Miao Xing

Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters

BackgroundsThe fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II.ResultsThe transcriptional profiles of 13 key genes that participate in glucose metabolism in T. reesei were analyzed by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). The results indicated that the mRNA levels of pdc (encoding pyruvate decarboxylase) and eno (encoding enolase) genes were much higher than other genes under high glucose conditions. Recombinant T. reesei strains that homologously expressed xylanase II were constructed by using the promoters of the pdc and eno genes, and they respectively produced 9266 IU/ml and 8866 IU/ml of xylanase activities in the cultivation supernatant in a medium with high glucose concentration. The productivities of xylanase II were 1.61 g/L (with the pdc promoter) and 1.52 g/L (with the eno promoter), approximately accounted for 83% and 82% of the total protein secreted by T. reesei, respectively.ConclusionsThis work demonstrates the screening of constitutive promoters by using RT-qPCR in T. reesei, and has obtained the highest expression of recombinant xylanase II to date by using these promoters.

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, β-1,4-exoglucanase activity (ρ-nitrophenyl-β-D-cellobioside as substrate), β-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.