Improvement surfactin production by substitution of promoters in Bacillus subtilis TD7

Surfactin is one of the most representative biosurfactants and exhibits excellent surface activity plus other biological effects. It has potential applications in microbial enhanced oil recovery, environmental bioremediation, agricultural bio-control, pharmacy, cosmetics and food industries. The low yield of the surfactant from wild strains is a key restriction for industrial applications. The construction of genetically engineered bacteria by promoter substitution is an effective method to enhance surfactin production, as the promoter is a key element in gene expression. This study focuses on constructing strains with efficient surfactin production by replacing the native srfA promoter by strong promoters. In this study, two different promoter patterns with different homology arm positions were used for srfA promoter substitution. The most efficient installation way was identified as the sequence between the transcriptions start site and ribosome binding site of srfA. Moreover, eight endogenous strong auto-inducible phase-dependent promoters were chosen to substitute the native promoter of srfA using an effective substitution by the CRISPR-Cas9 system. As a result, high surfactin yielding strains with potential application in industry were constructed. According to the results, three constructed strains with promoters P43, PspoVG, and PyvyD showed increased yields of 3.5, 2.8, and 2.3 times over the wild stain B. subtilis TD7.