methods have been described to isolate DNA from fungi suitable (exclusively) for PCR and appropriate for the simultaneous treatment of a large number of samples. The methods use minute quantities of starting material and are as short as possible. Some methods describe the use of a microwave oven to help in the alteration of cell walls and membranes so that lysis buffers are able to further break open cell and organelle membranes. The methods can be applied on mycelium or on spores and they are cheap, since they do not require any expensive reagents or equipment (240, 241). The DNA extracts are pure enough to amplify fragments of about 0.5 to 1 kb.
Chapter II: PCR-based analysis of transformants 85
The method described by Griffin et al. (239) uses tissue from freshly grown fungal isolates and subjects it to seven rounds of freezing/thawing using a crushed dry-ice/ethanol bath and a boiling water bath. Rapid freezing and boiling cycles were also applied by Manian et al. (242) in mycorrhizal fungi, but this method involves the use of liquid nitrogen. The latter method provided DNA suitable for the amplification of PCR fragments from 650 bp to more than 2 kb. Other methods use salt extraction buffers to weaken the cell wall (243), a universal method applicable to plant tissue, fruits, vegetables, fungi and insects. Fragments larger than 1000 bp could be obtained by PCR on these samples. Cold acetone treatments or the use of glass beads (sometimes in combination with LiCl) are also described to weaken the cell wall prior to lysis or DNA extraction (244, 245, 246). Some methods describe the direct use of mycelium (247, 248, 249) in the PCR-reaction mixture, and even the use of spores in PCR reactions with a prolonged initial denaturation step is described. When using mycelium, it is recommended to use a tiny amount of mycelium from the periphery of freshly grown fungal colonies (247). Roca et al. (249) conclude that only fragments smaller than 0.7 kb can be amplified using mycelium straight in the PCR-tube. In some cases the spores are pre-treated by freezing them in liquid nitrogen or at -80°C for a certain time (238). Nevertheless, direct PCR on fungal spores is not readily suitable to all fungi, mainly because of the difficulties in rupturing the cell wall (241). Next to mycelium and spores, fungal protoplasts can be used in PCR reactions (233, 249). The mycelium is treated with Novozyme TM 234, with Glucanex TM or with a mixture of glucanase and Driselase ®. Protoplasts are boiled and centrifuged or put on ice to settle the cell debris, and the supernatant is used as PCR-template. Using this method, Van Zeijl et al . (233) were able to amplify products of at least 3.2 kb, using standard thermostable polymerases and without much aspecific products. Longer fragments up to 6 kb were obtained using the Long Expand polymerase (Roche Diagnostics), but the amount of aspecific products was significantly increased. In the method of Cassago et al . (234), mycelium is grown on cellophane disks overlaid on solid medium and glass beads are used for cell wall disruption. The growth on the cellophane disks is assumed to result in softer cell walls than those of liquid culture cells. This method yields approximately 2 µg DNA per disk, which is of good quality, with high molecular weight and no apparent smear and which is suitable for PCR reaction (fragments of 0.75 kb) and even for Southern blot analysis. Several samples can be processed simultaneously, growing the fungus on multiple well cell culture plates.
We have compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR. Myrothecium gramineum is an Ascomycete used in our research as a new expression host. The Aspergillus strain was used as a reference. Ten methods were tested. In nine of these methods DNA was extracted from mycelium prior to PCR. A final assay used mycelium straight in the PCR- reaction mixture. For all procedures, tiny amounts of mycelium grown on solid medium were used.
Chapter II: PCR-based analysis of transformants 86
2. MATERIALS AND METHODS
2.1. Strains and culture conditions Myrothecium gramineum BCCM TM /MUCL 39210 and Aspergillus nidulans FGSC A722 (pyrG89 pabaA1; fwA1 uaY9 ), a defined OMPD negative strain, were used to extract their genomic DNA. M. gramineum was grown on Potato Dextrose Agar (PDA, Oxoid); Aspergillus nidulans A722 was grown on Aspergillus minimal medium (AMM, 231), supplemented with 10 mM uracil, 10 mM uridine (Acros) and 5 g/L yeast extract (Difco). Cultures were grown at 25°C for M. gramineum and at 30°C for A. nidulans .
2.2. Collection of material Freshly grown mycelium of Myrothecium gramineum and Aspergillus nidulans was harvested in a ‘standardised’ way: the head of an Eppendorf tube was pushed on PDA plates, thus creating a circle, and the mycelium within the circle was gathered. The mycelium was put in an 2 mL Eppendorf tube containing 500 µL mQ water and centrifuged for 20 minutes at 14000 rpm. The pellet was either used directly in the PCR mixture (248) or submitted to extraction procedures.
2.3. Extraction methods The DNA was extracted from the mycelium according to the tested procedures (233, 240, 243, 244, 245, 246, 250, 251). These are explained in Table II. 2-2. For method 10, mycelium was dissolved directly in 50 µL PCR reaction mixture.
2.4. PCR amplifications
2.4.1. Amplification of ‘small’ fragments The DNA samples of M. gramineum were used for 2 PCR reactions with standard Taq - polymerase (Roche Diagnostics), amplifying fragments smaller (PCR 1) and larger than 1 kb (PCR 2). The PCRs were performed in a Progene thermocycler (Techne): after an initial denaturation step at 94°C for 2 minutes, 35 cycles were run with denaturation at 94°C for 10 s, primer annealing at 50°C for 30 s and extension at 72°C for 2 minutes (3 minutes for PCR2 of M. gramineum ). The PCR was completed with a final extension step of 7 minutes at 72°C. The PCR-reaction mixture contained 1 unit Taq DNA polymerase, 1X PCR buffer, 0.2 mM dNTP mix (Sigma), 0.4 µM primers, template (5 µL if the concentration was lower than 100 ng/µL, 2 µL if it was between 100 and 200 ng/µL and 1 µL if it was higher than 200 ng/µL) and mQ-water up to 50 µL. The primers (Figure II. 2-1, Table II. 2-1, Sigma Genosys) were ‘forwardM<1kb’ and ‘reverseM<1kb’ for PCR1 and ‘forwardM>1kb’ and ‘reverseM>1kb’ for PCR 2 on genomic DNA of M. gramineum (for sequence details, see chapter VII). The same PCR as PCR 1 was carried out on samples of the Aspergillus strain. The primers were ‘forwardA<1kb’ and ‘reverseA<1kb’, based on the A. nidulans ompd gene sequence (Genbank accession number M19132 , Figure II. 2-1, Table II. 2-1).
Chapter II: PCR-based analysis of transformants 87
Table II. 2-1. Primers used in the colony PCR experiments Primer name Feature Primer sequence (5’-3’) forwardM<1kb PCR 1 Myrothecium ATCCGAGCCACAACGAATCC reverseM<1kb PCR 1 Myrothecium AATCAGAGCCGCCACGTATG forwardM>1kb PCR 2 Myrothecium CAGACCGCATGCATTCGTATGCC reverseM>1kb PCR 2 Myrothecium ACCAAGCACCAACA forwardA<1kb PCR 1 Aspergillus CAACGGCTTCGGTCGTATTG reverseA<1kb PCR 1 Aspergillus AGGTAGAAGCTACGATTCCGCC forwardM4.3kb PCR 3 Myrothecium GATCTTGCTACCTGCCAACTTC reverseM4.3kb PCR 3 Myrothecium GCTATTGGCGGGATATTCTG
2.4.2. Amplification of ‘longer’ fragments For PCR3 with the polymerases of the Expand TM Long Template PCR System (Roche Diagnostics) and the Elongase Mix (Invitrogen), an initial denaturation step at 94°C for 1 minute preceded 40 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 30 s and extension at 68°C for 5 minutes. The PCR was completed with a final extension step of 7 minutes at 68°C. PCR3 with the Goldstar TM polymerase (Eurogentec) was carried out with the same scheme of cycles, except that the elongation was carried out at 72°C. The PCR-reaction mixture of the Expand TM Long Template PCR System contained 1 unit of the DNA polymerase mix, 1X PCR buffer, 0.35 mM dNTP mix (Sigma), 0.3 µM primers, template and mQ-water up to 50 µL. The PCR-reaction mixture of the Elongase Mix was the same but it contained 2 µL buffer A, 8 µL buffer B and 0.2 mM dNTP mix (Sigma). The mix of the Goldstar TM polymerase contained also the same ingredients of that of the Expand TM
Long Template PCR System, with 1.8 mM MgCl 2 and 0.2 mM dNTP mix (Sigma). The primers for PCR3 on genomic DNA of Myrothecium gramineum transformants were ‘forwardM4.3kb’ and ‘reverseM4.3kb’. These transformants were obtained by transformation of the M. gramineum wild type with the plasmids pGPD-AMY and pCSN43 (see paragraph 2.1 and 3.2 of the previous section). Both primers are based on the plasmid pGPD-AMY.
Routine recombinant DNA methodology was performed as described in § 2.7.2 of Chapter I (p. 74).
Figure II. 2-1. Scheme of the primers used in the PCRs for M. gramineum and A. nidulans.
Chapter II: of PCR-basedtransformants analysis Table II. 2-2. Extraction procedures tested on mycelium of M. gramineum and A. nidulans Method 1 (4 hours) Method 2 (1.25 hours) Method 3 (1 hour) Salt extraction procedure (243) Glass bead procedure (245) LiCl procedure (246) mycelium + 700 µL salt homogenisation buffer mycelium + 700 µL buffer mycelium + 700 µL LETS-buffer (10 mM Tris pH 8.0, 2 mM EDTA, 0.4 M NaCl) (200 mM Tris pH 7.5, 10 mM EDTA, 0.5 M NaCl, 1 % SDS) (0.1 M LiCl, 10 mM EDTA, 10 mM Tris-Cl pH 8, 0.5 % SDS) vortex 1 minute + 40 mg glass beads and 0.2 mL 1:1 phenol:chloroform + 40 mg glass beads + 40 µL 20 % SDS, shake heavily vortex 6 minutes at top speed vortex 2 minutes at top speed 1 hour at 60°C + 300 µL buffer, 300 µL phenol:chloroform + 1 mL phenol:chloroform:isoamyl alcohol 25:24:1 + 300 µL 6 M NaCl, vortex 30 s vortex a few minutes and centrifuge 30 s vortex 20 s medium speed 30 minutes 1000 g transfer water phase to new Eppendorf tube 5 minutes 3000 rpm transfer supernatant to new Eppendorf tube fill Eppendorf tube with 95 % ethanol and invert 2 times transfer water phase to new Eppendorf tube + 1 volume isopropanol, shake heavily 30 minutes -20°C + 1 mL 100 % ethanol 1 hour at -20°C centrifuge 5 minutes max speed, remove supernatant keep 15 minutes on dry ice (at -80°C) 20 minutes 10000 g, remove supernatant wash pellet with 500 µL 70 % ethanol 15 minutes 3000 rpm wash pellet with 500 µL 70 % ethanol, dry 30 minutes dry in Speedvac remove supernatant, dry pellet + 300 µL mQ + 100 µL mQ + 40 µL TE (10 mM Tris, 0.1 mM EDTA, pH 8)
Method 4 (1.5 hours) Method 5 (1.5 hours) Method 6 (3 hours) Microwave oven procedure (240) Boiling procedure (250) Boiling procedure 2 (5 and 7 combined) mycelium + 700 µL lysis buffer mycelium + 700 µL lysis buffer mycelium + 500 µL lysis buffer (50 mM Tris pH 7.2, 50 mM EDTA, 3 % SDS, 1 % 2-mercaptoethanol) (0.25 mM Tris pH 8, 1.5 % SDS) (0.25 mM Tris pH 8, 1.5 % SDS) 15 s, 10 s, 5 s in microwave oven (open lid) 30 minutes 100°C 30 minutes 100°C, vortex 2 minutes + 325 µL lysis buffer vortex 2 minutes + 500 µL phenol:chloroform:isoamyl 10 minutes at 80°C + 500 µL phenol:chloroform:isoamyl alcohol 25:24:1 alcohol 25:24:1 + 400 µL 1:1 chloroform:phenol, vortex 30 s vortex 10 minutes vortex 10 minutes, 5 minutes 3000 rpm 15 minutes 10000 g 5 minutes 3000 rpm + 0.54 volumes isopropanol, invert transfer supernatant to new Eppendorf tube transfer water phase to new Eppendorf tube 30 minutes 4500 g, remove supernatant + 0.54 volumes isopropanol + 10 µL 5 M Na-acetate + 1 mL acetone, 5 minutes 13000 rpm wash pellet with 100 µL 70 % ethanol 88 2 minutes 10000 g, remove supernatant remove supernatant, dry pellet 30 minutes 4500 g, , remove supernatant wash pellet with 500 µL 70 % ethanol, dry pellet + 50 µL mQ dry 30 minutes + 50 µL TE (10 mM Tris, 0.1 mM EDTA, pH 8) + 100 µL TE (pH 8)
Chapter II: of PCR-basedtransformants analysis Table II. 2-2. Extraction procedures tested on mycelium of M. gramineum and A. nidulans (continued) Method 7 (6.5 hours) Method 8 (2.5 hours) Method 9 (0.75 hours) Acetone procedure (244) Liquid nitrogen procedure (251) Protoplast procedure (233) mycelium + 100 µL cold acetone mycelium + liquid nitrogen mycelium + 50 µL KC-buffer vortex, 20 minutes 14000 rpm let the nitrogen evaporate (0.8 M KCl, 10 mM citrate, pH 6.2) + 700 µL cold acetone repeat these 2 steps add 2.5 mg enzymes per mL KC-buffer mycelium + acetone + 700 µL lysis buffer (Driselase ® (Interspex products Inc.), ImmunO 3 hours at -20°C (50 mM Tris pH 8, 50 mM EDTA, 3 % SDS, Yeast Lytic Enzyme (MP Biochemicals Inc.), 20 minutes Speedvac 1 % 2-mercaptoethanol, 0.1 mg/mL ProteaseK) β-D-glucanase G Cell Wall Res. Enzyme 1 g dry mycelium+ 300 µL lysis buffer vortex (Interspex products Inc.) 1:2.3:1.6) (10 mM Tris-Cl pH 7.5, 0.5 M NaCl, 1 % SDS) 1 hour at 60°C, vortex after 30 and 60 minutes 30 minutes (25°C M. gramineum , 30°C A. nidulans + 40 mg glass beads + 500 µL phenol + 150 µL dilution buffer vortex 30 s 5 minutes 8000 rpm (10 mM Tris-Cl pH 7.5, 10 mM NaCl, 1 mM EDTA) + 400 µL phenol:chloroform:isoamyl alcohol 25:24:1 transfer 450 µL water phase to new Eppendorf tube 3 minutes at 95°C mix + 450 µL phenol 5 minutes on ice transfer to Corex tubes 5 minutes 8000 rpm store at -20°C 20 minutes at 65°C transfer 400 µL water phase to new Eppendorf tube
30 minutes 4500 g + 400 µL 24:1 chloroform:isoamyl alcohol transfer supernatant to new Eppendorf tube 5 minutes 8000 rpm + 4/5 v/v phenol:chloroform:isoamyl alcohol 25:24:1 transfer 350 µL water phase to new Eppendorf tube 30 minutes 4500 g + 50 µL 7.5 M ammonium acetate transfer supernatant to new Eppendorf tube mix softly + 0.54 volumes isopropanol, invert + 880 µL 95 % ethanol 2 minutes 10000 g, remove supernatant invert wash pellet with 100 µL 70 % ethanol 30 minutes at -20°C 2 minutes 10000 g 20 minutes 13000 rpm dry 30 minutes dry pellet + 100 µL TE (10 mM Tris, 0.1 mM EDTA, pH 8) + 20 µL TE (10 mM Tris, 0.1 mM EDTA, pH 8) 89
Chapter II: PCR-based analysis of transformants 90
3. RESULTS AND DISCUSSION
3.1. DNA extractions The results of the 9 DNA extraction methods are presented in Table II. 3-1. Each extraction was done 3 times. RNA and DNA are measured at 260 nm. Proteins are measured at 280 nm (peptide bond) and at 230 nm (presence of aromatic amino acids e.g. tyrosine and tryptophan). Phenol, other aromatic compounds, and buffer salts are measured at 230 nm. Good extractions result in a 260/280 ratio between 1.8 and 2.0 (higher than 1.8). The 260/230 ratio gives an indication of the contamination of the sample with phenol or other aromatic compounds: values lower than 1.8 are indicative for significant amounts of contaminants (252, 253, 254, 255, 256, 257, 258, 259). Table II. 3-1. Result DNA extraction procedures on mycelium of M. gramineum and A. nidulans Extraction Total amount of Concentration 260/280 260/230 method DNA obtained (µg) (ng/µL) mean standard mean standard mean standard mean standard deviation deviation deviation deviation M. gramineum 1 Salt extract 2.69 4.07 8.97 13.57 1.40 0.09 0.94 0.25 2 Glass bead 0.45 0.25 4.53 2.49 1.45 0.10 0.34 0.1 3 LiCl 0.04 0.03 1.10 0.79 1.03 0.27 0.35 2.9 4 Microwave 1.36 0.91 27.21 18.27 1.55 0.05 1.82 0.14 5 Boiling 9.12 1.41 182.36 282.15 1.88 0.15 2.13 0.25 6 Boiling 2 1.91 1.03 19.12 10.26 1.58 0.06 1.72 0.42 7 Acetone - - 0 2.49 - - 1.74 1.59
8 Liquid N 2 1.69 1.75 84.49 87.49 1.63 0.05 1.47 0.47 9 Protoplast 15.54 4.82 103.58 32.12 1.07 0.04 0.38 0.01 A. nidulans 1 Salt extract 1.20 0.27 3.99 0.89 2.71 0.32 0.88 0.24 2 Glass bead 33.79 10.48 337.89 104.80 1.80 0.12 1.90 0.13 3 LiCl 8.52 10.23 213.06 255.66 1.70 0.28 1.70 0.45 4 Microwave 2.95 3.38 58.98 67.57 1.05 1.02 0.85 0.87 5 Boiling 13.70 9.81 273.98 196.17 1.78 0.18 2.18 0.24 6 Boiling 2 0.89 0.17 8.88 1.75 1.98 0.17 - - 7 Acetone 0.06 0.04 0.64 0.35 - - 1.49 6.05
8 Liquid N 2 3.81 0.82 190.67 40.89 1.67 0.04 2.12 0.05 9 Protoplast 63.32 33.66 422.16 224.37 0.97 0.04 0.37 0.02 -: negative values
For M. gramineum , the highest DNA concentration was obtained with the extraction procedure 5. The highest amount of DNA was obtained with method 9. Concentrations lower than 10 ng/µL were obtained with the methods 1, 2, 3 and 7; concentrations between 10 and
Chapter II: PCR-based analysis of transformants 91
100 ng/µL were measured for the microwave procedure, the second boiling method (method 6) and the liquid nitrogen requiring method, and concentrations higher than 100 ng/µL were reached with methods 5 and 9. For the methods 1, 5, 7 and 8 the standard deviation of the concentrations (and obtained amounts of DNA) was higher than 100 %.
For A. nidulans , the highest DNA concentration and the highest amount of DNA were obtained with the extraction procedure 9. Concentrations lower than 10 ng/µL were measured for the methods 1, 6 and 7; concentrations between 10 and 100 ng/µL were obtained with method 4, and concentrations higher than 100 ng/µL were reached with methods 2, 3, 5, 8 and 9. For the methods 3 and 4 the standard deviation of the concentrations was higher than 100 %. Except for method 1 and 6, all procedures resulted in higher DNA concentrations for A. nidulans than for M. gramineum .
The 260/280 ratio of the Aspergillus extractions was in the correct range for all phenol including methods (method 2 to 8), except for method 4 and 7. For the extractions of the Myrothecium DNA, only the 260/280 ratio of method 5 was higher than 1.8. The ratio 260/230 was good for method 4 to 7 for the M. gramineum samples and for method 2, (3), 5 and 8 for the Aspergillus extractions.
3.2. PCR 1 and 2 The expected fragment lengths for PCR 1 and 2 for M. gramineum and for PCR 1 for A. nidulans were 762 bp, 2321 bp; and 420 bp, respectively. The results of PCR 1 for Myrothecium are presented in Figure II. 3-1. The PCRs of method 10 are the ones where the mycelium was put into PCR-tubes without previous treatment. In all cases, the PCRs resulted in one fragment if amplification occurred.
marker m10, P1 s1, m10, P1 s2, m10, P1 s3, m9, P1s1, m9, P1s2, m9, P1s3, marker m1, s1, P1 m1, s2, P1 m1, s3, P1 m2, s1, P1 m2, s2, P1 m2, s3, P1 marker m3, s1, P1 m3, s2, P1 m3, s3, P1 m4, s1, P1 m4, s2, P1 m4, s3, P1 marker m5, s1, P1 m5, s2, P1 m5, s3, P1 m6, s1, P1 m6, s2, P1 m6, s3, P1 m7, s1, P1 m7, s2, P1 m7, s3, P1 marker m8, s1, P1 m8, s2, P1 m8, s3, P1
Figure II. 3-1. Gel electrophoresis of PCR 1 on the samples of M. gramineum (P = PCR, m = method, s = sample). None of the 3 samples of the salt extraction procedure (method 1) and the protoplast based procedure (method 9) resulted in a PCR-fragment for M. gramineum . For methods 2, 3, 6, 7 and 8, all extraction samples were suited for PCR amplification, although the PCRs on the samples of methods 3 and 7 resulted in weak fragments. For methods 4 and 5 there was only amplification in two samples and for method 10 only one sample gave a clear fragment. As can be concluded from Table II. 3-1 and Figure II. 3-1, there is no correlation between the fact
Chapter II: PCR-based analysis of transformants 92 that the 260/280 ratio or the 260/230 ratio are higher than 1.8 and the possibility to amplify a fragment smaller than 1 kb. For example, method 2 and 8 resulted in a clear fragment for each sample while both the 260/280 and the 260/230 ratio were bad. Nevertheless, a good 260/280 (method 5) or 260/230 (method 4, 5, 7) resulted in amplified products in 2 or 3 of the samples. Methods 2, 5, 6, 8 and 10 were chosen for further investigation. Methods 3 and 7 were not further tested because they yield very low concentrations of DNA. Method 5 was included because it yields the highest concentrations. Although for method 10 only one sample gave a fragment, the method was not excluded because of its simplicity. Two new samples were taken to further test the possibilities of the procedure.
m6, s1, P2 m5, s3, P2 m8, s2, P2 m8, s1, P2 m2, s2, P2 m5, s2, P2 marker m5, s1, P2 m6, s3, P2 m6, s2, P2 m2, s3, P2 m10, P2 s1, m2, s1, P2 marker m10, P2 s2, m8, s3, P2
Figure II. 3-2. Gel electrophoresis of PCR 2 on the samples of M. gramineum (P = PCR, m = method, s = sample) .
Figure II. 3-2 represents the results of the PCR 2 on samples of M. gramineum . The expected fragment is 2319 bp long. None of the samples of method 2 gave a fragment. One sample of method 6 and two samples of method 5 were good enough for the amplification of the larger fragment. In the samples of method 10, a weak amplification band could be detected. All PCRs on the samples of method 8 resulted in one clear fragment of the expected size. This method includes the use of liquid nitrogen and of toxic compounds such as phenol. The procedure takes about 2.5 hours. Method 8 was chosen to test further in order to know if even larger fragments can be amplified. Because method 5 gave also two fragments, it was also included and again, method 10 was not excluded.
The results of the PCR 1 on the samples of Aspergillus are given in Figure II. 3-3. Again, in all cases, the PCRs resulted in one fragment if amplification occurred.
marker marker
m1, s1, P1 m1, s2, P1 m1, s3, P1 m3, s1, P1 m3, s2, P1 m3, s3, P1 marker m4, s1, P1 m4, s2, P1 m4, s3, P1 m6, s1, P1 m6, s2, P1 m6, s3, P1 marker m7, s1, P1 m7, s2, P1 m7, s3, P1 m8, s1, P1 m8, s2, P1 m8, s3, P1 m2, s1, P1 m2, s2, P1 m2, s3, P1 m5, s1, P1 m5, s2, P1 m5, s3, P1
Figure II. 3-3. Gel electrophoresis of PCR 1 on the samples of Aspergillus (P = PCR, m = method, s = sample) .
Chapter II: PCR-based analysis of transformants 93
The amplification reactions on all three samples of methods 5, 6 and 8 contain the expected fragment of 420 bp. For methods 1, 2 and 3, only two of the three samples were suitable to amplify the product. With methods 4, 7, 9 and 10, none of the samples gave a fragment (data not shown for method 9 and 10). Contradictory to the results obtained for M. gramineum , the ratios 260/280 and 260/230 of the extractions of Aspergillus give some indication of the suitability of the sample for PCR-amplification. For methods 4, 7 and 9, both ratios were lower than 1.8 and no products are obtained. Both ratios were fine for the procedures 2, 3, 5 and 8 and at least two PCR reactions on these samples resulted in an amplification product. Method 1 and 6 resulted in a low 260/230 ratio and a good 260/280 ratio, and in 2, respectively 3, samples an amplification product is detected.
3.3. PCR 3 Extraction procedures of genomic DNA of M. gramineum which proved to result in samples with a quality good enough to allow PCR amplifications of both fragments described in 2.4.1 were selected for a next round of PCR. For these reactions, two transformants of Myrothecium were used which were obtained by transformation of the M. gramineum wild type with the plasmids pGPD-AMY and pCSN43 (see paragraph 3.2 of the previous section, transformant 55 and 114). The primer pair for the amplification of a 4272 bp fragment (PCR 3) is based on the sequence of plasmid pGPD-AMY. The PCR reactions were carried out on the same amount of sample as described in 2.4.1.
The extractions of DNA from these strains according to procedures 5 and 8 contained between 100 and 940 ng/µL for method 5 and between 14 and 50 ng/µL for method 8. These results are according to the ones obtained with the wild type strain (3.1). The 260/280 and the 260/230 ratio of the samples of method 8 again were not higher than 1.8. The ratios for the extracts of method 5 were good, as with the wild type.
In order to check the results obtained with the wild type strain, two extraction samples of method 8 were used for PCR amplification of the 2319 bp fragment (with the Elongase Mix). At the same time, PCR 3 was carried out. Some of the results are given in Figure II. 3-4. The PCRs to amplify the larger fragment of 4.3 kb were carried out with long distance polymerase mixes, because Taq -DNA polymerase is only suitable for reliable amplification of fragments up to 3 to 5 kb in samples of high quality and purity. This limitation is believed to be largely due to the inability of Taq -DNA polymerase, which lacks proofreading activity, to correct nucleotide mis-incorporations and continue primer elongation. Other systems have been described which contain, in addition to the parent DNA polymerase, a proofreading enzyme, which edits the nascent strand to allow subsequent polymerisation by the parent enzyme (260). The polymerases tested are the Expand TM Long Template PCR System (Roche Diagnostics), the Elongase Mix (Invitrogen) and the Goldstar TM polymerase (Eurogentec). The Expand TM Long Template PCR System is a mix of the standard Taq polymerase and a proofreading polymerase and is suitable for long and accurate reactions (from 5 to 20 kb) (261). The Elongase Enzyme Mix is also an optimised
Chapter II: PCR-based analysis of transformants 94 mixture of Taq DNA polymerase and the proofreading enzyme Pyrococcus species GB-D polymerase. The system is able to amplify DNA fragments up to 30 kb from purified or crude samples (260). The Goldstar TM polymerase is a recombinant Taq -DNA polymerase that is isolated from extremely thermostable clones, which lacks the 3’-5’ proofreading activity. DNA fragments as long as 12 kb can be efficiently amplified in the presence of inhibiting impurities still present (i.e. cell lysate material) (262). The two amplification reactions of the 2.3 kb fragment with the Elongase Mix were successful, both for transformant 55 and transformant 114. Thereby, the results obtained with the wild type strain are confirmed: method 8 is suitable to extract genomic DNA which is suited for the reliable amplification of fragments up to 2.3 kb. The amplification of the product with the Elongase Mix was more efficient than with the standard Taq -DNA polymerase.
None of the reactions to amplify the 4.3 kb fragment with the Goldstar TM polymerase were successful. Thus, the Goldstar TM polymerase is unable to amplify the larger fragment in samples of method 5 and method 8, and when mycelium is used directly in the PCR-tube (method 10). The same is true for the Expand TM Long Template PCR System, although in one sample of method 10 the 4.3 kb fragment was present. The Elongase Mix was able to reliably produce the 4.3 kb PCR product in all samples of method 8 from transformant 114 and in two of the three samples of method 8 from transformant 55. For one sample of method 10 positive results were also obtained with this polymerase. The fragment bands were quite intense, and in most cases only the desired product was amplified. It has to be noted that the ratios 260/280 and 260/230 in the case of M. gramineum genomic DNA samples, again, are no good indication for the suitability of the sample for PCR amplifications, even with longer fragments to amplify. The Elongase Mix was able to amplify the 4.3 kb fragment in the samples of method 8, which had a mean 260/230 ratio of 1.2 instead of higher than 1.8.
4,3 kb
2,3 kb
m5, s2 55, P3 m5, s3 55, P3 m8, s2 55, P3 m8, s3 55, P3 m10,55, s2 P3 m10,55, s3 P3 marker m8, s1 55, P2 m8, s2 55, P2 m5, s2 114, P3 m5, s3 114, P3 m8, s2 114, P3 m8, s3 114, P3 marker m10,114, s2 P3 m10,114, s3 P3 m8, s1 114, P2 m8, s2 114, P2 m5, s1 55, P3 m8, s1 55, P3 m10,55, s1 P3 marker m5, s1 114, P3 m8, s1 114, P3 m10,114, s1 P3 Figure II. 3-4. Results of the PCRs 2 and 3 on the extraction samples of M. gramineum with the Elongase Mix. (P = PCR, m = method, s = sample).
A summary of these results is given in Table II. 3-2.
Chapter II: PCR-based analysis of transformants 95
Table II. 3-2. Extraction procedures suitable for PCR for Aspergillus and Myrothecium Aspergillus Myrothecium DNA extraction Highest amount DNA Protoplast Protoplast High concentration Glass bead, LiCl, boiling, Boiling, protoplast
liquid N 2, protoplast PCR PCR product < 1 kb Boiling, boiling 2, liquid Glass bead, LiCl, boiling
N2 2, acetone, liquid N 2
PCR product ≈ 2 kb - liquid N2, mycelium in PCR-mixture PCR product ≈ 4 kb - liquid N 2 with Elongase -: not investigated
4. CONCLUSION Ten methods were tested to rapidly produce genomic DNA samples which are suited for PCR applications. The methods were selected based on their cost and short duration and on the fact that no special equipment is required and a lot of samples can be processed at the same time. For all procedures, tiny amounts of mycelium grown on solid medium were used.
For Myrothecium gramineum , the highest DNA concentration was obtained with the protoplast procedure. Concentrations higher than 100 ng/µL were reached with the boiling procedure (method 5) and the protoplast based method. As for M. gramineum, for A. nidulans the highest DNA concentration was obtained with the protoplast extraction procedure. Concentrations higher than 100 ng/µL were reached with the glass bead method, the LiCl method, the boiling procedure, the liquid nitrogen method and the protoplast based method. Most extraction protocols resulted in higher DNA concentrations for A. nidulans than for M. gramineum .
Samples of M. gramineum resulting from the boiling procedure (1.5 hours) and the liquid nitrogen procedure (2.5 hours) were suitable for the amplification of fragments up to 2.3 kb. The direct use of mycelium from M. gramineum in the PCR tube can be employed for the amplification of fragments up to 1 kb and even weak signals of 2.3 kb can be obtained. Amplification of fragments up to 4.3 kb, however, requires the use of the long distance polymerase Elongase Mix on samples extracted with the liquid nitrogen procedure.
Part II: Development of a homologous expression system
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 99
Chapter III
Cloning and sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase ( gpd ) gene of M. gramineum
1. INTRODUCTION The use of strong promoters for the expression of (heterologous) proteins in suitable host organisms is of great importance for biotechnological applications. It is claimed that all other factors playing a role in transcription and translation are less important than the use of a strong promoter (263). However, it has been shown that there is not much homology between fungal promoters, not even in related fungi. Even different genes coding for the same enzyme, such as nitrate reductase, show nearly no homology in their promoter sequences in Aspergillus nidulans and Neurospora crassa . A study of Moraleyo et al . (150) which compared homologous and heterologous promoters indicated that the best results are obtained with homologous promoters. Because little is known about the critical parts of fungal promoters till today, a strong homologous promoter is usually searched for when developing a new expression system. It is assumed that this strong promoter will function ideally in the new expression host.
The most frequently used constitutive promoter is the glyceraldehyde-3-phosphate dehydrogenase (gpdA ) promoter of A. nidulans . This promoter is functional in different species, including industrially important Penicillium and Aspergillus species. The glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.12) promoter is a promising candidate (39) because in many eukaryotic microorganisms, the gpd -genes are expressed constitutively and in large amounts. The soluble cellular proteins of S. cerevisiae, A. nidulans and other higher eukaryotes can contain up to 5 % GPD (143, 136). The fact that GPD is present in large amounts indicates that it is regulated by a very active promoter (39). Other constitutive promoters which have been used for example in Aspergillus sp. are the trpC promoter from A. nidulans and the TEF1-alpha promoter of A. oryzae (4). Another recently developed expression system is based on the constitutive promoter of the histone H4 of P. funiculosum (158).
The constitutive gpd -promoter has been used in many expression systems, even in combination with prokaryotic genes (147, 148). A few examples are given in Table 8-1 of the Introduction p. 44. Other applications are based on the fact that the transformation efficiency of dominant selection systems can be improved by the use of a (homologous) gpd -promoter. Jungehülsing and co-workers (264) were able to significantly improve the transformation efficiency with the phleomycin resistance system of Claviceps purpurea by using the homologous gpd -promoter. First, the original A. nidulans trpC promoter pGW926 was exchanged by the strong heterologous gpdA -promoter of A. nidulans . This considerably improved the efficiency of the system, but the highest transformation efficiencies were
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 100 obtained with the homologous gpd -promoter of Claviceps purpurea . Similar results were obtained when the hph -gene was put under the control of the homologous gpd -promoter in Podospora anserina (265).
As described in § 8.1.1 of the Introduction, Punt et al. (145) found several boxes in the gpdA- promoter with similarities to other promoters, more precisely the pgk -box 600 bp upstream of the transcription start point ( tsp ), a CT-rich region immediately before the transcription start site and the gpd -box situated 250 bp upstream of the major tsp . Both the gpd -box and the pgk - box contain a considerable amount of direct or inverted repeats, which could mean that the pgk- and the gpd -box are part of regulatory sites (146). Deletion of the gpd -box caused a 50 % decrease in lacZ expression, deletion of the pgk -box decreased expression with 30 % and deletion of the CT-box lowered expression with 80 % (136). Deletion of the 5’ upstream intron did not have an effect on expression. Mini-promoter::lacZ fusions were studied in order to further elucidate the importance of the gpd -box (128). The studies were carried out in single copy transformants ( argB locus). The mini-promoter comprised the CT-box, which is important for the recognition of the correct transcription start point, and other sequences required for transcription initiation. With this promoter, expression levels of only 5 % of the levels obtained with the intact gpdA -promoter were reached. Introduction of activating sequences upstream from the mini-promoter did not alter the transcription initiation site. Introduction of the gpd -box (in either orientation) significantly increased the level of lacZ expression and introduction of a second copy of the box improved expression even further. Introduction of the gpd -box with flanking gpdA -sequences improved expression more than without the flanking regions, suggesting that, besides the gpd -box additional functional elements are present in these larger promoter fragments. Deletion of 18 bp at the 3’ end of the gpd -box clearly reduced the activity of the box, whereas deletion of 7 bp at the 5’ end did not significantly affect the level of gene expression. Introduction of the gpd -box into the upstream region of the amdS gene of A. nidulans increased the expression of a lacZ -gene fused to these expression signals 30-fold (232). This increase was dependent on the orientation of the introduced box and the site of introduction in the upstream amdS region. Introduction of the gpd -box with flanking gpdA -sequences reduced or even extinguished positive effects of the gpd -box. Catabolite regulation of the amdS promoter was retained after introduction of the gpd -box.
Although the gpd -promoter is a constitutive promoter, Redkar and collaborators (266) were surprised to find an increased transcript level of gpdA in NaCl adapted cultures of Aspergillus nidulans . The results were even more surprising because increased activity of GPD diverts carbon away from glycerol, which was previously shown to be accumulating in salt-adapted cultures, into the pathway leading to glycolysis and ATP formation. Cellular adjustment to elevated salinity requires additional energy for growth and regarding this, GPD interconnects stress response and ATP formation during growth under saline conditions. The cultures adapted to increasing concentrations of NaCl also had increased levels of GUS activity (gpdA-uidA construct), leading to the consideration that NaCl adaptation in strains expressing
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 101 proteins under the control of the gpd -promoter might be a cheap way to increase protein production. Salt stress regulated expression of the gpd -gene of the edible basidiomycete Pleurotus sajor-caju has been reported by Jeong et al . (267). Drought, heat, salt and cold stress induced eight-, five-, four- and two-fold induction of gpd expression in this fungus, respectively. Another indication for the fact that gpd expression is regulated came from a study of the expression of the gpd1 -gene of the zygomycete Mucor circinelloides (126, 268). This study shows that, in the presence of glucose, gpd1 expression levels are much higher than in the presence of glycerol or ethanol. Strong expressions were observed under anaerobic conditions with glucose or galactose as carbon sources and the mRNA gradually decreased as the sugar was consumed. However, subsequent addition of glucose to the medium resulted in a rapid increase in gpd1 expression. The level of reporter gene expression was also correlated with the concentration of glucose in the growth medium. Moreover, possible heat shock elements (AGAAN), an element possibly involved in development and differentiation (ATGAAAT) and four CATCAT repeats, which might be responsible for the carbon source dependent expression, were found in this gpd -promoter (126), confirming the possibility of regulated expression of the gene. Repression of the expression of the only gpd -gene of Hypocrea jecorina during conidiation and mycoparasitism has been found by Puyeski and co-workers (269). Similarly, the expression of the single glyceraldehyde-3-phosphate dehydrogenase gene of Pichia pastoris is constitutive, but the level of expression depends on the carbon source. The highest mRNA levels were observed in glucose-grown cells, followed by glycerol and methanol (270). Some filamentous fungi have more than one gpd -gene. Agaricus bisporus, for example, has two gpd -genes of which only one is expressed (271). Likewise, of the three different gpd - genes of M. circinelloides only one is expressed (268). Moreover, the expression of this gpd1 - gene strongly depends on the culture conditions (carbon source and/or anaerobiosis) and growth form: a high level of expression of gpd1 was observed in the yeast culture of this dimorph zygomycete, whereas low expression of the gene was detected after the shift to filamentous growth. In S. cerevisiae , three separate GPD-encoding genes are differentially expressed, indicating that the three isoforms may have distinct cellular roles.
A considerable number of GPD sequences are available and the enzyme and gene sequences are highly conserved throughout the kingdoms (264) which allows one to isolate the gene in genetically poorly studied organisms. Therefore, the gpd -gene of M. gramineum was cloned in order to be able to use its promoter for the (heterologous) expression of proteins. Part of the gpd -gene was already cloned, as described by Jonniaux et al . (29). Genomic walking was used to isolate the remainder of the gene, as discussed in this chapter.
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 102
2. MATERIALS AND METHODS
2.1. Strains and growth conditions Myrothecium gramineum BCCM TM /MUCL 39210 was used to isolate the glyceraldehyde-3- phosphate dehydrogenase gene. Escherichia coli DH5 α-F’ was used for all cloning experiments.
M. gramineum was grown on Potato Dextrose Agar (Oxoid) for sporulation. Cultures of 50 mL Potato Dextrose broth (Difco) were grown for 3 days prior to genomic DNA isolation. M. gramineum was grown for 2 days in a 500 mL Erlenmeyer flask containing 100 mL AMM prior to RNA isolation. AMM was used as described by Barrat et al. (231), supplemented with 5 g/L yeast extract (Difco). Liquid cultures were grown at 25°C and 125 rpm (50 mL culture) or 150 rpm (100 mL culture).
2.2. Standard DNA manipulation Routine recombinant DNA methodology was performed according to Sambrook & Russell (2001) (230). DNA concentrations were measured with the NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies). The Smartladder TM of Eurogentec was used to control the length of the amplified products. PCR reactions were performed with standard Taq -DNA polymerase (Roche Diagnostics), with the High Fidelity PCR Master kit (Roche Diagnostics) or with the BD Advantage 2 Polymerase Mix (BD Biosciences) according to the procedure described by the manufacturer. Gel fragments were purified with the QiaexII Gel Purification kit (Qiagen). Reactions were purified with the Bioline DNAce Quick-Clean kit (Bioline, Gentaur) or with the Minelute Cleanup kit (Qiagen). Plasmids were extracted with the Nucleospin QuickPure kit (Machery Nagel) or with the QIAprep ® Spin Miniprep kit (Qiagen). PCR-fragments were ligated in pGEM-T® (Promega) for proliferation and sequencing. The ligation mixtures were transformed into E. coli DH5 α-F’. The plasmids of the colonies resulting from these transformations were tested as to the presence of the insert in pGEM-T® by colony PCR with standard Taq -polymerase (Roche Diagnostics) and with the primers ‘T7’ and ‘SP6’ (Table III. 2-1). The plasmids were amplified in E. coli DH5 α-F’ and isolated with the HiSpeed Plasmid Maxi kit (Qiagen) when larger amounts were necessary. For Southern blot analysis, digested genomic DNA (20 µg) was blotted onto positively charged nylon filters (Hybond N +, GE Healthcare) with the Trans-Blot ® SD DNA/RNA Blotting Kit (Bio-Rad) following the instructions of the manufacturer. The DNA was hybridised to DIG-labelled probes which were constructed with the DIG High Prime Labelling and Detection Kit (Roche Diagnostics). Restriction enzymes were obtained from Roche Diagnostics or from New England Biolabs and were used as indicated by the manufacturer.
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 103
2.3. Cloning of the Myrothecium gramineum gpd -gene
2.3.1. Genomic DNA isolation DNA was isolated from freshly grown mycelium with the Dneasy Plant Mini/Maxi kit (Qiagen). The mycelium (1 g, wet weight) was lyophilised prior to homogenisation. All centrifugation steps were carried out at 5000 rpm (GSA rotor). The elution step was performed two times. The samples were concentrated with sodium acetate and ethanol according to the procedure described in § 2.3 of Chapter I.
2.3.2. Genome walking Based on the sequence available in Jonniaux et al. , 2004 (29), primers were developed to perform several genome walkings. These walkings were carried out in 4 genomic libraries, as described in the BD GenomeWalker TM Universal kit (BD Biosciences). Four gene specific primers (GSP) were designed (Table III. 2-1): ‘1 GPD’ and ‘nested 1 GPD’, and ‘3 1 GPD’ and ‘3 1 nested GPD’. The PCR reaction mixture and cycles were optimised for use with the Expand Long Template PCR System (Roche Diagnostics). The primary PCR-reaction mixture contained 3.75 units LD polymerase mix, 1X PCR buffer (1.75 mM MgCl 2), 0.35 mM dNTP mix (Sigma), 0.3 µM primers, 2 µl template and 37.5 µl milliQ-water. After initial denaturation at 92°C for 1 minute, 5 cycli of denaturation at 92°C for 10 s, primer annealing at 70°C for 30 s and extension at 68°C for 4 minutes were carried out, followed by 25 cycli of denaturation at 92°C for 10 s, primer annealing at 65°C for 30 s and extension at 68°C for 4 minutes. In the end, 10 cycli of denaturation at 92°C for 10 s, primer annealing at 60°C for 30 s and extension at 68°C for 4 minutes were run with a last extension step of 7 minutes at 68°C. The secondary PCR-reaction mixture was the same as the one described for the primary PCR, but 1 µl of the primary PCR mixture was used as template. The cycli were: initial denaturation at 92°C for 1 minute, 5 cycli of denaturation at 92°C for 10 s, primer annealing at 70°C for 30 s and extension at 68°C for 4 minutes, 20 cycli of denaturation at 92°C for 10 s and primer annealing and extension at 68°C for 4 minutes and a final extension of 7 minutes at 68°C.
2.3.3. Cloning and sequence analysis The complete sequence of the gpd -gene was amplified from genomic DNA with the High Fidelity PCR Master kit (Roche Diagnostics). The primers used for this reaction are ‘5 lp GPD’ and ‘3 GPD complete’ (Table III. 2-1). The obtained PCR-fragment was cloned in pGEM-T® and the resulting vector was called pGV. Subsequently, the gpd -gene on pGV was sequenced at the VIB Genetic Service Facility (Belgium). The sequence was analysed with the Clone Manager Professional Suite Software (Version 6.0) and its deduced amino acid sequence was blasted against the UNIPROT database (Release 5.0), using the WU-Blastp (v2.0) program on the website of the European Bio-informatics Institution (score matrix: PAM250).
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 104
Table III. 2-1. Primers used for the M. gramineum gpd -gene Primer name Feature Primer sequence (5’-3’) 1 1 GPD primary GSP upstream AGAAGCAGCACTTACAGCGTACTTGG nested 1 GPD nested GSP upstream GTCGTTGACGGCAACGATCTCGAC 2 GPD primary GSP upstream TCGACGGCGTTGCGGAAGACGATACGGT 3 GPD primary GSP upstream GGACAGCGTGCAGGAATTGATGAGAGGT nested 3 GPD nested GSP upstream AAGCTTTCGCTCGCTCCTAGCTTGG 4 GPD primary GSP upstream GGAGAGGATTGTTGGTGGTTATGG 4 nested GPD nested GSP upstream AAGCAAGCTGCATTGCCTGCCTGCCTGCCAAC 3 1 GPD primary GSP downstream CAAGGACAAGGCTGCTGCTCACCTGAAG 3 1 nested GPD nested GSP downstream GCAGCGCCGACGTCATCTCCAACGCTTCTT 5 lp GPD amplification gpd -gene gacgtcgac-TGGTCATGCTCATCATC 3 GPD complete amplification gpd -gene GTAGTCAAGGCTTGAGATGG 5 cDNA cDNA amplification GGCCGCATTGACCGTATCGT 3 cDNA cDNA amplification GCCTTGGAGGCATCGACCTT 3’ RACE CDS polyA signal localisation AAGCAGTGGTATCAACGCAGAGTAC(T) 30 5’ long GPD creation expression vector gacgtcgac-TGGTCATGCTCATCATC 5’ short GPD creation expression vector gacgtc-GACGCAGTGGACAGACGTTA 3’ prom GPD creation expression vector TGCCAACCTTGATG GcAtgCAT TTTGTG T7 control pGEM-T® ligation GGCGATTAAGTTGGGTAACG SP6 control pGEM-T® ligation CGCCAAGCTATTTAGGTGAC 1 Matching sequences are indicated with capitals, non-matching sequences are printed in small-type lettering and the multicloning site of the gpd -promoter is represented in bold .
The position of the intron was confirmed by cDNA analysis. RNA was extracted from freshly grown mycelium with the Rneasy Plant Mini kit (Qiagen). This RNA was treated with DNAse to remove all genomic DNA traces. The sample was incubated for 30 minutes at 37°C with 1 unit DNAse (Roche Diagnostics), 6 µl 10x DNAse buffer and RNAse free water. After that, the reaction was purified according to the protocol described in the BD Genome Walker Universal kit. The purified mixture was used for first strand cDNA synthesis, which was carried out with the RevertAid TM H Minus First Strand cDNA Synthesis kit (Fermentas, Germany). The synthesised cDNA was used directly for PCR with the High Fidelity PCR Master kit using the primers ‘5 cDNA’ and ‘3 cDNA’ (Table III. 2-1). The resulting fragment was cloned in pGEM-T® and sequenced. The position of the polyA signals was determined using the BD Smart TM Race cDNA Amplification kit (Clontech). RNA was isolated with the RNeasy Plant Mini Kit (Qiagen) as described above and treated with DNAse using the RNAse free DNAse Set (Qiagen). The cDNA was synthesised with the BD Smart TM Race cDNA Amplification kit (Clontech). The RACE-PCR reactions were performed with the BD TM Advantage TM 2 PCR kit (Clontech), containing a mixture of the BD TITANIUM Taq - polymerase and a proofreading polymerase. The PCR reactions and mixtures were used as described in the kit. The sequence of the 3’ RACE CDS primer is given in Table III. 2-1.
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 105
3. RESULTS AND DISCUSSION
3.1. Isolation of the full length gpd -gene A part of the gpd -gene was already isolated by degenerate PCR, as described by Jonniaux et al . (29). This part consists of 850 bp of the coding region of the gene and contains an intron sequence of 456 bp. The rest of the gene was cloned using the Universal GenomeWalker kit TM (Figure III. 3-1). After isolation, genomic DNA is cut with restriction enzymes which produce blunt ended DNA fragments ( Dra I, Eco RV, Pvu II, Stu I and Hinc II).
Figure III. 3-1. Strategy followed to clone the complete sequence of the gpd-gene. Four genomic libraries are constructed by cutting genomic DNA with 4 different restriction enzymes and ligating these fragments to the BD GenomeWalker Adaptor. Two gene specific primers (primary -GSP1- and nested -GSP2) are constructed based on the partially known sequence of the gene. These primers are used in PCR-reactions in combination with the adaptor primer AP1 and AP2, respectively, to amplify the unknown regions. Unknown sequences are represented by white boxes, known sequences by shaded boxes (resulting from degenerate PCR), by grey ones (resulting from genome walking) or by black ones (adaptor sequences). Primers are indicated with black arrows above their target site.
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 106
The restriction mixtures are purified and ligated to the BD GenomeWalker Adaptors, which are also blunt. In this way, genomic libraries are constructed on which PCR reactions are performed. Each PCR uses a primer designed to anneal at the known part of the gene and another primer designed to anneal at the known sequence of the adaptor sequences. In the primary PCR reaction, the outer adaptor primer (AP1) and an outer gene specific primer (GSP1) are used. In the second reaction, the nested PCR reaction, the nested adaptor primer (AP2) and a nested gene specific primer (GSP2) are used to amplify products obtained in the primary PCR.
3.1.1. Construction of the BD GenomeWalker banks Genomic DNA of M. gramineum was isolated using the 1 2 M Qiagen DNeasy Plant Maxi kit and the samples were concentrated with 3 M sodium acetate and ethanol. Two samples were obtained with a concentration of 0.145 µg/µL (elution 1) and of 0.382 µg/µL (elution 2). A total of 26.35 µg DNA was isolated. 2.5 µg of the DNA of Myrothecium was cut with Dra I, Eco RV, Pvu II, Stu I or Hinc II according to the protocol described in the kit. As a control, the same amount of a human DNA (supplied with the kit) was cut with Dra I. 5 µL of each 100 µL Figure III. 3-2. Restriction of DNA restriction mix was used for gel electrophoresis. The with Dra I (lane 1: M. gramineum , results for the restrictions with Dra I are given in Figure lane 2: human, lane M: TM III. 3-2. All restrictions were successful: a smear of Smartladder . genomic DNA was visible in each lane. The restricted fragments were purified and ligated to the adaptors.
3.1.2. Genomic walking Two gene specific primers were developed based on the sequence of the gpd -gene which was already known (29). Their position relative to this fragment is represented in Figure III. 3-3. Table III. 3-1 gives an indication of the length of the fragments which have to be sequenced in order to know the full (coding) sequence of the gpd -gene of M. gramineum . For the DNA sequence, only the exons and the introns were considered.
Binding site genome walking primer
nested 1 GPD 1 GPD Eco RV Stu I 3 1 GPD 3 1 nested GPD 200 400 600 800
UPSTREAM DOWNSTREAM (5') (3') Fragment resulting from degenerated PCR (850 bp) Figure III. 3-3. Position of the gene specific primers relative to the known 850 bp fragment.
