Molecular Analysis and Improvement of Protein Production by Aspergillus Oryzae Grown on Solid Substrates

Molecular Analysis and Improvement of Protein Production by Aspergillus oryzae Grown on Solid Substrates Rob te Biesebeke Promotoren Prof. Dr. W.M. de Vos Hoogleraar in de Microbiologie aan de Universiteit van Wageningen Prof. Dr. C.A.M.J.J. van den Hondel Hoogleraar in de Genetica aan de Universiteit van Leiden Co-promotor Dr. P.J. Punt Product manager TNO Life Sciences Promotiecommissie Prof. Dr. Ir. C.T. Verrips Universiteit Utrecht Prof. Dr. Ir. A.J.J. van Ooyen DSM Food Specialties Prof. Dr. H.A. Wosten Universiteit Utrecht Prof. Dr. D.B. Archer Universiteit van Nottingham, UK Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG (Voeding, Levensmiddelentechnologie, Agrobiotechnologie en Gezondheid) Molecular Analysis and Improvement of Protein Production of Aspergillus oryzae Grown on Solid Substrates Rob te Biesebeke Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit Prof. Dr. Ir. L. Speelman in het openbaar te verdedigen op dinsdag 29 maart 2005 des namiddags om vier uur in de Aula Molecular analysis and improvement of protein production by Aspergillus oryzae grown on solid substrates Rob te Biesebeke Thesis Wageningen University. – With ref. – With summary in Dutch ISBN 90-8504-144-9 Subject headings: Aspergillus / protease / amylase / protein production / solid state fermentation / morphology / Aspergillus hemoglobin Microbial Functionality and Safety The research program of Microbial Functionality and Safety is directed to understanding, improving and predicting the activity of microorganisms in bioreactors, food products and the gastro-intestinal tract. Project Solid State Food Fermentations WCFS researchers at Wageningen University and Research Centre have been studying the process of solid state fermentation at different levels: from the genome and transcriptome level to the level of large-scale fermenters. The research has been focused on two main areas. The first has been the development of non-empirical, model-based methods for designing and operating solid state fermentation processes. The other research focus was on physiological and molecular genetic aspects of the fungal response to specific conditions in the solid state fermentation process, such as temporal and spatial gradients in nutrient, oxygen and water availability. In these studies, Aspergillus oryzae has been used as the model organism with wheat and soybean as model substrates, and amylases and proteases as model products. Contents Chapter 1 General introduction 7 Chapter 2 Aspergillus oryzae in solid state and submerged fermentation 17 Chapter 3 Identification of secreted proteins of Aspergillus oryzae associated with conversion and penetration of solid cereal substrates 27 Chapter 4 Different control mechanisms regulate the glucoamylase and protease gene transcription in Aspergillus oryzae in solid state and submerged fermentation 51 Chapter 5 Identification of growth phenotype related genes of Aspergillus oryzae by using heterologous macroarray and suppression subtractive hybridisation 71 Chapter 6 Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates 93 Chapter 7 An Aspergillus oryzae gene expressed during hyphal growth is a member of an unusual family of fungal flavohemoglobins 109 Chapter 8 Overproduction of Aspergillus hemoglobin domains in Aspergillus oryzae grown on solid substrates improves biomass yield, growth rate and enzyme production 127 Chapter 9 General discussion and concluding remarks 141 References 149 Samenvatting 163 Curriculum Vitae List of Publications Dankwoord ____________________________________________________________Chapter 1 CHAPTER 1 General introduction Introduction to this thesis Filamentous fungi are able to grow on solid substrates like wheat and rice kernels. Conversion of complex carbohydrate biopolymers (e.g. starch, celluloses) and biomolecules (e.g. proteins) into oligo- and monomeric nutrients is accomplished by secretion of a wide variety of enzymes. The research on industrially relevant enzymes that are produced by filamentous fungi during growth on solid substrates has recently gained increasing attention. This thesis contributes to the basic knowledge of molecular biological mechanisms associated to the production of proteins by the filamentous fungus Aspergillus oryzae during growth on solid substrates and describes approaches for improvement of protein production. Hence, this introduction provides a historic overview of fungal solid state fermentation with specific attention for recent studies on protein secretion by and morphology of filamentous fungi. In addition a short outline of this thesis is given. - 7 - General introduction____________________________________________________ Mycotechnology: