GENETIC MANIPULATION OF ZYMOMONAS MOBILIS: Gene Transfer Systems, Cloning and Expression of Genes Involved in Cellulosic Bioconversion
A thesis submitted for the degree of DOCTOR OF PHILOSOPHY
by
PING SU
January, 1989 Department of Biotechnology University of New South Wales UNIVERSITY CF N.S.W. 2 8 MAR 1990
B'OMED’CAL LIBRARY ACKNOWLEDGEMENTS
I wish to thank Professor Pamela Rickard for allowing me to undertake a Ph.D. in the
Department and for her interest in my progress.
I am indebted to my supervisors, Dr.Stephen Delaney and Associate Professor Peter
Rogers, for their valuable time, advice and encouragement.
Special thanks are due to Associate Professor Noel Dunn and Dr. Amanda Goodman for their helpful suggestions and advice.
I would also like to thank all the academic staff and students of the Department, especially Liu, Rachel, Russell, Paul, Aneta, Pittaya, Glenda, Wolf, Janette, Geoff, Phil, Baha, Rose, John and Cho for their help and friendship.
I am grateful for the Postgraduate Scholarships provided by Mr. Martial Lawson of Lily vale Mushrooms Pty. Ltd. and by the University of New South Wales.
Finally, I thank Jenny for her excellent typing of this thesis. w
I ABSTRACT
Zymomonas mobilis has been evaluated as being "an ideal ethanol producer" if the problem of its narrow substrate range could be overcome. Cellobiose is one of the main products of cellulase-catalysed breakdown of cellulose, which is the most abundant organic compound in the world. This thesis reports the development of efficient recombinant DNA techniques in Z. mobilis and construction of Zymomonas strains capable of converting cellobiose to ethanol.
A rapid procedure achieving high transformation frequencies of Z. mobilis ZM6 by a range of plasmids was established. Using a hybrid plasmid, pNSW301, the highest efficiency of transformation obtained was 1.8 x 105 transformants per jig plasmid DNA. High frequency transformation was also achieved with a native Z. mobilis plasmid marked with a transposon, with large broad-host-range IncP-1 and IncW plasmids, and with small IncW cloning vectors. The kinetics of transformation and behaviour of the small IncW plasmids in Z. mobilis was investigated. A method for preparing Z. mobilis spheroplasts was developed and fusion of auxotrophic Z. mobilis ZM4 strains was achieved.
A pKT230 gene bank of the genome from the cellulolytic bacterium, Xanthomonas albilineans, was screened for p-glucosidase-producing clones. The p-glucosidase from one such clone which harboured the plasmid, pNSW904, and which grew efficiently on cellobiose was partially characterised. Transfer of the p-glucosidase gene to Z. mobilis ZM6 and ZM6100 was achieved by subcloning the p-glucosidase gene onto the small broad-host-range plasmid, pRK404, followed by three-way mating involving the helper plasmid, pRK2013. Enzyme assays showed that p- glucosidase was produced by the recombinant strains ZM6901, ZM6902 and
II ZM6903. Thin layer and gas chromatography of ZM6901 extracts indicated that cellobiose was consumed and glucose formed simultaneously. The glucose was further converted to ethanol yielding 13.3 mM ethanol from 5 mM cellobiose. Intact cells of ZM6901 were capable of producing 132 mM ethanol from 110 mM cellobiose after 11 days.
Genes encoding p-glucosidase and endoglucanase from X. albilineans were linked on the same vector, pRK404, and then transferred to E. coli HB101 and Z. mobilis ZM6. Simultaneous expression of p-glucosidase and endoglucanase in Z. mobilis was confirmed, thus completing a further step of construction a novel cellulytic pathway in Z. mobilis.
Ill LIST OF PUBLICATIONS JOURNALS Su, P. and Goodman, A.E. (1987) High frequency transformation of Zymomonas mobilis by plasmid DNA. J. Biotechnol. 6, 247-258. Su, P., Delaney, S.F. and Rogers, P.L. (1988) Kinetics of plasmid transformation in Zymomonas mobilis. J. Biotechnol. 8, 317-320. Su, P., Delaney, S.F. and Rogers, P.L. (1989) Cloning and expression of a p- glucosidase gene from Xanthomonas albilineans in Escherichia coli and Zymomonas mobilis. J. Biotechnol., in press.
CONFERENCE Su, P., Delaney, S.F. and Rogers, P.L. (1989) Cloning and expression of a p- glucosidase gene from Xanthomonas albilineans in Escherichia coli and Zymomonas mobilis. Proc. 8th Aust. Biotechnol. Conf.
IV TABLE OF CONTENTS Page ACKNOWLEDGEMENTS I ABSTRACT II LIST OF PUBLICATIONS IV TABLE OF CONTENTS V LIST OF TABLES X LIST OF FIGURES XI
CHAPTER 1: INTRODUCTION 1 1.1 Introduction to Zymomonas mobilis 1 1.1.1 Characteristics of Z. mobilis 1 1.1.2 Carbohydrate metabolism in Z. mobilis 2 1.1.3 Ethanol production by Z. mobilis and its industrial potential 6 1.2 Development of genetic techniques forZ. mobilis 10 1.2.1 Strain selection 10 1.2.2 Strain improvement 10 1.2.3 Cloning vehicles 11 1.2.4 Systems of gene transfer 13 1.3 Conversion of cellulosic materials to ethanol and other products 15 1.4 Cloning and expression of cellulase genes 20 1.4.1 Cellulose - degrading microorganisms 20 V 1.4.2 The cellulase enzyme system 21 1.4.3 Strategies for cloning and expressing cellulase genes 23 1.4.4 Survey of cloned cellulase genes 25 1.5 Major objectives of the investigation 33 CHAPTER 2: MATERIALS AND METHODS 34
2.1 General equipment 34 2.2 Reagents, solutions and media 35
2.2.1 Reagents 35 2.2.2 Solutions 36 2.2.2.1 Saline 36 2.2.22 Saline phosphate buffer (SPB) 36 2.2.2.3 Carbon sources 37 2.2.2.4 Mcllvaine’s buffer 37 2.2.2.5 DNS reagent 37 2.22.6 Gel electrophoresis buffer (TAE) 37 2.22.1 TBE buffer 38
V 2.2.2.8 TE buffer 38 2.2.2.9 Saline sodium citrate (SSC) 38 2.2.2.10 Dilution fluid 38 2.2.3 Media 38 2.2.3.1 Luria broth (LB) 39 2.2.3.2 Minimal medium (MM) 39 2.2.3.3 Rich medium (RM) 39 2.2.3.4 Basal medium (BM) 40 2.2.3.5 Nutrient yeast broth (NYB) 40 2.2.3.6 PM medium 40 223.1 Spheroplast regeneration medium 42 2.2.3.8 Esculin plates 42 2.23.9 Carboxymethyl cellulose (CMC) - Congo red plates 42 2.2.3.10 X-Gal plates 43
2.3 Biological materials 43 2.4 Microbiological techniques 49 2.4.1 Preparation of standard bacterial inocula for enzyme work 49 2.4.2 Growth of cultures 49 2.4.3 Antibiotic supplementation of media 50 2.4.4 Estimation of bacterial concentrations 52 2.4.5 Storage of bacterial cultures 52 2.4.6 Patching 52 2.4.7 Replica plating 53 2.4.8 Stability testing of introduced plasmids 53 2.5 Recombinant DNA techniques 54 2.5.1 Plasmid isolation 54 2.5.1.1 Plasmid isolation from Z. mobilis 54 2.5.1.2 Plasmid isolation from E. coli 55 2.5.2 Electrophoresis 55 2.5.3 Determination of plasmid DNA concentration 55 2.5.4 Size determination of DNA fragments - 56 2.5.5 Restriction endonuclease digests 56 2.5.6 Physical mapping 56 2.5.7 Ligations 57 2.5.8 Electroelution of DNA from gels 57 2.5.9 Southern blotting and hybridization 58 2.5.9.1 Southern transfer 58 2.5.9.2 Preparation of hybridization probes 58 2.5.9.3 Hybridization 59 2.5.10 Transformation 59 2.5.10.1 Transformation of E. coli 59 2.5.10.2 Transformation of Z. mobilis 59 2.5.10.3 Storage of competent cells 61 2.5.11 Conjugation 61 2.5.11.1 Spot mating 61 2.5.11.2 Filter mating 61 2.5.12 Formation of Z. mobilis spheroplasts and cell wall regeneration 62
VI 2.6 Analytical procedures 62 2.6.1 Quantitative analysis of cellobiose by high pressure liquid chromatography (HPLC) 62 2.6.2 Thin layer chromatography (TLC) 63 2.6.3 Gas chromatography (GC) 64 2.6.4 Enzyme preparation for assay 64 2.6.5 p-Glucosidase assay 65 2.6.6 Cellobiase assay 66 2.6.7 Endoglucanase assay 66 2.6.8 Total protein determination 66 CHAPTER 3: HIGH FREQUENCY TRANSFORMATION OF ZYMOMONAS M OBI US BY PLASMID DNA 68 3.1 Transformation of Z. mobilis ZM6 with pNSW301 68 3.2 Effect of various factors on transformation frequency 73 3.2.1 State of the recipient cells 73 3.2.2 Effect of DNA exposure time 76 3.2.3 The effect of one or two heat shocks on transformation frequency 78 3.2.4 Effect of storage time of competent cells on cell viability and transformation 80 3.3 Kinetics of plasmid transformation in Zymomonas mobilis 82 3.4 Transformation of Z. mobilis ZM6 by other plasmids 82 3.5 Behaviour of a set of plasmids derived from Sa in Z. mobilis 91 3.5.1 Stability test of pSal52, pSa727 and pSa747 in Z. mobilis 91 3.5.2 Re-arrangement of pSal52 and pSa747 in ZM6 after long periods of growth under selective conditions 95 3.5.3 Characterisation of pNSW901 and pNSW902 97
3.6 Discussion 99 CHAPTER 4: SPHEROPLAST FUSION IN ZYMOMONAS MOBILIS 102 4.1 Spheroplast formation and regeneration 102 4.2 Optimization of lysozyme treatment for spheroplast formation 105
4.2.1 Effect of incubation time with lysozyme on spheroplast formation and regeneration in ZM6 105 4.2.2 Effect of lysozyme concentration on spheroplast formation and regeneration in ZM6 107
4.3 Effect of different hypertonic stabilizers on spheroplast regeneration in ZM6 107 4.4 Spheroplast fusion in ZM4 109 4.5 Discussion 109
VII CHAPTER 5: CLONING AND EXPRESSION OF A p-GLUCOSIDASE GENE FROM XANTHOMONAS ALBIUNEANS IN ESCHERICHIA COU 111 5.1 Selection and screening procedures 112 5.1.1 Selection via Pseudomonas 112 5.1.2 Sequential selection procedure in E. coli 115 5.2 Stability of pNSW904 in E. coli ED8654 117 5.3 Characterization of the recombinant plasmid pNSW904 119 5.4 Expression of the p-glucosidase gene 124 5.5 Preliminary characterization of the enzyme 124 5.5.1 Temperature optimum 124 5.5.2 pH optimum 127 5.5.3 Metal ion requirements 127 5.5.4 Michaelis-Menten kinetics with pNPG 130 5.5.5 Optimum cellobiose concentration for the assay of p-glucosidase on cellobiose 130 5.6 Regulation of gene expression 130 5.7 Growth of the recombinant strain on cellobiose 134 5.8 Subcloning of the p-glucosidase-encoding fragment of pNSW904 to reduce its size 136 5.8.1 Construction of the subclone, pNSW910, with a 3.0 kb insert 136 5.8.2 Construction of the subclone, pNSW911, with a 1.5 kb insert 141 5.9 Discussion 146 CHAPTER 6: EXPRESSION OF A CLONED p-GLUCOSIDASE GENE FROM XANTHOMONAS ALBIUNEANS IN ZYMOMONAS MOB I LIS 150 6.1 Subcloning the p-glucosidase gene 150 6.2 Transconjugation of the p-glucosidase gene into Z. mobilis 153 6.3 Re-arrangement of pNSW906 in ZM6 transconjugants 156 6.4 Expression of the cloned p-glucosidase gene in Z. mobilis 159
6.4.1 p-glucosidase activity in Z. mobilis transconjugants 159 6.4.2 Time course of enzyme hydrolysis in cell extracts 161 6.4.3 Subcloning and expression of the p-glucosidase gene onto pSal52 164
6.5 Stability studies on the Z. mobilis transconjugants 166
6.6 Ethanol formation from cellobiose by recombinant Z. mobilis 168 6.6.1 Ethanol formation from cellobiose by cell extracts of ZM6901 168 6.6.2 Ethanol fermentation from cellobiose by whole cells of ZM6901 170
VIII 6.7 Discussion 172 CHAPTER 7: SUBCLONING AND CHARACTERISATION OF A p-GLUCOSIDASE GENE FROM PSEUDOMONAS pS2-2 175 7.1 Preliminary characterisation of the p-glucosidase-encoding plasmid, pND71 175 7.2 Subcloning the p-glucosidase gene from Pseudomonas into pRK404 178 7.3 Expression of the p-glucosidase gene of pNSW903 in E. coli HB101 180 7.3.1 p-Glucosidase assay of the E. coli HB101(pNSW 903) 180 7.3.2 Location of the product of the subcloned p-glucosidase gene 182 7.4 Mobilization of pNS W903 into Z. mobilis ZM6 184 7.5 Discussion 185 CHAPTER 8: LINKAGE OF GENES ENCODING P-GLUCOSIDASE AND ENDOGLUCANASE ON THE SAME VECTOR AND EXPRESSION IN E. COLI AND Z. MOBILIS 186 8.1 Linkage of genes encoding p-glucosidase and endoglucanase on the same vector 186 8.2 Restriction endonuclease analysis of the linked plasmids 190 8.3 Introduction of pNSW907 and pNSW908 into Z. mobilis 192 8.4 Expression of the cloned p-glucosidase and endoglucanase genes in E. coli and Z. mobilis 192 8.5 Discussion 195 CHAPTER 9: GENERAL DISCUSSION AND CONCLUSIONS 197. REFERENCES 201
IX LIST OF TABLES Page 1.1 Comparison of ethanol production by Zymomonas with yeast 7 1.2 Cloned genes encoding cellulase enzymes 26 2.1 Bacterial strains 44 2.2 Bacterial plasmids 46 2.3 Concentration of antibiotics added to media 51 3.1 Transformation efficiencies of Z. mobilis and E. coli by plasmid pNSW301 70 3.2 The effect of cell age on transformations frequency 74 3.3 Effect of incubation time on ice on the transformation frequency 77 3.4 The effect of heat shock on transformation frequency 79 3.5 Effect of storage on the viability and transformation of competent cells 81 3.6 Effect of altering the DNA concentrationon the recovery of ZM6(pNSW301) transformants 84 3.7 Transformation frequencies of Z. mobilis ZM6 using various plasmids 89 3.8 Maintenance of pSal52, pSa727 and pSa747 in ZM6 under non-selective conditions 93 3.9 Maintenance of pSal52, pSa727 and pSa747 in ZM6 under selective conditions 94 3.10 Maintenance of antibiotic resistance in ZM6(pNSW901) under non-selective conditions 98 4.1 Production and regeneration of Z. mobilis spheroplasts 103 4.2 Effect of different hypertonic stabilizers on spheroplast regeneration in ZM6 108 5.1 p-Glucosidase activities of the recombinant strain, ED8654(pNSW904), using either the synthetic substrate, pNPG, or the natural substrate, cellobiose 12^ 5.2 Determination of metal ion requirements 129 5.3 Effect of cellobiose and glucose concentrations on the specific activity of p-glucosidase in the recombinant strain ED8654(pNSW904) 133
6.1 Specific activity of p-glucosidase from E. coli transformants and Z. mobilis transconjugants assayed on pNPG 160 6.2 Maintenance of pNSW906 in Z. mobilis under batch culture conditions 167 6.3 Ethanol formation by ZM6901 in RMCe medium (110 mM cellobiose) 171
7.1 Enzyme assay of p-glucosidase clones 181 7.2 p-Glucosidase activity on different cell fractions of E. coli HB101(pNSW903) and P.putida ATCC17527(pND71) 183 8.1 Expression of the linked p-glucosidase and endoglucanase genes on the same vector 194
X LIST OF FIGURES
Page 1.1 Carbohydrate catabolism in Z. mobilis 4 1.2 Schematic presentation of sequential stages in enzymatic hydrolysis of cellulose 18 1.3 Mechanism of bacterial cellulolysis according to Ramasamy and Verachtert 24 3.1 Plasmid DNA from ZM6(pNSW301) and ZM6(pNSW60) transformants 72 3.2 The effect of pNSW301 DNA concentration on transformation frequency of Z. mobilis 86 3.3 Plasmid DNA from ZM6(pSal52), ZM6(pSa727) and ZM6(pSa747) transformants 90 3.4 Plasmid DNA from ZM6 harbouring a set of derivative plasmid from Sa after 200 generations of growth under selective conditions 96 4.1 Effect of incubation time in lysozyme on spheroplast formation and regeneration in ZM6 106 5.1 Extraction of plasmid DNA from the cellobiose-utilising ATCC17527 isolates 114 5.2 Plasmid DNA from ED8654 clones which turned esculin black 116 5.3 Extraction of plasmid DNA from esculin-positive isolates from the serial selection procedure 118 5.4 Restriction endonuclease digestion of pNSW904 120- 122 5.5 Physical map of pNSW904 123 5.6 Temperature profile of p-glucosidase activity of ED8654(pNSW904) with pNPG as substrate 126 5.7 pH profile of p-glucosidase activity of ED8654(pNSW904) with pNPG as substrate 128 5.8a Effect of pNPG concentration on activity of p-glucosidase from ED8654(pNSW904) 131w 5.8b Double reciprocal plot of p-glucosidase activity versus pNPG concentration from Figure 5.8a 131 5.9 Growth on cellobiose of E. coli with and without the p-glucosidase gene 135 5.10 Sau 3A complete digest of pNSW904 137 5.11 Sau 3A partial digest of pNSW904 138 5.12 Rapid disruption of colonies to test for inserts in plasmids 140 5.13 Restriction enzyme digestion of pNSW910 142 5.14 Restriction enzyme digestion of pNSW911 and pNSW910 143 5.15 Construction of a subclone, pNSW911, comprising pUC8 with a 1.5 kb p-glucosidase-encoding insert from XA1-1 145
6.1 Characterisation of pNSW906 152 6.2 Agarose gel electrophoresis of plasmid DNA from Z. mobilis transconjugants 154 6.3 Extraction of plasmid DNA from HB101 transformants transformed by the plasmid extracted from ZM6901 155 6.4 Agarose gel electrophoresis of the eluted p-glucosidase-encoding fragment 157 6.5 Gel electrophoresis and Southern hybridization of plasmids from ZM6, ZM6901, and the p-glucosidase-encoding fragment 158
XI 6.6 TLC analysis of the enzymic hydrolysis of cellobiose by cell- associated fractions 162 6.7 Agarose gel electrophoresis of ZM6903 plasmid DNA 165 6.8 Analysis by GC of the products of cellobiose hydrolysis by a whole cell extract of ZM6901 169
7.1 Restriction endonuclease digestion of pND71 177 7.2 Plasmids from subclones containing the insert from pND71 cloned intopRk404 179
8.1 Hind III partial digestion of pND82 188 8.2 Plasmids from clones carrying the linked endoglucanase and p-glucosidase genes 189 8.3 Hind III digestion of pNSW907 and pNSW908 191 8.4 Linkage of genes encoding p-glucosidase and endoglucanase on the same vector, pRK404. 192.
i
XII 1
CHAPTER 1
Introduction
1.1 Introduction to Zymomonas mobilis
\ 1.1.1 Characteristics of Z. mobilis
Zymomonas mobilis is a common Gram-negative microorganism and is found in tropical regions, in sugar-containing juices such as fermenting plant juices, spoiled beer, cider and perry.
According to recent taxonomy based on modern methods (De Ley and Swings, 1976) the genus Zymomonas includes only one species with two subspecies - Z. mobilis subsps. mobilis and Z. mobilis subsps pomaceae. It was also suggested that Z. mobilis either is of recent evolutionary origin and has not yet had time to develop genetic diversity or is genetically a very stable genus (Swings and De Ley, 1977).
The size of the genome of forty Zymomonas strains was examined by Swings and De Ley (1977). The genome size was similar in these strains, the average molecular mass being 1.53 (+ 0.19) x 109 daltons. This is equal to about 56% of the mass of the
Escherichia coli genome and could code for about 1500 genes. The G + C content of the cellular DNA is between 47.5 - 49.5%.
The cells of Z. mobilis normally occur singly or in pairs. Their shape is that of a plump rod with rounded ends, 2-6 }im long and 1-1.4 |im wide. Spores or other resting stages, capsules, intracellular lipids and glycogen are not found. Motility is 2
not an essential feature of Z. mobilis, but, if motile, movement is achieved by means of 1-4 lophotrichous flagella.
Besides the usual lipids and proteins, cis-vaccenic acid and unusual hopanoids consititute 70% of the membrane of Z. mobilis. It has been demonstrated that when E. coli was exposed to ethanol the amount of ds-vaccenic acid increased from 26% to 50% of the fatty acids (Ingram, 1986). The synthesis of hopanoids (1,2,3,4- tetrahydroxypentane-29-hoptane and its derivatives) and the production of stress proteins by Z. mobilis sustains the tolerance of this organism both to ethanol and increased temperature (Barrow et al., 1984; Michel and Starka, 1986).
er Z. mobilis is an anaerobic, Gram-negative bac^um that converts glucose and fructose, via Enter-Doudoroff pathway, to pyruvate followed by decarboxylation of pyruvate to ethanol. As such Z. mobilis appears to be the only true anaerobic organism that uses a pathway found mainly in aerobic bacteria (Kersters and De Ley, 1968).
1.1.2 Carbohydrate metabolism in Z. mobilis
Substrate range of Z. mobilis is limited to only glucose, fructose and sucrose. Glucose is fermented by all strains whilst most utilize fructose. Utilisation of sucrose is strain-dependend, and in some cases, appears to be inducible (Swings and De Ley, 1977). Some strains utilize raffinose poorly (Dadds et al., 1973) and occasionally sorbitol has been reported to be utilized by some strains (Milllis, 1956). No strains reported to date will grow on other carbohydrates tested - cellobiose, galactose, lactose, maltose, mannose, ribose, xylose or starch (Swings and De Ley, 1977). Also, amino acids will not support the growth of Zymomonas (Swings and De Ley, 1977). 3
It may be possible to extend this limited range of substrates by genetic manipulation to construct more useful and versatile strains for industrial purposes.
The D-glucose transport system of Z. mobilis was studied by Dimarco and Romano (1985). D-glucose is transported by a constitutive, stereospecific, carrier-mediated, facilitated-diffusion system, consequently its intracellular concentration quickly reaches a plateau close to but not exceeding the external concentration.'- The uptake of D-glucose by Z. mobilis is by a low-affinity, high-velocity, non-concentrative mechanism. D-Xylose appears to be transported by the D-glucose system because its uptake is inhibited by glucose. The D-glucose transport system has a low affinity for D-fructose since the uptake of D-glucose was not significantly inhibited by D- fructose.
Carbohydrate metabolism in Zymomonas has been reviewed recently by Viikari (1988). The pathway and enzymes involved are illustrated in Figure 1.1.
Glucose metabolism in Z. mobilis is not via the glycolytic pathway but rather via the Entner-Doudoroff pathway, anaerobically and in association with pyruvate carboxylase (Gibbs and DeMoss, 1951, 1954). All of the enzymes involved in glucose metabolism in Z. mobilis (Figure 1.1) have been purified and characterized (Viikari, 1988). ^ P NMR studies of the intracellular accumulation of fermentation intermediates suggest that glucose-6-P-dehydrogenase and phosphoglycerate mutase are the rate limiting enzymes (Viikari, 1988). Relatively high levels of pyruvate decarboxylase and alcohol dehydrogenase appear to be required for rapid ethanol formation (Barrow et al., 1984). A molar equation for the conversion of glucose to ethanol was established by Kluyver and Hoppenbrouwers (1931) as follows: 4
SUCROSE OR GLUCOSE - FRUCTOSE OLIGOMER
(fructose)
Levansucrase 1 Levansucrase GLUCOSE. 5LUCOSE 7 SUCROSE / GLUCOSE - FRUCTOSE OLIGCMER LEVAN
© - *PhoAphoghye.e.Aatz mutate!>e ^*[nadhJ
1,3-DIPHOSPHOGLYCERATE
|Si.(ATP)
PhotphoglyceAate 3-phosphoglycerate kinate I 2-PHOSPHOGLYCERATE
PHOSPHOENOLPYRUVATE ADP PyAuvate kinate fc(ATP) PYRUVATEJVATE
PyAu.va.te. decaAboxylate |s^co
ACETALDEHYDE ACcoho dehydAogenatet ETHANOL ]
Figure 1.1: Carbohydrate catabolism in Z. mobilis. *, rate limiting enzymes. 5
1 mol glucose—*-1.8 mol ethanol +1.9 mol CO2 + 0.15 mol lactic acid
The molor conversion of glucose to ethanol was found to vary from 1.5 to 1.9 mole depending on the culture conditions and strain used (Gibbs and De Moss, 1954).
Fructose is phosphorylated by a constitutive fructokinase which is highly specific for 1 fructose and ATP. Phosphorylation of fructose is regulated by the concentration of glucose. Phosphoglucose isomerase converts fructose-6-phosphate to glucose-6- phosphate, which then enters the common pathway of glucose metabolism. Glucose phosphate isomerase is present in amounts sufficient to maintain the maximum metabolic flux (Viikari, 1988). The ethanol yield obtained from fructose in batch fermentations is generally about 5% lower than that from glucose because of the formation of byproducts such as dihydroxyacetone, mannitol and glycerol (Viikari and Korhola, 1986).
Sucrose is hydrolyzed to glucose and fructose, either by invertase or by levensucrase, followed by metabolism of glucose and fructose. The formation of levan is a typical characteristic of Z. mobilis on sucrose-based media and is an alternative produce of levansucrase activity. In addition to the byproducts - levan, oligomers, sorbitol, some acids and other produces typical of fructose metabolism have also been detected (Viikari and Korhola, 1986). The byproduct pattern formed from sucrose is greatly affected by the fermentation conditions used and can be suppressed by controlling physical parameters (Doelle and Greenfield, 1985.).
Z. mobilis is not a strict anaerobe but can grow in the presence of oxygen. This leads increased formation of acetaldehyde, acetoin, dihydroxyacetone and acetic acid, which cause toxic effects and inhibit growth. 6
1.1.3 Ethanol production by Z. mobilis and its industrial potential
As Zymomonas mobilis is capable of producing 1.8 mol ethanol from 1 mol glucose (Swings and De Ley, 1977), there has been recent intense investigation of its potential for the production of ethanol on an industrial scale (Lawford et al., 1982; Rogers et al., 1982; Buchholz et al., 1987; Schenberg and Costa, 1987). i
Comparisons of the kinetics of batch and continuous fermentations of Z. mobilis with a variety of yeasts have repeatedly shown considerable advantages of the Zymomonas process for ethanol production when glucose was used as substrate (Table 1.1). The specific rates of sugar uptake and ethanol production in batch and continuous culture were considerably higher than those of yeast (Table 1.1). The higher ethanol yield of Z. mobilis is associated with lower biomass yield due to different carbohydrate metabolism.
The ethanol tolerance of Z. mobilis is the same as that of yeasts. Rogers et al. (1980) state that a concentration of 85 g 1_1 can be sustained in continuous culture and up to 127 g 1-1 in batch culture. The glucose tolerance of Z. mobilis is also similar to that of yeasts. It should be noted that most osmotolerant yeasts are not efficient ethanol producers (Haraldson and Bjorling, 1981).
