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In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters
Author(s) -
Jiao Song,
Li Xu,
Yu Huimin,
Yang Huan,
Li Xue,
Shen Zhongyao
Publication year - 2017
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26197
Subject(s) - surfactin , bacillus subtilis , promoter , biology , chemistry , titer , microbiology and biotechnology , biochemistry , gene , genetics , bacteria , gene expression , antibody
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.