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 107
Table III. 3-1. Overview of the length of the gpd -genes (coding sequence) and enzymes of some closely related fungi Organism Total length GPD Number of missing AA AA 1 bp 5’ AA 2 3’ AA 3 Neurospora crassa 338 1016 20 188 Trichoderma koningii 337 1016 20 187 Sordaria macrospora 338 1090 20 188 Colletotrichum lindemuthianum 337 1110 21 187 Colletotrichum gloesporioides 338 1016 21 187 Aspergillus oryzae 338 1398 21 187 Aspergillus nidulans 336 1402 20 186 Aspergillus niger 336 1437 20 186 1 AA = amino acids 2 Number of amino acids lacking upstream the known sequence 3 Number of amino acids lacking downstream the known sequence
Eco RV bank The primary PCRs were performed with the 1 2 M 3 4 M primers ‘1 GPD’ and ‘3 1 GPD’ for the upstream and downstream walking, respectively. The nested PCR reactions were performed with the ‘nested 1 GPD’ and the ‘nested 3 1 GPD’ primers, respectively. The upstream walking PCRs did not produce fragments, but the downstream walking resulted in a clear fragment of about 1300 bp after the primary PCR and of approximately 1200 bp after the nested PCR (Figure III. 3-4). In the nested Figure III. 3-4. Primary (left) and nested PCR mixture, a fragment of approximately 950 bp PCR (right) Eco RV bank. Lane 1 and 3: was also visible, but this was a result of aspecific downstream. Lane 2 and 4: upstream. amplification, which was confirmed by sequence analysis. The 1200 bp fragment was gel purified, ligated into pGEM-T® and E. coli DH5 α-F’ was transformed with the ligation mixture. Plasmids of the colonies which proved to contain the insert in pGEM-T® were sent to the VIB Genetic Service Facility for sequencing, which revealed that the sequence of the gpd -gene was extended with 1115 bp to a total of 1965 bp.
The sequence now comprised 1480 bp of the coding sequence (exons + introns) and 485 bp of the 3’ flanking sequences. It was decided to carry out no more walkings to the 3’ end of the gene.
Pvu II bank The walkings to the 5’ end of the gene in the Pvu II bank were carried out with the primer ‘2 GPD’. The primer ‘2 GPD’ anneals at the start of the known sequence. A fragment of
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 108 approximately 300 bp was sequenced. The known sequence was extended with 242 bp (63 bp of exon 1 and 179 bp of the 5’ flanking sequences), now comprising 2207 bp.
Dra I and Stu I bank The walkings to the 5’ end of the gene did not reveal additional information about the gpd - sequence.
Hinc II bank In the literature, different lengths of promoter regions are described for gpd -genes. For example, the well studied gpdA -promoter of Aspergillus nidulans is more than 1000 bp (136, 200) and the gpd -promoter of Flammulina velutipes is about 1400 bp (39). A 1400 bp fragment of the gpd1 -promoter of Claviceps purpurea gives higher expression than a 500 bp fragment of the same promoter (264). A fragment of the homologous gpd -promoter of 1.9 kb improved transformation efficiency of P. anserina 2.4 times while a fragment of 350 bp improved it only 1.6 times as compared to the heterologous A. nidulans gpd -promoter (265). Since only 179 bp of the promoter region (or 5’ UTR) were known up to now, it was decided to develop new walking primers and to perform an additional walking in a new GenomeWalker bank, constructed after restriction of the genomic DNA with Hinc II. The walkings with the primers ‘3 GPD’ (annealing at position 126 of the known sequence) and ‘3 GPD nested’ (annealing at position 85) resulted in a fragment of approximately 500 bp. Sequencing of the fragment revealed that the fragment was 462 bp long, covering 85 bp of the known sequence and extending this sequence with 377 bp to 2584 bp.
The 5’ flanking sequences of the gene known at this point comprised a possible TATA-box, two CAAT-boxes and two CT-rich sequences, in between which the TATA-boxes are situated. It was possible that a functional gpd -promoter was cloned, but since the known sequence of this promoter was only 556 bp long, it was decided to develop new walking primers (‘4 GPD’ annealing at position 206 and ‘nested 4 GPD’ annealing at position 67) and to perform an additional walking to the 5’ end in the Dra I, Pvu II, Eco RV and Stu I bank. The longest amplification fragment was obtained in the Dra I bank and proved to extend the length of the known sequence with 457 bp. A promoter region of 1013 bp was sequenced.
Finally, the gpd -gene comprises 3041 bp, containing (Figure III. 3-7): 1013 bp of the 5’ flanking sequences (promoter region and 5’ UTR), 1543 bp of the coding sequence and 485 bp of the 3’ flanking sequences.
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 109
3.1.3. Cloning and sequencing of the complete gpd -gene of M. gramineum Based on the obtained sequence, two primers to pick up the 1 M complete gpd -gene from the genome of M. gramineum were 3021 bp designed. The primers, ‘5 lp GPD’ and ‘3 GPD complete’ amplify a product of 3021 bp. The result of the High Fidelity PCR performed with these primers is presented in Figure III. 3-5. After PCR cleanup, the fragment was ligated into pGEM-T®. The resulting plasmid is further referred to as pGV. The complete sequence of the gpd -gene was confirmed Figure III. 3-5. High Fidelity PCR resulting in the amplification of the by sequencing at the VIB Genetic Service Facility. The gene gpd -gene of M. gramineum (lane was submitted to Genbank, with the accession number 1). Smartladder TM (lane M). EF486690 .
3.2. Nucleotide sequence analysis
3.2.1. Analysis of the coding sequence The number of introns in gpd -genes of different eukaryotic organisms varies between none in yeast genes and 11 in the gpd -gene of a chicken (272). Most gpd -genes of filamentous fungi contain one or more introns. Their number and position in the genes of some filamentous fungi and some yeasts is given in Figure III. 3-6 (adapted after 268). In the gpd -gene of A. nidulans (145) and Claviceps purpurea (264), an intron was found in the region upstream the start codon (not illustrated in Figure III. 3-6). The positions of the introns are conserved among the Ascomycota and the Basidiomycota , but only one intron is conserved between the two phyla (271). The number of introns present and their position in fungal genes relate well to their systematic relationship (264).
Figure III. 3-6. Position of the introns in the coding sequence of gpd -genes of some fungi and yeasts. I: Zygomycota ; II: Basidiomycota ; III: Ascomycota .
Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 110
1 catggtcaag gtcatggtca tgctcatcat catggtcgtg gccacggcca tggatggccc agtgtaaatg 71 tgccgcgagc gcagcatccc gtcgagaaag atagttattg gccgtgccct ctggggtacc tgtagggtgt 141 ccggtgtcgc agggcggcgt aggtactgga ggggtaccgt gctggctgca tgtacggtac tgcggcttgt 211 accgccatcg gacatggttg ctcgttgccc ctccacccgc tccagctaat gaggcaaggc gagggggggc 281 ggcggcggcg gcggaaaggg cccaagaggc aaagggtggt gcgttctggt acgtaagacg agataatctc 351 tctctctctc tct ggatggc cgcggtcgtg gtccctccat aggtccaggc gcaggcgcag gtgtgcgtag 421 gtctgggtcc aggtgtaggt ctacggtagg tccaggtgtt gacgcagtgg acagacgtta gcaggtcggt 491 aggttggcag gcaggcaggc aatgcagctt gcttgcttgg ttgcttgctt ggactgtcat ttcggacaaa 561 tattaagctt tggctcctcc catctactct tcctttccct cctctcttct tcttctcctt cttcttcttt 631 cttccctttc c ataaccacc aacaatcctc tccataccta taccctcaaa ccaacgtacg tacctcgtcc 701 tcctcgagct ctccctcgtc gatgattctc cctccctccc atcacgcgtt ccccctcctc tcttctc aat 771 ggcttgcttg gctttgctgc tgctgccatc gcccgctgtc tgctggacgg acccgatgct cctgcagctg 841 ctgagctttc tcgtctcctt ttcacccctc gctcc ggctg ggctcgaccc aggtccaagc taggagcgag 911 cgaaagcttt caccctcacc ccacctctca tcaattcctg cacgctgtcc caccttgggc cctgggctaa 981 cttggagctt ccctccct ac agaaattcac aaa ATG GCTC CCATCAAGGT TGGCATCAAC GGTTTCGGCC 1 M A P I K V G I N G F G 1051 GCATTGGCCG TATCGTCTTC CGCAACGCCG TCGAGCACTC CGACGTCGAG ATCGTTGCCG TCAACGACCC 13 R I D R I V F R N A V E H S D V E I V A V N D 1121 CTTCATTGAG CCCAAGTACG CT gtaagtgc tgcttctgct tcccctcagt cgacgagcga gcccaaagcc 36 P F I E P K Y A intron 1191 gagctgcagc tagcggagcc atgcgctgcc tgcatgccac tgcataacag cagctagagg aggggtacac 1261 ggccgcgcgc gcagacacac atacaacacc accaccacca aaaggagggg cagaaaaaat ccagcattgt 1331 ccgatttcac cccaccatct cacgtcaacc aatttgcccc tccatgatat catgtgtccg cgcccagctc 1401 aacacgtcca cctcctctgg ccaatggcga gcgcattgat gctttgatga gcggaaacga cgctgaggcc 1471 ctcagcctcg tcgtcgctgc cgctgccgcc gcgcgccgct cacgcatcgg cgggctcccg tcgctgggct 1541 tcaattgaca tgacatgatg catggccacc gtgctaacca cccctgtgtc tgtccgatag GCCTACATGC 44 A Y M 1611 TCAAGTATGA CTCTACCCAC GGTCTCTTCA AGGGTGAGGT CACCGTCGAT GGCGATGACC TGACCGTCAA 47 L K Y D S T H G L F K G E V T V D G D D L T V 1681 CGGCAAGAAG GTCCGCTTCT ACACTGAGCG TGACCCCGCC GCCATCCCCT GGAAGGAGAC TGGTGCCGAG 70 N G K K V R F Y T E R D P A A I P W K E T G A E 1751 TACATTGTCG AGTCCACCGG TGTCTTCACC ACCAAGGACA AGGCTGCTGC TCACCTGAAG GGTGGTGCCA 94 Y I V E S T G V F T T K D K A A A H L K G G A 1821 AGAAGGTCAT CATCTCTGCC CCCTCTGCCG ATGCCCCCAT GTACGTTATG GGTGTCAACG AGGAGACCTA 117 K K V I I S A P S A D A P M Y V M G V N E E T 1891 CGACGGCAGC GCCGACGTCA TCTCCAACGC TTCTTGCACC ACCAACTGCC TGGCTCCCCT CGCCAAGGTC 140 Y D G S A D V I S N A S C T T N C L A P L A K V 1961 ATCCACGACA AGTTCACCAT TGTCGAGGGT CTCATGACCA CTGTCCACTC CTACACCGCC ACCCAGAAGA 164 I H D K F T I V E G L M T T V H S Y T A T Q K 2031 CCGTCGATGG TCCTTCCGCC AAGGACTGGC GTGGTGGCCG TGGTGCTGCC CAGAACATCA TTCCCAGCAG 187 T V D G P S A K D W R G G R G A A Q N I I P S 2101 CACCGGTGCC GCCAAGGCTG TCGGCAAGGT CATCCCCGAC CTCAACGGCA AGCTCACCGG CATGTCCATG 210 S T G A A K A V G K V I P D L N G K L T G M S M 2171 CGTGTCCCCA CCGCCAACGT CTCCGTTGTC GACCTGACTG CCCGCATTGA GAAGGGTGCC AGCTACGACG 234 R V P T A N V S V V D L T A R I E K G A S Y D 2241 AGATCAAGCA GGTCATCAAG GATGCCGCCA ACGGTCCCCT CAAGG gtgag ttgaaccctc cagccgtgct 257 E I K Q V I K D A A N G P L K intron 2311 ccggctcagt tgcatctgct ttggctgacc agatttccag GCATTCTCGC CTACACTGAG GACGAGGTTG 272 G I L A Y T E D E V 2381 TCTCCAGCGA CATGATCGGC AACACCGCCT CCTCCATCTT CGATGCCAAG GCCGGTATCT CCCTCAACGA 282 V S S D M I G N T A S S I F D A K A G I S L N 2451 CAACTTTGTC AAGCTGGTCT CCTGGTACGA CAACGAGTGG GGTTACTCCC GCCGTGTCCT CGACCTCCTG 305 D N F V K L V S W Y D N E W G Y S R R V L D L L 2521 GCCCACGTTG CCAAGGTCGA TGCCTCCAAG GCT TAA gtta gcctagatta gcaacaccta atgattaaaa 329 A H V A K V D A S K A 2591 aaaaaaatga aagagagaac cagacgtctg cgagttgggc catcaaagac atggcagaaa agcctctcat 2661 ttgtcacgac caaagacgaa gtataaaatc aggagcacca cgcacttggg cggcacatgt tggaggtggg 2731 atcgagcaag atccgtgctc tgacatggca gctgctccga ggaacaaggt gcaatcctag tatagtgtat 2801 agccattggg aaataaagac cagtctgcct ctcacgcttg gatacgtttc actgctcgtc atcgttgctc 2871 gtagcccgtt ggactaacat ctgctgtggg attcacgtag accaactgcg tgttgccctg tgaggctctc 2941 gagtctcggc cttgctagta tagcctctgc aagaacaaaa ctctcgtacg tgcatacagt ccaccatctc 3011 aagccttgac tacttgacaa tgttggatat c Figure III. 3-7. Nucleotide and deduced amino acid sequence of the gpd -gene of M. gramineum . Nucleotides are represented in lower case lettering (non-coding) or by capital letters (coding); amino acids by bold capital letters. Base 1-1013: promoter region (and 5’UTR), base 1014-2556: coding sequence, base 2557-3041: 3’ flanking sequences. The boxes represent sequences important for transcription, sequences important for translation are shaded in black. CT-rich stretches in the promoter are underlined. The introns are underlined and in italic letters. Genbank number
EF486690 . Chapter III: Cloning and sequence analysis of the M. gramineum gpd-gene 111
In the gpd -gene of M. gramineum , two introns were found (Figure III. 3-8): one quite long intron (458 bp) in the beginning of the gene (nt 1143 to 1600, between AA 43 and 44) and a shorter one (65 bp) near the end (nt 2286-2350, between AA 271 and 272) (Figure III. 3-7). The position of the first intron corresponds to the conserved position for the Ascomycota .
3' cDNA 5' cDNA genomic DNA
exon 1 intron 1 exon 2 intron 2 exon 3 (129 bp) (458 bp) (685bp) (65bp) (203 bp)
Figure III. 3-8. Schematic representation of the exons and introns of the gpd -gene of M. gramineum.
The position and length of the introns was confirmed by a cDNA analysis. RNA was extracted from freshly grown mycelium and treated with DNAse. The purified mixture was used for first strand cDNA synthesis and the synthesised cDNA was used directly for PCR with the primers ‘5 cDNA’ and ‘3 cDNA’. This PCR resulted in a 991 bp fragment, as expected. Amplification of genomic DNA would have resulted in a 1506 bp fragment. The 991 bp fragment was cloned in pGEM-T® and sequenced. The length and the position of both introns were confirmed. They are characterised by the consensus splice sites (5’ ‘GT’ and 3’ ‘AG’) and splicing signal of filamentous fungi (Table III. 3-2). Table III. 3-2. Consensus splice sites for introns of filamentous fungal genes Non-Sordariomycetes Sordariomycetes 5’ splice site g|GT A(A/C/T/)GT(C/T) g|GT AAGT Splicing signal (T/A)(G/A) CT (A/G)AC (A/G)CT (A/G)A(C/A) 3’ splice site (A/C)(C/T) AG| g (T/A/G)(T/A/G)(T/C) AG| References 133 273
3.2.2. Analysis of the 5’ and 3’ flanking sequences
3.2.2.1. 3’ flanking sequence The most important elements associated with transcription termination and polyadenylation of the mRNA for eukaryotic genes are the YGTGTTYY and the AAUAAA motifs (14). These sequences have been found in filamentous fungal genes, but mostly they are present in a shorter form (AUAA) or even completely absent. As indicated in Figure III. 3-7, similar sequences were found in the gpd -gene of M. gramineum (two putative polyA signals, each followed by two putative transcription termination sites). The BD Smart TM Race cDNA Amplification kit (Clontech) was used to determine the true positions of the polyA signal of the gpd -gene of Myrothecium gramineum . cDNA is made from RNA with BD PowerScript TM Reverse Transcriptase (RT), which is a variant of the MMLV RT. In the 3’ RACE reaction, the 3’ RACE CDS primer serves both for the annealing to the polyA signal and for the incorporation of an oligo sequence. As such, the cDNA is prolonged with a known sequence which can be used for primer design. After the reverse transcription, the cDNA is used in 3’ RACE PCR-reactions (Rapid Amplification of cDNA Ends). The strategy is represented in Figure III. 3-9.
Chaptersequence III: andanalysis Cloning of the
N = A, C, G or T Poly A + RNA 5' NBAAAAAAA-3' V = A, G or T NVTTTTTTTTTTTTTTTTTTTT CATGAGACGCAACTATGGTGACGAA-5' B = T, G or C 3' RACE CDS primer BD SMART TM first strand synthesis 5' NBAAAAAAA-3' NVTTTTTTTTTTTTTTTTTTTT CATGAGACGCAACTATGGTGACGAA-5'
3’ RACE PCR
NBAAAAAAA-3' Gene specific primer NVTTTTTTTTTTTTTTTTTTTT CATGAGACGCAACTATGGTGACGAA-5' First round of gene specific amplification NBAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGATACCACTGCTT NVTTTTTTTTTTTTTTTTTTTT CATGAGACGCAACTATGGTGACGAA-5'
NBAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGATACCACTGCTT Long UP TGAGACGCAACTATGGTGACGAACGGGATATCACTCAGCATAATC NVTTTTTTTTTTTTTTTTTTTTCATGAGACGCAACTATGGTGACGAA-5'
Incorporation of suppression PCR inverted repeat elements gpd- CGGGATATCACTCAGCATAATC by the Long Universal Primer
NBAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGATACCACTGCTTGCCTATAGTGAGTCGTATTAG gene Gene specific primer Short UP CTATGGTGACGAACGGATATCACTCAGCATAATC-5’ NVTTTTTTTTTTTTTTTTTTTT CATGAGACGCAACTATGGTGACGAACGGATATCACTCAGCATAATC-5' Repetitive PCR rounds: amplification of the 3' fragment
Double stranded 3’ RACE fragment Figure III. 3-9. Strategy of the 3’ RACE reactions in the BD Smart TM Race cDNA Amplification kit.
112
Chapter III: Cloning and characterisation of the gpd -promoter 113
For the 3’ RACE reactions, the primer ‘3 1 GPD’ was used in combination with the kit’s ‘Long Universal Primer’ and 3’ CDS primer. When considering the position of the two possible polyA sites, a fragment of approximately 816 bp is expected when the first polyA site is used (most 5’) and a fragment of 1040 bp will be obtained when the second polyA site is used to start the polyadenylation of the mRNA (Figure III. 3-10).
Figure III. 3-10. RACE products 3’ end gpd.
The result of the RACE PCR reaction is given in Figure III. 3-11. A clear fragment of approximately 800 bp was detected. A weak fragment of about 1000 bp was seen on the gel (not visible in Figure III. 3-11). Both fragments were gel purified and cloned in pGEM-T®, the mixtures were transformed into E. coli and plasmids of positive clones were sequenced.
TM
Lane M: Smartladder 1 2 3 4 5 a b M c d M 1 Lane 1 left side: 3’ RACE GPD Lane 1 to 5 right side: clones with 800 bp fragment Lane a to d right side: clones with 1000 bp fragment
800 bp
Figure III. 3-11. Gel electrophoresis of the 3’ RACE PCR reactions (left) and the control of the clones containing the obtained fragments (right).
The sequence analysis revealed that both polyadenylation signals are used: the polyA-signal situated most closely to the 5’ end is the major polyA-signal, while the second one is a minor polyA signal. Multiple poly adenylation signals were also observed in other fungal genes, e.g. the gpd -genes of Cochliobolus heterostrophus (272), Lentinula edodes ( Lentinus edodes ) (274) and Xanthophyllomyces dendrorhous (Phaffia rhodozyma ) (275).
Chapter III: Cloning and characterisation of the gpd -promoter 114
3.2.2.2. 5’ flanking sequences In the following section, some typical promoter elements are discussed (see Figure III. 3-7): - TATAAA box In most eukaryotic systems, the TATA-box (consensus TATAAA) appears to be essential for a functional core promoter. The TATA-box is the site where the TBP (TATA Binding Protein) binds and where the initiation complex for transcription is formed. In higher eukaryotes, this sequence almost always lays between 25 and 30 bp (20-40 bp) upstream the transcription start point (tsp) and mutations within this sequence usually lead to aberrant initiation of transcription. In S. cerevisiae , however, both the presence and/or the siting of the TATA-element appear to be far less critical. Some genes, even highly expressed, do not have a TATA-box, while other genes have one or more copies of this element up to 120 bp upstream of the tsp (133). In this sense, filamentous fungal genes resemble those of yeast more closely: consensus TATA-boxes have been found in few fungal promoters (134). Although AT-rich sequences were observed upstream of the transcription initiation sites, their location is variable and their functional significance unknown. Deletion analysis of the A. nidulans oliC promoter (135) indicated that the TATA-box is important for the determination of the tsp , but does not affect the level of expression. Similar findings have been reported for the A. nidulans trpC promoter. Two TATA like sequences were found in the promoter region of the gpd -gene of M. gramineum , 347 bp and 453bp upstream the start codon. - CAAA, CCAAT box In many higher eukaryotic systems, the CAAT-box is another motif which shows core promoter activity and which is usually situated between 70/80 and 90 bp upstream the tsp . In fungal genes, the CAAT box often is absent, or when present, is situated at a variable distance of the tsp (60-120 bp). In the promoter region of the gpd -gene of M. gramineum , 4 CAAT sequences were found. Two of them are sited further downstream the TATA boxes. The other two are each situated upstream one of the two TATA-boxes, as expected, at positions - 363 and -506 from the ATG. - Transcription start site immediately downstream CT-rich regions The element which is most important in determining the tsp in filamentous fungal genes is the CT-box. This is a pyrimidine-rich sequence which is found directly upstream the tsp in a considerable number of fungal genes (136). The length of this pyrimidine stretch can vary, as particularly marked in the A. nidulans oliC gene, where it extends to 96/100 bases (134). Functional analysis of the gpdA promoter of A. nidulans has shown that deletion of this sequence results in aberrant initiation of transcription and suggests that this region alone is sufficient for the determination of the tsp (137). The fact that, in cases where the CT-box has been deleted, the alternative transcription start points used are often found downstream other CT-rich sequences, supports the involvement of this motif in determining the tsp . In the gpd -promoter of M. gramineum , 5 CT-rich stretches were found: - the first CT-box situated most upstream at position -656 is quite short (16 bp) - the second CT-rich stretch is 74 nucleotides long at position - 378 - the third CT-box is of similar length of the second one (70 nucleotides) and situated at
Chapter III: Cloning and characterisation of the gpd -promoter 115
position -252 - the fourth CT-box is 30 nucleotides long and lays at position - 144 - the fifth CT-box is sited at position -21 and is 11 nucleotides long The two TATA-boxes and their corresponding CAAT-boxes are situated in between the first and the second CT-box and the second and the third CT-box, respectively. Several attempts were made to determine the transcription start site with the BD SmartTM Race cDNA Amplification kit (Clontech), but unfortunately, none of them succeeded. Table III. 3-3. TATA-boxes, CAAT-boxes and CT-rich sequences of some gpd -genes
Organism TATA-box(es) CAAT-box(es) CT-stretch(es) Phylum 4 Ref. #1 Position 2, 3 # position # position
A. nidulans - 1 -218 to -268 A 136 A. niger 1 -244 to -254 A X99652 5 Claviceps -197 to -316 -330 to -337 2 A 264 purpurea -68 to -92 Cochliobolus 1 -76 to -81 1 -39 to -76 A 272 heterostrophus Cryphonectria 1 -305 to -308 A X53996 5 parasitica Erisphe 1 -257 1 -206 1 -233 A 276 graminis Podospora -14 to -30 1 -136 to -141 1 -191 to -195 2 A 265 anserina -80 to -114 Coprinopsis 1 -62 to -57 B AB094148 5 cinerea Cryptococcus -60 to -63 1 -267 to -270 2 1 -67 to -94 B 277 neoformans -291 to -294 Flammulina 1 ? 2 ? 1 ? B 39 velupites Ganoderma -467 to -470 1 -67 to -73 2 1 ? B 278 lucidum -601 to -605 Lentinula -204 to-200 1 - 98 to -92 2 1 -78 to -42 B 274 edodes -216 to -214 Pycnoporus 1 -89 to -85 1 -339 to -336 1 -81 to -37 B AB1947805 coccineus Trametes -64 to -58 B AY081189 5 versicolor Ustilago maydis 1 -231 to -235 1 -341 to -345 - - B X07879 5 Mucor -74 to -77 1 -84 to -89 2 1 ? to -34 Z 268 circinelloides -158 to -161 1 # = number 2 Position relative to the ATG start codon 3 If an AT-rich stretch is present instead of a TATA box, its position is indicated 4 A = Ascomycota , B = Basidiomycota (for more information about gpd -promoter sequences of Basidiomycota , see also reference 279), Z = Zygomycota 5 GenBank accession numbers
Chapter III: Cloning and characterisation of the gpd -promoter 116
The positions of the TATA-boxes, CAAT-boxes and CT-rich sequences of some other fungal gpd -genes are given in Table III. 3-3. It can be deduced from the table that there is quite some variation in the position (and length) of these promoter characterising sequences, even among the genes of fungi belonging to the same phylum.
- The Kozak sequence: ACAAA ATG GC The translation initiation site in filamentous fungal genes usually occurs at the first ATG in the sequence and almost always has a purine (usually A) at the -3 position. Translation in eukaryotes often can be modulated by the sequences flanking the start codon. Kozak (201) worked out a consensus sequence for the ATG environment, based on 31 fungal genes: TCA(A/C)(A/C) ATG (G/T)C. Bruchez et al. (280) analysed 77 known nuclear genes of Neurospora crassa (transcribed by the RNA Polymerase II) and deduced the following
consensus sequence: C 57 NNNC 77 A81 (A 44 /C 43 )"T" 3A99 T100 G99 G51 C53 . In this sequence “T” stands for the conserved absence of a thymine and the subscript number represents the percentage of appearance of the nucleotide at that site. These sequences are presumably involved in recognition of the correct AUG by the ribosome. Comparison of the Kozak sequence to the M. gramineum sequence reveals a similarity of 90 %
The gpd -promoter was also investigated regarding the presence of some gpd -promoter characteristic sequences, such as the gpd -box and the pgk -box (136), but no similar sequences were found.
Although the gpd -promoter is generally considered to be a constitutive promoter, some gpd - genes are regulated, as described in the introduction of this chapter. Sequence analysis of the full-length promoter fragment of the M. gramineum gpd -gene was carried out using Motif Finder (a program based on the TRANSFAC Database (141)). Two putative heat shock elements (HSE) were found (consensus sequence AGAAN, nt 95-99 and 201-205). Similar elements were found in the promoter region of the Mucor circinelloides gpd1 -promoter (126). The presence of such motifs suggests that the expression of the gene might be under stress regulation, but, in this case, this is only a theoretical speculation. Larsen et al . (126) speculate that CATCAC elements in the promoter region of the Mucor circinelloides gpd1 -gene could be responsible for the down regulation of the gene in conditions of low hexose concentration. A CATCAC element was found in the gpd -promoter of M. gramineum at nucleotide 740 to 745.
3.3. Determination of the gpd copy number The gpd copy number in the genome of M. gramineum was determined. The probe was constructed based on the DNA sequence corresponding to a conserved region, as indicated in Figure III. 3-12. Genomic DNA from the wild type strain was digested with restriction enzymes which have one ( Eco NI, Acu I, Sac I and Xho II) or two ( Xho I) restriction sites in the gene. Eco NI and Acu I cut within the probe region, the other enzymes cut outside this region. As shown in Figure III. 3-13, digestion with enzymes which do not cut in the probe-sequence
Chapter III: Cloning and characterisation of the gpd -promoter 117 results in one single hybridisation signal, whereas two bands are observed in DNA digested with restriction enzymes which cut once in the probe. Digestion with a restriction enzyme that cuts two times outside the probe also resulted in one signal, of the expected length. These results indicate that a single copy of the gpd -gene is present in the genome of M. gramineum , thus it is plausible that the gpd isolated from M. gramineum is a functional gene.
Xho I Xho I Sac I Acu I Eco NI Xho II
5' UTR ATG c stop polyA 1 polyA 2 GPD probe
GV (3021 bp) Figure III. 3-12. Schematic representation of the probe used for Southern blot analysis of the genomic DNA from Myrothecium . ‘Stop’ represents the stopcodon. The ‘c’ illustrates a very conserved amino acid region. The coding sequence is indicated by a grey shaded pointer, the probe by an open one.
Figure III. 3-13. Results obtained after restriction of the genomic DNA with Eco NI, Xho I, Sac I, Acu I and Xho II
and hybridisation with the gpd -probe. The marker used
is the DNA Molecular-Weight Marker III, DIG-labelled (Roche).
II I
Xho Xho II I I NI I+ I+ Acu Marker Xho Sac Eco Xho Sac
3.4. Protein sequence analysis The glyceraldehyde-3-phosphate dehydrogenase is a tetrameric enzyme depending on NAD + as a cofactor (281, 282). The gene of M. gramineum encodes an enzyme of 339 amino acids, which is similar to other GPD enzymes (see also Table III. 3-1). The enzyme contains the recognition pattern for all known GPD enzymes (PROSITE), which is [ASV]-S-C-[NT]-T- x(2)-[LIM]. In this pattern, the C (cysteine) is essential for the activity of the enzyme. It functions as the binding site of the enzyme in the catalytic region (278). The GPD-enzyme of M. gramineum contains the pattern in the form of ASCTTNCL. Thus, the obtained sequence is certainly representing a GPD-enzyme (glyceraldehyde-3-phosphate dehydrogenase, GPD, EC 1.2.1.12). Other conserved amino acids are a histidine, mostly found at position 179 of the amino acid sequence and two phosphate binding residues, lysine and arginine (position 191 and 231) (272). The histidine residue was found at the expected position, while the lysine and arginine residues are located at positions 194 and 234, respectively.
Chapter III: Cloning and characterisation of the gpd -promoter 118
The amino acid sequence was blasted against the UNIPROT database (Release 5.0). The WU- BLASTp (v2.0) program on the EBI-website was used with the standard parameters. The sequence shows most similarity with the GPD-enzymes of Beauveria bassiana (95 %), Colletotrichum gloeosporioides (94 %), Colletotrichum lindemuthianum (94 %), Neurospora crassa (94 %), Sordaria macrosporia (94 %), Metarhizium anisopliae (93 %) and Hypocrea jecorina (92 %). The amino acid sequence was also blasted against the CDD database (conserved domain database) using the CDART-tool (Conserved Domain Architecture Retrieval Tool) with the standard parameters (283). The results indicated that the sequence shows homology with protein families ‘pfam02800’ (Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain) and ‘pfam00044’ (Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain). ‘Pfam’ is a database of protein families that currently contains 7973 entries (release 18.0) (284). According to these results, the NAD binding site is situated from amino acid 3 to 152.
A phenomenon which is often seen in genes of filamentous fungi is codon bias (133). This results from the fact that 61 codons code for 20 amino acids and thus more than one codon can code for the same amino acid. Codon bias occurs when there is a clear preference for one or more codons of a certain amino acid. Codon bias in gpd -genes is normally high, because the genes are highly expressed (278). For example, the codon usage in the gpd1 and gpd2 genes of the zygomycete Mucor circinelloides is highly biased with 83 and 81 % pyrimidines at the third position, respectively, and 21 unused codons in both genes (268). In the gpd1 of the ascomycete Cochliobolus heterostrophus , 77 % of all codons have a pyrimidine at the third position and when a choice between a purine or a pyrimidine is allowed, a pyrimidine is chosen in 93 % of the cases (272). A strong preference for a pyrimidine in the third position was also observed in the gpd -gene of the basidiomycete Ganoderma lucidum (278).
The codon use in the gpd -gene of M. gramineum was examined (Table III. 3-4). Analysis shows that 80.9 % of all codons have a pyrimidine at the third position and when a choice between a purine or a pyrimidine is allowed, a pyrimidine is chosen in 97.2 % of the cases. This proves again codon that usage in gpd -genes is highly biased. This is an indication of the fact that this gene is highly expressed and it implies that the promoter of this gene is a strong one.
A parameter which is often used to determine the codon bias in a certain gene is the ‘ effective number of codons ’ (Nc) (285). Nc indicates at what account all 61 codons are used in a gene. Nc is about 20 (minimum) in a highly biased gene, while it comes close to 61 in genes without bias. The effective number of codons Nc was calculated for the gpd -gene of M. gramineum with the EMBOSS-program CHIPS to confirm the results described above. This resulted in an Nc = 27.73. Since this value approaches 20, it confirms the high bias in gpd - genes.
Chapter III: Cloning and characterisation of the gpd -promoter 119
Table III. 3-4. Analysis of the codon usage in the gpd -gene of M. gramineum (340 codons)
AA 1 Codon % AA 1 Codon % AA 1 Codon % AA 1 Codon %
Ala GCG 0 His CAT 0 Pro CCG 0 Ser AGT 0 GCA 0 CAC 100 CCA 0 AGC 22 GCT 26 Ile ATA 0 CCT 8 TCG 0 GCC 74 ATT 30 CCC 92 TCA 0 Cys TGT 0 ATC 70 Gln CAG 100 TCT 17 TGC 100 Lys AAG 100 CAA 0 TCC 61 Asp GAT 28 AAA 0 Arg AGG 0 Val GTG 0 GAC 72 Leu TTG 0 AGA 0 GTA 0 Glu GAG 100 TTA 0 CGG 0 GTT 18 GAA 0 CTG 35 CGA 0 GTC 82 Phe TTT 11 CTA 0 CGT 55 Trp TGG 100 TTC 89 CTT 0 CGC 45 Tyr TAT 8 Gly GGG 0 CTC 65 Thr ACG 0 TAC 92 GGA 0 Met ATG 100 ACA 0 STOP TGA 0 GGT 59 Asn AAT 0 ACT 22 TAG 0 GGC 41 AAC 100 ACC 78 TAA 100 1AA = amino acid
3.5. Phylogenetic analysis Alignments of the amino-acid sequences of the GPD enzymes of various fungi reveal long stretches of conserved amino acids, altered with differing nucleotides (265). This makes these proteins suitable for phylogenetic studies. (286). A phylogenetic tree was constructed based on fungal GPD enzymes available at the NCBI website. Only full length GPD genes were used and identical sequences of the same species were removed. The phylogenetic tree was constructed using the Maximum Likelihood method with the BioEdit software (v7.0.4.1; 287). The Jones-Taylor-Thornton model was used as evolutionary model for amino acid substitution. An unrooted tree with a Log Likelihood of -21047.48 was obtained. All nods were statistically significant (P < 0.05). The tree was rooted using the TreeIllustrator software (v0.5 beta, 288, 289). The human GPD enzyme was used as outgroup. The distances along the branches between GPD enzymes of different species are proportional to the calculated evolutionary distance between their amino acid sequences. So, the tree gives an indication of the evolution of the species. Based on its GPD sequence, M. gramineum is closely placed to other Sordariomycetes , as expected. The M. gramineum GPD fits into the pattern reported by Puyeski (269 and references therein) in which the GPD sequences of filamentous Ascomycota are closer to other filamentous fungi than to ascomycetous yeasts, or put otherwise: the tree reveals a separation of the filamentous fungi and the unicellular Ascomycota before the separation into Basidiomycota and Ascomycota (265, 276, 286). The closer relationship of the Zygomycota to the ascomycetous yeasts and the Basidiomycota than to the filamentous Ascomycota is confirmed (268).
Chapter III: Cloning and characterisation of the gpd -promoter 120
The obtained tree (Figure III. 3-15 2) corresponds well with the taxonomic classification of the fungi. As expected, the four classes of yeasts ( Saccharomycetes ), filamentous Ascomycetes , Basidiomycetes and Zygomycetes fall into four groups (268, 276, 278). Clusters containing species currently classified in the same families (subclasses, classes and phyla) are usually well supported, as, for example, also observed by Barbosa et al . (290). Exceptions are Ashbya gossypii , Candida albicans , Candida glabrata , Neosartorya fischeri , Aspergillus clavatus , Cordyceps bassiana , Gibberella zeae , Hypocrea koningii clone pT56, Lyophyllum shimeji , Armillariella tabescens and Coprinopsis cinerea.
Ashbya gossypii belongs to the family of the Eremotheaceae , but is clustered here within the Saccharomycetaceae . Both Candida species belong to the family of the Incertae sedis but are here also placed within the Saccharomycetaceae. Clone pT56 encodes for the GAPDH II of Hypocrea koningii , which is an unusual GPD as it is resistant to the mycotoxin koningic acid. Other phylogenetic analyses also proved this GPD is distant from other fungal sequences (264, 269). Lyophyllum shimeji belongs to the family of the Tricholamataceae , but is grouped here with the Pluteaceae . This result was also obtained by Kilaru and Kües (279), who constructed a phylogenetic tree with GPD enzymes of Basidiomycota . The position of the Agaricus bisporus GPD 2 and the Hypocrea koningii GAPDH II in the tree corresponds well to the results obtained in the phylogenetic analysis performed by Verdoes et al . (275).
3.6. Construction of expression vectors with the M. gramineum gpd -promoter The promoter of the gpd -gene was cloned into pGEM-T® in order to create an expression vector. The reverse primer used for the amplification of the promoter was extended with unique restriction sites, which allow cloning a coding sequence of interest in frame with the natural ATG codon of the gpd -gene. A schematic view of this multicloning site is given in Figure III. 3-14. Two vectors were created, one containing a shorter 581 bp fragment and the other containing a longer 1033 bp fragment of the gpd -promoter. Depending on the expression levels of the A. oryzae amylase gene obtained with both fragments, it will be decided which fragment will be used in further experiments (see also Chapter IV) (264). Bbu I Bst N SI N sp I Eco T22I Pae I M ph 1103I Spa H I N si I Sph I Zsp 2I Xce I
startcodon C A A A A T G C A T G C Figure III. 3-14. Schematic view of the multicloning site surrounding the natural ATG codon of the gpd -gene in the expression vectors created with its promoter.
2 Classification based on the Ainsworth and Bisby's Dictionary of the Fungi, 9 th edition, available at www.indexfungorum.org/Names/fundic.asp (June, 7 th , 2007)
Chaptercharacterisation III: and Cloning of the Figure III. 3-15 . Phylogenetic analysis of GPD sequences using the Maximum Likelihood method with human GPD as outgroup. Phylogenetic tree of 95 fungi and yeast derived from the amino acid sequences of their GPD enzymes. Family names are indicated at the right side of the tree. The genera which they include are correspondingly coloured in the tree. Subclasses are boxed, classes and phyla are indicated second most left and most left, respectively. Species indicated in gpd black lettering were expected at -promoter other places in the tree, based on their current classification.
121
Chapter III: Cloning and characterisation of the gpd -promoter 122
The forward primers used to clone the promoter in the expression vectors also contain short multicloning sites, in order to allow their isolation from the vectors in further experiments. The primer for the long fragment contains restriction sites for AatII, Sal I (both sticky) and Zra I (blunt), while the primer for the shorter fragment has the sites for restriction with Aat II, Acc I, Sal I (all three sticky) and Hinc II and Zra I (both blunt).
High Fidelity PCRs on the plasmid pGV with the primers “5’ long GPD” and “3’ prom GPD” and “5’ short GPD” and “3’ prom GPD” resulted in fragments of 1033 bp and 581 bp, respectively (Figure III. 3-16). The fragments were ligated into pGEMT ®, the ligation mixtures transformed in E. coli DH5 α. The plasmids of five colonies obtained with the long promoter fragment were controlled by restriction analysis with Alw NI. If the correct plasmids are present, fragments of 1063 bp and 2972 bp (pGEM-T® + 1033 bp) or of 2332 bp and 1703 bp (inverted pGEM-T® + 1033 bp) are expected.
M p p l
k lp col 1 lp col 2 lp col 3 lp col 4 lp col 5 1033bp 2332bp 1703bp
581bp
marker
marker Figure III. 3-16. Result High Fidelity PCR for the long promoter fragment (lp) and the short one (kp) and the control of the restriction of the plasmids of 5 colonies with the long fragment.
Colony 3 was chosen to use for further experiments and its plasmids will further be referred to as ‘pGPDlp’ (Figure III. 3-17). Sequence analysis at the VIB Genetic sequence facility revealed that the vector had the expected sequence, including the multicloning site.
Zra I Zsp 2I Zsp 2I Nsi I Nsi I Nco I Mph 1103I Mph 1103I Bst NSI Eco T22I Nsp I Eco T22I Sap I Nsp I Bst NSI Eco T22I Mph 1103I gpdKP Bbu I Pae I Nsi I gpdLP Sph I Zsp 2I Bbu I MCS Pae I Eco T22I Sph I Mph 1103I Nsi I pGPDlp Zsp 2I pGPDkp gpd'ATG Bbu I 4032 bps Nco I Bst NSI 3583 bps Bbu I Nsp I f1 Bst NSIPae I Nsp I Sph I Pae I f1 Sph I Zra I AmpR AmpR Bsa I
Figure III. 3-17. Molecular map of pGPDlp and pGPDkp.
Chapter III: Cloning and characterisation of the gpd -promoter 123
The same strategy was followed for the plasmids of ol 10 ol
the colonies with the short fragment. Fragments of c
marker
® kp 1col kp 2col kp 3col kp 4col kp 5col kp 6col kp 7col kp 8col kp 9col 1063 bp and 2535 bp (pGEM-T + 581 bp) or of kp 2332 bp and 1251 bp (inverted pGEM-T® + 2332bp 581 bp) were expected. After restriction analysis of the plasmids of 10 colonies (Figure III. 3-18), 1251bp colony 7 was picked out. Its plasmid will be further referred to as ‘pGPDkp’ (Figure III. 3-17).
Figure III. 3-18. Control of the colonies with pGEM-T® + short promoter with Alw NI.
4. CONCLUSIONS The complete sequence of the glyceraldehyde-3-phosphate dehydrogenase gene of Myrothecium gramineum was determined and submitted to the Genbank ( EF486690 ). The gene was cloned into the pGEM-T vector, resulting in the plasmid pGV. In the promoter region, two TATA-boxes, four CAAT-similar sequences and five CT-rich stretches are found. The coding region contains two introns, the first of which is situated at a position conserved within gpd -genes of Ascomycota . Two polyA-signals are each followed by a transcription termination site.
The 3041 bp gene encodes an enzyme of 339 amino acids. The amino acid sequence shows most similarity with the GPD-enzymes of Beauveria bassiana (95 %), Neurospora crassa (94 %), Colletotrichum lindemuthianum (94 %), Colletotrichum gloeosporioides (94 %), Sordaria macrosporia, (94 %), Metarhizium anisopliae (93 %) and Hypocrea jecorina (92 %). Phylogenetic analysis based on the amino acid sequence of the M. gramineum GPD confirmed the current classification of M. gramineum within the class of the Sordariomycetes .
Analysis shows that 80.9 % of all codons have a pyrimidine at the third position and when a choice between a purine or a pyrimidine is allowed, a pyrimidine is chosen in 97.2 % of the cases. This proves that the codon usage in this gpd -gene is highly biased. It is an indication for the fact that this gene is highly expressed and it implies the promoter of this gene is a strong one. Only one copy of the gene is present in the genome of Myrothecium gramineum .
Two expression vectors were created containing a short (581 bp) and a long (1033 bp) fragment of the M. gramineum gpd -promoter sequence followed by a multicloning site. The vectors are called ‘pGPDkp’ and ‘pGPDlp’, respectively.
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 124
Chapter IV
Expression of an Aspergillus oryzae amylase gene ( amy3 ) in Myrothecium gramineum
1. INTRODUCTION Filamentous fungi have found application in many technologies, such as the production of enzymes and peptides (homologous and heterologous), antibiotics, organic acids, polysaccharides, foods and beverages, etc. The attraction of the filamentous fungi as hosts for enzyme production are built upon the capacity to genetically transform an increasing number of different species, the well known abilities of some of these species to secrete substantial quantities of proteins, metabolites and organic acids, their growth on cheap media under well documented conditions and their eukaryotic characteristics (2, 14, 16, 20). The range of commercially available enzymes is broad and they are produced by a number of different species, such as Aspergillus species, Penicillium species and Trichoderma species.
In order to explore its capabilities to produce (heterologous) enzymes, Myrothecium gramineum , which has been developed as a new fungal expression host during our research, was tested as to its production of an α-amylase of A. oryzae . α-Amylases constitute an important class of enzymes which find many biotechnological applications in processes which involve, for example, the degradation of starch and the determination of soluble and insoluble dietary fiber in rice and wheat bran. Such applications include baking, brewing, detergents and desizing (in textile industries) (291). In baking, for example, α-amylases are used because they increase the bread volume, because they improve the crumb grain, crust, and crumb colour, and for their flavour development promoted in the final product (292). The α-amylase is an endo-enzyme that randomly hydrolyses the α-1,4 glucosidic linkages in polysaccharides. Some fungal systems expressing the α-amylase of A. oryzae are given in Table IV. 1-1. Table IV. 1-1. TAKA-amylase production by fungal expression systems
Production organism Expression signal Copy number Yield 1 Reference A. oryzae amy3 ( A. oryzae ) ? 49000 U/g 216 amy3 ( A. oryzae ) ? 12 g/L (F) 293 A. awamori amy3 ( A. oryzae ) >1 10.3 U/g 216 T. viride amy3 ( A. oryzae ) >1 4 mg/L 216 cbhI ( T. reesei ) >1 1 g/L 294 A. nidulans amy3 ( A. oryzae ) ? 2070 U/g 216 1 Yields are indicated as the amount of protein (g or mg) per L of culture fluid or as the enzyme activity (units) per gram dry weight mycelia. If no further indications are given, production was determined in shake-flask cultures, when ‘F’ follows the yield, the production was performed in a controlled fermentation process.
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 125
The use of strong promoters for the expression of (heterologous) proteins in suitable host organisms is of great importance for biotechnological applications. Several strong promoters involved in primary and secondary metabolism are now available. These include, among others, the promoters of the glyceraldehyde-3-phosphate dehydrogenase gene ( gpdA ) of A. nidulans , the glutamate dehydrogenase gene ( gdhA ) of A. awamori , the isopenicillin N- synthase gene ( pcbC ) of Penicillium chrysogenum and the B2 wide-spectrum esterase gene (cesB ) of Acremonium chrysogenum (150). The most frequently used constitutive promoter is the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA ) promoter of A. nidulans . This promoter is functional in different species, including industrially important Penicillium and Aspergillus species. The glyceraldehyde-3-phosphate dehydrogenase (GPD; EC 1.2.1.12) promoter is a promising candidate (39) because in many eukaryotic microorganisms, the GDP genes are expressed constitutively and in large amounts.
Gpd promoters have been successfully used for the expression of enzymes in filamentous fungi (147, 148). For example, the gpdA promoter of A. nidulans has been used as a homologous promoter for the expression of an E. coli aspartase (151) and a synthetic elastomer in A. nidulans (152), as a heterologous promoter for the expression of the A. niger glucoamylase (149) and for the plant thaumatin in A. awamori (150), of the barley α-amylase (147) and chicken lysozyme (155) in A. niger and for the E. coli β-galactosidase (156) in P. chrysogenum . The A. niger gpd -promoter has been successfully used as a homologous promoter for the expression of a H. jecorina xylanase (153) and a laccase (154).
Because homologous promoters are generally more efficient than heterologous ones for the expression of enzymes and proteins (14, 77, 150), the gpd -promoter of M. gramineum was isolated (chapter III) and characterised (GenBank accession number EF486690 ). In this chapter, the use of this gpd -promoter for the expression of an A. oryzae α-amylase is investigated (TAKA-amylase A, EC 3.2.1.1., encoded by amy3 ).
2. MATERIALS AND METHODS
2.1. Strains, plasmids and growth conditions Myrothecium gramineum BCCM TM /MUCL 39210 was used as the wild type strain. Escherichia coli JM109 or DH5 α were used for plasmid amplification.
Plasmid pCSN43 contains the Escherichia coli hygromycin B resistance gene ( hph ) (see § 2.1 of Chapter I). The hygromycin B resistance gene was used as a selection marker when transforming M. gramineum . Plasmid p2G-S has 2 copies of the A. oryzae Taka-amylase gene (amy3 , under the transcriptional control the promoter of the gpdA -gene of A. nidulans and its own terminator) (29). This plasmid was used to isolate the A. oryzae Taka-amylase gene (amy3 , Genbank accession number X12727 ). Plasmids pGPDkp and pGPDlp (chapter III) were used as expression vectors. The A. oryzae amylase gene was ligated into these plasmids, resulting in the vectors ‘pGPDkpAmyAO’ and ‘pGPDlpAmyAO’, respectively.
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 126
Standard cultivation techniques were performed as described in § 2.2.1 of Chapter I. After transformation, the protoplasts were inoculated on PDAG (PDA + 1 M glucose) and grown for 2 hours. Thereafter, a top layer of 8 mL PD + 0.8 % agar + 0.5 g/L hygromycin was poured over the plates, which were further incubated until growth appeared. M. gramineum was grown in 100 mL AMM + 3% sucrose for amylase assays (final concentration of 2.10 6 spores/mL). AMM was used as described by Barrat et al. (231), supplemented with 5 g/L yeast extract (Difco). Liquid cultures were grown at 25°C and 175 rpm.
2.2. Standard DNA manipulation Routine recombinant DNA methodology was performed as described in § 2.2 of Chapter III. The ‘InFusion Cloning Kit’ of Clontech was used to ligate the A. oryzae amylase into the vectors pGPDlp and pGPDkp. Plasmids were concentrated prior to the transformation of M. gramineum according to the procedure described in § 2.3 of chapter I. Table IV. 2-1. Primers used for the construction of the vectors pGPDkpAmyAO and pGPDlpAmyAO and for the control of M. gramineum transformants Primer name Feature Primer sequence (5’-3’) 2 AmyAO 5 amplification amy3 A. oryzae gctcttcc-ATGGTCGCGTGGTGGTCTCTAT AmyAO 3bis amplification amy3 A. oryzae acagcggaagagc-GCAACCACCAGGTCAAAC F IF AmyAO forward InFusion primer gaaattcacaaaatgatgGTCGCGTGGTGG R IF AmyAO reverse InFusion primer ttgggcccgacgtCGGCAACCACCAGGTCAAAC hphF control hph in tr. ATGCCTGAACTCACCGCGACG hpfR control hph in tr. CTATTCCTTTGCCCTCGG 5’ short GPD forward control gpd-amy in tr. gacgtc-GACGCAGTGGACAGACGTTA AMY reverse control gpd-amy in tr. GTCGATGATGCCCTGCC T7 control pGEM-T® ligation GGCGATTAAGTTGGGTAACG SP6 control pGEM-T® ligation CGCCAAGCTATTTAGGTGAC 1 tr. = transformants 2 Matching sequences are indicated with capitals, non-matching sequences are printed in small-type lettering
2.3. Transformation of M. gramineum and control of transformants M. gramineum transformations were performed as described in § 2.4 of chapter I. Amylase enzyme tests and cell dry weight measurements were performed according to the procedure described in § 2.5 of chapter I. The extracellular protein concentration was measured with the Advanced Protein Kit (Sigma), according to the procedure described by the producer. The concentration of sucrose, glucose and fructose was determined by HPLC analysis (ELSD- detector).
PCR analysis of the transformants was performed as described in chapter II (method 10). The reaction mix consisted of 5 µL Taq -polymerase buffer (Roche Diagnostics), 2 µL primers (10 pmol/µL, Sigma Genosys), 1 µL nucleotides (10 mM, Sigma), 0.2 µL Taq -polymerase (5 u/µL, Roche Diagnostics) and mQ-water (up to 50 µL). One primer pair (primers ‘hphF’ and ‘hphR’) is based on the hph -gene of the plasmid pCSN43 (not present in the wild type). The second primer pair (primers ‘5’ short GPD’ and ‘AMY’) is based on the A. oryzae amy3 gene
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 127 preceded by the M. gramineum gpd -promoter. One primer anneals to the gene, the other to the promoter, forming a combination not present in the wild type genome. A first denaturation step of 4 minutes at 94°C was followed by 25 cycles of 30 s denaturation at 94°C, 30 s annealing at 55 °C and 1.5 minutes elongation at 72°C. The PCR was completed with a final extension of 7 minutes at 72°C.
