短小芽孢杆菌(Brevibacillus choshinensis)是一种潜在的高效表达系统,但由于高效强启动子的缺乏严重限制了该表达系统的应用。该研究的主要目的是为B.choshinensis表达系统提供更多新高效强启动子。在该研究中,首先通过转录组测序技术获得B.choshinensis的转录组数据。然后基于基因的转录水平及功能,筛选高表达目的基因。基于筛选到的目的基因的启动子序列,以α-淀粉酶为报告蛋白构建了强启动子筛选文库。将含有不同启动子的重组菌摇瓶发酵后,以胞外α-淀粉酶活性为筛选指标,获得了内源性强启动子PE和PF,对应的重组菌BCWES和BCWFS胞外α-淀粉酶活性分别是常用的强启动子P2介导的对照菌BCWPS胞外α-淀粉酶活性的2.3和1.3倍。最后以蔗糖异构酶和角质酶为新的报告蛋白证明了PE启动子具有通用性,且经3 L罐发酵进一步验证了重组菌BCWES高分泌生产α-淀粉酶的能力。该研究丰富了可用于B.choshinensis表达系统的强启动子数量,促进了B.choshinensis作为一种新高效表达系统的开发与应用。
Brevibacillus choshinensis is a potentially efficient expression system, however, the lack of efficient strong promoters severely limits the application of this expression system. To provide more new efficient strong promoters for B. choshinensis expression system, firstly, the transcriptome data of B. choshinensis was obtained by RNA-Seq technology. Then, based on the transcription level and function of the genes, the highly expressed target genes were screened. Based on the promoter sequences of selected target genes, a strong promoter screening library was constructed using α-amylase as the reporter protein. After shake flask fermentation of recombinant strains with different promoters, the endogenous strong promoters PE and PF were obtained using extracellular α-amylase activity as the screening index. The extracellular α-amylase activities of recombinant strains BCWES and BCWFS, mediated by promoter PE and PF respectively, were 2.3 and 1.3-fold greater than that of the control strain BCWPS, which was mediated by the well-characterized promoter P2. Finally, sucrose isomerase and cutinase were used as new reporter proteins to prove the universality of the PE promoter, and the ability of BCWES to efficiently produce α-amylase was further confirmed in 3 L fermenter fermentation. This study enriched the number of strong promoters available for B. choshinensis expression system, and promoted the development and application of B. choshinensis as a new efficient expression system.
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