The pH range for the growth of most Z. mobilis strains was 3.9-7 (Swings and De
CL Ley, 1977). This is similar to the pH range for ettyiol production of 3.5-7.5 (Lawford, et al., 1983). Therefore, the risk of growth of contaminating bacteria in the fermentation vessel during industrial production can be reduced by fermentation at acid pH. No significant contamination or bacteriophage infection problems have been found during batch and continuous studies in this laboratory. 7
Table 1.1: Comparison of ethanol production by Zymomonas with yeast
FACTOR ZYMOMONAS YEAST
Specific glucose uptake rates 5.45 2.08 (Qs, g.g-lh-1) Specific ethanol productivity 2.53 0.87 (Qp, g.g-lh-1) Productivity in continuous processes with cell recycle 120-200 30-40 (g.Hh-1)- Ethanol yields 92.5 85.9 (% theoretical yield)
Maximum ethanol tolerance (g 1“1) 127 120 Glucose tolerance (g 1-1) >40 >40 ATP yield (per mole glucose) 1 2 (Enter-Doudoroff vs. Emden-Meyerhof) pH range for ethanol production 3.5-7.5 2-6.5 Optimum temperature (°C) 25-30 30-38 Growth conditions Grows rapidly Requires in the absence controlled of oxygen addition of 02 to maintain viability at high cell densities (Cysewski and Wilke, 1977)
Genetic manipulation Prokaryotic- Eukarytic - more simple more complex
Data in this table was collected from Rogers et al., 1979, 1982 and Lawfrod et al., 1983. 8
Since Z. mobilis is a prokaryote the wide range of in vivo and in vitro techniques developed for the genetic manipulation of bacteria can be readily applied. Genetic manipultion including recombinant DNA techniques is being undertaken in this and other laboratories to broaden the range of substrates utilized by Z. mobilis to include lactose, starch and cellulosic raw materials.
The potential of a variety of commercial substrates for ethanol production by Z. mobilis has been evaluated in two step processes or by sequential microbial fermentations. In the only commercial Z. mobilis pilot plant in operation (Bringer et al., 1984) starch from wheat flour was enzymatically hydrolysed and used for ethanol production using a flocculent strain in continuous culture. High ethanol producitivity (70.7 g 1-1 h~l), an ethanol yield 98% of theoretical, 99% substrate conversion and an ethanol concentration of 49.5 g 1-1 were obtained (Torres and Baratti, 1988).
Direct fermentation to ethanol by Z. mobilis was investigated using alternative crude sucrose substrates, for example, sugar-beet and raisins, which showed promise as raw substrates for a Z. mobilis process (Rogers, 1984). A Z. mobilis strain, developed by mutation and selection, produces ethanol from 25% sugar-cane juice or sugar-cane syrup, efficiently and without the need for nutritional supplementation or for sterilization of the medium (Doelle and Greenfield, 1985).
A variety of high productivity systems using Z. mobilis have been developed. A continuous culture system with cell recycle was developed to exploit the high specific rate of ethanol production of Z. mobilis. Strain ZM4 was used in a cross-flow microfiltration unit with polyamide membranes. Productivities as high as 200 g 1-lh-l were reached (Lee et al., 1980). Vacuum fermentation was used to remove 9
ethanol directly from the culture vessel, so that higher sugar concentrations could be used in the medium and consequently higher volumetric productivities were achieved (Rogers et al., 1982). Immobilized cell techniques, including attaching a flocculent strain to glass fibre pads (Amin and Verachtert, 1982), were applied to reduce the cost and complexities associated with maintaining high cell concentrations and ethanol toxicity associated with cell recycle.
To sum up Z. mobilis has excellent potential for industrial ethanol production but can utilize only a restricted range of fermentable substrates. Work is being carried out to extend this narrow range by genetic manipulation. Transfer of the lactose operon from E. coli to Z. mobilis results in the expression of p-galactosidase (Carey et al., 1983; Goodman et al., 1984; Yanase et al., 1988), expression of xylose catabolic genes in Z. mobilis has been achieved (Liu et al., 1987) and three endoglucanase genes have been expressed in Z. mobilis (Misawa et al., 1987; Lucas et al., 1987, Yoon et al., 1988). No reports of the expression of a p-glucosidase (cellobiase) gene in Z. mobilis have been published to date. 10
1.2 Development of genetic techniques for Zymomonas
1.2.1 Strain selection
Studies of Zymomonas strains revealed considerable variation between them,
including ethanol tolerance, temperature tolerance, growth at high sugar
1 concentrations, use of different sugars and flocculation (for a review, Swings and De
Ley, 1977). The wide variation suggested that better strains of Zymomonas may have
been available than those originally chosen for study. Thus several groups isolated
strains from a number of different locations and compared them for their suitability
for ethanol production. Viikari et al. (1980) found several brewery isolates produced
more ethanol and were more temperature tolerant than ATCC 10988, a strain used for
comparison. After a series of studies of batch cultures, continuous culture and
continuous culture with cell recycle, ZM4 was judged the best overall for ethanol production in terms of growth rate, ethanol-tolerance (80 g 1~1), temperature tolerance
(produces ethanol at 420Q and faster rates of ethanol production (200 g 1-1 h-1) in
continuous fermentation with cell recycle sustaining ethanol concentrations of 60-65
g 1-1 (Rogers et al., 1982).
1.2.2. Strain improvement
Several methods of mutagenesis were investigated to improve Zymomonas
genetically for ethanol production (Skotnicki et al., 1982). Nitrosoguanidine (NTG)
was found to be a good mutagen for Zymomonas. There was considerable strain
variation in the level of sensitivity to this mutagen and, depending on the strain, 25 mg 1-1 to 100 mg 1-1 NTG for 60 min achieved the best levels of mutagenesis. 11
Adding NTG directly to growing cultures rather than in buffer gave the best rates of mutagenesis.
Subsequently, Zymomonas mutants with altered characteristics were isolated following NTG mutagenesis. Strain ZM4 is non-flocculent, but after NTG mutagenesis a highly-flocculent mutant, ZM401, was isolated. ZM401 forms granular floes in the fermentor which settle out as floes up to several millimetres in i diameter within a minute after agitation is stopped. ZM401 shows promise for use in semi-batch fermentations and continuous culture systems with cell recycle (Lee et al., 1982). An ethanol-tolerant strain, ZM481, selected from 15% v/v ethanol plates, retained a higher viability at high ethanol concentrations compared to ZM4 so that ethanol concentrations could be maintained at 85 g H in the cell recycle system (Lee, 1981). Mutants showing improved growth on molasses, inability to produce hydrogen sulphide and temperature tolerance were also obtained (Skotnicki et al., 1982; Goodman et al., 1984; Rogers et al., 1982). Auxotrophic mutants were isolated to facilitate genetic studies. Mutants requiring cysteine, adenine or methionine were found to be isolated more commonly than others (Goodman et al., 1982). By selection in continuous culture for extended periods spontaneous flocculent mutants were obtained (Fein et al., 1983).
1.2.3 Cloning vehicles
For commercial exploitation of this organism, genetic engineering can be used to increase its narrow substrate range. For this, suitable cloning vectors are necessary.
The desired characteristics of suitable vectors for Z. mobilis are: 12
1. Stable replication in both E. coli and Z. mobilis.
2. Ability to be transferred by conjugation or transformation to E. coli and Z. mobilis.
3. They should encode characters (such as tetracycline and chloramphenicol
i resistance) which are selectable in both E. coli and Z. mobilis.
4. Unique cleavage sites for a number of common restriction enzymes.
5. One or more strong promoters which can be used to optimize the expression of cloned genes.
6. Small size.
Vectors of Z. mobilis can be divided into three major groups:
1. Broad-host-range plasmids. Cloning vectors commonly used in E. coli such as pBR322 and phage vectors cannot be transferred to and/or maintained in Z. mobilis. The plasmids most commonly used for cloning in Z. mobilis are the IncP-T group broad-host-range plasmids. The transfer of IncP-1 plasmids and the possibility of using such plasmids as cloning vectors to introduce foreign DNA to Z. mobilis have been investigated by several groups (Dally et al., 1982; Stokes et al., 1982;
Carey et al., 1983; Eveleigh et al, 1983; Skotiniki et al., 1983). Behaviour of the IncW plasmid, Sa, in Z. mobilis has been studied by Strzelecki et al. (1987). The transformation of a group of small, Tra-, Mob+ derivatives of Sa to, and their 13
behaviour in, Z. mobilis formed part of this study and the results will be discussed in detail in Chapter 3.
2. Shuttle vectors. Novel shuttle vectors were constructed as cloning vehicles of Z. mobilis, by fusion of small native plasmids of Z. mobilis with small E. coli plasmids (Yoon and Park, 1987; Mukundn et al., 1988). The Z. mobilis component with its origin of replication provides stability in Z. mobilis while’the E. coli component allows plasmid constructions to be carried out and tested in E. coli before transfer to Z. mobilis.
3. Modified broad-host-range vectors which incorporate further Z. mobilis features, such as promoter functions (Byun et al., 1986; Misawa et al., 1988). These plasmids are also called expression plasmids.
1.2.4 Systems of gene transfer
To genetically engineer Z. mobilis methods of transferring recombinant DNA into the organism need to be developed. There are three naturally-occurring mechanisms of bacterial DNA transfer, transduction, conjugation and transformation.
Transduction is a form of bacterial gene transfer mediated by bacteriophage (Stent and Calendar, 1978), but as yet there have been no reports of any bacteriophage that infect Z. mobilis.
At the present time, transfer of plasmids to Z. mobilis from E. coli or pseudomonads is routinely achieved by conjugation. For efficient conjugation, the plasmid must be either self-mobilizable or aided by a mobilizing plasmid such as pRK2013. The 14
transfer of broad-host-range plasmids to Z. mobilis by conjugation has been reported by several laboratories (Carey et al., 1983; Dally et al., 1983; Skotnicki et al., 1983).
Yields of 1 x 10-6 transconjugants per donor from plate or filter matings are common. However, strains are found to vary in their ability to maintain R plasmids
(Skotnicki et al., 1983).
There have been few reports on the transfer of DNA to Z. mobilis by transformation. Transformation of Z. mobilis was first reported by Browne et al. (1984), but the number of transformants obtained was low, and the success of the method was thought to be associated with the use of a particular hybrid plasmid, isolated in low yields from Z. mobilis. More recently, Yanase et al. (1986) reported the development of a method for transforming partial spheroplasts of Z. mobilis, using hybrid plasmids constructed from a Z. mobilis native plasmid and the Escherichia coli cloning vector pACYC184 (Chang and Cohen, 1978). Although the highest frequency of transformation obtained was 2 x 105 transformants per |ig of DNA, the transformation frequency varied markedly, depending upon the Z. mobilis strain used and the conditions of spheroplast formation (Yanase et al., 1986). This study (Chapter 3, Su and Goodman, 1987) established a simple and rapid transformation procedure that reproducibly achieved high numbers of Z. mobilis ZM6 transformants using a range of plasmids. These plasmids included: a Z. mobilis-derived antibiotic- resistance hybrid plasmid (pNSW301, Strzelecki et al., 1987); a native Z. mobilis plasmid (pNSW60), marked with a transposon; large, conjugative, broad-host-range IncP-1 (RPl::Tn507; Bennett et al., 1978) and IncW (Sa; Ward and Grinsted, 1982) plasmids; and small, non-conjugative, broad-host-range cloning vectors (pSal52, pSa727, pSa747; Tait et al., 1983). 15
Spheroplast fusion has been developed as an alternative means of DNA transfer.
Spheroplast formation and regeneration in Z. mobilis was reported by Lee and Seong (1984). Yanase et al. (1984) investigated spheroplast fusion in Z. mobilis and used mutants that had lost the ability to ferment sucrose or fructose. In this study an improved method to produce spheroplasts of Z. mobilis was investigated and fusants were produced from two auxotrophic ZM4 mutants (Chapter 4). \
1.3 Conversion of cellulosic materials to ethanol and other products.
Cellulose is the most abundant renewable resource available for conversion to fuel, food and chemical feedstocks. The annual worldwide production of cellulose through photosynthesis is estimated to approach 100 x 109 tonnes (Ghose, 1977). As much as 25% of this could be made readily available for the conversion processes. A significant fraction of the available cellulose, i.e., 4-5 x 109 tonnes per year, occurs as waste, mainly as agricultural and municipal wastes. If only a fraction of these materials were to be converted into fuel, sugar or feed protein, a significant contribution could be made to resource recycling and conservation.
The recent worldwide energy crisis following the Arab oil embargo of the 1970’s has sparked an interest in the development of alternative sources of energy, particularly for liquid transportation fuels. One alternative that has been studied widely and shown to be promising is the microbial fermentation of ethanol from renewable agricultural resources, i.e., cellulosic materials. The ethanol can be used directly as a fuel and also as an octane booster (Bungay, 1981; Ferchak and Pye, 1981; Lipinsky,
1978, 1981). The use of ethanol in the U.S.A. as a gasoline extender and octane enhancer is considerable today, with a rise in production from 80 million gallons in
1980 to 880 million gallons in 1985. Production has been mainly by yeast 16
fermentation of hydrolysed starch (com) feedstocks (Maiorella, 1985) which is relatively expensive. Therefore, it would be economically advantageous to use cellulose materials and other biomass which is less expensive. Research on the utilization of biomass has focussed on cellulosic materials because of its abundance
(Douglas, 1982; Detroy et al., 1981). Ethanol has been focussed on as one major product because of its convenience as a liquid transportation fuel or a versatile chemical feedstock.
As discussed in Section 1.1.2. Z. mobilis has shown great potential for ethanol production. Esser and Karch (1984) have evaluated Z. mobilis as being an "ideal ethanol producer" if the problem of its narrow substrate range could be overcome. A long-term research project in this laboratory is to extend the fermentable substrate range of Z. mobilis to include cellulosic materials and the study reported in the thesis fulfils part of this work. Presently conversion of cellulose to ethanol using Z. mobilis can be achieved either via a two-step process involving hydrolysis of pretreated cellulosic substances to glucose, then the use of Z. mobilis to ferment glucose to ethanol, or by growing Z. mobilis in sequential culture with other cellulolytic organisms (Saddler et al., 1981). If the aim of this long-term project is achieved then genetically-engineered Z. mobilis strains would be expected to ferment pretreated cellulosic substances to ethanol directly.
There have been many reviews published covering aspects of the bioconversion of cellulosic biomass to useful products (e.g. Bisaria and Ghose, 1981; Enari, 1987; Ladisch et al., 1983; Ladisch and Tsao, 1986; Tangnu, 1982; Tsao et al, 1982; Tsao,
1987). Cellulose hydrolysis and its product, glucose, play a central role in the conversion of this renewable resource. Glucose then can be fermented to different products either in a separate process or simultaneously with enzymatic hydrolysis. 17
The basic molecular structure of cellulose is a linear homopolymer of 8000-12000 anhydroglucose units linked by (3-D-l, 4-glucosidic bonds (Chang et al., 1987). In nature, cellulose forms insoluble fibrils consisting of several parallel cellulose molecules held together by hydrogen bonds. The cellulose fibers contain ordered crystalline and less well-ordered amorphous regions (Figure 1.2). In wood, these fibrils are embedded in lignin. Lignin consists of an aromatic polymer based on three dimensional phenylpropane units held together by ether and carbon-carbon bonds (Taso et al., 1987). Hemicellulose is an alkali-soluble and highly branched heteropolymer, which commonly comprises two or more of the following sugars: D- xylose, L-arabinose, D-mannose, D-glucose, D-galactose and D-glucuronic acid (Taso et al., 1987). The composition of cellulosic materials varies depending on the source, and the range is (as percentage of dry cellulosic material) cellulose 40-60; hemicellulose, 20-40; and lignin, 10-25.
Since the major structural features of cellulose that determine its susceptibility to enzymatic degradation are the association of the cellulose with lignin, the surface area accessible to the enzymes and the degree of crystallinity, the native lignocellulosic substrates must first be pretreated to increase the direct physical contact between the cellulosic microfibrils and the cellulose complex so that enzymatic hydrolysis can proceed efficiently. Methods of physical, chemical and biological pretreatment have been reviewed by Fan et al. (1982) and Marsden and
Gray (1986).
The enzyme hydrolysis process has been studied by Reese (1972) and Mandels (1974). There are many sources of cellulolytic enzymes. However, the fungus Trichoderma viride has proven to be the most effective source to date (Reese, 1972). 18
CRYSTALLINE AMORPHOUS REGIONS H3T*.REGIONS t A. E^o-3-J&jCYm6e (EG:Cx) . i.
/
Ce-Ptobiohyd/iotaM. (CBH:C^)
*
>V«V4__
1 EG/CBH
*—•—• - - - - - 9 n t « ♦ ■« GLUCOSE
Figure 1.2: Schematic presentation of sequential stages in enzymatic hydrolysis of cellulose (Montenecourt and Eveleigh, 1979). 19
With proper pretreatment a cellulase preparation from T. viride is able, in a reasonable time (<100 h), to break down completely a pure cellulose substrate. It is usually difficult to obtain much more than 50% conversion from natural sources and the problem may be solved by removing lignin and hemicelluloses from the native cellulose or addition of hemicellulases. In a typical enzymatic hydrolysis of a 5% suspension of ball-milled newsprint, a sugar syrup containing 1.6% glucose, 1.4%
! cellobiose and 0.2% xylose is readily obtained (Mandels, 1974). ’•
Microbial hydrolysis of cellulose can often be very direct, fast and complete. This is because cellulolytic organisms grow on the cellobiose or glucose produced, continuously removing them from solution and relieving their inhibitory effects. An excellent volumetric cellulose hydrolysis rate of nearly 2 g 1-lh-l was obtained by growing a Thermoactinomyces sp. at 60OC on Avicel for 8-10 h (Armiger et al., 1977). Since microorganisms produce primarily extracellular cellulases, it is possible to separate the organism from the enzyme-containing supernatant. However, hydrolysis is usually faster in the presence of organisms than the cellulase-containing solution alone. It is possible that engineered strains of Z. mobilis will ferment ethanol efficiently since produced ethanol can be removed continuously from the reaction mixture.
The acid hydrolysis process has been studied for many years (Grethlein, 1975) and is by far the most commonly used hydrolysis system in practical application. Although it is a relatively straightforward process, it has some major disadvantages such as decomposition of the component sugars, corrosion of reactor components, environmental pollution and production of by-products (Ghose and Ghosh, 1978). 20
1.4 Cloning and expression of cellulase genes
1.4.1 Cellulose-degrading microorganisms
Cellulolytic enzymes are produced by a large number of microorganisms including fungi, actinomycetes and bacteria (Coughlan, 1985).
The ability to produce extracellular cellulase is prevalent among fungi, but only relatively few species have been considered as good producers of cellulase. Such fungi include: Trichoderma reesei, Trichoderma konigii, Penicillium funiculosum, Penicillium iriensis, Pennicillium verruculosum, Fusarium solani, Ajpergillus terreus, Phanerochaete chrysosporium, Polyporus adustus, Myrothecium verrucaria, Pellicularia filamentosa and Eupenicillium javanicum. Thermophilic fungi including Chaetomium thermophile var. dissitum, Sporotrichum thermophilicum and Thermoascus aurantiacus have been studied bec|C$se of their temperature tolerance.
Bacteria studied for cellulase synthesis belong to various taxonomic groups n including: Myxobacteriales - Sporocytophaga, Cytophaga; Acti^iomycetales - Micromonospora, Streptomyces, Thermoactinomyces, Thermomonospcra, Thermopolyspora; Eubacteriales - Bacillus, Cellulomonas, Pseudomonas,
Clostridium, Bacteroides, Ruminococcus.
Gray et al (1980) have summerised the advantages of bacterial systems as follows:
1. Bacterial species have greater long-term potential for genetic inanimation.
2. They require a much shorter time for enzyme production. 21
3. Relatively low levels of protein are needed for cellulotic activity.
The gene donor in this study, Xanthomonas albilineans XA1-1, is a Gram-negative cellulolytic bacterium isolated from rotting sugar cane bagasse samples (Opolski,
1984). Xanthomonas and Pseudomonas belong to the family Pseudomonadaceae and they are taxonomically and genetically closely related (De Ley et al., 1966).
Xanthomonas was chosen as the source of the p-glucosidase gene because studies on the expression of cloned Xanthomonas D-xylose catabolic genes (Liu et al., 1988) suggest that Xanthomonas promoters may be efficiently recognised by Z. mobilis and so direct expression of the Xanthomonas gene in Z. mobilis may be possible. Also the Xanthomonas strain used, XA1-1, exhibited a high level of p-glucosidase, was found to clear holocellulose repaidly, was not sensitive to catabolite repression by glucose or cellobiose, and the p-glucosidase activity seems to be a true cellobiase.
1.4.2 The cellulase enzyme system
A knowledge of the cellulase enzyme system is essential to the cloning of cellulase genes. The properties, characteristics, biosysthesis and regulation of cellulase have been reviewed in many publications (Bisaria and Ghose, 1981; Brown and Gritzali, 1984; Coughlan, 1985; Enari, 1983; Enari and Niku-Paavola, 1987; Ladisch et al.,
1983; Lee and Fan, 1980; Mandels, 1982; Marsden and Gray, 1986; Wood, 1985).
The degradation of crystalline cellulose in fungi is a complex process and involves the interaction of at least three enzymes: endoglucanase (endo-1, 4-p-D-glucan 4- glucanohydrolase, EC 3.2.1.4), cellobiohydrolase (1,4-p-D-glucan cellobiohydrolase, 22
EC 3.2.1.91) and p-glucosidase (p-D-glucoside glucohydrolase, EC 3.2.1.21), with all three components acting synergistically and no one component being able to completely hydrolyse cellulose independently.
The currently-favoured model (Figure 1.2) for the enzymatic hydrolysis of cellulose incorporates observations from many laboratories (Montenecourt and Eveleigh,
i 1979). According to this model endoglucanase acts on amoiphous regions in the cellulose fibres to reveal new chain-ends for attack by cellobiohydrolase, which then splits off cellobiose units from the non-reducing ends of the cellulose chains, p- glucosidase hydrolyses cellobiose and soluble cello-oligosaccharides to glucose. Since cellobiose, the end-produce inhibitor of cellobiohydrolase and endoglucanase, is removed, the overall hydrolysis is further enhanced.
Synergism between cellobiohydrolase and endoglucanase is clearly established (Selby, 1969; Maoris and Galiotou-Panayotou, 1986) and supported by electron microscopic observation (White and Brown, 1981). Because endoglucanase can only act on exposed glucan chains it alone hydrolyses amorphous regions of glucan chains on the surface of the cellulose fibrils and the random cleaving action does not remove surface layers or expose new glucan chains. Cellobiohydrolase then removes cellobiose from the exposed non-reducing chain-ends until all nicked chain-ends have been removed. Cellobiohydrolase cannot attack the newly-exposed glucan chains unless endoglucanase is also present. In this way endogulcanase and cellobiohydrolase act synergistically to create sites of action for each other. Wood (1975, 1980) found that the effect of synergistic action was maximal when the components were used in the same ratios found in the original fermentation filtrates and suggested that the synergism between cellobiohydrolase and endoglucanase may be directed primarily by the endoglucanase. 23
In addition to the three major classes of cellulases, glucohydrolase and cellobiose oxidase were found in some organisms. Glucohydrolase removes single glucose units successively from the non-reducing ends of cellulose chains (Mullings, 1985). Cellobiose oxidase oxidizes cellobiose and cello-oligosaccharides to the corresponding acids using molecular oxygen (Eriksson, 1981).
1
The degradation of cellulose by bacteria differs from the fungal cellulolytic system and has been studied less in detail. A major distinctive feature in the bacterial system is the high amount of endoglucanase relative to cellobiohydrolase compared to the ratio in Trichoderma cellulases (Ladish et al., 1983), and furthermore the bacteria do not, with a few exceptions, form extracellular p-glucosidase. Thus adherence of bacterial cells to the cellulosic substrate is generally necessary for extensive hydrolysis. Bacterial extracellular endoglucanases degrade cellulose to short-chain oligosaccharides, which are then hydrolysed to glucose by a cell-associated p- glucosidase (Figure 1.3). In some bacteria the metabolism of cellobiose is via phosphorylase oxidize systems (Schimz et al., 1983).
1.4.3 Strategies for cloning and expressing cellulase genes
Cloning strategies firstly involve construction of gene banks. All of the cloned prokaryotic cellulase genes have been screened from banks of genomic DNA from cellulolytic bacteria and fungal cellulase genes were cloned from banks of genomic
DNA or derived from cDNA.