3. RESULTS AND DISCUSSION
3.1. Cloning of the A. oryzae amy3 gene into pGEM-T®
Sph I A High Fidelity PCR with the primers ‘AmyAO 5’ Sap I Sph I and ‘AmyAO 3bis’ was performed using the plasmid pGPD-AMY as sample material. A pA fragment of 2584 bp was obtained, as expected. f1 This fragment was cloned into pGEM-T®. The sequence of the amy3 gene of this plasmid was
AmpR pAMYAObis confirmed at the VIB Genetic Service Facility. It 5550 bps AmyAO will further be referred to as ‘pAmyAObis’ (Figure IV. 3-1). pAmyAObis contains the DNA sequence encoding the mature A. oryzae TAKA-amylase, preceded by its natural secretion signal (20 AA) and secsig followed by its own transcription termination signals (± 500 bp). Sap I Sap I
Figure IV. 3-1. Molecular map of
pAmyAObis.
3.2. Construction of pGPDkpAmyAO and pGPDlpAmyAO The ‘InFusion Cloning Kit’ of Clontech (Figure IV. 3-2, (after 295)) was used to ligate the A. oryzae amylase into the vectors pGPDlp and pGPDkp. This kit is designed for the cloning of PCR products without the need for compatible restriction fragments, ligase or blunt-end polishing. Fragments that have at least 15 bases of homology with sequences flanking the desired site of insertion in the cloning vector are combined with the linearised cloning vector in the InFusion cloning reaction. The InFusion Enzyme ‘ligates’ the two fragments in the correct orientation and the right frame.
The vectors pGPDlp and pGPDkp were cut with Sp HI (Figure IV. 3-3, right side). Sp HI is one of the restriction enzymes of the multicloning site at the 3’ end of the gpd -promoter on the plasmids pGPDlp and pGPDkp. Fusion with the InFusion Enzyme at this site results in an in frame ligation of the start codon of gpd with the (coding) sequence of the A. oryzae amylase gene (natural secretion signal, DNA sequence encoding mature A. oryzae TAKA-amylase, natural transcription termination signals).
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 128
Figure IV. 3-2. The InFusion cloning method.
The amylase gene was PCR amplified with the BD Advantage 2 Polymerase Mix using the primers ‘F IF AmyAO’ and ‘R IF AmyAO’ (Table IV. 2-1). A product of 2558 bp is obtained (Figure IV. 3-3, left side), which comprises the coding sequence (including the secretion signal) of the amy3 gene and its 3’ flanking sequences (transcription termination site and poly adenylation signal) (Figure IV. 3-4).
The linearised vector fragments and the PCR product were gel purified, their concentration was determined and they were mixed according to the indications given in the InFusion manual. The ‘ligation’ mixtures were transformed into BD Fusion-Blue™ Competent cells, as described in the kit.
Lane M: Smartladder TM 1 M 2 3
Lane 1: PCR AmyAOIF
Lane 2: pGPDkp cut with Sp HI
Lane 3: pGPDlp cut with Sp HI
Figure IV. 3-3. PCR amplification of the amy3 gene (coding sequence and 3’ UTR) and restriction of pGPDkp and pGPDlp with SpH I.
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 129
e1 e2 e3 e4 e5 e6 e7 e8 e9 3' flanking sequences
secretion signal
amy3
Figure IV. 3-4. Fragment of the amy3 gene used for the construction of pGPDlp/kpAmyAO (coding sequence (pointer) and 3’ flanking sequences (grey box)). Exons are indicated with an open arrow (e).
The plasmids of ten colonies of both constructs were controlled by restriction analysis with Bsp HI (pGPDkpAmyAO) or with Sp HI (pGPDlpAmyAO). Three fragments are expected after restriction of pGPDkpAmyAO with Bsp HI, i.e. 2725 bp, 2329 bp and 1008bp, while one fragment of 6514 bp is expected after restriction of pGPDlpAmyAO with Sp HI (Figure IV. 3-5). Colony 1 of ligation for GPDlpAmyAO and colony 3 of the ligation for pGPDkpAmyAO were chosen for further use, and their plasmids are further referred to as ‘pGPDlpAmyAO’ and ‘pGPDkpAmyAO’, respectively (Figure IV. 3-6). Their sequence was confirmed by sequencing at the VIB Genetic Service Facility.
Lane M: Smartladder TM 1 2 3 4 5 6 M 7 8 9 10 11 6514 bp Lane 1 to 10: colonies 1 to 10 for the 2725 bp construction of pGPDlpAmyAO ( Sp HI) 2329 bp Lane 11: colony 3 for the construction of
1008 bp pGPDkpAmyAO ( Bsp HI)
Figure IV. 3-5. Control of the plasmids for the construction of pGPDlpAmyAO and pGPDkpAmyAO.
Psp OMI Ava I
gpdKP gpdLP Psp OMI sec
sec
pGPDlpAmyAO pGPDkpAmyAO AmpR AmyAO 6511 bps AmpR 6063 bps Ava I AmyAO
f1 f1 Ava I 3' UTR 3' UTR
Psp OMI Figure IV. 3-6. Molecular map of pGPDlpAmyAO and pGPDkpAmyAO.
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 130
3.3. Transformation of Myrothecium gramineum with pGPDkpAmyAO and pGPDlpAmyAO and analysis of the transformants The plasmids pGPDkpAmyAO and pGPDlpAmyAO were used for the transformation of Myrothecium gramineum , in order to test the strength of the promoter and to compare the strength of the longer fragment to that of the shorter one. The hygromycin resistance gene hph on the plasmid pCSN43, which was co-transformed, was used as the selection marker. The ratio of pGPDAmyAO/pCSN43 was about 1.5.
After 7 days, 8 colonies were obtained which were transformed with the vector pGPDlpAmyAO (referred to as ‘LA’) and 2 colonies grew on the plates of the protoplasts transformed with pGPDkpAmyAO (referred to as ‘KA’). The colonies were transferred to PDA + hygromycin for a second round of selection. All of them grew further, which proves they had stably integrated the hygromycin resistance gene. The colonies were subjected to colony PCR (Figure IV. 3-7). One PCR was performed with the primer pair primers ‘hphF’ and ‘hphR’, amplifying a fragment of 1 kb of the hph -gene (Table IV. 2-1). The second primer pair (primers ‘5’ short GPD’ and ‘AMY’) is based on the A. oryzae amy3 gene preceded by the M. gramineum gpd -promoter (Table IV. 2-1). A fragment of 818 bp is expected with these primers. All PCRs with the hygromycin primers resulted in the expected fragment, which confirms the 10 transformants carry at least one (functional) copy of the hph - gene (results not shown for LA 8). Additional bands could have resulted from rearrangements of the pCSN43 plasmid upon integration. The PCRs with the primer pair based on the construct of the M. gramineum gpd -promoter followed by the A. oryzae amylase resulted in a fragment of the expected size for the colonies LA 2, LA 4, LA 5, LA 6, LA 7 and KA 2, meaning they also carry at least one copy of this construct. Again, additional bands could have resulted from rearrangements of the plasmids upon integration or from mis-priming. For colonies LA 1, LA 3, LA 8 and KA 1 no products were amplified, not even after a second attempt of colony PCR and reamplification of these PCRs. This could indicate that these colonies have only integrated the plasmid pCSN43, rendering them hygromycin resistance, but not plasmid pGPDlpAmyAO or pGPDkpAmyAO.
A
A
LA 1 H LA 2 H LA 3 H LA 4 H LA 5 H LA 6 H LA 7 H KA 1 H KA 2 H Marker LA 1 A LA 2 A r LA 2 A LA 3 A LA 4 A r LA 5 LA 5 Marker LA 6 A r LA 6 A LA 7 A LA 8 A KA 1 A KA 2 A r KA 2 A
1 kb 0.8 kb
Figure IV. 3-7. Colony PCR with the hph primer pair (H) and the primer pair based on the gpd-amy construct (A). (r = reamplification).
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 131
The 8 colonies with pGPDlpAmyAO and the 2 colonies with pGPDkpAmyAO were inoculated in 100ml AMM + sucrose (0.5.10 6 spores/mL) in order to test their amylase production. The amylase production of the cultures was followed during 252 hours. The result of this test is given in Figure IV. 3-8. In these graphs, the background amylase production was neglected. The maximum production obtained for each strain was: 1.68 units for KA 1, 2.6 units for LA 1, 81.25 units for LA 2, 2.93 units for LA 3, 57.35 units for LA 4, 19.94 units for LA 5, 162.74 units for LA 6, 4.06 units for LA 7 and 0.63 units for LA 8. After 110 hours of incubation, a contamination occurred in the culture of transformant KA 2 and this culture was not further tested.
These preliminary results indicate that transformants LA 2, LA 4, LA 5, LA 6 and LA 7 (carrying the GPDlpAmyAO construct) produce more amylase (4 units/mL to 153 units/mL) than the wild type strain, which produces maximally 2.51 ± 0.52 units amylase per mL. These results confirm the ones obtained by colony PCR: colonies LA 2, LA 4, LA 5, LA 6 and LA 7 carry at least one functional copy of the GPDlpAmyAO construct, while colonies LA 1, LA 3 and LA 8 do not. The transformant KA 1 with the short fragment of the promoter followed by the amylase gene (GPDkpAmyAO construct) produces similar levels of amylase as the wild type. This also is in accordance with the PCR results, which could not confirm the presence of a copy of the construct in the genome of this strain.
180 1,8 160 1,6 140 1,4 120 1,2 100 1 80 0,8 60 0,6
Units amylase/mL Units amylase/mL 40 0,4 20 0,2 0 0 0 50 100 150 200 250 0 50 100 150 200 250 Time (h) Time (h) LA1 LA2 LA3 LA4 LA5 LA6 LA7 LA8 KA1 KA2 Figure IV. 3-8. Amylase production by the transformants with the GPDlpAmyAO construct (LA, left side) and with the GPDkpAmyAO construct (KA, right side).
In order to confirm the results of the enzyme tests, new cultures were set up with transformants LA 2, LA 4, LA 5, LA 6, KA 1 and KA 2. In addition, the wild type strain and transformant 114 (chapter II) were inoculated in 100 mL AMM + 3% sucrose. Transformant 114 is the highest amylase producer obtained after transformation of M. gramineum with plasmid p2G-S, containing the Aspergillus nidulans gpdA -promoter followed by the amylase of A. oryzae . All cultures were inoculated such that a final concentration of 0.5.10 6 spores/mL was obtained. The cultures were followed during 305 hours, during which the amylase production, the cell dry weight, the extracellular protein concentration, the glucose concentration, the sucrose concentration, the fructose concentration and the pH were followed. The initial glucose concentration was 10 g/L and the one of sucrose 30 g/L. The
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 132 initial pH of the cultures was about 6.3. The results of this experiment are presented in Figures IV. 3-9 to IV. 3-16.
For all strains, it can be observed that during growth the pH increases from the initial pH of about 6.3 to 9. The cell dry weight increases to about 20 to 22 g/L after 100 to 140 hours of incubation. These facts, taken together with the fact that all cultures grew under the form of tiny pellets, indicate that the growth was comparable for all strains. Generally speaking, glucose and sucrose are consumed together. In the cultures of the wild type strain and the transformants 114 and KA 2, the fructose accumulates until glucose concentrations drop below the detection limit.
In this experiment, the wild type strain reaches its maximum amylase production of 1.37 units/mL after 233 hours of culture. The units of amylase production per mL indicated in the figures for the transformants represent the value measured for the transformants reduced with the background amylase production of the wild type at corresponding times of incubation. All transformants reach their maximum production after 210 or 233 hours of growth, which corresponds with the trend observed in the wild type. The maximum units of recombinant amylase produced by the transformants are given in Table IV. 3-1.
300 35 300 35 30 250 30 250 25 25 200 200 20 20 150 150
proteins 15
proteins 15 100 100 10 10 Amylase, extracellular pH, CDW, glu, fru, suc Amylase, extracellulair pH, CDW, glu, fru, suc 50 50 5 5 0 0 0 0 18 18 117 136 210 233 42,5 68,5 93,5 117 136 210 233 42,5 68,5 93,5 162,5 186,5 256,5 304,5 162,5 186,5 256,5 304,5 Time (h) Time (h)
Extracellular proteins (mg/L) Amylase production (units/mL) Extracellular proteins (mg/L) Amylase production (units/mL) CDW (g/L) pH CDW (g/L) pH Glucose (g/L) Sucrose (g/L) Glucose (g/L) Sucrose (g/L) Fructose (g/L) Fructose (g/L) Figure III. 3-9. Wild type strain. Figure III. 3-10. Transformant 114. 450 35 300 35 400 30 250 30 350 25 25 300 200 250 20 20 150 200 proteins
proteins 15 15 150 100 10 10 Amylase, extracellulair Amylase, extracellulair pH, CDW, glu, fru, suc 100 pH, CDW, glu, fru, suc 50 5 50 5 0 0 0 0 18 18 117 136 210 233 117 136 210 233 42,5 68,5 93,5 42,5 68,5 93,5 162,5 186,5 256,5 304,5 162,5 186,5 256,5 304,5 Time (h) Time (h)
Extracellular proteins (mg/L) Amylase production (units/mL) Extracellular proteins (mg/L) Amylase production (units/mL) CDW (g/L) pH CDW (g/L) pH Glucose (g/L) Sucrose (g/L) Glucose (g/L) Sucrose (g/L) Fructose (g/L) Fructose (g/L) Figure IV. 3-11. Transformant KA 1. Figure IV. 3-12. Transformant KA 2.
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 133
250 35 600 35 30 550 200 500 30 25 450 25 400 150 20 350 20 300
proteins 100 15 proteins 250 15 10 200 10 Amylase, extracellulair50 pH, CDW , glu, fru, suc 150 Amylase, extracellulair pH, CDW, glu, fru, suc 5 100 5 50 0 0 0 0
1 8 18 1 1 7 1 3 6 2 1 0 2 3 3 42 ,5 68 ,5 93 ,5 117 136 210 233 42,5 68,5 93,5 1 62 ,5 1 86 ,5 2 56 ,5 3 04 ,5 162,5 186,5 256,5 304,5 Time (h) Time (h)
Extracellular proteins (mg/L) Amylase production (units/mL) Extracellular proteins (mg/L) Amylase production (units/mL) CDW (g/L) pH CDW (g/L) pH Glucose (g/L) Sucrose (g/L) Glucose (g/L) Sucrose (g/L) Fructose (g/L) Fructose (g/L) Figure IV. 3-13. Transformant LA 2. Figure IV. 3-14. Transformant LA 4.
300 35 300 35
250 30 250 30 25 25 200 200 20 20 150 150
proteins 15
proteins 15 100 100 10 10 Amylase, extracellulair pH, CDW , glu, fru, suc Amylase, extracellulair pH, CDW, glu, fru, suc 50 50 5 5 0 0 0 0
8 5 5 5 7 6 3 7 0 3 7 5
, , ,
1 1 3 6 8 1 3 5 0 18
2 8 3
1 1 1 1 2 2 2 3 117 136 210 233
4 6 9 42,5 68,5 93,5
162,5 186,5 256,5 304,5 Time (h) Time (h)
Extracellular proteins (mg/L) Amylase production (units/mL) Extracellular proteins (mg/L) Amylase production (units/mL) CDW (g/L) pH CDW (g/L) pH Glucose (g/L) Sucrose (g/L) Glucose (g/L) Sucrose (g/L) Fructose (g/L) Fructose (g/L) Figure IV. 3-16. Transformant LA 5. Figure IV. 3-15. Transformant LA 6. Table IV. 3-1. Units of recombinant amylase produced by the transformants obtained by transformation with pGPDlpAmyAO (LA), pGPDkpAmyAO (KA) or p2G-S (114)
Strain Construct Units amylase/mL Units amylase/(mL.g CDW) 1, 2 KA 1 pGPDkpAmyAO 1.21 75.6 KA 2 pGPDkpAmyAO 3.51 195.0 LA 2 pGPDlpAmyAO 101.34 7238.6 LA 4 pGPDlpAmyAO 43.68 2426.7 LA 5 pGPDlpAmyAO 75.24 5374.3 LA 6 pGPDlpAmyAO 117.08 5854.0 114 p2G-S 38.01 2715.0 1 CDW= cell dry weight 2 For the wild type, a background production of 76.6 units/(mL.g CDW) or 1.37 units/mL was obtained
As indicated in Table IV. 3-1, the two transformants carrying the A. oryzae amylase under the control of the 581 bp fragment of the gpd -promoter of M. gramineum produce much fewer (Taka-) amylase than the transformants carrying the amy3 gene under the control of the 1033 bp fragment of the M. gramineum gpd -promoter or of the A. nidulans gpd -promoter (2 kb). It
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 134 has to be noted though, that two transformants are not enough to conclude that the 581 bp fragment is weaker as a promoter sequence than the 1033 bp fragment. Moreover, the presence of a copy of the GPDkpAmyAO construct in transformant KA 1 was not confirmed by PCR. According to the colony PCR, this construct indeed is present in the genome of transformant KA 2, resulting in recombinant amylase production, as proven by the enzyme tests (Table IV. 3-1).
When comparing the production levels obtained with the 1033 bp gpd -promoter sequence of M. gramineum with the gpd -promoter of A. nidulans , it can be concluded that with the homologous promoter higher amylase production is obtained than with the heterologous A. nidulans gpd -promoter. While with the A. nidulans promoter (transformant 114) a maximum of 2715 units amylase per mL and per gram CDW is obtained, transformants LA 2 and LA 6 produce 7238.6 and 5854.3 units per mL and per gram CDW. The results for the highest producers (LA 2 and LA 6) obtained in this experiment are in the same order of magnitude as obtained in the first test (117 units/mL and 162 units/mL for LA 6, respectively, and 101 units/mL and 81 units/mL for LA 2, respectively). The increase in production observed for strain LA 6 might be explained by the fact that more aerobic conditions were used this time (175 rpm instead of 140 rpm in the first experiment). A correlation between the extracellular enzyme production and the units of α-amylase can be observed for both transformants. Although the results are not obtained with single copy transformants having the constructs at a defined place in the genome, they confirm observations reported in literature (14, 77). For example, Moraleyo et al . (150) compared homologous and heterologous promoters to produce thaumatin in A. awamori and found that the best results are obtained with homologous promoters. Also in this report, no single copy strains with defined integration sites were studied.
Two samples of each culture were chosen to perform SDS-PAGE (Figure IV. 3-17). The first sample is the one taken after 18 hours of growth, when no amylase production was yet observed, in none of the strains. The second sample is the sample corresponding with the maximal amylase production. The ‘Low Molecular Weight Calibration Kit for SDS Electrophoresis’ (Amersham Biosciences) was used to estimate the size of the proteins. The Aspergillus oryzae amylase appears at 52 to 54 kDa in SDS-PAGE (BRENDA, 296), which is situated between the second and the third band of the marker (66 and 45 kDa, respectively). The protein concentration of the first sample of each strain was about 1 tenth of its corresponding maximal sample, which might be the reason why no bands are visible in the lanes of the first samples. The amount of protein loaded was similar for all samples with maximal amylase activity.
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 135
Marker WT 1 WT max 114 1 114 max LA 2 1 LA 2 max LA 6 1 LA 6 max Marker Marker LA 4 1 LA 4 max LA 5 1 LA 5 max KA 1 1 KA 1 max KA 2 1 KA 2 max Marker
Figure IV. 3-17. SDS-PAGE of the samples collected after 18 hours of culture (1) and at the time of maximal production (max) of the wild type (WT) and of the tranformants obtained with pGPDkpAmyAO (KA 1 and 2), pGPDlpAmyAO (LA 2, 4, 5 and 6) and p2G-S (114).
In all samples corresponding with the maximal amylase production of the transformants which produce high amounts of amylase (LA 2, LA 4, LA 5, LA 6 and 114), a band of about 54 kDa appears, which is not detectable in the wild type lanes or in the lanes of KA 1 and KA 2. These results correspond with the ones obtained in the enzyme tests and colony PCRs and confirm that the A. oryzae amylase is produced.
Although production of homologous fungal proteins is usually quite efficient and can reach the g/L levels, such as reported for glucoamylase production by A. niger , equally successful production of heterologous proteins from filamentous fungi can be (but is not always) achieved and the production levels may lay within the mg/L range (14, 166, 205). Production levels of non-fungal enzymes often are even lower. For example, Gouka et al . (214) report in a comparative study (single copy transformants with integration at the pyrG locus) the production in A. awamori of ± 23 mg/L A. awamori 1,4-β-endoxylanase, of ± 30 mg/L Thermomyces lipase, of > 100 mg/L A. niger glucoamylase and of < 0.1 mg/L human interleukin 6. All expressions in this report were controlled by the exlA -promoter of A. awamori . Van Gemeren et al. (166) obtained maximally 70 mg/L cutinase of Fusarium solani pisi produced in A. awamori (copy number 5-10, A. awamori exlA -promoter, Erlenmeyer culture). Higher values were obtained when the glaA -promoter was used for the expression of M. miehei aspartic protease in A. awamori (150 mg/L, Erlenmeyer culture). Similar production levels of this protease were reached using the amy- or glaA -promoter in A. oryzae . Even higher levels were reached when the A. niger glucoamylase was expressed in A. awamori under the control of its own promoter (4.6 g/L), but this level was obtained in a production strain (multiple copies, Erlenmeyer culture) (216). Regarding Taka-amylase production, only in a review article of Verdoes et al. (216) some data were found. Production of the A. oryzae α-amylase reached 4 mg/L in T. viride when placed under the control of its own promoter (and secretion signal) and 1 g/L when placed under the control of the cbhI promoter (and secretion signal) of Hypocrea jecorina (Trichoderma reesei ). These production levels were obtained in Erlenmeyer cultures of production strains in which multiple copies were introduced. The relative amount of Taka-
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 136 amylase produced by the strains LA 2 and LA 6 was estimated with the LabImage Software (LabImage 1D 2006, Version 3.3.0, Kaplelan Bio-Imaging Solutions). By this analysis, the Taka-amylase appears to be about 20 % of the total amount of protein secreted, for both strains. This means that about 40 mg/L of Taka-amylase is secreted by the M. gramineum transformants. The value corresponds to those reported in the literature for the production of heterologous (fungal) proteins in fungal expression systems, although it is rather low (compared to 30 mg/L Thermomyces lipase, 70 mg/L Fusarium cutinase, 150 mg/L M. miehei protease, 1 g/L TAKA-amylase). On the other hand, lower production values were also reported, for example for the expression of the M. miehei aspartic protease in M. circinelloides (1.2 mg/L) and for the production of the A. niger glucoamylase in A. nidulans (0.2 mg/g CDW under the control of the A. nidulans gpdA -promoter). The latter value corresponds to 2 mg/g CDW Taka-amylase produced by strain the M. gramineum LA 6 strain (under the control of the M. gramineum gpd -promoter).
Transformant LA 6 was grown a third time in 100 mL of AMM + 3% sucrose 250 to confirm the high α-amylase 200 production obtained with this strain. /mL The result is given in Figure IV. 3-18. 150
This time the strain produced a 100 maximum of 194.84 units/mL (9278 amylase Units 50 units/(mL.g CDW)), which is about two times more than obtained in the 0 0 20 40 60 80 100 120 140 160 180 200 first experiment. Again, the increase in Time (h) production might be explained by the LA6 AMM+3%sucrose fact that the culture was shaken more Figure IV. 3-18. Confirmation of a-amylase production intensively (200 rpm instead of 175 by transformant LA 6 (units TAKA-amylase/mL rpm), thus creating more aerobic corrected for wild type background production). conditions. All other culture conditions were exactly the same. An increase in Taka-amylase production by Trichoderma viride as a result of more aerobic conditions was also observed by Cheng et al . (294). The amount of amylase produced increased from 0.6 g/L to 1.0 g/L. In their study, the amylase of A. oryzae was placed under the control of the cbhI -promoter and secretion signal.
To construct a probe for a Southern analysis of the transformants obtained after transformation of M. gramineum with pGPDkpAmyAO and pGPDlpAmyAO, a High Fidelity PCR was performed on plasmid pGPDkpAmyAO with the primers “5’ short GPD” and “3’ prom GPD”. These primers amplify the short fragment of the promoter (581 bp) (§ 3.6 in Chapter III), such that the probe based on this fragment can be used for both the analysis of the transformants obtained with pGPDkpAmyAO and with pGPDlpAmyAO (Figure IV. 3-19). The PCR fragment was gel purified and labelled with the DIG High Prime Labelling and Detection Kit (Roche Diagnostics).
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 137
1033 bp promoter (lp) amy3 coding region 3' flanking sequences amy3 581 bp promoter (kp)
probe
Figure IV. 3-19. Schematic view of the probe, used for Southern analysis of the transformants obtained with pGPDkpAmyAO and pGPDlpAmyAO, in relation to the promoter-reporter enzyme construct on these plasmids.
Genomic DNA (gDNA) of the wild type and the transformants LA 2, LA 4, LA 6 and KA 2 was isolated with the HiSpeed Plasmid Maxi kit (Qiagen). The gDNA samples were cut with Bam HI and Nsi I. Bam HI does not cut in the plasmids pGPDlpAmyAO and pGPDkpAmyAO, while Nsi I linearises them. The result of the Southern analysis is presented in Figure IV. 3-20. As expected, in the lane of the wild type, one band is observed. Nsi I cuts one time within the known gpd -sequence, after 1558 bp, which means the observed fragment has to be at least 1558 bp, corresponding with the results of the Southern analysis. The fact that only one band occurs is consistent with previous results (chapter III), which indicated that M. gramineum has only one copy of the gpd -gene in its genome. The band of the wild type copy of the gene is also observed in all transformants, at the same position. This might give an indication for the fact that the expression vectors did not integrate at the homologous gpd -locus in the genome, but that integration took place by illegitimate recombination (Type II, ectopic integration, see also Introduction).
The plasmid pGPDlpAmyAO is 6511 bp long and plasmid pGPDkpAmyAO is 6063 bp. Tandem integration events (of the intact plasmids) should be visible at these positions in the lanes with the NsiI restrictions. For strains LA2 and LA6 a band of approximately that size is visible, but since it is not more intense than the wild type band, it probably does not correspond with tandem integration events.
LA 2 B LA 2 N LA 4 B LA 4 N Marker LA 6 B LA 6 N Marker KA 2 B KA 2 N WT N 21226bp
5148bp/4973bp 4268bp 3530bp WT 2027bp 1904bp 1584bp 1375bp
Figure IV. 3-20. Southern analysis of the transformants LA 2, LA 4 and LA 6 (carrying pGPDlpAmyAO), KA 2 (carrying pGPDkpAmyAO), 114 (carrying p2G-S) and wild type (WT). (N = restriction gDNA with Nsi I, B = restriction gDNA with Bam HI, WT band = wild type copy gpd - gene).
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 138
In the lanes of the higher producers (LA2, LA4 and LA6) additional bands are visible next to that of the wild type gpd -gene. The presence of (multiple) copies of the expression plasmids in the genome of these transformants corresponds with the results of the amylase enzyme tests. No correlation could be found between the copy number and the amylase activity.
Finally, one can state that the following observations confirm the fact that the amylase production observed in the transformants indeed is encoded by the copy of the amy3 -gene on the pGPDAmyAO plasmids: - recombinant amylase production was observed only in the transformants where the presence of at least one copy of the expression vector was confirmed by colony PCR - the presence of (multiple copies of) the expression plasmids in the genome of the transformants was confirmed in Southern analysis by the appearance of additional bands hybridising with the gpd -probe - the recombinant amylase production by the transformants differs significantly from the background amylase production of the wild type - in SDS-PAGE analysis, an extra band corresponding with the molecular size of the A. oryzae α-amylase is present in the lanes of the high-amylase producers, which is not detected in the wild type - the increase in the extracellular protein concentration in cultures of transformants LA 2 and LA 6 corresponds with the increase in extracellular amylase production.
4. CONCLUSION In order to explore its capabilities to produce (heterologous) enzymes, Myrothecium gramineum was tested as to its production of an α-amylase of A. oryzae . Therefore, M. gramineum was transformed with the plasmids pGPDlpAmyAO and pGPDkpAmyAO, containing the full length gpd -promoter of M. gramineum and a shorter fragment thereof, respectively, followed by the amy3 gene of A. oryzae , with its natural secretion signal and terminator sequences. Eight transformants were obtained with the plasmid pGPDlpAmyAO and two with the plasmid pGPDlpAmyAO. Colony PCR confirmed that all 10 transformants carry at least one (functional) copy of the hph -gene. The colony PCR with the primer pair based on the M. gramineum gpd -promoter-A. oryzae amylase construct resulted in a fragment of the expected size for the colonies LA 2, LA 4, LA 5, LA 6, LA 7 and KA 2, meaning they also carry at least one copy of this construct.
The 10 colonies were tested as to their amylase production. The preliminary results obtained in this experiment indicated that transformants LA 2, LA 4, LA 5, LA 6 and LA 7 (carrying the GPDlpAmyAO construct) produce more amylase than the wild type strain. These results confirmed the ones obtained by colony PCR: colonies LA 2, LA 4, LA 5, LA 6 and LA 7 carry at least one functional copy of the GPDlpAmyAO construct, while colonies LA 1, LA 3 and LA 8 do not. The transformant KA 1 with the GPDkpAmyAO construct produced no more amylase than the wild type, which was also in accordance with the PCR results.
Chapter IV: Expression of an A. oryzae amylase in M. gramineum 139
In order to confirm the results of the enzyme tests, new cultures were set up with transformants LA 2, LA 4, LA 5, LA 6, KA 1, KA 2, the wild type strain and transformant 114 (highest amylase producer obtained after transformation of M. gramineum with the Aspergillus nidulans gpdA -promoter-A. oryzae amylase construct. The two transformants carrying the A. oryzae amylase under the control of the 581 bp fragment of the gpd -promoter of M. gramineum produced much fewer (TAKA-) amylase than the transformants carrying the amy3 gene under the control of the 1033 bp fragment of the M. gramineum gpd -promoter or of the A. nidulans gpd -promoter (2 kb). It has to be noted though, that two transformants are not enough to conclude that the 581 bp fragment is weaker as a promoter sequence than the 1033 bp fragment. When comparing the production levels obtained with the 1033 bp gpd - promoter sequence of M. gramineum with the gpd -promoter of A. nidulans , it can be concluded that with the homologous promoter higher amylase production was obtained than with the heterologous A. nidulans gpd -promoter (7238.6 and 2715 units amylase per mL and per gram CDW, respectively).
After SDS-PAGE, a band of about 54 kDa appears in all samples of transformants which produce high amounts of amylase (LA 2, LA 4, LA 5, LA 6 and 114). These results correspond with the ones obtained in the enzyme tests and colony PCRs and confirm that the A. oryzae amylase is produced. The high production of transformant LA 6 was confirmed a third time. This time the strain produced a maximum of 194.84 units/mL (9278 units/(mL.g CDW)). The increase in production might be explained by the fact more aerobic culture conditions were applied. The presence of (multiple copies of) the expression plasmids in the genome of the transformants was confirmed in Southern analysis by the appearance of additional bands hybridising with the gpd -probe.
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 140
Chapter V
Expression of a Penicillium griseofulvum endo-1,4-βββ-xylanase gene in Myrothecium gramineum
1. INTRODUCTION Lignocellulose is the most abundant and renewable biomass source on earth. It comprises three major groups of polymers: cellulose, hemicellulose and lignin. These compounds are present in the cell wall and in middle lamella of plant cells. Xylan, which is the major component of hemicellulose, is a heterogeneous polysaccharide consisting of β-1,4-linked D- xylosyl residues on the backbone, and containing arabinose, glucuronic acid and arabinoglucuronic acids linked to this D-xylose backbone. Due to the heterogeneity of xylan, its enzymatic hydrolysis requires a complex of different enzymes, but the main enzymes involved are endo-1,4-β-xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) (10). Endo- 1,4-β-xylanases attack the internal main-chain xylosidic linkages and β-xylosidases release xylosyl residues by endwise attack of the xylooligosaccharides (297, 298). For the complete hydrolysis of the molecule, side-chain cleaving enzyme activities are also necessary. The first two enzymes are the major components of xylanolytic systems produced by biodegradative microorganisms, such as Trichoderma , Aspergillus , Humicola and Schizophyllum species. The principal commercial source of these enzymes are filamentous fungi (for review, see Polizeli et al. , 2005 (10)), but they are also produced by bacteria ( Bacillus , Clostridium and Streptomyces species), yeast, marine algae, protozoans, snails, crustaceans, insects, seeds of land plants, etc.
Recently, there has been much industrial interest in xylan and its hydrolytic complex. Xylanases and cellulases, together with pectinases, currently account for 20 % of the world enzyme market (10). For example, increasing concern over preserving the environment from industrial wastes has initiated a growing interest in applying microbial enzyme systems in the paper and pulp industry. The aim of these biotechnological processes is to reduce or replace the harmful alkaline extraction of hemicellulose and the need for chlorine in the bleaching process (299). Besides in the paper and pulp industry, xylanases find their applications as supplements in animal feed, for the manufacturing of bread, food, juice and wine, in the textile industry, and for the production of ethanol and xylitol (10, 298). In particular, glucoside hydrolase (GH) family 11 xylanases from Aspergillus niger and Hypocrea jecorina (Trichoderma reesei ) have been extensively studied, and the three-dimensional structures were shown to have the shape of a right hand (298). The majority of xylanases fall into the GH families 10 (MW > 30kDa) or 11 (MW between 19 and 25 kDa). In general, endoxylanases show peak activity between 40°C and 80°C and between pH 4.0 and 6.5, but also optimal conditions outside these ranges have been found (10).
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 141
A few examples of the heterologous production of fungal endo-1,4-β-xylanases are given in Table V. 1-1. Both filamentous fungi, such as Aspergillus and Penicillium , and yeast have been used as host organisms for the production of fungal endo-xylanases. As can be observed in the table, the secretion signals of the xylanases under investigation are used most commonly to obtain secretion of the xylanases into the culture supernatant. Even in yeast, the fungal secretion signals are efficiently processed (300). Table V. 1-1. Heterologous production systems of fungal endo-1,4-β-xylanases
Expression/secretion/ Host organism Source organism Yield 3,4 Ref termination signals 1, 2 Pichia pastoris Penicillium citrinum aox1 , natural, aox1 0.017 3 298 Pichia stipitis Trichoderma reesei xyl1 , natural, n.s. 143 4 301 tkl, natural, n.s. 156 4 301 pgk1 , natural, n.s. 0 301 S. cerevisiae Trichoderma reesei adh2 , natural, adh2 1200 4 302 pgk1 , natural, pgk1 160 4 302
S. cerevisiae T73 Aspergillus nidulans Actin gene, natural, n.s. n.s. 300 Aspergillus niger Aspergillus awamori natural, natural, natural 140 3 216 glaA , n.s., n.s. 72 3 216 gpdA , n.s., n.s. 12 3 216 Aspergillus niger Trichoderma reesei gpd , natural, glaA 8000 4 153 Coprinus cinereus Aspergillus oryzae priA , natural 5/mnp , priA 0.3 3 303 Penicillium 0.464 3 Penicillium funiculosum his4.1 , natural, his4.1 158 funiculosum (37mg/L) Trichoderma reesei Humicola grisea cbhI , cbhI , n.s. 12700 4 188 1 Promoter, secretion signal, terminator (natural = promoter/secretion signal/terminator of the expressed xylanase) 2 aox1 = alcohol oxidase 1 gene, glaA = Aspergillus niger glucoamylase gene, gpdA = Aspergillus sp. glyceraldehyde-3-phosphate dehydrogenase gene, adh2 = Saccharomyces cerevisiae alcohol dehydrogenase II, pgk1 = S. cerevisiae phosphoglycerate kinase, xyl1 = Pichia stipitis xylose reductase gene, tkl = P. stipitis transketolase gene, gpd = Aspergillus niger glyceraldehyde-3-phosphate dehydrogenase gene, his4.1 = Penicillium funiculosum histone 4.1 gene, priA = Lentinula edodes , mnpc = Pleurotus otreatus manganese peroxidase, n.s. = not specified 3 Yield expressed as kU/mL 4 Yield expressed as nkat/mL 5 Natural secretion signal, but cleavage site changed from serine-arginine into lysine-arginine
Until recently, little was known about the genetic regulation of xylanase expression (2, 26). In order to address this lack of knowledge, studies were initiated in A. nidulans, A. niger and Trichoderma . When grown in the presence of xylan or xylooligosaccharides, A. nidulans produces a complex of at least three endo-1,4-β-xylanases and a β-xylosidase. All four
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 142 enzyme expression levels are elevated in the presence of xylan and repression occurs when glucose is added. Carbon catabolite repression is achieved through the action of the major regulator CreA, which can act as a direct and indirect regulator of the expression. Expression of the xln structural genes requires an induction event, not simply derepression, the first being performed by a specific activator of the xln -genes, XlnR. XlnR binds to the GGCTAAR motif, which appears as single sites in the promoter of the structural genes (26, 304). After induction, distinct expression profiles were found for the different genes. Expression of the genes is also differentially regulated in response to ambient pH. This response is mediated by the wide domain transcription factor PacC. For H. jecorina (T. reesei ), similar results were found. When this strain is grown on xylan, the formation of two specific xylanases (XynI and XynII) and of one unspecific endoglucanase is observed. The expression of all three enzymes is differentially regulated in the presence of sophorose, xylobiose and glucose (26). Also for T. reesei , glucose repression is mediated by the major carbon catabolite repressor, Cre1. An XlnR-homologue, Xyr1 was recently isolated. Xyr1 binds to a GGCTAA motif, which has to be present as an inverted repeat in order to be functionally recognised.
In order to explore its capabilities to produce (heterologous) enzymes, Myrothecium gramineum , which was developed as a new fungal expression host during this research, was tested as to its production capacity of an endo −1,4−β-xylanase of Penicillium griseofulvum MUCL 41920. This xylanase has previously been isolated and characterised (305). The enzyme has a molecular mass of 24 kDa. It is characterised by an optimum pH around 5.0 and a temperature profile having its maximum activity at about 50°C. For example, the enzyme could increase the specific volume of a baked product and reduce the viscosity of flour suspended in water.
2. MATERIALS AND METHODS
2.1. Strains, plasmids and growth conditions Myrothecium gramineum BCCM TM /MUCL 39210 was used as the wild type strain. Escherichia coli JM109 or DH5 α strains were used for plasmid amplification.
Plasmid pCSN43 contains the Escherichia coli hygromycin B resistance gene ( hph ) (see § 2.1 of Chapter I). The hygromycin B resistance gene was used as a selection marker when transforming M. gramineum . Plasmid pPGXYN1E-X contains an endo-1,4-β-xylanase (EC 3.2.1.8) of Penicillium griseofulvum MUCL 41920 (305). This plasmid was used to isolate the coding sequence of the Penicillium xylanase gene (including the prepropeptide) and its natural termination signals. Plasmids pGPDkp and pGPDlp (chapter III) were used as expression vectors. The P. chrysogenum xylanase gene was ligated into these plasmids, resulting in the vectors ‘pGPDkpXylPG’ and ‘pGPDlpXylPG’, respectively.
Standard cultivation techniques were performed as described in § 2.2.1 of Chapter I. After transformation, the protoplasts were inoculated on PDAG (PDA + 1 M glucose) and grown
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 143 for 2 hours. Thereafter, a top layer of 8 mL PD + hygromycin was poured over the plates, which were further incubated until growth appeared. Initial screenings for xylanase were performed on PDA plates containing 2 % 4-O-methyl-D-glucurono-D-xylan-remazol brilliant blue R (RBB-xylan, Sigma). For xylanase assays, M. gramineum was grown in 100 mL AMM, but the glucose as a carbon source was reduced to 1 g/L and replaced by 10 g/L mannitol (25°C, 175 rpm). Lower concentrations of glucose were used to reduce the reducing- sugar background at the start of the growth, enabling the assessment of β-xylanase activity in the culture supernatant (302). The low glucose concentrations had to be maintained because the spores were not able to germinate when no glucose at all was used (results not shown).
2.2. Standard DNA manipulation Routine recombinant DNA methodology was performed according to the procedures described in chapters III or IV. Table V. 2-1. Primers used for the construction of the vectors pGPDkpXylPG and pGPDlpXylPG and for the control of M. gramineum transformants Primer name Feature 1 Primer sequence (5’-3’) 2 F IF XylPG forward InFusion primer gaaattcacaa-AATGGTCTCTTTCTCAAGC R IF XylPG reverse InFusion primer ttgggcccgacgtcg-AGTTGTGGTTTCAC hphF control hph in tr. ATGCCTGAACTCACCGCGACG hpfR control hph in tr. CTATTCCTTTGCCCTCGG 5’ long GPD control constr. pGPDlpXylPG gacgtcgac-TGGTCATGCTCATCATC 5’ short GPD forward control gpd-xyl in tr. gacgtc-GACGCAGTGGACAGACGTTA XylPG-colR reverse control gpd-xyl in tr. GAGTAGAAGTAGCCGCCGTTGGTTCCTC T7 control pGEM-T® ligation GGCGATTAAGTTGGGTAACG SP6 control pGEM-T® ligation CGCCAAGCTATTTAGGTGAC 1 tr. = transformants, constr. = construction 2 Matching sequences are indicated with capitals, non-matching sequences are printed in small-type lettering
2.3. Transformation of M. gramineum and control of transformants M. gramineum transformations were performed as described in § 2.4 of chapter I. Xylanase enzyme activities were measured as followed: 450 µL culture supernatant was incubated during 30 minutes with 450 µL 2 % birch wood xylan (Sigma, in 100 mM acetate buffer, pH 5.0) at 50°C, the mixture was centrifuged for 30 s at 4000 rpm and 450 µL of it was incubated with 450 µL DNS reagens at 95 °C; 450 µL stop solution was added and the mixtures were cooled on ice during 5 minutes, followed by immediate measurement of the absorbance at 540 nm. The DNS reagens consisted of 10 g/L dinitrosalicylic acid, 0.5 g/L sodium sulfite and 10 g/L sodium hydroxide. The stop solution contained 40 % potassium sodium tartrate. One unit of xylanase was defined as the amount of enzyme able to release 1 µmol xylose per minute under the above defined reaction conditions. The DNS method was adapted from the method described by Miller et al . (306). The xylanase enzyme test was performed as reported by Bailey et al . (307). The activity was measured at 50°C (pH 5.0) because this is the optimum temperature (and corresponding pH) for activity of the P. griseofulvum xylanase
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 144
(305). The extracellular protein concentration and the CDW were determined as described in chapter IV.
PCR analysis of the transformants was performed as described in chapter II (method 10) and in chapter IV. One primer pair (primers ‘hphF’ and ‘hphR’) is based on the hph -gene of the plasmid pCSN43 (not present in the wild type). The second primer pair (primers ‘5’ short GPD’ and ‘XylPG-colR’) is based on the P. griseofulvum xylanase gene preceded by the M. gramineum gpd -promoter. One primer anneals to the gene, the other to the promoter, forming a combination not present in the wild type genome.
3. RESULTS AND DISCUSSION
3.1. Cloning of the P. griseofulvum xylanase gene into pGEM-T® A PCR with the BD Advantage 2 Polymerase Mix using the primers ‘F IF XylPG’ and ‘R IF XylPG’ was performed on the plasmid pPGXYN1E-X. A fragment 3' fl seq f1 of 1186 bp was obtained, as expected. The fragment was cloned into pGEM-T®. The sequence of the pXylPGIF xylPG xylanase gene on the plasmid of colony 1 was 4188 bps AmpR sec confirmed at the VIB Genetic Service Facility. The plasmid was called ‘pXylPGIF’ (Figure V. 3-1). Plasmid pXylPGIF contains the DNA sequence encoding the mature P. griseofulvum xylanase (623 bp), preceded by its natural secretion signal (81 bp) and Figure V. 3-1. Molecular map of followed by its own transcription termination signals pXylPGIF (3’ fl seq = 3’ flanking (470 bp) (Figure V. 3-2). sequences, sec sig = secretion signal).
The gene has an intron of 50 bp. It encodes a presumed prepropeptide of 27 amino acids and a mature protein of 190 amino acids. The signal peptidase recognition site is predicted between amino acids 19 and 20 (SignalP 3.0 Server, for references and associated URL’s, see reviews Dönnes, P. and Höglund, A., 2004 (308) and Emanuelsson, O., 2002 (309)). Similar properties were found, for example, for the Penicillium citrinum endo-1,4-β-xylanase gene (accession number AB198065 , 298).
start stop codon codon
secsig polyA?
xylanase Penicillium griseofulvum Figure V. 3-2. Fragment of the P. griseofulvum xylanase gene used for the construction of pGPDlp/kpXylPG (coding sequence (arrow) and 3’ flanking sequences). Start and stop codon are represented by vertical lines, a putative poly A signal is indicated as ‘polyA?’ and the secretion signal is marked as ‘secsig’.
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 145
3.2. Construction of pGPDkpXylPG and pGPDlpXylPG Because of the ease of the procedure, it was decided to use the ‘InFusion Cloning Kit’ of Clontech, as described in § 3.2 of chapter IV, to ligate the P. griseofulvum xylanase into the vectors pGPDlp and pGPDkp. These two expression vectors were created because in some cases longer promoter sequences can give stronger expression signals than shorter ones. This was for example reported by Jungehülsing et al . (264), who observed significantly higher transformation efficiencies of Claviceps purpurea when using a 1400 bp fragment of the homologous gpd -promoter than when a fragment of only 500 bp was used. They concluded that the 500 bp fragment is sufficient to allow efficient expression, but nevertheless, the additional sequence in the longer fragment significantly enhances expression. Considering the fact that with the plasmids pGPDlpAmyAO and pGPDkpAmyAO only 8, respectively 2 transformants were obtained (chapter IV), which is not sufficient to make any conclusions regarding the strength of the two promoter fragments of M. gramineum , it was decided to investigate this matter further with the xylanase encoding gene.
As described in chapter IV, the vectors pGPDlp and pGPDkp were cut with Sp HI (Figure V. 3-3). Sp HI is one of the restriction enzymes of the multicloning site at the 3’ end of the gpd -promoter on the plasmids pGPDlp and pGPDkp. Fusion with the InFusion Enzyme at this site results in an in frame ligation of the start codon of the M. gramineum gpd -gene with the (coding) sequence of the P. griseofulvum xylanase gene (natural secretion signal, DNA sequence encoding the mature enzyme, natural transcription termination signals). The xylanase gene sample (Figure V. 3-3) described in § 3.1 was used in the InFusion reaction. The linearised vector fragments and the PCR product were gel purified, their concentration was determined and they were mixed according to the indications given in the InFusion manual. The ‘ligation’ mixtures were transformed into BD Fusion-Blue™ Competent cells, as described in the kit. HI HI HI
Left: Xylanase gene fragment and linearised Sp Sp pGPDkp and pGPDlp vectors for the InFusion Marker col 1 col 2 col 3 col 4 col 5 col 6 reaction Marker xylPGIF pGPDkp pGPDlp
Right: Colony PCR on the 6 colonies for the construction of pGPDlpXylPG by exchanging the short promoter on pGPDkpXylPG with the long one
Figure V. 3-3. Gel electrophoresis performed for the construction of pGPDkpXylPG and pGPDlpXylPG.
The plasmids of ten colonies of both constructs were controlled by restriction analysis with Pst I (pGPDkpXylPG) or with Sca I (pGPDlpXylPG). Three fragments are expected after restriction of pGPDkpXylPG with Pst I, i.e. 3390 bp, 899 bp and 401 bp. Also with pGPDlpXylPG three fragments are expected, more precisely 3223 bp, 1766 bp and 153 bp. Unfortunately, only colony 7 of the ligation for the GPDkpXylPG-construct contained the
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 146 expected plasmid. Analysis of 50 extra colonies for the construction of pGPDlpXylPG also did not result in a positive colony. It was decided to exchange the short promoter fragment on the plasmid of colony 7 with the long promoter fragment of pGPDlp.
Restriction of both plasmids with Xho I and Dra III provided following fragments: - for pGPDkpXylPG: 1905 bp (part of the short promoter fragment, full xylanase sequence and vector flanking sequences) and 2785 bp (rest of the vector) - for pGPDlp: 3237 bp (part of the vector sequences missing in the 1905 bp fragment and part of the promoter sequences missing to obtain the long promoter fragment) and 798 bp Ligation of the 1905 bp fragment of pGPDkpXylPG with the 3237 bp fragment of pGPDlp with the T 4-DNA ligase (Fermentas) would normally result in the vector pGPDlpXylPG. The plasmids of 6 colonies were analysed by colony PCR with the primers ‘5’ long GPD’ and ‘T7’, which should result in a fragment of 2277 bp. All of the six colonies were positive (Figure V. 3-3). The plasmids of colony 7 for the construction of pGPDkpXylPG and colony 2 for the construction pGPDlpXylPG were sent to the VIB Genetic Service Facility for sequencing.
Unfortunately, the sequencing report showed that about ten nucleotides after the start codon of the xylanase gene an A was missing in the sequence of both the plasmids of colony 7 and colony 2. Since this results in a frame shift almost immediately at the start of the coding sequence, translation of this product would have resulted in an incorrect protein. Therefore, the sequence encoding for the xylanase on the plasmids was exchanged with the (correct) coding sequence of the plasmid pXylPG (Figure V. 3-4). Ligation of the gel purified fragments of 723 bp to 4424 bp and to 3019 bp would result in the plasmids pGPDlpXylPG and pGPDkpXylPG, respectively; this time with the correct sequence.
Sph I Sph I Sph I Nhe I Nhe I Nhe I 3' fl seq gpdKP gpdLP f1 sec ori ori sec xyl PG pXylPGIF xylPG Plasmid colony 2 Plasmid colony 7 xyl PG 4188 bps 5139 bps 4690 bps AmpR secsig Sph I AmpR Sph I Sph I polyA? polyA? AmpR
f1 f1
Cut with Sp HI and Nhe I Cut with Sp HI and Nhe I Cut with Sp HI and Nhe I
446 bp + 723 bp + 3019 bp 714 bp + 4424 bp 714 bp + 3019 bp
polyA secsig gpdLP' ? polyA? gpdKP'
xylPG f1 AmpR f1 AmpR
723 bp 4424 bp 3019 bp
Figure V. 3-4. Strategy for the correction of the missing A-residue on the plasmids for the construction of pGPDkpXylPG and pGPDlpXylPG.
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 147
After transformation of E. coli DH5 α with the ligation mixtures, six colonies of both transformations were chosen for analysis by colony PCR. For control of the ligation for pGPDlpXylPG, the primers ‘5’ long GPD’ and ‘T7’ were used, while the ligation for pGPDkpXylPG was checked with the primers ‘5’ short GPD’ and ‘T7’.
The result of this PCR is given in Figure V. 3-5. For the constructs of pGPDlpXylPG, a fragment of 2277 bp was expected, while for the constructs of pGPDkpXylPG a fragment of 1825 bp should be obtained. As can be seen in the figure, colonies 2, 5 and 6 of the construction of pGPDkpXylPG carry the correct plasmid. The same is true for the colonies 5 and 6 of the construction of pGPDlpXylPG. The sequence of colony 5 and 6 of each construct was investigated at the VIB Genetic Service Facility.
col 1 KX col 2 KX col 3 KX col 4 KX col 5 KX col 6 KX Marker col LX 1 col 2 LX col 3 LX col 4 LX col 5 LX col 6 LX Marker
Figure V. 3-5. Control of the colonies obtained for the correction of the missing A-residue on the plasmids for the construction of pGPDkpXylPG (KX) and pGPDlpXylPG (LX).
All four plasmids have the correct sequence, and the plasmids of colony 5 of pGPDkpXylPG and of colony 6 of pGPDlpXylPG were chosen to further work with. They will further be referred to as ‘pGPDkpXylPG’ and ‘pGPDlpXylPG’ (Figure V. 3-6).
gpdKP gpdLP sec ori ori xyl PG sec
pGPDkpXylPG pGPDlpXylPG xyl PG 4690 bps 5139 bps
polyA? AmpR
AmpR polyA?
f1 f1
Figure V. 3-6. Molecular map of the plasmids pGPDkpXylPG and pGPDlpXylPG.
