过表达TatAdCd转位酶对枯草芽孢杆菌脂肪酶分泌的影响

Effect of TatAdCd translocases overexpression on the secretion of lipase in Bacillus subtilis

摘要:

【目的】研究过表达枯草芽孢杆菌Tat运输途径的TatAdCd转位酶对促进脂肪酶分泌的影响。【方法】用cdd基因的串联启动子和前导区，替换tatAD-CD操纵子的启动子和前导区，并在染色体sacB基因位点整合表达；采用qRT-PCR方法表征tatAD-CD操纵子的表达水平；用脂肪酶表达质粒pHP13L转化TatAdCd转位酶过表达菌株，构建产脂肪酶重组菌。通过测定脂肪酶活性，以及聚丙烯酰胺凝胶电泳，考察TatAdCd转位酶过表达对脂肪酶分泌的影响。【结果】tatAD-CD操纵子被过表达，其胞内mRNA相对水平提高了185倍。TatAdCd转位酶的过表达，使脂肪酶发酵单位提高了40%。【结论】使用cdd基因的串联启动子和前导区，能够有效地过表达目的基因；枯草芽孢杆菌脂肪酶可以同时经由Sec途径和Tat途径分泌；过表达TatAdCd转位酶，能够显著提高脂肪酶的分泌量。

Abstract:

[Objective] This study focuses on the influence of overexpression of TatAdCd translocases in Tat pathway on the secretion of LipA in Bacillus subtilis. [Methods] The promoter and mRNA leader region of the operon tatAD-CD were replaced by the tandem promoter and mRNA leader region of cdd gene and this modified gene was inserted into sacB gene. The expression level of the tatAD-CD operon was characterized by qRT-PCR. We transformed the plasmid pHP13L which can express lip gene of Bacillus subtilis excessively into the recombinant strain. The influence of overexpression of TatAdCd translocases on the secretion of LipA was assessed by SDS-PAGE and measuring the enzyme activity of the recombinant in fermentation broth. [Results] The modified tatAD-CD operon was expressed successfully. Compared with the control strain, the relative transcription level was improved by 185 times. The enzyme activity of recombinant in fermentation broth was increased by 40% due to overexpression of TatAdCd translocases. [Conclusion] The tandem promoter and mRNA leader region of the cdd gene can increase the expression level of the target gene effectively. LipA can be secreted to extracellular through Sec pathway and Tat pathway in Bacillus subtilis. The overexpression of tatAD-CD operon can promote the secretion of LipA.