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Organization and characterization of genetic regions in Bacillus subtilis subsp. krictiensis ATCC55079 associated with the biosynthesis of iturin and surfactin compounds.

ABSTRACT: Bacillus subtilis subsp. krictiensis ATCC55079 produces the cyclic lipopeptide antibiotics iturin A-F as well as several surfactins. Here, we analyzed and characterized the biosynthetic genes associated with iturin and surfactin production in this strain. We aligned the sequences of each iturin and surfactin synthetase ORF obtained from a genomic library screen and next generation sequencing. The resulting 37,249-bp and 37,645-bp sequences associated with iturin and surfactin production, respectively, contained several ORFs that are predicted to encode proteins involved in iturin and surfactin biosynthesis. These ORFs showed higher sequence homologies with the respective iturin and surfactin synthetase genes of B. methylotrophicus CAU B946 than with those of B. subtilis RB14 and B. subtilis ATCC6633. Moreover, comparative analysis of the secondary metabolites produced by the wild-type and surfactin-less mutant (with a spectinomycin resistance cassette inserted into the srfAB gene within the putative surfactin gene region) strains demonstrated that the mutant strain showed significantly higher antifungal activity against Fusarium oxysporum than the wild-type strain. In addition, the wild-type strain-specific surfactin high performance liquid chromatography (HPLC) peaks were not observed in the surfactin-less mutant strain. In contrast, the iturin A peak detected by HPLC and liquid chromatography-mass spectrometry (LC/MS) in the surfactin-less mutant strain was 30% greater than that in the wild-type strain. These results suggested that the gene cluster we identified is involved in surfactin biosynthesis, and the biosynthetic pathways for iturin and surfactin in Bacillus strains producing both iturin and surfactin may utilize a common pathway.

SUBMITTER: Kim YT

PROVIDER: S-EPMC5739386 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Organization and characterization of genetic regions in Bacillus subtilis subsp. krictiensis ATCC55079 associated with the biosynthesis of iturin and surfactin compounds.

Kim Young Tae YT Park Byung Keun BK Kim Sung Eun SE Lee Won Jung WJ Moon Jae Sun JS Cho Min Seop MS Park Ho-Yong HY Hwang Ingyu I Kim Sung Uk SU

PloS one 20171221 12

Bacillus subtilis subsp. krictiensis ATCC55079 produces the cyclic lipopeptide antibiotics iturin A-F as well as several surfactins. Here, we analyzed and characterized the biosynthetic genes associated with iturin and surfactin production in this strain. We aligned the sequences of each iturin and surfactin synthetase ORF obtained from a genomic library screen and next generation sequencing. The resulting 37,249-bp and 37,645-bp sequences associated with iturin and surfactin production, respect ...[more]

PMID: 29267290

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Project description:BackgroundSurfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (L-glutamate, L-aspartate, L-leucine and L-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear.ResultsIn this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of L-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of L-leucine and L-valine, not only improved surfactin production but also increased the proportion of the C14 isoform.ConclusionsThis study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals.

Project description:The anaerobic growth of B. subtilis to synthesize surfactin poses an alternative strategy to conventional aerobic cultivations. In general, the strong foam formation observed during aerobic processes represents a major obstacle. Anaerobic processes have, amongst others, the distinct advantage that the total bioreactor volume can be exploited as foaming does not occur. Recent studies also reported on promising product per biomass yields. However, anaerobic growth in comparison to aerobic processes has several disadvantages. For example, the overall titers are comparably low and cultivations are time-consuming due to low growth rates. B. subtilis JABs24, a derivate of strain 168 with the ability to synthesize surfactin, was used as model strain in this study. Ammonium and nitrite were hypothesized to negatively influence anaerobic growth. Ammonium with initial concentrations up to 0.2 mol/L was shown to have no significant impact on growth, but increasing concentrations resulted in decreased surfactin titers and reduced nitrate reductase expression. Anaerobic cultivations spiked with increasing nitrite concentrations resulted in prolonged lag-phases. Indeed, growth rates were in a similar range after the lag-phase indicating that nitrite has a neglectable effect on the observed decreasing growth rates. In bioreactor cultivations, the specific growth rate decreased with increasing glucose concentrations during the time course of both batch and fed-batch processes to less than 0.05 1/h. In addition, surfactin titers, overall Y P/X and Y P/S were 53%, ∼42%, and ∼57% lower than in serum flask with 0.190 g/L, 0.344 g/g and 0.015 g/g. The Y X/S, on the contrary, was 30% lower in the serum flask with 0.044 g/g. The productivities q were similar with ∼0.005 g/(g⋅h). However, acetate strongly accumulated during cultivation and was posed as further metabolite that might negatively influence anaerobic growth. Acetate added to anaerobic cultivations in a range from 0 g/L up to 10 g/L resulted in a reduced maximum and overall growth rate μ by 44% and 30%, respectively. To conclude, acetate was identified as a promising target for future process enhancement and strain engineering. Though, the current study demonstrates that the anaerobic cultivation to synthesize surfactin represents a reasonable perspective and feasible alternative to conventional processes.

Dataset Information

Organization and characterization of genetic regions in Bacillus subtilis subsp. krictiensis ATCC55079 associated with the biosynthesis of iturin and surfactin compounds.

Publications

Organization and characterization of genetic regions in Bacillus subtilis subsp. krictiensis ATCC55079 associated with the biosynthesis of iturin and surfactin compounds.

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