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.

Genome and transcriptome analysis of Bacillus velezensis BS‐37, an efficient surfactin producer from glycerol, in response to d‐/l‐leucine

“…Furthermore, many studies on replacing the promoter of the srfA operon to abrogate the effect of quorum sensing on the synthesis of surfactin have been reported (Jiao et al, ; Sun et al, ; Willenbacher et al, ). Unfortunately, the results of these attempts were far from satisfactory and did not lead to high surfactin yields, indicating that a complex regulatory network controls srfA expression, so that modifying the srfA operon is not a good choice (Jung et al, ).…”

“…Therefore, improving its yield and reducing its production cost will be of great significance. Most of the efficient surfactin producers reported to date prefer sucrose as the carbon source (Jiao et al, ; Yan, Wu, & Xu, ). By contrast, glycerol, which is a by‐product of biodiesel production, is a widely available and inexpensive source of carbon, making its conversion into value‐added products of great interest (Clomburg & Gonzalez, ; Faria et al, ).…”

A combination of genome reduction and promoter engineering can enhance surfactin production by Bacillus amyloliquefaciens LL3

“…The biosynthesis pathway of surfactin was presented in Figure S4, which mainly contains three parts: the synthesis and activation of branched-chain fatty acids; the synthesis of the four amino acids; and the assembly of peptide chain. For surfactin, it can hardly achieve a signi cant breakthrough in production only through traditional fermentation optimization because of its low yield in wild strains [16,35]. Strategies for surfactin overproduction were focused on strain modi cation recent years, such as substitution of the native promoter P srf of srfA operon [15,16], overexpressing transporters to enhance surfactin e ux [18], and modifying the regulators ComX and PhrC [35].…”

“…Considering the heterologous expression of srfA is challenging for which large genetic sequence (over 25 kb), substitution of the native srfA promoter by strong promoters is considered more bene cial for enhanced transcription of srfA operon [15,16,20]. In this study, two promoters PR suc and PR tpx with better transcription and expression levels were integrated into upstream of the srfA operon in GR167ID, generating mutant strains GR167IDS and GR167IDT, respectively.…”

Enhanced Expression of Pullulanase in Bacillus subtilis by New Strong Promoters Mined From Transcriptome Data, Both Alone and in Combination

“…Using this system, the obtained enzyme activity was 88.86 U/mL in shake flasks and 205 U/mL in a 5 L fermenter. In addition, the core elements of promoters, including the −35 and −10 regions (Jiao et al, 2017 ) and ribosome recognition site (Wang and Doi, 1984 ), have been optimized to enhance the promoter strength.…”