里氏木霉双基因同步缺失菌株的构建与甘露聚糖酶表达研究

在里氏木霉中建立了一个快速的双基因位点同步同源重组新方法，较好解决了里氏木霉基因逐个敲除周期长等问题。研究以里氏木霉自身甘露聚糖酶基因（man5A）为重组表达的报告基因，通过一步转化，将该基因定点整合入纤维二糖水解酶Ⅰ（cbh1）基因位点，同时缺失主要的两个纤维素酶基因（cbh1、cbh2），得到重组工程菌Man12。将重组工程菌Man12与出发菌株Tu6Δku70进行摇瓶发酵，结果显示，重组菌株的甘露聚糖酶产量比出发菌株提高10倍，而纤维素酶产量降低了60%，胞外总蛋白分泌水平降低了40%。Real-time PCR检测甘露聚糖酶基因（man5A）的转录水平，发现重组菌株较出发菌株提高了25倍。在里氏木霉中首次报道了通过一步转化实现两个基因同步定点整合的方法，对利用基因工程手段构建高效表达重组蛋白的里氏木霉工程菌株具有一定的指导意义。

Expression of mannanase in Trichoderma reesei by construction of a simultaneous double-gene disruption strain

A new rapid method for simultaneous homologous recombination was constructed in Trichoderma reesei for better solving long-cycle gene manipulation issues. The Trichoderma reesei recombinant strain Man12 was obtained by replacing the cellobiohydrolase Ⅰ(cbh1) ORF with the homologous mannanase gene (man5A) as a reporter. In addition, two major cellulases genes (cbh1, cbh2) were deleted simultaneously. The shake flask fermentation indicated that as compared with the parent strain, the production of mannanase activity of the recombinant strain Man12 was increased 10-fold, while the production of cellulases activity was reduced by 60% and the production of extracellular secreted protein was reduced by 40%. Real-time PCR analysis showed that mRNA level of the mannanase gene (man5A) in Man12 exhibited about 25-fold increase. This work will be the first report of a method, by which site-specific integration of two genes could be achieved simultaneously in one-step transformation in Trichoderma reesei, and should provide valuable knowledge for the engineering of high-level recombinant protein expression in Trichoderma reesei.