The impact of putative methyltransferase overexpression on the Trichoderma harzianum cellulolytic system for biomass conversion.

Trichoderma harzianum has attracting attention for its potential alternative use in biofuel production, due to a recognized competence for high diversity glycoside hydrolases (GH) enzyme complex, including higher ß-glucosidases and auxiliary proteins, using low-cost carbon sources. This strain constitutively overexpressed the global regulator putative methyltransferase - LAE1, in order to improve the GHs production. The recombinant strain achieved 79-fold increase in lae1 expression and high GHs productivity. The evaluation of the LAE1 impact to induce the GHs used soluble and lignocellulose inexpensive carbon sources in a stirred-tank bioreactor. Using sugarcane bagasse with sucrose, the overexpression of lae1 resulted in significantly increment of gh61b (31x), cel7a (25x), bgl1(20x) and xyn3 (20x) genes expression. Reducing sugar released from pretreated sugarcane bagasse, which hydrolyzed by recombinant crude enzyme cocktail, achieved 41% improvement. Therefore, lae1 overexpression effectively is a promising improving GHs target for biomass degradation by T. harzianum.

The relation between xyr1 overexpression in Trichoderma harzianum and sugarcane bagasse saccharification performance.

This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and ß-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum.