title: In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters.
abstract: 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, PgroE, Pcdd, PrplK, and PsspE, 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, PgroE, was selected to replace the native THY-7 surfactin synthase (SrfA) promoter through single-cross homologous recombination; however, the resulting engineered strain containing the PgroE substitution did not synthesize surfactin. The sucrose-inducible promoters PsacB and PsacP were then substituted in place of PsrfA, 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 PgroE and PsacB ribonucleic antiterminator (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 PgroE-lacO fusion and then substituted for the chromosomal PsrfA 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.
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.|Key Laboratory of Industrial Biocatalysis (Tsinghua University), Ministry of Education, Beijing, 100084, China.
authors.forename: Song
authors.initials: S
authors.lastname: Jiao
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.|Key Laboratory of Industrial Biocatalysis (Tsinghua University), Ministry of Education, Beijing, 100084, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.|Key Laboratory of Industrial Biocatalysis (Tsinghua University), Ministry of Education, Beijing, 100084, China.|Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.|Key Laboratory of Industrial Biocatalysis (Tsinghua University), Ministry of Education, Beijing, 100084, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.|Key Laboratory of Industrial Biocatalysis (Tsinghua University), Ministry of Education, Beijing, 100084, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.|Key Laboratory of Industrial Biocatalysis (Tsinghua University), Ministry of Education, Beijing, 100084, China.
authors.forename: Zhongyao
authors.initials: Z
authors.lastname: Shen
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D020869:Gene Expression Profiling; D055666:Lipopeptides / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013501:Surface-Active Agents; D059467:Transcriptome
chemical_list: D055666:Lipopeptides; D013501:Surface-Active Agents
keywords: Bacillus subtilis; artificial inducible promoter; biosurfactant; surfactin synthesis