Bacillus licheniformis DW2 is an important industrial strain for bacitracin production, and it is also used for biochemical production, however, the lack of effective toolkit for precise regulation of gene expression hindered its application seriously. Here, a gradient strength promoter library was constructed based on bacitracin synthetase gene cluster promoter P_bacA. First, different P_bacA promoter variants were constructed via coupling P_bacA with various 5′-UTRs, and expression ranges of 32.6–741.8% were attained among these promoters. Then, three promoters, P_Ubay (strong), P_bacA (middle), and P_Undh (weakest), were applied for red fluorescent protein (RFP) and keratinase expression assays, and these promoters were proven to have good universality for different proteins. Second, the promoter of bacitracin synthetase gene cluster was replaced by these three promoters, and bacitraicn titer was enhanced by 14.62% when P_Ubay was applied, which was decreased by 98.05% under the mediation of P_Undh compared with that of the original strain DW2. Third, promoters P_Ubay, P_UyvgO, and P_Undh were selected to regulate the expression levels of critical genes that are responsible for pucheriminic acid synthesis, and pucheriminic acid yield was increased by 194.1% via manipulating synthetic and competitive pathways. Finally, promoters P_Ubay, P_bacA, and P_Undh were applied for green fluorescent protein (GFP) and RFP expression in Escherichia coli, and consistent effects were attained based on our results. Taken together, a gradient strength promoter library was constructed in this research, which provided an effective toolkit for fine-tuning gene expression and reprogramming metabolite metabolic flux in B. licheniformis.

中文翻译：

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地衣芽孢杆菌微调基因表达梯度强度启动子文库的构建与表征

Bacillus licheniformis DW2是杆菌肽生产的重要工业菌株，也用于生化生产，但缺乏有效的基因表达精准调控工具包严重阻碍了其应用。在此，基于杆菌肽合成酶基因簇启动子P _bacA构建了梯度强度启动子文库。首先，通过将 P _{bacA与各种 5'-UTR 偶联构建不同的 P bacA}启动子变体，在这些启动子中达到了 32.6-741.8% 的表达范围。然后，三个启动子，P _Ubay（强）、P _bacA（中）和 P _Undh（最弱），用于红色荧光蛋白（RFP）和角蛋白酶表达测定，这些启动子被证明对不同的蛋白质具有良好的普遍性。其次，将杆菌肽合成酶基因簇的启动子替换为这三个启动子，与原菌株DW2相比，在P _{Ubay作用下，杆菌肽滴度提高了14.62%，在P Undh}的介导下降低了98.05%。. 三、发​​起人 P _Ubay、 P _UyvgO和 P _Undh被选择来调节负责合成pucheriminic酸的关键基因的表达水平，通过操纵合成和竞争途径，pucheriminic酸产量增加了194.1%。最后，将启动子 P _Ubay、 P _bacA和 P _Undh应用于大肠杆菌中的绿色荧光蛋白 (GFP) 和 RFP 表达，根据我们的结果获得了一致的效果。综上所述，本研究构建了梯度强度启动子文库，为地衣芽孢杆菌基因表达微调和代谢物代谢通量重编程提供了有效的工具包。