Detection of genes encolding cellulase is an important step in the cloning strategy. The most straightforward method of screening is by enzyme activity. For screening 24
CELLULOSE
______ExtAaczllula^ undoglucancun
v LONG OLIGOSACCHARIDES
V Ve.A.iplaAmic $-glucoA'i.dcu> CELLOBIOSE VoAiptaAvnic. $-glucoAidoA GLUCOSE Figure 1.3: Mechanism of bacterial cellulolysis according to Ramasamy and Verachtert (1980). 25 P-glucosidase genes the cleavage of chromogenic or fluorogenic p-glucosides is used to monitor P-glucosidase activity. p-Nitrophenyl p-D-glucoside (pNPG) (Raynal and Guerineau, 1984) which releases the yellow compound, p-nitrophenol, 5-bromo-4- chloro-3-indolyl P-D-glucopyranoside (X-glu) (Penttila et ah, 1984) which releases indigo (dark blue) and 4-methylumbelliferyl p-D-glucoside (MUG) (Wakarchuk et al., 1984) which releases fluorescent methylumbelliferone were used to detect P- glucosidase genes. A problem with these screening methods is'that aryl-P- glucosidases do not always cleave cellobiose (Woodward, 1982) and so it is advantageous to select for cellobiase genes by plating on minimal medium with cellobiose as sole carbon source (Armentrout and Brown, 1981). However, expression of cellobiase is not always sufficient to allow growth on cellobiose, because transport either of the enzyme to the periplasm or of the substrate into the cytoplasm appears to be necessary. Other screening methods include immunological detection and nucleic acid hybridization. The cloned genes are then isolated and characterised (for a review, see Andrews et al., 1988). 1.4.4. Survey of cloned cellulase genes Genes encoding cellulase have been isolated from a number of bacteria and fungi and these are summarised in Table 1.2. The table shows that the cloned cellulase genes from bacteria were all readily expressed in E. coli. It is possible to improve cloned gene expression by further genetic manipulation such as using an appropriate high copy number vector containing a strong promoter. Wakarchuk et al. (1986) reported that when a P-glucosidase gene from Agrobacterium was cloned into E. coli, the level of expression was only 6.8% of that of parent strain. The expression level was then Table 1.2: C loned genes encoding cellulase enzymes £ O .5 0 P^ T> jD X < £ Td Q £ b 00 bO V 3 O D on O cd o c O 3 2 5 o 1-1 U M < PQ < P^ oo VO on x: 13 u £ ■a -si -s: o o To Isj •2 •2 • G 'H XI- - .2 1 0 2 o o "X* p o v. o P 3 o o &C g a s. h • O U-i 3 Oo o o P Pi £ -g O PI X) -s; ^ 3 PI 0^ *s» Ck X3 To jd kj •2 •2 6 on c/T O on o 3 o P h VO T O oo r-^. .2 • -S cq “ & ^ <>, — s 2 n > ' • T3 13 ^ VO xf •g-G PQ kj ’ II Q xd o h Cd -2 cq 3 a oo VO O j ij Pk -s: d <£ r- -s; ■ •2 * 'O kj ^- **--k CQ 13 c c <-^k &n o ^ — O bO c n „ j h i O Tj" oo t CO xj i— Pi * cn Pi -s; -si -2 PQ Oi r- Isj (N T — s cd S £ g-e co V-> o c — O V. P bO c O C 3 3 -k n ■< < — 3 i h -~2 vo ON OO r_ Pk g o <3 3 g I -* 1 VO ^ on oo O 13 r-H o x: lO • t3 •s: •«•§ To ^3 td ki -s: ■c - £ ■§ § 3 bO h o_r ON oo 00 ^ ^ r- »^k ks cq -a b •2 p O o > $ C3 vj 3 On 3 ^ O oo vo ‘ 13 o ON oo r-H Ud •2 -si ki -s; •2 'O To J kj •2 b 53 CS cn *-^k *^~k -k *0 -c cq '-3 . h ON oo pf XJ • — cn cs h ON OO no 13^ O 5=3 Os 2? h ^ cd ; ON OO H X) - 26 1 endoglucanase Saccharomyces pOP, pN 3 S kipper et al., 1985 cerevisiae Rhodobacter pJAJ103, Johnson et al., 1986 capsulatus pJAJ21 1 endoglucanase Aspergillus G w ynneet al., 1987 I I co K X Os ob-u CO (N ***■4 PQ -s; •2 •2 •-* £ -P S « P CO co co < V. O o o d h TVO r H h Pv T _j — SS-3 cj « O cj co < - H d - vo - 0 G\ OO *-< I h CO -C P * co 5 «J cj C/5 'z'z'z O 00 vo v CO tjq 72 45 H-> (N 2 *^3 rtX) O -2 -3 — cj CD cj cj 8 q t P CO CO »4 >3 cj (O co V. o 00 00 o h VO 00 T"H 1 *04 * * Pq H-> 2 cj cj cj V *-> < OV VO 00 ' r o cj Cj W cj cu -c -c •2 *44 •2 bJ f *0 3 *■^4 -Q 1 5 - < W y m -q 2 45 2 c/5 O 0 W) 1 ■ 1 9 8 6 r-H ON 00 vo 73 CS in N W P^ *-4* -s; *~4 -Sj •2 •2 t*} T3 0 G> r~t l& Cj P P CQ. -q 72 45 -2 C/5 ID C/5 bO s 0 ex a « l * 73 U 00 k-i < CJ * * -sc .2 •2 ^q T3 'Hb — *"*»4 -2 -2 vU O -2 *4 •2 cj 2 al •2 q P co — CO o Co co V. ■*4 Cj Cj C/5 q (D fH 0 CD wj CO CO 5 4 5 H 4 h 1 9 8 8 UU^ O -2 T3 ffi O **4«» *-4 -s: •2 •2 QJ 'tb J3 III *-*4» -SC O P Cj ^3 .2 C/5 £ P O co CO co o Co 2 (j cj G Cj C/5 (D 0 h 1 . ,1 9 8 3 a ^ CC3 11 9 , 8 3 b XT OO 7 uu T <<^ UUCJ >^^00 — O P ^ C h L, 1 9 8 5 OO Tf H — P H h 73 ►2 is , 1 9 8 6 h DO 7 ^4 •2 *4 CQ 3 0 0 Co $ CO 3 s <3 - B a u & e r CO ^ ■§ 1 •*4 *^4 *4 Cq 2 c u e r1 , 9 8 7 t OV C50 •q — 1 CO sy (Sis « c^o 44 • -sc •4 -sc .2 ^- kj -H T3 w O 0 Cj II O P co 4 CO Co Q> Co Co S. o Cj q cj H ^OO - ■< h Cv 1/-J _ T OO Tt t < *4»* * _ -sc -sc *4 -2 H-> kj • PS -H — ccj < 4 P d co CO CO Co <3 Co r-H o O h T3 71 JG 2 Cj >s 1 ■s 73 ON to co c r ' * h -2 . ’ xr cN PQ > *44 '4 -si • toq §8 43 S '4* cj G P a Co 05 co co ^ — Co <3 o 00 h h 00 ON — $ Cj 5 u 2 q 27 1 Escherichia 1 p-glucosidase Escherichia coli pB R 322 A rm e n tro uand t adecarhoxylate B ro w n ,1981 a chrysanthemi 1 endoglucanase Escherichia coli cosmid Barras et al., 1984 pMMB4 Boyer et al., 1987 1 endoglucanase Escherichia coli RP4::mini-Mu van Gijsegem et al., 1985 1 endoglucanase Escherichia coli X phage L47.1 Kotoujansky et al, 1985 VO O oo Tj- 'a? 13 00 t O -3 .2 p I til nd V) *§ ft. — c ^ *§ ft. § 2 3 oo 3 oo c O oo no & V CO (N O cd c/3 ' *§ ft. C/3 to 3 s 2 g g 13 ON 00 ^■*4 r r- (N -3 • -3 •2 II til £ TD •^4 ►3 £ C< •5 — cd 3 3 3 P ^ Oo 3 3 © a a 00 o toba o c ON 00 00 nd V -3 * -3 ■ CO. -2 £ 12 £ *^<4 O s cd 0 3 3 3 i P Oo 3 3 3 — o <£ nd < fv 00 £3 co oo 00 wo | to nd ’ js o cd a a 00 0 2 c o bb o^ ^ v r-H O Z ^ 5 (N r-~ p r t O 00 b p r- o (N X nd *^4 H -5: 3 • •2 •2 5 od Vo — -3 -3 E- O cd O cd C 3 p 3 — 3 3 3 2 cd c a 00 Thermomonospora fusca 2 endoglucanases Escherichia coli pBR322Collm & Wilson, 1983 er 1 endoglucanase Streptomyces lividans Ghangas & Wilson, Bacillus subtilis 1987 28 2 endoglucanases Escherichia coli pKT230 Lucas, 1987 Pseudomonas putida T3 XJ • H o co. • t c/3 co P PH -H 00 cb C/3 ►"--A -O * •5 r-A to a O © B o C 5 5 29 Aspergillus niger 1 P-glucosidase Saccharomyces Cosmidp3030 Penttila et al., 1984 cerevisiae -d -3 CJ o CO • to X u -s: •2 •2 kj 'Hb § £ &!■§ * O C/5 e t C on OO Tl- i -Si — CO . S |S 05 g* Co CO co 2 5.2 C c - „oo 3 < on n ^4 ’ — -2 o a CO ^ I ON OO CO 13 •c H 44 5-g Co • | CO r- -O ef C cu 5 <4 cj 00 o) C/5 ctf SX l , O OO CO ^ 44 •3 00 43 CJ X i— °6 CQ « i h ^ J ; 13 O oo *C r- H P CJ OO _^T o O 4 lO ^CJCJ o C n 4 OO O ▼-H r-~ T3 4 * oo PP 4 cd ° a O a > p< -* n ! r _4 ON OO NO • ^ P -» oo 5-g Co r J 1-H <3 y 5.2 O d J ctf bX) h 3 C ) » 4 - 4 n TD t-H ON OO 4 r- < 4 cd rO < » — c > o CO 31 increased by subcloning the gene downstream from the E. coli lacZ promoter in pUC18. The level of activity upon induction was 1,869 times higher than before. y P-Glucosidase is usually cell-associated in cellulol/tic bacteria (Gong and Tsao, 1979) and p-glucosidase from genetically-engineered strains is also generally found in the cell-associated enzyme fractions (Armentrout and Brown, 1981; Love and Streiff, 1987). In contrast, Cellulomonas uda excretes p-glucosidase and cell-free extracts of recombinant strains carrying the cloned genes also showed considerable p- glucosidase activity (Nakamura et al., 1986a). Two types of p-glucosidase can be distinguished according to substrate specificity, physical properties and genetic control of enzyme biosynthesis (Gong and Tsao, 1979). These are the true cellobiases and aryl p-glucosidases. Aryl p-glucosidases from Stachybotrys atra, Schizophyllum commune, Chaetomium sp. and Alcaligenes faecalis do not degrade cellobiose while p-glucosidases from other fungal and bacterial sources, such as T. reesei and P. chrysosporium show activity towards both substrates. Genes encoding true cellobiase and aryl p-glucosidase were found to exist together in Pseudomonas PS2-2 and Xanthomonas XA1-1 (Lucas, 1986; this study). During cloning two kinds of clones were obtained; one showed activity to both substrates and the other did not cleave cellobiose. p-glucosidase genes were successfully expressed in Saccharomyces cerevisiae and the engineered strains producing high level p-glucosidase (Raynal and Guerineau, 1984). Under aerobic conditions these strains could utilise cellobiose as the sole carbon source, while under anaerobic conditions, although the strains still produced p-glucosidase, fermentation of cellobiose was not observed. This difference may be 32 caused by problems of anaerobic transport of the disaccharide into yeast (Leclerc et al., 1986). A major aim of the present project was to clone and express P-glucosidase genes in order to establish a cellobiose catabolic pathway in Z. mobilis. The first part of this work, cloning and expression p-glucosidase genes in E. coli, is presented in Chapters i 5 and 7. Chapter 6 describes the cloning and expression a P-glucosidase gene in Z. mobilis. The expressed p-glucosidase gene was further linked with an endoglucanase gene on the same vector; cloning and expression of the linked genes are reported in Chapter 8. 1.5 Major objectives of the investigation Because some important methods for gene transfer were still at a rudimentary stage in Z. mobilis, one of the major goals was to investigate and improve these systems in order to provide more efficient means for genetic manipulation in Z. mobilis. The other major objective was to use recombinant DNA techniques to clone a p- glucosidase (cellobiase) gene in Z. mobilis. Expression of this gene extends the fermentable substrate range of Z. mobilis to include cellobiose. 34 CHAPTER 2 Materials and Methods 2.1 General equipment \ Media, glassware and other materials were sterilised by autoclaving at 103.4 kPa (15 p.s.i., 1210C) for 20 minutes. A model MREC1016 Autoclave, obtained from Athertons Pty. Ltd., was employed. Cellulose nitrate membrane filters (0.45 Jim pore size, Gelman Sciences Inc., USA) were used routinely for filter-sterilising amino acids and D-cellobiose. A Mettler (Switzerland) AE166 analytical balance was used for accurate weightings and a top loading Sartorius L2200P was used for approximate weightings. A Pye Unicam SP6-550 spectrophotometer with a 1 cm light path or a Bausch and Lomb (USA) Spectronic 21 spectrophotometer was used for optical density measurements. For all pH measurements, a PHM 83 Standard pH meter (Radiometer, Copenhagen) was used. The centrifuges used were: a Heraeus Christ Biofuge A bench centrifuge (Australia); a BHG Heka bench centrifuge (Australia); a Janetzki Model 75 centrifuge (West Germany); a Sorvall RC5-B refrigerated superspeed centrifuge (Dupont Instruments, USA) and an L8-M Ultracentrifuge (Backman, USA). A voxtex mixer (Scientific Industries Inc., USA) was used for mixing and for resuspending pellets after certrifugation. 35 Electrophoresis was powered by an LKB Bromma or BioRad power supply. Gels were photographed on a Gelman Clemco Pty. Ltd. (USA) Model C-61 transilluminator through a yellow UV filter with a Polaroid (USA) MP-4 land camera. Film used for photographing gels after electrophoresis was Polaroid, either type 665 (positive-negative) or type 107 (positive only) black and white film. Autoradiography was carried out in Kodak autoradiography cassettes using Kodak XRP-l X-ray film. '• An Olympus (Japan) BHA microscope with phase contrast lenses was used to observe and count cells. An IBI (USA) model UEA unidirectional electroeluter was used to isolate DNA fragments. 2.2 Reagents, solutions and media 2.2.1 Reagents All reagents used were of analytical reagent grade. Amino acids, vitamins, D(+) cellobiose, antibiotics, ethylenediaminetetraacetic acid a (EDTA), Tris-HCl Trizma base, boving serum albumin, p-nitrophenyl p-D- glucopyranoside (pNPG), esculine hydrate (6,7-dihydroxycoumarin 6-glucoside), N.N. - dimethyl formamide, isopropyl p-D-thiogalactopyranoside (IPTG), 5-bromo- 4-chloro-3-indolyl p-D-galactoside (X-gal), ethidium bromide, bovine serum albuny, RNA-ase, calf thymus DNA and dithiothreitol were purchased from the Sigma Chemical Company(USA). Cesium chloride and Wako phenol (special grade) were 36 supplied by Novachem Pty.Ltd. Agarose was from Difco (USA). Toluene, potassium tartrate, Congo red, carboxymethyl cellulose, and formamide were from BDH Chemicals Ltd. (England). T4 ligase, restriction endonucleases and DNA d standards came from New England Biolabs (USA). Radioactively labelledj!ATP ([a- 32P]ATP) came from Amersham International (England) and sodium dodecylsulphate (SDS) was from Tokyo Scientific Company (Japan). D(+)-glucose and glycerol were from BDH (Australia). Ferric ammonium citrate, Folin- Ciocalteu’s reagent and ethanol were supplied by Ajax Chemicals (Australia) and p- nitrophenol (pNP) came from the Tokyo Chemical Industry Co. Ltd. (Japan). Methylene blue came from G.T.Gurr (London) and bromophenol blue was supplied by H.B. Selby & Co. Pty. Ltd. (Australia). All microbiological media ingredients were from Oxoid Ltd. (England). Glucose test combination kits and lysozyme were purchased from Boehringer Mannheim (West Germany). 2.2.2 Solutions All aqueous solutions were prepared with distilled water. 2.2.2.1 Saline NaCl was added to water to give a final concentration of 8.5 g 1-1 and was sterilized by autoclaving. 22.2.2 Saline Phosphate Buffer (SPB) SPB, used for serial dilutions of Z. mobilis suspensions, contained: NaCl 8.5 g 1~ 1; K2HPO4 7 g 1-1; KH2PO4 3 g 1-1. 37 2223 Carbon sources Glucose, cellobiose and carboxymethyl cellulose (CMC) were sterilised separately from other media components. Glucose was prepared as a 500 g 1-1 solution and sterilised by autoclaving at 103.4 kPa (1210C) for 15 minutes. Cellobiose was i prepared as a lOOg 1-1 solution and was filter-sterilized. CMC was prepared as a 100 g 1-1 solution and sterilised in the same way as glucose. 2.2.2.4 Mcllvaine’s buffer Mcllvaine’s buffer, used in enzyme assays, was prepared by mixing 18.2 ml 0.1 M citric acid and 81.8 ml 0.2 M Na2P04, and up to one litre with water. The pH was 7.2. 2.2.2.5 DNS reagent DNS reagent, used to quantitate reducing sugars, was prepared according to Miller (1959). NaOH (lOg), sodium potassium tartrate (182g), dinitrosalicylic acid (lOg), phenol (2g) and sodium sulphite (0.5g) were dissolved in 600 ml distilled water in sequence and the final volume adjusted to 1L with distilled water. 2.2.2.6 Gel electrophoresis buffer (TAE) TAEG was prepared as a 50-fold stock solution containing Trizma-base (193.76 g 1- 1), anhydrous sodium acetate (16.4 g 1-1) and disodium EDTA (14.89 g 1-1). The pH was adjusted to 8.0 by the addition of glacial acetic acid. 38 22.2.1 TBE buffer TBE was prepared as a 10-fold stock solution containing Trizma-base (108 g 1-1), boric acid (55 g 1-1) and disodium EDTA (9.3 g 1~1). The pH was 8.3. 2.22.8 TE buffer • TE consisted of 0.05M Tris-HCl and 0.01M EDTA. The pH was 8.0. 22.2.9 Saline Sodium Citrate (SSC) SSC buffer consisted of 0.15 M NaCl, 0.015 M Sodium Citrate and the pH was adjusted to 7.0 using HC1. 2.2.2.10 Dilution fluid Dilution fluid, used for serial dilution of spheroplasts of Z. mobilis, consisted 0.02 M maleate buffer (pH 6.5), 0.5 M sorbitol and 0.02 M MgCl2 (Lee and Seong, 1984). 2.2.3 Media All media were prepared with distilled water and where necessary solidified by the addition of 15 g 1_1 Difco Bacto-agar. Luria broth and minimal medium were prepared for the growth of E. coli. 39 2.2.3.1 Luna Broth (LB) (Miller, 1972). Tryptone (10 g 1-1), yeast extract (5 g 1_1) and NaCl (5 g 1-1) were dissolved in distilled water and autoclaved. 2.2.3.2 Minimal Medium (MM) (Clowes and Hayes, 1968) 1 The minimal salts solution was prepared as a 4 x concentrate by dissolving, in order, the following salts in water: NH4CI (20 g 1-1), NH4NO3 (4 g 1-1), Na£04 anhydrous (8 g 1-1), K2HPO4 anhydrous (12 g 1-1), KH2PO4 (4 g 1-1) and MgSC>4 .7 H2O (0.4 g 1-1). The pH was adjusted to 7.2 and the medium autoclaved. For enzyme assay the appropriate carbon source was added to the minimal salts solution and the solution made up to final volume with sterile water. The pH was readjusted to 7.2. Minimal agar (MA) was prepared by mixing water agar, 100 ml minimal salts solution and 1.6 ml 500 g 1-1 glucose or 80 ml 100 g 1-1 cellobiose. Water agar was made by dissolving 6g agar in 300 ml (glucose MA) or 220 ml (cellobiose MA) water, adjusting the pH to 7.2 and autoclaving. 2.2.3.3 Rich Medium (RM) (Goodman et al., 1982) RM for the growth of Z. mobilis strains contained glucose (100 g 1-1 or 20 g 1-1), yeast extract (10 g 1“ 1) and KH2PO4 (2 g 1-1). A solution of 200 g 1-1 KH2PO4 was sterilized separately and then added aseptically to the autoclaved, cooled medium at 1:100. 40 2.2.3.4 Basal Medium (BM) (Goodman et al., 1982) Basal medium, for Z. mobilis contained KH2PO4 (1 g 1~ 1), K2HPO4 (1 g 1-1), NaCl (0.5 g 1-1) and (NH4)2S04 (1 g 1-1). After autoclaving and allowing the BM to cool to about 550C the following filter-sterilized solutions (mg 1-1 final concentration) were added: MgS04.7H20, 200; CaCl2-2H20,200; Na2Mo04-2H2O,25; and FeS04.7H20,25. One ml of a filter-sterilized vitamin solution which Contained the following (mg 1-1) was also added: calcium pantothenate, 5; thiamine hydrochloride, 1; pyridoxine hydrochloride, 1; biotin, 1; and nicotinic acid, 1. Glucose was added to a final concentration of 20 g 1-1. The pH was 6.0. Growth factors were added at concentrations recommended by Davis et al. (1980). 2.2.3.5 Nutrient Yeast Broth (NYB) Oxoid Nutrient Broth No. 2 (25 g 1_1) and yeast extract (5 g 1-1) were dissolved in water and autoclaved. Glucose (3g 1-1 final concentration) was added after autoclaving, for growth of XA1-1 (NYBG). 2.2.3.6 PM Medium For enzyme assays on XA1-1 and P. putida the growth medium consisted of PM stock salts (Paul Morjanoff, personal communication) prepared by diluting the following stock solutions to 1L. 4 ml MgS04 • 7H20 (20 g 1-1) 20 ml Nitrate solution 1 20 ml Phosphate buffer2 41 10ml ZnS04.7H20(l gl'1) 4 ml Trace element solution3 1. Nitrate solution (170 g H mixed nitrates! KNO3 (229.4 g) and NaNC>3 (64.6 g) were dissolved in water and made up to 1L. 2. Phosphate buffer (1 M) NaH2P04 (44.5 g) and K2HPO4 (124.4 g) were dissolved in water, made up to 1L, and pH adjusted to 7.2. 3. Trace elements solution Concentration Salt (gl-1) FeS04 .7H20 0.5 Q1SO4 .5H2O 0.01 H3BO4 0.007 MnS04 .7H20 0.05 ZnS04 -7H20 0.05 Na2Mo04 0.01 CaCl2 -2H20 1.324 C0CI2 .6H2O 0.01 Carbon sources were sterilized separately and added after autoclaving and cooling the PM stock salts. 42 2.23.1 Spheroplast regeneration medium Regenration medium (Lee and Seong, 1984) was prepared for the regeneration of spheroplasts of ZM6. The medium consisted of glucose (20 g 1-1), yeast extract (10 g 1-1), sorbitol (91 g 1-1) and agar (15 g 1-1). After autoclaving a sterilized solution of KH2PO4, MgS04 .7H20, MgCl2 .6H2O Casamino acid and CaCl2 .2H20 was added to give a final concentration of 2,1,1,1 and 1 g 1-1 respectively. \ 2.2.3.8 Esculin plates Esculin (6,7-dihydroxycoumarin 6-glucoside, Montenecourt and Eveleigh, 1979) plates (Esc), were designed to show a colour difference between colonies which produce p-glucosidase and those which do not, the former causing the agar around the colony to turn black. Esculin is cleaved by p-glucosidase, releasing esculetene, which forms a black product with ferric ions. The plates were made by adding 0.4 g 1-1 of esculin hydrate and 1 g 1-1 of feme ammonium citrate to LB or MM agar. 2.2.3.9 Carboxymethyl Cellulose (CMC) - Congo Red Plates (Teather and Wood, 1982). CMC - Congo red plates provided a method of screening for endoglucanase production and were prepared by adding 0.5% CMC to LB agar. After incubation at 370C overnight the plates were flooded with 0.1% aqueous Congo red for 15 min. and then washed with 1 M NaCl. Congo red stains CMC red and so clear zones around colonies against a red background indicated endoglucanase activity. 43 2.2.3.10 X-Gal plates X-gal and LPTG solutions were prepared and stored as described by Maniatis et al. (1982). When used for screening E. coli 20 pil of the IPTG stock and 40 p.1 of the X- gal stock were spread onto an LB plate and allowed to dry before use. 2.3 Biological materials \ The bacterial strains and plasmids in this study are given in Table 2.1 and 2.2 respectively. The gene banks (provided by R. Lucas) were described by Lucas et al. (1987). These gene banks were constructed by ligation of chromosomal DNA, incompletely digested with Hind III, into the Hind III restriction site of the plasmid, pKT230, followed by transformation of Escherichia coli ED8654 which is auxotrophic for methionine and thiamine. 44 Table 2.1: Bacterial Strains Bacterial strains Characteristics* Source or reference Zymomonas mobilis ZM6 Isolated from fermenting Skotnicki et al., 1983 Elaecis sap, Zaire (ATCC29129, Z6) ZM6100 met-1 contains only pNSWl Goodmaii et al., 1984 and pNSW2 obtained by NTG muta-genesis of ZM6 ZM6901 TcR p-glucosidase-producing This study ZM6 containing pNSW906 ZM6902 TcR p-glucosidase-producing This study ZM6100 containing pNSW906 ZM6903 KmR CmR P-glucosidase-producing This study ZM6 containing pNSW905 ZM6904 TcR p-glucosidase- and This study endoglucanase-producing ZM6 containing pNSW907 ZM6905 TcR P-glucosidase- and This study endoglucanase-producing ZM6 containing pNSW908 ZM4 Isolated from fermenting Skotnicki et al., 1984 sugarcane juice, Brazil (CP4) ZM4751 ade met Goodman, 1984 NTG mutagenesis of ZM4 ZM4761 cys arg RpR Goodman, 1984 NTG mutagenesis of ZM4 Escherichia coli K12 ED8654 met thi Murray et al., 1977 HB101 pro leu thi xyl-5 recA Boyer & Roulland- SmR Dussoix, 1969 JM101 supE thi A(lac-proAB) Yanisch-Perron et [F’traD36 proAB lacl^Z al., 1985 AM15] JM101 supE thi A(lac-proAB) [F* traD36 proAB lacIQZ AM 15] Xanthomonas albilineans XA1-1 wild type Opolski, 1984 Psedomonas putida ATCC17527 wild type Stanier et al., 1966 Pseudomonas \ PS2-2 wild type Hendy, 1980 * Antibiotic symbols are given inTable 2.3. 46 Table 2.2: Bacterial Plasmids Plasmid Characteristics* Source or reference Sa 37 kb, IncW, Ward & Grinsted,1982 CmR KmR SpR RPl::Tn507 65 kb, IncP-1, Bennett et al., 1978 CbR KmR TcR HgR pSal52 15 kb, IncW, Tait et al.1, 1983 CmR KmR SpR, Derivative of Sa pSa727 14.7 kb, IncW, Tait et al., 1983 CmR KmR SpR, derivative of Sa pSa747 15 kb, IncW, Tait et al., 1983 KmR SpR, Derivative of Sa pNSWl 14.5 kb cryptic plasmid Skotnicki et al., 1984 of strain ZM6 and ZM6100 Goodman, 1985 pNSW2 15.5 kb cryptic plasmid Skotnicki et al., 1984 of strain ZM6 and ZM6100 Goodman, 1985 pNSW3 34 kb cryptic plasmid Skotnicki et al., 1984 of strain ZM6 Goodman, 1985 pNSW60 20.5 kb plasmid formed Su and Goodman, by transposition of 1987 Tn7 from RP1 onto pNSW2 from ZM6100, CbR pNSW301 51 kb hybrid plasmid Strzelecki et al., formed between pNSWl 1987 from ZM6100 and Sa, CmR KmR SpR pKT230 11.9 kb, KmR SmR Bagdasarian et al., 1981 pRK404 10.6 kb, TcR, IncP-1 Ditta et al., 1985 pRK2013 48 kb, KmR, Inc P-1, Figurski and Helinski, RK2 transfer genes cloned 1979 onto a Col El replicon pUC8 2.7 kb, ApR lac? lacZ Vieira and Messing, 1982 pND71 23.8 kb, SmR, (3-Glu+, Lucas, 1987 fragment of pS2-2 DNA encoding P-glucosidase cloned into pKT230 pND82 18.3 kb, Tcr, End+, Lucas, 1987 fragment of XA1-1 DNA encoding endogluconase cloned into pRK404 pNSW901 29.5 kb, hybrid plasmid This study formed between pNSWl and pSal52 in vivo, CmR KmR SpR pNSW902. 14 kb deletion of pSa747 This study in vivo, KmR SpR pNSW903 22.5 kb, TcR P-Glu+, insert This study fragment from pND71 subcloned into pRK404 pNSW904 23.9 kb, SmR p-Glu+, 12.0 kb This study fragment of XA1-1 DNA encoding P-glucosidase inserted into KmR site ofpKT230 pNSW905 26 kb, CmR KmRp-Glu+, This study insert fragment of pNSW904 subcloned into SpR site of pSal52 pNSW906 22.6 kb, TcR P-G1u+, This study insert fragment of pNSW904 subcloned into pRK404 pNSW907 29 kb, TcR p-Glu+ End+, This study linked genes encoding p-glucosidase and endoglucanase from XA1-1 subcloned from pNSW904 and pND82 into pRK404 pNSW908 41 kb, TcR, p-Glu+ End+, This study linked genes encoding p-glucosidase and endoglucanase from XA1-1 subcloned from pNSW904 and pND82 into pRK404 pNSW910 5.7 kb, ApR,p-Glu+, deletion This study subclone ofXAl-1 DNA from pNSW904 in pUC8 pNSW911 4.2 kb, ApR,p-Glu+, deletion This study subclone of XA1-1 DNA from pNSW910 in pUC8 * Antibiotic symbols are given in Table 2.3. 49 2.4 Microbiological techniques 2.4.1 Preparation of standard bacterial inocula for enzyme work A standard procedure was used to prepare bacterial inocula in studies of enzyme expression and regulation, and other tests related to enzyme synthesis. t A single colony from a culture plate was transferred to 10 ml of nutrient broth and incubated overnight under appropriate conditions. The culture was then centrifuged. The cells were washed twice with sterile saline and the absorbance was adjusted to 0.6 at 600 nm with sterile saline. 1% (v/v) of this was used to inoculate the growth medium. 2.4.2 Growth of cultures E. coli strains were grown at 370C with shaking (200-280 rpm) in Luria broth or in minimal medium supplemented with proline and leucine (40 pig ml“l) and thiamine (2 fig ml'l) for HB101 or with methionine and threonine (50 fig ml“l) and thiamine (1.0 fig ml"*) for ED8654. XA1-1 was grown at 30°C in NYBG medium or in PM medium prepared by adding methionine and glutamate at 20 fig ml"* and yeast extract (0.04% w/v) to PM stock salts solution. P. putida cultures were grown at 30°C in NYBG medium or in PM medium. Z. mobilis was cultured at 30OC. Liquid cultures were incubated statically in tightly-capped centrifuge tubes. 50 2.4.3 Antibiotic supplementation of media To maintain plasmids and to select transconjugants or transformants media were supplemented with antibiotics. The concentrations used are shown in Table 2.3. 51 Table 2.3: Concentrations of antibiotics added to media Strain Antibiotic Concentration (pg/ml) Ap Cb Cm Km Rp Sm Sp Tc Tp E. coli 50 300 50 50 _ 100 100 50 l 20 Z. mobilis 600 100 100 100 100 20 100 X. alibilineans 50 50 40 10 Pseudomonas 50 40 20 Ap: ampicillin Cb: carbenicillin Cm: chloramphenicol Km: kanamycin Rp: rifampicin Sm: streptomycin Sp: spectinomycin Tc: tetracycline Tp: trimethoprim When Tc was used together with esculin the concentration of antibiotic was doubled 52 2.4.4. Estimation of bacterial concentrations Concentration of bacteria were estimated by three methods: (a) Cultures were checked by optical density (O.D.) measurements at 600 nm. i (b) Cells were observed and counted using a Helber Bachena Chamber and phase contrast microscope. % (c) Viable cell counts were made by serially diluting cultures in SPB. Aliquots (0.1 ml) were then spread over RM or LB plates and incubated until single colonies appeared. Each single colony was assumed to have grown from a single bacterial cell and counted accordingly. 2.4.5 Storage of bacterial cultures Bacterial strains were stored on plates sealed with parafilm at 40C for up to one month for Z. mobilis and up to three months for E. coli, X. alibilineans and P. putida. For longer storage, cultures were grown on the appropriate plates, scraped off, resuspended in fresh RM (for Z. mobilis) or LB (for E. coli), and an equal volume of 80% glycerol was added. The glycerol stocks were stored at -20OC or -70OC. 2.4.6 Patching Patching was used to test the growth of colonies on different media. Single colonies were picked from a plate using a sterile stick and patched onto corresponding 53 segments of different test plates. Patches were positioned with the aid of templates divided into 50 rectangular areas. After incubation, growth of corresponding patches was compared. 2.4.7 Replica plating Replica plating was done to screen large numbers of bacterial colonies. A fully grown master plate, ideally containing approximately 100 colonies, was pressed lightly onto a clean, sterile velvet square secured over a replicating block with a rubber band. The replica plates were then pressed lightly onto the velvet, thereby transferring an exact replica of the master plate. 