Construction and Characterization of a Gradient Strength Promoter Library for Fine-Tuned Gene Expression in Bacillus licheniformis.

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 PbacA. First, different PbacA promoter variants were constructed via coupling PbacA with various 5'-UTRs, and expression ranges of 32.6-741.8% were attained among these promoters. Then, three promoters, PUbay (strong), PbacA (middle), and PUndh (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 PUbay was applied, which was decreased by 98.05% under the mediation of PUndh compared with that of the original strain DW2. Third, promoters PUbay, PUyvgO, and PUndh 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 PUbay, PbacA, and PUndh 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.