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

摘要： 丝状真菌里氏木霉（Trichoderma reesei）产生的纤维素酶主要包括纤维二糖水解酶和内切葡聚糖酶。在使用里氏木霉表达外源基因时，它们会形成较高的纤维素酶背景，而且其表达还可能会导致对细胞转录、翻译和分泌等相关资源的占用。应用CRISPR/Cas9技术，在体外组装Cas9/gRNA复合物并转化里氏木霉以定点敲除主要的纤维二糖水解酶cbh1基因，同时结合RNAi干扰技术来沉默主要的内切葡聚糖酶eg2基因，以期一步构建里氏木霉低纤维素酶背景的表达系统。获得了一株纤维素酶表达显著下降的11号转化子SUS6。该转化子中，cbh1的表达完全消失，而eg2基因的表达水平较出发菌株SUS5降低了98%。以该转化子为宿主表达外源基因NfBgl3A，两个代表性转化子的β-葡萄糖苷酶酶活最高分别为172.4和79.3 U·mL^-1，远高于以出发菌株（高纤维素酶背景）为宿主表达β-葡萄糖苷酶酶活（两个代表转化子分别为11.6和31.9 U·mL^-1）。因此，构建低纤维素酶背景的菌株作为表达宿主，还可明显的提高某些异源基因的表达水平。

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