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 148
3.3. Transformation of Myrothecium gramineum with pGPDkpXylPG and pGPDlpXylPG and analysis of the transformants The plasmids pGPDkpXylPG and pGPDlpXylPG were used for the transformation of Myrothecium gramineum , in order to test the capability of M. gramineum to produce the P. griseofulvum xylanase and to compare the strength of the longer promoter fragment to that of the shorter one. The hygromycin resistance gene hph on the plasmid pCSN43, which was co- transformed, was used as the selection marker. The ratio of pGPDXylPG/pCSN43 was about 1.5.
After 5 days, 404 colonies were obtained with the vector pGPDlpXylPG and 534 colonies grew on the plates with the protoplasts of the pGPDkpXylPG transformation. The colonies which were clearly grown through the top layer were picked and inoculated onto PDA + hygromycin plates for a second round of selection. These were 240 colonies transformed with pGPDlpXylPG and 175 colonies obtained by transformation with pGPDkpXylPG. All colonies grew further on the selective plates. The colonies which grew fastest were inoculated a third time on selective plates (90 for pGPDlpXylPG and 66 for pGPDkpXylPG) to check their stability. Mycelium of 36 of the colonies with pGPDkpXylPG and 44 colonies with pGPDlpXylPG was picked to perform a colony PCR with the primers ‘ hph F’ and ‘ hph R’ (control of the presence of pCSN43). A second PCR with the primers ‘5’ short GPD’ and ‘XylPG-colR’ (control of the presence of pGPDkp/lpXylPG) was performed on mycelium of 96 colonies. A fragment of 1 kb was expected for the PCR with the primers annealing to the hph -gene, and one of 687 bp for the PCR with the primers annealing to the gpd -promoter- xylanase constructs. The results of these PCRs are given in Figure V. 3-7. Out of the 80 colonies tested as to the presence of pCSN43, 69 gave a positive result. Additional bands could have resulted from rearrangements of the pCSN43 plasmid upon integration. The fact that no fragment of 1 kb is detected in the lanes of some colonies which grow on the selective plates could be explained by the fact that the sample of mycelium taken for the PCR was not of sufficient quality, or too much impurities (such as medium from the plates) were taken along with the mycelium.
The PCRs with the primer pair based on the constructs of the M. gramineum gpd -promoter followed by the P. griseofulvum xylanase resulted in a fragment of the expected size for 74 of the 96 analysed colonies, meaning they also carry at least one copy of this construct. The fact that no fragment is visible in the lanes of some colonies could again be caused by the sample quality, but it could also mean that they only integrated the plasmid pCSN43 and not the gpd - promoter-xylanase construct. Rearrangements could also have caused the impossibility to amplify the expected fragment.
Because the amplification of the promoter-xylanase fragment in the colony PCR does not necessarily mean that a functional copy of the xylanase has integrated in the genome of the transformants, the colonies which gave a positive result in the PCR were further analysed on PDA + 2 % RBB-xylan. Colonies which express a functional xylanase are able to produce a
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 149 zone of clearing in the blue background (302, 310, 311) when grown on RBB-xylan containing plates. The size of the zone of clearing can be used to estimate the xylanase activity. An example of these plates after one day of incubation at 25°C is given in Figure V. 3-8. Some of the colonies produced relatively larger clearing zones, which is an indication for the fact that more xylanase is expressed by these transformants. The clearing zone around the wild type strain was about the size of the zone around transformant L43.
All colonies which formed only small- to moderate-sized clearing zones were eliminated from further investigation. For the transformants obtained after transformation with plasmid pGPDlpXylPG, 30 colonies had formed significantly larger clearing zones than the wild type, and for the transformants with plasmid pGPDkpXylPG 12 colonies were selected for further investigation. The strains were inoculated in 100 mL AMM (0.1 % glucose, 1 % mannitol) and grown at 25°C (175 rpm).
KX1 KX2 KX3 KX4 KX5 KX6 KX8 KX9 Marker KX10 KX12 KX14 KX17 KX20 KX21 KX25 KX27 KX30 KX36 KX39 KX40 Marker KX41 KX43 KX44 KX45 KX47 KX48 KX49 KX50
KX51 KX53 KX54 KX55 KX56 KX59 KX65 KX66 Marker LX7 LX20 LX21 LX22 LX23 LX24 LX25 LX26 LX27 LX28 LX29 LX30 Marker LX31 LX32 LX33 LX34 LX39 LX40 LX41 LX42
LX44 LX45 LX46 LX50 LX51 LX52 LX53 LX54 Marker LX57 LX58 LX59 LX62 LX63 LX64 LX67 LX68 LX69 LX72 LX73 LX74 Marker LX82 LX83 LX84 LX87 LX88 LX90
KX2 KX8 KX9 KX12 KX15 KX16 KX17 KX18 Marker KX19 KX24 KX26 KX27 KX28 KX29 KX30 KX31 KX32 KX33 Marker KX36 KX37 KX39 KX40 KX41 KX43 KX44 KX45 KX47 KX49
KX54 KX55 KX56 KX59 KX66 LX4 LX5 LX7 Marker LX10 LX12 LX13 LX14 LX18 LX20 LX21 LX22 LX23 LX25 Marker LX28 LX30 LX31 LX32 LX35 LX36 LX37 LX39 LX40 LX41
LX54 LX56 LX57 LX60 LX63 LX65 LX68 LX70 LX73 LX74 LX42 LX43 LX44 LX45 LX46 LX49 LX51 LX53 Marker Marker LX78 LX82 LX83 LX88 LX89 KX14 KX25 KX61 KX63
LX11 LX17 LX29 LX38 LX47 Marker LX50 LX52 LX64 LX69 LX72 Marker LX79 LX80 LX81
Figure V. 3-7. Colony PCRs performed for the control of the presence of the plasmid pCSN43 (upper part) and the plasmids pGPDlpXylPG (LX) or pGPDkpXylPG (KX) (lower part).
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 150
K 36 L 49 L 47 K 33 K 40 L 51 L 45 K 32 K41 L 52 L 44 K31 K28 K29 L 53 L43
Figure V. 3-8. Transformants of M. gramineum after transformation with pGPDkpXylPG (K, left part) or with pGPDlpXylPG (L, right part), grown on PDA with RBB-xylan. The numbers correspond with the number of the colony.
The result of the xylanase enzyme tests performed on supernatant of these cultures is presented in Figure V. 3-9 (only the highest producers are shown). All values given for the transformants are corrected for the xylanase background production of the wild type strain.
250
200
150
100
50
0
Strain Figure V. 3-9. Xylanase production by the transformants obtained by transformation of M. gramineum with pGPDkpXylPG (K) or pGPDlpXylPG (L). The numbers correspond with the number of the colony.
As can be observed in the figure, the transformants of the group with the longer promoter fragment produce between 8.30 and 219.86 units of recombinant P. griseofulvum xylanase per mL culture and per gram CDW. The transformants of the group with the shorter promoter fragment produce between 46.55 and 130.14 units of recombinant xylanase per mL culture and per gram CDW. The background xylanase production of the wild type was 71.93 units/(mL.g CDW). No clear distinction can be made between the group of transformants with the shorter promoter fragment and the group with the longer one. Nevertheless, twice as much transformants with the longer fragment (15) are situated in the group of the higher xylanase producers as compared to the ones with the shorter fragment (7). Moreover, the highest producers are situated in the group with the longer promoter sequence. The highest producer
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 151 with the shorter fragment produces 130 units/(mL.g CDW) recombinant xylanase, while the highest producer with the longer promoter produces 220 units/(mL.g CDW), which is about 1.7 times more. It has to be noted though that these transformants are no single copy transformants carrying the expression cassette at a defined place in the genome. The differences among them can thus result from integration events at different sites in the genome or events resulting in a different copy number of the cassette in the genome.
The strains K5, K15 and K24 obtained after transformation with pGPDkpXylPG and the strains L63, L65 and L78 obtained after transformation with pGPDlpXylPG were chosen for further investigation. They were inoculated a second time in 100 mL AMM (0.1 % glucose, 1% mannitol) and grown at 25°C (200 rpm) in order to confirm the recombinant xylanase production. At the same time, the strains were grown in 100 mL AMM (0.1 % glucose, 1 % mannitol) with 15 g/L xylan (birch wood xylan (Sigma)) in order to test the effect of this component, which is reported to be a major inducer of xylanolytic activity (10, 26). It would be expected that the addition of xylan to the culture medium does not affect the amount of recombinant xylanase produced, since this xylanase production is under the control of the constitutive gpd -promoter of M. gramineum . The result of this experiment is given in Figure V. 3-10. The values for the transformants indicated with the shaded bars represent xylanase activity measurements in the culture supernatant of these transformants reduced with the background xylanase production of the wild type strain. The culture of strain L78 was contaminated.
1200
1000 800 M MX 600 M rec 400 MX rec
activity Xylanase (Units/(mL.g CDW) (Units/(mL.g 200
0 WT L63 L65 K5 K15 K24 Strain Figure V. 3-10. Xylanase activity of the M. gramineum wild type strain and of the transformants obtained with pGPDkpXylPG (K) and pGPDlpXylPG (L), grown on mannitol (M) and on mannitol + xylan (MX). Total xylanase activity (M and MX) and recombinant xylanase activity (M rec and MX rec) in units per mL and per g CDW.
This time the wild type produced 200 units of (background) xylanase (per mL and per g CDW) in the medium with mannitol. Addition of xylan increased the wild type xylanase activity with 280 units, or 2.3 times, which confirms that xylan induces xylanase activity, as reported in the literature. The higher activity on the mannitol medium as compared to the result presented in Figure V. 3-9 could be a combination of higher aeration (200 rpm and 175 rpm, respectively) and the fact that the cultures were inoculated with (higher amounts of)
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 152 freshly harvested spores in stead of with spores conserved in a cryovial. The same counts for the transformants. When comparing the P. griseofulvum xylanase production with and without induction, one can see that strains L63, L65 and K5 produce the same amount of xylanase on both media. This confirms the fact that this part of the measured xylanase activity is indeed encoded by the xylanase-gene copy or copies driven by the gpd -promoter. Indeed, the gpd -promoter is constitutive and thus not influenced by the addition of xylan. Strain L63 produced ± 600 units/(mL.g CDW) P. griseofulvum xylanase, strain L65 produced ± 440 units and strain K5 ± 360 units. The increase in extracellular enzymes was also reflected in the determination of the amount of extracellular proteins (95 µg/ml for the wild type, and 125, 115 and 105 µg/mL for strains L63, L65 and K5, respectively).
For strain K15 and K24 one can see that the recombinant xylanase activity is influenced by the addition of xylan. The fact that there is a considerable increase (K15) or decrease (K24) in ‘recombinant’ production upon induction is an argument to claim that the ‘recombinant’ production is not encoded by the xylanase-gene copy or copies driven by the gpd -promoter, since this promoter is considered to be constitutive. For example, integrations of plasmids or DNA fragments at the homologous xylanase-gene (regulatory) sites could also have resulted in a change of the amount of ‘wild type’ xylanase production.
The samples of the experiment ‘MX’ of L63, L65 and K5 were used for SDS-page (Figure V. 3-11). Similar amounts of extracellular protein were loaded for all strains (± 3 µg). Detection was performed by silver staining. The P. griseofulvum endo-1,4-β-xylanase is expected at 24 kDa (305). The ‘Low Molecular Weight Calibration Kit for SDS Electrophoresis’ (Amersham Biosciences) was used to estimate the size of the proteins. Analysis with the LabImage Software (LabImage 1D 2006, Version 3.3.0, Kaplelan Bio- Imaging Solutions) indicated the presence of a 24 kDa protein band in the lanes of L63 and L65, which was not detectable in the lane of the wild type or in the lanes of the other transformants. Based on the enzyme activity tests, one would also expect a band in the lane of K5.
The amount of xylanase relative to the total amount of extracellular protein produced by the strains L63 and L65 was also estimated with the software, based on the relative intensities of the bands. For strain L63, the xylanase was estimated to be about 28 % (35 mg/L) of the total amount of extracellular protein, while for strain L65 it was about 40 % (46 mg/L). According to the enzyme test, strain L63 produced more active P. griseofulvum xylanase than strain L65, meaning the strain L65 might accumulate some inactive ( P. griseofulvum ) xylanase.
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 153
L63 L65 WT LMW K5 kDa 97
66
45
30 24 kDa 20 14
Figure V. 3-11. SDS-PAGE followed by silver staining of the samples ‘MX’ of the transformants of M. gr amineum with the GPDlpXylPG-construct (L63, L65 and L78) or with the GPDkpXylPG- construct (K5, K15 and K24). The P. griseofulvum xylanase is 24 kDa (left). The numbers on the right side correspond with the bands of the protein marker (LMW).
The presence of a protein band of the expected size of the recombinant P. griseofulvum xylanase (24 kDa) in the lanes of the samples with high enzyme activity indicates that the xylanase is encoded by the xylanase-gene copy or copies driven by the gpd -promoter. It also gives an indication for the fact that M. gramineum is able to process the secretion signal of the P. griseofulvum xylanase. The cleavage by the signal peptidase is predicted to take place between amino acid 19 and 20 of the protein (ALA LP). This cleavage site is similar to the one of the Taka-amylase of A. oryzae (ALA AT), which is also processed by M. gramineum (chapter IV).
4. CONCLUSION In order to explore its capabilities to produce (heterologous) enzymes, M. gramineum was tested as to its production of an endo-1,4-β-xylanase of Penicillium griseofulvum . Therefore, M. gramineum was transformed with the plasmids pGPDlpXylPG and pGPDkpXylPG, containing the full length gpd -promoter of M. gramineum and a shorter fragment thereof, respectively, followed by the xylanase gene of P. griseofulvum , with its natural secretion signal and terminator sequences.
After transformation, 404 colonies were obtained with the vector pGPDlpXylPG and 534 colonies grew on the plates with the protoplasts of the pGPDkpXylPG transformation. Mycelium of colonies with pGPDkpXylPG and with pGPDlpXylPG was picked to perform a colony PCR to control of the presence of pCSN43 and of the GPDkp/lpXylPG construct. 69 out of the 80 colonies tested as to the presence of pCSN43 gave a positive result. The PCR’s with the primer pair based on the construct of the M. gramineum gpd -promoter followed by the P.griseofulvum xylanase revealed that 74 colonies carry at least one copy of this construct. The colonies where grown on RBB-xylan containing plates. Some of the colonies produced relatively larger clearing zones: 30 colonies obtained after transformation with plasmid
Chapter V: Expression of a P. griseofulvum xylanase in M. gramineum 154 pGPDlpXylPG and 12 colonies with plasmid pGPDkpXylPG were selected for further investigation. The transformants of the group with the shorter promoter fragment produce between 46.55 and 130.14 units of recombinant xylanase per mL culture and per gram CDW, while the ones with the longer promoter fragment produce between 8.30 and 219.86 units. Twice as much transformants with the longer fragment (14) are situated in the group of the higher xylanase producers as compared to the ones with the shorter fragment (7).
The strains K5, K15 and K24 obtained after tranfsormation with pGPDkpXylPG and the strains L63, L65 and L78 obtained after tranfsormation with pGPDlpXylPG were chosen for further investigation. They were inoculated in 100 mL AMM (0,1% glucose, 1% mannitol) and in 100 mL AMM (0,1% glucose, 1% mannitol) with 15 g/L birchwood xylan in order to test the effect of this component, which is reported to be a major inducer of xylanolytic activity. Addition of xylan increased the wildtype xylanase activity with 280 units, or 2.3 times, which confirms that xylan induces xylanase activity, as reported in the liteature. When comparing the P. griseofulvum xylanase production with and without induction, it becomes clear that strains L63, L65 and K5 produce the same amount of xylanase on both media. This confirms the fact that this part of the measured xylanase activity is indeed encoded by the xylanase-gene copy or copies driven by the gpd -promoter. Strain L63 produced ± 600 units/(mL.g CDW) P. griseofulvum xylanase, strain L65 produced ± 440 units and strain K5 ± 360 units.
SDS-PAGE indicated the presence of a 24 kDa protein band, which is the expected size of the P. griseofulvum xylanase, in the lanes of L63 and L65, which was not detectable in the lane of the wildtype or in the lanes of the other transformants. For strain L63, the xylanase was estimated to be about 28% (35 mg/L) of the total amount of extracellular protein, while for strain L65 it was about 40% (46 mg/L).
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 155
Chapter VI
Expression of a Bacillus subtilis xylanase gene ( xynA ) in Myrothecium gramineum
1. INTRODUCTION Efficient and cost-effective production of an enzyme having properties suitable for use at high process temperatures and pH is a challenge, because these enzymes mainly originate from relatively unstudied bacteria (microorganisms) in which the production level is low. There may be little or no experience with cultivating these microbes in a fermentor, or they may otherwise be unsuitable for industrial scale production (206). Filamentous fungi, such as Aspergillus sp., Penicillium sp. and Trichoderma sp. are used as producers of industrial enzymes, and (genetically modified) strains can produce high levels of both homologous and heterologous fungal enzymes. Although production of homologous fungal proteins is usually quite efficient and can reach the g/L levels, equally successful production of heterologous proteins from filamentous fungi can be (but is not always) achieved and the production levels may lay within the mg/L range (14, 166, 205). Unfortunately, the published yields of non- fungal enzymes from filamentous fungi often are even lower, not exceeding a few tens of milligrams per litre or micrograms per litre, and in many of the studies, the enzymes were detected only intracellularly (14, 206). The only reported exception has been the Streptomyces hindustanus phleomycin-binding protein which was produced in Tolypocladium geodes at the high yield of 1.5 g/L (206).
Several factors which influence the production levels of non-fungal enzymes have been described, showing that production can be limited at any level, be it transcription, translation, secretion and/or extracellular degradation (22). The limiting factor(s) often depend(s) on the protein to be expressed and/or the fungal host strain. On the basis of limitations observed for the production of non-fungal enzymes, several strategies have been developed to improve protein yields (see Introduction, § 8.5). The application of a gene fusion strategy has been especially successful and has resulted in a change in type of expression cassettes for non- fungal enzymes (22). Originally, the coding region of the non-fungal gene was fused to efficient expression signals. This signal sequence codes for a signal peptide of 13 to 50 amino acids, which directs the protein to the endoplasmatic reticulum and subsequently to the secretory pathway. In spite of lacking consensus sequences, signal sequence structures among different organisms are conserved and consist of three parts: a stretch of mainly positively charged (basic) amino acids (n-region) is followed by an uninterrupted region of hydrophobic amino acids (h-region), whilst the cleavage site for the signal peptidase which removes the signal peptide after translocation is located at the more polar C-terminal end (c-region) (20, 312). The structural determinants for cleavage of the signal sequence from the mature protein seems to reside in the n- and h-regions, with positions -3 and -1 relative to the cleavage site being the most important ones. The signal is derived in most cases from well-secreted
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 156 homologous (fungal) proteins, but, when the heterologous gene is secreted per se , the endogenous secretion signal can be used.
Nowadays, the heterologous gene is often fused to the complete coding region of a highly expressed (homologous) gene coding for an efficiently secreted enzyme. In this way, the secreted gene serves as a carrier for the heterologous protein, which in some cases leads to improved protein yields. The coding sequence of extracellular proteins contains information which promotes the secretion by improving the translocation of the protein into the endoplasmatic reticulum, aiding folding and protecting the protein from degradation. To date, this approach is probably the most successful modification to increase non-fungal protein production (19). It has resulted in 5 to 100 fold increases in the secretion of heterologous proteins (202), giving protein levels of 5 to > 250 mg/L (206). For example, the yield of calf chymosin in A. awamori was significantly improved by the fusion of the prochymosin gene to the complete coding region of the fungal glucoamylase, instead of only to the glucoamylase leader sequence (203). A similar strategy in A. awamori where Korman et al. (204) used α- amylase-prochymosin fusions also resulted in higher yields. Gouka et al. (205) showed that the limitation of the production of non-fused human interleukin-6 ( hil6 ) and guar galactosidase ( aglA ) could be resolved by fusing the genes to the A. niger glucoamylase gene (glaA ).
The problem of splicing the heterologous protein and the carrier can mostly be solved by inserting a recognition site for a KEX2-type endopeptidase (20). This endopeptidase splices proteins endoproteolytically between two adjacent basic amino acids, preferably lysine (K) and arginine (R). Often the propeptide sequence of the glucoamylase of A. niger is used as the proteolytic processing site (NVISKR) in Aspergillus systems (16), while in T. reesei ( H. jecorina ) systems the sequences PMDKR or RDKR are used for KEX2 cleavage (220). Surprisingly, recent results have also shown that in some cases the presence of the proteolytic processing site between the carrier and the target protein is not essential for the cleavage of the fusion protein, indicating that the KEX2-type processing is not essential for splicing the carrier and the target in these cases (16).
The glucoamylase of A. niger and A. awamori and the cellobiohydrolase of Hypocrea jecorina are the most commonly used fusion partners (22). Interestingly, both enzymes can be divided into three domains: an N-terminal catalytic domain, a C-terminal starch or cellulose binding domain and a flexible O-glycosylated linker region. The C-terminal domain can efficiently be replaced by the heterologous protein, although full-length fusions are also successful. It has been suggested that the positive effect of the fusion is caused by the fact that the linker region (hinge) permits the catalytic domain and the rest of the fusion protein to fold independently. The linker sequences of microbial β-1,4-glycanases can considerably vary in length (6 to 59 amino acids), they are rich in prolines and/or hydroxyamino acids (Ser (S), Thr (T) and Tyr (Y)) and can contain aspartate (Asp, D) or glutamate (Glu, E) or both (313). Paloheimo et al. (206) studied the effect of various carrier polypeptides on the expression of a
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 157 bacterial xylanase derived from Nonomuraea flexuosa in the Hypocrea jecorina , controlled by the cbhI promoter. The study was performed in single-copy isogenic transformants. They conclude that high-yield production requires a carrier with an intact domain structure and that a flexible hinge region (connection) between the carrier and the xylanase has a positive effect on both the production of the xylanase and the efficiency of cleavage of the fusion polypeptide. The transformants could be divided into two groups, according to the type of carrier encoded by the expression cassette: those having carriers with intact domain structures produced eight- to nine-fold more xylanase activity than the strains containing the incomplete carrier structures. The best yield of bacterial xylanase in 1 L fermentors was 820 mg/L. The strain with the xylanase fused to the signal sequence alone produced the least xylanase. The authors suggest that the structure of the carrier has an important role in obtaining increased yields of the heterologous product, while the actual size of the carrier is not as meaningful. This is supported by the fact that with shorter polypeptides, prosequences and additional N- terminal amino acids of well-secreted fungal proteins as carriers improvements have usually not been obtained or they have been low compared to the yields with a long carrier polypeptide (203). The hinge permits separate folding of the two independent domains and prevents the cleavage site from being embedded in the fusion protein structure and thus not to be efficiently recognised by the protease.
In order to explore its capabilities to produce bacterial enzymes, Myrothecium gramineum , was tested as to its production of an endo −1,4−β -xylanase of Bacillus subtilis . The enzyme is characterised by an optimum pH around 5.0 and has maximum activity at about 50°C (314). In one construct, the coding sequence of the mature bacterial xylanase was fused to the secretion signal and transcription termination sequences of the A. oryzae Taka-amylase. In an other construct, it was fused using a linker with a KEX2 processing site to the C-terminal end of the sequence of the A. oryzae Taka-amylase (including the secretion signal). The Taka- amylase could act as a carrier to guide the bacterial xylanase through the fungal secretion pathway. The gpd -promoter of M. gramineum was used to drive expression in both constructs. The use of the A. oryzae Taka-amylase as a carrier protein was previously reported by Nakajima et al . (315) for the production of a heterodimeric fruit protein, neoculin, in A. oryzae . The amylase was fused to the N-terminus of both subunits. The carrier and the target protein were separated by a KEX2 cleavage site (KR) followed by 3 glycine residues. The triglycine motif was not added in the constructs used in our research, since the correct release of the target protein was also achieved without the motif, e.g. when producing antibodies in A. oryzae .
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 158
2. MATERIALS AND METHODS
2.1. Strains, plasmids and growth conditions Myrothecium gramineum BCCM TM /MUCL 39210 was used as the wild type strain. Escherichia coli JM109 or DH5 α strains were used for plasmid amplification.
Plasmid pCSN43 contains the Escherichia coli hygromycin B resistance gene ( hph ) (see § 2.1 of Chapter I). The hygromycin B resistance gene was used as a selection marker when transforming M. gramineum . Plasmid pAPXyn7 contains the endo-1,4-β-xylanase gene of Bacillus subtilis (EC 3.2.1.8, Uniprot reference P18429 , GenBank reference M36648 ). This plasmid was used to isolate the coding sequence of the Bacillus xylanase gene. Plasmid pGPDlpAmyAO (chapter IV) was used as a source for the M. gramineum gpd -promoter, and the A. oryzae amylase secretion signal and termination signals. The B. subtilis xylanase gene was ligated into (parts of) this plasmid, resulting in the vectors ‘pGPDcarXylBS’ and ‘pGPDnocarXylBS’.
Standard cultivation techniques were performed as described in § 2.2.1 of Chapter I. After transformation, the protoplasts were inoculated on PDAG (PDA + 1 M glucose) and grown for 2 hours. Thereafter, a top layer of 8 mL PD + hygromycin was poured over the plates, which were further incubated until growth appeared. Initial screenings for xylanase activity and cultures for xylanase assays were set up as described in § 2.1 of Chapter V.
2.2. Standard DNA manipulation Routine recombinant DNA methodology was performed according to the procedures described in chapters III or IV. The primers used for the constructions and control of the plasmids and transformants are given in Table VI. 2-1. Table VI. 2-1. Primers used for the construction of the vectors pGPDcarXylBS and pGPDnocarXylBS and for the control of M. gramineum transformants Primer name Feature 1 Primer sequence (5’-3’) 2 gcaggtagcaagatcacgtcctcctccaatgtcatttccaagcg F IF XylBS forward InFusion car t-GCTAGCACAGACTAC F IF XylBS no car forward InFusion no car ctgctttggctgca-AGCACAGACTACT gagctactacagatc- R IF XylBS reverse InFusion TTACCACACgGTgACGTTAGAAC hphF control hph in tr. ATGCCTGAACTCACCGCGACG hpfR control hph in tr. CTATTCCTTTGCCCTCGG XylBS-colcarF forward gpd-xyl in tr. Car ACCACCAAGGATGTGGTCAAG 5’ short GPD forward gpd-xyl in tr. no car gacgtc-GACGCAGTGGACAGACGTTA XylBS-colR reverse control gpd-xyl in tr. GCCCAAACTCCGGCATTATAG T7 control pGEM-T® ligation GGCGATTAAGTTGGGTAACG SP6 control pGEM-T® ligation CGCCAAGCTATTTAGGTGAC 1 tr. = transformants, constr. = construction, car = carrier 2 Matching sequences are indicated with capitals, non-matching sequences are printed in small-type lettering
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 159
2.3. Transformation of M. gramineum and control of transformants M. gramineum transformations were performed as described in § 2.4 of chapter I. Xylanase enzyme activities were measured as described in § 2.3 of Chapter V.
PCR analysis of the transformants was performed as described in chapter II (method 10) and in chapter IV. One primer pair (primers ‘hphF’ and ‘hphR’) is based on the hph -gene of the plasmid pCSN43 (not present in the wild type). For the control of the presence pGPDcarXylBS and pGPDnocarXylBS a combination of a primer which anneals to the xynA gene (XylBS-colR) and to the carrier (XylBS-colcarF), respectively to the promoter (5’ short GPD), were used, forming a combination not present in the wild type genome.
3. RESULTS AND DISCUSSION
3.1. Construction of the plasmids pGPDnocarXylBS and pGPDcarBS In order to be able to test the capacity of M. gramineum to secrete a prokaryotic enzyme, more precisely the xynA of B. subtilis , two vectors were created (Figure VI. 3-1 and Figure VI. 3-2). The vector pGPDnocarXylBS consists of the coding sequence for the mature xylanase of B. subtilis , under the control of the M. gramineum gpd -promoter sequences (1033 bp) and the A. oryzae Taka-amylase termination signals (433 bp). The natural secretion signal of the B. subtilis xylanase was exchanged by the A. oryzae Taka-amylase secretion signal. The vector pGPDcarXylBS consists of the same elements of pGPDnocarXylBS, but the coding sequence for the mature xylanase of B. subtilis is preceded by the complete coding sequence of the A. oryzae Taka-amylase (including the secretion signal) and a linker sequence with a KEX2 processing site. In this case, the amylase serves as a carrier protein to guide the efficient secretion of the B. subtilis xylanase.
The length of the secretion signals of the A. oryzae Taka-amylase and the B. subtilis xylanase was found in their Uniprot Database files (P0C1B3 and P18429 , respectively). These correspond with the results obtained by several online prediction servers, such as SignalP and TargetP (for references and associated URLs, see reviews Dönnes, P. and Höglund, A., 2004 (308) and Emanuelsson, O., 2002 (309)).
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 160
Figure VI. 3-1. A. Bacillus subtilis xylanase. B. Aspergillus oryzae amylase. C. Construct for xylanase secretion in pGPDnocarXylBS. D. Construct for xylanase secretion in pGPDcarXylBS. B. subtilis sequences are represented by open boxes, A. oryzae sequences by shaded ones. Secretion signals are pictured as grey boxes, sequences for mature proteins as white boxes and linker sequences as black boxes. The signal cleavage sites are indicated with arrows, the KEX2 processing site is indicated with a dotted arrow.
It was decided to use the ‘InFusion Cloning Kit’ of Clontech, as described in § 3.2 of chapter IV, to ligate the B. subtilis xylanase into the expression vectors. The strategy for the construction of the vectors is given in Figure VI. 3-2. For the construction of pGPDnocarXylBS, a PCR with the BD Advantage 2 Polymerase Mix using the primers ‘F IF XylBS no car’ and ‘R IF XylBS’ was performed on the plasmid pAPXyn7. A fragment of 584 bp was obtained, as expected, containing the coding sequence of the mature B. subtilis xylanase. The fragment was gel purified. Likewise, for the construction of pGPDcarXylBS, a PCR was performed with the primers ‘F IF XylBS’ and ‘R IF XylBS’ on pAPXyn7. A fragment of 618 bp was gel purified, containing the coding sequence of the mature B. subtilis xylanase preceded by the linker sequence (L).
The linearised vector fragments (4555 bp and 6507 bp) were gel purified and ligated to their corresponding xylanase fragments (54 bp and 618 bp, respectively) in the InFusion reaction according to the indications given in the InFusion manual. The ‘ligation’ mixtures were transformed into BD Fusion-Blue™ Competent cells, as described in the kit. The plasmids of 19 colonies of both constructs were controlled by colony PCR. For the construction of pGPDnocarXylBS, the primers ‘5’ short GPD’ and ‘T7’ were used, while for the construction of pGPDcarXylBS, the primers ‘XylBS-colcarF’ and ‘T7’ were used. A fragment of 1788 bp and of 3091 bp, respectively, was expected. The results of these colony PCRs are given in Figure VI. 3-3. For the construction of pGPDnocarXylBS, the colonies 8, 9 and 14 carried the correct plasmid. This was also the case for colony 6 for the construction of pGPDcarXylBS.
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 161
Bst API
gpdLP Bst API sec
pGPDlpAmyAO AmpR 6511 bps AmyAO
f1 3' flank
Bgl II Cut with Bgl II and partially with Cut with Bgl II Bst API
3' flanking gpdLP sec 3' flanking gpdLP sec
f1 AmpR AmyAO'' f1 AmpR AmyAO' 4555 bp 6507 bp
Ligate to 584 bp PCR fragment Ligate to 618 bp PCR fragment
'xylanase L 'xylanase
3' UTR 3' UTR
XylBS sec XylBS L
f1
f1 AmyAO' gpdLP
pGPDnocarXylBS pGPDcarXylBS 5102 bps 7103 bps AmpR
AmpR
sec
gpdLP
Figure VI. 3-2. Strategy for the construction of pGPDnocarXylBS (left) and pGPDcarXylBS (right).
The sequence of the plasmids of colonies 9 and 6 was analysed at the VIB Genetic Service Facility (Belgium). This analysis confirmed the sequence of both plasmids, further referred to as ‘pGPDnocarXylBS’ and ‘pGPDcarXylBS’, respectively.
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 162
M M c1 c2 c3 c4 c5 c6 c7 c8 c9 nc1 nc2 nc3 nc4 nc5 nc6 nc7 nc8 nc9 c10 c11 c12 c13 c14 c15 c16 c17 c18 c19 nc10 nc11 nc12 nc13 nc14 nc15 nc16 nc17 nc18 nc19
Figure VI. 3-3. Colony PCR for the control of the constructions of pGPDnocarXylBS (nc) and pGPDcarXylBS (c). The numbers correspond with the numbers of the colonies. The Smartladder TM of Eurogentec was used to determine the length of the fragments (M).
3.2. Transformation of Myrothecium gramineum with pGPDnocarXylBS and pGPDcarXylBS and analysis of the transformants The plasmids pGPDnocarXylBS and pGPDcarXylBS were used for the transformation of Myrothecium gramineum , in order to test the capability of M. gramineum to produce the B. subtilis xylanase. The hygromycin resistance gene hph on the plasmid pCSN43, which was co-transformed, was used as the selection marker. The ratio of pGPDXylBS/pCSN43 was about 1.5.
After 6 days, 448 colonies grew on the plates with the protoplasts of the pGPDcarXylBS transformation and 303 colonies were obtained with the vector pGPDnocarXylBS. Based on the hygromycin resistance, a transformation efficiency of 3.3 transformants per µg DNA and per 10 6 viable protoplasts was obtained for the co-transformation with pGPDcarXylBS. After a second round of selection on PDA + hygromycin plates, 60 colonies of each transformation were chosen for investigation by colony PCR (Figure VI. 3-4). A PCR with the primers ‘ hph F’ and ‘ hph R’ was performed to check the presence of pCSN43. For the control of the presence of pGPDnocarXylBS, a PCR with the primers ‘5’ short GPD’ and ‘XylBS-colR’ was performed, while for the integration of pGPDcarXylBS the primers ‘XylBS-colcarF’ and ‘XylBS-colR’ were used. A fragment of 1 kb should be found with the primers based on the hph -gene. A fragment of 798 bp was expected for the PCR on pGPDnocarXylBS and one of 1364 bp would indicate the presence of pGPDcarXylBS.
Out of the 60 colonies tested as to the presence of pCSN43, 59 colonies co-transformed with pGPDcarXylBS and 56 colonies co-transformed with pGPDnocarXylBS gave a positive result. Additional bands could have resulted from rearrangements of the pCSN43 plasmid upon integration. The fact that no fragment of 1 kb is detected in the lanes of a few colonies which grow on the selective plates could be explained by the fact that the sample of mycelium taken for the PCR was not of sufficient quality, or too much impurities (such as medium from the plates) were taken along with the mycelium.
The PCRs with the primer pair based on the construct of the M. gramineum gpd -promoter followed by the B. subtilis xylanase resulted in a fragment of the expected size for 78 % of the colonies with pGPDcarXylBS and for 87 % of the colonies with pGPDnocarXylBS. Based on
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 163 these PCR results, it can be concluded that a ratio of 1.5 times more µg co-transformed plasmid (in this case, pGPDcar/noXylBS) than selected plasmid (in this case pCSN43) thus guarantees high co-transformation efficiencies for M. gramineum .
Because the amplification of the promoter-xylanase fragment in the colony PCR does not necessarily mean that a functional copy of the bacterial xylanase has integrated in the genome of the transformants or that the bacterial xylanase is secreted, the 120 colonies were further analysed on PDA + 2 % RBB-xylan. Colonies which express a functional xylanase are able to produce a zone of clearing in the blue background (310, 311, 302) when grown on RBB-xylan containing plates. The size of the zone of clearing can be used to estimate the xylanase activity. The colonies did not produce large clearing zones beyond the border of mycelial growth (as was observed for the colonies with the fungal xylanase (chapter V)), but they did decolour the medium covered by the mycelium.
M M c1 X c2 X c3 X c4 X c5 X c7 X c8 X c9 X
c11 X c12 X c13 X c14 X c15 X c16 X c17 X c18 X c19 X c20 X c21 X c22 X c23 X c24 X c25 X c26 X c27 X c28 X c29 X c30 X
M M c31 X c32 X c33 X c34 X c35 X c36 X c37 X c38 X c39 X c40 X c41 X c42 X c43 X c44 X c45 X c46 X c47 X c48 X c49 X c50 X c51 X c52 X c53 X c54 X c55 X c56 X c57 X c58 X
M c59 X c60 X nc1 X nc2 X nc3 X nc4 X nc5 X nc6 X nc7 X nc8 X nc9 X nc26 X nc10 X nc12 X nc13 X nc14 X nc15 X nc16 X nc17 X nc18 X nc19 X nc20 X nc21 X nc22 X nc23 X nc24 X nc25 X
nc 11 X
nc27 X nc28 X nc29 X nc30 X nc31 X nc32 X nc33 X nc34 X nc35 X nc36 X M nc37 X nc38 X nc39 X nc40 X nc41 X nc42 X nc43 X nc44 X nc45 X nc46 X nc 47X nc48 X nc49 X nc50 X nc51 X nc52 X nc53 X nc54 X nc55 X nc56 X nc57 X nc58 X nc59 X M nc60 X c1 H c2 H c3 H c4 H
c10 H c11 H c12 H c13 H c14 H c15 H c16 H c17 H c18 H c19 H c20 H M c21 H c22 H c23 H c24 H c25 H c26 H c27 H c28 H c29 H c30 H c31 H c32 H c33 H c34 H c35 H c36 H c37 H c38 H c39 H M c40 H c41 H c42 H c43 H c44 H c45 H c46 H
c47 H c48 H c49 H c50 H c51 H c52 H c53 H c54 H c55 H c56 H c57 H c58 H c59 H M c60 H nc1 X nc2 H nc3 H nc4 H nc5 H nc6 H nc7 H nc8 H nc9 H Figure VI. 3-4. Colony PCR fo r the control of the presence of pCSN43 (H) and pGPDcarXylBS (c, X) or pGPDnocarXylBS (nc, X) in the transformants of M. gramineum .
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 164
Twenty colonies of each transformation were selected for further investigation. The strains were inoculated in 100 mL AMM (0.1 % glucose, 1 % mannitol) and grown at 25°C (175 rpm). The results of the xylanase enzyme tests performed on the culture supernatant of these strains are given in Figure VI. 3-5. All values given for the transformants are the measured values reduced with the xylanase background production of the wild type strain (3.06 units/mL).
25 25
20 20
15 15
10 10
5 5 Xylanase activity (Units/mL) activity Xylanase 0 (Units/mL) activity Xylanase 0 2 4 6 8 10121416183013262831323334 2 8 13 14 18 20 22 24 26 32 34 36 38 42 44 48 54 56 58 60 Strain Strain no carrier carrier Figure VI. 3-5. Xylanase activity tests on the culture supernatant of the transformants obtained with pGPDnocarXylBS (left) and pGPDcarXylBS (right).
The strains obtained by transformation of M. gramineum with the plasmid pGPDnocarXylBS produce between 0.18 and 1.99 units of (recombinant) xylanase per mL. Those obtained with the plasmid pGPDcarXylBS produce between 0.31 and 19.20 units/mL B. subtilis xylanase, and only 8 of them produce less xylanase than the highest producer without the carrier.
Although these results are preliminary, the fact that higher production is achieved using a carrier protein (with linker and KEX2 processing site) to guide the B. subtilis xylanase through the secretion pathway of M. gramineum as compared to fusion with the secretion signal alone, confirms results described in the literature (19, 202, 203, 206). The amylase production of the strains with the carrier protein was measured, but no Taka-amylase activity was detected in the cultures. Further research will be necessary in order to confirm the obtained results. SDS-PAGE analysis will have to be performed to know whether or not the carrier protein is efficiently separated from the target protein and to determine the relative amount of bacterial protein in the extracellular proteins of M. gramineum cultures. Moreover, intracellular accumulation of the fusion or the processed carrier and target will have to be assessed.
Chapter VI: Expression of a B. subtilis xylanase in M. gramineum 165
4. CONCLUSION Myrothecium gramineum was tested as to its production of a bacterial enzyme, more precisely an endo −1,4−β -xylanase of Bacillus subtilis . In one construct, the coding sequence of the mature bacterial xylanase was fused to the secretion signal and transcription termination sequences of the A. oryzae Taka-amylase (pGPDnocarXylBS). In an other construct (pGPDcarXylBS), it was fused using a linker with a KEX2 processing site to the C-terminal end of the sequence of the A. oryzae Taka-amylase (including the secretion signal). The Taka- amylase could act as a carrier to guide the bacterial xylanase through the fungal secretion pathway. The gpd -promoter of M. gramineum was used to drive expression in both constructs.
Based on the hygromycin resistance, a transformation efficiency of 3.3 transformants per µg DNA and per 10 6 viable protoplasts was obtained for the co-transformation with pGPDcarXylBS, confirming the results obtained in chapter I. The PCR’s to control the presence of the B. subtilis xylanase gene resulted in a fragment of the expected size for 78 % of the colonies with pGPDcarXylBS and for 87% of the colonies with pGPDnocarXylBS. Thus, it can be concluded that a ratio of 1.5 times more µg co-transformed plasmid than selected plasmid guarantees high co-transformation efficiencies for M. gramineum .
The 120 colonies analysed on PDA + 2 % RBB-xylan did not produce large clearing zones beyond the border of mycelial growth, but they did decolourise the medium covered by the mycelium. Twenty colonies of each transformation were selected for further investigation. The strains obtained by transformation of M. gramineum with the plasmid pGPDnocarXylBS produce between 0.18 and 1.99 units of recombinant xylanase per mL, while those obtained with the plasmid pGPDcarXylBS produce between 0.31 and 19.20 units. Only 8 of them produced less xylanase than the highest producer without the carrier.
Although the results are preliminary, the fact that higher production is achieved using a carrier protein (with linker and KEX2 processing site) to guide the B. subtilis xylanase through the secretion pathway of M. gramineum as compared to fusion with the secretion signal alone, confirms results described in the literature. Further research will be necessary in order to confirm the obtained results.
Part III: Development of a new selection system for M. gramineum based on an auxotrophic marker
Chapter VII: Cloning of the ompd -gene of M. gramineum 169
Chapter VII
Cloning, sequence analysis and heterologous expression of the Myrothecium gramineum orotidine-5’-monophosphate decarboxylase gene
1. INTRODUCTION Myrothecium gramineum is an asexual filamentous Ascomycete fungus belonging to the class of the Sordariomycetes . To be able to use this fungus for the industrial production of enzymes, an efficient expression system is required, as well as an easy selection method for clones of this fungus carrying the introduced expression cassette.
The literature describes many different methods to select transformants of fungi (25, 208). One can use dominant selection markers, such as antibiotic resistance markers, or auxotrophic markers, which are, for example, based on the nitrate reductase gene. Since antibiotic resistance markers cannot be used for some industrial applications (e.g. in the food industry) and homologous transformation systems are more efficient, the orotidine monophosphate decarboxylase gene ( ompd -gene) was chosen as selection marker and isolated from the genome of Myrothecium gramineum .
The OMPD selection system was first developed in yeast (316) and has since been successfully applied in some industrially important fungi, e.g. Aspergillus sp. (317), Trichoderma sp. (318), Claviceps purpurea (319), Blakeslea trispora (320) and Penicillium nalgiovense (321). It is based on the fluoro-orotic acid resistance of mutants defective in the enzymatic conversion of orotate to uridine-5’-monophosphate (UMP), which is catalysed by the combined action of OMPD and orotic acid phosphoribosyltransferase (OPRT). These mutants are uracil or uridine auxotrophic. Thus, an advantage of the system is that both positive and negative selection is possible for OMPD-wild type strains as well as for OMPD- deficient strains: mutants are resistant to fluoro-orotic acid (5-FOA) (positive selection) and uracil auxotrophic (negative selection), while wild type strains are 5-FOA sensitive (negative) and uracil prototrophic (positive). This is very useful to perform successive transformations on the same strain (321). Another advantage of this selection marker is that it can be isolated easily from genomes of poorly studied organisms because it has a conserved amino acid sequence (322 and references therein). The de novo pyrimidine biosynthetic pathway is one of the oldest metabolic pathways and the six enzymatic steps are nearly identical in eubacteria, archaebacteria and eukaryotes, making the OMPD-enzyme suitable for phylogenetic studies. Any construction can be introduced in a single copy at the ompd locus by gene targeting using a mutant ompd -gene, an asset which is important when performing expression studies (119, 323).
Chapter VII: Cloning of the ompd -gene of M. gramineum 170
In this part, the cloning of the ompd -gene of Myrothecium gramineum is discussed. A vector containing this gene was used to complement a defined OMPD-negative strain of Aspergillus nidulans . As further described, the gene will be used to develop a homologous transformation system for Myrothecium gramineum .
Many of ompd -genes of filamentous fungi are isolated via heterologous hybridisation with a genomic library. Examples are the pyrG genes of Aspergillus awamori , A. parasiticus , A. niger , Blakeslea trispora, Hypocrea jecorina, Mucor circinelloides and Phycomyces blakesleeanus , and the pyr4 gene of Claviceps purpurea (Table VII. 1-1). Nevertheless, the technique of heterologous hybridisation is not always successful. Often, only weak hybridisation signals are obtained which are difficult to distinguish from non-specific hybridisation signals (324). In such cases, a homologous probe is needed, which can be obtained by amplifying part of the gene via degenerate PCR. Table VII. 1-1. Isolation of ompd -genes by heterologous hybridisation with a genomic library
Organism ompd -gene Heterologous probe Reference Aspergillus awamori pyrG pyrG (Aspergillus niger) 119 Aspergillus niger pyrG pyr4 (Neurospora crassa) 317 Aspergillus parasiticus pyrG pyrG (Aspergillus nidulans) 325 Blakeslea trispora pyrG pyrG (Mucor circinelloides) 320 Claviceps purpurea pyr4 pyr4 (Neurospora crassa) 319 Hypocrea jecorina pyrG pyr4 (Neurospora crassa) 318 Mucor circinelloides pyrG pyrG (Phycomyces blakesleeanus) 322 Penicillium nalgiovense pyrG pyrG (Penicillium chrysogenum) 321 Phycomyces blakesleeanus pyrG pyrG (Aspergillus niger) 326
The ompd -genes of e.g. Aspergillus fumigatus (327), Rhizomucor pusillus (328), Rhizopus oryzae (42) en Mortierella alpina (329) were isolated by using a degenerate PCR based strategy. Degenerate primers were developed based on conserved amino acid sequences (Table VII. 1-2). The rest of the genes was isolated by homologous hybridisation with a genomic or a cDNA library. Although hybridisation with a genomic library is frequently used to clone genes, the technique is laborious, difficult and time consuming. The use of PCR based methods for the cloning of whole genes is a more convenient strategy. Table VII. 1-2. Conserved sequences in the OMPD amino acid sequences used for cloning
Organism Conserved region Reference Aspergillus fumigatus FLIFEDRKF 327 KGDKLGQQY Rhizopus pusillus FEDRKFADIG 328 DGLGQQYRTP Mortierella alpina KFADIGNTV 329 RGVIIVDAG
Chapter VII: Cloning of the ompd -gene of M. gramineum 171
In our research, PCR based genome walking was used to clone the ompd -gene of M. gramineum . Degenerate PCR was combined with the procedures described in the BD GenomeWalker Universal Kit (see also Figure III. 3-1 in Chapter III, BD Biosciences).
2. MATERIAL AND METHODS
2.1. Strains, plasmids and growth conditions Myrothecium gramineum BCCM TM /MUCL 39210 was used to isolate the orotidine-5’- monophosphate decarboxylase gene. Aspergillus nidulans FGSC A722 ( pyrG89 pabaA1; fwA1 uaY9 ), a defined OMPD-negative strain, was complemented with the ompd -gene of Myrothecium gramineum to prove it codes for a functional enzyme. Escherichia coli DH5 α- F’ was used for all cloning experiments.
Standard cultivation techniques were performed as described in § 2.2.1 of Chapter I. Aspergillus nidulans A722 was grown on Aspergillus Minimal Medium (231), supplemented with 10 mM uracil, 10 mM uridine (Acros) and 5 g/L yeast extract (Difco).
2.2. Standard DNA manipulation The plasmid pOV was amplified in E. coli DH5 α-F’ and isolated with the HiSpeed Plasmid Maxi kit (Qiagen) before transformation of A. nidulans . All other standard procedures were performed as described in § 2.2 of chapter III.
2.3. Cloning of the Myrothecium gramineum ompd -gene
2.3.1. Genomic DNA isolation DNA was isolated from freshly grown mycelium with the Dneasy Plant Mini/Maxi kit (Qiagen). The mycelium was lyophilised prior to homogenisation.
2.3.2. Degenerate PCR Part of the ompd -gene of M. gramineum was amplified by the use of degenerate oligonucleotides, based on conserved amino acid sequences. The degenerate primers were obtained from Sigma Genosys (Table VII. 2-1): ‘OMPDforward’ (based on ‘FLIFEDR’) and ‘OMPDreverse’ (based on ‘GDXXGQQY’). The PCR-reaction mixture used for degenerate PCR was composed of 2 units Taq -DNA polymerase (Roche Diagnostics), 1x Taq - polymerase buffer (Roche Diagnostics), 0.2 mM dNTP mix (Sigma), 1 µM primers, 187 ng template and 32.6 µl milliQ-water. The PCR was performed in a Progene thermocycler (Techne): after initial denaturation at 94°C for 4 minutes, 40 cycli were run with denaturation at 94°C for 30 s, primer annealing at 50°C for 60 s and extension at 72°C for 2 minutes. The PCR was completed with a final extension step of 7 minutes at 72°C.
2.3.3. Genome walking Based on the sequence obtained with the degenerate PCR, primers were developed to perform several genome walkings. These walkings were carried out in 4 genomic libraries, as
Chapter VII: Cloning of the ompd -gene of M. gramineum 172 described in the BD GenomeWalker TM Universal kit (BD Biosciences). Four gene specific primers (GSP) were designed (Table VII. 2-1): ‘1 OMPD’ and ‘nested 1 OMPD’, and ‘3 1 OMPD’ and ‘3 1 nested OMPD’. The PCR reaction mixture and cycli were optimised for use with the Expand Long Template PCR System (Roche Diagnostics) and performed as described in § 2.3.2 of chapter III.
2.3.4. Cloning and sequence analysis The complete sequence of the ompd -gene was amplified from genomic DNA with the High Fidelity PCR Master kit (Roche Diagnostics). The primers used for this reaction are ‘5 OMPD’ and ‘3 OMPD’ (Table VII. 2-1). The obtained PCR-fragment was cloned in pGEM- T® and the resulting vector was called pOV. Subsequently, the sequence of the ompd -gene on pOV was determined at the VIB Genetic Service Facility (Belgium). The sequence was analysed with the Clone Manager Professional Suite Software (Version 6.0) and its deduced amino acid sequence was blasted against the UNIPROT database (Release 5.0), using the WU-Blastp (v2.0) program on the website of the European Bio-informatics Institution (score matrix: PAM250).