fungi in biotechnology Filamentous fungi have been used for centuries in diverse biotechnological processes. This so-called mycotechnology (Bennett 1998) is traditionally used in relation to food production. The fermentation technology in Europe involved Aspergillus species for the fermentation of cacao beans and Penicillium species for the preparation of meat sausages (e.g. salami) and cheeses (e.g. roquefort and camembert). A. oryzae and A. sojae are being used for centuries for the preparation of koji in the Japanese traditional fermentation industry. Koji is used as a starter for the production of sake (rice wine), shoyu (soy sauce), miso (soybean paste) and shochu (distilled spirits) (Machida 2002, Kitamo 2002). The industrial application of filamentous fungi has expanded rapidly last century since the discovery that A. niger was an excellent citric acid producer in submerged fermentation (Wainwright 1992, Grewal and Kalra 1995). Also other fungal products have contributed to the rapid expansion of filamentous fungal applications; excellent examples of this are the antibiotic penicillin and purified enzymes. Amylase from A. oryzae was the first microbial enzyme used in industrial processes (Takamine 1894, Bennett 1998). Takamine grew A. oryzae cultures in thin layers on steamed rice and wheat bran and suggested new uses for the partially purified fungal diastase as a substitute for malting enzyme, and as a digestive aid for the treatment of dyspepsia (Takamine 1894, Takamine 1914, Bennett 1998). Also other filamentous fungi have been explioted because of their enormous potential to secrete a variety of proteins (van den Hondel et al. 1992, Punt et al. 1994, Pandey et al. 1999). Currently, a wide range of industries use (partly) purified filamentous fungal proteins and these include proteases, cellulases, dextranases, galactosidases, - 8 - ____________________________________________________________Chapter 1 glucoamylases, glucose oxidases, xylanases, lipases, invertases, pectinases, tannases, catalases, asparaginases, hesperidinases, naringinases, phytases (Bennett 1998, Pandey et al. 1999). In the last decades, the development of recombinant DNA technology has enabled the construction of filamentous fungal strains for the production of heterologous proteins (Jeenes et al. 1991, Conesa et al. 2001, Ishida et al. 2001, Machida 2002, Joosten et al. 2003). In parallel to this development, the research on protein secretion by Aspergillus species has focussed on (post-)transcriptional regulation of protein production, the transit of proteins through the secretion pathway and the structure of the proteins produced (reviewed in Archer and Pederby 1997 and Conesa et al. 2001). In the past three years the sequencing of the genomes of A. niger and A. oryzae has been completed (reviewed by Archer and Dyer 2004, Machida 2002). The available genome sequence enables identification of genes and functionally important regions of the genome. It has directed the research focus towards development of genome-wide analysis methods for exploiting that information (Archer and Dyer 2004, Hofmann et al. 2003, Maeda et al. 2004), also in relation to fungal product formation and protein secretion (Archer and Dyer 2004, Askenazi et al. 2003). Secretion of proteins and growth phenotype of filamentous fungi In its natural environment A. niger is involved in degradation of plant cell material by producing highly specialised enzymes like pectinases, (hemi)cellulases, and xylanases (de Vries and Visser 2001, de Vries et al. 2002, de Vries 2003). Enzymes that are produced by filamentous fungi are transported to the exterior of the cell via the secretion pathway. Compared to the knowledge of other eukaryotic - 9 - General introduction____________________________________________________ organisms, little is known about the filamentous fungal secretion pathway through which secretory proteins are transported to the exterior of the cell (reviewed by Conesa et al. 2001, Archer and Peberdy 1997, Punt et al. 1994). However, it is generally accepted that the secretion pathway is similar to those of yeasts and higher eukaryotes (Mior and Mao 1990, Conesa et al. 2001). Therefore, in the secretion pathway, posttranslational modifications such as formation of disulfide bond, glycosylation, and folding of the amino acid chain, are expected to involve similar protein activities in all eukaryotic organisms. Filamentous fungi have a better capacity to secrete proteins compared to Saccharomyces cerevisiae. A yield of 30 g/l of extracellular protein can be obtained with certain Aspergillus and Trichoderma strains (Durand et al. 1988, Finkelstein et al. 1989). In contrast, yields in the grams-per-liter range are only obtained

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