2.4.8 Stability testing of introduced plasmids Plasmid-carrying Z. mobilis strains were grown statically in RM in a tightly-capped centrifuge tube at 30°C, and subcultured 1:1C)3 every 24 h into fresh medium with or without antibiotics. Grown cultures were serially diluted and plated onto RM plates for single colonies. To test for plasmid maintenance the colonies were then replica plated or patched onto appropriate plates to select for plasmid-encoded characters and/or characters encoded by cloned DNA. Rapid plasmid extraction were done on several colonies picked randomly from the plates to test for maintenance of a plasmid of the expected size. For E. coli strains, cultures were grown in LB with or without antibiotics at 370C. The cells were subcultured 1:105 every 24 h. The cultures were then screened as described above. 54 2.5 Recombinant DNA techniques 2.5.1 Plasmid isolation 2.5.1.1 Plasmid isolation from Z. mobilis Plasmid DNA was isolated from Z. mobilis by the alkaline sodium dodecyl sulphate 1 method of Portnoy and White as reported by Nester (Nester, 1981). An overnight culture (5 ml) was centrifuged, washed with TEH and resuspended in 150 jil of TEII. Cells were lysed by adding 3 ml of lysis buffer, consisting of TE buffer at pH 12.65 (adjusted with 10 M NaOH immediately prior to use) plus SDS at 40 g 1-1. This mixture was incubated at 37°C for 20 min , then neutralised by the addition of 150 \i\ of 2M Tris (pH7.0), and mixed by inverting the tube gently several times. 1.2 ml 5M NaCl was added and mixed gently until a milky consistency was observed. The mixture was held on ice for 1-4 h and then centrifuged at 15,000 rpm for 20 min. The supernatant was transferred to another sterile centrifuge tube and 2.75 ml of isopropanol was added. The solution was stored at -20OC for at least 30 min and then centrifuged at 15,000 rpm for 10 min. The supernatant was removed and the tube was inverted on paper towelling and drained for 10-30 min. The plasmid DNA was resuspended in 200 jil of sterile double-distilled water. Residual chromosomal DNA was removed by second alkali treatment (Marko et al., 1982). The plasmid DNA was finally resuspended in 100 p.1 of sterile glass-distilled water and stored at 4oC. This method could be scaled up to isolate DNA from 200 ml of overnight Z. mobilis cultures. In this case, all the solutions were added at ten times the original volume. 55 2.5.1.2 Plasmid isolation from E. coli Plasmids were isolated from E. coli using the alkaline lysis method of Bimboim and Doly (1979). Sterile glass-distilled water instead of TE buffer was used to suspend plasmids. Plasmids for subcloning was purified by cesium chloride/ethidium bromide equilibrium density gradient centrifugation (Maniatis et al., 1982). The modified alkaline lysis method of Bimboim and Doly described by Maniatis et al. (1982) was used for rapid mini scale plasmid isolation. Rapid disruption of colonies was used to test for inserts in plasmids (Barnes, 1977). 2.5.2 Electrophoresis Electrophoresis buffer contained 32 mM Trizma base, 0.8 mM EDTA, 4 mM sodium acetate. Plasmids were characterised on horizontal gels containing 0.5% to 1.5% agarose in electrophoresis buffer. Electrophoresis was carried out horizontally at 2 V/cm for 24 h for a 30 cm long gel apparatus, at 4 V/cm for 5 h for a 20 cm gel apparatus, or at 5 V/cm for 5 h for a 15 cm minigel apparatus. 2.5.3 Determination of plasmid DNA concentration The concentration of a pure plasmid after ultracentrifugation in cesium chloride/ethidium bromide gradients was determined by measuring the optical density of the appropriately diluted plasmid. One optical density unit at 260 nm through a 1 cm light path is equivalent to 50 jig ml'l DNA. 56 Using agarose gel electrophoresis, the concentration of a particular plasmid was determined by comparison with a //mdlll digest of X DNA of known concentration, as described by Maniatis et al. (1982). 2.5.4 Size determination of DNA fragments DNA fragments were run on agarose gels together with a Hind III digested X DNA or Bst N1 digested pBR322 as standard size markers. X DNA digested with Hind III contains fragments of the following size (kb): 23.15, 9.42, 6.56, 4.38, 2.32, 2.02, 0.56 and 0.125. Bst NI digested pBR322 contains fragments of the following size (kb): 1.86, 1.06, 0.93, 0.38, 0.12 and 0.013. The size of an unknown linear DNA fragment was estimated by reference to a standard curve which was constructed by plotting size of the DNA standard in kb against the reciprocal of mobility as reported by Southern (1979). 2.5.5 Restriction endonuclease digests Restriction endonuclease digests were carried out under the conditions described by the suppliers. Reactions were terminated by heating to 650C for 10 minutes or by addition of EDTA (to 15 mM) followed by phenol/chloroform extraction and ethanol precipitation. Agarose gel electropheresis was used to monitor the extent of digestion. 2.5.6 Physical mapping A restriction enzyme map was generated by standard procedures involving analysis of single and double digests by agarose gel electrophoresis. DNA of bacteriophage X 57 digested with Hind III (Philippsen et al., 1978) and plasmid pBR322 digested with Bst N1 (Sutcliffe, 1978) were used as molecular size markers. 2.5.7 Ligations Each ligation mixture contained, per 20 Al: 0.2 ytg - 1 jig DNA (the vector to insert ratio was about 1 to 2), water to 18/U, 2 JU\ lOx ligation buffer and 5 units of T4 ligase. The mixture was incubated at 4°C for 16-20 h before transformation into competent E. coli. lOx ligation buffer consisted of 0.2 M Tris-HCl (pH 7.8), 0.1 M MgCl2, 0.2 M dithiothreitol (DTT), 10 mM spermidine, 1 mg ml-1 BSA and 10 mM ATP. 2.5.8 Eletroelution of DNA from gels Eletroelution of DNA from gels was carried out using an IBI model UEA unidirectional eletroeluter (New Haven, USA). DNA was electrophoresed on an agarose gel (0.5% for large fragments around 12 kb and 0.8% for small fragments, about 2 kb) or a polyacrylamide gel (0.5% for fragments around 2 kb). The gel was then stained with ethidium bromide. The gel slice containing the DNA fragment of interest was excised and cut into small pieces and placed into the circular gel receptacle. The electroelutor was run with 0.5 x TBE buffer at 80 volts for 60-90 min depending upon the size of the DNA fragment and the type of gel from which it was eluted. The DNA was eluted into the V-shaped channel and trapped within a small volume of high salt solution (7.5 M ammonium acetate and 0.1 g 1-1 bromophenol blue). This solution was carefully removed 2.5 volume of 95% ethanol was added to precipitate the DNA. After washing with 70% ethanol the pellet was resuspended in double distilled water. This method was found to give a good yield of DNA at 58 least up to a fragment size of 12 kb. DNA eluted from agarose gels was used as a hybridisation probe and DNA eluted from polyarylamide gels was used for sub cloning DNA fragments. 2.5.9 Southern blotting and hybridisation 2.5.9.1 Southern transfer (Southern, 1975; Smith and Summers, 1980). The DNA for hybridisation analysis was separated on agarose gels, photographed, depurinated in 0.25 M HC1 for 5 min, rinsed in water, denatured in 0.5 M NaOH, 0.5M NaCl for 30 min, rinsed and neutralised in 1M NFLpAc, 0.02 M NaOH for 30 min. The denatured DNA was transferred to two nitrocellulose filter sheets (BA85, Schleicher and Schull, West Germany) by sandwiching the gel between the two nitrocellulose sheets and two Whatman 3MM papers which were wetted with neutralization buffer. This was placed between two stacks of dry paper towel. The whole stack was covered with a glass plate and a 1 kg weight placed on top. The gel was blotted in 10 x SSC for 18-24 h at room temperature. After blotting, the nitrocellulose filter was rinsed in 2 x SSC, dried between two filter papers and baked in vacuo at 80°C for 2 h. 2.5.9.2 Preparation of hybridization probes Radioactive probes were made using radioactively-labelled [a-^^P]-ATP (Amersham, Australia) and the nick translation method of Rigby et al. (1977). A specific activity of greater than 10$ c.p.m./Ug'^ was obtained. 59 2.5.9.3 Hybridization Nitrocellulose filters of Southern blots were placed in a plastic bag and prehybridization buffer (5 x SSC, 20 mM Tris pH7.4, 0.5% SDS, 250 Jig/ml sonicated calf thymus DNA) was added. The bag was sealed and rocked gently at 420C for 24 h. The prehybridization buffer was removed and hybridisation buffer (prehybridization buffer containing 50% formamide) and about 106 ppm ml-1 of DNA probe were added. The bag was sealed and rocked at 37°C for 24 h. Filters were washed in 2 x SSC containing 0.1% SDS and then in 2 x SSC for 30 min at 37°C with rocking. Filters were air-dried and autoradiographed with intensifying screens at -70OC for 1-7 d. 2.5.10 Transformation 2.5.10.1 Transformation of E. coli For E. coli the CaCl2 transformation method of Dagert and Ehrlich (1979), without the extended preincubation in CaCl2, was used. 2.5.10.2 Transformation of Z. mobilis The CaCl2 procedure of Cohen et al. (1972) for transformation of E. coli was modified for Z. mobilis, as follows. 100 jil of cells from a glycerol stock held at - 20°C was inoculated into 5 ml 100 g 1-1 glucose RM, and incubated for 24 h. 10 |il of this culture was subcultured into 10 ml of 150 g 1-1 glucose RM, for a further 24 h, then 1 ml was further subcultured into 20 ml 150 g H glucose RM. This culture was incubated for 3-4 h until the cell density was 6-9 x 107 ml-1 (OD6Q0 = 0.28-0.31). 60 The cells were then centrifuged at 7000 x g for 7 min. at 4°C, washed in cold 100 mM NaCl, resuspended in 10 ml (50% of initial volume) of ice-cold transformation buffer containing 100 mM CaCl2, 250 mM KC1, 5 mM MgCl2, 5 mM Tris-HCl, pH 7.6 and placed on ice for 30 min. The cells were centrifuged and resuspended in cold transformation buffer and the cell density was adjusted to 2 x 109 ml-1 (about 5% of initial volume). 100 |il aliquots were transferred to 1.5 ml sterile Eppendorf micro centrifuge tubes. DNA was added in a volume of 50 |il, and 5 |il of 1M CaCl2 was added to bring the CaCl2 concentration in the mixture to 100 mM. As a control, 100 pi of cells was similarly treated but without the addition of DNA. The mixture was swirled and incubated on ice for 1 h. The mixture was heat-pulsed at 42°C for 2 min, placed on ice for 2 min, then heat-pulsed and cooled on ice again. The mixture was transferred to 1 ml of RM and incubated for 16-18 h at 30°C. Serial dilutions were then plated onto RM medium with and without the addition of an appropriate antibiotic. After incubation for 72 h at 30°C, the total number of viable cells was determined from the RM plates lacking antibiotics, and the number of transformants was scored on the antibiotic-supplemented plates. All measurements were made in triplicate. The frequencies of transformation are expressed as transformants per viable cell at the end of the expression period. Transformation efficiency is defined as the number of transformants per microgram of input plasmid DNA. Transformants obtained on plates containing one antibiotic were replica plated onto RM plates containing other antibiotics as appropriate to monitor the antibiotic resistance markers carried by the transforming plasmid. Plasmid DNA from Z. mobilis transformants was extracted and analysed by agarose gel electrophoresis to confirm the presence of the intact plasmids. 61 2.5.10.3 Storage of competent cells Cells made competent for transformation by the above procedure were stored at - 70°C after the addition of glycerol to a final concentration of 14% (Morrison, 1977). Stored cells were thawed on ice before the addition of transforming DNA. 2.5.11 Conjugation i 2.5.11.1 ' Spot mating Spot mating was performed to test for the transfer of plasmids from Z. mobilis to E. coli. E. coli strains were grown to mid-log phase, 200 jil plated onto an RM plate, and incubated at 37°C for 2 h. 5 pi of a fresh mid-log phase culture of Z. mobilis was spotted onto the RM plate with the E. coli lawn. After incubation at 30°C for 20-24 h the plate was replicated onto selection media. 2.5.11.2 Filter mating Plasmids were conjugated into E. coli and Z. mobilis recipients using a filter mating technique. Cultures of the donor, recipient and (if necessary) helper strains were grown overnight with the appropriate antibiotics. Donor and helper strains were subcultured one in ten and incubated for another 3 h. 1 ml of each culture were mixed and collected on a 25 mm, 0.45 pm nitrocellulose membrane filter. The filter was placed on RM agar for Z. mobilis recipients or Luria agar for E. coli recipients and incubated for 4 to 24 h. The cells were then washed from the filter into SPB and after serial dilution were plated onto appropriate selection media. As 62 controls, recipient, donor and helper strains individually were treated in the same way. After mating, the number of recipient organisms in the mating mixture was estimated by plating the mixture onto a medium in which only the recipient was able to grow. The transfer frequency was expressed as transconjugants/recipient. 1 2.5.12 Formation of Z. mobilis spheroplasts and cell wall regeneration Lysozyme was used for spheroplast formation of Z. mobilis. 100 |il of fresh mid-log phase culture was inoculated to 20 ml RM medium and incubated at 30°C overnight. The cells were centrifuged at 7,000 rpm for 10 minutes at 4oC and washed with Tris- Maleate buffer (pH 6.5) which contained 0.125 M CaCl2 and then resuspended in 4 ml Tris-HCl buffer (0.08 M, pH8) containing 0.5M sorbitol as the osmotic stabilizer, lml of lysozyme was added to give a final concentration of 2.5 mg ml-1. After incubation in a 37°C water bath with gentle shaking for 2 h, the mixture was serially diluted in dilution fluid which contained stabiliser and plated onto regeneration plates. As a control, the number of osmotically stable cells was determined by serially diluting the spheroplast preparation in SPB and plating onto RM plates. Under these conditions spheroplasts will lyse and only intact cells can survive. Plates were incubated at 30OC for 4 d after which the colonies were counted. 2.6 Analysis procedures 2.6.1 Quantitative analysis of cellobiose by High Pressure Liquid Chromotography (HPLC) 63 Cultures grown in cellobiose minimal medium were pretreated by deproteinization with an Amicon micropartition system MPS-1 using a YMT membrane (14 mm diameter) and desalting using a desalting unit (Baird & Tatlock, London Ltd.). This was followed by high pressure liquid chromatography (HPLC) to determine cellobiose quantitatively. HPLC analysis was carried out using a Waters Associates Model 440 HPLC fitted with differential refractometer R401 and Spectrophysics SP 4270 integrator. The column used was an Aminex PHX-87C column fitted with an Aminex micro-guard column holder containing two Bio-Rad micro-guard deashing cartridges." The column was inserted into a column heater and heated to 75°C. The mobile phase was double-distilled, deionized, degassed water heated to 750C. The flow rate was 0.6 ml min-1 and the run time was 20 min per sample. The injection volume was 10 p.1. 2.6.2 Thin Layer Chromatography (TLC) Bacterial extracts were incubated together with 5 mM cellobiose and Mcllvaine buffer in small hermatically-sealed tubes in a 45°C water bath. At intervals samples were removed and examined for the hydrolysis of cellobiose using thin layer chromatography (TLC) on Merck DC-Alufolien Kieselgel 60 (Type 5553) plates which were developed with isopropanol: ethanol: water (15:3:2 v/v). Spots were detected by spraying with diphenylamine/aniline/H3P04 in acetone and heating at 105OC for 10 min (Daly et al., 1983). 64 2.6.3 Gas Chromatography (GC) To detect the production of ethanol, samples prepared for TLC were also subjected to gas chromatography (GC) using a Packard model 427 gas chromatograph equipped with a hydrogen flame ionization detector and a 5 mm (internal diameter) x 1800 mm glass column packed with Porapak Q 100/120 mesh (Alltech Aust. Pty. Ltd.). Temperature programming was used with the sample being injected at 170OC and increasing to 190°C at 5°C min-1. The carrier gas flow rate was 30 ml min-1. The amounts of ethanol were quantified by peak heights relative to known standards. 2.6.4 Enzyme preparations for assay E. coli and X. albilineans strains to be tested for enzyme production were grown in the appropriate minimal medium containing 0.05% (w/v) glucose, 0.04% yeast extract, and 0.5% (w/v) cellobiose for P-glucosidase assay or 0.2% CMC for endoglucanase assay. The medium was also supplemented with any amino acids required and antibiotics to maintain the plasmid. A modified rich medium, containing 2% (w/v) glucose and 0.5% (w/v) cellobiose for p-glucosidase assay or 0.2% CMC for endoglucanase assay with the pH adjusted to 7.2 after autoclaving, was used for P-glucosidase and endoglucanase assays of Z. mobilis. After incubation of the cultures to an optical density of 0.6 at 600 nm the cells were centrifuged. The supernatant formed the extracellular fraction. The cell pellet was washed and resuspended in Mcllvaine buffer and lysed by addition of toluene to a final concentration of 5% (v/v). The cell suspension was gently mixed and left to stand at room temperature for 20 min prior to assay. This formed the cell-associated fraction. Each sample was assayed in duplicate. Buffer, substrate and diluted 65 enzyme preparations were preincubated separately for 5 min at the assay temperature. Specific activity was defined as U/mg of protein. 2.6.5 P-Glucosidase assay p-Glucosidase activity was estimated using the artificial substrate, pNPG. The method of Choi et al. (1978) was used to assay P-glucosidase from E\ coli and X. albilineans. 1 ml each of buffer, pNPG (1 g 1-1) and enzyme preparation were mixed in a 25 ml Erlenmeyer flask. The flask was then incubated in a water bath for 60 min at 37oC with orbital shaking. The reaction was stopped by the addition of 2 ml of 1M NaC03. NaC03 was added to controls prior to the enzyme addition. Standards were prepared from nitrophenol at concentrations ranging from 20 to 100 jiM. These solutions were treated in the same way as the assays, i.e., by incubating and then adding 2 ml of 1M Na2C03. The absorbance was read at 400 nm. One unit of enzyme activity on pNPG is defined as that which liberates one nanomole of pNPG per minute. Since the p-glucosidase encoded by the recombinant plasmid was confirmed to be active both on pNPG and cellobiose, enzyme assays in Z. mobilis were carried out using pNPG as the substrate and the method was modified after characterization of the enzyme. The reaction mixture, consisting of 0.5 ml cell extract, 0.5 ml Mcllvaine buffer (Mcllvaine, 1921) and 0.5 ml 20 mM pNPG, was incubated in a water bath at 45°C for 60 min, with orbital shaking. The reaction was terminated by adding 1 ml 1M Na2C03 and the mixture centrifuged at 10,000 g for 15 min. 66 2.6.6 Cellobiase assay For assaying cellobiose activity a similar method to the p-glucosidase assay was employed but using 1 g 1-1 cellobiose in place of the pNPG, and the reaction was terminated by boiling at 100°C for 10 min. One unit of enzyme activity on cellobiose is defined as the activity required to liberate two nanomoles of glucose per minute at 370C, with the glucose estimated by the glucose oxidase method using; a diagnostic kit (Boehringer Mannheim). 2.6.7 Endoglucanase assay Endoglucanase activity was measured by the release of reducing sugars from CMC (Reese et al., 1950). Appropriately diluted enzyme extract (1 ml) was added to 1 ml Mcllvaine buffer containing 10 g 1-1 CMC and incubated at 37°C for 30 min. DNS reagent (3 ml) was added to the mixture to terminate the reaction and react with the released reducing sugars. The DNS reagent was added to controls prior to the enzyme reaction. A standard curve was constructed by mixing 1 ml glucose solution (0.1 - 0.7 mg ml-1) together with 1 ml CMC solution and 3 ml DNS reagent. The assay mixtures were boiled for 15 min and then cooled in water. The absorbance was read at 575 nm. One unit of enzyme activity is defined as the amount of enzyme which releases one nanomole of glucose equivalent per minute. 2.6.8 Total protein determination The protein concentration of the extracts was measured using a modified Lowry method (Bergmeyer et al., 1974) and the Folin-Ciocalteu reagent. The following reagents were used: 67 Reagent A: Na2C03 (20 g I'1), NaKC4H406.4H20 (0.2 g l'1) in 0.1N NaOH Reagent B: CuS04.5H20 (5g 1-1) in water Reagent C: 50 ml Reagent A + 1 ml Reagent B (prepared freshly) Reagent D: 5 parts of Folin-Ciocalteu reagent + 9 parts water (prepared freshly) 0.5 ml of washed cell suspension containing 25 to 150 jig protein was incubated with 0.5 ml of 1M NaOH in a boiling water bath for 5 min to extract protein in a soluble form. After cooling in cold water 5 ml of reagent C was added and the mixture allowed to stand for 10 min before rapid addition of 0.5 ml of reagent D. The solution was mixed and left at room temperature for 10 min after which the O.D. was read at 750 nm against a blank using 0.5 ml of distilled water instead of cell suspension. A set of standard protein solutions containing 25 to 200 jig ml"* of bovine serum albumin was treated in the same manner, including the heating stage. Protein content was estimated from the standard curve. 68 CHAPTER 3 High Frequency Transformation of Zymomonas mobilis by Plasmid DNA 3.1 Transformation of Z. mobilis ZM6 with pNSW301 \ To establish an efficient transformation system, it is necessary to have a plasmid which is easily selected and can be stably maintained in the host. The plasmid pNSW301, is a cointegrate formed between the native 14.5 kb plasmid,pNSWl,of ZM6100 and the 37 kb IncW R plasmid, Sa. The formation and characterization of pNSW301 are described fully in Strzelecki et al. (1987). pNSW301 is 51 + 3 kb; confers resistance to Cm, Km and Sp, is capable of replication in both E. coli HB101 and Z. mobilis ZM6100 and can be maintained stably in ZM6100 for at least 300 generations in batch culture without antibiotic selection (Strzelecki et al., 1987). For these reasons, pNSW301 was used initially to establish an efficient procedure for a transformation of Z. mobilis. pNSW301 was extracted by the alpine sodium dodecyl sulphate method and residual chromosomal DNA was removed by a second alkali treatment. Since it was found that the quality of the plasmid was an important factor affecting the efficiency of transformation, the plasmid was checked by running a 2 p.1 sample on an agarose gel. A sharp clear band should be visualised at the appropriate position. A ZM6 glycerol stock (the stock period should not be longer than 3 months) was transferred to 100 gl-1 glucose RM and incubated to mid log phase. The culture was then subcultured twice in 150 gl-1 glucose RM to get a quick start of vigorous growth 69 to an optical density of 0.28 - 0.31 at 600 nm (cell density 6 - 9 x 10^ ml'l). After harvesting and washing the cells were resuspended in 10 ml chilled transformation buffer and kept on ice for 30 minutes. The cells were centrifuged again and cold transformation buffer was added to adjust the cell density to 2 x 109 ml-1. 100 pi of the cells, 50 pi of plasmid pNSW301 solution and 5 pi of 1 M CaCl2 were mixed together and kept on ice for 1 hour. The mixture was heat-shocked at 42°C for 2 minutes, placed on ice for 2 minutes and heat-shocked once more. The’mixture was transferred to 1 ml RM and incubated for 16 - 20 hours. Serial dilutions were plated onto RM to determine the number of viable cells and RM Sp plates to determine the number of transformants. Plates were incubated for 3 d at 30°C. 100 colonies were picked from the RM Sp 100 plates and patched onto RM Cm 100 and RM Km 100 plates to test for the coinheritance of the other antibiotic resistance markers carried by pNSW301. The frequencies of transformation of ZM6, by plasmid isolated from Z. mobilis and from E. coli are given in Table 3.1. E. coli HB101 was also transformed with pNSW301 (Table 3.1). It was found that the highest efficiency of transformation was obtained using plasmid DNA isolated from ZM6100 (pNSW301) (Table 3.1). However, for further work, unless otherwise stated, experiments were carried out using pNSW301 isolated from E. coli HB101 (pNSW301), because the amount of plasmid DNA recovered from E. coli cells was higher than that from Z. mobilis cells. In all experiments the number of viable cells after transformation was between 4 - 9 x 1C)8 ml-1. Patching showed that all of the SpR colonies were also CmR and KmR, indicating 100% coinheritance of the resistances encoded by pNSW301 in the transformants. For control cells, no antibiotic resistant colonies were obtained, even without dilution. 70 TABLE 3.1: Transformation efficiencies of Z. mobilis and E. coli by plasmid pNSW301. Bacterial Plasmid Transformation Number of strain source efficiency repeats transformed (transformants per jig pNSW301) ZM6 ZM6100 1.8 x 105 1 HB101 4.4 x 104 5a \ HB101 HB101 2 x 104 b 2 a Standard deviation = 1.5 x 1C)4. b To check the competence of HB101 cells, transformation of HB101 was carried out using a 4.4 kb E. coli cloning vector, pBR322 (Bolivar et al., 1977) containing a 1.6 kb fragment of Z. mobilis DNA; the transformation efficiency was 3 x 10$, with selection for Cb* 71 To confirm the presence of intact pNSW301 in the transformants, six colonies were purified, plasmid extractions carried out, residual chromosomal DNA removed and the plasmids analysed by agarose gel electrophoresis. Plasmid profiles of ZM6, ZM6100, HB101 (pNSW301), ZM6100 (pNSW301) and a typical ZM6 (pNSW301) transformant are shown in Figure 3.1. From the figure, it can be seen that when transformed into ZM6, pNSW301 replaced the 14.5 kb native plasmid, pNSWl. This is because plasmid pNSW301 was formed between Sa and the native plasmid pNSWl in vivo; hence, pNSW301 is incompatible with pNSWl. 72 1 2 3 4 5 6 7 e c d b a Figure 3.1: Plasmid DNA from ZM6 (pNSW301) and ZM6 (pNSW60) transformants. Lane 1, ZM6 standard plasmid profile Lane 2, ZM6 (pNSW301) transformant Lane 3, HB101 (pNSW301) transformant Lane 4, ZM6100 (pNSW301) Lane 5, ZM6 (pNSW60) transformant Lane 6, ZM6100 (pNSW60) Lane 7, ZM6100 standard plasmid profile a, pNSWl; b, pNSW2; c, pNSW3; d, pNSW60; e, pNSW301. 73 3.2 Effect of various factors on transformation frequency The effect of various components of the transformation system on the frequency of transformation of ZM6 was investigated. 3.2.1. S tate of the recipient cells i The state of the recipient cells was found to be a critical factor in transformation. Two sets of cells at different growth stages were made competent using the standard transformation protocol. One was in early exponential phase, with an optical density of 0.292 at 600 nm, and the other was an overnight culture (stationary phase) with an optical density of 1.05 at 600 nm. Both were transformed by the same batch of pNSW301. Table 3.2 indicates that when the cells grew to the stationary phase, the transformation frequency was reduced by about 700-fold. It was found that the seed culture used to prepare competent cells needed to be fresh. Glycerol stocks were made from fresh plates and the glycerol stocks for transformation need to be made every three months. The stock culture was subcultured twice to ensure vigorous growth, enabling the final subculture to grow to an optical density of 0.28 - 0.31 at 600 nm in less than 4 hours. When an old seed culture was used, it took longer to grow to early exponential phase and the transformation frequency was reduced despite the final optical density being the same. 74 Table 3.2: The effect of cell age on transformations frequency Cell &ge Transformation frequency (Transformants per viable cell) 3.5 h,OD600 0.292 1.9 x 10-5 overnight culture OD600 1-05 2.7 x 10-8 75 Because of the importance of the state of recipient cells, the growth of the cells was monitored both by optical density measurements at 600 nm and by counting the cell number. Also, the morphology of the cells was carefully monitored under oil immersion. 