The position of the intron was confirmed by cDNA analysis as described in § 2.3.3 of chapter III. The synthesised cDNA was used directly for PCR with the High Fidelity PCR Master kit using the primers ‘5 cDNA’ and ‘3 cDNA’ (Table VII. 2-1). The resulting 703 bp fragment was cloned in pGEM-T® and sequenced. Table VII. 2-1. Primers used for the isolation and the sequence analysis of the M. gramineum ompd -gene
Primer name Feature Primer sequence (5’-3’) OMPDforward degenerate primer CCTSATCTTYGARGAYCGC OMPDreverse degenerate primer GGHGTSTKGTACTGCTGHCC 1 OMPD primary GSP upstream CGATAGGGAAACGTCTCGTCGGTAATG nested 1 OMPD nested GSP upstream GGTACTGTTTCTGGGCTGTTGAGCCAAT 3 1 OMPD primary GSP downstream CCCTGCTTCTGCTTGCTGAGATGAC 3 1 nested OMPD nested GSP downstream CTGGTATCAACAGCAGCCAATCTGAC 5 OMPD isolation of 2050 bp fragment CAGACCGCATGCATTCGTATGCC 3 OMPD isolation of 2050 bp fragment ACCAAGCACCAACAC 5 cDNA cDNA amplification ATCCGAGCCACAACGAATCC 3 cDNA cDNA amplification AATCAGAGCCGCCACGTATG
The accession numbers of the DNA and the amino acid sequences used as references in this work are: - DNA sequences: Acremonium chrysogenum (Cephalosporium acremonium) (X15937 ), Aspergillus awamori ( AY530810 ), Aspergillus kawachii ( AB064659 ), Aspergillus niger (X06626 ), Aspergillus oryzae ( AB017705 ), Aureobasidium pullulans ( AF165169 ), Blakeslea trispora ( AJ534694 ), Cladosporium fulvum ( AF288696 ), Cryptococcus humicola ( AB084379 ), Emericella nidulans (Aspergillus nidulans) ( M19132 ), Epichloe typhina x Neotyphodium lolii
Chapter VII: Cloning of the ompd -gene of M. gramineum 173
pyr4-1 (U14564), Epichloe typhina x Neotyphodium lolii pyr4-2 (U14565 ), Hypocrea jecorina (Trichoderma reesei ) ( X55880 ), Mortierella alpina ( AB109469 ), Neurospora crassa ( X05993 ), Penicillium camemberti ( AB100244 ), Penicillium chrysogenum ( X08037 ), Penicillium nalgiovense ( AF510725 ), Phycomyces blakesleeanus ( X53601 ), Rhizopus niveus (D17362 ), Rhizopus oryzae ( AF497632 ), Schizophyllum commune ( M26019 ), Solorina crocea ( AF206163 ), Sordaria macrospora ( Z70291 ), Trichoderma harzianum ( U05192 ). - Amino acid sequences: Acremonium chrysogenum (Cephalosporium acremonium ) ( P14017 ), Aspergillus awamori ( Q5J2D0 ), Aspergillus fumigatus ( O13410 ), Aspergillus kawachii (Q96WP7 ), Aspergillus niger ( P07817, Q9HGS5 ), Aspergillus oryzae ( O13416 ), Aureobasidium pullulans ( Q9P8X9 ), Auxarthron zuffianum strain UAMH 3079 (Q9C155 ), Auxarthron zuffianum strain UAMH 4082 (Q9C156), Auxarthron zuffianum strain UAMH 1875 (Q9C158), Blakeslea trispora ( Q6X1B7 ), Cladosporium fulvum ( Q9HFV8 ), Coccoides immitis strain CA3 (Q9C144 ), Coccoides immitis strains CA4, S and MX1 (Q9C0T0 ), Cryptococcus humicola ( Q7Z7W9 ), Emericella nidulans ( Aspergillus nidulans ) ( P10652 ), Epichloe typhina x Neotyphodium lolii pyr4-1 ( Q12565 ), Epichloe typhina x Neotyphodium lolii pyr4-2 ( Q12566 ), Hypocrea jecorina (Trichoderma reesei) (P21594 ), Mortierella alpina ( Q6F5H7 ), Mucor circinelloides ( P32431 ), Neurospora crassa ( P05035, Q7RV44 ), Penicillium camemberti ( Q7Z8L4 ), Penicillium chrysogenum ( P09463 ), Penicillium nalgiovense ( Q8J269 ), Phycomyces blakesleeanus ( P21593 ), Rhizomucor pusillus ( Q9Y720 ), Rhizopus oryzae ( Q71HN5 ), Rhizopus niveus ( P43230 ), Schizophyllum commune ( P14964 ), Solorina crocea ( Q9HF68 ), Sordaria macrospora ( P78748 ), Trichoderma harzianum ( Q12709 ), Ustilago maydis ( P15188 ).
2.4. Complementation of the uracil auxotrophic Aspergillus nidulans A722 strain
2.4.1. Transformation conditions A transformation experiment with the isolated ompd -gene was performed as described in chapter I. STC-solution was used to stabilise the protoplasts osmotically instead of GTC. This solution contains 1,2 M D-sorbitol (Sigma), 50 mM CaCl 2.2H 2O (UCB), 10 mM Tris pH 7.5 (Merck Eurolab) and 35 mM NaCl. A 7.5mL suspension of 1.6.10 7 protoplasts/mL was transformed with 61.8 µg purified pOV. These protoplasts were plated on AMM medium, which was osmotically stabilised with 1.2 M sorbitol.
2.4.2. Verification of the transformants The uracil prototrophic colonies obtained in the transformation experiment were tested as to their 5-FOA sensitivity by plating them on AMM with 1 g/L 5-FOA (pH 4.5). The control medium was AMM (pH 4.5). The primer pair ‘5 cDNA’ and ‘3 cDNA’, specific for the ompd -gene of M. gramineum was used to control the integration of this gene in the genome of the A. nidulans colonies.
Chapter VII: Cloning of the ompd -gene of M. gramineum 174
3. RESULTS AND DISCUSSION
3.1. Cloning of the Myrothecium gramineum ompd -gene
3.1.1. Development of a degenerate primer pair The degenerate primers for the isolation of the M. gramineum ompd -gene were designed based on conserved sequences in the DNA and the amino acid sequences of the ompd -genes and enzymes of related filamentous fungi and yeasts. First, a multiple sequence alignment was carried out with known amino acid sequences of 50 closely related fungi. All of them are Ascomycota , like Myrothecium . Three conserved sequences were found (Figure VII. 3-1): - FLIFEDRKF-DIG-TV (most closely to the 5’ end) - RGLLILA-M - GD--GQQY-T (most closely to the 3’ end)
PYRF_CLAFU 60 VIKTHIDILS--DFGPETING--LNALAEKHN FLIFEDRKF IDIG NTV QKQYHGGALKISEWAHIINCAV PYRF_ASPFU 60 VIKTHIDILT--DFSVDTING--LNVLAQKHN FLIFEDRKF IDIG NTV QKQYHGGALRISEWAHIINCSV PYRF_ASPNG 60 VIKTHIDILS--DFSDETIEG--LKALAQKHN FLIFEDRKF IDIG NTV QKQYHRGTLRISEWAHIINCSI PYRF_ASPOR 60 VIKTHIDILS--DFSEETITG--LKALAEKHN FLIFEDRKF IDIG NTV QKQYHGGTLRISEWAHIINCSI PYRF_PENCH 63 VIKTHIDILS--DFSQETIDG--LNALAQKHN FLIFEDRKF IDIG NTV QKQYHNGTLRISEWAHIINCSI PYRF_NEUCR 68 VLKTHYDLITGWDYHPHTGTGAKLAALARKHG FLIFEDRKF VDIG STV QKQYTAGTARIVEWAHITNADI PYRF_SORMA 66 VLKTHYDLITGWDYHPHTGTGAKLAALARKHG FLIFEDRKF VDIG STV QKQYTAGTARIVEWAHITNADI PYRF_CEPAC 62 VLKTHYDMVAGWDFTPETGTGARLAKLARKHG FLIFEDRKF GDIG NTV ELQYTQGAARIIEWAHIVNVNM PYRF_TRIHA 62 VLKTHYDMVSGWDFHPDTGTGAKLASLARKHG FLIFEDRKF GDIG HTV ELQYTSGSARIIDWAHIVNVNM PYRF_TRIRE 62 VLKTHYDMVSGWTSHPETGTGAQLASLARKHG FLIFEDRKF GDIG HTV ELQYTGGSARIIDWAHIVNVNM * *** * * * * ** ** ********* *** ** ** * * **** *
PYRF_CLAFU 147 ------GS------ERGLLILA EMTSKGSLATGEY PYRF_ASPFU 147 ------GP------ERGLLVLA EMTSKGSLATGEY PYRF_ASPNG 147 ------GP------ERGLLILA EMTSKGSLATGQY PYRF_ASPOR 147 ------GS------ERGLLILA EMTSKGSLATGQY PYRF_PENCH 150 ------GS------ERGLLILA EMTSKGSLATGAY PYRF_NEUCR 201 -KDTDGRKSSIVSITTVTQTYEPADSPRLVKTISEDDEMVFPGIEEAPLD RGLLILA QMSSKGCLMDGKY PYRF_SORMA 199 EKDTDGRKGSIVSITTVTQTYEPADSPRLAKTISEGDEAVFPGIEEAPLD RGLLILA QMSSKGCLMDGKY PYRF_CEPAC 201 -ESSDGRKGSIVSVTTVTQQYESAHSPRLTKTIAEEGDMLLAGLEEPPLN RGLLILA QMSSAGNFMNAEY PYRF_TRIHA 199 GRNGDGRKGSIVSITTVTQQYESAASPRLGKTIAEGDESLFPGIEEAPLN RGLLILA QMSSEGNFMTGEY PYRF_TRIRE 201 NRSGDGRKGSIVSITTVTQQYESVSSPRLTKAIAEGDESLFPGIEEAPLS RGLLILA QMSSQGNFMNKEY **** ** * * * *
PYRF_CLAFU 221 ----- GD KL GQQY QTPQ SAIG-RGADFIIAGRGIYTAPDPVEAAKQYQQQGWEAYLARVGGASQ PYRF_ASPFU 222 ----- GD KL GQQY QTPA SAIG-RGADFIIAGRGIYAAPDPVEAAQRYQKEGWEAYMARVCGKS- PYRF_ASPNG 222 ----- GD KL GQQY QTPA SAIG-RGADFIIAGRGIYAAPDPVQAAQQYQKEGWEAYLARVGGN-- PYRF_ASPOR 222 ----- GD KL GQQY QTPE SAVG-RGADFIIAGRGIYAAPDPVEAAKQYQKEGWDAYLKRVGAQ-- PYRF_PENCH 223 ----- GD KL GQQY QTPQ SAVG-RGADFIISGRGIYAAADPVEAAKQYQQQGWEAYLARVGAQ-- PYRF_NEUCR 340 ----- GD GL GQQY NTPD NLVNIKGTDIAIVGRGIITAADPPAEAERYRRKAWKAYQDRRERLA- PYRF_SORMA 339 ----- GD GL GQQY NTPD NLVNIKGTDIAIVGRGIITASDPPAEAERYRRKAWKAYQDRRERLA- PYRF_CEPAC 317 DAELR GD GK GQQY NTPE KLIGVCGADIVIVGRGILKAGDLQHEAERYRSAAWKAYTERVR---- PYRF_TRIHA 319 NGKVG GD GQ GQQY NTAH KIIGIAGSDIAIVGRGILKASDPVEEAERYRSAAWKAYTERLLR--- PYRF_TRIRE 321 NGSVG GD GQ GQQY NTPH KLIGIAGSDIAIVGRGILKASDPVEEAERYRSAAWKAYTERLLR--- ** **** * * * * **** * * * * * ** * Figure VII. 3-1. Multiple sequence alignment of the OMPD amino acid sequences of closely related fungi. The sequences FLIFEDRKF-DIG-TV and GD--GQQY-T were also used in previous research to develop the degenerate primers (Table VII. 1-2). It was decided to use these two sequences for the design of degenerate primer pair for M. gramineum . The corresponding DNA sequences of these amino acid sequences were identified and compared in order to seek consensus sequences and to allow the designed primers to be as specific as possible (see Table VII. 3-1 for the forward primer and Table VII. 3-2 for the reverse primer). The sequence of the designed primer pair is also given in these tables. The theoretical length of the expected amplification products is given in Table VII. 3-3. For the Sordariomycetes , a fragment of 700 to 750 bp is expected, while for all other fungi the fragment is about 430 bp. Because M. gramineum belongs to the Sordariomycetes , a fragment of 700 to 750 bp is expected.
Chapter VII: Cloning of the ompd -gene of M. gramineum 175
Table VII. 3-1. Corresponding DNA sequences of the FLIFEDRKF-DIG-TV conserved sequence used to design the forward primer
Organism Conserved sequence F L I F E D R K F X D I G Sordariomycetes Acremonium chrysogenum C TTC CTC ATC TTT GAG GAC CGC AAG TTT GGC GAT ATT GG Epichloe typhina A TTC TTG ATA TTC GAA GAT CGC AAG TTC GCT GAC ATT GG Neurospora crassa C TTC CTC ATC TTC GAG GAC CGC AAG TTC GTC GAC ATT GG Sordaria macrospora C TTC CTC ATC TTC GAG GAC CGC AAG TTC GTC GAC ATT GG Trichoderma harzianum C TTC CTC ATT TTC GAA GAC CGC AAG TTC GGG GAC ATT GG Hypocrea jecorina C TTC CTC ATC TTC GAG GAC CGC AAG TTT GGC GAC ATT GG Eurotiomycetes Aspergillus awamori C TTC CTC ATC TTC GAG GAC CGC AAA TTC ATT GAC ATC GG Aspergillus fumigatus C TTT TTG ATC TTC GAG GAC CGC AAA TTC ATC GAC ATC GG Aspergillus kawachii C TTC CTC ATC TTC GAG GAC CGC AAA TTC ATT GAC ATC GG Aspergillus nidulans C TTC CTC ATC TTT GAG GAC CGC AAG TTC ATC GAC ATC GG Aspergillus niger C TTC CTC ATC TTC GAG GAC CGC AAA TTC ATC GAC ATT GG Aspergillus oryzae T TTC CTC ATC TTC GAA GAT CGC AAG TTC ATC GAT ATC GG Aspergillus parasiticus T TTC CAC ATC TTC GAA GAT CGC AAG TTC AGA GAT ATC GG Penicillium camemberti C TTC CTC ATC TTC GAA GAC CGC AAA TTC ATC GAC ATC GG Penicillium chrysogenum C TTC CTT ATC TTC GAA GAC CGC AAA TTC ATC GAC ATC GG Penicillium nalgiovense C TTC CTC ATC TTC GAA GAC CGC AAA TTC ATC GAC ATC GG Auxarthron zuffianum G TTC TTG ATT TTC GAG GAC CGC AAG TTC GTC GAT ATT GG Coccidioides immitis C TTC CTG ATC TTT GAG GAC CGG AAA TTC GTT GAT ATT GG Uncinocarpus reesei C TTC CTC ATC TTC GAA GAC CGG AAG TTC GTC GAT ATC GG Other Cladosporium fulvum C TTT CTC ATC TTT GAG GAC CGC AAG TTC ATC GAC ATC GG Solorina crocea C TTT CTC ATC TTC GAG GAC CGG AAA TTC GTT GAT ATC GG Clustal consensus ** ** ** ** ** ** ** ** ** ** ** Consensus sequence N TTY YTB ATH TTY GAR GAY CGS AAR TTY RBN GAY ATY GG OMPDforward C CTS ATC TTY GAR GAY CGC AA Table VII. 3-2. Corresponding DNA sequences of the GD--GQQY-T conserved sequence used to design the reverse primer
Organism Conserved sequence G D X X G Q Q Y Q/N T P/A Sordariomycetes Acremonium chrysogenum GGC GAT GGG AAG GGC CAG CAG TAC AAC ACC CCC GA Epichloe typhina GGC GAC GGA AAA GGC CAG CAG TAT AAT ACA CCA CA Neurospora crassa GGC GAC GGA CTG GGT CAG CAG TAC AAC ACG CCG GA Sordaria macrospora GGT GAC GGA CTT GGT CAG CAG TAC AAC ACG CCG GA Trichoderma harzianum GGA GAT GGC CAA GGC CAG CAG TAC AAC ACG GCG CA Eurotiomycetes Aspergillus kawachii GGA GAT AAG CTC GGT CAG CAG TAC CAG ACG CCC GC Aspergillus nidulans GGG GAT AAG CTG GGG CAG CAG TAT CAG ACA CCT GG Aspergillus niger GGA GAT AAG CTC GGT CAG CAG TAC CAG ACT CCC GC Aspergillus fumigatus GGA GAT AAG CTT GGA CAG CAA TAC CAG ACT CCT GC Aspergillus oryzae GGT GAC AAG CTG GGA CAG CAG TAC CAA ACT CCT GA Aspergillus parasiticus GGA GAC AAG CTG GGA ACA CAA TAC CAG ACT CCT GA Penicillium camemberti GGC GAT AAG CTC GGT CAG CAG TAC CAG ACA CCG CA Penicillium chrysogenum GGC GAT AAG CTC GGT CAG CAG TAC CAG ACG CCG CA Penicillium nalgiovense GGC GAT AAG CTC GGT CAG CAG TAC CAG ACG CCG CA Clustal consensus ** ** ** ** ** * ** * Consensus sequence GGN GAY RRV MWN GGN MMR CAR TAY MAN ACN SCN SV W GGN CAG CAG TAC MAV ACD CC OMPDreverse GG HGT BTK GTA CTG CTG NCC W
Chapter VII: Cloning of the ompd -gene of M. gramineum 176
Table VII. 3-3. Theoretical length of the amplification products obtained with the designed primer pair
Organism PCR fragment Organism PCR fragment (bp) (bp) Eurotiomycetes Sordariomycetes Aspergillus awamori 432 Acremonium chrysogenum 714 Aspergillus kawachii 432 Neurospora crassa 750 Aspergillus nidulans 426 Sordaria macrospora 753 Aspergillus niger 432 Trichoderma harzianum 720 Aspergillus oryzae 432 Hypocrea jecorina 726 Penicillium camemberti 429 Penicillium chrysogenum 426 Penicillium nalgiovense 429 Other Cladosporium fulvum 429
3.1.2. Degenerate PCR on genomic DNA of M. gramineum The designed primers were used for a PCR on 187 ng of genomic DNA of M. gramineum (Figure VII. 3-2). The gel shows 3 fragments: one of 900 bp, one of 700 bp and one of 400 bp. All three fragments were gel purified and sequenced at the VIB Genetic Service Facility.
From the sequencing results, it became clear that only the 441 bp fragment was part of the ompd -gene of Myrothecium gramineum . Contradictory to the expectations, the 700 bp fragment was not a part of the gene.
The 441 bp fragment contains the strongly conserved amino-acid sequences of ompd -genes on which the degenerate primers were based and the consensus pattern which (according to the PROSITE website) all OMPD-enzymes (EC 4.1.1.23) contain: [LIVMFTAR]-[LIVMF]-x-D-x-K-x(2)-D-[IV]- [ADGP]-x-T-[CLIVMN TA], in this case IFEDRKFVDIGSTA. The DNA sequence of this fragment was blasted against the EMBL database (WU-Blast2) and showed most similarity with the pyrG gene of Aspergillus oryzae (64 %) an Aspergillus Figure VII. 3-2. Degenerate PCR fumigatus (58 %). Based on these results, it could be on genomic DNA of M. gramineum. concluded that part of the ompd -gene of Myrothecium was isolated.
Chapter VII: Cloning of the ompd -gene of M. gramineum 177
3.1.3. Genome walking
3.1.3.1. Genomic walking Four gene specific primers were designed, based on the 441 bp fragment. Their position relative to this fragment is schematically represented in Figure VII. 3-3. Table VII. 3-4 gives an indication of the length of the fragments which still have to be sequenced to know the full sequence of the ompd -gene of M. gramineum . For the DNA sequences, only the exons and the introns were considered. It can be concluded that approximately 100 amino acids (300 bp) to the 5’ end and 40 amino acids (120 bp) to the 3’ end are still required to know the full sequence of the OMPD-enzyme (gene) of M. gramineum . The walkings were performed on the genomic libraries described in chapter III for the isolation of the gpd -gene of M. gramineum .
Figure VII. 3-3. Position of the genome walking primers relative to the 441 bp fragment amplified by degenerate PCR. Table VII. 3-4. Overview of the length of the ompd -genes and enzymes of the most closely related fungi
Organism Total length OMPD Number of missing AA AA bp (exons and introns) 5’ AA 3’ AA Aspergillus nidulans 274 883 89 39 Aspergillus fumigatus 278 904 89 43 Aspergillus oryzae 277 898 89 42 Aspergillus niger 277 901 89 42 Cladosporium fulvum 278 897 89 43 Penicillium chrysogenum 278 882 92 40 Trichoderma harzianum 379 1139 93 144 Neurospora crassa 397 1193 100 133
Dra I library The first walking was carried out in the Dra I genome walking library. The primers ‘1 OMPD’ and ‘nested 1 OMPD’ were used for the upstream walking and ‘3 1 OMPD’ and ‘3 1 nested OMPD’ for the downstream walking. The primary PCRs resulted in fragments of 4 kb, 2 kb and 1650 bp for the upstream walking. The primary PCR for the downstream walking resulted in a smear of fragments, containing one weak band of 700 bp.
Chapter VII: Cloning of the ompd -gene of M. gramineum 178
The nested (secondary) PCR for the upstream walking was carried out with a 50 times diluted primary PCR mix and with the undiluted mix as samples. For the 3’ walking, the undiluted sample was used. Figure VII. 3-4 shows that the upstream walking resulted in one fragment of approximately 1.9 kb. The fragment was 100 bp smaller than the one from the primary PCR, as expected. The nested PCR for the downstream walking resulted also in a smear, and one very weak fragment at 0,6 kb. The 1.9 kb and the 0.6 kb fragment were gel purified and cloned into the pGEM-T cloning vector according to the supplier’s instructions and their sequence was determined at the VIB Genetic Service Facility.
1 2 3 M
Lane 1: upstream (5’) - nested PCR diluted primary PCR - 4.0 k b
mix
- 1.9 k b Lane 2: upstream (5’) - nested PCR undiluted op primary - 1.5 k b PCR mix
Lane 3: downstream (3’) - nested PCR undiluted primary
PCR mix - 0.6 kb Lane M: marker (SmartLadder TM )
Figure VII. 3-4. Result of the PCR walkings in the Dra I genome walking library .
The sequence of 1.9 kb fragment (1894 bp) was similar to ompd sequences of other fungi, as expected. From the 0.6 kb fragment, only 326 bp were sequenced. Both fragments contained the expected sequences overlapping with the known 441 bp fragment. After deleting the overlapping sequences and joining all three sequences together, the total known sequence of the ompd -gene of M. gramineum was now 2549 bp. This sequence contained 1478 bp of the 5’ flanking sequences, 908 bp from start to stop codon and 163 bp of the 3’ flanking sequences. Since the known sequence of the 3’ flanking region was only 163 bp and contained no polyA-signal or transcription termination signal, it was decided to perform additional walkings to the 3’ end of the gene. The PCRs of these walking were carried out with the same primers as used for the Dra I library, but this time the walkings were done in the Eco RV library and the Pvu II library. The Stu I library was not used, since there is a Stu I restriction site between the binding site of the primary primer and the one of the nested primer. It was expected to obtain fragments larger than 360 bp, since there are no recognition sites for Eco RV or Pvu II downstream the annealing site of the primers within the sequence known this far. Since the promoter region (with 5’ UTR) was already 1.5 kb, no more upstream walkings were carried out.
Pvu II library The primary PCR resulted in a 1 kb fragment and the nested PCR in a 800 bp fragment, which was, as expected, about 200 bp smaller than the first fragment (Figure VII. 3-5). The 800 bp fragment was sequenced after it was cloned into the pGEM-T vector. The sequencing results revealed that the fragment comprised 730 bp, which extended the known sequence with 367 bp up to a total of 2918 bp.
Chapter VII: Cloning of the ompd -gene of M. gramineum 179
M ’ 1 2 M TM Lane M’ : SmartLadder SF
Lane 1: primary PCR Pvu II library
Lane 2: nested PCR
TM Lane M: marker (SmartLadder )
- 1 .0 k b
- 0 .8 k b
Figure VII. 3-5. Results of the primary and nested PCR with the Pvu II library.
3.1.3.2. Cloning and sequencing of the complete ompd-gene of M. gramineum Based on the obtained sequence, primers to pick up the complete gene were developed (primers 5’ OMPD and 3’ OMPD, Figure VII. 3-6). The primers amplify a product of 2050 bp (Figure VII. 3-7). The PCR reaction mix was cleaned up and the fragment was cloned into pGEM ®-T. The plasmid is referred to as ‘pOV’ (5052 bp). The sequence of the ompd -gene on the pOV plasmid was checked at the VIB Genetic Service Facility.
Figure VII. 3-6. Schematic view of the ompd -gene of M. gramineum and the primers used to clone the gene. 1 M
Lane 1: High Fidelity PCR resulting in the amplification of the
OMPD-gene
TM Lane M: SmartLadder 2050 bp -
Figure VII. 3-7. Amplification of the ompd -gene of M. gramineum. pOV contains 632 bp of the promoter region (with 5’ UTR); 908 bp from start to stop codon and 510 bp of the 3’flanking regions of the ompd -gene of M. gramineum , an ampicillin - resistance gene ( β-lactamase), an interrupted lacZ gene, an origin of replication, a phage f1- origin and the T7 and SP6 phage promoters. The complete nucleotide sequence and deduced amino acid sequence have been deposited at the GenBank database (accession no. DQ359751 ).
Chapter VII: Cloning of the ompd -gene of M. gramineum 180
1 tttaaaggca gagagaaagt gacggacgga agcatgccgg tgccgtaccc gattgaagtg ctgcactggc 71 tctatgcccg ggccttggtt gtaagcggtg ctcaggcttt gaagtgattc gatgccttct gtacgacgcc 141 wtttactata tacgttgatc aagcgtaggg gttgatgtat caaattcaaa cwtttagacg agtacacgcg 211 tcagtcaaca ggcakttttg tagagctgaa tgtwttttta agaccaggat taraaraatc tccctcaagt 281 ctcgtttccc gaagtagacg ccagttgaga gagggaagaa ggacaaaaag tgttaggggc ctgatatcga 351 ggctctatat agctggatat tagggatatt cggccagaga gccgctattg ctgaatcttt tggttcttgt 421 tatggcgtag tggttaaacc taatgccggc cctgacaaaa agcatcttgg gtatagcgat ggatgcaagg 491 tggatggtaa agtggttttg aaaaatatga cttagttatt attaaatgat atcaaagctc tttagggtag 561 ccagaatcaa ctggagagaa tgcagctctc tacaagccag cttctatggc ttcccagcag atgcggatga 631 ttgtgcaatc acctatgagt tgttgttgga ttccacaatg agtcttggac ccccccctcc caccaggttc 701 ttcgtagaaa cagagcgttc tctgaagata cgacggtgat ccatctgtcc ataagtcgca tggggtagca 771 cgtatcactg ccctgatggg atcctatgac agtttttgac agtttttctg aagaatcagt ggtgggtaat 841 catcgtggca gaccgcatgc attcgtatgc cgagctcttc cgcttctagc tcagatgcgt agtcgtcgcg 911 tcaagaaatt tactaaagtt tctcaccagc acatgcagtg cttctacggg gttgtatgat gccaaaatct 981 gccaagggct gcttctggat tacacgaagc cgtgtcaccg gacaacccgt ggtgaaaccc actcgttggc 1051 gaggtttctg gatggcgctc ggcgttgaat ggacttccga cggttgtgac gaatagtgct cattagtgaa 1121 tagacatgtt ccgattccca accattgttg ctgaggaata gtgaatcgga aatcgccaat tttcggtcac 1191 tttttcacgt cgaacattgc atattgatac gctggccaag acgcgagtag agtctgatta gggtatgctc 1261 ctcagcctga tagtttggat ctgcataagg ggttatccgc tcctgaaacc ctctcccctt tcgttttctt 1331 ctcaacatcc ttgaattatc ctactcccct ctctcacgcc tgcagtcaag aa tata cgca cgcacactgt 1401 ctgcttttcc cacctctc ta ta ccctcttc aatcccccac tt gttatgga gaagaacgac agcgaccgca 1471 gaaaccaagg ATG AGTTCAA AATCGGGACT CCCGTATCAT ATCCGAGCCA CAACGAATCC GCACCCAGTG 1 M S S K S G L P Y H I R A T T N P H P V
1541 GCTCGAAAGC TTTTCGCCGT TGCGGAACGC AAAAAGTCGA ACCTGATCAT ATCCGCTGAT TTGACCGATA 21 A R K L F A V A E R K K S N L I I S A D L T D
1611 CAAAGAGTCT CTTGGAGTGT GCTGATG gta gagtagccaa caaacccatt gcatgctcac tcgctaactt 44 T K S L L E C A D intron
1681 atatgcgcct gtttag AGCT CGGGCCCTTC ATCTCAGTGT TCAAGACCCA TATCGACATC ATTCACGATT 53 E L G P F I S V F K T H I D I I H D
1751 TTGGCGACGA GACGGTTCGG GGCCTCAAGA GTCTGGCAAG AAAGCACGAC TTCCTCATCT TCGAGGATCG 71 F G D E T V R G L K S L A R K H D F L I F E D
1821 CAAGTTTGTG GATATTGGCT CAACAGCCCA GAAACAGTAC CATGGTGGTG CCCTGCGCAT CTCCGAGTGG 94 R K F V D I G S T A Q K Q Y H G G A L R I S E W
1891 GCCGACATTG TCAACGTCAG TCTCCTAGGG GGTGATGGCG TTGTGAATGC ACTGGGCCAG GTCATTACCG 118 A D I V N V S L L G G D G V V N A L G Q V I T
1961 ACGAGACGTT TCCCTATCGC GGTCAACGGG CCCTGCTTCT GCTTGCTGAG ATGACCACGG CCGGTAGCCT 141 D E T F P Y R G Q R A L L L L A E M T T A G S
2031 TGCCGTTGAA AAATACACAG AGCGCTGCGT CGCTGTCGCA CGAAAGAATG GCATTGCCGC TGGAGTCATG 164 L A V E K Y T E R C V A V A R K N G I A A G V M
2101 GGCTTCGTTG CCACGCGAGG CCTCGAAGAC GTTGGCAACG AACCTGGGGT CGAGAGAGAC GAGGACGAAG 188 G F V A T R G L E D V G N E P G V E R D E D E
2171 ACTTTGTCGT TTTTACTACT GGTATCAACA GCAGCCAATC TGACAATGTG CTGGGACAGA AGTATCAGAC 211 D F V V F T T G I N S S Q S D N V L G Q K Y Q
2241 ACCCGAGGCA GCCATACGTG GCGGCTCTGA TTTCATCATC GCCGGCAGGG GCATATATGC CTCTGAGGAT 234 T P E A A I R G G S D F I I A G R G I Y A S E D
2311 CGTGTTCAGG CTGCGAAGCA ATATCAAGCC GAGGGTTGGG CTGCCTATGT GGCTCGCATA GGGCCAGGGC 258 R V Q A A K Q Y Q A E G W A A Y V A R I G P G
2381 ATTAT TGA gg ggactctaag gttcaatata ccgaaacaat attgtgtcta cttgcctggt cagttgcaga 281 H Y *
2451 gcacacgtaa tctacttgtg agttggggtt cgactccaat gcatatcgtt tctaatcaaa ggatctcagt 2521 ccagggcttc tctctgtaga tcttggcctt gaatttggcc ggaaaggcca tttattaact gagtgacggg 2591 ctcgaagtcg gcttaggctt gatcggagct tttttcaccg gcattcgcct tctactgtag cccatactgg 2661 tcgtttgcag tatgcaacga gagccttaga gcgagtagcg agattcaacc agagatgatg aagccctcta 2731 cagagcagct tctaggggcg tctcgcagat taatatagat tcatctcgtc ccgagcatat caccacacca 2801 actcggcagt cgtcaaatct aacagataat caggactgac tttacgcttc cgagccacgc tcgggaagtc 2871 tttctttttg cacgtgttgg tgcttggttg attcactgag cgcagctg Figure VII. 3-8. Nucleotide and deduced amino acid sequence of the OMPD-gene of M. gramineum . Nucleotides are represented in lower case lettering (non-coding) or by capital letters (coding); amino acids by bold capital letters. Base 1-1480: promoter region, base 1481-2388: coding sequence, base 2389-2918: 3’ flanking sequences. The boxes represent sequences important for transcription, sequences important for translation are shaded in black. A CT-rich stretch in the promoter is underlined. The intron is underlined and in italic letters. An uncommon lysine residue (K) is shaded in grey. Chapter VII: Cloning of the ompd -gene of M. gramineum 181
3.2. Nucleotide sequence analysis
3.2.1. Analysis of the coding sequence Genes of filamentous fungi mostly have short introns (48-240 bp) (133). This gene contains an intron of 59 bp, which was confirmed by cDNA analysis. RNA was extracted from freshly grown mycelium and treated with DNAse. The purified mixture was used for first strand cDNA synthesis and the synthesised cDNA was used directly for PCR with the primers ‘5 cDNA’ and ‘3 cDNA’. As expected, this PCR resulted in a 703 bp fragment (Figure VII. 3-9). Another fragment of 762 bp was visible on the gel. This fragment results from the amplification of genomic DNA, of which some remainders were left in the mRNA sample. The 703 bp fragment was cloned in pGEM-T®, sequenced and its sequence was compared with the genomic sequence. The length and the position of the intron were thus confirmed. The intron is characterised by the consensus splice sites (5’ ‘GT’ (GGTAGAG) and 3’ ‘AG’ (TTTAG)) and splicing signal of filamentous fungi (GCT AAC 22 nucleotides upstream the 3’ splice site).
31 792 1 2 M
cDNA OMPD 5' cDNA OMPD 3' genomic DNA -762 bp -703 bp exon 1 intron exon 2 (157 bp) (59 bp) (689 bp) Figure VII. 3-9. Left: schematic view of the position of the intron within the coding region of the ompd -gene of Myrothecium gramineum . The positions of the primers ‘5 cDNA’ and ‘3 cDNA’ are indicated with shaded arrows. Right: lane 1: PCR on genomic DNA of M. gramineum with the cDNA primers, lane 2: PCR on a TM cDNA sample with the cDNA primers, lane M: SmartLadderSF .
The presence of an intron is unique, because -to the best of our knowledge- it is the first ompd -gene of a Sordariomycete which has an intron. Normally, only ompd -genes of non- Sordariomycetes contain introns, corresponding in length and position with the intron found here (320). Figure VII. 3-10 shows that the ompd -genes of most Ascomycota , except the class of Sordariomycetes , have one intron at a conserved position (after 157 bp). An exception is Aureobasidium pullulans , which has two introns. The Sordariomycetes have no intron in their ompd -gene. Remarkably, the Myrothecium gene, in contrast to all other ompd -genes of Sordariomycetes , has the intron at the conserved position. Zygomycota and Basidiomycota have two introns, which are also strongly conserved in position (and length).
Chapter VII: Cloning of the ompd -gene of M. gramineum 182
ZYGOMYCOTA 180 56 112 58 506 Rhizopus oryzae 265 180 56 112 58 506 Rhizopus niveus 265 180 77 112 64 509 Blakeslea trispora 266 186 65111 67 507 Phycomyces blakesleeanus 267 180 111 112 173 497 Mortierella alpina 262
BASIDIOMYCOTA 186 50 48 539 Schizophyllum commune 112 278 50 112 187 506 Cryptococcus humicola 192 269
ASCOMYCOTA 366 310 60 131 372 Aureobasidium pullulans 270 157 69 677 Aspergillus awamori 277 157 65 677 Aspergillus oryzae 277 157 69 677 Aspergillus kawachii 277 68 Aspergillus niger 157 677 277 59 668 Emericella nidulans 157 274 157 59 674 Penicillium camemberti 276 157 61 674 Penicillium nalgiovense 276 157 46 680 Penicillium chrysogenum 278 157 60 680 Cladosporium fulvum 278 157 132 686 Solorina crocea 280
ASCOMYCOTA (Sordariomycetes) 157 59 692 Myrothecium gramineum 282 1194 Neurospora crassa 397 Sordaria macrospora 1191 396 1131 Acremonium chrysogenum 376 1146 Hypocrea jecorina 381 1140 Trichoderma harzianum 379
Epichloe typhina x 1104 367 Neotyphodium lolii (1) 1101 Epichloe typhina x 366 Neotyphodium lolii(2) Figure VII. 3-10. Comparison of different fungal ompd -genes. The numbers above the lines (exons) and boxes (introns) indicate the number of nucleotides. The last column shows the length of the corresponding proteins.
3.2.2. Analysis of the promoter region and the 3’ flanking sequences With higher eukaryotes, 2 sequences are important for the initiation of transcription: the CAAT-box, normally between 70 and 90 nucleotides (nt) upstream the transcription start point (tsp), and the TATA-box, situated between 20 and 40 nucleotides upstream the tsp (133). Some genes of filamentous fungi may contain TATA-like sequences, others only have AT-rich sequences. Likewise, CAAT-sequences are described for some fungal genes, but often are absent. As indicated in Figure VII. 3-8, a potential CCAAT-fragment and 2 potential
Chapter VII: Cloning of the ompd -gene of M. gramineum 183
TATA-boxes were found in the gene of Myrothecium , but the distance between them is rather large (-305 nt, respectively -98 nt and -62 nt from the start codon).
Next to the TATA-box and the CAAT-box, the transcription initiation in genes of fungi is predominantly determined by a CT-rich sequence immediately upstream the tsp. A pyrimidine rich region was found in the promoter of this ompd -gene between 172 and 39 nucleotides upstream the start codon (100/134 = 74.6 %) (1309-1442). The two TATA-boxes are included in the CT-rich region. In this CT-rich region, two regions with a very high CT content can be distinguished: - CCCTCTCCCCTTTCGTTTTCTTCTCAACATCCTTGAATTATCCTACTCCCCTCTCTCACGCCT from -172 to -110 (1309-1371) (52/63=82.5 %) - CTTTTCCCACCTCTC TATA CCCTCTTCAATCCCCCACTT from -77 to -39 (1404-1442) (33/39=84.6 %) One of the two TATA boxes is located in the second region. This could indicate that this is a functional TATA box and that the tsp is situated closely downstream this TATA box.
The distance between the tsp and the start codon varies for filamentous fungi from 25 to 400 bp (133). The Kozak sequence is a consensus sequence surrounding the translation start point (start codon). For nearly all eukaryotic genes, a purine residue is found at position -3 of the start codon. In the majority of the cases this is an A, as for the ompd -gene of M. gramineum .
Genes of higher eukaryotes have AATAA polyadenylation signals, but these are often shorter or missing in filamentous fungi. A potential polyA signal was found 208 nucleotides downstream the stop codon. The consensus sequence for transcription termination in eukaryotes is YGTGTTYY (208), positioned downstream the polyA signal. A similar sequence (CGTGTT) was found in the ompd -gene of Myrothecium 124 nucleotides downstream the polyA sequence (Figure VII. 3-8).
3.3. Determination of the copy number of the ompd -gene As a preliminary step to the isolation of OMPD mutants, the ompd copy number in the genome of M. gramineum was determined. The probe was designed based on the DNA sequence corresponding to a conserved region, as indicated in Figure VII. 3-11. Genomic DNA from the wild type strain was digested with restriction enzymes which have no (Bam HI), one ( SmL I and Nco I) or two ( Nsi I) restriction sites in the gene. SmL I and Nco I cut within the probe region, the other enzymes cut outside this region. As shown in Figure VII. 3-12, digestion with enzymes that do not cut in this gene results in one single hybridisation signal, whereas two bands were observed in DNA digested with restriction enzymes that cut once in the probe. Digestion with a restriction enzyme that cuts twice outside the probe also resulted in one signal. These results indicate that a single copy of the ompd -gene is present in the genome of M. gramineum . This allows the isolation of OMPD-
Chapter VII: Cloning of the ompd -gene of M. gramineum 184 negative mutants as a means towards the establishment of a genetic transformation system for M. gramineum , based on the use of the homologous ompd -gene as a selection marker.
Figure VII. 3-11. Schematic representation of the probe and the expected fragments for Southern blot analysis of the genomic DNA from Myrothecium . ‘i’ stands for the intron and ‘stop’ for the stopcodon. The marker ‘100’ shows the position were the OMPD-genes of Sordariomycetidae normally have 100 amino acids extra. The ‘c’ illustrates a very conserved amino acid region. The coding sequence is indicated by a grey shaded pointer, the probe by an open one.
Bam HI Nco I Marker SmL I Nsi I Figure VI I. 3-12 . Results obtained after restriction of the genomic DNA with Bam HI, Nco I, SmL I and Nsi I and hybridisation with the
5148 ompd -probe. The marker used is the DNA 4973 Molecular-Weight Marker III, DIG-labelled 3530 (Roche Diagnostics). The numbers on the right side of the marker correspond with the length of 2027 1904 its fragments (bp). 1584 1375
3.4. Protein sequence and comparison to other OMPD sequences The gene codes for an enzyme of 282 amino acids, with a calculated molecular mass of 30 kDa. The enzyme contains the recognition site found in all known OMPD-enzymes. This sequence is represented by the consensus [LIVMFTAR]-[LIVMF]-x-D-x-K-x(2)-D-[IV]- [ADGP]-x-T-[CLIVMNTA] (PROSITE – website). In this pattern, the K (lysine residue) is absolutely essential for the activity of the enzyme. In the amino acid sequence of the OMPD- enzyme of M. gramineum , the pattern is found as IFEDRKFVDIGSTA. Thus, the obtained sequence is certainly representing an OMPD-enzyme [orotidine 5'-phosphate decarboxylase (OMPdecase), EC 4.1.1.23]. OMPD-enzymes of Sordariomycetes normally consist of about 360-400 amino acids, while those of other Ascomycota enclose approximately 280 (330). Thus, on top of the fact that the ompd -gene of M. gramineum has an intron, its protein also
Chapter VII: Cloning of the ompd -gene of M. gramineum 185 lacks the 100 amino acid insert while other ompd -genes of Sordariomycetes lack an intron and their enzymes do have the insert of 100 amino acids (Figure VII. 3-10). The amino acid sequence of the OMPD-enzyme of M. gramineum was blasted against the UNIPROT database (Release 5.0). The WU-BLASTp (v2.0) program on the EBI-website was used with the standard parameters, except for the score matrix (PAM250). Comparison of the OMPD-enzyme of Myrothecium with other OMPD-enzymes revealed -as expected- a higher similarity with enzymes of non-Sordariomycetes and even with those of yeast than with OMPD’s of other Sordariomycetes , a class that includes the Myrothecium genus. This is why the gene here will be referred to as pyrG and not pyr4 . Pyr4 is normally used for ompd -genes of Sordariomycetes , while pyrG is used for ompd -genes of non-Sordariomycetes. Blast results with the amino acid sequence revealed that the enzyme shows the highest similarity with OMPD-enzymes of Aspergillus sp. (79 % similarity with the OMPD of A. oryzae ), Penicillium sp., Cladosporium fulvum and Solarina crocea .
Another finding is that this enzyme has a lysine residue at position 231 of the amino acid sequence (K, Figure VII. 3-6) instead of a strongly conserved glutamine residue (Q, Figure VII. 3-13).
Candida albicans GDGLGQQYRTVD Zygosaccharomyces bailii SDALGQQYRTVE Candida boidinii GDALGQQYRTVS Acremonium chrysogenum GDGKGQQYNTPE Candida dubliniensis GDGLGQQYRTVN Aspergillus awamori GDKLGQQYQTPA Candida glabrata GDALGQQYRTVD Aspergillus fumigatus GDKLGQQYQTPA Candida lusitania GDALGQQYRTVQ Aspergillus niger GDKLGQQYQTPA Candida maltosa GDGLGQQYRTVD Aspergillus oryzae GDKLGQQYQTPE Candida parapsilosis GDNLGQQYRTVD Aureobasidium pullulans GDGLGQQYNTPE Candida rugosa GDALGQQYRTVD Cladosporium fulvum GDKLGQQYQTPQ Candida tropicalis GDALGQQYRTVD Emericella nidulans GDKLGQQYQTPG Kluyveromyces lactis GDALGQQYRTVD Epichloe typhina GDGKGQQYNTPQ Kluyveromyces marxianus GDALGQQYRTVD Hypocrea jecorina GDGQGQQYNTPH Pichia angusta GDSLGQQYRTVD Neurospora crassa GDGLGQQYNTPD Pichia fabianii GDGLGQQYRGVD Penicillium camenbertii GDKLGQQYQTPQ Pichia farinosa GDGLGQQYRTVD Penicillium chrysogenum GDKLGQQYQTPQ Pichia jadinii GDSLGQQYRTVD Penicillium nalgiovense GDKLGQQYQTPQ Pichia pastoris GDALGQQYRTVS Solorina crocea GDPLGQQYQTPE Pichia stipitis GDSLGQQYRTVD Sordaria macrospora GDGLGQQYNTPD Saccharomyces cerevisia GDALGQQYRTVD Trichoderma harzarium GDGQGQQYNTAH Saccharomyces exiguus GDALGQQYRTVD Myrothecium gramineum DNVLGQKYQTPE Figure VII. 3-13. Strongly conserved glutamine residue in the amino acid sequence of yeast and fungal OMPD-enzymes (amino acids 224 to 236). Filamentous fungi are indicated in bold printing.
These novel findings can be interesting for further research related to the taxonomical classification of Myrothecium and to the transformation efficiency of systems based on the use of this pyrG gene.
Chapter VII: Cloning of the ompd -gene of M. gramineum 186
A phenomenon which is often seen in genes of filamentous fungi is codon bias (133). Codon bias is most pronounced in genes which are highly expressed. In those cases, there is a preference for the most abundant tRNA’s. The codon use in the ompd -gene of M. gramineum was examined (Table VII. 3-5). Table VII. 3-5. Analysis of the codon use in the ompd -gene of M. gramineum (283 codons)
AA 1 Codon %2 AA Codon % AA Codon % AA Codon % Ala GCA 13 His CAC 43 Pro CCA 25 Ser AGC 18 GCC 50 CAT 57 CCC 38 AGT 24 GCG 7 Ile ATA 21 CCG 25 TCA 18 GCT 30 ATC 53 CCT 12 TCC 12 Cys TGC 50 ATT 26 Gln CAA 36 TCG 12 TGT 50 Lys AAA 31 CAG 64 TCT 18 Asp GAC 58 AAG 69 Arg AGA 12 Val GTA 0 GAT 42 Leu CTA 5 AGG 6 GTC 36 Glu GAA 28 CTC 33 CGA 24 GTG 27 GAG 72 CTG 33 CGC 35 GTT 36 Phe TTC 58 CTT 19 CGG 12 Trp TGG 100 TTT 42 TTA 0 CGT 12 Tyr TAC 22 Gly GGA 11 TTG 10 Thr ACA 31 TAT 78 GGC 46 Met ATG 100 ACC 25 STOP TAA 0 GGG 18 Asn AAC 50 ACG 31 TAG 0 GGT 25 AAT 50 ACT 13 TGA 100 1 AA = amino acid 2 % = procentual use of codon per amino acid
Analysis of the codon use shows that there is a preference for codons with a pyrimidine (C or T) at the third position and more precisely C is chosen above T. Codons ending on A are apparently omitted. This can be seen very clearly for Leu and Val, but also for Ala, Glu, Gly, Ile and Lys. The preferred use of codons ending on C and the avoidance of codons ending on A is also observed in other genes of filamentous fungi (133, 331, 273). Furthermore it can be noticed that two codons are not used at all, i. e. TTA (Leu) and GTA (Val). Similar observations were found in Sordaria macrospora , Neurospora crassa and Acremonium chrysogenum (331, 273).
A parameter which is often used to determine the codon bias in a certain gene is the ‘ effective number of codons ’ (Nc) (285). Nc is about 20 in a highly biased gene, while it comes close to 61 in genes without bias. The effective number of codons Nc was calculated for the ompd - gene of M. gramineum with the EMBOSS-program CHIPS. This resulted in an Nc = 58.06. Because this value is close to the maximum, it can be concluded that little bias occurs for the ompd -gene. It is known that ompd -genes are not highly expressed; thus, no severe codon bias was expected.
Chapter VII: Cloning of the ompd -gene of M. gramineum 187
3.5. Phylogenetic analysis Several articles describe the use of the OMPD-enzyme as a phylogenetic marker (330, 332, 333, 334, 335). Radford (332), for example, constructed a fungal phylogeny based on 14 fungal OMPD amino acid sequences. It was comparable with the taxonomy and analyses based on the 18s rRNA sequences known then. Also Kimsey and Kaiser (330) found the OMPD-enzyme to be an ideal protein for molecular evolutionary studies because of the following reasons: - all organisms have the same de novo pyrimidine biosynthesis pathway and thus must have OMPD activity - the OMPD-enzyme is a universal enzyme and nearly all investigated organisms have only one ompd -gene - the OMPD amino acid sequences contain some strongly conserved regions with amino acids which have been conserved over more than a billion years Kimsey and Kaiser compared a set of 20 amino acid sequences of vertebrate animals, slime molds, different fungi and Gram-positive and Gram-negative bacteria. Four regions of homology between all investigated sequences were found. These regions were confirmed by Rodriguez et al. (333) who performed the analysis with 50 sequences. The second region is most strongly conserved with six strictly conserved amino acids ( DxKxx DI(P/G)(S/T/N)T). The lysine residue is essential for catalytic activity of the enzyme. The distance between the four regions is also conserved and most OMPD-enzymes are about 270 amino acids long (Zygomycota , Basidiomycota and most Ascomycota (320)). Only the OMPD-enzymes of Sordariomycetes such as Hypocrea jecorina , Neurospora crassa and Acremonium chrysogenum have an insertion of 90 to 100 amino acids between region 2 and 3. Traut and Temple (335) compared the OMPD amino acid sequence of more than 80 species and found 8 amino residues on 270 residues to be very strongly conserved.
It was decided to build a fungal phylogenetic tree based on 35 fungal amino acid sequences which are currently known: 27 sequences of Ascomycota (including M. gramineum ), 2 sequences of Basidiomycota and 6 sequences of Zygomycota . The tree (Figure VII. 3-14) was rooted with Saccharomyces cerevisiae as an outgroup in the program TreeView (v1.6.6, 289). The amino acid sequences were retrieved from the UniProt database (Release 5.0). The BioEdit software (v7.0.4.1, Hall, 1999) was used for phylogenetic analysis. The phylogenetic analysis was done according to the Maximum Likelihood (ML) method. This is statistically the most robust method for these kind of analyses (336). Nara et al. (334) also used the ML method for proteins to build a phylogeny based on OMPD.
The unrooted tree has a Log Likelihood of -8770. All nodes and branches were statistically significant ( P<0.05 ). The tree corresponds well with the current taxonomy of filamentous fungi 3.
3 Classification based on the Ainsworth and Bisby's Dictionary of the Fungi, 9 th edition, available at www.indexfungorum.org/Names/fundic.asp (June, 7 th , 2007)
Chapter VII: Cloning of the ompd -gene of M. gramineum 188
The phylogeny based on the OMPD amino acid sequences divides these organisms clearly in three groups: the Ascomycota , the Basidiomycota and the Zygomycota . Within the Ascomycota three groups can be distinguished: the Eurotiomycetes , the Dothideomycetes and the Sordariomycetes . Aspergillus sp . and Penicillium sp . belong to the Eurotiomycetes , while Neurospora crassa and Trichoderma sp . belong to the Sordariomycetes . Remarkably, M. gramineum , taxonomically belonging to the Sordariomycetes , is classified here in the class of the Eurotiomycetes . This result is in accordance with the fact that the M. gramineum OMPD- enzyme resembles more to OMPD-enzymes of Eurotiomycetes than to those of Sordariomycetes : - the Ascomycota , except the Sordariomycetes , have ompd -genes with one intron at a strongly conserved position, while the Sordariomycetes (except M. gramineum ) have no introns - the OMPD amino acid sequences are normally between 262-282 amino acids long, except those of the Sordariomycetes , which have an insert of 100 amino acids. The OMPD- enzyme of M. gramineum lacks these 100 amino acids
In order to know whether other M. gramineum strains would have similar OMPD-enzymes to that of the M. gramineum strain MUCL 39210, which is used during our research, attempts were made to isolate the ompd -gene of M. gramineum strain MUCL 44829, 44487 and 11831. Degenerate PCR was performed on genomic DNA of these strains, using the primers ‘OMPD forward’ and ‘OMPD reverse’ (Table VII.2.1.). Unfortunately, only a fragment of the ompd - gene of strain MUCL 44829 was obtained. The sequence of the OMPD-enzyme of this strain also differs from the OMPD-enzyme sequences of Sordariomycetes : like the enzyme of strain 39210, it has an intron and it lacks the 100 amino acids. Accordingly, strain 44829 was placed close to strain MUCL 39210, in the class of the Eurotiomycetes, after phylogenetic analysis based on the OMPD sequences. Further analysis will be necessary in order to know if other strains or other Myrothecium species also have OMPD amino acid sequences differing from those of Sordariomycetes .
