In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters

Surfactin‐family lipopeptides are green biosurfactants with substantial industrial potential. The major problem prohibiting surfactin use is the low titer of the wild producer, Bacillus subtilis . Using transcriptomic analysis, four strong promoters, P groE , P cdd , P rplK , and P sspE , were identified and cloned from the genome of B. subtilis THY‐7, a novel surfactin producer that has been identified from soil with a 0.55 g/L surfactin titer. An optimal promoter, P groE , was selected to replace the native THY‐7 surfactin synthase (SrfA) promoter through single‐cross homologous recombination; however, the resulting engineered strain containing the P groE substitution did not synthesize surfactin. The sucrose‐inducible promoters P sacB and P sacP were then substituted in place of P srfA , and the resulting engineered strains produced 1.09 and 0.22 g/L surfactin, respectively. An artificial, sucrose‐inducible Pg1 promoter was produced through fusion of the P groE and P sacB ribonucleic a nti t erminator (RAT), and the engineered strain containing the Pg1‐substitution produced a surfactin titer of 1.44 g/L. An artificial IPTG‐inducible promoter, Pg2, was constructed from a P groE ‐lacO fusion and then substituted for the chromosomal P srfA locus, and the surfactin titer of the resulting THY‐7/ Pg2 ‐ srfA increased to 5.98 g/L. The driving capacity of Pg2 was further improved by the inclusion of two point mutations in the −35 and −10 regions to produce the novel promoter Pg3. Pg3 exhibited super‐strong activity as measured by lacZ reporter gene overexpression (approximately 3000 U). The Pg3‐substitution strain THY‐7/ Pg3 ‐ srfA produced up to 9.74 g/L surfactin in a 5 L fermentor. The maximum productivity was 0.30 g/L/h, and the greatest yield reached 0.14 g surfactin/g sucrose. Biotechnol. Bioeng. 2017;114: 832–842. © 2016 Wiley Periodicals, Inc.