76 3.2.2. Effect of DNA exposure time After adding plasmid pNSW301 to competent cells the mixture was held on ice for different times to allow DNA to adsorb to the cell envelope before heat shocking. The results are shown in Table 3.3. Increasing the incubation time on ice from 5 minutes to 1 hour increased the transformation frequency by 42%. However, increasing the incubation time on ice from 1 hour to 1.5 hours resulted in no further increase in transformation frequency. Therefore, incubation on ice for 1 hour was chosen for the standard procedure. 77 Table 3.3: Effect of incubation time on ice on the transformation frequency Incubation time Transformation frequency on ice (transformants per viable cell) 5 minutes 1.9 x 10-5 1 hour 2.7 x 10-5 1.5 hours 2.7 x 10-5 78 3.2.3 The effect of one or two heat shocks on transformation frequency After incubation on ice, one mixture of competent cells and DNA was heat-shocked at 420C for 2 minutes then cooled on ice. A duplicate mixture was heat-pulsed at 42°C for 2 minutes, placed on ice for 2 minutes, then heat-pulsed and cooled on ice again. Table 3.4 shows that a double heat shock increased the transformation frequency about twofold. \ 79 Table 3.4: The effect of heat shock on transformation frequency Number of heat shocks Transformation frequency (transformants per viable cell) 1 1.1 X 10-5 2 2.0 x 10-5 80 3.2.4 Effect of storage time of competent cells on cell viability and transformation Competent cells of Z. mobilis could be stored conveniently at -70OC. Glycerol was added, to a final concentration of 14%, to competent cells which were then stored at - 70OC. After 1 and 5 wk of storage, the cells were transformed, using plasmid DNA isolated from ZM6100 (pNSW301), and the numbers of transformants per pig plasmid DNA were found to be 4 x 104 and 3.5 x 1C)4, respectively, a reduction of about fourfold. Table 3.5 compares the cell viability and the transformation frequency of stored cells. Cell viability was tested by viable counting on RM plates and transformation frequency was expressed as transformants per viable cell. Cell viability was reduced by 1/4 during the first week of storage but changed little after that. Similarly the transformation frequency fell by 2/3 during the first week of storage, but decreased only slightly in the next month. These results show that the transformation frequency fell at a faster rate than the viability. Since the transformation frequency after storage was adequate for most purposes, this provided a quick and convenient method for transforming Z. mobilis routinely. The rapid initial decline in cell vaibility and transformation frequency suggested that these were more likely due to freezing and thawing the cells than to the period of storage. 81 Table 3.5: Effect of storage on the viability and transformation of competent cells Period of Storage Viability Transformation Transformation at -70°C Frequency Efficiency (weeks) (100%) (Transformants (Transformants per viable per jig DNA) cell) 0 100 1.9 x 10-5 1.8 x .105 1 74.1 6.0 x 10-6 4 x 104 5 72.4 5.3 x 10-6 3.5 x 104 82 3.3 Kinetics of plasmid transformation in Zymomonas mobilis The transformation process can be divided into three stages: firstly, the DNA in the transformation mixture binds to the outside of the cell; secondly, the DNA is transported across the cell envelope; thirdly, the plasmid markers are inherited either by plasmid replication, or if this is not possible, by recombination with a resident replicon. ’• During the early part of this work, a dramatic effect of plasmid DNA concentration on transformation was noticed. In an initial experiment, two different concentrations (310 pg ml"!, 31 pg ml-1) of plasmid DNA were used to transform the same lot of competent cells. After incubation of the transformation mixture in RM and culture of them, on RM and RM SplOO it was found the higher DNA concentration gave a transformation frequency of 2.2 x 10_5 but the lower one only gave a transformation frequency of 1.6 x 10-2. The concentration of plasmid DNA, isolated from HB101 (pNSW301), used in the ZM6 transformation experiments described before this section was either 250 ng or 300 ng per 50 pi. The results of altering this DNA concentration, either by dilution, or concentration, are shown in Table 3.6. Reduction in the amount of plasmid DNA decreased the number of transformants obtained, but not in a directly proportional manner. For example, when the amount of plasmid DNA was reduced by 5- and 10- fold, the number of transformants was reduced 40- and 100-fold, respectively, and the transformation efficiency was correspondingly reduced 7.5- and 10-fold respectively. However, increasing the DNA concentration up to 10-fold did not increase the number of transformants significantly. Because the number of transformants remained about the same, increasing the DNA concentration decreased the 83 transformation efficiency. The number of viable cells was between 5.5 and 9.5 x 10^ ml_l. When the plasmid concentration was maintained at 300 ng per 50 fil, but the volume of plasmid DNA added to competent cells was increased 2-fold (thus giving 600 ng of plasmid in the mixture), the transformation efficiency was reduced about 5-fold. In E. coli the relationship between transformation frequency and plasmid DNA concentration is linear, passing through zero and indicating first order kinetics (Cohen et al., 1972). Unlike this situation in E. coli, the frequency of transformation in Z. mobilis did not show first-order kinetics with respect to the concentration of DNA. With increasing amounts of DNA, the transformation frequency increased more than expected for first-order kinetics. Therefore, a dose response curve relating the concentration of plasmid DNA to the transformation frequency was constructed using various amounts of pNSW301 with the standard transformation protocol. 84 Table 3.6: Effect of altering the DNA concentration on the recovery of ZM6 (pNSW301) transformants DNA Number of Number of Transformation (ng) transformants viable efficiency in 50 |il cells (transformants per |ig plasmid DNA) Expt. 1 25 4 x 101 7.5 x 108 1.6x'l03 50 1.2 x 102 5.5 x 108 2.4 x 103 100 9 x 102 9.5 x 108 9 x 103 . 150 1.3 x 103 6 x 10& 8.7 x 103 250 4.4 x 103 7.5 x 108 1.8 x 104 Expt.2 250 1.1 x 104 7.5 x 108 4.4 x 104 1000 7.4 x 103 8 x 108 7.4 x 103 2500 1.3 x 104 7 x 108 5 x 103 85 Figure 3.2 shows (first experiment) that at low DNA concentrations, plasmid transformation activity increased with the square of the DNA concentration. The correlation coefficient for this relationship in the range 0.5 - 5 p.g mT^ DNA (i.e. before DNA saturation) for the line of best fit was 0.951 and gave a relationship of exponential function y = *xA power index = 2.0479. The capacity to transform was saturated at a DNA concentration of 10 mg ml-1. At a DNA concentration of 5 fig ml-1, one in 1.7 x 105 of the viable cells was transformed. In the second experiment, the curve gave a relationship of exponential function y = *xA power index = 2.0676. Figure 3.2 shows that transformation by pNSW301 followed second-order kinetics. Since the transforming plasmid DNA consisted of plasmid monomers, there being no detectable multimeric molecules, this suggests that two plasmid molecules reacted cooperatively to produce a single transformant (Saunders et al., 1984). A similar phenomenon was observed in Streptococcus pneumoniae (Saunders and Guild, 1981) where transformation by the covalently closed monomer form of the plasmid, pMV158, followed second-order kinetics. These workers suggested that transformation involves binding and non-specific cleavage of duplex DNA at the cell surface, followed by entry of one strand while the complement is degraded and released into the medium. Consequently, covalently closed circular duplexes are converted to linear single strands by this process. Two strands that have entered separately can associate to form a duplex, probably with gaps which can be repaired, thereby regenerating an intact replicon. Different preparations of competent cells differed in their transformation efficiencies, shifting the does response curve laterally (Figure 3.2). While this caused the absolute transformation frequencies to vary, the gradient of the does response curve remained the same and indicated second-order kinetics. 86 > LU r) o LU tr CQ UL < 2 > CO o H H Z < < cc DTP O LL LL z (/) < iZ cc < cc h- O ' 10-1 1 101 102 103 DNA CONCENTRATION (^g ml'1) Figure 3.2: The effect of pNSW301 DNA concentration on transformation frequency of Z. mobilis. First experiment data (■), second experiment data (□). 87 3.4 Transformation of Z. mobilis ZM6 by other plasmids Having established a method that reproducibly yielded high numbers of Z. mobilis transformants by using the plasmid pNSW301, the method was applied to ZM6 using several other plasmid types. The plasmid pNSW60 was isolated from a strain of ZM6100(RP1>) which had received the IncP-1 plasmid RP1, which confers resistance to Cb, Km and Tc (Riess et al., 1980), after conjugation with E. coli HBlOl(RPl) (see Skotnicki et al., 1983 for conjugation of IncP-1 group plasmids to Z. mobilis’, Goodman, 1985). After 50 generations of growth of ZM6100(RP1) in liquid RM containing 500 jig ml'l Cb, serial dilutions were plated onto RM plates. Colonies from these plates were replica plated to RM plates containing either Cb, Km or Tc. Ten colonies which were CbR, but KmS and TcS were purified, and plasmid DNA was isolated from these strains. Four strains contained a plasmid profile the same as ZM6100, but 6 strains contained a plasmid profile in which the 15.5 kb native ZM6100 plasmid, pNSW2 (Goodman, 1985), was missing and a new plasmid, of about 20.5 kb, was present (data not shown). One of these strains was picked for further work, and the new 20.5 kb plasmid designated pNSW60. It was thought that the 5 kb transposon, Tn7 (encoding CbR (Riess et al., 1980), may have transposed from RP1 and to 15.5 kb native ZM6100 plasmid, to form the new plasmid pNSW60. To test this, transformation and conjugation experiments were carried out. Attempts to transform E. coli HB101 to Cb resistance with pNSW60 were not successful. Conjugation experiments, selecting for CbR, between the donor ZM6100 (pNSW60) and either ZM6 or HB101 recipients were also unsuccessful. 88 Transformation of ZM6 by pNSW60, isolated from ZM6100 (pNSW60), with selection for CbR was successful (Table 3.7). For control cells, no antibiotic resistant colonies were obtained. The plasmid profiles of a typical ZM6 (pNSW60) transformant and the original ZM6100 (pNSW60) are shown in Figure 3.1 (lanes 5 and 6 respectively). It can be seen from Figure 3.1 that pNSW60 replaced the native 15.5 kb plasmid pNSW2 in the transformant ZM6 (pNSW60). t The ability of other non-conjugative plasmids to transform ZM6 was investigated. The plasmids pSal52, pSA727 and PSa747 are small (14-15 kb), Tra~, Mob+ derivatives of Sa (Tait et al., 1983). Although these plasmids can be mobilized, using the helper plasmid pSa322 (containing the Sa tra region) in a three way conjugation, among E. coli, Rhizobium and Pseudomonas species (Tait et al., 1983), no conjugation of these plasmids from E. coli donors to either ZM6 or Zm6100 had been obtained (A. Goodman, unpublished results). The plasmids pSal52, pSa727 and pSa747, isolated separately from plasmid-containing E. coli strains, were used to transform ZM6, with selection for SpR. Transformation of Z. mobilis ZM6 with these plasmids was successful. The results are shown in Table 3.7. Plasmid profiles of transformed ZM6 strains are shown in Figure 3.3. ZM6 was also transformed with the plasmid Sa (to compare with the results obtained for the smaller derivative plasmids), and with RPl::Tn5(97, a broad host range plasmid belonging to a different incompatibility group (IncP-1), with selection for TcR. The results are shown in Table 3.7. The transformation frequency for the small pSa vectors was about the same as that of pNSW301, while that of Sa, RPl::Tn507 and pNSW60 was lower. For control cells, no antibiotic resistant colonies were obtained. 89 Table 3.7: Transformation frequencies of Z. mobilis ZM6 using various plasmids Plasmid Plasmid DNA No.of Transformation Initial source type conctration trans efficiency antibiotic (ng per formants (transformants selection 50 pi) per pg plasmid DNA) ZM6100 pNSW60 120 1.1 X 102 9.3 x 102 Cb 350 1.7 x 103 4.9 x 103 Cb E. coli pSal52 150 1.6 x 103 1.1 x 104 Sp pSa727 600 5.4 x 103 9 x 103 Sp pSa747 480 4.4 x 103 9.2 x 103 Sp Sa 5 000 3.2 x 103 6.2 x 102 Sp RPl::Tn 501 1 100 4.5 x 102 4x 102 Tc Control ZM6 cells, treated in the same way, but without addition of DNA, produced no antibiotic resistant colonies. 90 1 2 3 4 5 6 8 Figure 3.3: Plasmid DNA from ZM6(pSal52), ZM6(pSa727) and ZM6(pSa747) transformants. Lane 1 = pSa747 Lane 2 = ZM6(pSa747) transformant Lane 3 = ZM6 standard plasmid profile Lane 4 = pSal52 Lane 5 = ZM6(pSal52) transformant Lane 6 = pSa727 Lane 7 = ZM6(pSa727) transformant Lane 8 = ZM6 standard plasmid profile. a, pNSWl; b, pNSW2; c, pNSW3; ch, chromosomal DNA. The gel was run at 2 V cm-1 for 24 hours. Note that under these conditions the bands of the small plasmids are sharp, but the 34 kb native ZM6 plasmid band is diffuse and slightly bowed. 91 3.5 Behaviour of a set of derivative plasmids from Sa in Z. mobilis A set of broad host range cloning vectors pSal52, pSa727 and pSa747, has been constructed from the Inc W plasmid pSa by Tait et al. (1983). These vectors have been constructed from the transfer defective deletion derivative pSal51, which encodes resistance to kanamycin and spectinomycin^streptomycin. pSa727 also contains the chloramphenicol resistance gene of TnP and pSal52 ’contains the chloramphenicol resistance gene of pSa. Together these vectors contain cloning sites for Sst II, Hind III, EcoR I, Kpn I, Pvu II, Bam HI, Sma I and Bgl II, and recombinants at certain of these sites can be detected by insertional inactivation of a drug resistance phenotype. The success of transformation of these plasmids into Z. mobilis (see section 3.4) makes them promising cloning vectors in this organism. To test if these plasmids would be suitable vectors with which to introduce foreign DNA into Z. mobilis their behaviour in Z. mobilis was investigated. 3.5.1 Stability tests of pSal52, pSa727 and pSa747 in Z. mobilis pSal52, pSa727 and pSa747 were introduced into ZM6 by transformation. Purified ZM6 transformants showed all the antibiotic resistances encoded by the plasmids. Agarose gel electrophoresis of plasmid DNA demonstrated that in addition to the native plasmids of ZM6, pNSWl, pNSW2 and pNSW3, there was a clear band corresponding to the transformed plasmid (Figure 3.3). Transformants ZM6(pSal52), ZM6(pSa727) and ZM6 (pSa747) were grown in batch culture either without antibiotic, or with 100 jig ml-! cm for ZM6 (pSal52) and ZM6 (pSa727), or 100 |ig ml“l Sp for ZM6 (pSa747). Every 24 hours the cultures were subcultured 1:100 or 1:1000. Before subculture the cultures were serially diluted and 92 spread onto Rm plates. After single colonies had developed ZM6 (pSal52) and ZM6 (pSa727) colonies were patched onto Sp 100, Km 100 and Cm 100 and ZM6 (pSa747) colonies were patched onto Sp 100 and Km 100 plates. The maintenance of pSal52, pSa727 and pSa747 under nonselective and selective conditions is shown in Tables 3.8 and 3.9. Under nonselective conditions the plasmid was lost gradually. Although unstable, these plasmids were not lost as quickly as the parent plasmid, Sa, in Z. mobilis. Strzelecki et al. (1987) reported that under nonselective conditions Sa was not stably maintained in Z. mobilis and the whole of the plasmid was lost after only 10 generations. The size of Sa is 37 kb (Ward and Grinsted, 1982) and pSal52, pSa727 and pSa747 are 15 kb, 14.7 kb and 15 kb respectively (Tait et al., 1983). It is likely that with the reduction in plasmid size the deletion derivatives, pSa 152, pSa 727 and pSa 747 became more stable than the parent in Z. mobilis. All three plasmids were 100% stable in ZM6 when the selective pressure was Cm for pSal52 and pSa727 and Sp for pSa747. All of the isolates tested maintained all of the drug markers encoded by the plasmid. 93 Table 3.8: Maintenance of pSal52, pSa727 and pSa747 in ZM6 under non-selective conditions Plasmid Number of % maintenance of generations plasmid markers pSa 152 0 100 3 88 6 78 23 35 \ 33 18 66 2 * 107 0.3 pSa 727 0 100 3 71 6 51 23 30 33 17 66 1 107 0.1 pSa 747 0 100 3 72 6 59 23 32 33 17 66 1 107 0.1 94 Table 3.9: Maintenance of pSal52, pSa727 and pSa747 in ZM6 under selective conditions Plasmid Number of % maintenance generations pSa 152 100 100 200 100 l pSa 727 100 100 200 100 pSa 747 100 100 200 100 95 3.5.2 Re-arrangement of pSal52 and pSa747 in ZM6 after long periods of growth under selective conditions Under selective conditions the smaller Sa derivative plasmids showed full maintenance of all drug markers after 200 generations (3.4.1). After 200 generations under selective conditions plasmid extractions were performed on several single colonies of each strain. Figure 3.4 shows that the plasmid profile of ZM6(pSa727) remained the same as the control but in ZM6 (pSal52) and ZM6 (pSa747) the plasmid profile had changed. In ZM6 (pSal52) the original pSal52 and pNSWl bands had disappeared. Instead a new plasmid with a size of 29.5 kb was observed. Strzelecki et al. (1987) reported that when the parent plasmid, Sa, was transferred into ZM6100, it cointegrated with pNSWl, after growth under selective condition, to form a new plasmid, pNSW301. It is suggested that pSal52 cointegrated with pNSW 1 in vivo under selective conditions to form the new plasmid which was designated to pNSW 901. A new plasmid band was seen in the plasmid profile of ZM6 (pSa747) after 200 generations of growth under selective conditions. The size of this plasmid was 14 kb. It is suggested that a deletion occurred in pSa747 in vivo to form the new plasmid which was designated to pNSW 902. 96 1234567 89 10 ch c b £ -41 a Figure 3.4: Plasmid DNA from ZM6 harbouring a set of derivative plasmid from Sa after 200 generations of growth under selective conditions. Lane 1, ZM6 (pSa747) Lanes 2,3, ZM6 harbouring pSa747 after 200 generations of growth under selective conditions Lane 4, ZM6 (pSa727); Lanes 5,6,7, ZM6 harbouring pSa727 after 200 generations of growth under selective conditions Lanes 8,9,10, ZM6 harbouring pSal52 after 200 generations of growth under selective conditions a, pNSWl; b, pNSW2; c, pNSW3; ch, chromosomal DNA. The gel was run at 2V cm-1 for 24 hours. 3.5.3 Characterisation of pNSW901 and pNSW902 When ZM6(pNSW901) was patched onto SplOO, Cm 100 and Km 100 plates and ZM6(pNSW902) was patched onto SplOO and Km 100 plates, the results showed complete maintenance of the drug markers of the parent plasmids, pSal52 and pSa747. pNSW901 and pNSW902 were then used to transform competent HB101. The transformation efficiencies were 4.5 x 104 and 5.1 x 104 transformants per pg plasmid DNA respectively. Plasmid DNA extractions were performed on several transformants and confirmed the presence of intact plasmids. It was found that pNSW901 and pNSW902 from E. coli could transform ZM6 and the transformation efficiencies were 1.2 x 104 and 1.0 x 104 transformants per pg plasmid DNA respectively. The stability of pNSW901 in ZM6 was tested by growing ZM6(pNSW 901), under non-selective conditions for 200 generations and then testing for drug resistance. Table 3.10 indicates that all of the antibiotic resistances encoded by pSal52 were maintained stably in ZM6 (pNSW901) for at least 200 generations and plasmid extraction confirmed the presence of intact pNSW901. The high stability of pNSW901 under non-selective conditions and the smaller size of pNSW902 together with other properties, e.g., ability to transform and replicate in both E. coli and Z. mobilis and expression of all drug markers of the parent plasmids make the two new plasmids promising candidates for the construction of Z. mobilis cloning vehicles. 98 Table 3.10: Maintenance of antibiotic resistance in ZM6 (pNSW901) under non- selective conditions Number of Maintenance (%) generations Km resistance Sp resistance Cm resistance 50 100 100 100 100 100 100 100 200 100 100 100 99 3.6 Discussion Using the procedure described in this chapter, transformation of Z. mobilis ZM6 at consistently high frequencies was achieved using several plasmid species. The highest transformation frequency achieved, using the plasmid pNSW301, was about 40-fold higher than the best transformation frequency previously reported for Z. mobilis ZM6 (Browne et al., 1984), and about the same as the maximum achieved by transformation of Z. mobilis partial spheroplasts (Yanase et al., 1986). The present study is the first report of high frequency transformation of Z. mobilis by plasmid species, isolated from E. coli, that do not contain a Z. mobilis plasmid origin of replication. Conditions which affected the transformation frequency to a small extent included the source of the plasmid DNA, the storage of competent cells in glycerol at -70OC, the use of either one or two heat pulses and the length of time the plasmid plus competent cell mixture was left on ice before the heat pulse step. Conditions which significantly affected the transformation frequency are discussed below. The total volume of the plasmid plus competent cell mixture was important, in that an increase in volume, either by increasing the amount of added plasmid, or the amount of competent cells, markedly reduced the transformation frequency. This is in agreement with results found previously by Browne et al. (1984) and was thought to be caused by a dilution effect such that there was less contact between competent cells and plasmid molecules. ZM6 cultures grown to an OD600 of 0.28 - 0.31 (6-9 x 107 cells per ml) were transformed at a significantly higher frequency than those grown to higher OD. This is similar to the situation in E. coli where the best transformation frequencies are obtained using cells grown to early exponential phase (Dagert and Ehrlich, 1979; Maniatis et al., 1982). 100 The concentration of plasmid DNA was also a significant factor. From the results it appeared that at a concentration of about 250 ng of pNSW301 saturation occurred, such that increasing the plasmid concentration did not increase the number of transformants recovered. This is similar to results found for E. coli where Hanahan (1983) showed saturation of E. coli cells occurred at about 200 ng of pBR plasmids. At plasmid concentrations below saturation it has been found for E. \coli that the number of transformants is directly proportional to plasmid concentration (Cohen et al., 1972; Dagert and Ehrlich, 1979; Hanahan, 1983). In contrast, the results of the present study indicate second-order kinetics in the relationship between the concentration of DNA and the number of Z. mobilis transformants obtained. This would explain the low transformation frequencies found for the low DNA concentrations used by Browne et al. (1984). The ability of the plasmid pNSW60 to transform ZM6 to Cb resistance that transposition of the Cb resistance transposon, Tn7, from RP1 to the native 15.5 kb Z. mobilis plasmid had occurred in ZM6100(RP1). This is the first report of a native Z. mobilis plasmid, which is not a cointegrate with an R plasmid, carrying a selectable marker. Because of this selectable marker, the plasmid’s relatively small size and ability to transform Z. mobilis, pNSW60 has the potential for use as a cloning vector for Z. mobilis. It is interesting that the transformation frequency of ZM6 was lower with pNSW60 than with pNSW301, as pNSW60 is about 2.5 times smaller than pNSW301. This is in contrast to E. coli for which it has been shown that transformation frequency is proportional to plasmid size such that the frequency decreases as plasmid size increases (Hanahan, 1983). 101 The transformation of ZM6 with the plasmids pSal52, pSa727 and pSa747 is significant in that these plasmids, developed as small broad host range cloning vectors (Tait et al., 1983), can now be used as such for Z. mobilis. Their inability to be conjugated to Z. mobilis, using the appropriate helper plasmid, had previously excluded their use as cloning vectors for Z. mobilis. To date, transfer of DNA to Z. mobilis has been limited to vectors capable of conjugal transfer to Z. mobilis. Using the high frequency transforamtion procedure established in this study if will now be possible to use a much wider range of plasmids as cloning vectors for Z. mobilis. 102 CHAPTER 4 Spheroplast Fusion in Zvmomonas mobilis 4.1 Spheroplast formation and regeneration The term spheroplast refers to an osmotically sensitive Gram-negative organism, retaining part of the membranous lipopolysacchride layer. A new method for preparing Z. mobilis spheroplasts was developed as follows. Z. mobilis strain ZM6 or ZM4 was grown at 30OC in 20 ml RM to early exponential phase (5 ~ 9 x 107 cell ml-1). The cells were centrifuged and washed with Tris-maleate buffer (pH 6.5) containing 0.125 M CaCl2, then centrifuged and resuspended in 4 ml 0.067 M Tris- HC1 buffer (pH8) containing 0.5 M sorbitol. 1 ml lysozyme solution (2.5 mg ml-1) was added to give a final concentration of 5000 }ig ml"l. The mixture was incubated in a 37°C water bath with shaking for 2 hours. The cells were examined under oil immersion by phase contract microscopy. More than 99% of the cells changed their shape from a long rod to an ovoid. The frequency of spheroplast formation was measured by counting the number of osmotically-fragile cells determined as the difference between the number of osmotically-resistant colonies before and after spheroplast formation. Z. mobilis strains ZM6 and ZM4 both produced more than 99% spheroplasts since the CFU ml-1 after dilution in dilution fluid was about 1,000 times the CFU ml-1 after dilution in SPB (Table 4.1). The spheroplasts formed were washed in dilution buffer to remove lysozyme and regeneration was investigated. Osmotically resistant cells were determined by resuspension and dilution of the spheroplasts in dilution fluid or SPB. 100 |il of this spheroplast suspension was added to 3 ml regeneration soft agar or RM soft agar and 103 Table 4.1: Production and regeneration of Z. mobilis spheroplasts CFU ml-1 for spheroplasts diluted in Cells Dilution SPB Counted fluid % Re under gene micro ration scope- l Strain Expt. (ml"l) ZM6 1 2.4 x 109 1.1 X 108 5.3 x 104 4.6 2 2.8 x 109 1.3 x 108 2.3 x 105 4.7 3 5.4 x 109 2.0 x 108 1.4 x 105 3.7 ZM4 1 1.4 x 109 4.6 x 10? 8.0 x 104 3.2 2 1.8x109 7.0 x 107 1.0 x 104 3.6 104 poured onto a regeneration plate or RM plate. The plates were incubated at 30°C for four days, and spheroplast regeneration frequencies were calculated by the formula: 100 x (b-c)/a, where, a is the cell count before spheroplasts were prepared, b is the colony count on the regeneration plates after dilution in dilution Suffer, and c is the colony count of the residual shock-resistant bacteria on the RM plate after dilution in SPB. The average regeneration frequency of ZM6 was 4.3% and the average regeneration frequency of ZM4 was 3.4% (Table 4.1). Also after spheroplast formation the spheroplast suspension was spread onto regeneration plates instead of using soft agar overlays. Regeneration was observed although the regeneration frequency was reduced to about 50%. 