以枯草芽孢杆菌(Bacillus subtilis)为表达平台,海藻糖合酶为表达蛋白,研究了单启动子和多个启动子串联对外源蛋白表达的影响。分别选择6个启动子构建PsrfA、P43单启动子和PabrB-spoVG-LytR-mmgA四个时期特异性启动子串联表达系统进行验证。根据实验结果分析,组成型启动子P43转录强度最高,摇瓶中酶活达到3 381 U/g,串联启动子PabrB-spoVG-LytR-mmgA强度次之。针对验证酶活最高的重组菌pHT01-P43-treS进行发酵优化,并在5 L发酵罐中进行放大实验,酶活达到7 215 U/g。实现了海藻糖合酶在枯草芽孢杆菌中自诱导高效表达,为获得高效率制备海藻糖合成酶的表达系统奠定了基础。
The effects of single promoter and multiple promoters on the expression of foreign proteins were studied using Bacillus subtilis as an expression platform and trehalose synthase as an expression protein. Six promoters were selected to construct single promoters PsrfA and P43. A four-stage specific promoter tandem expression system PabrB-spoVG-LytR-mmgA was also constructed for validation. According to the experimental results, the constitutive promoter P43 had the highest transcriptional strength, the enzyme activity in the shake flask reached 3 381 U/g. The tandem promoter PabrB-spoVG-LytR-mmgA had the second highest strength. The fermentation was optimized for recombinant enzyme pHT01-P43-treS, which was verified to have the highest activity, and the scale-up experiment was carried out in a 5 L fermentor, and the enzyme activity reached 7 215 U/g. The self-induced and high-efficiency expression of trehalose synthase in B. subtilis was achieved, which laid a foundation for obtaining a highly efficient expression system of trehalose synthase.
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