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Enhancement of the Trichoderma reesei expression system
thesisposted on 2022-03-28, 23:46 authored by Shingo Miyauchi
Filamentous fungi are attractive as cell factories for recombinant protein production. Trichoderma reesei, an industrially-important filamentous fungus, has been used for the expression of recombinant gene products for over a decade and a variety of molecular genetics strategies has been applied to increase the yields of these products; yet no substantial improvements have been made and a significant increase in the yields remains a priority for economic production of non-fungal recombinant proteins. In this work, a new approach was developed to increase further recombinant protein production in T. reesei, featuring simultaneous use of multiple promoters for gene expression. Reliance on a single strong promoter, such as the one from the cbh1 gene encoding the main cellobiohydrolase I (CBHI), for driving expression of the genes encoding proteins of interest may not utilise the full capacity of protein expression by the organism. Hence, the novel concept of a multiple promoter platform (MPP) was put into practice with the objective of improving the efficiency of recombinant (heterologous) protein production. Instead of using one type of promoter for gene expression, the strategy behind MPP was to increase overall transcription of the gene of interest, here xynB encoding a thermophilic bacterial xylanase enzyme (Xylanase B), by employing multiple different fungal gene promoters. -- Enhancement of heterologous protein production was explored by combined proteomic, molecular genetic and glycoproteomic approaches. Major secreted proteins from T. reesei, including EGLII, CBHII and XYNII, were identified by 2-DE and MALDI-TOF analyses and the promoter sequences of the genes encoding these proteins were used to construct expression vectors for the production of the heterologous Xylanase B (XynB) from the thermophilic bacterium, Dictyoglomus thermophilum. Two generations of transformants carrying either a single promoter (SP) or multiple promoters (MP) controlling the expression of the xynB gene were examined. The best performing SP and MP recombinants were selected based on Xylanase B activity. The increase of activity in the best strain carrying the three different promoters to drive the expression of the xynB gene was 170 % compared to that of the best SP strain (transformation host for the MP strains). The copy number of the integrated genes and the mode of integration of the expression cassettes by homologous or non-homologous recombination were not significant factors in the production of Xylanase B, suggesting the presence of additional, as yet unidentified elements were involved in heterologous protein production. Examination of the glycosylation of Xylanase B fused to the linker region of EGLII revealed considerable amounts of O-linked oligosaccharides attached to the protein. One of the sugars identified by LC-MS/MS was hexuronic acid, which presents a sugar not previously found in Trichoderma glycoproteins. Overall, production of Xylanase B was enhanced by using a combination of three promoters, xyn2, cbh2 and egl2 inducing xynB gene expression.