Genetic Engineering Of Trichoderma Reesei To Improve The Capacity Of Cellulose Degradation

Plant biomass is one of the most abundant resources on the earth and it has been considered as a promising material for producing renewable biofuels and other bio-products.With the high capacity of protein secretion,Trichoderma reesei has become the most commonly used industrial strain for cellulases prouction.It is reported that several industrial strains secrete cellulase quantities exceeding 100 g/L of culture.Based on this property,in recent years,T.reeesei has been also widely used as an excellent host for recombinant protein expression.Among the secreted proteins profiles,cellobiohydrolase I(CBHI)accounts for 64%-80%of the total amounts,based on which,the cbhl promoter is usually considered as the strongest promoter in T.reesei.However,the activity of CBHI from T.reesei is not consistent with its large secretion amount,which suggests that T.reesei CBHI might have lower specific activity.Considering that the major bottleneck in the economic feasibility of cellulosic fuels is the cost efficiency of converting complex carbohydrate polymer found in lignocellulose into soluble sugars for fermentation,in this thesis,the following studies were conducted to modify the specific activity of CBHI and further improve the secretion capacity of T.reesei.1.Deciphering the role of drs2 gene in the protein secretion in T.reeseiOur preliminary data showed that deletion of drs2 gene in Neurospora crassa resulted in increase in protein secretion.To further investigate if the function of drs2 gene is reserved in T.reesei,the drs2 gene deletion strais Adrs2 were constructed and the protein secretion levels were measured.Our data showed that the protein secretion levels in Adrs2 strains were 2-3 folds higher than that in the parental strains.These data indicated that deletion of drs2 gene in T.reesei could affect the growth rate and lead to increase the protein secretion,which was similar to what was observed in N.crassa.2.Comparative analysis of CBHI enzyme activity in C.thermophilum and T.reeseiOur preliminary data showed C.thermophilum dispayed a relatively higher cellulose degradation efficiency while with lower protein secretion level.Therefore,we hypothesized that cellulases in C.thermophilum might have better enzyme properties.To evaluate this hypothesis,we first investigated CBHI,which is the major cellulase.C.thermophilum CBHI and T.reesei CBHI were respectively expressed in T.reesei by using cDNAl promoter and CBHI signal peptide,from which,CBHI would be the only secreted cellulase under glucose,enabling us to perform the enyme activity assay without the interference of other cellulases.Our data demonstrated that the specific activity of C.thermophilum CBHI was about 4-5 folds higher than T.reesei CBHI.In addition,the optimum temperature of T.reesei CBHI and C.thermophilum CBHI was 60 0C and 70 ?,respectively,while the optimum pH for both is 4.These data indicated that C.thermophilum CBHI had better property for industrial application compared to T.reesei.3.Replacement of T.reesei CBHI with C.thermophilum CBHI in T.reesei to improve the cellulose degradation efficiencyConsidering that the specific activity of C.thermophilum CBHI was significantly higher than T.reesei CBHI,we subsequently constructed recombinant T.reesei strains,in which T.reesei CBHI was replaced by C.thermophilum CBHI to investigate whether this combination could increase the cellulose degradation efficiency.Our results showed that the filter paper activity in the recombinant strain were 3 folds increased compared to the parental strains.Our data provided a promising strategy to perform genetic engigeering of T.reesei strains to improve the cellulose degradation efficiency.