105 4.2 Optimisation of lysozyme treatment for spheroplast formation Lysozyme was prepared as a stock solution (10 mg ml-1) in distilled water, filter- sterilized and stored at -20OC. To optimise the lysozyme treatment, the effect of treatment time and lysozyme concentration on spheroplast formation and regeneration were investigated. 1 4.2.1 Effect of incubation time with lysozyme on spheroplast formation and regeneration in ZM6 Early exponential phase cells of ZM6 were harvested, washed and resuspended in Tris-HCl buffer containing 0.5 M sorbitol. Lysozyme solution was added to a final concentration of 500 pig ml“l. Samples were taken at intervals and spheroplast formation and regeneration frequencies were determined as described in Section 4.1. The results are shown in Figure 4.1. With increasing lysozyme treatment the spheroplast frequency increased dramatically, reaching 95% after 2 hours. In the same time, the regeneration frequency decreased from 11.4% to 5% after 2 hours. Since the absolute numbers of regenerants was the most important parameter a treatment time of 2 hours was chosen for subsequent experiments. 106 - 100 r 500 -8 of Nos. Relative Hours Figure 4.1: Effect of incubation time in lysozyme on spheroplast formation and regeneration in ZM6, the final concentration of lysozyme was 500 jig ml" ^. m, spheroplast formation frequency; ♦, regeneration frequency;a, relative nos. of regenerants/ml. m, values for 5,000 jig ml'* lysozyme. 107 4.2.2 Effect of lysozyme concentration on spheroplast formation and regeneration in ZM6 Early exponential phase cells (20 ml) were harvested, washed and resuspended in 4 ml Tris-HCl buffer containing 0.5 M sorbitol. The mixture was divided into two portions. 0.5 ml 2.5 mg ml-1 lysozyme solution was added to one half to make the final concentration of 500 fig ml'l and 0.5 ml 2 5 mg ml"! lysozyme was added to the other half to get a final concentration of 5,000 fig ml'*. After 2 hours incubation the spheroplast formation and regeneration frequencies were compared (Figure 4.1). Using lysozyme to a concentration as high as 5,000 fig ml"* still gave a good regeneration frequency. The spheroplast formation frequency increased to 99.9% and the absolute number of regenerants was the same. Because the area of exposed cytoplasmic membrane of individual spheroplasts might be greater at higher lysozyme concentrations, (To confirm this hypothesis, electron microscopy could be used to compare the exposed membrane of spheroplasts), a final concentration of 5,000 fig ml"* lysozyme was used to prepare spheroplasts for fusion. 4.3 Effect of different hypertonic stabilizers on spheroplast regeneration in ZM6 Different hypertonic stabilizers were used to prepare dilution fluid and regeneration medium. When ZM6 was grown on medium containing sucrose sticky colonies were formed due to accumulation of levan. Thus sucrose was not chosen as the stabilizer. Spheroplasts were washed and overlayed on regeneration plates with sorbitol or mannitol as the stabilizer. After four days incubation the regeneration frequencies on both types of plates were the same (Table 4.2). Consequently, sobitol and mannitol were considered to be good stabilizers for Z. mobilis spheroplast formation and regeneration. Table 4.2: Effect of different hypertonic stabilizers on spheroplast regeneration in ZM6 Hypertonic stabilizer Regeneration frequency (%) Sorbitol 4.3 Mannitol 4.7 The concentration of sorbitol and mannitol was 0.5M. I 109 4.4 Spheroplast fusion in ZM4 Two auxotrophic strains of ZM4 were used to test for spheroplast fusion in Z. mobilis. ZM4751 is ade met and ZM4761 is cys arg RpR 2.5 ml spheroplast suspension prepared as described previously (Section 4.1), of each strain was mixed, centrifuged at 3,000 rpm for 15 minutes at 40C, washed in TMSC buffer (0.1 M Tris- Maleate buffer containing 0.5 M sorbitol and 50 mM CaCl2) and then resuspended in 0.2 ml TMSC buffer. 2 ml TMSC buffer containing 40% PEG 6,000 was added to the spheroplast suspension and mixed. The mixture was incubated at 30°C for 10 minutes after which 10 ml dilution fluid was added, mixed well and the spheroplasts filtered through a nitrocellulose membrane with a pore size of 0.45 jam. The membrane was placed on regeneration agar and incubated at 30OC for four days. As controls, the parental strains were subjected separately to the same treatment. The regenerated cells were washed from the membrane with SPB, spun down and washed with SPB twice. The cells were then resuspended in SPB and plated onto BM plates to select recombinants. Viable cells were determined on RM plates. After five days incubation a few colonies appeared on each fusion plate, while none of the control plates produced colonies. The fusion frequency was 1.5 x 10'8 per viable cell. 4.5 Discussion Spheroplast formation of Z. mobilis by sequential treatment with lysozyme, EDTA and glycine was reported by Lee and Seong (1984). A similar method based on glycine or penicillin G treatment was reported by Yanase et al.(1984)? who used an 8- 12 hour exposure time to the spheroplasting agent. A different method for preparing Z. mobilis spheroplasts using lysozyme is described in this chapter. The spheroplast formation frequency was greater than 99.9% and the regeneration frequency was 110 about 4.3%. The exposure time to the spheroplasting agent (lysozyme) was 2 hours and the regeneration frequency was higher than achieved by other methods. The increase in regeneration frequency may be the result of fewer steps in the procedure, less contact with harmful reagents and shorter treatment times. During the course of this work a method for high frequency transformation of Z. mobilis by plasmid DNA was developed (Chapter 3). Consequently, thi3 preliminary investigation of spheroplast fusion in Z. mobilis was not developed further. For future work in this area the untreated and lysozyme-treated cells should be examined by electron microscopy to determine whether the osmotically-sensitive forms are true protoplasts (complete removal of the cell wall) or spheroplasts (retention of part or all of the membranous lipopolysaccharide layer). The mechanism of fusion using the membrane method reported here should be investigated further, a better understanding of the mechanism perhaps leading to improved fusion frequencies. 111 CHAPTER 5 Cloning and Expression of a p-glucosidase Gene from Xanthomonas albilineans in Escherichia coli The cellulolytic bacterium, Xanthomonas albilineans XA1-1 served as the source of the p-glucosidase gene. Gene banks of XA1-1 were constructed by Lucas (1987). Chromosomal DNA, incompletely digested with Hind III was ligated into the Hind III restriction site of the plasmid, pKT230, followed by transformation of E. coli ED8654. Direct selection of clones expressing p-glucosidase from these gene banks was attempted by Lucas (1987). Xanthomonas albilineans XA1-1 and Pseudomonas PS 2- 2 gene banks in E. coli were in^oculated into PM liquid minimal medium containing 1% cellobiose and the appropriate amino acids, and incubated for several weeks at 3(PC with shaking. Samples were then plated out from each flask onto the corresponding solid medium. After incubation for 1 week the plates were examined for growth. One of the ED8654 clones of PS2-2 DNA showed some growth on minimal agar plates containing cellobiose as the sole carbon source (Lucas, 1987), while none of the XA1-1 clones showed any growth on minimal agar plates containing cellobiose as the sole carbon source (Lucas, personal communication). Indirect selection with pNPG was also attempted by Lucas. Gene banks of XA1-1 DNA in ED8654 were screened for the presence of clones encoding P-glucosidase using the pNPG plate screening procedure. Single colonies from the gene bank were grown on PM agar plates containing 4 mM sodium succinate, thiamine and 112 methionine and overlaid with 3 mis soft agar made with Mcllvaine Buffer and containing 0.5 mg ml-1 pNPG. Several colonies gave positive results, //mdlll digestion of plasmids extracted from these clones indicated that they contained insert DNA of different sizes ranging from 2 kb to 4 kb. However, none of the clones showed any significant p-glucosidase or cellobiase activity. No further work was done on any of these clones from the XA1-1 gene bank (Lucas, 1987). Lucas’ results showed that E. coli ED8654 clones encoding p-glucosidase from XA1-1 were not easy to isolate on minimal medium with cellobiose as sole carbon source and thus direct selection is difficult. On the other hand, indirect selection with pNPG was not specific enough to select the p-glucosidase (cellobiase) gene. Therefore, two alternative strategies were designed to screen for ED8654 clones encoding p- glucosidase (cellobiase) from XA1-1. 5.1 Selection and screening procedures e 5.1.1 Selection via Pseudomonas t— * — De Ley et al. (1966) has pointed out that the nearest taxonomic neighbours of the genus Xanthomonas are in the genus Pseudomonas. Consequently, Pseudomonas was chosen as an intermediate host with the expectation that Xanthomonas genes would be expressed more efficiently in Pseudomonas. For screening these gene banks for clones encoding p-glucosidase, host strains were required which were streptomycin sensitive and p-glucosidase negative. Pseudomonas ATCC17527 proved to be a suitable strain. The vector, pKT230, could be mobilised into ATCC17527 using the mobilising plasmid pRK2013. ATCC17527 was sensitive to kanamycin and streptomycin, contained no plasmids, maintained pKT230 and grew 113 vigorously on simple sugars but was unable to utilise any substrates related to cellulose or cellulosic materials (Lucas, 1987). The gene banks in ED8654 were mobilised into ATCC17527 by filter mating using the helper plasmid, pRK2013. ATCC17527 was infioculated in NYBG and incubated at 30°C overnight. HB101(pRK2013) was grown overnight and subcultured for another 3 hours in LB Km50. 20 |il glycerol stock of each gene bank was revived by incubating in 1 ml LB SmlOO for 3 hours. 1 ml ATCC17527, 1 ml HB101(pRK2013) and 1 ml gene bank were well mixed and filtered. pKT230 was also conjugated into ATCC17527 as control. After incubation on NYG agar plates at 3(X>C overnight, the cells were washed from the filter with SPB and serial dilutions o were spread on}/PM Sm50 agar plates containing 0.5% cellobiose as the sole carbon source. Plates were incubated for 48 hours at 3(X>C after which colonies were purified by streaking twice on plates of the same medium. Plasmids were extracted from 36 clones. Agarose gel electrophoresis (Figure 5.1) showed five different plasmid patterns among these clones. All possessed more than one plasmid the sizes of which ranged from 7 kb - 35 kb. This suggested that some considerable rearrangement of the recombinant plasmids had occurred in P^udomonas ATCC 17527. It was also observed that although ATCC 17527 was unable to utilise any substrates related to cellulose or cellulosic materials (Lucas 1987), ATCC17527 (pKT230) could turn esculin black after a prolonged incubation (more than 2 days) and grew slowly on PM agar with cellobiose as the sole carbon source. Because other strategies for isolating p-glucosidase-producing clones were producing encouraging results the reasons for the above observations were not explored. 114 1 2 3 4 5 6 7 8 9 10 11 y.Mwaifl < j. ; - 4 — UNI — Figure 5.1: Extraction of plasmid DNA from the cellobiose-utilising ATCC17527 isolates. Lanes 1-6, 8-11, plasmid extracted from the cellobiose-utilising ATCC17527 strains; lane 7, ccc DNA size maker, a, 14.5 kb; b, 15.5 kb; c, 24 kb; d, 34.5 kb. The gel was run at 80V for 5 hours. 115 5.1.2 Sequential selection procedure in E. coli As E. coli ED8654 does not have any detectable p-glucosidase activity and cannot grow on cellobiose as the sole carbon source, the conversion of E. coli to cellobiose utilization was used to detect the presence of the p-glucosidase gene and direct selection was modified to serial selection. The XA1-1 gene banks from glycerol stocks were activated by growing for three hours in Luria broth supplemented with antibiotic, washed in saline and plated onto esculin plates. After two days incubation, about 100 colonies on each plate turned esculin black. Plasmids were extracted from 5 black colonies and agarose gel electrophoresis (Figure 5.2) showed that the size of the plasmids these clones contained ranged from 13 kb - 17 kb. Enzyme assay were done on these clones, all of which showed positive activity on pNPG but no activity on cellobiose. A significant difference between the size of colonies on the esculin plates was noticed. Most colonies were about 0.5 mm in diameter, but some were much larger, being about 2.5 mm in diameter. 116 1 2 3 4 5 6 7 Figure 5.2: Plasmid DNA from ED8654 clones which turned esculin black. 1, Plasmid from ZM6 as CCC plasmid size marker (14.5 kb, 15.5 kb, 34 kb); 2, Plasmid pND71(23.8 kb); 3, Hind III digested A. DNA 4-7. Plasmids extracted from ED8654 clones of XA1-1 DNA. 117 The above plates were replicated onto BM agar containing 3 g 1“^ of cellobiose plus streptomycin. It was expected that only those recombinant clones which contained the jB-glucosidase gene would be able to use cellobiose as the sole carbon source. After 5 d incubation, approximately 2% of the replicated colonies had grown on the cellobiose plates. Thirty colonies, picked at random from these plates, were purified and rapid plasmid DNA extractions were performed. Each of these extracts gave the same plasmid pattern after agarose gel electrophoresis (Figure 5.3). One of the (3- glucosidase-positive transformants was purified twice on LB Esc and the plasmid it contained was designated pNSW904. pNSW904 could transform competent ED8654 to the esculin-positive phenotype with an efficiency of 2.7 x 10^ transformants per jig plasmid DNA. 5.2 Stability of pNSW904 in E. coli ED8654 ED8654 (pNSW904) was grown in LB for 100 generation or in LB SmlOO for 50 generations. The cultures were then serially diluted and plated onto LB plates and incubated overnight. Four plates with about 100 colonies on each plate were*3hen replicated onto LB SmlOO to monitor the maintenance of the antibiotic-resistance marker of pKT230 and onto LB esculin to monitor the maintenance of the cloned (3- glucosidase gene. Mini-scale plasmid extractions were carried out on the colonies to determine the size of the plasmid they contained. All of the colonies tested showed resistance to streptomycin and turned esculin black. Plasmid extraction showed the presence of intact pNSW904. This suggested that pNSW904 was stably maintained in ED8654 for at least 100 generations under selective conditions and for at least 50 generations under non-selective pressure. 118 9 8 7 6 5 4 3 2 1 Figure 5.3: Extraction of plasmid DNA from esculin-positive isolates from the serial selection procedure. Lane 3, CCC DNA size marker, a, 12 kb; b, 14.5 kb; c, 15.5 kb; d, 34 kb; lane 1,2, 4-9, plasmid extracted from esculin-positive isolates from the serial selection procedure. The gel was run at 100 V for 3 h. 119 5.3 Characterization of the recombinant plasmid pNSW904 pNSW904 was extracted from HB101(pNSW904) by the alkaline lysis method followed by cesium chloride/ethidium bromide equilibrium density gradient centrifugation. The purified DNA was digested with different restriction endonucleases for two hours under the condition described by the manufacturers. EDTA was added to a final conjerjcjtration of 15 mM to stop the reaction and the DNA extracted with phenol/chloroform. After ethanol precipitation the DNA was redissolved and some subjected to digestion with a second enzyme for two hours. Single and double digests were run on agarose gels together with X DNA digested with Hind III and pBR322 digested with Bst N1 as size markers (Figure 5.4). Single digests using the restiction endonucleases, BamWl, FcoRI, HindlW, Smal, SstI and Xhol were performed and revealed one site for BamWl, six sites for EcoRl, two sites for Hindlll, three sites for Smal, two sites for SstI and one site for Xhol. Double digests allowed mapping of the 15 restriction sites for these six enzymes. The size of the fragments generated in single and double digests allowed an estimation of the total size of pNSW904 (23.9 kb) and the construction of a physical map fcf the plasmid (Figure 5.5). The plasmid consisted of a unique copy of the vector, pKT230, plus an insert of about 12 kb. 120 13 12 11 10 9 8 7 6 5 4 3 2 1 Figure 5.4(a): Restriction endonuclease digestion of pNSW904 (1) Plasmid pNSW904 was digested with various restriction enzymes. The DNA was then run in a 1% agarose gel using a horizontal electrophoresis unit. The gel was run in TAE buffer for 20 h at 20 V. Digests were: Lane 1, Hind III and Sst I Lane 8, Sma I Lane 2, Hind III and Xho I Lane 9, Sma I and Sst I Lane 3, Pst I Lane 10, Sma I and Xho I Lane 4, Pst I and Sma I Lane 11, Sst I Lane 5, Pst I and Sst I Lane 12, Sst I and Xho I Lane 6, Pst I and Xho I Lane 13, Xho I Size markers were: X DNA digested with Hind III and pBR322 digested with Bst NI (lane 7). 121 13 12 11 10 9 8 7 6 5 4 3 2 1 Figure 5.4(b): Restriction endonuclease digestion of pNSW904 (2) Plasmid pNSW904 was digested with various restriction enzymes. The DNA was then run in a 1% agarose gel using a horizontal electrophoresis unit. The gel was run in TAE buffer for 20 h at 20V. Digests were: Lane 1, Bst Eli and Sma I lane 8, Eco RI and Sma I lane 2, Bst Eli and Sst I lane 9, Eco RI and Sst I lane 3, Bst Eli and Xho I lane 10, Eco RI and Xho I lane 4, Eco RI lane WJUnd III lane 5, Eco RI and Hind III lane 12, Hind III and Pst I lane 6, Eco RI and Pst I lane 13, Hind III and Sma I Size marker were: X DNA digested with Hind III and pBR322 digested with Bst NI (lane 7). 122 13 12 11 10 9 8 7 6 5 4 3 2 1 Figure 5.4(c): Restriction endonuclease digestion of pNSW904 (3). Plasmid pNSW904 was digested with various restriction enzymes. The DNA was then run in a 1% agarose gel using a horizontal electrophoresis unit. The gel was run in TAE buffer for 20 h at 20V. Digests were: Lane 1, Bam HI Lane 8, Bam HI and Sst I Lane 2, Bam HI and Bst Eli Lane 9, Bam HI and Xhol Lane 3, Bam HI and EcoR I Lane 10, Bst Eli Lane 4, Bam HI and Hind III Lane 11, Bst Eli and £cr;RI Lane 5, Bam HI and Pst I Lane 12, Bst Eli and Hind III Lane 6, Bam HI and Sma I Lane 13, Bst Eli and Pst I Size marked were X DNA digested with Hind III and pBR322 digested with Bst Nl (lane 7) 123 23 0 pNSW904 - 18 23.9kb H E S Figure 5.5: Physical map of pNSW904. The numbers indicate size in kb. The abbreviations used are as follows: B, BamWI; E, EcoR\\ H, Hindill; S, Sma\\ Ss, Sjrd; X, Xhol Xanthomonas DNA insert;______, vector pKT230. 124 5.4 Expression of the p-glucosidase gene In order to identify the expression of the p-glucosidase gene encoded by pNSW904 assays for p-glucosidase activity were performed, with pNPG as substrate, on the cell-bound fractions from the donor strain, X. alibilineans XA1-1 the recipient strain, E. coli ED8654, the recipient strain with vector, ED8654(pKT230), and the recombinant strain, ED8654(pNSW904). Since activity towards the natural substrate is essential for the purposes of this project assays for enzyme activity were also performed on these strains with cellobiose as substrate. XA1-1 was grown in PM medium containing methionine and glutamate at 20 jig ml-* and 0.4 g l"* yeast extract. E. coli strains were grown in MM containing the appropriate amino acid. Cellobiose was added at 0.5 g 1-1 to each medium as inducer and the pH was adjusted to 7.2 after autoclaving. Cells harvested in log phase were used to make enzyme preparations. The results are presented in Table 5.1. The recipient strain and the recipient strain with vector displayed no detectable p-glucosidase activity. The E. coli transformant, ED8654(pNSW904) showed a specific activity which was 18.7% that of the donor on pNPG and 166.2% that of donor on cellobiose. 5.5 Preliminary characterization of the enzyme 5.5.1 Temperature optimum The activity of p-glucosidase was determined at various temperatures ranging from 21«C to 62QC. Taking the highest activity as 100%, the relative activities of the other values were plotted against temperature (Figure 5.6). The temperature optimum of the 125 Table 5.1: P-Glucosidase activities of the recombinant strain, ED8654(pNSW904), using either the synthetic substrate, pNPG, or the natural substrate, cellobiose. Donor Recipient Recipient Recombinant strain strain strain with strain XA1-1 ED8654 vector ED8654 ED8654 (pNSW904) (pKT230) Specific activity 100.8 <0.01 <0.01 18.8 on pNPG (U mg-1) Specific activity on 114.2 0.01 0.01 189.8 cellobiose (U mg-1) 126 o 03 CJ (X Figure 5.6: Temperature profile of (Tglucosidase activity of ED8654(pNSW904) with pNPG as substrate. 127 p-glucosidase expressed in E. coli was found to be between 50°C and 54°C with a sharp decline in enzymatic activity at higher temperatures. 5.5.2 pH optimum The optimal pH was determined over a pH range of 6.2 to 9.0. To get a wide pH range 0.05M phosphate buffer was used for pH 6.2-7.5 and 0.05M Tris buffer was used for pH 7.5-9.0. Taking the highest activity as 100%, the relative activities of the other values were plotted against pH. The pH profile of enzyme activity showed an optimum value between pH7.5 and pH8.0 (Figure 5.7). 5.5.3 Metal ion requirements Determination of metal ion requirements was carried out by adding metal ions to the reaction mixture at a final concentration of 5 mM. No significant change of enzyme activity was found after adding Mn2+, Mg2+ or Co2+ ions (Table 5.2). 128 pH Figure 5.7: pH profile of |B-glucosidase activity of ED8654(pNSW904) with pNPG as substrate. 129 Table 5.2: Determination of metal ion requirements Co2+ Mg2+ Mn2+ No additional metal ion p-glucosidase activity on pNPG (U mg-1) 19.1 18.7 18.4 18.8 p-glucosidase activity on cellobiose (U mg-1) 192.3 190.6 185.1 189.8 Metal ion was added to the reaction mixture at a final concentration of 5 mM. 130 5.5.4 Michaelis-Menten kinetics with pNPG In order to determine the affinity of the enzyme from the recombinant strain towards pNPG, the Michaelis-Menten kinetics were investigated. The Michaelis-Menten plots in Figure 5.8(a)show the effect of pNPG concentration on the activity of p- glucosidase from the recombinant strain ED8654(pNSW904). A double reciprocal Lineweaver-Burk plot of the same data is shown in Figure 5.8(b). From the Lineweaver-Burk plots a Km value of 6.5 mM for pNPG was determined. 5.5.5 Optimum cellobiose concentration for the assay of p-glucosidase on cellobiose The effect of cellobiose concentration in the assay of p-glucosidase on cellobiose was investigated. The enzyme preparation was incubated in Mcllvaine’s buffer with various concentrations of cellobiose ranging from 5 mM to 60 mM. The substrate concentration giving the highest enzyme activity for cellobiose was 15 mM and at cellobiose concentrations greater than 20 mM there appeared to be substrate inhibition. 5.6 Regulation of gene expression Once in a new host, expression of the gene could be controlled by regulatory genes on either the host DNA or the plasmid or both. Such genes may be responsible for phenomena such as induction and repression. Variation of the cellobiose and glucose concentrations in growth media were used to obtain information on factors likely to influence gene expression. 131 Figure 5.8a.: Effect of pNPG concentration on activity of p-glucosidase from ED8654(pNSW904). 2.0 " 1 /mM pNPG Figure 5.8b: Double reciprocal plot of P-glucosidase activity versus pNPG concentration from Figure 5Bcl 132 Table 5.3 shows that the p-glucosidase activity of the cells remained the same in the presence or absence of cellobiose. This suggested that, unless an unknown inducer was present in the medium, the enzyme was synthesized constitutively. Increasing glucose concentration in the growth medium caused little decline in the specific activity of the enzyme. This suggested that p-glucosidase synthesis was not repressed by glucose. It is possible that native genes controlling p-glucosidase expression had been separated from the P-glucosidase structural gene by the molecular cloning procedures, or that such regulatory genes were not functionally expressed in E. coli. It was also found that there were increases in cell growth and p-glucosidase activity when the parent and recombinant strains were grown in the presence of yeast extract although ED8654(pNSW904) could grow efficiently in minimal medium without adding yeast extract. For XA1-1 it is important to supplement with yeast extract as a growth promoter. 133 Table 5.3: Effect of cellobiose and glucose concentrations on the specific activity of P-glucosidase in the recombinant strain ED8654(pNSW904) Glucose (% w/v) 0.05 0.5 2 - 0.05 0.5 2 0.05 Cellobiose (% w/v) - - - 0.5 0.5 0.5 0.5 3 Specific activity 19.2 18.7 19.3 19.0 18.8 18.6 19.6 19.1 on pNPG (U/mg-1) Specific activity 193.3 189.6 179.1 219.7 189.8 180.6 200.5 201.4 on cellobiose (U mg-1) 134 5.7 Growth of the recombinant strain on cellobiose To investigate the growth of the recombinant strain on cellobiose, ED8654(pKT230) and ED8654(pNSW904) were inoculated using a 1% (v/v) inoculum, into minimal medium containing 0.5% (w/v) cellobiose and streptomycin (100 jig ml"*), and grown at 37°C with shaking. At intervals the optical density was measured at 600 nm to monitor cell growth and HJPLC was used to measure the concentration of cellobiose in the growth medium. The results are shown in Figure 5.9. The host strain containing the vector, ED8654(pKT230) did not grow at all while the recombinant strain, ED8654(pNSW904), grew efficiently on cellobiose. At the same time the cellobiose was consumed by ED8654(pNSW904) but not by ED8654(pKT230). 135 ■a 0.5 U Time (h) Figure 5.9: Growth on cellobiose of E. coli with and without the p-glucosidase gene. Cultures were infloculated with 1% (v/v) of an overnight culture and grown in cellobiose minimal medium containing 100 |ig ml"* Sm at 37°C with shaking, a growth of ED8654(pNSW904), & growth of ED8654(pKT230), cellobiose concentration in the culture supernatant of ED8654(pNSW904), k cellobiose concentration in the culture supernatant of ED8654(pKT230). 136 5.8 Subcloning of the p-glucosidase-encoding fragment of pNSW904 to reduce its size For further studies on the p-glucosidase gene from Xanthomonas such as gene sequencing and improving the level of gene expression it is necessary to subclone pNSW904 to obtain a plasmid with a small insert fragment encoding p-glucosidase. For subcloning, pUC8 was chosen as the vector. 5.8.1 Construction of the subclone, pNSW910, with a 3.0 kb insert To get a smaller P-glucosidase-encoding fragment, pNSW904 was both completely and partially digested with the restriction enzyme, Sau 3A. This enzyme recognises the tetranucleotide sequence, GATC, which occurs within hexanucleotide target sequence of Bam HI. Assuming that restriction endonuclease sites are distributed randomly along the DNA, the tetranucleotide will occur 16 times more frequently than the hexanucleotide. For complete digestion four units of Sau 3A were added to 1 jig of pNSW904 DNA in 20 |il digestion buffer and incubated at 37°C for 2 hbrFor partial digestion, 0.08 U of Sau 3A was added to 1 jig DNA in 20 |il digestion buffer and incubated for either 20 min. or 60 min. The digestions were checked by agarose gel electrophoresis (Figure 5.10 and Figure 5.11). The complete digest and the 60 min. partial digest were ligated with pUC8, after the plasmid had been completely digested with Bam HI and phenol/chloroform extracted. The ligation mixture was used to transform competent E. coli JM101 cells. The transformants were selected by plating onto LBX-gal plates and LB Esc AplOO plates which were incubated overnight at 37oC. Using X-gal plates, a transformation frequency of about 2 x 106 transformants |ig'l DNA was obtained for both ligations. On LB Esc AplOO plates 137 2 1 Figure 5.10: Sau 3A complete digest of pNSW904. 