关键词: 里氏木霉, CRISPR/Cas9, RNAi, 纤维素酶, CBH1, EG2

Abstract: The cellulase produced by the filamentous fungus Trichoderma reesei is mainly composed of cellobiohydrolases and endoglucanases. When using T. reesei to express a heterologous gene, these enzymes form a high cellulase background. Additionally, their expression used the engery for the cellular transcription, translation, and secretion apparatus. In this study, the CRISPR/Cas9 technique, specifically assembly of the Cas9/gRNA complex in vitro, was employed to transform T. reesei for disruption of the major cellobiohydrolase gene cbh1. The RNAi technique was used at the same time to silence the major endoglucanase gene eg2. The combined manipulations were expected to construct a low cellulase background T. reesei expression system. By this way, a transformant No. 11(designated SUS6) was obtained with significantly reduced cellulase expression. In this transformant, the expression of cbh1 was completely abolished, while the transcript level of eg2 decreased by 98% compared to that in the parental strain. Using this strain as a host microbe for expressing a heterologous gene NfBgl3A, it was found that the maximal b-glucosidase activities of two representative transformants were 172.4 and 79.3 U·mL^-1, respectively, much higher than 11.6 and 31.9 U·mL^-1 obtained when SUS5  (the parental strain of SUS6) was used as the recipient strain. Therefore, using low cellulase background strain as the host can significantly improve the expression of some heterologous genes.

Key words: Trichoderma reesei, CRISPR/Cas9, RNAi, cellulase, CBH1, EG2