Genetic improvement of enzyme production in industrially important fungal strains: Dissertation

Filamentous fungi have a number of significant advantages for production of enzymes for industrial applications.In this study, enzyme producing filamentous fungal trains used in biotechnology have been improved genetically.Strains producing up to four times more enzyme than the initial parent, were obtained.The work is divided in two parts.The first part deals with mutagenesis and screening for induction and isolation of fungal strains with enhanced enzyme production and the second part concerns the application of recombinant DNA in developing new industrial strains.Different fungi have been selected for production of particular enzymes.Through mutagenesis and screening, improved strains of Aspergillus awamori and A. niger well suited to industrial scale production of amyloglucosidase and ß-galactosidase, respectively, have been isolated and characterised.The mesophilic cellulolytic filamentous fungus Trichoderma reesei is an important organism in studies of enzymatic hydrolysis of cellulose for production of fuels and chemicals.In this work, a series of hypercellulolytic T. reesei strains capable of production of cellulases on a relatively cheap fermentation medium has been induced and isolated.The genetic regulation of cellulase biosynthesis in T. reesei was studied through induction and isolation of mutant strains unable to hydrolyze Walseth cellulose.Cellulase negative mutants were enriched on plates using the antibiotic nystatin.Results suggest a separate regulation of actual cellulases to that of the ß-glucosidase enzyme in this fungus. Recombinant DNA methods have been introduced to study the expression of filamentous fungal genes in yeast and to gain important basic information of the structure and function of fungal cellulase genes.For further investigations in Trichoderma, a transformation system is needed and work towards this goal has been started by isolation of auxotrophic mutants of T. reesei.Cloning of genes from filamentous fungi by expression in the yeast Saccharomyces cerevisiae, as a general method, was studied.Yeast transformants carrying A. niger DNA were screened for functional expression of genes coding for the secreted enzymes ß-galactosidase, ß-glucosidase and amyloglucosidase and for the expression of Aspergillus genes complementing the yeast leu 2 and ura 3 mutations.Only the gene for ß-glucosidase was obtained in this way its expression being detected as blue coloured transformant colonies on plates containing the Xglu reagent.In addition to A. niger ß-glucosidase, the T. reesei CBH I gene coding for cellobiohydrolase I, the most abundant enzyme present in culture fluids of the fungus, is shown to express in yeast.CBH I enzyme produced by recombinant yeast shows activity comparable to that of T. reesei enzyme.However, the expression of genomic filamentous fungal genes in yeast seems to be rare and when it occurs the level of expression is very low. For efficient production of filamentous fungal enzymes in yeast, yeast regulatory signals and cDNAs of the genes to be expressed, need to be used.In future, both approaches, mutagenesis and screening and recombinant DNA, will be needed in development of new fungal strains for industry.