1, Hind III digest of X DNA; 2, Sau 3A complete digest of pNSW904. 138 1 2 3 Figure 5.11: Sau 3A partial digest of pNSW904. 1, Hind III digest of X DNA; 2, Sau 3A partial digest of pNSW904, incubation time 60 min; 3, Sau 3A partial digest of pNSW904, incubation time 20 min. 139 no transformants from the complete digest turned the agar black, while 2% of the transformants from the partial digest did so. Rapid disruption of colonies to test for inserts in plasmids were done on 40 black colonies randomly selected from the Esc plates. Selected plasmid profiles are shown in Figure 5.12. The clone with smallest insert (3 kb) was purified twice on LB Esc AplOO plates and the plasmid was designated pNSW910. 140 Figure 5.12: Rapid disruption of colonies to test for inserts in plasmids. 1, pUC8, a, monomer 2.7 kb, b, dimer 5.4 kb, c, trimer 8.1 kb; 2, subclone of pNSW904 with smallest insert (3 kb), designated pNSW910; 3-7, subclone of pNSW904. 141 5.8.2 Construction of subclone, pNSW911, with a 1.5 kb insert The insert of pNSW910 was excised by digestion with EcoRl and Pstl. Electrophoresis (Figure 5.13) showed that EcoRl cut pNSW910 into two fragments of 5.2 kb and 0.5 kb (lane 6). Pstl produced two fragments also, 4.7 kb and 1 kb in size (lane 8). This indicated that there was one EcoRl site and one Pstl site in the insert. pNSW910 was double digested with EcoRl and Pstl and the digested DNA was extracted with phenol/chloroform, precipitated with ethanol and dissolved in double- distilled water. This digest should release the insert DNA as three fragments of 0.5 kb, 1 kb and 1.5 kb, together with the vector, pUC8, that has one Pstl and one EcoRl end. The DNA was divided into two. Ligase and ligation buffer were added to one half. Ligase, ligation buffer and Pstl digested pUC8 were added to the other half. After incubation at 40C overnight the ligation mixtures were used to transform competent JM101 and transformants were selected on LB Esc AplOO plates. The former ligation mixture gave black colonies while the latter one did not. Four black colonies were picked, purified and rapid plasmid isolations were carried out. The plasmid from one clone was designated pNSW911. This was digested witfirEstl alone and double digested with EcoRl and Pstl. The uncut and digested plasmid was visualised by agarose gel electrophoresis along with uncut and digested pNSW910. The result is shown in Figure 5.14. The monomer of pNSW911 (lane 4) was far smaller than the monomer of pNSW910 (lane 1) and significantly larger than the pUC8 monomer (lane 8). The Pstl digest of pNSW911 (lane 5) showed that the size of pNSW911 was 4.2 kb. Double digest of pNSW911 with EcoRl and Pstl (lane 6) indicated that pNSW911 consisted of the 2.7 kb vector fragment and a 1.5 kb insert. By comparing the double digest pattern of pNSW910 (lane 3) with that of pNSW911, together with the fact of pNSW911 gave a 142 8 7 6 5 4 3 2 1 Figure 5.13: Restriction enzyme digestion of pNSW910. 1, undigested pUC8; 5, undigested pNSW910; 2, EcoK\ digest of pUC8; 6, Eco\W digest of pNSW910; 3, Pst\ digest of pUC8; 7, Him1 III digest of pNSW910; 4, Hind III digest of X DNA; 8, Pstl digest of pNSW910 143 987 654321 Figure 5.14: Restriction enzyme digestion of pNSW911 andpNSW910. 1, pNSW910 2, /^rl digested pNSW910 3, Pstl and EcoRl digested pNSW910 4, pNSW911 5, kyd digested pNSW911 6, Pstl and EcoRI digested pNSW911 7, Hind III digested X DNA 8, pBR322 digested with toNI 9, pUC8 144 positive test on esculin, it was concluded that the (3-glucosidase gene from XA1-1 was located on the 1.5 kb EcoRI and Pstl fragment of pNSW910. The derivation of pNSW911 from pNSW904 is shown in Figure 5.15. It was observed that, when pNSW904 and pNSW911 were streaked on esculin plates for single colonies, pNSW911 turned the agar blacker and more quickly, indicating that the p-glucosidase activity in the pNSW911 clone was higher than in the pNSW904 clone. This may be accounted for by the higher copy number of the vector, pUC8, compared to pKT230. 145 H pNSW 904 23.9kb Sau 3 A partial digest of pNSW904 ligated with BamHI-digested pUC8. pNSW 910 5.7kb Digestion of pNSW910 with Pst\ and EcoRl, followed by religation. Figure 5.15: Constniction of a subclone, pNSW911, comprising pUC8 with a 1.5 kb P-glucosidase-encoding insert from XA1-1. E, £c«RI; P, Pstl. —vector, pKT230 or pUC8; •—XA1-1 DNA insert. 146 Discussion To select a clone encoding the p-glucosidase (cellobiase) gene from Xanthomonas albilineans XA1-1 gene banks direct selection (Lucas, 1987) indirect selection with pNPG (Lucas, 1987), selection via Psfudomonas, indirect selection with esculin and sequential selection (this work) were attempted. For sequential selection, indirect selection with esculin was used first. This gave similar results to indirect selection with pNPG. Several clones containing recombinant plasmids with different insert sizes were obtained. These were smaller than 4 kb and showed no activity on cellobiose. It was noticed that colonies of different size were obtained on the esculin plates. It is possible that clones encoding p-glucosidase could degrade esculin to yield glucose, an efficiently utilized carbon source, so that these clones were able to grow larger. It was expected that these colonies would grow on cellobiose more easily than single cells and so for the second selection these plates were replicated onto MM plates with cellobiose as the sole carbon source. Thus the first selection overcame the difficulty of the clone encoding p-glucosidase growing up on cellobiose as sole carbon source and the second selection overcame the complication of indirect selection with the artificial substrate, esculin. As was confirmed later, degradation of esculin was caused by more than one p-glucosidase encoded by the donor. Serial selection turned out to be a successful method for isolating p-glucosidase-encoding clones. E. coli clones producing p-glucosidase were detected initially by their ability to turn esculin agar black. However, only a minority of these clones were able to grow on cellobiose as the sole carbon^ source. This suggested that the parent Xanthomonas albilineans, encoded more than one p-glucosidase gene. If this is the case then X. albilineans is similar to Cellulomonas fimi and Pseudomonas spri, cellulolytic 147 organisms which possess two p-glucosidases, one which functions as a cellobiase and a second as an aryl-P-glucosidase (Wakarchuk et al., 1984, Lucas et ah, 1986). Because the emphasis of this work was on cellobiose degradation, subsequent work concentrated on strains which showed enzyme activity towards both pNPG and cellobiose. The presence of more than one P-glucosidase in XA1-1 would explain the higher pNPGase activity of the donor compared to the clone, ED8654(pNSW904) (Table 5.1). This could occur if the donor contained two types of p-glucosidase, one which was a true cellobiase and the other which was an aryl-P-glucosidase and showed no activity on cellobiose. Since activity towards the natural substrate is essential for the purposes of this project only the true cellobiase gene was cloned. i.e.: donor XA1-1: (1) cellobiase (p-glucosidase) + (2) aryl-p-glucosidase clone ED8654(pNSW904): (1) cellobiase (P-glucosidase) let: the activity of (1) on cellobiose = x the activity of (1) on pNPG = y the activity of (2) on pNPG = z y the activity of (2) on cellobiose = 0 The ratio of activity on cellobiose to activity on pNPG is: 148 donor: x + 0 x clone: x + 0 x y + z y + z y + 0 y y + z > y x x < y + z y i.e., the activity ratio on cellobiose and pNPG in the clone is higher compared to that in the DNA donor. This is in agreement with the data in Table 5.1. Therefore, the difference between the activity ratio on cellobiose and pNPG in the clone and that in the donor supports the concept that not all the p-glucosidase genes from the donor have been cloned. As in the parent (Dunn, personal communication), the enzyme activity in ED8654(pNSW904) was cell-bound since no detectable p-glucosidase was found in the culture fluid. This agrees with the results of the cloning of the p-glucosidase gene of E. adecarboxylata (Annentrout and Brown, 1981), Caldocellum saccharolyticum (Love and Streiff et al., 1987) and Pseudomonas sp. (Lucas et al., 1986). Thin layer chromatography at intervals during the course of cellobiose hydrolysis by cell extracts of ED8654(pNSW904) revealed a gradual accummulation of glucose. From this it was concluded that the major p-glucosidase activity is likely to be the simple hydrolysis of cellobiose to glucose (Sternberg et al., 1977; Shewale, 1982). Preliminary characterization and regulatory studies of the p-glucosidase were carried out in order to optimize the assay conditions with respect to substrate concentration, cofactors, temperature and pH. 149 Comparison between enzyme activities obtained on the different substrates, pNPG and cellobiose, indicated that the recombinant strain hydrolysed cellobiose faster than it did pNPG. This, together with other characteristics of the enzyme, e.g., low sensitivity to catabolite repression and constitutive expression, are considered to be advantageous in subsequent strain construction work. The cloning of the gene for (3-glucosidase from Xanthomonas albilineans will allow it to be introduced into organisms which could benefit from this additional catabolic activity. This gene has subsequently been transferred to and expressed in Zymomonas mobilis. 150 CHAPTER 6 Expression of a Cloned [3-plucosidase Gene from Xanthomonas albilineans in Zvmomonas mobilis 6.1 Subcloning the (3-glucosidase gene The (3-glucosidase gene from the cellulolytic organism Xanthomonas XA1-1 was present on the Hind III fragment cloned into the vector pKT230 (Chapter 5). As this vector is not suitable for use in Z. mobilis (Eveleigh et al., 1983), the gene had to be subcloned onto a suitable vector before it could be transferred into Z. mobilis. The broad-host-range plasmid, pRK404, has been successfully mobilized by pRK2013 into, and stably maintained in, Z. mobilis ZM6100 (K. Smith and A. Goodman, unpublished results). The plasmids pNSW904 and pRK404 were isolated from E. coli ED8654 and ftBlOl respectively and were purified by centrifugation to equilibrium on cesium chloride/ethidium bromide gradients. Both plasmids were digested with Hind III. Oj\d The restriction enzyme was removed by phenol extraction^the two Hind III digests were then ligated using T4 DNA ligase. The ligation mixture was transformed into competent E. coli HB101. HB101 is recA (Boyer and Roulland-Dussoix, 1969), and so transformed DNA cannot integrate into the host chromosome. Transformants were selected on LBTc and were then patched onto EscTc. One Esc+TcR clone was purified by subculturing twice on EscTc. The plasmid harboured by this clone was designated pNSW906. Hind III digestion of 151 pRK404, pKT230, pNSW904 and pNSW906 showed that pNSW906 comprised pRK404 and the 12 kb XA1-1 DNA insert from pNSW904 (Figure 6.1). 152 1 2 3 4 5 6 23.15 9*42 6.56 4.38 Figure 6.1: Characterisation of pNSW906. 1, Hind III digested pKT230 4, Hind III digested pNSW906 2, Hind III digested pNSW904 5, intact pNSW906 3, Hind III digested pRK404 6, Hind III digest of X DNA 153 6.2 Transconjugation of the p-glucosidase gene into Z. mobilis The plasmid, pNSW906, was mobilised into Z. mobilis strains ZM6 and ZM6100 by three-way filter matings using HB101(pRK2013). Z. mobilis transconjugants were selected on RMTmTc, since Z. mobilis is inherently TmR but HB101 is not. The transconjugation frequency was approximately 10-6 per donor cell. Tc& transconjugants were purified on the selection medium. One ZM6 TcR transconjugant was designated ZM6901 and one ZM6100 TcR transconjugant was designated ZM6902. Plasmids were isolated from ZM6901 and ZM6902 and the plasmid profiles showed, besides the native plasmids of ZM6 and ZM6100, several novel plasmid bands with weaker intensity than the native plasmids (Figure 6.2). These plasmid preparations from ZM6901 and ZM6902 were then used to transform competent HB101. HB101 transformants selected on LBTc were patched onto LBEsc and 100% of the transformed colonies turned the agar black. This indicated that the p-glucosidase Z gene was encoded by the plasmids from ZM6901 and $M6902. Plasmid extraction was then performed on these HB101 transformants and the plasmid profile revealed that the transformants harboured plasmids of different sizes which corresponded in size to the faint bands in the plasmid preparations of ZM6901 and ZM6902 (Figure 6.3). 154 7 6 5 4 3 2 1 ch c b a Figure 6.2: Agarose gel electrophoresis of plasmid DNA from Z. mobilis transconjugants. 1-3, plasmids from ZM6901 4, ZM6 standard plasmid profile 5-7, plasmids from ZM6902 a, pNSWl; b, pNSW2; c, pNSW3; ch, chromosomal DNA. Note the novel plasmid bands with weaker intensity than the native plasmids. 155 6 5 4 3 2 1 Figure 6.3: Extraction of plasmid DNA from HB101 transformants transformed by the plasmids extracted from ZM6901. 1-5, plasmids from HB101 after transformation with plasmids extracted from ZM6901; 6, Hind III digest of X DNA. 156 6.3 Re-arrangement of pNSW906 in ZM6 transconjugants It appeared that after mobilization into Z. mobilis the plasmid, pNSW906 (pRK404 containing the p-glucosidase encoding fragment from Xanthomonas) re-arranged itself to form several novel plasmids. DNA hybridization studies were carried out on the plasmids extracted from strains ZM6 and ZM6901. Plasmids were separated by agarose gel electrophoresis and then probed with the 32p-iabelled XA1-1 fragment encoding p-glucosidase which was eluted from Hind III digested pNSW904 after agarose gel electrophoresis (Figure 6.4). No plasmid of ZM6 showed cross- hybridization to the p-glucosidase gene fragment, but at least two plasmid bands in ZM6901 hybridized with the XA1-1 fragment (Figure 6.5). One of these corresponded in size to pNSW906 and could only be transformed into HB101. The other, larger size than pNSW906, could be transformed or conjugated into HB101. This latter plasmid appeared to be similar to that described by Liu et al., (1988) who reported that when the plasmid, pND90 (pRK404 containing an insert encoding the D-xylose catabolic genes), was mobilized into ZM6100 using the mobilizing plasmid, pRK2013, it became larger in size and self-transmissible as a result of recombination between pND90 and pRK2013. 157 2 1 p cr Figure 6.4: Agarose gel electrophoresis of the eluted P-glucosidase-encoding fragment. 1, Hind III digested pNSW906; 2, eluted P-glucosidase-encoding fragment; a, p-glucosidase-encoding fragment (12 kb); b, pRK404 vector fragment (10.6 kb). 158 Figure 6.5: Gel electrophoresis and Southern hybridization of plasmids from ZM6, ZM6901, and the p-glucosidase-encoding fragment. J\,Hind III digested pNSW906; a, I,near pRK404; 2-4, ZM6; b, XA1 -1 DNA insen encoding P-glucosidase; 5, ZM6901 c, |,near pNSW906 159 6.4 Expression of the cloned p-glucosidase gene in Z. mobilis 6.4.1 p-Glucosidase activity in Z. mobilis transconjugants Z. mobilis strains were found to grow slowly in defined medium therefore a modified rich medium was used for enzyme assay. ZM6, ZM6100, ZM6901 and ZM6902 were grown in 5 ml RM medium for 24 h. When necessary, tetracycline was added to maintain the plasmid. The cultures were then added to 200 ml RM containing 20 g 1-1 glucose and 0.5 g 1-1 cellobiose (with the pH adjusted to 7.2 after autoclaving) and incubated for another 24 h. The cells were then harvested, washed and resuspended in 2.5 ml Mcllvaine buffer. 0.625 ml toluene was added, mixed well and the cell suspension left at room temperature for 20 min. p-glucosidase assays were performed on the cell-associated fractions with pNPG as substrate because it has been confirmed that the cloned p-glucosidase gene was active towards both pNPG and natural substrate cellobiose (See Chapter 5). Enzyme assays confirmed that the P-glucosidase gene was expressed in HB101(pNSW906), ZM6901 and ZM6902. Table 6.1 shows that P-glucosidase was not detected in HB101, ZM6 or ZM6100. The level of expression of the enzyme by pNSW906 harboured in HB101 was approximately the same as that from pNSW904 harboured in ED8654. The p-glucosidase activities of Z. mobilis ZM6901 and ZM6902 were 7.5% and 10% of that of E. coli HB101(pNSW906) respectively. 160 Table 6.1: Specific activity of p-glucosidase from E. coli transformants and Z. mobilis transconjugants assayed on pNPG Strain Specific activity on pNPG (Umg-l) ED8654 <0.01 ED8654(pNSW904) 18.8 HB101 <0.01 HB101(pRK404) <0.01 HB101(pNSW906) 16.0 ZM6 <0.01 ZM6901 1.2 ZM6100 <0.01 ZM6902 1.6 pNSW904, the p-glucosidase-encoding fragment from X ant homo nas inserted into pKT230; pNSW906, the p-glucosidase gene subcloned into pRK404; ZM6901, ZM6 transconjugant containing the p-glucosidase gene; ZM6902, ZM6100 transconjugant containing the p-glucosidase gene. 161 6.4.2 Time course of enzymic hydrolysis in cell extracts Thin layer chromatography was shown to be an effective technique for observing the time course of hydrolysis of a substituted xylan by crude enzyme preparations of Cellulomonas (Daly et al., 1983). In order to get more information about the enzyme hydrolysis of cellobiose with time TLC was used to reveal a sequential pattern of appearance of hydrolysis products. 200 pi of enzyme preparation for p-glucosidase assay, 200 pi Mcllvaine buffer and 200 pi cellobiose solution in an hermetically- sealed tube were incubated in a 45°C water bath. The final concentration of cellobiose was 5 mM. At intervals small samples were taken and the products resolved by TLC, with cellobiose and glucose solution as standards. The results (Figure 6.6) revealed that for ED8654(pNSW904) cellobiose was degraded gradually and almost completely disappeared by the third day. This degradation was accompanied by an increase in glucose, and glucose was the only product of cellobiose degradation. Cellobiose can be degraded in a number of ways (Schimz et al., 1983) to its component sugars or their derivatives by: 1. direct hydrolysis to two molecules of glucose; 2. oxidation to cellobionic acid, via cellobiono-5-lactone, followed by hydrolysis to glucose and gluconic acid; 3. ATP-dependent phosphorylation of the C6 hydroxyl at the non-reducing moiety, followed by hydrolysis to glucose and glucose-6-phosphate; and 4. inorganic phosphate dependent phosphorolysis leading to glucose and glucose-1 -phosphate. 162 unknown ^ ^compound^ glucose _ cel lob lose • «t »t*»i ft1111 0 O 0 © ~4- C G 0 1 3 0 1 0 1 3 0 1 3 0 1 3 ED8654 ZM6 ZM6901 ZM6100 ZM6902 (pNSW904) Figure 6.6: TLC analysis of the enzymic hydrolysis of cellobiose by cell-associated fractions. Samples were taken for analysis at: zero time (0); 1 day (1); 3 days (3). G, glucose; C, cellobiose. 163 In the above mechanisms cellobiose degradation via oxidation, ATP dependent phosphorylation and inorganic phosphate dependent phosphorolysis produces one molecule of glucose and one molecule of a glucose derivative, either gluconic acid, glucose-6-phosphate or glucose-1-phosphate. The charge and molecular weight of these derivatives are different from that of glucose thus their migration rate when chromatographed should be different. If intermediate products such as cellobiono-5- lactone and cellobionic acid are also considered, cellobiose degradation by these mechanisms should, when chromatographed, give at least one extra spot besides glucose. Because the product of cellobiose degradation by the recombinant strain ED8654(pNSW904) is only glucose it suggested that the mechanism of cellobiose degradation in the recombinant strain was by hydrolysis to two molecules of glucose and so the P-glucosidase must be a true cellobiase as defined by Sternberg et al. (1977) and Shewale (1982). The enzymic hydrolysis of cellobiose by recombinant Z. mobilis was observed by TLC. For the control strains, ZM6 and ZM6100, cellobiose remained at almofr the same level as at zero time and no glucose was produced. With the ZM6-derived strain, ZM6901, glucose and another two unknown compounds were produced during the first day but disappeared by the third day and at the same time the amount of cellobiose decreased significantly. Using the semi-quantitative method reported by Ghani (1987), the relative intensity of the spots was judged by eye and is recorded in legends to photographs of the chromatograms. It was estimated that about 80% (i.e., 4 mmol) of the cellobiose was consumed by the third day of incubation. With the ZM6100-derived strain, ZM6902, glucose increased in concentration and cellobiose decreased simultaneously. 164 6.4.3 Subcloning and expression of the (3-glucosidase gene onto pSal52 The (3-glucosidase gene from Xanthomonas was also subcloned onto the IncW group plasmid, pSal52, and the level of expression compared with pNSW906. pNSW904 and pSal52 were digested with Hind III, phenol/chloroform extracted and ligated overnight at 4oC. The ligation mixture was used to transform HB101 and transformants were selected on LB EScCmlOO plates. Ten black colonies were picked from the plate and miniscale plasmid extractions showed that all of these harboured a 26 kb plasmid. This is consistent with their carrying pSal52 into which the (3-glucosidase encoding fragment had been cloned. One clone was purified and the plasmid which it contained designated pNSW905. pNSW905 was extracted from the recombinant strain HB101(pNSW905) and purified by a second alkaline treatment. It was then transformed into ZM6 using the method of Su and Goodman (1987; see also Chapter 3) and the transformants were selected on RM CmlOO plates. Ten transformants were purified and plasmids extracted from them. Agarose gel electrophoresis showed that besides the native plasmids, pNSWl, pNSW2 and pNSW3, the transformants harboured a novel plasmid (Figure 6.7). The size'of the novel plasmid was estimated by reference to a standard curve which was constructed by plotting size in kb of pNSWl, pNSW2 and pNSW3 against the reciprocal of mobility. Calculations indicated that the size of the novel plasmid was 26 kb which was the same as the size of pNSW905. The intensity of the pNSW905 band in ZM6903 was about the same as that of the native plasmid bands. This contrasts with pNSW906 which, when transferred toZ. mobilis, produced novel plasmid bands with weaker intensity than the native bands (Figure 6.2). This suggests that the copy number of pNSW905 was higher than that of pNSW906 in the recombinant Z. mobilis. ZM6903 was assayed for (3-glucosidase activity and the specific activity on pNPG was 0.7 U mg-1. This was the same as the levels in ZM6901 and ZM6902. 165 Figure 6.7: Agarose gel electrophoresis of ZM6903 plasmid DNA. 1-10, plasmids extracted from ZM6903; 11, Hind III digest of X DNA. a, pNSWl; b, pNSW2; c, pNSW905; d, pNSW3; e, chromosomal DNA. 166 6.5 Stability studies on the Z. mobilis transconjugants The maintenance of pNSW906 in the Z. mobilis transconjugant strains was studied under batch culture conditions. Each strain was grown for 100 generations in 10% glucose RM medium with or without tetracycline. Cultures were diluted 1:103 every 24 h during which time the cultures passed through ten generations. Serial dilutions were then plated onto RM plates. After 3 d incubation the colonies were patched onto RMTc20 plates to test for the maintenance of the antibiotic resistance marker. Table 6.2 reveals that the cultures grown in the presence of tetracycline showed complete maintenance of antibiotic resistance, while tetracycline resistance was lost gradually in the absence of antibiotic. Plasmid extractions were carried out on several TcR colonies and the plasmids were used to transform competent HB101. Rapid plasmid extractions and agarose gel electrophoresis were carried out on the HB101 transformants and all showed the presence of the intact plasmid. The stability of pNSW906 in the ZM6 transconjugant (ZM6901) was similar to that of pNSW906 in the ZM6100 transconjugant (ZM6902). 167 Table 6.2: Maintenance of pNSW906 in Z. mobilis under batch culture conditions % Maintenance No selection With selection (20 [ig ml-1 Tc) \ No. of Strain \ Generations 30 40 ZM6901 ZM6902 60 42 25 19 12 100 100 200 colonies were tested in each case. 168 6.6 Ethanol formation from cellobiose by recombinant Z. mobilis 6.6.1 Ethanol formation from cellobiose by cell extracts of ZM6901 As observed by TLC, for the recombinant Z. mobilis strain ZM6901, glucose and two other unknown compounds were formed during the first day and disappeared by the third day. To determine the direction of flow of the products, a third-day sample of ZM6901 prepared for TLC was analyzed by GC with 7.6 mM ethanol as standard and ZM6 as control. Figure 6.8 reveals that for ZM6 almost no ethanol had been formed by the third day. By contrast an obvious peak at the retention time for ethanol appeared with the recombinant strain, ZM6901. Calculations indicated that 13.3 mM ethanol was formed. Since about 4 mM cellobiose was consumed (Section 6.4.2) the yield of ethanol was approximately 83%. This estimate was based on the maximum conversion of one mole of cellobiose to two moles of glucose which in turn are converted to four moles of ethanol. It is clear that cell extracts of the recombinant strain, ZM6901, were capable of converting cellobiose to ethanol. 169 E E ♦ t t Ethanol ZM6901 ZM6 n tandard (7.6mM) Figure 6.8: Analysis by GC of the products of cellobiose hydrolysis by a whole cell extract of ZM6901. The cell extract was incubated with 5 mM cellobiose in Mcllvaine buffer at 45^C for 3 days. The arrows indicate the time at which the samples were injected and E indicates the peaks of ethanol in the samples. 170 6.6.2 Ethanol fermentation from cellobiose by whole cells of ZM6901 To investigate the formation of ethanol by whole cells of the recombinant Z. mobilis, ZM6(pRK404) and ZM6901 were grown in RM plus 1.4 mM cellobiose to mid- logarithmic phase. The cultures were then washed twice in buffer and resuspended in the original volume of RM containing 110 mM cellobiose as sole carbon source (RMCe), pH7.2 (5 x 10$ cells/ml). Samples were taken at intervals and ethanol production was measured by GC.Table 6.3 shows that after 6 d incubation 72 mM ethanol was produced in the medium by ZM6901 and after 11 d, 132 mM ethanol. ZM6(pRK404) was used as a control strain and no detectable ethanol was found. 