Construction, Model-based Analysis and Characterization of a Synthetic Promoter Library for Fine-tuned Gene Expression in Bacillus subtilis

Bacillus subtilis is the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. To extend its productivity, Bacillus subtilis needs to undergo further engineering, including the development of applicable promoter libraries. We constructed constitutive promoter library based on -10 -35 consensus sequences recognized by sigA, Bacillus subtilis -16 consensus and an AT-rich region upstream. Using green fluorescent protein as a reporter, a total of 5000 promoters were randomly picked and the GFP expression level was investigated. The strength of the library covers a range of 200 fold. Firstly, 225 potential promoters of various strengths were isolated and sequenced for promoter analysis. A model describing the promoter strength was built and validated. Secondly, the library was employed to precisely regulate adenylosuccinate (sAMP) synthase, which is encoded by purA, one of the most important enzymes involved in the de novo biosynthesis of purine metabolites. We assess the impact of sAMP levels on inosine production. Finally, we developed a strategy to improve promoter strength by constructing the promoter clusters consisted of multiple promoters in tandem stemmed from our constructed library. Application of the promoter clusters in secretory production of endoxylanase proved that it was efficient. This is the first report of synthetic promoters constructed in Bacillus subtilis, and our screening strategy together with the use of synthetic promoters of various strengths will contribute to the future engineering of Bacillus subtilis.