Highly expressed Trichoderma reesei genes: Cloning, characterization and use in protein production on glucose-containing media: Dissertation

The filamentous fungus Trichoderma reesei is probably the best studied cellulolytic organism and is widely used by the biotechnical industry for production of cellulolytic and xylanolytic enzymes. Accumulating data on the production of mammalian and heterologous fungal proteins by T. reesei suggest that it is also applicable for production of heterologous proteins. In this work, T. reesei was further developed as a versatile host organism by using the techniques of molecular biology. The work is divided into two parts. A method was first developed which allows the isolation of genes that are strongly expressed in the desired culture conditions, and the promoters of two of the isolated genes were used to construct an expression system for glucose-based cultures in T. reesei. The second part dealt with the biochemical and genetic characterization of hydrophobins in T. reesei. Five genes were isolated that were abundantly expressed when T. reesei was growing on glucose as the only carbon source. Two of the genes were identified as a translation elongation factor 1a, tef1, and a hydrophobin, hfb1. Three out of the five genes (cDNA1, cDNA12 and cDNA15) highly expressed on glucose medium could not be characterized. A second hydrophobin gene, hfb2, was also isolated from a cDNA bank induced with complex plant polysaccharides by using the hfb1 gene as a probe. The primary structure of HFBI and HFBII were highly similar to each other and to the class II type hydrophobins cryparin of Cryponectria parasitica and cerato-ulmin of Ophiostoma ulmi. The expression of hfb1 and hfb2 hydrophobin genes was divergent, hfb1 being expressed on glucose-containing media but not on media containing plant polysaccharides and their derivatives inducing cellulase expression, whereas hfb2 was expressed on the latter media. hfb2 expression on glucose-based media was variable. HFBI protein was secreted into the culture medium when T. reesei was growing in cultures inducing its expression. HFBI was purified by bubbling or freezing the culture medium or by extracting the induced vegetative hyphae with trifluoroacetic acid - acetonitrile solution. A hydrophobin was also found in the culture medium of cultures induced for hfb2 expression but the conclusive identity of the protein is still unknown. HFBII was extracted from the outer walls of aerial spores of T. reesei. As a spore hydrophobin hfb2 expression was also detected in conidiating aerial hyphae and its expression was shown to be induced by light and by starvation of carbon and nitrogen sources. The promoters of the tef1 and cDNA1 genes were tested in protein production on glucose-containing medium using T. reesei CBHI and EGIcore cellulases as model proteins. The obtained product yields ranged from approximately 2 to 100 mg per liter of culture medium. cDNA1 promoter gave the best yields, 50 mg/l in the shake flask cultivations and 100 mg/l in a laboratory scale fermentor cultivation. No leakage of endogenous cellulases was observed on glucose medium, and cellulases produced under the control of the cDNA1 promoter constituted up to half of the total secreted protein on glucose-based cultures. However, the amount of cellulases produced under the control of the cDNA1 promoter was not comparable to the amount of endogenous cellulases on complex media and also the amount of total secreted protein on glucose medium remained low.