| In this article, a rapid system was constructed for double-gene simultaneous site-specific integration in the filamentous fungus Trichoderma reesei to further improve the homologous recombine expression level of mannanase, through disruption of the two highest endogenous expression cellobiohydrolase(CBH Ⅰ, CBHⅡ) and deletion of the transcription repressor Crel. The Trichoderma reesei recombinant strain TrMan12was obtained by replacing the cellobiohydrolase I (cbhl) ORF with the homologous mannanase gene (man5A) as a reporter, in addition, the two major cellulases genes (cbh1,cbh2) were disrupted simultaneously. Also, the recombinant strain TrMan11which was cbh1-negative was constructed by replacing the cellobiohydrolase I (cbh1) ORF with the homologous mannanase gene (man5A) under the cbhl promoter. Furthermore, The strain TrMan12was employed and deleted the transcription repressor Crel, thus obtained the recombinant strain TrMan13whose cbhl,cbh2,crel gene were deleted in the meantime. The shake flask fermentation indicated that compared to the parent strain, the production of mannanase activity of the recombinant strain TrManll was increased7-fold, where as the strain TrMan12was increased10-fold. While the production of cellulases activity and the production of extracellular secreted protein was reduced in varying degrees. Real-time PCR analysis showed that mRNA level of the mannanase gene (man5A) in the three different recombinant strain, also exhibited varied increase extent compared to the parent strain. This work will be the first report of a rapid system by which site-specific integration of two genes could be achieved simultaneously in one-step transformation in Trichoderma reesei, and further disrupt the major cellobiohydrolase and the transcription factor. Our study herein at least should provide valuable knowledge for the engineering of high-level recombinant protein expression in Trichoderma reesei. |
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