It is noteworthy that, in the case of M. gramineum strains MUCL 39210 and 44829, the results of phylogenetic research based on the OMPD-enzyme would be misleading: these Myrothecium strains would not have been classified under the Sordariomycetes , even though, for strain MUCL 39210, morphological characteristics and ITS sequence analysis prove it belongs to this class (29, 48). Moreover, phylogenetic analysis based on the GPD sequence of this strain confirmed its current classification (chapter III). A possible explanation for the discrepancy between the classification based on OMPD and earlier classifications is that horizontal gene transfer has occurred at some stage in the evolution of this Myrothecium strain, which might explain the uncommon structure of this gene as compared to other genes of Sordaryomycetes . However, as discussed in the Introduction, the Myrothecium genus is an artificial genus and it is currently under discussion (46).
Chapter VII: Cloning of the ompd -gene of M. gramineum 189
BASIDIOMYCOTA Ustilago maydis Schizophyllum commune Rhizomucor pusillus Phycomyces blakesleeanus ZYGOMYCOTA Mucor circinelloides Blakeslea trispora Rhizopus oryzae Rhizopus niveus Aureobasidium pullulans Dothideomycetes Sordaria macrospora Neurospora crassa Hypocrea jecorina Sordariomycetes Trichoderma harzianum Acremonium chrysogenum Epichloe typhina x Neotyphodium lolii pyr4-1 Epichloe typhina x Neotyphodium lolii pyr4-2 Uncinocarpus reesii
ASCOMYCOTA Coccidioides posadasii Coccidioides immitis strain CA3 Coccidioides immitis strains S, MX1 and CA4 Emericella nidulans Eurotiomycetes Paracoccidioides brasiliensis Myrothecium gramineum MUCL 44829 Myrothecium gramineum MUCL 39210
Auxarthron zuffianum strain UAMH 3079 Auxarthron zuffianum strain UAMH 4082 Auxarthron zuffianum strain UAMH 1875 Cladosporium fulvum Penicillium camenbertii Penicillium nalgiovense Penicillium chrysogenum Aspergillus oryzae Aspergillus kawachii Aspergillus niger Aspergillus fumigatus 10 Figure VII. 3-14. Phylogenetic analysis of OMPD sequences using the Maximum Likelihood method with the OMPD of S. cerevisiae as outgroup. 3.6. Functional complementation of a defined OMPD-deficient Aspergillus strain To prove that this cloned gene codes for a functional enzyme, the vector pOV was used to complement a defined OMPD-negative strain of A. nidulans : this strain, A722, can only grow when uracil is present in the medium (Figure VII. 3-15) and it is 5-FOA-resistant (resistant to up to 3 g/L 5-FOA when 10 mM uracil and uridine are added, see also Figure VIII. 3-2).
A total of 1.2x10 8 protoplasts was transformed with 61.8 µg pOV. After 6 days, 117 colonies were transferred from the AMM + sorbitol plates to AMM plates. During the next days, more colonies appeared and those were also transferred. As a result, a total of 597 colonies was obtained. The first 117 colonies were replica-plated a second time on AMM to investigate their stability: 113 colonies (= 97 %) were able to grow. A transformation efficiency of 0.08 transformants per µg DNA per 10 6 protoplasts was reached. It is difficult to compare transformation efficiencies of different studies because they are dependent on, for example, the transformation method, the strain under investigation and the nature of the transforming DNA (integrative, replicative, linear, circular). In Table VII. 3-6 the transformation efficiency of some systems is compared. Compared to these results, the transformation efficiency of this system is rather low. However, it has to be noticed that in this case A. nidulans was
Chapter VII: Cloning of the ompd -gene of M. gramineum 190 transformed with the ompd -gene of M. gramineum , a Sordariomycete , and that there is only 76% similarity and 56 % identity between the proteins of both fungi. A decrease of 12.5% of the efficiency can be observed when the homology drops from 100 to 15% with H. jecorina . Another reason for the low efficiency could be the fact that A. nidulans is unable to efficiently express the ompd -gene of M. gramineum , regulated by the Myrothecium promoter. In this case, the exchange of the Myrothecium promoter by a homologous promoter of A. nidulans could result in a higher transformation efficiency. Table VII. 3-6. Comparison of transformation efficiencies obtained with the OMPD selection marker for a few filamentous fungi ( A. = Aspergillus, H. = Hypocrea, N. = Neurospora )
Transformed strain Origin OMPD Similarity (%) # transformants Reference per µg plasmid A. fumigatus A. fumigatus 100 150 327 A. nidulans N. crassa 49 0.33 – 0.66 337 A. nidulans M. gramineum 76 9.6 This study A. nidulans A. fumigatus 86 50 327 A. nidulans A. niger 100 1000 317 A. niger N. crassa 50 2 338 A. niger A. niger 100 8-50 317 A. niger A. niger 100 20-80 319 A. niger A. niger 100 40 338 A. oryzae A. niger 98 16 339 H. jecorina A. niger 50 700-800 340 H. jecorina N. crassa 75 1500 340 H. jecorina H. jecorina 100 12000 318 Rhizomucor pusillus Rhizomucor pusillus 100 5 328
A selection of 18 complemented colonies was tested as to their 5-FOA sensitivity by plating them on AMM with 1 g/L 5-FOA (pH 4.5). AMM was the control medium (pH 4.5) (Figure VII. 3-15). All colonies, except colony 103, grew on the AMM medium (without uracil) and thus were stable. None of the transformants was able to grow on the medium with 5-FOA (results shown in Figure VII. 3-15 for colonies 38, 39, 107 and 111), which again proofs that a functional OMPD is present. The wild type A. nidulans was able to grow on this 5-FOA- medium (3 g/L) with uracil and uridine.
A PCR, specific for the ompd -gene of M. gramineum was carried out to check the integration of this gene in the genome of 18 A. nidulans transformants. The primers which were used are the ‘5 cDNA’ and the ‘3 cDNA’ primer (Table VII. 2-1). Genomic DNA of A. nidulans was prepared according to the method of Chow and Käfer (245), as described in chapter II. All PCRs resulted in the expected 762 bp fragment while the wild type strain did not yield any fragment (Figure VII. 3-16). The fragments can not clearly be noticed for the colonies 57, 63 and 90, but they were detectable on the gel. The wild type result is not shown. These results confirm the presence of at least one copy of the ompd -gene of M. gramineum in the genome of the Aspergillus strain.
Chapter VII: Cloning of the ompd -gene of M. gramineum 191
Aspergillus nidulans FGSC A722
+ uracil - uracil
39 38 111 107
Figure VII. 3-15. Upper left: growth of Aspergillus nidulans A722 on AMM with and without uracil. Upper right: test of the 5-FOA- sensitivity of the transformants. Lower part: growth of transformants on AMM without uracil. The numbers correspond to the number of the colony.
Tr 38 Tr 39 Tr 41 Tr 49 Tr 57 Tr 63 Tr 66 Tr 72 Tr 77 Tr 86 Tr 90 Tr 93 Tr 95 Tr Tr 106 Tr Tr 100 Tr 103 Tr
Marker
Figure VII. 3-16. PCR analysis of transformants with primers specific for the OMPD-gene of
Myrothecium gramineum . 4. CONCLUSION The orotidine-5’-monophosphate decarboxylase gene of Myrothecium gramineum was cloned. It is the second gene of Myrothecium gramineum that has ever been cloned and analysed. The 2050 bp gene codes for an enzyme of 282 amino acids and has an intron of 59 bp, which is unique for an ompd -gene of a Sordariomycete . Accordingly, phylogenetic analysis based on the amino acid sequence of the M. gramineum OMPD did not confirm the current classification of M. gramineum within the class of the Sordariomycetes . The gene and its amino acid sequence show high similarity with OMPD-enzymes of Aspergillus sp., Penicillium sp., Cladosporium fulvum and other fungi. It was shown that this Myrothecium strain has only one ompd copy. The functionality of the enzyme as a selection marker was proven by complementation of the uracil auxotrophy of A. nidulans A722 and by the fact that it renders the strain 5-FOA-sensitive. The transformants were uracil prototroph and 5-FOA sensitive. This enzyme will be used to develop a homologous transformation system for M. gramineum (chapter VIII).
Chapter VIII: A new selection system for M. gramineum 192
Chapter VIII
Development of a new selection system based on the ompd -gene of Myrothecium gramineum
1. INTRODUCTION Myrothecium gramineum is a filamentous Ascomycete fungus belonging to the class of the Sordariomycetes . To use this fungus for the industrial production of enzymes, an efficient expression system is required, as well as an easy selection method for clones of this fungus carrying the introduced expression cassette.
The literature describes many different methods to select transformants of fungi (25, 208). On the one hand, one can use dominant selection markers, such as antibiotic resistance markers. However, since antibiotics cannot be used for some industrial applications (e.g. in the food industry) or are not suitable regarding their cost price and stability when large scale cultures possibly requiring long cultivation periods, are needed. For those reasons, other non- antibiotic, dominant selection markers were developed. An example is the acetamidase gene (amdS ) which was used as a selection marker for the first time by Tilburn and co-workers (1983) for the transformation of Aspergillus nidulans (51) and has since been widely used for the transformation of filamentous fungi. Transformants which express the amdS gene are able to grow on media with acetamide as the only nitrogen source. Counter selection for amdS positive strains can be performed using their sensitivity for fluoroacetamide (94). Because Myrothecium gramineum MUCL 39210 can not grow on minimal medium with acetamide as the sole nitrogen source, the amdS gene could theoretically be used as a selection marker for M. gramineum (29). However, protoplast regeneration problems on the minimal media which are required for the selection and the repression of the amdS gene promoter by high glucose concentrations which are needed for the osmotic stabilisation of the protoplasts hampered the development of a transformation system based on this marker (see also chapter I).
Besides dominant selection markers, auxotrophic markers can be used. Unfortunately, these have the disadvantage that the desired genotypes have to be created. For example, arginine auxotrophic mutants can be complemented with the an argB gene (54), tryptophan requiring mutants can be transformed with a trpC gene or with a trp-1 gene (96, 97, 98). An extremely useful characteristic of some auxotrophic selection systems is that the marker is both selectable and counter selectable. These systems allow the use of positive screening methods for certain auxotrophic mutants and thus can be used in genetically poorly characterised fungi. One method uses selenate resistant mutants which are ATP sulphurylase negative and can be complemented with the sC gene of A. nidulans (103). Homologous transformation systems based on the sulphurylase selection marker have only been described for A. fumigatus, A. nidulans and A. niger (103, 341, 342). Also fluoroacetate selection to obtain acetyl-CoA synthetase mutants was described (104), but only for P. chrysogenum as a (homologous)
Chapter VIII: A new selection system for M. gramineum 193 transformation system. Another system which is used more commonly is the chlorate resistance system. Mutants resistant to chlorate are nitrate reductase negative, unable to grow on media with nitrate as the sole nitrogen source and can be transformed with the niaD gene of A. nidulans as heterologous selection marker (101) (e.g. transformation of Beauveria bassiana (343) and Fusarium oxysporum (344)) or with the gene of A. niger (P. canascens (345), Gibberella fujikori (346) and F. oxysporum (347)). Heterologous selection systems have also been described based on the F. oxysporum nitrate reductase gene (for P. griseoroseum (348) and for Botrytis cinerea (349)). Homologous systems are described for A. niger (350), A. oryzae (351), A. parasiticus (352), Botryts cinerea (349), A. chrysogenum (353), F. oxysporum (354), Gibberella fujikori (355), Leptosphaeria maculans (356), P. chrysogenum (357), P. griseoroseum (358) and Staganospora nodorum (359).
Another procedure which is used a lot is the selection of orotidine monophosphate decarboxylase (OMPD) negative mutants which are resistant to 5-fluoro-orotic acid (5-FOA) and are uracil and/or uridine auxotrophic. The OMPD selection system was first developed in yeast (316) and has since been successfully applied in some industrially important fungi, e.g. Aspergillus sp. (317), Trichoderma sp. (318), Claviceps purpurea (319), Blakeslea trispora (320) and Penicillium nalgiovense (321). The mutants can be complemented with the homologous ompd -gene or with e.g. the heterologous pyrG ( A. nidulans ), pyr4 ( Neurospora crassa ) or ura3 ( S. cerevisiae , for yeasts) genes which code for orotidine monophosphate decarboxylase (105). An overview of the homologous and heterologous systems using the OMPD enzymes of filamentous fungi as selection markers is given in Table VIII. 1-1.
The orotidine monophosphate decarboxylase gene was chosen as selection marker and isolated from the genome of Myrothecium gramineum (chapter VII, GenBank accession no. DQ359751 ). In this part, the development of a homologous transformation system based on the gene is described for Myrothecium gramineum .
As with the previously described systems, both positive and negative selection is possible for OMPD-wild type strains as well as for OMPD-deficient strains: mutants are resistant to fluoro-orotic acid (5-FOA) (positive selection) and uracil auxotrophic (negative selection), while wild type strains are 5-FOA sensitive (negative) and uracil prototrophic (positive). This is very useful to perform successive transformations on the same strain (321).
Chapter VIII: A new selection system for M. gramineum 194
Table VIII. 1-1. Homologous and heterologous selection systems using the OMPD enzyme Homologous systems Heterologous systems Host Ref. Host Source organism Ref. Aspergillus awamori 119 Aspergillus aculeatus Aspergillus nidulans 360 Aspergillus fumigatus 327 Aspergillus fumigatus Neurospora crassa 122 Aspergillus nidulans 361 Aspergillus nidulans Neurospora crassa 362 Aspergillus niger 338 Aspergillus niger Neurospora crassa 363 Aspergillus oryzae 364 Aspergillus oryzae Aspergillus niger 339 Aspergillus parasiticus 325 Hypocrea jecorina Aspergillus niger 340 Aureobasidium pullulans 365 Hypocrea jecorina Neurospora crassa 340 Claviceps purpurea 319 Hypocrea jecorina Trichoderma harzianum 366 Hypocrea jecorina 367 Mucor circinelloides Blakeslea trispora 320 Mucor circinelloides 322 Mucor circinelloides Phycomyces blakesleeanus 368 Penicillium chrysogenum 369 Pleurotus ostreatus Hypocrea jecorina 370 Penicillium nalgiovense 321 Rhizopus delemar Rhizopus niveus 371 Phanerochaete chrysosporium 372 Rhizopus oryzae Rhizopus niveus 373 Rhizomucor pusillus 328 Ustilago maydis 374 Wangiella dermatitidis 375
The availability of an OMPD-negative strain and a transformation system based on the complementation of the uracil/uridine auxotrophy with the homologous ompd -gene offers several other advantages over other selection markers such as antibiotic resistance markers or the acetamidase ( amdS ) system (321): - the transformation efficiency is higher than with any other system - the pyrG -blaster can be used to knock-out several genes in successive transformation rounds (105) - any construction can be introduced in a single copy at the ompd locus by gene targeting using a mutant ompd -gene, an asset which is important when performing expression studies (119, 323) - recently, there is much concern about the use of heterologous genes to develop improved strains of microorganisms used in the food industry, and in many countries, strict regulations on this issue have been implemented. The use of a homologous selection system such as the ompd -gene rules out these apprehensions. Moreover, homologous selection systems are mostly more efficient than heterologous ones. This selection marker can be isolated easily from genomes of poorly studied organisms because it has a conserved amino acid sequence (322 and references therein). The gene coding for OMPD has been cloned and characterised from a variety of organisms from E. coli to Homo sapiens . - OMPD encoding genes in fungi are responsible for a single function and the encoded enzyme is essential for genome replication and for gene expression (376, 377). The de novo pyrimidine biosynthetic pathway is one of the oldest metabolic pathways and the six
Chapter VIII: A new selection system for M. gramineum 195
enzymatic steps are nearly identical in eubacteria, archaebacteria and eukaryotes, making the OMPD-enzyme suitable for phylogenetic studies.
The enzyme orotidine-monophosphate decarboxylase (OMPD; EC 4.1.1.23) catalyses the last step in the de novo biosynthesis pathway of pyrimidines, more precisely the decarboxylation of orotidine-5’-monophosphate (OMP) to uridine-5’-monophosphate (UMP) (330) (Figure VIII. 1-1).
Figure VIII. 1-1. De novo biosynthesis pathway of pyrimidines of filamentous fungi.
A mechanism by which the 5-FOA kills the cells was proposed to be resulting from the conversion of 5-FOA to 5-fluoro-uridine-5’-monophosphate (5-FUMP) (105, 316, 325). In A. fumigatus and A. niger it was shown by 19 F NMR spectroscopy that 5-FUMP is converted into 5-fluoro-uridine-5’-triphosphate (5-FUTP) and 5-fluoro-2’-deoxyuridine-5’-monophosphate (5-FdUMP) (378). The 5-FUTP is incorporated in RNA, while 5-FdUMP is a potential
Chapter VIII: A new selection system for M. gramineum 196 inhibitor of the enzyme thymidylate synthase (TS). For the plant Nicotiana plumbaginifolia it was also proven that 5-FUMP is converted to 5-FdUMP by the cell metabolism, which inhibits TS (379). The inhibition of TS causes a strong reduction of the thymine biosynthesis resulting in a lethal thymine deficit.
5-FOA appears to have a broad action spectrum. Boeke et al. (316) already found that 5-FOA inhibits the growth of the yeasts Saccharomyces , Schizosaccharomyces and Candida . E. coli HB101 also proved to be sensitive, while E. coli DB6507, a mutant in the pyrF gene coding for OMPD is resistant. Meanwhile, the toxicity of 5-FOA has been reported for many different organisms: certain concentrations of 5-FOA inhibit the growth of bacteria, yeasts, filamentous fungi, and plant and animal cells. However, Boeke and co-workers also found that the growth inhibition of URA + S. cerevisiae cells by 5-FOA is at least partially reversible. Of the 40 URA + cells inoculated on 5-FOA medium (3 days incubation at 30°C) 15 cells were able to recover and grow further after transfer to YPD medium.
O O F F orotate HN HN phosphoribosyltransferase (OPRT) OH O O OH N O NH HO P O O O O PRPP PPi OH 5-fluoro-orotine acid (5-FOA) OH OH 5 fluoro orotidine 5' monophosphate (5 FOMP) O
F HN orotidine-5'-monophosphate decarboxylase (OMPD) O O N HO P O O CO 2 OH
OH OH 5 fluoro uridine 5' monophosphate (5 FUMP)
5 FUTP5 FUDP 5 FdUDP 5 FdUMP
incorporation in RNA Inhibition of dUMP dTMP thymidylate synthase (TS)
Figure VIII. 1-2. Metabolism and toxic action of 5-FOA.
Different OMPD-negative mutants of filamentous fungi were isolated with the 5-FOA resistance system. For that purpose, mostly large amounts of fungal spores (10 8) are mutated via ultraviolet (UV) irradiation or with the aid of chemical mutagentia and are subsequently plated on 5-FOA containing medium. The 5-FOA concentrations in the media vary between 0.06-2.5 g/L. Uracil and/or uridine (5-20 mM) is added to allow growth of the auxotrophic
Chapter VIII: A new selection system for M. gramineum 197 mutants. An overview of the mutagenic agents which are used and the most important components of the selective media is given in Table VIII. 1-2. Table VIII. 1-2. Induction of OMPD mutants: mutagentia and medium composition
Organism Method S1 5-FOA 2 Uracil 3 Uridine 3 FOA R 4 OMPD - 5 Ref.
Aspergillus UV 5-80 0.8 - 10 11 2 317 niger DEO* 10 Aspergillus MNNG* 66 2 - 10 46 3 325 parasiticus Claviceps UV 40-50 0.06 - 10 144 9 319 purpurea Colletotrichum Screening WT - 0.87 - 5 1 0 380 graminicola Aspergillus Transformation - 0.75 - 10 27 16 119 awamori Trichoderma UV 10 1 0.89 0.41 ? 8 381 harzianum Aspergillus 4-nitroquino- 75 1 5 5 100 >3 105 fumigatus line-N-oxide Rhizomucor UV 1 1.2 2.2 - 18 1 328 pusillus Aureobasidium EMS* 20 0.8 0.45 - 65 2 365 pullulans Rhizopus MNNG ? 2.5 4.5 - 17 5 42 oryzae Aspergillus UV 10-20 1.2 - 20 200 1 360 aculeatus Penicillium UV + filtration- 20 1 - 0.57 1 1 321 nalgiovense enrichment Mortierella MNNG ? 1 0.45 - >6 0 329 alpina * DEO = 1,2,7,8-diepoxyoctane; MNNG = N-methyl-N’-nitro-N-nitrosoguanidine; EMS = ethyl methane sulfonate 1 Survival (%) 2 Concentration in g/L 3 Concentration in mM 4 Number of 5-FOA resistant colonies 5 Number of OMPD-negative colonies
Some procedures describe the use of the ‘filtration enrichment’ method to select OMPD- negative mutants. This method differs from the direct plating on 5-FOA medium after mutagenesis in that treated spores are inoculated in minimal medium without 5-FOA and without uracil/uridine. After a certain period of incubation, only the prototrophs, which do not require uracil/uridine, will have grown. The grown mycelium is harvested by filtration and the filtrate, containing spores of the OMPD-negative mutants, is again inoculated in minimal medium without 5-FOA and without uracil/uridine. Several rounds of filtration and plating result in an enrichment of the spores of the OMPD mutants. Finally, the enriched filtrate is plated on medium with 5-FOA and with uracil/uridine, or plated on medium which allows growth of both auxotrophs and remaining prototrophs and replica plated on medium with 5- FOA and with uracil/uridine. An advantage of the method is that mostly low doses of mutagenic agentia are used, resulting in less mutations at other sites than the site of interest.
Chapter VIII: A new selection system for M. gramineum 198
The method for the enrichment of auxotrophic mutants via filtration of mutated spores was optimised for N. crassa by Case (382) and has since been used for the isolation of auxotrophic mutants of Ascolobus immersus (383), T. harzianum (384) and P. nalgiovense (321). OMPD-negative mutants can also efficiently be obtained by homologous recombination of the wild type gene with a deficient gene via transformation followed by selection on 5-FOA medium. The creation of mutants by homologous recombination is preferred compared to the use of mutagenic agentia because in this way, defined mutants can be selected e.g. by Southern analysis which only have changed their DNA sequence at the site of interest. The use of defined strains is necessary for certain industrial applications. Moreover, unidentified changes in crucial enzymes can not influence the metabolism of the strains or the synthesis of the product of interest. Gouka and co-workers (119) created in this way defined OMPD- negative strains of Aspergillus awamori which were used for the development of a site- specific integration system. The construction of these strains was realised in 2 steps: first, in vitro mutations were introduced in the cloned ompd -gene of A. awamori . Next, these plasmids were used to transform A. awamori and thus to exchange the chromosomal ompd -gene by the deficient one via homologous recombination. The resulting transformants were plated on 5- FOA medium and they were tested as to their uridine-auxotrophy. Finally, the integration pattern of the transforming DNA in the auxotrophs was examined. This procedure proved to be very efficient for the isolation of OMPD-negative mutants: 60% of the 5-FOA resistant strains was obtained by gene replacement.
The 5-FOA resistance system, which allows both positive and negative selection, can be used for the development of new strategies to manipulate fungal genomes (105, paragraph 7.2 in Literature review). Alani et al. (385) developed the so-called ‘ ura -blaster’ system for yeasts and D’Enfert (105) constructed a ‘ pyrG - blaster’ for Aspergillus fumigatus and proved it could be used for knocking out several genes in the same fungal strain. The pyrG -blaster is a cassette containing the pyrG gene of A. niger , flanked by two identical elements (the neomycin phosphotransferase gene ( neo )) which form a direct repeat ( Figure VIII. 1-3). Figure VIII. 1-3. The pyrG -blaster.
The blaster is flanked by sequences homologous to the gene of interest. A uracil-auxotrophic strain ( pyrG negative) is transformed with this construct and prototrophic strains carrying the construct at the site of interest are selected. Next, these strains are submitted to a round of negative selection by plating them on medium with 5-FOA and uracil. Only the strains that
Chapter VIII: A new selection system for M. gramineum 199 lose their pyrG gene by recombination between the two direct repeats are able to grow on this medium. The desired mutation in the gene of interest is preserved, because one copy of the direct repeat remains in the target gene. Thus, this strategy results again in an auxotrophic strain with a deletion of the gene of interest. The strain can be used again for a deletion of a second gene of interest or another copy of the same gene.
Several tests can be performed in order to differentiate between OPRT- and OMPD-negative strains, which are both 5-FOA resistant. A first test is to inoculate the strains both on minimal medium and on uracil/uridine containing medium. Indeed, not all 5-FOA resistant strains are uracil or uridine auxotrophic. Skory and his team (325) observed that only 22 % of their 5- FOA resistant A. parasiticus strains were stable uridine auxotrophs. d’Enfert (105) found with A. fumigatus 79 uracil/uridine auxotrophs in 100 5-FOA resistant mutants. For Claviceps purpurea 12.5 % of the 5-FOA resistant colonies appeared to be pyrimidine auxotrophic (319). In further examination rounds, the uracil/uridine auxotrophic mutants are submitted to more specialised tests to distinguish between OPRT and OMPD-negative mutants. By the measurement of the enzymatic activity of OPRT and/or OMPD one is able to set apart OPRT- negative strains from OMPD negative mutants. These enzyme tests are based on the conversion of orotate to OMP (OPRT) and the conversion of OMP to UMP (OMPD). Such tests were used by Skory et al. (325), Smit & Tudzynski (319), Rasmussen et al. (380), Skory (42) and Takeno et al. (329).
Another method to distinguish between OPRT- and OMPD-negative mutants is via complementation with a wild type ompd -gene. OMPD-negative strains can be complemented with such a gene. If not, the mutant is probably OPRT-negative. Table VIII. 1-3 gives a view of the ompd -genes which were used to prove via complementation that obtained strains were OMPD-negative and not OPRT negative. Heerikhuisen et al . (386) observed that A. sojae pyrG mutants were not able to grow on medium only containing uridine and no uracil. The pyrG negative strains needed uracil for growth. This has never been observed before in related Aspergillus strains. Table VIII. 1-3. ompd -genes used to distinguish between OMPD and OPRT negative strains
Host ompd -gene used for complementation Reference Trichoderma harzianum pyr4 gene of Neurospora crassa 381 Aspergillus fumigatus pyrG gene of Aspergillus niger 105 Rhizomucor pusillus homologous pyr4 gene of Rhizopus pusillus 328 Aspergillus sojae pyrG gene of Aspergillus niger 386 Aspergillus aculeatus pyrG gene of Aspergillus nidulans 360 Penicillium nalgiovense pyrG gene of Penicillium chrysogenum 321
Chapter VIII: A new selection system for M. gramineum 200
In this part, the development of a homologous transformation system based on the gene is described for Myrothecium gramineum . The development of a selective medium, the construction of vectors containing defective ompd -genes and the creation of OMPD-negative strains of M. gramineum are described.
2. MATERIALS AND METHODS
2.1. Strains and standard cultivation techniques Myrothecium gramineum BCCM TM /MUCL 39210 was used as wild type strain. Aspergillus nidulans FGSC A722 ( pyrG89 pabaA1; fwA1 uaY9 ), a defined OMPD-negative strain and Aspergillus oryzae MUCL 144.92, an OMPD positive strain, were used as controls. Escherichia coli DH5 α-F’ was used for all cloning experiments. Myrothecium gramineum BCCM TM /MUCL 44487 was used as a reference Myrothecium strain and was kindly provided by the MUCL.
Standard cultivation techniques were performed as described in § 2.2.1 of Chapter I. Aspergillus nidulans A722 was grown on Aspergillus Minimal Medium (231), supplemented with 10 mM uracil, 10 mM uridine (Acros) and 5 g/L yeast extract (Difco).
Mycelium was harvested by filtration through a Mirocloth filter (Calbiochem) and the cell dry weight as determined with the Moisture Analyser XM60 (Led Techno N.V., boost program).
2.2. Standard DNA manipulation The plasmids pOVmut3’ and pOVmut5’ were amplified in E. coli DH5 α-F’ and isolated with the HiSpeed Plasmid Maxi kit (Qiagen) before transformation. Sticky ends of fragments were blunted with T 4 DNA polymerase (Roche Diagnostics) and ligations were carried out with T 4 DNA ligase (Roche Diagnostics) as specified by the supplier. All other standard procedures were performed as described in § 2.2 of chapter III.
2.3. Selective media
2.3.1. Selection against OMPD deficient strains The medium ‘anti-Oneg’ was used to select against OMPD deficient strains. This medium has the same composition as the Aspergillus Minimal Medium (231), but with Yeast Nitrogen
Base (YNB without amino acids, Difco) as nitrogen source instead of NaNO 3, i.e. (g/L) 10 glucose; 6.7 YNB; 1.52 KH 2PO 4; 0.52 MgSO 4.7H 2O; 0.52 KCl; 15 agar (Oxoid) supplemented with 1 mL/L Hutner’s trace solution.
2.3.2. Selection against OMPD positive strains The composition of the medium used to select against the OMPD positive wild type strain is given in Table VIII. 2-1. YNB (without amino acids) and Agar Noble were purchased from Difco; 5-FOA was obtained from Fermentas.
Chapter VIII: A new selection system for M. gramineum 201
Table VIII. 2-1. Composition of the medium ‘anti-Opos’ and ‘anti-OposG’ (G stands for osmotically stabilised with Glucose), concentrations are given in g/L Component Anti-Opos Anti-OposG Glucose 10 180 YNB 6.7 6.7 FOA 4 4 Uracil (2mM) 0.23 0.23
KH 2PO 4 1.52 1.52 KCl 0.52 0.52
MgSO 4.7H 2O 0.52 0.52 Agar Noble 20 20 Hutner’s trace elements 1 mL 1 mL pH 3.5 3.5
2.4. Creation of OMPD-negative M. gramineum strains
2.4.1. Transformation and homologous recombination Transformations were performed as described in chapter I for M. gramineum . Colony PCRs were performed with the primers given in Table VIII. 2-2 as described in chapter II. PCR-amplifications to prepare linear DNA for the transformation of M. gramineum were performed in four-fold with the High Fidelity PCR Master kit (Roche Diagnostics) using the primers ‘5 OMPD’ and ‘3 OMPD’ for the amplification of the 5’ mutated OMPD-gene and ‘T7’ and ‘T3’ for the amplification of the hygromycin resistance gene (Table VIII. 2-2). The reactions were carried out on plasmid preparations of pOVmut5’ and pCSN43 as described in the kit. In order to break down all remaining plasmid DNA, the samples were treated with Dpn I, where after they were purified. Table VIII. 2-2. Primer description Primer name Feature Sequence (5’-3’) U OMPD anneals upstream ompd -gene AGCCAGCTTCTATGGCTTCC D OMPD anneals downstream ompd gene CTGCGCTCAGTGAATCAACC T7 amplification of hph GGCGATTAAGTTGGGTAACG T3 amplification of hph CGCGCAATTAACCCTCAC 5 OMPD amplification of ompd mut5’ CAGACCGCATGCATTCGTATGCC 3 OMPD amplification of ompd mut5’ ACCAAGCACCAACAC 3’del anneals at 3’deletion site CCAGCGGCAATGCCATTCTT
2.4.2. UV mutagenesis The protocol used is based on the procedure described by Bos & Stadler (1996) (387). Spores were harvested in 16 mL physiological solution (0.9c% NaCl), centrifuged at 5000 rpm during 4 minutes and resuspended in physiological solution to obtain a concentration of 1 or 2.10 7 spores per mL. The spores (12 mL) were UV-irradiated with an UV lamp (UVM-57 handheld UV lamp, Upland CA) placed 30 cm above the suspension. Samples of 200 µL were
Chapter VIII: A new selection system for M. gramineum 202 taken at different time intervals and appropriate dilutions of them were plated on PDA in order to determine the percentage of survival. The plates were incubated in the dark at 25°C. Suspensions of 2.10 7 spores per mL (10mL) were treated 4, 6 or 10 minutes and plated on selective medium or submitted to several rounds of filtration enrichment. For the filtration enrichment, a sample of 10 8 spores was inoculated in 150 mL Aspergillus Minimal Medium (AMM). After 24 hours of growth in the dark (25°C, 150 rpm), the culture was filtered for the first time over a Miracloth filter (Calbiochem, 22 – 25 µm pores). Fresh AMM (50 mL) was added to the filtrate and this culture was again incubated during 24 hours. The culture was filtered, but no more fresh AMM was added. A last period of 24 hours of growth was followed by filtration. The filtrate was centrifuged (4000 rpm, 5 minutes) and resuspended in 4 mL 0.9 % NaCl, where after 100 µL of spores were inoculated on AMM and 100 µL on AMM + 10 mM uracil + 10 mM uridine. The rest of the spores were inoculated on 5-FOA plates.
3. RESULTS AND DISCUSSION
3.1. Development of selective media
3.1.1. Selection against OMPD deficient strains OMPD-negative strains are unable to grow on media which lack uracil or uridine. A defined OMPD-negative strain was used to test the selectivity of the medium ‘anti-Oneg’ against OMPD deficient strains; more precisely, the strain Aspergillus nidulans FGSC A722 was used. This strain is described several times in literature as a control during the development of a transformation system based on the OMPD selection marker (325, 337, 362). A. nidulans FGSC A722 was not able to grow on the medium ‘anti-Oneg’, as expected, since this medium does not contain any uracil or uridine. When supplemented with 10 mM uracil and 10 mM uridine, the strain was able to grow (Figure VIII. 3-1).
Aspergillus nidulans FGSC A722 + uracil and uridine uracil and uridine
Figure VIII. 3-1. Growth of A. nidulans A722 on ‘anti-Oneg’ (left) and on ‘anti-Oneg’ supplemented with uracil and uridine (right).
Chapter VIII: A new selection system for M. gramineum 203
3.1.2. Selection against OMPD positive strains The composition of the initial medium which was used to select against OMPD positive strains (5-FOA added) and which allows growth of OMPD-negative strains (uridine and uracil added) is given in Table VIII. 3-1 (‘anti-Opos’). It is based on the minimal medium normally used for growth of M. gramineum ( Aspergillus Minimal Medium (231), supplemented with 5 g/L yeast extract (Difco)) and on medium compositions used for the development of a transformation system based on the OMPD selection marker found in literature (e.g. 42, 119, 317, 328, 360, 381). Table VIII. 3-1. Initial composition of the ‘anti-Opos’ medium (pH 4.0) Component Concentration (g/L) Glucose 10
NaNO 3 6 Uracil (10 mM) 1.12 Uridine (10 mM) 2.44 5-fluoro-orotic acid (5-FOA) 3
KH 2PO 4 1.52
MgSO 4.7H 2O 0.52 KCl 0.52 Agar 15 Hutner’s trace elements 1 mL/L
Growth tests on this medium with and without 5-FOA proved that M. gramineum was sensitive to 5-FOA, but it is still able to grow on the medium with 5-FOA, even at concentrations of 3 g/L (Figure VIII. 3-2). The Aspergillus control strains performed as expected: the OMPD-negative strain was able to grow in the presence and absence of 5-FOA, while the OMPD positive strain grew vigorously on the plates without 5-FOA but not on the plates with 5-FOA. Aspergillus nidulans M. gramineum Aspergillus oryzae A722 OMPD WT OMPD + WT OMPD +
without FOA
with FOA
Figure VIII. 3-2. Growth of M. gramineum and Aspergillus control strains on the initial medium (pH 4, 3 g/L 5-FOA, 10 mM uracil and uridine).
Chapter VIII: A new selection system for M. gramineum 204
Further experiments were performed to increase the selectivity of the plates for M. gramineum . Several parameters were optimised: the concentration of uracil and uridine, the pH of the medium and the nitrogen source of the medium. Since uracil and uridine can possibly be used to synthesise UMP, without the use of 5-FOA, lowering their concentration could lead to a higher sensitivity for 5-FOA. Experiments proved that the plates are more selective when only uracil is added, and not both uridine and uracil or only uridine (results not shown). The concentration of uracil was lowered to 2 mM, which increased the selectivity of the plates: the plates were no longer completely covered with mycelium (as in Figure VIII. 3-2), but individual colonies appeared. Below that concentration, wild type M. gramineum was still able to grow on the 5-FOA plates, but the defined OMPD- negative Aspergillus strain was not (Figure VIII. 3-3). The Aspergillus strain was able to grow on media with other nitrogen sources at a concentration of 2 mM uracil and up to 3 g/L 5- FOA (results not shown). Since the plates have to allow growth of an OMPD-negative Myrothecium , it was decided not to decrease the concentration further.
Figure VIII. 3-3. Effect of decreasing the uracil concentration (mM) on the selectivity of the 5-FOA medium and the growth of M. gramineum and A. nidulans A722. Another parameter suitable for optimisation is the pH of the medium, which was up to now set at 4.0. Several protocols which describe the use of 5-FOA in a medium press on the fact that the pH of the medium can not be adjusted with NaOH because 5-FOA loses its toxic effect when the pH is too high. Indeed, only the non-protonated form of 5-FOA can enter the cells and be metabolised. The influence of the pH on the deprotonation of 5-FOA is given in Table VIII. 3-2. When the pH of the medium is not adjusted, it is about 2.8. The influence of the initial pH of the medium on the growth of M. gramineum is given in Figure VIII. 3-4. The test was carried out in complete liquid and solid medium (Potato Dextrose Broth/Agar). The cultures were grown for 4 days. From Table VIII. 3-2, it can be deduced that the pH of the medium should be kept lower than 3.5 in order to keep the necessary concentration of 5-FOA reasonable (5-FOA is an expensive compound and concentrations higher than 3 g/L are not found in literature). However, in Figure VIII. 3-4, it can be observed that growth of M. gramineum is hampered severely at pH values below 4 in a rich medium. Sporulation does only occur when the pH is higher than 4. Selection and regeneration of protoplasts on a minimal medium with pH values lower than 4 could thus be difficult. Selection on complete medium with 5-FOA was not possible, since growth of M. gramineum occurred even at concentrations higher than 4 g/L (results not shown).
Chapter VIII: A new selection system for M. gramineum 205
Table VIII. 3-2. Influence of the pH on the dissociation of fluoro-orotic acid
Formulas pH medium 2.5 2.86 3.1 3.5 4.0 4.5 5.0 pKa= ± 2.07 6 A1 10^(pH-pKa) 3 6.166 9 27 85 269 851 B2 100/(A+1) 27.1 14.0 10.5 3.6 1.2 0.4 0.1 C3 100-B 72.9 86.0 89.5 96.4 98.8 99.6 99.9 D4 B/100*0.75 0.203 0.105 0.079 0.027 0.009 0.003 0.001 5-FOA needed (g/L) 5 0.105/B*100 0.39 0.75 0.99 2.92 9.01 28.27 89.19 1 Dissociated 5-FOA/non-dissociated 5-FOA 2 % Non-dissociated 5-FOA 3 % Dissociated 5-FOA 4 Toxic concentration 5-FOA (g/L) (= amount of non-dissociated 5-FOA) 5 pH 2.86 as a reference (0.105 g/L 5-FOA effectively toxic when 0.75 g/L added to the medium) 6 see 388
6
5 4 3 pH 6,5 pH 5,0 pH 4,5 (g/L) CDW 2 1 0 0 2 4 6 8 pH pH 4,0 pH 3,5 pH 3,0 Figure VIII. 3-4. Influence of the pH on the growth of M. gramineum . Left: cell dry weight in function of the pH after growth in liquid medium (50 mL PD broth, 25 °C, 125 rpm). Right: g rowth of M. gramineum on PDA plates with different initial pH’s. It was decided to check the growth of M. gramineum on minimal media with different nitrogen sources at pH values between 3 and 4. Since the pH can change during growth of the fungus, this value was measured before and after cultivation. Increases of the pH value of the medium during growth of the fungus could lead to changes in the toxic concentrations of 5- FOA and thus it was important to investigate them. Table VIII. 3-3 shows the composition of the media which were used to evaluate the growth of Myrothecium at low pH. Table VIII. 3-3. Different nitrogen sources tested to optimise growth at low pH
NaNO 3 NH 4Cl NH 4NO 3 (NH 4)2SO 4 YNB Component (g/L) (g/L) (g/L) (g/L) (g/L) Glucose 10 10 10 10 10 NaNO 3 6 - - - - NH 4Cl - 4 - - - NH 4NO 3 - - 5.65 - - (NH 4)2SO 4 - - - 9.33 - YNB - - - - 6.7 KH 2PO 4 1.52 1.52 1.52 1.52 1.52 MgSO 4.7H 2O 0.52 0.52 0.52 0.52 0.52 KCl 0.52 0.52 0.52 0.52 0.52 Hutner’s trace solution 1 mL 1 mL 1 mL 1 mL 1 mL
Chapter VIII: A new selection system for M. gramineum 206
The concentration of the nitrogen sources was calculated in such a way that the amount of N- mol/L was the same as in the original medium with NaNO 3. The Yeast Nutrient Broth (YNB) does not contain any amino acids. The cultures were grown for 6 days. Each condition was tested 3 times. The results of this test are given in Figure VIII. 3-5. NaNO 3 is the only nitrogen source causing an increase in the pH of the medium on metabolisation. Literature sources describing the use of different nitrogen sources and their effect on the pH change of the medium confirm this observation: fungi normally acidify the medium when grown on ammonium (or derivates) while the pH of the cultures increases during growth on nitrate (23,
389, 390, 391). Slight increases are also seen with (NH 4)2SO 4, NH 4NO 3 and YNB at initial pH values 3.
2,5 3 2,5 2 2 1,5 1,5 1 0,5 1 CDW (g/L) 0 -0,5 0,5 (end-pH) - (initial-pH) -1 NaNO3 NH4Cl (NH4)2SO4 NH4NO3 YNB 0 -1,5 NaNO3 NH4Cl (NH4)2SO4 NH4NO3 YNB -2 N-source N-source pH 3 pH 3,5 pH 4 pH 3 pH 3,5 pH 4 Figure VIII. 3-5. Right. Growth of M. gramineum with different nitrog en sources and with different initial pH values. Left. Effect of the growth of M. gramineum on the pH of the medium.
The highest cell dry weight amounts at pH 3.0 are obtained for NH 4Cl, followed by YNB and
NaNO 3. Only little growth is observed on (NH 4)2SO 4 and NH 4NO 3 with initial pH 3.0. At initial pH 3.5, the best growth is also seen with NH 4Cl, closely followed by (NH4) 2SO 4,
NH 4NO 3 and YNB. The best growth is seen with YNB and NaNO 3 with initial pH 4.0, followed by (NH4) 2SO 4, NH 4NO 3 and NH 4Cl. The results confirm the fact that growth is severely hampered when the initial pH is set below 4.0.
The following media were tested as to their selectivity against OMPD positive stains. In order to allow growth of OMPD-negative strains, 2 mM uracil was added and suitable 5-FOA concentrations were used to select against the OMPD wild type cells: - YNB initial pH 3 (2 g/L 5-FOA, 2 mM uracil) - YNB initial pH 3.5 (4 g/L 5-FOA, 2 mM uracil)
- NH 4Cl initial pH 3.0 (2 g/L 5-FOA, 2 mM uracil)
- NH 4Cl initial pH 3.5 (4 g/L 5-FOA, 2 mM uracil)
- NH 4NO 3 initial pH 3.5 (4 g/L 5-FOA, 2 mM uracil)
- (NH4) 2SO 4 initial pH 3.5 (4 g/L 5-FOA, 2 mM uracil)
The plates were incubated for 9 days at 25°C. Pictures of these plates after the incubation are presented in Figure VIII. 3-6. Growth of M. gramineum is severely hindered at pH 3.0 on
Chapter VIII: A new selection system for M. gramineum 207 solid media: even without 5-FOA, almost no mycelium grew on the plates with YNB or
NH 4Cl. This confirms the results obtained with the PDA medium. Thus, these media can not be used for the regeneration of protoplasts after transformation. Sporulation occurred on none of the plates. The best results were obtained with YNB and (NH 4)2SO 4 at pH 3.5: clear mycelium formation is visible on the plates without 5-FOA, while no mycelium grew on the plates with 5-FOA. Since the best (and the fastest) growth without 5-FOA was observed on the YNB plates, further experiments were done with this medium.
Figure VIII. 3-6. Growth of the M. gramineum wild type strain on different N- sources, wi th different initial pH values and different 5-FOA concentrations.
It was decided to test the effect of the addition of 1 M glucose on the selectivity of these YNB plates. The high concentrations of glucose are required to stabilise the plates osmotically and thus to prevent bursting of the protoplasts. The addition of glucose did not alter the selectivity of the medium. This medium was used in further experiments to create the OMPD-negative mutant of M. gramineum (5-FOA resistant, uracil auxotrophic). It is referred to as ‘anti-Opos’ medium and its composition is given in § 2.3.2.
3.2. Construction of OMPD knock-out vectors
3.2.1. Principle By transforming a filamentous fungus with a mutant ompd -gene, OMPD-negative strains of the fungus can be obtained efficiently after homologous recombination. In this research, a strategy to create a mutant which allows the use of the obtained mutant strain in expression studies was developed. In such expression experiments, it is important that the expression cassette integrates at a defined site in the genome, in single copy and preferably at a high frequency. Gouka et al. (119) developed a strategy based on mutant pyrG genes to obtain single copy transformants at a high frequency. Two knock-out vectors are required: a vector containing an ompd -gene with a mutation at the 3’ end of the gene and one with an ompd -
Chapter VIII: A new selection system for M. gramineum 208 gene with a mutation at the 5’ end. When knock-outs, created with one vector are complemented with the other vector (with the mutation at the 5’ end), only integration at the homologous locus leads to the repair of the ompd -gene and the co-integration of an expression cassette (see also paragraph 8.4 in ‘Literature review’). The use of two different mutations within one gene so that a selectable phenotype is only generated by a single cross over at the site of interest increases the chance of obtaining 1 copy of the vector at a specific site. Gouka et al. obtained with this system an efficiency of 35 % of the transformants having the desired single copy at a specific site. Similar strategies for the creation of single copy transformants were followed by Gouka et al. in later studies for the study of heterologous protein production in A. awamori (214), for the analysis of the effect of gene fusions on mRNA and protein levels (205), by van Gemeren et al. to study the expression of the Fusarium solani pisi cutinase gene in A. awamori (166) and by Weidner et al. in A. fumigatus (327).
It was decided to follow this strategy for the creation of a defined OMPD-negative mutant of M. gramineum which will be appropriate for expression studies. For both the 5’ and 3’ mutations, the deletion of conserved amino acid regions was aimed at, in combination with frameshifts resulting in incorrect translation of the mRNA downstream of the mutation, in order to ensure the enzyme inactivation after recombination.
3.2.2. Knock-out vector with mutation at 3’ end of the ompd -gene The plasmid pOV containing a functional copy of the ompd -gene of M. gramineum (§ 3.1.3.4 in the previous chapter) was used to introduce a deletion at the 3’ end of the ompd -gene. The plasmid (2 µg) was cut with Eco47 III, Spe I and Stu I. Ligation the fragments of 4202 bp and 783 bp results in a deletion of 67 bp 0.5 kb downstream the start codon and a frame shift resulting in incorrect translation of the mRNA downstream this deletion (Figure VIII. 3-7).
Figure VIII. 3-7. Strategy for the creation of a 3’ deletion in the ompd-gene of M. gramineum .
Chapter VIII: A new selection system for M. gramineum 209
The result of the gel electrophoresis of the restrictions is given in Figure VIII. 3-8 (left part). It is clear that StuI cut the fragment of 850 bp into 783 bp and 67 bp. After ligation of the 4202 bp fragment to the 783 bp fragment, E. coli DH5 α− F’ was transformed with the ligation mixture and the plasmids of the resulting colonies were checked by restriction analysis (Figure VIII. 3-8, right part). Restriction of pOV with Nsi I and Eco47 III results in fragments of 440 bp, 467 bp, 1192 bp and 2953 bp, while restriction of the knock-out vector should result in fragments of 467 bp, 1565 bp and 2953 bp. Colonies 1, 3 and 5 displayed the expected restriction pattern. The additional fragments can be explained by incomplete restriction. Colony 3 was chosen to use in further experiments and its plasmid is referred to as ‘pOVmut3’. Sequence analysis at the VIB Genetic Service Facility indicated that the 67 bp were correctly deleted.
The vector pOVmut3’ contains a deletion of 67 bp 0.5 kb downstream the start codon resulting in a frame shift which causes incorrect translation of the mRNA downstream this site.
4202 bp M 1 2
850 bp
783 bp 1565 bp 1192 bp
M 1 2
kol 1 kol 2 kol 3 kol 4 kol 5 M pOV Figure VIII. 3-8. Left: Smartladder TM (SF) (lane M), restriction of pOV with Eco47 III and Spe I (lane 1) and with Eco47 III, Spe I and Stu I (lane 1). Right: restriction of pOV and knock-out plasmids with Nsi I and Eco 47III.
3.2.3. Knock-out vector with mutation at 5’ end of the ompd -gene The plasmid pOV (2 µg) was cut with Pst I and Dra III (Figure VIII. 3-10, left part). After the fragments were blunted with T 4-DNA polymerase, they were ligated and the ligation mixture was used for the transformation of E. coli DH5 α− F’. The restriction enzymes were chosen in this way that a mutation at the 5’ end of the ompd -gene would arise after the blunted fragments are ligated (Figure VIII. 3-9). This mutation consists of a deletion of the end of the promoter (including the putative TATA-boxes and transcription start site), the ATG start codon and an N-terminal part of the gene, which theoretically means transcription and translation can not occur and if they would begin at other sites, the enzyme would not be functional. As a control, the resulting plasmids were cut with Nsi I. The results of the restrictions are given in Figure VIII. 3-10. When the knock-out vectors are correct, restriction with Nsi I results in fragments of 2953 bp, 1465 bp and 462 bp. Only colony 9 has the correct
Chapter VIII: A new selection system for M. gramineum 210 restriction pattern. Its plasmid was sequenced at the VIB Genetic Service Facility and proved to have the correct deletion. The plasmid is further referred to as ‘pOVmut5’.
Figure VIII. 3-9. Strategy for the creation of a 5’ deletion in the OMPD-gene of M. gramineum.
1 M 2 M col 1 col 2 col 3 col 4 col 5 col 6 M col 7 col 8 col 9 col 10 col 11 3505 bp 3900 bp 2953 bp 1465 bp 1547 bp 1152 bp 462 bp
Figure VIII. 3-10. Left: pOV cut with Pst I (lane 1), Smartladder TM (lane M). Middle: Smartladder TM (lane M), pOV cut with Dra III (lane 2). Right: restriction of plasmids from colonies 1 to 11 with Nsi I (Smartladder TM between colony 6 and 7).
3.3. Creation of OMPD-negative M. gramineum strains
3.3.1. Via transformation and homologous recombination
3.3.1.1. Transformation with plasmid pOVmut3’ Plasmid pOVmut3’ was used to create an OMPD knock-out by transformation of the Myrothecium gramineum wild type strain. Homologous recombination between the mutant
Chapter VIII: A new selection system for M. gramineum 211 gene on the plasmid and the wild type gene should lead to a deficient copy of the ompd -gene in the genome. After cultivation 4.91 g mycelium was treated with the cell wall degrading enzymes. A suspension of 0.5 mL protoplasts was obtained, with a concentration of 4.10 7 protoplasts/mL. These protoplasts were transformed with 49.5 µg plasmid. After the transformation, a suspension of 4 mL protoplasts (1.10 7 protoplasts/mL) was plated on selective medium (anti-OposG). After 6 days, 399 colonies were transferred to ‘anti-Opos’ medium for a second selection round. 381 colonies grew within 2 days, and after 5 days most of them were sporulated.
