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

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