RNA interference with carbon catabolite repression in Trichoderma koningii for enhancing cellulase production.

The cellulase and xylanase genes of filamentous Trichoderma fungi exist under carbon catabolite repression mediated by the regulator carbon catabolite repressor (CREI). Our objective was to find the role of CREI in a cellulase-hyperproducing mutant of Trichoderma koningii, and address whether enzyme production can be further improved by silencing the cre1 gene. cre1 partially silenced strains were constructed to improve enzyme production in T. koningii YC01, a cellulase-hyperproducing mutant. Silencing of cre1 resulted in derepression of cellulase gene expression in glucose-based cultivation. The cre1 interference strain C313 produced 2.1-, 1.4-, 0.8-, and 0.8-fold higher amounts of filter paper activity, ß-1,4-exoglucanase activity (ρ-nitrophenyl-ß-D-cellobioside as substrate), ß-1,4-endoglucanase activity (sodium carboxymethyl cellulose as substrate), and xylanase activity, respectively, than the control strain, suggesting that silencing of cre1 resulted in enhanced enzyme production capability. In addition, downregulation of cre1 resulted in elevated expression of another regulator of xylanase and cellulase expression, xyr1, indicating that CREI also acted as a repressor of xyr1 transcription in T. koningii under inducing conditions. These results show that RNAi is a feasible method for analyzing the regulatory mechanisms of gene expression and improving xylanase and cellulase productivity in T. koningii.

Enhancing cellulase production in Trichoderma reesei RUT C30 through combined manipulation of activating and repressing genes.

To investigate whether enzyme production can be enhanced in the Trichoderma reesei industrial hyperproducer strain RUT C30 by manipulation of cellulase regulation, the positive regulator Xyr1 was constitutively expressed under the control of the strong T. reesei pdc promoter, resulting in significantly enhanced cellulase activity in the transformant during growth on cellulose. In addition, constitutive expression of xyr1 combined with downregulation of the negative regulator encoding gene ace1 further increased cellulase and xylanase activities. Compared with RUT C30, the resulting transformant exhibited 103, 114, and 134 % greater total secreted protein levels, filter paper activity, and CMCase activity, respectively. Surprisingly, strong increases in xyr1 basal expression levels resulted in very high levels of CMCase activity during growth on glucose. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression, and suggest an attractive new single-step approach for increasing total cellulase productivity in T. reesei.