In order to test the colonies on their uracil-auxotrophy, they were inoculated on ‘anti-Oneg’ medium, lacking uracil and uridine. In order to preserve the colonies, they were also inoculated on PDA supplemented with 10 mM uracil and 10 mM uridine. Unfortunately, all of the colonies were able to grow on the ‘anti-Oneg’ medium, which means none of them is OMPD-negative. In order to confirm this, a PCR was carried out on DNA of 24 transformants. This PCR was done with ( Figure VIII. 3-11 ): - a forward primer annealing to the genomic DNA just upstream the part of the ompd -gene on the vector (‘U OMPD’) - a reverse primer annealing to the part of the ompd -gene which was deleted on the vector - a second reverse primer annealing to the genomic DNA just downstream the part of the ompd -gene on the vector (‘D OMPD’)
Figure VIII. 3-11. Possible ways of integration of the plasmid pOVmut3’ at the genomic ompd site. Genome sequences are indicated with full lines, plasmid sequences with dotted lines and primers with marker points. The part of the ompd -gene cloned on the vector pOVmut3’ is represented by a rectangle.
Chapter VIII: A new selection system for M. gramineum 212
In this way, all changes at the genomic ompd site could be detected. Ectopic integration was not considered, since this does not lead to OMPD-negative mutants. Knock-out of the ompd - gene as a result of a double crossover was desired and results in a PCR pattern of only one fragment, the 2264 bp fragment. For the wild type, two fragments are expected: 2331 bp and 1499 bp. Single crossovers, resulting from type I integration, would lead to a defect copy and an intact copy of the ompd -gene present at the ompd genomic site. This recombination was not desired and normally is excluded, because the intact copy of the ompd -gene should render the transformants sensitive to the 5-FOA present in the selective plates.
All PCRs on the colonies resulted in one fragment of 1499 bp, meaning a wild type copy of the ompd -gene was present in their genome (Figure VIII. 3-12). The fact that none of the longer fragments was amplified can be explained by the fact that the colony-PCR method was used here. Amplification of larger fragments with the use of mycelium directly in the PCR- tubes is difficult, but it is a very easy and quick method to control the transformants. Since none of them resulted from a double cross over between the mutated and the wild type gene, no further tests were carried out which could have more precisely determined the integration pattern. The transformants can be wild type strains or can have originated from type I integration events (part D in Figure VIII. 3-11).
marker 7col 18col 23col 32col 68col 73col marker 76col 77col 79col 99col 102col 117col
119col 172col 173col 271col 343col ckol 353 marker 44col 60col wildtype
1499 bp
1499 bp
Figure VIII. 3-12. Control PCR on the transformants obtained after transformation of the wild type with plasmid pOVmut3’.
From these results, it was clear that problems had arisen with the selection on the ‘anti-Opos’ plates and that the efficiency of type III integration events had to be improved. Double crossovers can be enhanced by transformation with linearised vectors and even more by the use of PCR amplified replacement cassettes (14, 117, 120, 121). Before testing the possibility of transformation with linear cassettes, experiments were set up to find the cause of the selection of false positive colonies.
Because too many false positive colonies were obtained, it was decided to test if there is a difference between inoculating mycelium or inoculating spores on the selective medium. The protoplasts which are inoculated on the selective plates after transformation are derived from mycelium and therefore may physiologically resemble to it more. Until now, the selectivity of
Chapter VIII: A new selection system for M. gramineum 213 the medium was always tested by inoculating plates with spore suspensions. In this experiment, ‘anti-Opos’ plates were inoculated with spores and mycelium of M. gramineum MUCL 39210 (wild type) and MUCL 44487 (as a reference).
As can be seen in Figure VIII. 3-13, there is a clear difference between the growth of mycelium and that of the spores. Mycelium of the strain MUCL 39210 is able to grow on the ‘anti-Opos’ plates, while spores are not.
For M. gramineum MUCL 44487, this difference is not observed: no growth of mycelium was visible on the ‘anti-Opos’ plates. A possible explanation for the fact that mycelium is able to grow may be that this mycelium already contains RNA at the time it is plated on the selective medium. Spores, however, still have to synthesise all their RNA (or most of it), which explains the fact that 5-FOA is toxic to them. Mycelial growth is only hampered when new RNA synthesis is required and thus mycelium can be able to form small colonies before the 5- FOA becomes lethal. However, this explanation does not hold for strain MUCL 44487, since this strain’s mycelium is sensitive to 5-FOA. Another possible explanation is that M. gramineum MUCL 39210 only expresses its ompd -gene in the early stages of germination and/or that it expresses other enzyme(s) which can catalyse a reaction similar to that of OMPD. However, the de novo biosynthesis pathway of pyrimidines consists of 6 enzymatic steps which are identical in Eubacteria , Archaea and eukaryotes. The OMPD-enzyme is considered to be an essential enzyme. Moreover, nearly all organisms investigated have only one ompd -gene, as M. gramineum MUCL 39210 does. Only one exception to this rule is known, namely Acremonium sp. has two different functional pyr-4 genes (330, 332, 334, 392). The authors were able to show that the duplication was the result of an interspecific hybridisation between Epichloë typhina and Acromonium lolii .
In the following transformation experiments, the protoplasts were inoculated on complete medium (osmotically stabilised Potato Dextrose Agar (PDAG) supplemented with 10 mM uridine and 10 mM uracil) and the plates were incubated until sporulation occurred. The spores were harvested then and inoculated on ‘anti-Opos’ plates.
Chapter VIII: A new selection system for M. gramineum 214
anti-Opos anti-Opos anti-Opos -FOA pH 3 -FOA pH 4
Myrothecium gramineum MUCL 39210
mycelium
spores
Myrothecium gramineum MUCL 44487
mycelium
spores
Figure VIII. 3-13. Mycelium and spores inoculated on ‘anti-Opos’ plates (left), on this medium without FOA at pH 3 (middle) and at pH 4 (right).
3.3.1.2. Transformation with PCR amplified mutant ompd-gene In order to increase the appearance of double crossover events in the transformants, a transformation experiment was performed with a PCR-amplified fragment of the vector pOVmut5’. Because M. gramineum was never transformed with linear DNA before, the strain was also transformed with a PCR-amplified fragment containing the hygromycin B resistance marker gene. As expected, the PCR yielded a fragment of 1883 bp for the 5’ mutated ompd - gene ( ompdmut5’ ) and of 2639 bp for the hygromycin resistance marker gene ( hph ).
Two solutions of 5 g mycelium were treated with cell wall degrading enzymes, resulting in suspensions of 4 mL protoplasts, with a concentration of 4.2.10 7 protoplasts/mL and 5.3. 10 7 protoplasts/mL. Both solutions were transformed with 21 µg linear DNA. After the transformation, a suspension of 2.73 mL protoplasts (1.7.10 7 protoplasts/mL) was obtained for the transformation with hph and one of 2.1 mL protoplasts (0.8.10 7 protoplasts/mL) was obtained for the transformation with ompdmut5’ . The protoplasts of the hph transformation were plated on PDAG, incubated for 2 hours and then covered with a top layer containing hygromycin. The protoplasts of the ompdmut5’ transformation were plated on PDAG supplemented with 10 mM uridine and 10 mM uracil. The viability of the protoplasts was tested before and after transformation and proved to be rather low (2.85 % before and 0.73 %
Chapter VIII: A new selection system for M. gramineum 215 after transformation for the hph protoplasts, and 0.6 % before and 0.93 % after transformation for the ompdmut5’ protoplasts).
The transformation with the linear fragment of hph yielded only 14 transformants, which means a transformation efficiency of 1.97 transformants per µg DNA and per 10 6 viable protoplasts was obtained (41.4 transformants per 106 viable protoplasts). In chapter I, a transformation efficiency of 5.6 transformants per µg DNA and per 10 6 viable protoplasts was obtained after the transformation of M. gramineum with circular DNA carrying the hph gene. Thus, the use of linear DNA for the transformation of M. gramineum approximately reduces the transformation efficiency 3 times.
After the regeneration and sporulation on the PDAG plates supplemented with 10 mM uridine and 10 mM uracil, 1370 spore droplets were inoculated on ‘anti-Opos’ medium. None of them was able to germinate and form mycelium. Because the number of spore droplets inoculated might have been too small to yield a transformant, all spores were harvested from all of the plates and inoculated on ‘anti-Opos’ Unfortunately, no colony grew on the plates. Based on the viability, and the transformation efficiency with the hph fragment, spores of 0.16.10 6 viable protoplasts could have yielded 6.6 transformants. This number is probably too small to obtain an OMPD-negative strain, because only a double crossover event at the homologous locus yields such a knock-out. It has to be noted, that the problems with the selectivity of the ‘anti-Opos’ plates are solved. Further experiments yielding more candidates will have to be performed.
3.3.1.3. Transformation with plasmid pOVmut5’ Because the transformation efficiency with linear DNA fragments was so low, a transformation experiment with plasmid pOVmut5’ was carried out. The protoplasts were grown until sporulation on PDAG with uracil and uridine, as in § 3.3.1.2. A suspension of 0.54.10 8 protoplasts/mL (1100 µL) was obtained after incubation of 8.05 g mycelium with cell wall degrading enzymes. The transformation was carried out with 7.6 µg DNA. After transformation, a solution 0.22.10 8 protoplasts/mL was obtained (1500 µl). The viability of the protoplasts was tested before and after transformation by plating an equivalent of 500 and 100 protoplasts on PDAG + uracil + uridine. Again, the viability of the protoplasts was very low (0.95 % before and 0.1 % after transformation). 1327 individual spore droplets were inoculated on ‘anti-Opos’, as well as a harvested (mixed) spore suspension of all other spores. Twelve of the inoculated spore droplets produced tiny amounts of mycelium and 67 very small mycelial colonies grew on the plates inoculated with the spore mix. Unfortunately, the colonies were all able to grow on ‘anti-Oneg’ medium (inoculated with their mycelium). All of them were transferred to PDAG + uracil + uridine in order to enable them to sporulate. Since the toxic effect of 5-FOA on M. gramineum MUCL 39210 is only visible when germination is required, it could be possible that the uracil auxotrophy is only visible when spores are inoculated on the medium instead of mycelium. But, all colonies were able to germinate on the medium and large mycelial colonies appeared readily (Figure VIII. 3-14).
Chapter VIII: A new selection system for M. gramineum 216
Figure VIII. 3-14. Thirteen transformants obtained after transformation with plasmid pOVmut5’ are able to germinate on ‘anti-Oneg’.
In order to be able to obtain a defined OMPD-negative strain of M. gramineum , i.e. by homologous recombination, it will be necessary to increase the efficiency of the system by either increasing the efficiency of transformation with linear DNA fragments or by the investigation of the dominant pathways by which DNA is incorporated into the genome of M. gramineum . Knocking out of the genes responsible for exogenous DNA integration increases the efficiency of homologous recombination.
3.3.2. UV mutagenesis Because of the difficulties arising with the creation of an OMPD-negative strain by homologous recombination, some attempts were made to isolate such a strain after mutagenesis. Different mutagenic agentia can be used to create a mutant and it is important to chose the most suitable one (393). The choice depends on two factors: the type of the desired deletion (base substitution, deletion or frameshift) and the relative efficiency by which the mutagenic agent creates this mutation (Table VIII. 3-4). Since deletions give the least problems with revertants and the chance of complete inactivation of the enzyme is higher (e.g. frameshift by deletion of a number of basepairs not divisible by 3), it was decided to use the mutagen most suitable for causing deletions.
Table VIII. 3-4 shows that X-rays, UV-radiation, acridine mustards and nitrous acids cause deletions with medium to high efficiencies. Since the equipment to work with X-rays was not available at the laboratory and acridine mustards are difficult to obtain, it was decided to use UV-mutation. This technique is most widely used for fungi (Bos & Stadler, 1996). Since photo-reactivation needs to be avoided (blue and white light), all irradiation experiments and other manipulations were performed under red light. Plates were incubated in the dark.
Chapter VIII: A new selection system for M. gramineum 217
Table VIII. 3-4. Features of some generally used mutagenic agentia (after 393) Type mutagen Type mutation Relative efficiency Radiation X-rays, neutrons deletion, inversion high UV transition (GC to AT), deletion medium Chemicals Base-analogues transition (AT to GC), low Hydroxylamine transition (GC to AT), low Nitrous acid transitions, deletions medium NTG transition (GC to AT), very high EMS transition (GC to AT), medium Acridine half mustards frameshifts, insertions, deletions high Acridine colouring agents frameshifts low Novobiocine Blocks DNA replication high
The chance to create and select a desired mutant is usually quite low: often the UV-treatment is prolonged until only 0.1 % of the treated spores is still viable and moreover, the (desired) mutant frequencies in a population can be as low as 1 per 10 6 of 10 7. The survival curve obtained after UV-mutagenesis of M. gramineum is given in Figure VIII. 3-15. This graph presents the mean survival percentage obtained in 10 treatments. Killing percentages of 90 % were reached, but, as can be seen in the graph, the spores are quite resistant to the UV- irradiation. According to the literature, it is not always necessary to kill 99.9% of the spores (317, 319, 321). The longer the UV-treatment and the higher the dose, the more non-target genes will be affected too. A compromise has to be found between the yield of mutants (as high as possible) and the chance of affecting other genes than the one of interest (as low as possible).
140 y = 117,04e -0,1521x 120 R2 = 0,8908 100 80 60
(%) Survival 40 20
0 0 2 4 6 8 10 12 14 16 Time (min)
Mean Exponentieel (Mean)
Figure VIII. 3-15. Survival curve of M. gramineum after UV-irradiation of the spores.
Chapter VIII: A new selection system for M. gramineum 218
In the first experiment, 22.7.10 7 spores, 31.96.10 7 spores and 6.8.10 7 spores treated for 4, 6 and 10 minutes, respectively, were inoculated directly on selective plates. Unfortunately, no colonies grew on the plates. Therefore, in a second experiment, the spores were plated on a non-selective medium (PDA + uracil + uridine) in order to let them sporulate before inoculating them on selective medium. The reasons for incorporating such a step in the protocol after UV-mutagenesis are twofold: on the one hand, even a low doses of a mutagenic agent can cause so much physiological damage to a cell that inhibition of growth occurs and the spores need time to ‘recover’. On the other hand, such a sporulation also allows the segregation of different alleles and more important, the mutations need time before they are expressed (387). A total of 4.8.10 7 spores and 13.2.10 7 spores treated 6 and 10 minutes, respectively, were inoculated on the non-selective medium. After sporulation, the spores were harvested and inoculated on selective medium. This time, 369 colonies grew on the 5-FOA- plates, but none of them were uracil or uridine auxotrophic.
In the third experiment, filtration enrichment was tested in order to increase the number of 5- FOA resistant colonies and thus to increase the chance on obtaining an OMPD-negative mutant. The percentage of surviving spores obtained after 6 minutes of UV-treatment (approximately 50 %) is ideal for testing the filtration enrichment technique (387). 10 mL of a spore solution of 10 7 spores/mL were irradiated with UV during 6 minutes. In this experiment, a survival percentage of 48.5 % was obtained, confirming the results presented in Figure VIII. 3-15. The filtration enrichment was carried out as described in 2.4.2.
Inoculation of the spores on AMM + uracil + uridine (prototrophs and auxotrophs) and AMM (prototrophs) allows to determine the viable spores after the filtration enrichment and to estimate the number of uracil/uridine auxotrophs. A clear difference was observed between the AMM + uracil + uridine and the AMM plates: there was a lot of growth on both plates, and more colonies were present on the AMM + uracil + uridine plates than on the other plates, meaning still a lot of prototrophic spores were present, but also auxotrophs were formed. 2 mL of the suspension of the spores was diluted 10 times and 2 mL 100 times and these were inoculated on 5-FOA-medium. Only OPRT- and/or OMPD-negative strains are able to grow on this medium. Several hundreds of colonies grew on the plates, but none of them were uracil auxotrophic.
Despite the fact that a reasonable number of 5-FOA-resistant colonies was obtained, none of them were uracil auxotrophs. In literature, low frequencies of OMPD-negative strains among 5-FOA resistant ones are also reported. Rose et al. (2000) (365) found only 6.2 % of the 5- FOA resistant colonies of Aureobasidium pullulans to be true uracil auxotrophs. Similar results were obtained for Claviceps purpurea , where 12.5 % was OMPD-negative. Considering these observations, it might be necessary to further increase the number of 5- FOA resistant colonies in order to obtain a desired OMPD-negative strain.
Chapter VIII: A new selection system for M. gramineum 219
4. CONCLUSION The orotidine monophosphate decarboxylase gene was chosen as selection marker and isolated from the genome of Myrothecium gramineum (chapter VII, GenBank accession no. DQ359751 ). In this part, the development of a homologous transformation system based on the gene is described for Myrothecium gramineum .
Two selective media were developed, more precisely the ‘anti-Oneg’ medium selective against OMPD deficient strains (no uracil or uridine) and the ‘anti-Opos’ medium, selective against OMPD wild type strains (4 g/L 5-FOA, 0.23 g/L uracil, pH 3.5). This medium was used in further experiments to select for the OMPD-negative mutant of M. gramineum (5- FOA resistant, uracil auxotrophic).
By transforming a filamentous fungus with a mutant ompd -gene, OMPD-negative strains of the fungus can be obtained efficiently. In this research, a strategy to create a mutant which allows the use of the obtained mutant strain in expression studies was developed. Therefore, it is important that the expression cassette integrates at a defined site in the genome, in single copy and preferably at a high frequency. The use of two different mutations within one gene increases the chance of obtaining 1 copy of the vector at a specific site. It was decided to follow this strategy for the creation of a defined OMPD-negative mutant of M. gramineum . For both the 5’ and 3’ mutations, the deletion of conserved amino acid regions was aimed at, in combination with frameshifts. The vector pOVmut3’ contains a deletion of 67 bp 0.5 kb downstream the start codon resulting in a frame shift which causes incorrect translation of the mRNA downstream this site. The mutation on vector pOVmut5’ consists of a deletion of the end of the promoter (including the putative TATA-boxes and transcription start site), the ATG start codon and an N-terminal part of the gene.
Plasmid pOVmut3’ was used to create an OMPD knock-out by transformation of the Myrothecium gramineum wild type strain. 381 colonies grew ‘anti-Opos’ medium, but they were all able to grow on the ‘anti-Oneg’ medium, which means none of them is OMPD- negative, as confirmed by PCR control. From these results, it was clear that problems had arisen with the selection on the ‘anti-Opos’ plates. Before testing the possibility of transformation with linear cassettes, experiments were set up to find the cause of the selection of false positive colonies. A clear difference between the growth of mycelium and that of the spores on the ‘anti-Opos’ was observed. Mycelium of the strain MUCL 39210 is able to grow on the ‘anti-Opos’ plates, while spores are not. Thus, in the following transformation experiments, the protoplasts were inoculated on complete medium (10 mM uridine and 10 mM uracil) and the plates were incubated until sporulation had occurred. After that, the spores were harvested and inoculated on ‘anti-Opos’ plates.
In order to increase the chance on double cross-over events, a transformation experiment was performed with a PCR-amplified fragment of the vector pOVmut5’. The strain was also transformed with a PCR-fragment containing the hygromycin B resistance marker gene
Chapter VIII: A new selection system for M. gramineum 220 because it was never transformed with linear DNA before. After the regeneration and sporulation on the PDAG plates with uridine and uracil, 1370 spore droplets were inoculated on ‘anti-Opos’ medium. None of them was able to germinate and form mycelium. Based on the viability, and the transformation efficiency with the hph fragment, spores of 0.16.10 6 viable protoplasts could have yielded 6.6 transformants. This number is probably too small to obtain an OMPD-negative strain, because only a double cross-over event at the homologous locus yields such a knock-out. Because the transformation efficiency with linear DNA was so low, a transformation experiment with plasmid pOVmut5’ was carried out. Twelve of the inoculated spore droplets produced tiny amounts of mycelium on ‘anti-Opos plates’ and 67 very small mycelial colonies grew on the plates inoculated with the spore mix. Unfortunately, the colonies were all able to grow on ‘anti-Oneg’ medium (inoculated with their mycelium).
In order to be able to obtain a defined OMPD-negative strain of M. gramineum , i.e. by homologous recombination, it will be necessary to increase the efficiency of the system by either increasing the efficiency of transformation with linear DNA fragments or by the investigation of the dominant pathways by which DNA is incorporated into the genome of M. gramineum . Knocking out of the genes responsible for exogenous DNA integration increases the efficiency of homologous recombination.
Because of the difficulties arising with the creation of an OMPD-negative strain by homologous recombination, attempts were made to isolate such a strain after UV- mutagenesis. Despite the fact that a reasonable number of 5-FOA-resistant colonies was obtained, none of them were uracil auxotrophs. In the literature, low frequencies of OMPD- negative strains among 5-FOA resistant ones have also been reported. Considering these observations, it might be necessary to further increase the number of 5-FOA resistant colonies in order to obtain a desired OMPD-negative strain.
Part IV: General discussion, conclusion and research perspectives
General discussion, conclusion and research perspectives 223
General discussion, conclusion and research perspectives
Fungi and their enzymes are used since thousands of years in the food industry, e.g. for making beer, wine, bread and cheese and in several other fermented foods and drinks. Recently, they are also increasingly applied in other industrial fields. The rising interest in fungal enzymes and expression systems is illustrated in Figure 1. Nevertheless, until now, only a limited number of fungal host species has been explored for recombinant protein production. Since the field of modern fungal biotechnology is competitive and is attracting considerable interest from industrial companies interested in the applications of enzymes and other proteins, it is not surprising that several parties started to explore the alternatives to those covered by several patent applications. Patents and intellectual property rights have necessitated searching for expression hosts other than the fungal species traditionally used. Some examples of fungal hosts and/or expression systems which are covered by patent applications are given in Table 1. Recent research in several groups has focused on developing Fusarium venenatum , Aspergillus sojae, Aspergillus japonicus, Neurospora crassa, Mortierella alpinis and Chrysosporium lucknowense as production hosts.
fungi and enzyme fungi and enzyme expression
1800 350 1600 300 1400 1200 250 1000 200 800 150 600 100 400 200 50 #citations in Web of Science # citations # Webin Science of 0 0 1972- 1977- 1982- 1987- 1992- 1997- 2002- 1972- 1977- 1982- 1987- 1992- 1997- 2002- 1976 1981 1986 1991 1996 2001 2007 1976 1981 1986 1991 1996 2001 2007
Figure 1. Number of hits for the search terms ‘fungi and enzyme’ (left) and ‘fungi and enzyme expression’ (right) on Web of Science over the past 35 years.
The fact that many currently used fungal expression hosts are protected by patent applications, taken together with the consideration that, undoubtedly, filamentous fungi exist which have new characteristics and produce enzymes with new application possibilities, was an inspiration for exploring the BCCM/MUCL agro-industrial fungi- and yeast collection in order to find a potential new expression host. A preliminary study of the company Beldem in collaboration with the Laboratory of Microbiology of the UCL indicated that Myrothecium gramineum MUCL 39210 (syn. Xepiculopsis graminea ) seemed to possess all desired qualities necessary to perform as an interesting expression host. These qualities are, for example, a good expression of homologous and heterologous enzymes, poor protease production, a good transformability and good fermentation characteristics.
General discussion, conclusion and research perspectives 224
Table 1. Examples of fungal strains and expression systems covered by patent applications
Claims Patent application number(s) The glutamate dehydrogenase gene ( gdh ) promoters from Aspergillus awamori and related WO9951756, EP1084262 Aspergilli and their use for the expression of recombinant proteins in filamentous fungi An Aspergillus promoter , wherein said promoter comprises a creA binding site and other US5674707 regulatory sequences; and wherein said promoter variant lacks the creA binding site, comprises the other regulatory sequences and mediates expression of said protein-encoding DNA in the presence of glucose ( alcA , aldA , and alcR ) The use of Aspergillus japonicus and Aspergillus aceulatus as a hosts for enzyme WO9515391 expression A regulatory fungal sequence inducible by ethanol, said ethanol inducability being mediated EP0284603, by the alcR gene product, especially derived from Aspergillus nidulans or A. niger ( e.g. US5728547, US5503991 glaA promoter A. niger , alcA and aldA promoter A. nidulans ) A DNA construct comprising the A. niger glucoamylase gene (glaA ) promoter US5198345 DNA molecules comprising the Aspergillus niger pki promoter , hybrid vectors useful for EP0439997, PT96310, the expression of structural genes under the control of said promoter, hosts transformed with FI906123, IE904481, the vectors, and transformed hosts and the production of recombinant polypeptides by means JP5192150 of said transformed hosts An expression cassette comprising the promoter of the Aspergillus niger bpHA gene BE1005432 (benzoate-para-hydroxylase ) or a functionally equivalent sequence and filamentous fungi with such an expression cassette The manganese-superoxide dismutase (Mn-SOD ) gene promoter of Aspergillus niger JP2003164283 The use of Aspergillus oryzae as a host for enzyme expression and the use of the Aspergillus US5863759 oryzae TAKA-amylase promoter for the expression of enzymes in Aspergilli The promoters of the Chrysosporium cbhI , xyl1 and gpd1 genes and their use in vectors and AU782105B, the organisms transformed with these vectors CA2405954, US2003187243 A vector with a promoter and a terminator to use in Lyophyllum shimeji JP11127863 Use of the Magnaporthe grisea nitrate reductase promoter EP1674577 Use of a filamentous fungus of the genus Monascus for transformation and/or expression US2004072325, EP1325959, WO0200898 The Monascus acuF promoter FR2863627 The promoter sequence of the hex gene of Penicillium chrysogenum (and vectors based on US6558921, WO9839459, it) that is capable of directing expression of DNA downstream of it ES2149707 A vector plasmid which has a promoter being derived from a strain relative to Penicillium JP3262486 citrinum , a chemical-resistant gene and a terminator and which is capable of transforming P. citrinum Trichoderma viride cbhI promoter and termination sequences, including all derived US6277596, EP0952223, expression vectors and cells harbouring the vectors WO9811239 Any process for the production of recombinant protein using a strain of the genus FR2649120, EP0433431, Tolypocladium , into which a DNA sequence coding for a protein under the control of WO9100357 elements guaranteeing the expression has been introduced The use of Pseudozyma spp . for the production of recombinant polypeptides, proteins or US2003232413 peptides, by genetically transforming fungi with an expression vector A recombinant DNA molecule comprising the promoter, the signal sequence, the structural EP0353188, FI893541, gene or the terminator of anyone of the PL expression systems , or any combination of these JP3108487 fragments (pectin lyases PLA, PLB, PLC, PLE or PLF) A promoter and a terminator ( Abp1 ) which co-ordinately function with the expression of an US6913905, EP1221489, endogenous gene in a filamentous fungus that belongs to Agonomycetes , particularly in AU782525B, Mycelia sterilia , , including all derived expression vectors and cells harbouring the vectors WO0118219, CA2384000
General discussion, conclusion and research perspectives 225
A taxonomical reidentification of the strain Myrothecium gramineum MUCL 39210 confirmed that it presents all morphological characteristics of the species Myrothecium gramineum . The genus Myrothecium is actually an artificial genus and is liable to criticism. Not only the results of the molecular characterisation (ITS and 28S rDNA sequences) of 5 Myrothecium gramineum strains by Jonniaux et al. (29) indicated problems with the classification of certain strains, but Tulloch (30) also recognised difficulties to delimit Myrothecium morphologically from other genera. Moreover, based on a study of the composition of the cell wall polysaccharides of different Myrothecium species, Ahrazem and co-workers (46) concluded that Myrothecium is a heterogeneous genus. Briefly, one can state that the classification of certain species within the Myrothecium genus remains uncertain.
The aim of our research was to further develop a transformation system and to develop an expression system to enable us to use the filamentous fungus M. gramineum as a new, universal expression host.
In chapter I , further research was performed on the development of a transformation system for the filamentous fungus Myrothecium gramineum . In 1973, Mishra and Tatum (52) reported the first successful transformation of Neurospora crassa and since then, many fungi were transformed with success. Five different techniques for the transformation of filamentous fungi are currently described in the literature: protoplast transformation, electroporation, lithium acetate mediated transformation, biolistical transformation and Agrobacterium tumefaciens mediated transformation. The basic transformation protocol used for M. gramineum is the one described by Punt and Van den Hondel, which uses protoplast generation for the transformation of filamentous fungi. This protocol was used in preliminary research by Jonniaux et al. (29) for the transformation of Myrothecium . Transformants were selected based on their resistance to hygromycin B and their ability to overproduce the Taka amylase of Aspergillus oryzae , used as a reporter gene.
During this research, the transformation protocol was modified in order to obtain more transformants, which would allow screening for higher production strains. The preculture was no longer carried out in medium containing 2 % malt extract, 1 % peptone and 3 % glucose, but it was performed in Aspergillus minimal medium + V 8. In this medium, the strain grew under the form of filamentous mycelium which is more accessible for the cell wall digesting enzymes than pellets and thus more suitable for protoplast production. A heat shock was introduced after DNA was added to the protoplasts. With these modifications, a transformation efficiency of 5.6 transformants per µg DNA and per 10 6 viable protoplasts was achieved. It is difficult to compare transformation efficiencies of different studies because they are dependent on, for example, the transformation method, the strain under investigation and the nature of the transforming DNA (integrative, replicative, linear, and circular). Generally, with protoplast transformation, the efficiencies of transformation range between 1 and 10 3 transformants/ g DNA when dominant selection markers are used, and between 10 2 and 10 5 transformants/ g DNA when auxotrophic markers are used. Thus, since between 1
General discussion, conclusion and research perspectives 226 and 1000 transformants per µg DNA could be obtained with the hygromycin resistance gene, 5.6 is still low, although it is 9.6 times higher than the efficiency reported by Jonniaux et al . (29). In chapter V, based on the hygromycin resistance, a transformation efficiency of 3.3 transformants per µg DNA and per 10 6 viable protoplasts was obtained for the co- transformation with pGPDcarXylBS.
The transformation efficiency might be further increased by the use of the homologous gpd - promoter, described in chapter III, to control the expression of the hph -gene. Jungehülsing and co-workers were able to significantly improve the transformation efficiency with the phleomycin resistance system of Claviceps purpurea by using the homologous gpd -promoter. After the original A. nidulans trpC promoter was exchanged by the strong heterologous gpdA - promoter of A. nidulans , which considerably improved the efficiency of the system, the homologous gpd -promoter of Claviceps purpurea was used, which resulted in the highest transformation efficiencies. Similar results were obtained when the hph -gene was put under the control of the homologous gpd -promoter in Podospora anserina.
When one tries to develop a transformation system for a new fungal host or to study the molecular systematics of an organism, a quick and reliable method for screening fungal transformants for specific genetic modifications is essential. Unfortunately, while rapid and high-throughput PCR-techniques or other molecular procedures are amply available for bacteria and even for yeast, this is not the case for filamentous fungi. As discussed in chapter II , many methods for DNA extraction from fungi have been described in the past few years, but these methods often are tedious, time consuming, costly or limited to a small number of samples per run. Lately, a number of methods have been described to isolate DNA from fungi suitable (exclusively) for PCR and appropriate for the simultaneous treatment of a large number of samples. The methods use minute quantities of starting material and are as short and cheap as possible. They can be applied on mycelium or on spores. In chapter II, we compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR. The Aspergillus strain was used as a reference. Ten methods were tested. In nine of these methods DNA was extracted from mycelium prior to PCR. A final assay used mycelium straight in the PCR- reaction mixture. The methods were selected based on their cost and short duration and on the fact that no special equipment is required and a lot of samples can be processed at the same time. A summary of the results is given in Table 2.
General discussion, conclusion and research perspectives 227
Table 2. Extraction procedures suitable for PCR for Aspergillus and Myrothecium Aspergillus Myrothecium DNA extraction Highest amount DNA Protoplast Protoplast Highest Glass bead, LiCl, boiling, Boiling, protoplast
concentration liquid N 2, protoplast PCR PCR product < 1 kb Boiling, boiling 2, liquid Glass bead, LiCl, boiling
N2 2, acetone, liquid N 2
PCR product ≈ 2 kb - liquid N2, mycelium in PCR-mixture PCR product ≈ 4 kb - liquid N 2 with Elongase -: not investigated
Both for Myrothecium gramineum and A. nidulans , the highest DNA concentration was obtained with the protoplast procedure. Most extraction protocols resulted in higher DNA concentrations for A. nidulans than for M. gramineum . Samples of M. gramineum resulting from the boiling procedure (1.5 hours) and the liquid nitrogen procedure (2.5 hours) were suitable for the amplification of fragments up to 2.3 kb. The direct use of mycelium from M. gramineum in the PCR tube can be employed for the amplification of fragments up to 1 kb and even weak signals of 2.3 kb can be obtained. Amplification of fragments up to 4.3 kb requires the use of the long distance polymerase Elongase Mix on samples extracted with the liquid nitrogen procedure.
The use of strong promoters for the expression of proteins in suitable host organisms is of great importance for many biotechnological applications. However, there is not much homology between fungal promoters, not even in related fungi. Even different genes coding for the same enzyme show nearly no homology in their promoter sequences in Aspergillus. nidulans and Neurospora crassa . Since homologous promoters often give the best and because little is known about the critical parts of fungal promoters till today, a strong homologous promoter is usually searched for when developing a new expression system. It is assumed that this strong promoter will function ideally in the new expression host. The most frequently used constitutive promoter is the glyceraldehyde-3-phosphate dehydrogenase. This promoter is functional in different species, including industrially important Penicillium and Aspergillus species. The glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.12) promoter is a promising candidate because in many eukaryotic microorganisms, the gpd - genes are expressed constitutively and in large amounts.
During this research, the complete sequence of the glyceraldehyde-3-phosphate dehydrogenase gene of Myrothecium gramineum was determined and submitted to the Genbank ( EF486690 ), as described in chapter III . In the promoter region, two TATA-boxes, four CAAT-similar sequences and five CT-rich stretches are found. Consensus TATA-boxes have been found in few fungal promoters. Although AT-rich sequences were observed upstream of the transcription initiation sites, their location is variable and their functional
General discussion, conclusion and research perspectives 228 significance unknown. In fungal genes, the CAAT box often is absent, or when present, is situated at a variable distance of the transcription start point (tsp). The element which is most important in determining the tsp in filamentous fungal genes is the CT-box. This is a pyrimidine-rich sequence which is found directly upstream the tsp in a considerable number of fungal genes. The fact that, in cases where the CT-box has been deleted, the alternative transcription start points used are often found downstream other CT-rich sequences, supports the involvement of this motif in determining the tsp. In the gpd -promoter of M. gramineum , 5 CT-rich stretches were found. The two TATA-boxes and their corresponding CAAT-boxes are situated in between the first and the second CT-box and the second and the third CT-box, respectively. The gpd -promoter was also investigated regarding the presence of some gpd - promoter characteristic sequences, such as the gpd -box and the pgk -box, but no similar sequences were found. Although the gpd -promoter is generally considered to be a constitutive promoter, some gpd -genes are regulated. Sequence analysis of the full-length promoter fragment of the M. gramineum gpd -gene was carried out using Motif Finder. Two putative heat shock elements (HSE) were found. Similar elements were found in the promoter region of the Mucor circinelloides gpd1 -promoter. The presence of such motifs suggests that the expression of the gene might be under stress regulation, but, in this case, this is only a theoretical speculation. Another regulatory sequence, a CATCAC element, was found in the gpd -promoter of M. gramineum at nucleotide 740 to 745. Larsen et al . (126) speculate that CATCAC elements in the promoter region could be responsible for the down regulation of the gene when the hexose concentration is low.
Most gpd -genes of filamentous fungi contain one or more introns. Their positions are conserved among the Ascomycota and the Basidiomycota , and thus the number of introns present and their position in fungal genes relate well to their systematic relationship. In the gpd -gene of M. gramineum , two introns were found: one quite long intron (458 bp) in the beginning of the gene and a shorter one (65 bp) near the end. The position and length of the introns was confirmed by a cDNA analysis. The position of the first intron corresponds to the conserved position for the Ascomycota .
Codon bias in gpd -genes is normally high, because the genes are highly expressed. Analysis of the codon use in the gpd -gene of M. gramineum shows that 80.9 % of all codons have a pyrimidine at the third position and when a choice between a purine or a pyrimidine is allowed, a pyrimidine is chosen in 97.2 % of the cases. This is an indication of the fact that this gene is highly expressed and it implies that the promoter of this gene is a strong one.
Elements associated with transcription termination and polyadenylation of the mRNA, such as the YGTGTTYY and the AAUAAA motifs, have been found in filamentous fungal genes, but mostly they are present in a shorter form (AUAA) or even completely absent. In the gpd- sequence of M. gramineum, two polyA-signals were found, which are each followed by a transcription termination site. cDNA-analysis revealed that both polyadenylation signals are used: the polyA-signal situated most closely to the 5’ end is the major polyA-signal, while the
General discussion, conclusion and research perspectives 229 second one is a minor polyA signal. Multiple poly adenylation signals were also observed in other fungal genes, e.g. the gpd -genes of Cochliobolus heterostrophus , Lentinula edodes and Xanthophyllomyces dendrorhous .
The 3041 bp gene encodes an enzyme of 339 amino acids. The GPD-enzyme of M. gramineum contains the recognition pattern for all known GPD enzymes in the form of ASCTTNCL. Thus, the obtained sequence is certainly representing a GPD-enzyme. In this pattern, the C (cysteine) is essential for the activity of the enzyme. Other conserved amino acids are a histidine and two phosphate binding residues, lysine and arginine. The histidine residue was found at the expected position, position 179, while the lysine and arginine residues are located at positions 194 and 234, respectively. A blast of the amino acid sequence revealed that it shows most similarity with the GPD-enzymes of Beauveria bassiana , Neurospora crassa , Colletotrichum lindemuthianum , Colletotrichum lindemuthianum, gloeosporioides , Sordaria macrosporia , Metarhizium anisopliae and Hypocrea jecorina, which belong all to the class of the Sordariomycetes . Accordingly, phylogenetic analysis based on the amino acid sequence of the M. gramineum GPD confirmed the current classification of M. gramineum within the class of the Sordariomycetes .
The cloned gpd -gene was used for the creation of two expression vectors : one containing a short (581 bp) fragment of the M. gramineum gpd -promoter sequence and one containing the complete known 5’ flanking sequence (1033 bp). In both vectors, the promoter sequences were followed by a multicloning site. The vectors are called ‘pGPDkp’ and ‘pGPDlp’, respectively.
Although production of homologous fungal proteins is usually quite efficient and can reach the g/L levels, equally successful production of heterologous proteins from filamentous fungi can be (but is not always) achieved and the production levels may lay within the mg/L range. Unfortunately, the published yields of non-fungal enzymes from filamentous fungi often are even lower, not exceeding a few tens of milligrams per litre or micrograms per litre, and in many of the studies, the enzymes were detected only intracellularly. In order to explore its capabilities to produce heterologous fungal enzymes , Myrothecium gramineum was tested as to its production of an α-amylase of A. oryzae ( chapter IV ) and of an endo −1,4−β -xylanase of Penicillium griseofulvum (chapter V ). In the case of the amylase, the results obtained with the gpd -promoter of A. nidulans (chapter I) were compared with the results obtained with the homologous M. gramineum gpd -promoter (chapter IV). ααα-Amylases constitute an important class of enzymes which find many biotechnological applications. Such applications include baking, brewing, detergents and desizing (in textile industries). Xylanases and cellulases, together with pectinases, currently account for 20 % of the world enzyme market. For example, increasing concern over preserving the environment from industrial wastes has initiated a growing interest in applying microbial enzyme systems in the paper and pulp industry. Furthermore, xylanases find their applications as supplements in animal feed, for the
General discussion, conclusion and research perspectives 230 manufacturing of bread, food, juice and wine, in the textile industry, and for the production of ethanol and xylitol.
In order to enable M. gramineum to produce the Taka-amylase of A. oryzae (encoded by amy3 ), it was transformed with the plasmids pGPDlpAmyAO and pGPDkpAmyAO, containing the full length gpd -promoter of M. gramineum and a shorter fragment thereof, respectively, followed by the amy3 sequence coding for the immature Taka-amylase (i. e. with its natural secretion signal) and its terminator sequences. Eight transformants were obtained with the plasmid pGPDlpAmyAO (LA) and two with the plasmid pGPDkpAmyAO (KA). The 10 colonies were tested as to their amylase production. The two transformants carrying the A. oryzae amylase under the control of the 581 bp fragment of the gpd -promoter of M. gramineum produced much fewer (TAKA-) amylase than the transformants carrying the amy3 sequence under the control of the 1033 bp fragment of the M. gramineum gpd -promoter or of the A. nidulans gpd -promoter (2 kb). When comparing the production levels obtained with the 1033 bp gpd -promoter sequence of M. gramineum with the gpd -promoter of A. nidulans , it can be concluded that with the homologous promoter higher amylase production was obtained than with the heterologous A. nidulans gpd -promoter (7238.6 and 2715 units amylase per mL and per gram CDW, respectively). After SDS-PAGE, a band of about 54 kDa, corresponding with the size of the recombinant amylase, appears in all samples of transformants which produce high amounts of amylase, confirming the results obtained in the enzyme tests and colony PCRs. The presence of (multiple copies of) the expression plasmids in the genome of the transformants was confirmed in Southern analysis. Myrothecium gramineum was transformed with the plasmids pGPDlpXylPG and pGPDkpXylPG in order to test its production of an endo −1,4−β -xylanase of Penicillium griseofulvum . The enzyme has a molecular mass of 24 kDa. It is characterised by an optimum pH around 5.0 and a temperature profile having its maximum activity at about 50°C. The plasmids pGPDlpXylPG and pGPDkpXylPG contain the full length gpd -promoter of M. gramineum and a shorter fragment thereof, respectively, followed by the xylanase encoding sequence of P. griseofulvum , with its natural secretion signal and terminator sequences. The transformants were grown on RBB-xylan containing plates. Colonies which express a functional xylanase are able to produce a zone of clearing in the blue background in this medium. For the transformants obtained after transformation with plasmid pGPDlpXylPG, 30 colonies had formed significantly larger clearing zones than the wild type, and for the transformants with plasmid pGPDkpXylPG 12 colonies were selected for further investigation. No clear distinction could be made between the group of transformants with the shorter promoter fragment and the group with the longer one, although twice as much transformants with the longer fragment are situated in the group of the higher xylanase producers. The three best strains obtained after transformation with pGPDkpXylPG (K) and the three best strains obtained after transformation with pGPDlpXylPG (L) were inoculated in AMM and in AMM with 15 g/L birch wood xylan in order to test the effect of this inducer of xylanolytic activity. Addition of xylan increased the wild type xylanase activity 2.3 times, which confirms that xylan induces xylanase activity. When comparing the P. griseofulvum
General discussion, conclusion and research perspectives 231 xylanase production with and without induction, it becomes clear that strains L63, L65 and K5 produce the same amount of xylanase on both media. This confirms the fact that this part of the measured xylanase activity is indeed encoded by the xylanase-gene copy or copies driven by the gpd -promoter. Indeed, the gpd -promoter is constitutive and thus not influenced by the addition of xylan. Strain L63 produced ± 600 units/(mL.g CDW) P. griseofulvum xylanase, strain L65 produced ± 440 units and strain K5 ± 360 units. SDS-PAGE indicated the presence of a 24 kDa protein, corresponding with the size of the P. griseofulvum xylanase in the lanes of L63 and L65, which was not detectable in the lane of the wild type or in the lanes of the other transformants.
The relative amount of Taka-amylase produced by the best strains obtained after transformation of M. gramineum with the ‘homologous gpd -promoter – A. oryzae amylase’- construct appears to be about 20% of the total amount of protein secreted, for both strains (chapter IV). This means that about 40 mg/L of Taka-amylase is secreted by the M. gramineum transformants. The amount of xylanase relative to the total amount of extracellular protein produced by the strains L63 and L65 was also estimated (chapter V). It was also estimated to be about 40 mg/L . These values correspond to those reported in the literature for the production of heterologous (fungal) proteins in fungal expression systems, although they are rather low as compared to 30 mg/L Thermomyces lipase (214), 70 mg/L Fusarium cutinase (166), 150 mg/L M. miehei protease, 1 g/L TAKA-amylase (216). On the other hand, lower production values were also reported, for example for the expression of the M. miehei aspartic protease in M. circinelloides (1.2 mg/L) and for the production of the A. niger glucoamylase in A. nidulans (0.2 mg/g CDW). The latter value corresponds to 2 mg/g CDW Taka-amylase produced by strain the M. gramineum LA 6 strain.
As discussed in chapter VI , efficient and cost-effective production of an enzyme having properties suitable for use at high process temperatures and pH is a challenge, because these enzymes often originate from relatively unstudied bacteria in which the production level is low. While (genetically modified strains of) filamentous fungi can be used to produce high levels of both homologous and heterologous fungal enzymes, the published yields of non- fungal enzymes from filamentous fungi often are low. On the basis of limitations observed for the production of non-fungal enzymes, several strategies have been developed to improve protein yields. One of them, the application of a gene fusion strategy, has been especially successful: the heterologous gene is often fused to the coding region of a highly expressed gene coding for an efficiently secreted enzyme. In this way, the secreted gene serves as a carrier for the heterologous protein. These strategies have resulted in 5 to 100 fold increases in the secretion of heterologous proteins, giving protein levels of 5 to > 250 mg/L. A study of the effect of various carrier polypeptides on the expression of an Nonomuraea flexuosa xylanase in Hypocrea jecorina showed that that high-yield production requires a carrier with an intact domain structure and that a flexible hinge region (connection) between the carrier and the xylanase has a positive effect on both the production of the xylanase and the efficiency of cleavage of the fusion polypeptide (206). In chapter VI , Myrothecium
General discussion, conclusion and research perspectives 232 gramineum was tested as to its production of a bacterial enzyme , more precisely an endo −1,4−β -xylanase of Bacillus subtilis . In one construct, the coding sequence of the mature bacterial xylanase was fused to the secretion signal and transcription termination sequences of the A. oryzae Taka-amylase (pGPDnocarXylBS). In another construct (pGPDcarXylBS), it was fused using a linker with a KEX2 processing site to the C-terminal end of the sequence of the A. oryzae Taka-amylase. The gpd -promoter of M. gramineum was used in both constructs. The 120 colonies analysed on PDA + 2 % RBB-xylan decoloured the medium covered by the mycelium, but they did not produce large clearing zones beyond the border of mycelial growth. The strains obtained by transformation of M. gramineum with the plasmid pGPDnocarXylBS produce between 0.18 and 1.99 units of xylanase per mL. Those obtained with the plasmid pGPDcarXylBS produce between 0.31 and 19.20 units/mL B. subtilis xylanase. Only 8 of them produce less xylanase than the highest producer without the carrier. Although these results are preliminary, they indicate that M. gramineum is able to secrete a bacterial enzyme. Moreover, the fact that higher production is achieved using a carrier protein as compared to fusion with the secretion signal alone, confirms results described in the literature. Further research will be necessary in order to confirm the obtained results. For example, SDS-PAGE analysis will have to be performed to know whether or not the carrier protein is correctly cleaved from the target protein and intracellular accumulation of the fusion or the processed carrier and target will have to be investigated. Fusion to a highly expressed and efficiently secreted homologous enzyme might improve the production of the bacterial enzyme. Furthermore, a study of the codon usage of the bacterial xylanase showed that there are a lot of differences between the preferred codons of Bacillus subtilis and those of Myrothecium gramineum . While both in the gpd- and the ompd-gene of Myrothecium a (strong) preference for a cystosine or a guanine at the third position is found, the codon usage in the xylanase gene of Bacillus indicates a preference for adenine or thymine. Moreover, the codons ‘GTA’ and ‘TTA’ are not used in the gpd- or the ompd-gene of Myrothecium , while they appear 4, respectively 5 times in the Bacillus gene. For example, optimalisation of the codon usage of the Dictyoglomus xynB to suit expression in Trichoderma resulted in a dramatic increase in the production of the enzyme.
Since the transformants obtained during this research and discussed in literature are not (always) single copy transformants with integration at a defined place in the genome, it is difficult to compare the results. Different copy numbers and different sites of integration in the genome can have large effects on the expression levels. Moreover, some of the values reported are obtained in production strains. In the literature, different strategies have been developed to improve the (heterologous) protein yield. For example, the use of strong promoters and secretion signals are mentioned. The exchange of the heterologous A. nidulans promoter by the homologous gpd -promoter already improved the production of proteins in M. gramineum . In a next step, the enzymes under investigation could be fused to a secretion signal (or a complete coding sequence) of a highly secreted homologous enzyme . Although SDS-PAGE analysis gives an indication for the fact that M. gramineum is able to process the secretion signal of the P. griseofulvum xylanase (ALA/LP) (chapter V) and the A. oryzae
General discussion, conclusion and research perspectives 233
Taka-amylase (ALA/AT) (chapter IV), the exact cleavage has not been checked. Moreover, no measurements of intracellular accumulation of the enzymes were made. Optimalisation of the secretion of the proteins could thus lead to further improvements of the production. Another way to improve production is the introduction of a high copy number of the expression cassette in the genome, preferably at active sites in the genome. As demonstrated by Southern analysis, multiple copies of the vector carrying the amylase are present in the genome of the transformants. Nevertheless, the site of integration was not controlled. Although it can generally be said that a higher copy number results in a higher protein yield, these two phenomena are not strictly correlated. To prevent the recombinant DNA from integrating at a site in the chromosome with low transcription efficiency, site specific integration vectors can be. These vectors contain the expression cassette flanked by sequences of a non-essential gene which is known to be highly expressed. The expression cassette is integrated in transcriptionally active regions via homologous recombination. It is also possible to engineer artificially high copy number strains, for example by using a cosmid vector containing up to 10 copies of an expression cassette. Unfortunately, even when high copy numbers are present and when heterologous proteins are successfully excreted, a substantial part of the production can be lost via degradation by extracellular proteases. Moreover, heterologous proteins are more sensitive to proteolysis than homologous proteins. Proteolytic degradation by fungal proteases is considered to be one of the major problems with heterologous protein production. Thus, the reduction or the elimination of protease activity has been a major area of interest in host strain improvement. For example, the gene encoding the major secreted aspartyl protease ( pepA ) of A. awamori and A. niger has been successfully deleted from the strains, resulting in 2-fold higher yields. The expression of antisense RNA of the carboxypeptidase O gene led to a more stable and higher expression of lysozyme in Aspergillus oryzae . Besides the extracellular proteases, the large amounts of organic acids can be a problem once the proteins are secreted. As a result of the secretion, the external pH drops, which can alter the structure of certain proteins or lead to increased sensitivity to acid proteases active in the culture broth. Altering the culture conditions or genetic engineering to avoid the secretion of certain organic acids thus can be another objective in improving the protein production yield. Finally, classical methods such as random mutagenesis followed by a screening of the resulting mutants were already successfully applied to improve protein production.
To be able to use this fungus for the industrial production of enzymes an easy selection method for clones of this fungus carrying the introduced expression cassette. The literature describes many different methods to select for transformants of fungi, using dominant selection markers or auxotrophic markers. Since antibiotic resistance markers cannot be used for some industrial applications and homologous transformation systems are more efficient, the orotidine monophosphate decarboxylase gene ( ompd -gene) was chosen as selection marker and isolated from the genome of Myrothecium gramineum . The OMPD selection system is based on the fluoro-orotic acid resistance of mutants defective in the enzymatic conversion of orotate to uridine-5’-monophosphate (UMP), which is catalysed by
General discussion, conclusion and research perspectives 234 the combined action of OMPD and orotic acid phosphoribosyltransferase (OPRT). These mutants are uracil or uridine auxotrophic. Thus, both positive and negative selection is possible for OMPD-wild type strains as well as for OMPD-deficient strains: mutants are resistant to fluoro-orotic acid (5-FOA) and uracil auxotrophic, while wild type strains are 5- FOA sensitive and uracil prototrophic. This is very useful to perform successive transformations on the same strain.