171 Table 6.3: Ethanol formation by ZM6901 in RMCe medium (110 mM cellobiose) Ethanol Production (mM) Strain 3 days 6 days 11 days ZM6(pRK404) ND ND ND ZM6901 28 72 132 ND = Not detected 172 6.7 Discussion From the results presented here it can be seen that the P-glucosidase gene from Xanthomonas albilineans can be expressed in Z. mobilis. The introduction of a plasmid encoding p-glucosidase completed the cellobiose catabolic pathway in this organism and enabled the recombinant Z. mobilis to convert cellobiose to ethanol in a crude cell extijbts and whole cells. This is the first report of the expression of a p- glucosidase gene in Z. mobilis and the first report of the formation of ethanol from cellobiose by Z. mobilis. The addition of cellobiose to the range of sugars metabolized by Z. mobilis is an important step in the construction of a functional cellulase complex using recombinant DNA techniques, since p-glucosidase is frequently a rate-limiting enzyme in the microbial degradation of cellulose to glucose. Comparing the data for ethanol formation by crude cell extracts of ZM6901 and with whole cells, it was noticed that ethanol productivity of whole cells was much lower than crude cell extracts. In other words, there is still a great potential in the cellobiose catabolic enzyme system in ZM6901. Several improvements are necetoary to realize its full potential. Firstly, the transport of cellobiose across the cell membrane may be the rate-limiting step for cellobiose metabolism. Romano (1986) reported that in Z. mobilis glucose is transported by a low affinity facilitated diffusion system which requires no energy. However, the uptake of cellobiose by ZM6901 has not been investigated. It might be expected that a greater understanding of the mechanism of cellobiose transport in ZM6901 will facilitate improved transport efficiency. Secondly, conditions for ethanol formation by ZM6901 have not been optimized, e.g., the incubation temperature was lower than the optimum temperature for p-glucosidase activity (50-54°C) and the composition of fermentation medium may be less than ideal for p-glucosidase gene expression. Thirdly, the level of the 173 cloned p-glucosidase expressed in ZM6901 may be improved by further manipulation of the cloned gene. When the gene bank was screened for the p-glucosidase gene, E. coli clones producing p-glucosidase were detected initially by their ability to turn esculine agar black. However, only a minority of these clones were able to grow on cellobiose as the sole carbon source. This suggested that the parent Xanthomonas albilineans, encoded more than one p-glucosidase gene - one which functions as a cellobiase and at least another one as an aryl-p-glucosidase. Because the emphasis of this work was on cellobiose degradation, this study has concentrated on strains which showed enzyme activity towards both pNPG and cellobiose. As in the parent (N.W. Dunn, unpublished results), the enzyme activity in ED8654(pNSW904) was cell-bound since no detectable p-glucosidase was found in the culture fluid. Comparison between enzyme activities obtained on the different substrates, pNPG and cellobiose, indicated that the recombinant strain hydrolysed cellobiose faster than it did pNPG. This, together with other characteristics Of the enzyme, e.g., low sensitivity to catabolite repression and constitutive expression, are considered to be advantageous in subsequent strain construction work. This may have enabled the recombinant strain derived from ZM6 to convert cellobiose to ethanol more successfully. The ethanol concentration of 132 mM (6.1 gl~l) reported after extended incubation of intact cells in the presence of cellobiose, was similar to that found by Goodman et al. (1984) for a strain of Z. mobilis expressing P-galactosidase activity with lactose as the sole carbon source. In a further study (Chun & Rogers, 1986) lactose uptake was increased considerably in the presence of glucose, suggesting that a certain level of 174 metabolism may be required to facilitate transport of disaccharides such as lactose and cellobiose. In the case of lactose, a maximum specific uptake rate of 0.9 g g-1 h~ 1 was achieved in continuous culture suggesting that a similar strategy might be pursued for cellobiose utilization. 175 CHAPTER 7 Subcloning and Characterisation of a p-Glucosidase Gene from Pjsucfyomonas pS2-2 The P-glucosidase gene from the cellulolytic bacterium, Pseudomonas strain PS2- 2, was present on the 11.9 kb Hind III fragment in the vector, pKT230. The plasmid was designated pND71 and was harboured in E. coli ED8654(pND71) and P. putida ATCC 17527(pND71) (Lucas, 1986). This chapter describes the sub-cloning of the p-glucosidase gene from Pseudomonas PS2-2 onto the broad- host-range plasmid, pRK404, and characterisation of the cloned p-glucosidase gene. 7.1 Preliminary characterisation of the p-glucosidase-encoding plasmid, pND71 The plasmid, pND71, was isolated from E. coli ED8654(pND71) and purified by centrifugation to equilibrium on cesium chloride/ethidium bromide density gradients. The plasmid was digested with Hind III (Figure 7.1). As the insert fragment is approximately the same size as the vector, pKT|230, only one band was visible when Hind III - digested pND71 was electrophoresed. This band represents linear DNA of about 11.9 kb rather than 23.8 kb, indicating that the enzyme had in fact cut at two sites. Hind III was removed by phenol/chloroform extraction and the Hind Ill-digested pND71 DNA was then digested with a second enzyme (Bam HI, Eco RI or Pst I). The double digests and single digest were separated by agarose gel electrophoresis (Figure 7.1). The number and length of fragments from these digests were determined. Comparison of these data with the physical map of pKT 230 (Bagdasarian et al., 1981) showed that the insert was cut by Hind III and Bam HI into three fragments of 5.3 kb, 4.5 kb and 2.1 kb, by Hind 176 III and Eco RI into one fragment of 11.9 kb and by Hind III and Pst I into four fragments of 4.0 kb, 4.0 kb, 2.8 kb and 1.1 kb. Hence, the insert contained two Bam HI sites, no Eco RI site and three Pst I sites. 177 Figure 7.1: Restriction endonuclease digestion of pND71. 1, Hind. Ill and Bam HI digested pND71 2, Hind III and Eco RI digested pND71 3, Hind III and Pst I digested pND71 4, Hind III digested nND71 5, Hind III digested A DNA 178 7.2 Subcloning the p-glucosidase gene from Pseudomonas into pRK404. Plasmid, pRK404, was isolated from HB101(pRK404) and plasmid pND71 was isolated from ED8654 (pND71). Both plasmids were digested with Hind III. The restriction enzyme was removed by phenol/chloroform extraction, and the two digests were then ligated using T4 DNA ligase. The ligation mixture was used to transform competent E. coli HB101. Transformants were selected on LB Esc Tc plates. Competent HB101 cells were also spread on the same plates as a control. After incubation at 37°C for two days a transformation frequency of 5.6 x 10'6 per recipient cell was obtained and about one in ten of the transformants turned esculin black. Mini-scale plasmid extraction was done on five black colonies and the DNA electrophoresed (Figure 7.2). All plasmids harboured in these subclones were of the same size (22.3 kb) being much larger than pRK404 and a little smaller than pND71. One of the esculin-positive colonies was purified by subculturing twice on LB Esc Tc plates and the plasmid it harboured was designated pNSW903. Hind III digestion of pNSW903 was carried out and agarose gel electrophoresis showed that it was cut by Hind III into two fragments, 11.9 kb and 10.4 kb (data not shown). 179 1 2 3 4 5 6 7 Figure 7.2: Plasmids from subcloncs containing the insert from pND71 cloned into pRK404. 1-5, plasmids extracted from subclones of pND71; 6, pRK404; 7, pND71. The DNA was run in a 0.5% agarose gel at 80V for 1.6 h using a minigel electrophoresis unit. 180 7.3 Expression of the p-glucosidase gene of pNSW903 in E. coli HB101 7.3.1. P-Glucosidase assay of the E. coli HB101 (pNSW903) Expression of p-glucosidase by the subclone E. coli HB101(pNSW903) was firstly investigated by enzyme assay. E. coli strains were grown in MM medium containing the appropriate amino acids. P. putida ATCC 17527 (pND71) was grown in PM medium. Cellobiose was added at 0.5 gl“l to each medium as inducer. Log,phase cells were harvested and cell-bound fractions were used for p- glucosidase, assays on pNPG and cellobiose. HB101 containing only the vector was used as a negative control and the parent strains ED8654 (pND71) and ATCC 17527(pND71) were used as positive controls. Table 7.1 indicates that the expression of the subcloned p-glucosidase gene in HB101 (pNSW903) was about half of that of ED8654 (pND71) and P. putida (pND71). 181 Table 7.1: Enzyme assay of p-glucosidase clones Strains p-glucosidase activity U (mg protein)-! on pNPG on cellobiose HB101 (pRK404) <0.01 <0.01 ED8654 (pND71) 31.3 34.1 ATCC 17527 (pND71) 36.1 37.4 HB101 (pNSW903) 16.5 16.9 182 7.3.2 Location of the product of the subcloned p-glucosidase gene To localise the p-glucosidase enzyme, assays were performed on different cell fractions of the subclone,£. coli HB101(pNSW903), and compared with P.putida ATCC 17527(pND71). Log. phase cultures were centrifuged at 7000 rpm for 10 min at 40C, the cell pellet was collected and the supernatant centrifuged again at 15000 rpm for 10 min at 40C. The cleared supernatant was used as the extracellular fraction. The cell pellet was resuspended in Mcllvaine buffer and sonicated (16 x 15 sec pulses) in an ice-water bath. The broken cells were used as the cell-associated fraction. The sonicated cell suspension was centrifuged at 15000 rpm for 20 min at 40C. The supernatant was used as the cell-free extract and the pellet as the membrane-bound fraction. p-Glucosidase assays were performed on these different fractions using both pNPG and cellobiose as substrates. No detectable p-glucosidase activity was found in extracellular fraction of HB101(pNSW903), although an activity of 0.4 U ml-1 culture was recorded for Pseudomonas ATCC 17527(pND71) (Table 7.2). Since glucose, which would interfere the p-glucosidase assay on cellobiose, was present in the medium, p-glucosidase assays of the extracellular fraction were done only on pNPG. The major p-glucosidase activity was found to be cell-associated (Table 7.2) and when the broken cells were separated into cell-free extract and membrane-bound fraction, increased specific activity was found in the membrane- bound fraction. Therefore, it appeared that the p-glucosidase in the subclone, HB101(pNSW903), was cell-associated and membrane-bound (Table 7.2). 183 Table 7.2: p-Glucosidase activity on different cell fractions of E. coli HB101(pNSW903) and P. putida ATCC 17527(pND71) Fraction Strain P-Glucosidase activity on pNPG on cellobiose U(ml U(mg U(mg culture)-! protein)-! protein Extra- HB101 ND cellular (pNSW903) fraction ATCC 17527 (pND71) 0.4 Cell associated HB101 14.28 14.39 fraction (pNSW903) ATCC 17527 (pND71) 31.62 33.31 Cell-free HB101 extract (pNSW903) 2.17 1.81 ATCC 17527 (pND71) 25.95 19.49 Membrane- HB101 20.29 38.94 bound (pNSW903) fraction ATCC17527 (pND71) 98.01 174.6 184 7.4 Mobilization of pNSW903 into Z. mobilis ZM6 The plasmid from HB101(pNSW903) was mobilized intoZ. mobilis ZM6 by filter mating involving HB101(pRK 2013). One ml each of HB101(pNSW903), HB101(pRK2013) and ZM6 cultures were mixed well in a Petri dish, filtered through a membrane and the membrane incubated on an RM plate for 18 h. The cells were washed off the filter, diluted with SPB and plated onto RMTcTm plates to select ZM6 recombinants. A recombinant frequency of 2.16 x 10‘6 per recipient cell was obtained after incubation at 30°C for 3 d. Six ZM6 transconjugant colonies were purified and spot mating was carried out to confirm the existence of the p-glucosidase gene in them. 200 fil of mid-log. phase HB101 was spread onto an RM plate and incubated at 37°C for 2 h. 3 p.1 each of fresh ZM6 transconjugant culture was spotted onto the HB101 lawn. After incubation at 30°C overnight the plate was replicated onto LBEscTc agar and this was incubated at 37°C overnight. All of the replicated ZM6 transconjugant spots turned the esculin black although none of the control spots of ZM6 did so. This spot-mating test confirmed that the p-glucosidase gene had indeed been transferred into ZM6. The reason for pNSW903 becoming self-transmissable after mobilization into ZM6 by pRK2013 was presumed to be the same as the case reported by Liu et al. (1988) (See also Section 6.3). p-glucosidase assays were carried out on one of the ZM6 transconjugants, using the assay condition for E. coli strains. No detectable p-glucosidase activity was found in this ZM6 transconjugant. 185 7.5 Discussion A p-glucosidase gene from Pseudomonas strain PS2-2 was subcloned onto the broad-host-range plasmid pRK404 and the plasmid designated pNSW903. The insert fragment was characterised by restriction enzyme digestion. The expression of the subclone was about 50% of that of the parent plasmid in ED8654 and the product of the P-glucosidase gene appeared to be cell-associated and membrane- bound in the subclone. Expression of the p-glucosidase gene from Pseudomonas strain pS2-2 in Zymomonas mobilis ZM6 was not detected by enzyme assay. The plasmid pNSW903 was subsequently transferred to P. putida ATCC 17527 by Lucas (1987) to construct a P. putida strain expressing both endoglucanase and p-glucosidase. The constructed strain maintained activity of both of the enzymes when grown in the presence of the antibiotics. The enzyme levels found in the cell-bound enzyme preparations were 31.1 U (mg protein)-! for p-glucosidase and 12.8 U (mg protein)-1 for endoglucanase after incubation in the presence of 0.2% cellbiose for 14 h. This was similar to the levels of these enzymes expressed in the host strains harbouring each plasmid separately. y 186 CHAPTER 8 Linkage of Genes Encoding p-Glucosidase and Endoglucanase on the Same Vector and Expression in E. coli andZ. mobilis Recent models describing the cellulase system include three enzymes chiefly responsible for cellulose degradation. These are cellobiohydrolase, endoglucanase and p-glucosidase (Ghose and Ghosh, 1978). Lucas (1987) reported expression of endoglucanase by a clone of Z. mobilis ZM6100 and used enzyme kinetics to show that the increased levels of activity of the recombinant, ZM6100(pND82), over the parent ZM6100, were the result of the expression of the cloned DNA. In Chapter 6 the successful expression of a cloned p- glucosidase gene from X. albilineans in Z. mobilis ZM6 was described. With the goal of constructing a novel cellulolytic pathway in Z. mobilis,genes encoding p- glucosidase and endoglucanase from X. albilineans were linked on the same vector, pRK404, and introduced into E. coli and Z. mobilis. The expression of the p-glucosidase and endoglucanase genes in E. coli and Z. mobilis was investigated. w 8.1 Linkage of genes encoding p-glucosidase and endoglucanase on the same vector E. c<9//HB101 harbouring the plasmid pNSW906 (Chapter 6), which contains a 12 kb Hind. Ill fragment from Xanthomonas albilineans strain XA1-1 cloned into the Hind III site of pRK404, was used as the donor of the p-glucosidase gene. The source of the endoglucanase gene was E. coli HB101 containing pND82 which consisted of a 6.9 kb Hind III /Bam HI fragment from XA1-1 plus another small Hind \\\JBam HI fragment of approximately 0.8 kb, from the plasmid, pKT230, inserted into Bam HI site of pRK404 (Lucas, 1987). 187 The plasmid pNSW906 was extracted from HB101(pNSW906) and completely digested with Hind III. pND82 was isolated from HB101(pND82). Partial digests were prepared by using a limited amount (0.3U fig DNA'*) of Hind III and stopping the enzyme reaction at 10 min, 30 min and 60 min intervals. Agarose gel electrophoresis (Figure 8.1) showed that after 10 min pND82 was converted from the CCC to the linear form. This digest was chosen for ligation. pNSW906 completely digested with Hind III and pND82 partially digested with Hind III were extracted with phenol/chloroform, precipitated and resuspended in double distilled water. They were then mixed in a 1:1 ratio and ligation buffer and T4 DNA ligase were added. The ligation mixture was incubated at 4°C for 18 h and then used to transform competent HB101. The transformants were selected on LBTcEsc plates. Tc was used to select the vector, pRK404, linked with the endoglucanase gene in the partial digest and esculin was used to select (3- glucosidase gene from pNSW906. Thirty-five black colonies were picked from the LBTcEsc plates and streaked for single colonies on LBTcCMC plates which, after incubation, were flooded with 0.1% Congo red solution as described in Materials and Methods (2.3.8.3). Twenty-three colonies were confirmed to be TcR Esc+ CMC+. Mini-scale plasmid extractions wen* performed on five of these colonies and the plasmids characterised on a minigel electrophoresis apparatus. Four of the plasmids were of the same size while the fifth one was larger than the others (Figure 8.2). The small plasmid from one clone was designated pNSW907 and the larger plasmid was designated pNSW908. T88 Figure 8.1: Hind III partial digestion of pND82. 1, Hind III digested pND82, 10 min incubation time; 2, Hind III digested pND82, 30 min incubation time; 3, Hind III digested pND82, 60 min incubation time; 4, undigested pND82 (monomer and dimer). 189 5 4 3 2 1 m **■ a*; Figure 8.2: Plasmids from clones carrying the linked endoglucanase and (5- glucosidase genes. 1-5, plasmids extracted from TcR Esc+ CMC+ colonies. The gel was run for 2 h at 80V using a minigel electrophoresis unit. 190 8.2 Restriction endonuclease analysis of the linked plasmids pNSW907 and pNSW908 were completely digested with Hind III and the fragments run on an agarose gel, together with the uncut plasmids, ZM6 (pNSW905) as CCC DNA size markers and Hind III digested X DNA (Figure 8.3). The plasmids were 30.3 kb and 42.3 kb respectively. The pattern of Hind III digested pNSW907 showed a 12 kb fragment corresponding to the XA1-1 DNA fragment encoding p-glucosidase, an 11.4 kb band corresponding to the vector, pRK404 (10.6 kb), plus the 0.8 kb pKT230 fragment from pND82 and a 6.9 kb band corresponding to the XA1-1DNA fragment encoding endoglucanase. The Hind III digest pattern of pNSW908 showed the same three bands as Hind III digested pNSW907 but the 12 kb band of XA1-1DNA encoding p-glucosidase was relatively more intense than the other two bands. Together with the total size of pNSW908 this suggested that two fragments encoding p-glucosidase were in present J pNSW908. The linkage of genes encoding P-glucosidase and endoglucanase on the same vector, pRK404, is illustrated in Figure 8.4. 191 Figure 8.3: Hind III digestion of pNSW907 and pNSW908. 1, X DNA digested with Hind III; 2, ZM6(pNSW905) as CCC DNA size markers; a, 14.5 kb; b, 15.5 kb; c, 26 kb; d, 34 kb; 3, pNSW908; 4, pNSW907; 5, 6, Hind III digested pNSW907; 7, Hind III digested pNSW908. 192 Hind III partial Hind III complete digestion digestion ▼ ------pRK404 H i ...... "i pRK404 + endoglucanase gene (3-glucosidase gene 1. Ligated 2. Trasformed into HB101 3.Tcr CMC+ Esc+HBlOl transformants selected. pNSW907 pNSW908 29kb 41 kb Figure 8.4: Linkage of genes encoding p-glucosidase and endoglucanase on the same vector, pRK404.--- , pRK404 sequence; —,XA 1-1 DNA insert encoding endoglucanase; 1----J,XA1-1 DNA insert encoding P-glucosidase.Ht Hindlll. 193 8.3 Introduction of pNSW907 and pNSW908 into Z. mobilis Plasmids pNSW907 and pNSW908 which contain p-glucosidase and endoglucanase genes on the same vector, pRK404, were introduced into Z. mobilis ZM6 by three-way conjugation of this strain with HB101(pNSW907) or HB101 (pNSW908), using pRK2013 as the mobilizing plasmid. After incubation at 30°C overnight transconjugants were selected on RM plates containing Tc and Tp. After 3 d incubation at 30°C transconjugants were visible and colonies from each cross were purified on RMTpTc plates. One ZM6(pNSW907) transconjugant was designated ZM6904 and one ZM6(pNSW908) transconjugant was designated ZM6905. Plasmids were extracted from ZM6904 and ZM6905 and the plasmid preparations were used to transform HB101. Transformation efficiencies of 2.5 x 1()5 and 1.1 x 105 transformants p.g'1 DNA for the plasmids from ZM6904 and ZM6905 respectively were obtained. Ten colonies from each cross were purified and tested on esculin and Congo red plates. All tested positive showing that the cloned p- glucosidase and endoglucanase genes had been transferred to Z. mobilis intact. 8.4 Expression of the cloned p-glucosidase and endoglucanase genes in E. coli and Z. mobilis The expression of the cloned p-glucosidase and endoglucanase genes in E. coli and Z. mobilis was further confirmed by enzyme assay (Table 8.1). Since the p- glucosidase encoded by pND70 was confirmed to be active on both pNPG and cellobiose (see Chapter 5) the p-glucosidase assays of clones 194 containing the linked genes were carried out using pNPG as the substrate. Also because the identity of endoglucanase encoded by the parent plasmid, pND82, was confirmed by viscosity assays (Lucas et al., 1987), endoglucanase was measured using CMC as substrate. 195 Table 8.1: Expression of the linked p-glucosidase and endoglucanase genes on the same vector Activity IU (mg protein)-!] Strain p-glucosidase endoglucanase on pNPG on CMC HB101 <0.01 <2 HB101(pNSW906) 16.3 <2 HB101(pND82) <0.01 85.6 HB101(pNSW907) 16.1 83.2 HB101(pNSW908) 27.9 84.4 ZM6 <0.01 5.1 ZM6904 1.3 30.2 ZM6905 2.2 29.9 196 The donor strains, HB101(pNSW906) and HB101(pND82), exhibited activity only of the enzyme which was encoded by the plasmid in that strain. HB101 (pNSW907) and HB101(pNSW908) harbouring the linked genes on the same vector expressed significant activity of p-glucosidase and endoglucanase. In Z. mobilis the control strain, ZM6, displayed no detectable p-glucosidase and a low level of endoglucanase. Significant p-glucosidase activity and a six-fold increase in endoglucanase activity were found in the recombinant Z. mobilis strains, ZM6904 and ZM6905. Comparing the enzyme activities sustained by pNSW907 and pNSW908 in E. coli and Z. mobilis it can be seen that the level of endoglucanase expressed by the two plasmids was the same in each host, while the level of p-glucosidase expressed by pNSW908 was significantly higher (1.7 times) than that expressed by pNSW907. This increased level of p-glucosidase activity was most likely due to the increased dosage of the p-glucosidase gene in pNSW908. 8.5 Discussion Genes encoding p-glucosidase and endoglucanase from X. albilineans XA1-1 were linked on the same vector pRK404 and transformed into E. coli HB10I and Z. mobilis ZM6. Expression of the cloned P-glucosidase and endoglucanase genes was confirmed in both E. coli and Z. mobilis strains by enzyme assay. The significant feature of this work is the simultaneous expression of the P- glucosidase and endoglucanase genes in Z. mobilis. This is the first report of such simultaneous expression and, since cellulose degradation involves the synergistic action of endoglucanase, cellobiohydrolase and p-glucosidase, is a further step in the construction of a novel cellulolytic pathway in Z. mobilis. The size of pNSW908 was 12 kb larger than that of pNSW907. Hind III digestion of pNSW908 showed an intensity of the fragment encoding p-glucosidase which 197 was almost double that of the other fragments. The expression level of p- glucosidase in ZM6 harbouring pNSW908(ZM6905) was 1.7 times of that of ZM6 harbouring pNSW907(ZM6904). This increased level of p-glucosidase sustained by pNSW908 was most likely due to the increased dosage of the p- glucosidase gene in pNSW908. Enhancement of P-glucosidase activity is important in raising the cellulose hydrolysis efficiency, since p-glucosidase is frequently the rate limiting enzyme in the pathway. 198 CHAPTER 9 General Discussion and Conclusions The general aims of this project were the further development of recombinant DNA techniques in Z. mobilis and the use of these techniques to construct Z. mobilis strains which convert cellobiose to ethanol. The following were achieved: A simple and rapid transformation procedure was established. With this procedure a range of plasmids was introduced into Z. mobilis at reproducible and high transformation frequencies (up to 2.7 x 10_5 transformants per viable cell). Conditions tested to achieve high transformation frequencies included: the physiological condition of the cells to be made competent, the concentration of plasmid DNA in the transformation mixture, the source of plasmid DNA, storage of competent cells in glycerol at -70OC, the use of either one or two heat shocks and the length of time the transformation mixture was left on ice before the heat shock step. Among them the first two factors effected transformation frequency most significantly and second-order kinetics were observed for the formation of transformants. A native Z. mobilis plasmid marked with a transposon (pNSW60) and a group of small, non-conjugative, broad host range plasmids (pSal52, pSa727, pSa747) were transformed into Z. mobilis. The transformed pSal52 cointegrated with the native plasmid, pNSWl, in vivo to form a novel plasmid pNSW901 which was stably maintained in ZM6 for at least 200 generations without selection. This work enabled a much wider range of plasmids to be chosen for development of cloning vectors in Z. mobilis. Future work in this area could involve incorporation of Z. mobilis promoters into some of these plasmids to form expression vectors which could be used to increase the expression of foreign genes in Z. mobilis. With the method based on lysozyme, a spheroplast formation frequency of 99.9% and a regeneration frequency of 4.3% were obtained. Fusion of two auxotrophic ZM4 strains was achieved and the fusion frequency was 1.5 x 10-8 per viable cell. Future work should be aimed at fusion of two strains with different characteristics, for example, ZM4 and ZM6306. ZM6306 is a derivative of ZM6100(RP1 :: Tn 957), in which RP1 :: Tn 951 has integrated into the chromosome, and is a lactose-fermenting strain (Strzelecki et al., 1985). ZM4 was judged the best strain to produce ethanol from glucose (Rogers et al., 1982; Section 1.2.1.), but transferred Inc P-1 plasmids were not stable in ZM4 even with selection (Goodman, 1985). This made it difficult to construct a ZM4 lac+ strain using methods similar to those used for the construction of ZM6306. Fusion of ZM6306 and ZM4 may yield a strain which is superior in producing ethanol from lactose. A (3-glucosidase gene from X. albilineans was cloned and expressed in E. coli. The (3-glucosidase gene was present on a Hin d III fragment approximately 12 kb in size. This was inserted into the vector pKT230 to produce the recombinant plasmid, pNSW904. The p-glucosidase expressed by E. coli harbouring pNSW904 was shown to be active towards several p-glucosides. The specific activity in E. coli compared to that of the parent X. albilineans was 18.7% with pNPG as substrate and 166.2% with cellobiose as substrate. Evidence suggested that the enzyme was a true cellobiase, was produced constitutively and synthesis was not sensitive to catabolite repression. The recombinant strain grew efficiently on cellobiose. 200 Future work will involve sequencing the gene. A 1.5 kb fragment of pNSW904 encoding p-glucosidase has been subcloned into pUC8. This fragment was then cloned into Ml3 in both orientations and four hundred bases of the 1.5 kb fragment were sequenced (data not shown). Gene sequencing might provide detailed information for further genetic manipulation of this gene. For example, it might be placed directly after a Z. mobilis promoter to maximize gene expression. The cloned P-glucosidase gene from X. albilineans was successfully expressed in Z. mobilis by introduction of pNSW906, which was constructed by subcloning the 12 kb fragment encoding p-glucosidase onto pRK404. The recombinant strains were designated ZM6901 and ZM6902 and p-glucosidase was detected in both. The cell-associated fraction of ZM6901 converted 5 mM cellobiose via glucose to 13.3 mM ethanol. Cultures of ZM6901 produced 132 mM ethanol from 110 mM cellobiose after a prolonged incubation. Batch and continuous culture are now being used to study ZM6901 fermentations (Bin Wang, personal communication). Future studies should be aimed at determining the rate of cellobiose transport in ZM6901 and investigating means to increase cellobiose uptake. This, together with other investigations, e.g., conditions of fermentation, and further genetic manipulation if necessary, should further increase the overall rate and final yiald of ethanol formation from cellobiose. The p-glucosidase gene was then linked with an endoglucanase gene, also from X. albilineans, on the same vector and transferred to E. coli and Z. mobilis. Simultaneous expression of p-glucosidase and endoglucanase was observed in both E. coli and Z. mobilis. 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