In chapter VII , the cloning of the ompd -gene of Myrothecium gramineum is discussed. It is the second gene of Myrothecium gramineum that has ever been cloned and analysed. The gene was submitted to the GenBank (GenBank accession no. DQ359751 ). In the 5’ flanking region of the cloned gene, a potential CCAAT-fragment and 2 potential TATA-boxes were found. A pyrimidine rich region was found in the promoter between 172 and 39 nucleotides upstream the start codon (74.6 %). The two TATA-boxes are included in the CT-rich region. This gene contains an intron of 59 bp, which was confirmed by cDNA analysis. The presence of an intron is unique, because it is the first ompd -gene of a Sordariomycete which has an intron. Normally, only ompd -genes of non-Sordariomycetes contain introns, corresponding in length and position with the intron found here. The ompd -genes of most Ascomycota , except the class of Sordariomycetes , have one intron at a conserved position. Remarkably, the Myrothecium gene, in contrast to all other ompd -genes of Sordariomycetes , has the intron at the conserved position. While genes of higher eukaryotes normally have AATAA polyadenylation signals, these are often shorter or missing in filamentous fungi. A potential polyA signal was found 208 nucleotides downstream the stop codon. A sequence similar to other transcription termination signals (CGTGTT) was found in the ompd -gene of Myrothecium 124 nucleotides downstream the polyA sequence.
The M. gramineum ompd -gene codes for an enzyme of 282 amino acids, with a molecular mass of 30 kDa. The enzyme contains the recognition site found in all known OMPD- enzymes: [LIVMFTAR]-[LIVMF]-x-D-x-K-x(2)-D-[IV]-[ADGP]-x-T-[CLIVMNTA]. In the amino acid sequence of the OMPD-enzyme of M. gramineum , the pattern is found as IFEDRKFVDIGSTA. Thus, the obtained sequence is certainly representing an OMPD- enzyme (orotidine 5'-phosphate decarboxylase (OMPdecase), EC 4.1.1.23). OMPD-enzymes of Sordariomycetes normally consist of about 360-400 amino acids, while those of other Ascomycota enclose approximately 280. On top of the fact that the ompd -gene of M. gramineum has an intron, its protein thus also lacks the 100 amino acid insert. Comparison of the OMPD-enzyme of Myrothecium with other OMPD-enzymes revealed a higher similarity with enzymes of non-Sordariomycetes and even with those of yeast than with OMPD’s of other Sordariomycetes , a class that includes the Myrothecium genus. This is why the gene here will be referred to as pyrG and not pyr4 .
Determination of the ompd copy number in the genome of M. gramineum indicated that a single copy of the ompd -gene is present in the genome of M. gramineum . This allows the isolation of OMPD-negative mutants as a means towards the establishment of a genetic
General discussion, conclusion and research perspectives 235 transformation system for M. gramineum , based on the use of the homologous ompd -gene as a selection marker. The functionality of the enzyme as a selection marker was proven by complementation of the uracil auxotrophy of Aspergillus nidulans FGSC A722 and by the fact that it renders the strain 5-FOA-sensitive. The transformants were uracil prototroph and 5- FOA sensitive.
Several articles describe the use of the OMPD-enzyme as a phylogenetic marker. The OMPD- enzyme is an ideal protein for molecular evolutionary studies because all organisms have the same de novo pyrimidine biosynthesis pathway and thus must have OMPD activity, because the OMPD-enzyme is a universal enzyme and nearly all investigated organisms have only one ompd -gene and because the OMPD amino acid sequences contain some regions which have been conserved over more than a billion years. It was decided to build a fungal phylogenetic tree based on 35 fungal amino acid sequences. The obtained tree corresponds well with the current taxonomy of filamentous fungi: it divides these organisms in three groups, more precisely the Ascomycota , the Basidiomycota and the Zygomycota . Within the Ascomycota three groups can be distinguished: the Eurotiomycetes , the Dothideomycetes and the Sordariomycetes . Remarkably, M. gramineum , which taxonomically belongs to the Sordariomycetes , is classified here in the class of the Eurotiomycetes . This result is in accordance with the fact that the M. gramineum OMPD-enzyme resembles more to the OMPD-enzymes of Eurotiomycetes than to those of Sordariomycetes . In order to know if other M. gramineum strains have similar OMPD-enzymes to that of the M. gramineum strain MUCL 39210, which is used during our research, attempts were made to isolate the ompd - gene of M. gramineum strain MUCL 44829, 44487 and 11831. Unfortunately, only a fragment of the ompd -gene of strain MUCL 44829 was obtained. Like the enzyme of strain 39210, it has an intron and it lacks the 100 amino acids. Accordingly, after phylogenetic analysis based on the OMPD sequences, strain 44829 was placed close to strain MUCL 39210, in the class of the Eurotiomycetes . It is noteworthy that, in the case of M. gramineum strains MUCL 39210 and 44829, the results of phylogenetic research based on the OMPD - enzyme would be misleading : these Myrothecium strains would not have been classified under the Sordariomycetes , even though, for strain MUCL 39210, morphological characteristics and ITS sequence analysis prove it belongs to this subclass (29, 48). Moreover, phylogenetic analysis based on the GPD sequence of this strain confirmed its current classification (chapter III).
In chapter VIII , the development of a homologous transformation system based on the orotidine monophosphate decarboxylase gene is discussed. Two selective media were developed, one selective against OMPD deficient strains (no uracil or uridine) and the other selective against OMPD wild type strains. The latter was used in further experiments to select for the OMPD-negative mutant of M. gramineum (5-FOA resistant, uracil auxotrophic). In this research, a strategy to create a mutant which allows the use of the obtained mutant strain in expression studies was developed. In such expression experiments, it is important that the expression cassette integrates at a defined site in the genome, in single copy and preferably at
General discussion, conclusion and research perspectives 236 a high frequency. The use of two different mutations within one gene so that a selectable phenotype is only generated by a single cross over at the site of interest increases the chance of obtaining 1 copy of the vector at a specific site. In the vectors ceated for M. gramineum , both for the 5’ and 3’ mutations, the deletion of conserved amino acid regions was aimed at, in combination with frameshifts in order to ensure the enzyme inactivation after recombination. The vector pOVmut3’ contains a deletion of 67 bp 0.5 kb downstream the start codon and the mutation on vector pOVmut5’ consists of a deletion of the end of the promoter, the ATG start codon and an N-terminal part of the gene.
Plasmid pOVmut3’ was used to create an OMPD knock-out by transformation of the Myrothecium gramineum wild type strain. Homologous recombination between the mutant gene on the plasmid and the wild type gene should lead to a deficient copy of the ompd -gene in the genome. 381 colonies grew ‘anti-Opos’ medium, but all of the colonies were able to grow on the ‘anti-Oneg’ medium, which means none of them is OMPD-negative. It became that problems arose with the selection on the ‘anti-Opos’ plates. Before testing the possibility of transformation with linear cassettes, experiments were set up to find the cause of the selection of false positive colonies. A difference between the growth of mycelium and that of the spores on the ‘anti-Opos’ was observed: mycelium was able to grow on the ‘anti-Opos’ plates, while spores were not. Thus, in the following transformation experiments, the protoplasts were inoculated on complete medium and the plates were incubated until sporulation had occurred. After that, the spores were inoculated on ‘anti-Opos’ plates.
Since experiments showed that the transformation efficiency with linear DNA was to low, a transformation experiment with plasmid pOVmut5’ was carried out. Twelve of the inoculated spore droplets produced tiny amounts of mycelium on ‘anti-Opos plates’ and 67 very small mycelial colonies grew on the plates inoculated with the spore mix. Unfortunately, the colonies were all able to grow on ‘anti-Oneg’ medium (inoculated with their mycelium).
Because of the difficulties arising with the creation of an OMPD-negative strain by homologous recombination, some attempts were made to isolate such a strain after UV- mutagenesis. None of the obtained 5-FOA-resistant colonies were uracil auxotrophs, despite the fact that a reasonable number of them was obtained. In the literature, low frequencies of OMPD-negative strains among 5-FOA resistant ones have also been reported. Considering these observations, it might be necessary to further increase the number of 5-FOA resistant colonies or to improve the efficiency of homologous recombination in order to obtain a desired OMPD-negative strain.
Regarding the problems appearing with the development of a selection system based on the OMPD enzyme, it might be interesting to look for another system . An example is the acetamidase gene ( amdS ) which has been widely used for the transformation of filamentous fungi. Transformants which incorporate the amdS gene and which express it are able to grow on media with acetamide as the only nitrogen source. Counter selection for amdS positive
General discussion, conclusion and research perspectives 237 strains can be performed using their fluoroacetamide sensitivity. The degree of acetamide metabolisation reflects the gene copy number and thus can be used for the selection of transformants with a high gene copy number. Because Myrothecium gramineum MUCL 39210 can not grow on minimal medium with acetamide as the sole nitrogen source, the amdS gene could be used as a selection marker for M. gramineum . However, the repression of the amdS gene promoter by high glucose concentrations which are needed for the osmotic stabilisation of the protoplasts hampered the development of a transformation system based on this marker (see also chapter I). The isolation of the homologous gpd -promoter creates the possibility to exchange the A. nidulans amdS promoter by this homologous and constitutive promoter. Since all required media for the amdS system were already developed, the system might become more quickly available as compared to a completely new one.
Another possibility is to develop a system based on the nitrate reductase gene. In this system, like in the OMPD and the AmdS system, the marker is both selectable and counter selectable. These systems allow the use of positive screening methods for auxotrophic mutants of a defined gene and thus can be used for genetically poorly characterised fungi. Nitrate reductase negative mutants are resistant to chlorate and they are unable to grow on media with nitrate as the sole nitrogen source. Homologous systems are described for A. niger, A. oryzae, A. parasiticus, Botryts cinerea, A. chrysogenum, F. oxysporum, Gibberella fujikori, Leptosphaeria maculans, P. chrysogenum, P. griseoroseum and Staganospora nodorum.
It can be concluded that a homologous expression system for the fungus Myrothecium gramineum was successfully developed. The system is based on the homologous glyceraldehyde-3-phosphate dehydrogenase (Genbank accession number EF486690 ). Using this system, recombinant Aspergillus oryzae Taka-amylase and Penicillium griseofulvum endo-1,4-β-xylanase production was obtained. Preliminary results indicate that the fungus is also able to produce a bacterial enzyme, more precisely, the Bacillus subtillis endo-1,4-β- xylanase. In order to be able to screen for transformants of the M. gramineum wild type without the need for antibiotics, other selective markers were searched for. The orotidine monophosphate decarboxylase gene (GenBank accession no. DQ359751 ) was cloned and characterised. The gene was successfully used to complement a defined Aspergillus nidulans ompd deficient strain. When an OMPD deficient strain of M. gramineum is obtained, the suitability of the selection system can be further investigated.
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Summary
Summary 267
Summary For centuries, humans have unwittingly used microorganisms for their own benefit. Filamentous fungi, yeasts and their enzymes are indeed instrumental since thousands of years in the making of beer, wine, bread and cheese and in several other fermented foods . Nowadays, fungi and their enzymes are still widely used in the food industry, but they are also increasingly applied in other industrial fields. Despite the fact that fungi already have a broad spectrum of industrial applications, until now, only a limited number of fungal host species has been explored for recombinant protein production. Therefore, it is not surprising that several parties started to explore the alternatives to those covered by several patent applications.
The fact that many currently used fungal expression hosts are protected by patent applications, taken together with the consideration that, undoubtedly, filamentous fungi exist which have new characteristics and produce enzymes with new application possibilities, was an inspiration to start exploring the BCCM/MUCL agro-industrial fungi- and yeast collection for a potential new expression host. The aim of our research was to further develop a transformation system and to develop a homologous expression system to enable us to use the filamentous fungus Myrothecium gramineum MUCL 39210 (syn. Xepiculopsis graminea ) as a new, universal expression host.
As discussed in chapter I , further research was performed on the development of a transformation system which would enable us to use the filamentous fungus Myrothecium gramineum as a new, universal expression host. The basic transformation protocol to transform M. gramineum used in preliminary experiments at the Beldem company is the one described by Punt and Van den Hondel, which uses protoplast generation for the transformation of filamentous fungi. During this research, the transformation protocol was modified in order to increase the transformation efficiency, which would allow screening for higher production strains. The preculture was carried out in medium in which the strain grows under the form of filamentous mycelium instead of in pellets. A heat shock was introduced after DNA was added to the protoplasts. With these modifications, a transformation efficiency of 5.6 transformants per µg DNA and per 10 6 viable protoplasts was achieved. Transformants were selected based on their resistance to hygromycin B and their ability to overproduce the Taka amylase of Aspergillus oryzae , which was used as a reporter gene. From a selection of 80 transformants, 19 produced significantly more amylase than the wild type strain.
A quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications. While rapid and high throughput PCR-methods or other molecular techniques are amply available for bacteria and even for yeast, this is not the case for filamentous fungi. In chapter II , we compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR. Both for Myrothecium gramineum and for
Summary 268
Aspergillus nidulans , the highest DNA concentration was obtained with the protoplast extraction procedure. Most extraction protocols resulted in higher DNA concentrations for A. nidulans than for M. gramineum . Samples of M. gramineum resulting from the boiling procedure (1.5 hours) and the liquid nitrogen procedure (2.5 hours) were suitable for the amplification of fragments up to 2.3 kb. The direct use of mycelium from M. gramineum in the PCR tube can be employed for the reproducible amplification of fragments up to 1 kb and even weak signals of 2.3 kb can be obtained. Amplification of fragments up to 4.3 kb requires the use of the long distance polymerase Elongase Mix on samples extracted with the liquid nitrogen procedure.
The use of strong promoters for the expression of proteins in suitable host organisms is of great importance for biotechnological applications. However, it has been shown that there is not much homology between fungal promoters, not even in related fungi. Because little is known about the critical parts of fungal promoters till today, a strong homologous promoter is usually searched for when developing a new expression system. It is assumed that this strong promoter will function ideally in the new expression host. The glyceraldehyde-3-phosphate dehydrogenase promoter is a promising candidate because in many eukaryotic microorganisms, the gpd -genes are expressed constitutively and in large amounts. Thus, the complete sequence of the glyceraldehyde-3-phosphate dehydrogenase gene of Myrothecium gramineum was determined and submitted to the Genbank ( EF486690 ), as described in chapter III . In the promoter region, two TATA-boxes, four CAAT-similar sequences and five CT-rich stretches are found. The coding region contains two introns, the first of which is situated at a position conserved within gpd -genes of Ascomycota . Two polyA-signals are each followed by a transcription termination site. The 3041 bp gene encodes an enzyme of 339 amino acids. The amino acid sequence shows most similarity with the GPD-enzymes of Beauveria bassiana (95 %) and other filamentous fungi. Phylogenetic analysis based on the amino acid sequence of the M. gramineum GPD confirmed the current classification of M. gramineum within the class of the Sordariomycetes . Two expression vectors were created containing a short (581 bp) and a long (1033 bp) fragment of the M. gramineum gpd -promoter sequence followed by a multicloning site.
In order to explore its capabilities to produce (heterologous) enzymes, Myrothecium gramineum , was tested as to its production of an α-amylase of Aspergillus oryzae . This aspect of the research is discussed in chapter IV . α-Amylases constitute an important class of enzymes which find many biotechnological applications including baking, brewing, detergents and desizing (in textile industries). M. gramineum was transformed with the plasmids pGPDlpAmyAO and pGPDkpAmyAO, containing the full length gpd -promoter of M. gramineum and a shorter fragment thereof, respectively, followed by the amy3 gene of A. oryzae , with its natural secretion signal and terminator sequences. Eight transformants were obtained with the plasmid pGPDlpAmyAO (LA) and two with the plasmid pGPDkpAmyAO (KA). The two transformants carrying the A. oryzae amylase under the control of the 581 bp fragment of the gpd -promoter of M. gramineum produced much fewer amylase than the
Summary 269 transformants carrying the amy3 gene under the control of the 1033 bp fragment of the M. gramineum gpd -promoter or of the A. nidulans gpd -promoter (2 kb). When comparing the production levels obtained with the 1033 bp gpd -promoter sequence of M. gramineum with the gpd -promoter of A. nidulans , it can be concluded that with the homologous promoter higher amylase production was obtained than with the heterologous A. nidulans gpd -promoter (7238.6 and 2715 units amylase per mL and per gram CDW, respectively).
In chapter V , Myrothecium gramineum was tested as to its production of an endo −1,4−β - xylanase of Penicillium griseofulvum MUCL 41920. M. gramineum was transformed with the plasmids pGPDlpXylPG and pGPDkpXylPG, containing the full length gpd -promoter of M. gramineum and a shorter fragment thereof, respectively, followed by the xylanase gene of P. griseofulvum , with its natural secretion signal and terminator sequences. Some of the colonies produced relatively larger clearing zones on RBB-xylan containing plates, which is an indication for the fact that more xylanase is expressed. 30 colonies with pGPDlpXylPG (L) and 12 colonies with pGPDkpXylPG (K) were further invesigated. Twice as much transformants with the longer fragment (15) were situated in the group of the higher xylanase producers as compared to the ones with the shorter fragment (7). The strains K5, K15 and K24 and the strains L63, L65 and L78 were inoculated in AMM and in AMM with 15 g/L birch wood xylan in order to test the effect of this inducer of xylanolytic activity. Addition of xylan increased the wild type xylanase activity 2.3 times, which confirms that xylan induces xylanase activity. When comparing the P. griseofulvum xylanase production with and without induction, one can see that strains L63, L65 and K5 produce the same amount of xylanase on both media. This confirms the fact that this part of the measured xylanase activity is indeed encoded by the xylanase-gene copy or copies driven by the gpd -promoter. Strain L63 produced ± 600 units/(mL.g CDW) P. griseofulvum xylanase, strain L65 produced ± 440 units and strain K5 ± 360 units.
In chapter VI , Myrothecium gramineum was tested as to its production of a bacterial enzyme, more precisely an endo −1,4−β -xylanase of Bacillus subtilis . In one construct, the coding sequence of the mature bacterial xylanase was fused to the secretion signal and transcription termination sequences of the A. oryzae Taka-amylase (pGPDnocarXylBS). In an other construct (pGPDcarXylBS), it was fused using a linker with a KEX2 processing site to the C- terminal end of the sequence of the A. oryzae Taka-amylase. The gpd -promoter of M. gramineum was used to drive expression in both constructs. The 120 colonies analysed on PDA + 2 % RBB-xylan did not produce large clearing zones beyond the border of mycelial growth, but they did decolour the medium covered by the mycelium. The strains obtained by transformation of M. gramineum with pGPDnocarXylBS produced between 0.18 and 1.99 units of xylanase per mL and those obtained with pGPDcarXylBS between 0.31 and 19.20 units/mL B. subtilis xylanase. Only 8 of the latter produced less xylanase than the highest producer without the carrier. Although these results are preliminary, they indicate that M. gramineum is able to secrete a bacterial enzyme. Moreover, the fact that higher production is achieved using a carrier protein (with linker and KEX2 processing site) to guide the B. subtilis
Summary 270 xylanase through the secretion pathway of M. gramineum as compared to fusion with the secretion signal alone, confirms results described in the literature.
To be able to use this fungus for the industrial production of enzymes, an efficient expression system is required, as well as an easy selection method for clones of this fungus carrying the introduced expression cassette. Since antibiotic resistance markers cannot be used for some industrial applications and homologous transformation systems are more efficient, the orotidine monophosphate decarboxylase gene ( ompd -gene) was chosen as selection marker and isolated from the genome of Myrothecium gramineum , as discussed in chapter VII . It is the second gene of Myrothecium gramineum that has ever been cloned and analysed (GenBank accession no. DQ359751 ). The 2050 bp gene codes for an enzyme of 282 amino acids and has an intron of 59 bp, which is unique for an ompd -gene of a Sordariomycete . Accordingly, phylogenetic analysis based on the amino acid sequence of the M. gramineum OMPD did not confirm the current classification of M. gramineum within the class of the Sordariomycetes . The gene and its amino acid sequence show high similarity with OMPD- enzymes of Aspergillus sp., Penicillium sp., Cladosporium fulvum and other fungi. It was shown that this Myrothecium strain has only one ompd copy. The functionality of the enzyme as a selection marker was proven by complementation of the uracil auxotrophy of Aspergillus nidulans FGSC A722 and by the fact that it renders the strain 5-FOA-sensitive. The transformants were uracil prototroph and 5-FOA sensitive.
In chapter VIII , the development of a homologous transformation system based on the orotidine monophosphate decarboxylase gene is discussed. Two selective media were developed, more precisely the ‘‘anti-Oneg’ medium selective against OMPD deficient strains (no uracil or uridine) and the ‘anti-Opos’ medium, selective against OMPD wild type strains (addition of 5-FOA). This medium was used in further experiments to select for the OMPD- negative mutant of M. gramineum (5-FOA resistant, uracil auxotrophic). In this research, a strategy to create an OMPD deficient strain which can be used in expression studies was developed. The use of two different mutations within one gene so that a selectable phenotype is only generated by a single cross over at the site of interest increases the chance of obtaining 1 copy of the vector at a specific site. Thus, two vectors were created (pOVmut3’ and pOVmut5’). For both the 5’ and 3’ mutations, the deletion of conserved amino acid regions was aimed at. The vector pOVmut3’ contains a deletion of 67 bp 0.5 kb downstream the start codon. The mutation on vector pOVmut5’ consists of a deletion of the end of the promoter, the ATG start codon and an N-terminal part of the gene. Plasmid pOVmut3’ was used to create an OMPD knock-out by transformation of the Myrothecium gramineum wild type strain. Homologous recombination between the mutant gene on the plasmid and the wild type gene should lead to a deficient copy of the ompd -gene in the genome. Unfortunately, problems arose with the selection on the ‘anti-Opos’ plates. A clear difference between the growth of mycelium and that of the spores on the ‘anti-Opos’ was observed. Mycelium of the strain MUCL 39210 is able to grow on the ‘anti-Opos’ plates, while spores are not. Thus, in the following transformation experiments, the protoplasts were inoculated on complete
Summary 271 medium and the plates were incubated until sporulation had occurred. After that, the spores were inoculated on ‘anti-Opos’ plates. Because the transformation efficiency with linear DNA fragments was too low, a transformation experiment with plasmid pOVmut5’ was carried out. Seventy nine spore droplets produced tiny amounts of mycelium on ‘anti-Opos plates’. Unfortunately, the colonies were all uracil prototrophic. Because of the difficulties arising with the creation of an OMPD-negative strain by homologous recombination, some attempts were made to isolate such a strain after UV-mutagenesis. Despite the fact that a reasonable number of 5-FOA-resistant colonies was obtained, none of them were uracil auxotrophs.
It can be concluded that a homologous expression system for the fungus Myrothecium gramineum was successfully developed. The system is based on the homologous glyceraldehyde-3-phosphate dehydrogenase (Genbank accession number EF486690 ). Using this system, recombinant A. oryzae amylase and P. griseofulvum xylanase production was obtained. Preliminary results indicated that M. gramineum is able to produce a bacterial enzyme, namely a B. subtilis xylanase. In order to be able to screen for transformants of the M. gramineum wild type without the need for antibiotics, other selective markers were searched for. The orotidine monophosphate decarboxylase gene (GenBank accession no. DQ359751 ) was cloned and characterised. The gene was successfully used to complement a defined Aspergillus nidulans OMPD deficient strain. When an OMPD deficient strain of M. gramineum is obtained, the suitability of the selection system can be further investigated.
Samenvatting
Samenvatting 275
Samenvatting Filamenteuze schimmels, gisten en hun enzymen worden sinds duizenden jaren door de mens gebruikt voor het produceren van bier, wijn, brood en kaas, en in verschillende andere fermented foods . Tegenwoordig worden schimmels en hun enzymen nog steeds gebruikt in de voedingsindustrie, maar zij worden ook steeds meer ingezet voor andere industriële toepassingen. Ondanks het feit dat schimmels in een breed spectrum van industriële toepassingen worden ingeschakeld, wordt slechts een beperkt aantal fungale gastheren gebruikt voor de productie van recombinante eiwitten en bovendien worden de klassieke fungale gastheren beschermd door patenten. Het is dan ook niet verrassend dat verschillende groepen zoeken naar andere gastheren dan die beschermd door patenten. Vandaar het idee om de BCCM/MUCL agro-industriële schimmel- en gistcollectie te screenen naar potentieel nieuwe expressie-gastheren. Het doel van dit onderzoek was een transformatiesysteem verder uit te werken en een expressiesysteem te ontwikkelen dat ons in staat zou stellen de schimmel Myrothecium gramineum MUCL 39210 (syn. Xepiculopsis graminea ) te gebruiken als een nieuwe, universele expressie-gastheer.
Zoals beschreven in hoofdstuk I werd onderzoek verricht naar de verdere ontwikkeling van een transformatiesysteem voor Myrothecium gramineum . Het basisprotocol dat in preliminair onderzoek door de firma Beldem werd gebruikt voor de transformatie van deze nieuwe gastheer werd afgeleid van het protocol beschreven door Punt en Van den Hondel en maakt gebruik van protoplasten. Tijdens dit onderzoek werd het transformatieprotocol gewijzigd om de transformatie-efficiëntie te verhogen, wat het screenen naar betere productiestammen mogelijk zou maken. Voor het bereiden van de protoplasten werden de preculturen gegroeid in medium waarin de stam groeit in filamenteuze vorm in plaats van in pellets, die minder toegankelijk zijn voor de celwand-afbrekende enzymen en dus minder geschikt zijn voor de bereiding van protoplasten. Nadat het DNA werd toegediend aan de protoplasten werd een hitteschok toegediend. Met deze wijzigingen werd een transformatie-efficiëntie bekomen van 5.6 transformanten per µg DNA en per 10 6 leefbare protoplasten. Transformanten werden geselecteerd op basis van hun resistentie aan het antibioticum hygromycine B en op basis van hun capaciteit om het Taka-amylase van Aspergillus oryzae te overproduceren. Van een selectie van 80 transformanten produceerden er 19 significant meer amylase dan de wild type stam.
Een snelle en betrouwbare methode voor het screenen van fungale stammen naar specifieke genetische modificaties is essentieel voor vele moleculaire toepassingen, zoals bijvoorbeeld wanneer men een transformatie- of expressie-systeem voor een nieuwe gastheer ontwikkelt. Terwijl snelle en high-throughput PCR-methoden of andere moleculaire technieken ruimschoots ter beschikking zijn voor bacteriën en zelfs voor gisten, is dit niet het geval voor filamenteuze schimmels. In hoofdstuk II wordt het testen van 10 methoden voor de snelle extractie van DNA voor Myrothecium en voor Aspergillus beschreven . De bekomen DNA stalen werden getest op hun bruikbaarheid voor PCR. Zowel voor M. gramineum als voor A.
Samenvatting 276 nidulans werden de hoogste concentraties bekomen met de protoplast extractiemethode. De meeste extractiemethoden leverden stalen met hogere concentraties bij Aspergillus dan bij Myrothecium . Stalen bekomen via de ‘opkook’ methode (1.5 uur) en via een methode gebruik makend van vloeibare stikstof (2.5 uur) waren geschikt voor de vermenigvuldiging van fragmenten tot 2.3 kb in PCR. Bij direct gebruik van mycelium in de PCR reactiemix konden slechts fragmenten tot 1 kb vermenigvuldigd worden op een reproduceerbare manier. Voor de vermenigvuldiging van fragmenten tot 4.3 kb is het gebruik van de long distance polymerase Elongase Mix nodig, en dit op stalen geëxtraheerd met behulp van vloeibare stikstof.
Het gebruik van sterke promotoren voor de expressie van eiwitten in geschikte expressie- gastheren is van groot belang voor biotechnologische toepassingen. Het werd echter aangetoond dat er weinig homologie bestaat tussen fungale promotoren, zelfs van verwante schimmels. Omdat er algemeen weinig geweten is over de kritische onderdelen van fungale promotoren, wordt er meestal een sterke homologe promotor gezocht als men een nieuw expressiesysteem ontwikkelt. Er wordt verondersteld dat deze promotor ideaal zal functioneren in de nieuwe expressie-gastheer. De glyceraldehyde-3-fosfaat dehydrogenase (GPD, EC 1.2.1.12) promotor is een veelbelovende kandidaat omdat in vele eukaryote microorganismen de gpd -genen in hoge mate en constitutief tot expressie komen. Daarom werd tijdens dit onderzoek het glyceraldehyde-3-fosfaat dehydrogenase gen van Myrothecium gramineum gekloneerd (Genbank nummer EF486690 ), zoals beschreven in hoofdstuk III . In de promotor regio werden twee TATA-boxen, vier CAAT sequenties en vijf CT-rijke sequenties gevonden. Twee polyA-signalen worden elk gevolgd door een transcriptie terminatie plaats. Het 3041 bp lange gen codeert voor een enzyme van 339 aminozuren. De aminozuursequentie vertoont de grootste gelijkenissen met het GPD-enzym van Beauveria bassiana (95 %) en dat van andere filamenteuze schimmels. Fylogenetische analyse gebaseerd op de aminozuursequentie van het M. gramineum GPD bevestigde de huidige classificatie van M. gramineum in de classe van de Sordariomycetes . Twee expressievectoren met de M. gramineum gpd -promotor werden gecreëerd, waarvan 1 de volledige (gekende) sequentie van de promotor bevat (1033 bp, pGPDlp) en de andere een 581 bp fragment ervan (pGPDkp).
Om de capaciteit te testen van Myrothecium gramineum om (heterologe) enzymen te produceren, werd de schimmel onderzocht naar zijn productiemogelijkheden voor recombinant amylase, meer bepaald het α-amylase van A. oryzae . Dit deel van het onderzoek wordt besproken in hoofdstuk IV . α-Amylasen vormen een belangrijke klasse van enzymen die talrijke biotechnologische toepassingen kennen, bijvoorbeeld in het bakken, in het brouwen, in detergenten en in de textielindustrie. M. gramineum werd getransformeerd met de plasmiden pGPDlpAmyAO en pGPDkpAmyAO, die de volledige gpd -promotor van M. gramineum , respectievelijk een korter fragment ervan, bevatten gevolgd door het amy3 gen van A. oryzae , inclusief het natuurlijke secretiesignaal en de transcriptieterminatiesignalen. Er werden 8 transformanten bekomen met pGPDlpAmyAO (LA) en twee met pGPDkpAmyAO (KA). De 10 kolonies werden gecontroleerd op hun amylaseproductie en de resultaten
Samenvatting 277 bevestigden die van de kolonie PCR. De stammen LA 2, LA 4, LA 5, LA 6, KA 1, KA 2, de wild type stam en transformant 114 werden verder onderzocht. De twee transformanten bekomen met het korte promotorfragment produceerden veel minder amylase dan diegene met de volledige homologe gpd -promotor of met de gpd -promotor van A. nidulans . Met de homologe promotor werden hogere amylaseproducties bekomen dan met de gpd -promotor van A. nidulans .
In hoofdstuk V wordt besproken hoe de capaciteit van Myrothecium gramineum om een endo −1,4−β -xylanase van Penicillium griseofulvum MUCL 41920 te produceren onderzocht werd. M. gramineum werd getransformeerd met de plasmiden pGPDlpXylPG en pGPDkpXylPG, die de volledige gpd -promotor van M. gramineum bevatten, respectievelijk een korter fragment ervan, gevolgd door het xylanasegen van P. griseofulvum , inclusief het natuurlijke secretiesignaal en de transcriptieterminatiesignalen. De transformanten werden gegroeid op RBB-xylaan bevattende platen. Sommige kolonies vormden relatief grote klaringszones, wat een aanwijzing was voor het feit dat deze meer xylanase produceren. Dertig kolonies met pGPDlpXylPG en 12 kolonies met pGPDkpXylPG werden geselecteerd voor verder onderzoek naar hun (recombinante) xylanaseproductie. Dubbel zoveel transformanten met de lange promotor (15) als met de korte promotor zaten in de groep van geselecteerde overproducenten. De stammen K5, K15 en K24, bekomen na transformatie met pGPDkpXylPG, en de stammen L63, L65 en L78, bekomen na transformatie met pGPDlpXylPG werden gegroeid in AMM en in AMM met 15 g/L xylaan om het effect van deze inducer te controleren. Het toevoegen van xylaan aan het medium verhoogde de wild type xylanase activiteit 2.3 keer, wat bevestigt dat xylaan xylanase activiteit induceert. Wanneer de productie van recombinant xylanase in de stammen L63, L65 en K5 in medium met en zonder xylaan werd vergeleken, kon men vaststellen dat op beide media even veel recombinant xylanase werd geproduceerd. Dit bevestigt dat de gemeten recombinante xylanase activiteit inderdaad gecodeerd wordt door 1 of meerdere kopijen van het P. griseovulvum xylanase gecontroleerd door de gpd -promotor. Deze promotor is immers constitutief en wordt dus door xylaan niet beïnvloed. Stam L63 produceert ± 600 units/(mL.g CDW) P. griseofulvum xylanase, stam L65 produceert ± 440 units en stam K5 ± 360 units.
Hoewel de productie van homologe fungale enzymen het gram per liter niveau kan bereiken, is de productie van heterologe fungale enzymen (soms) veel minder rendabel en jammer genoeg worden met niet-fungale enzymen nog lagere productieniveaus bereikt. In hoofdstuk VI wordt de productie van een bacterieel enzym, meer bepaald het endo −1,4−β -xylanase van Bacillus subtilis door Myrothecium gramineum besproken. In een eerste construct werd de sequentie coderend voor het mature bacteriële enzym gekoppeld aan het secretiesignaal en de transcriptieterminatiesignalen van het A. oryzae Taka-amylase (pGPDnocarXylBS). In een tweede construct (pGPDcarXylBS) werd dezelfde sequentie via een ‘linker’ met een KEX2 protease knipplaats gekoppeld aan het C-terminale einde van het A. oryzae Taka-amylase. In beide constructen werd de gpd -promotor van M. gramineum gebruikt. Er werden 120 kolonies getest op platen met 2 % RBB-xylaan. Zij konden geen klaringszone maken verder dan de
Samenvatting 278 rand van de kolonie, maar onder het mycelium werd het medium wel ontkleurd. Twintig kolonies werden verder getest. De stammen met het pGPDnocarXylBS plasmide produceerden tussen 0.18 en 1.99 units xylanase per mL, terwijl die met het pGPDcarXylBS plasmide tussen 0.31 en 19.20 units/mL B. subtilis xylanase produceerden. Slechts 8 van deze stammen produceerden minder xylanase dan de hoogste producent bekomen met het construct zonder de amylase ‘drager’. Hoewel deze resultaten slechts voorlopig zijn tonen zij aan dat M. gramineum een bacterieel eiwit kan produceren en bevestigen zij de resultaten beschreven in de literatuur in verband met de fusie van bacteriële eiwitten aan fungale dragers.
Naast een efficiënt expressiesysteem is voor de ontwikkeling van een nieuwe expressie- gastheer ook een geschikt selectiesysteem nodig. Aangezien antibioticum resistentiemerkers niet gebruikt kunnen worden voor bepaalde industriële toepassingen (zoals bijvoorbeeld in de voedingsindustrie) en omdat homologe systemen vaak betere resultaten geven dan heterologe, werd er voor gekozen om het orotidine monofosfaat decarboxylase gen ( ompd -gen) als selectiesysteem te ontwikkelen voor Myrothecium gramineum . Dit systeem laat positieve selectie toe van zowel de auxotrofe stam (5-FOA resistentie) als van de gecomplementeerde stam (niet meer auxotroof voor uracil). In hoofdstuk VII wordt de klonering van het ompd - gen van Myrothecium gramineum besproken. Het is het tweede gen dat ooit van Myrothecium gramineum gekloneerd en geanalyseerd werd. Het 2050 bp gen (GenBank nummer DQ359751 ) codeert voor een enzyme van 282 aminozuren en heeft een intron van 59 bp, wat uniek is voor een ompd -gen van een Sordariomyceet . Het gen en de overeenkomstige aminozuursequentie vertonen de hoogste similariteit met OMPD-genen en enzymen van Aspergillus sp., Penicillium sp., Cladosporium fulvum en van andere schimmels. Deze Myrothecium stam bevat slechts 1 kopij van het ompd -gen. De functionaliteit van het enzym werd bewezen via de complementatie van een gedefinieerde uracil auxotrofe stam, meer bepaald Aspergillus nidulans FGSC A722 en door het feit dat het deze stam gevoelig maakt aan 5-FOA.
In hoofdstuk VIII wordt de ontwikkeling van een transformatiesysteem gebaseerd op het gekloneerde orotidine monofosfaat decarboxylase besproken. Twee selectieve media werden ontwikkeld, namelijk het ‘anti-Oneg’ medium dat selectief is tegen OMPD deficiënte stammen (geen uracil of uridine) en het ‘anti-Opos’ medium, selectief tegen OMPD wild type stammen (bevat 5-FOA). Tijdens dit onderzoek werd voor de creatie van een uracil auxotrofe (OMPD negatieve) stam gekozen voor een strategie die zou toelaten om de OMPD deficiënte stam te gebruiken voor complementatie tijdens expressiestudies. In zulke studies is het belangrijk dat de expressiecassette op een welbepaalde plaats in het genoom kan integreren, en dit in single copy . Het gebruik van twee verschillende mutaties in 1 gen zodat een selecteerbaar fenotype na complementatie alleen ontstaat als er een enkele crossover plaatsvond op de beoogde plaats in het genoom verhoogt de kans om 1 kopij van een vector te integreren op die bepaalde plaats. Er werd besloten om zo een strategie te volgen bij de creatie van een gedefinieerde OMPD-negatieve mutant van M. gramineum . Zowel voor de 5’ als voor de 3’ mutaties werden geconserveerde aminozuren gedeleet en werden ook frame shift
Samenvatting 279 mutaties beoogd. De vector pOVmut3’ bevat een deletie van 67 bp 0.5 kb downstream van het startcodon en de vector pOVmut5’ heeft een deletie aan het einde van de promotor, inclusief het startcodon en het N-terminale deel van het gen. Het plasmide pOVmut3’ werd gebruikt om een OMPD knock-out te creëren via transformatie en homologe recombinatie. Deze resultaten toonden aan dat er problemen waren met de selectie op het ‘anti-Opos’ medium. Er werd gezocht naar de oorzaak van de valse selectie van kolonies. Een duidelijk verschil tussen de groei van mycelium en van sporen op het medium werd vastgesteld. Het bleek dat mycelium op de platen kon verder groeien, terwijl sporen niet in staat waren te kiemen. In de volgende transformatie-experimenten werden de protoplasten daarom eerst op compleet medium uitgeplaat en geïncubeerd tot ze gesporuleerd waren. Vervolgens werden de sporen geoogst en overgeënt op ‘anti-Opos’ platen. Omdat gebleken was dat de transformatie- efficiëntie met lineaire DNA-fragmenten te laag was, werden de experimenten uitgevoerd met intacte plasmiden, meer bepaald met pOVmut5’. Twaalf sporendruppeltjes konden ontkiemen en kleine hoeveelheden mycelium vormen. Er werden ook 67 zeer kleine kolonies bekomen op de platen waarop de rest van de geoogste sporen werd geënt. Jammer genoeg waren zij allemaal in staat om te groeien op ‘anti-Oneg’ medium. Ook pogingen om via UV-mutatie een OMPD-negative mutant te bekomen faalden. Ondanks het feit dat een redelijk aantal 5- FOA resistente kolonies werd bekomen, bleek geen enkele van deze kolonies uracil auxotroof te zijn. Het zal dus nodig zijn om het aantal 5-FOA resistente kolonies nog te verhogen om de gewentste OMPD-negative stam te bekomen.
Er kan besloten worden dat tijdens dit onderzoek met succes een homoloog expressiesysteem voor een nieuwe fungale expressie-gastheer, nl. Myrothecium gramineum werd ontwikkeld. Het systeem is gebaseerd op de homologe glyceraldehyde-3-fosfaat dehydrogenase promotor (Genbank nummer EF486690 ). Met dit expressiesysteem werd recombinant A. oryzae amylase en P. griseofulvum xylanase geproduceerd. Voorlopige experimenten toonden ook aan dat M. gramineum in staat is om het bacteriële B. subtilis xylanase te produceren. Om M. gramineum transformanten te kunnen selecteren zonder gebruik te maken van een antibioticum werd geprobeerd om een ander selectiesysteem te ontwikkelen. Het orotidine monofosfaat decarboxylase gen (GenBank nummer DQ359751 ) werd gekloneerd en gekarakteriseerd. Het gen werd met succes gebruikt om een gedefinieerde Aspergillus nidulans OMPD negatieve stam te complementeren. Indien men een OMPD negatieve mutant van deze schimmel kan creëren, zal de geschiktheid van het nieuwe selectiesysteem voor M. gramineum onderzocht kunnen worden.
Curriculum vitae
Curriculum vitae
Curriculum Vitae
1. PERSONALIA De Maeseneire Sofie Leen Office address: Laboratory of Industrial Microbiology and Biocatalysis Department of Biochemical and Microbial Technology Ghent University Coupure links 653 B-9000 Gent Tel.: +32-9-264-60-28 Fax: +32-9-264-62-31 Email: [email protected]
2. EDUCATION
1991-1997: Science-Mathematics Mariacollege, Ronse, Belgium 1997-2002: Bio-engineer in Cell and Gene Biotechnology (great distinction) Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Thesis: “Construction and metabolic characterisation of gltA - and ppc -mutants of Escherichia coli ” Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium (Promotor: Prof. dr. ir. Erick Vandamme) 2002-2006: Doctoral training in Applied Biological Sciences Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium
Curriculum vitae
3. PROFESSIONAL ACTIVITIES
2002-2006: Doctoral research financially supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT- Vlaanderen), Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Research topic: “ Myrothecium gramineum as a novel fungal expression host” Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium (Promotors: Prof. dr. ir. Erick Vandamme and Prof. dr. ir. Wim Soetaert) 2007-present: Scientific research: ‘SANTS-project’, Synthesis and Application of Nanostructured Tethered Silicates Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium (Prof. dr. ir. Erick Vandamme and Prof. dr. ir. Wim Soetaert)
4. PUBLICATIONS
A1 publications • De Maeseneire, S. L. , De Groeve, M. R. M., Dauvrin, T., De Mey, M., Soetaert, W. & Vandamme, E. J. (2006). Cloning, sequence analysis and heterologous expression of the Myrothecium gramineum orotidine-5’-phosphate decarboxylase gene. FEMS Microbiology Letters , 261 , 262-271. • De Maeseneire, S. L. , De Mey, M., Vandedrinck, S. & Vandamme, E. J. (2006). Metabolic characterisation of E. coli citrate synthase and phosphoenolpyruvate carboxylase mutants in aerobic cultures. Biotechnology Letters , 28 , 1945-1953. • De Mey, M., De Maeseneire, S . L., Soetaert, W. & Vandamme, E. J. (2007). Minimizing acetate formation in Escherichia coli fermentations. Journal of Industrial Microbiology & Biotechnology , ( in press ).
• De Mey M., Lequeux, G. J., Maertens, J. , De Maeseneire, S. L. , Soetaert, W. K. & Vandamme, E. J. (2006). Comparison of DNA and RNA quantification methods suitable
Curriculum vitae
for parameter estimation in metabolic modelling of microorganisms. Analytical Biochemistry , 353 ,198-203. • De Muynck, C., Leroy, A. I. J., De Maeseneire, S. , Arnaut, F., Soetaert, W. & Vandamme, E. J. (2004). Potential of selected lactic acid bacteria to produce food compatible antifungal metabolites. Microbiological Research , 159 , 339-346. • De Muynck, C., Van der Borght, J., De Mey, M., De Maeseneire, S. L. , Soetaert, W. & Vandamme, E. J. (2007). Development of a selection system for detection of L-ribose isomerase expressing mutants of Escherichia coli . (accepted for publication in Applied Microbiology and Biotechnology ) • Van Bogaert, I. N. A., De Maeseneire, S. L. , De Schamphelaire, W., Develter, D., Soetaert, W. & Vandamme, E. J. (2006) Cloning, characterisation and functionality of the orotidine-5-phosphate decarboxylase gene (URA3) of the glycolipid producing yeast Candida bombicola . Yeast , 24(3) , 201-208. • De Maeseneire, S. L. , Jonniaux, J.-L., Dauvrin, T., De Groeve, M., De Muynck, C., , Soetaert, W. & Vandamme, E. J. (2007). Cloning and characterisation of the glyceraldehyde-3-phosphate dehydrogenase gene and the use of its promoter for heterologous expression in Myrothecium gramineum , a novel expression host. (submitted to Journal of Biotechnology ) • De Maeseneire, S. L. , Van Bogaert, I. N. A., Dauvrin, T., Soetaert, W. & Vandamme, E. J. (2007). Rapid sample preparation for long distance PCR on genomic DNA. (submitted to Biotechnology Letters ) • De Muynck, C., Van der Borght, J., De Groeve, R. M., De Maeseneire, S. L. , Soetaert, W. & Vandamme, E. J. (2007). Modification of the substrate specificity of L-arabinose isomerase by directed evolution. (submitted to Biotechnology Letters ) Other publications • De Maeseneire, S. L. , De Mey, M., De Groeve, M., Dauvrin, T., Soetaert, W. & Vandamme, E. J. (2005). The Orotidine Monophosphate Decarboxylase Gene of Myrothecium gramineum : Cloning and Sequence Analysis. Communications in Applied Biological Sciences, Ghent University, 70/2 , p. 91-95. • De Mey, M., De Maeseneire, S. , Van Nieuland, K.; Lequeux, G., Soetaert, W. & Vandamme, E. J. (2005). Evaluation of different methods for nucleic acids quantification. Communications in Applied Biological Sciences, Ghent University, 70/2 , p. 97-100.
Curriculum vitae
• Van Bogaert, I. N. A., De Maeseneire, S. L. & Vandamme, E. J. (2006). Extracellular polysaccharides produced by yeasts and yeast-like fungi. In: Kunze, G. & Satyanarayana, T. (eds) Diversity and potential biotechnological applications of yeasts. Amsterdam, Elsevier Publishers B.V., (accepted). • Vandedrinck, S., De Maeseneire, S. , Deschamps, G., Sablon, E. and Vandamme, E. J. (2002). Construction and enzymatic characterization of E. coli GLTA and PPC mutants. Communications in Applied Biological Sciences, Ghent University, 67/4, p. 261-264.
5. CONGRESSES AND WORKSHOPS
5.1. International congresses with poster presentation
• De Maeseneire, S. L. , De Groeve, M., Dauvrin, T., Soetaert, W. & Vandamme, E. J. Transformation of Aspergillus nidulans using the orotidine monophosphate decarboxylase gene of Myrothecium gramineum . (2006). Abstract Book of the 8 th European Congress on Fungal Genetics, Wien, Austria, April 8-12, PVIIp-34, p289. • De Maeseneire, S. L. , De Mey, M., De Groeve, M., Dauvrin, T., Soetaert, W. & Vandamme, E. J. (2005). Cloning and Sequence Analysis of the OMP-Decarboxylase Gene of Myrothecium gramineum . Abstract Book of the Renewable Resources and Biorefineries Conference, Ghent, Belgium, September 19-21, P27 • De Mey, M, Lequeux, G., Van Nieuland, K., Beauprez, J., De Maeseneire, S. , Maertens, J., Soetaert, W. & Vandamme, E. (2005). Evaluation of current methods for nucleic acid quantification. Abstract Book of the Renewable Resources and Biorefineries Conference, Ghent, Belgium, September 19-21, P28 • De Maeseneire, S. L. , De Mey, M., De Groeve, M., Dauvrin, T., Soetaert, W. & Vandamme, E. J. (2005). Comparison of rapid extraction methods for high quality fungal DNA for PCR-applications. Abstract Book of the 12th European Conference on Biotechnology: Bringing Genomes to Life (ECB12), Copenhagen, Denmark, August 21- 24, P IB44 ( Journal of Biotechnology , 118, S1 , p. 106). • De Mey, M., De Maeseneire, S. , Beauprez, J., Van Nieland, K., Soetaert, W. & Vandamme, E. (2005). Comparison of methods for nucleic acid quantitation. Abstract Book of the 12th European Conference on Biotechnology: Bringing Genomes to Life (ECB12), Copenhagen, Denmark, August 21-24 ( Journal of Biotechnology , 118, S1 ) • Van Bogaert, I. N. A., De Maeseneire, S. , Vandamme, E. J. & Soetaert, W. (2005). Cloning and partial sequencing of the NADPH-cytochrome P450 reductase gene of
Curriculum vitae
Candida bombicola . Abstract Book of the 12th International Conference on Yeast Genetics and Molecular Biology, Bratislava, Slovak Republic, August 7-12, P2-11 ( Yeast , 22 , S41). • De Mey, M., De Maeseneire, S. , Beauprez, J., Van Nieland, K., Soetaert, W. & Vandamme, E. (2005). Quantification of nucleic acids. World Congress of Industrial Biotechnology and Bioprocessing, Orlando, Florida, USA, April 20-22. • Vandedrinck, S., De Maeseneire, S. , Deschamps, G, Sablon, E. and Vandamme, E. J. (2002) Construction and enzymatic characterization of E. coli gltA and ppc mutants. Abstract book of the International Symposium on Genetics of Industrial Microorganisms (GIM), Gyeongju, Z. Korea, July 1-5, P-149.
5.2. International congresses with passive participation
• 17 th Forum for Applied Biotechnology, Ghent, Belgium, September, 17-19, 2003. • 15 th Forum for Applied Biotechnology, Ghent, Belgium, September, 24-25, 2001.
5.3. National congresses with poster presentation
• 11 th PhD Symposium on Applied Biological sciences. Leuven, Belgium, October, 6, 2005.
5.4. National congresses with passive participation
• Microbial Immune Evasion Strategies. Belgian Society for Microbiology, Brussels, Belgium, November, 21, 2003. • 8th PhD Symposium on Applied Biological sciences. Ghent, Belgium, October, 9, 2002.
5.5. Workshops and Lectures
• Workshop ‘The production and characterisation of foreign proteins in fungal hosts’, Dranouter, Belgium, August, 25, 2004. • Manager training MFCS/WIN-Fermentation B.Braun. Biotech International-Sartorius Group. Vilvoorde, Belgium, February, 11, 2004. • Seminar Biosafety: Faculty of Bioscience Engineering, Ghent University, January, 23-30, 2004. • Industrial Biotechnology and Sustainable Chemistry. Perspectives and Policy issues. Brussels, Belgium, January, 15, 2004. • From Gene to Functional Protein. Luik, Belgium, February, 20, 2003.
Curriculum vitae
• Seminar Biosafety: Faculty of Bioscience Engineering, Ghent University, January, 24-31, 2003. • Green Chemistry and Bio-energy: a sustainable solution! 51 st Post-Universitary Education day. Faculty of Bioscience Engineering, Ghent University, December, 4, 2002.
6. OTHER ACTIVITIES
- Student guidance practical exercises “General Microbiology’ - M.Sc. thesis guidance: - Buyle, Veerle. 2005-2006. Optimalisatie van transformatie en expressie met nieuwe vectoren in Myrothecium gramineum . Kaho Sint-Lieven. - De Groeve, Manu. 2004-2005. Klonering en genexpressie in de schimmel Myrothecium gramineum . Universiteit Gent, Faculteit Bio- ingenieurswetenschappen. - Gyselinck, Eline. 2004-2005. Homologe transformatie van Myrothecium gramineum . Kaho Sint-Lieven. - Van Liefferinge, Edward. 2003-2004. Ontwikkeling van een transformatiesysteem voor een filamenteuze schimmel gebaseerd op het OMPD-enzym. Kaho Sint-Lieven. - Vandermeersch, Bert. 2005-2006. Ontwikkeling van een transformatiesysteem voor Myrothecium gramineum gebaseerd op het homologe OMPD-gen. Hogeschool Gent. - Vernaillen, Tom. 2005-2006. Optimalisatie van kolonie-PCR voor Myrothecium gramineum . Hogeschool Gent. - Co-promotor thesis: - De Schamphelaire, Wouter (2006). Microbiële synthese van kortketen sophorolipiden. Scriptie, Gent, Faculteit Bio-ingenieurswetenschappen. 139p. - Member of the First Intervention Unit, Faculty of Bioscience Engineering, Ghent University - Co-worker of the ‘Forum for Applied Biotechnology’, Ghent, September, 18-19, 2003 - Member of the organising committee ‘Industrial Biotechnology and Sustainable Chemistry. Perspectives and Policy Issues’, Brussels, January, 15, 2004 - Member of the organising committee ‘Renewable Recourses and Biorefineries’, Ghent, September, 19-21, 2005 - Member of the organising committee ‘Renewable Recourses and Biorefineries’, Ghent, June, 4-6 2007