Scarless Genomic Point Mutation to Construct a Bacillus subtilis Strain Displaying Increased Antibiotic Plipastatin Production

Jeong, Dong‐Hoon; So, Younju; Lim, Hayeon; Park, Seung-Hwan; Choi, Soo Keun

doi:10.4014/jmb.1710.10034

Cited by 7 publications

(7 citation statements)

References 25 publications

(35 reference statements)

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“…The fermentation product was extracted and analyzed by HPLC. The HPLC profile showed that the production of two main components of plipastatin and surfactin, for which the retention time of plipastatin was 8− 22 min and that of surfactin was 28−38 min 34,35 (Figure 2). This was consistent with the previously reported data that plipastatin was in the range of 10−25 min and surfactin was in the range of 30−45 min.…”

Section: Analysis Of Fermentation Products Of the Gene-modified Strainmentioning

confidence: 99%

Mining New Plipastatins and Increasing the Total Yield Using CRISPR/Cas9 in Genome-Modified Bacillus subtilis 1A751

Zou

Maina

Zhang

et al. 2020

J. Agric. Food Chem.

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CRISPR/Cas9 is one of the robust and effective gene manipulation tools which has been widely applied in various organisms. In this study, the plipastatin gene cluster was successfully expressed in genome-modified Bacillus subtilis 1A751 by disrupting the surfactin operon (srf) through CRISPR/Cas9 technology. The presumed plipastatin biosynthetic pathway was proposed based on the analysis of its biosynthetic gene cluster. Two new plipastatins were identified by a combination of ultra-high performance liquid chromatography-coupled electron spray ionization-tandem mass spectrometry and gas chromatography−mass spectrometry analyses, together with nine known plipastatins or their derivatives. The yield of plipastatin was as high as 1600 mg/L which is the highest reported to date. Antimicrobial experiments revealed that its methanolic extracts exhibited powerful inhibitory effects on the growth of the tested pathogens and fungi. The results from this investigation highlight the remarkable utility of CRISPR/Cas9 in mining new plipastatins and increasing the total plipastatin yield, providing a new pipeline for the industrial application of plipastatin.

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Section: Analysis Of Fermentation Products Of the Gene-modified Strainmentioning

confidence: 99%

Mining New Plipastatins and Increasing the Total Yield Using CRISPR/Cas9 in Genome-Modified Bacillus subtilis 1A751

Zou

Maina

Zhang

et al. 2020

J. Agric. Food Chem.

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“…In fact, DegQ stimulates autophosphorylation of DegS sensor kinase resulting in enhanced phospho-transfer to DegU response regulator [29,35]. As a result, the phosphorylated and activated DegU-P version causes higher expression of ppsABCDE operon and increases the plipastatin production [36]. In this study, repair of degQ expression (strain BMV10) ensured DegU-P activation, which doubled the production of plipastatin compared to parental BMV9 strain (3NA sfp+).…”

Section: Discussionmentioning

confidence: 63%

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

Vahidinasab

Lilge

Reinfurt

et al. 2020

Preprint

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Background: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.Results: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain ensuring stimulation of DegU-P regulon (strain BMV10). Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition.Conclusions: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, it seems that surfactin synthetase or one of its subunits positively regulates plipastatin production by an unknown mechanism. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

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“…In fact, DegQ stimulates autophosphorylation of DegS sensor kinase resulting in enhanced phosphotransfer to DegU response regulator [29,35]. As a result, the phosphorylated and activated DegU-P version causes higher expression of ppsABCDE operon and increases the plipastatin production [36]. In this study, repair of degQ expression (strain BMV10) ensured DegU-P activation, which doubled the production of plipastatin compared to parental BMV9 strain (3NA sfp +).…”

Section: Discussionmentioning

confidence: 65%

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

Vahidinasab

Lilge

Reinfurt

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.Results: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition.Conclusions: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

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Scarless Genomic Point Mutation to Construct a Bacillus subtilis Strain Displaying Increased Antibiotic Plipastatin Production

Cited by 7 publications

References 25 publications

Mining New Plipastatins and Increasing the Total Yield Using CRISPR/Cas9 in Genome-Modified Bacillus subtilis 1A751

Mining New Plipastatins and Increasing the Total Yield Using CRISPR/Cas9 in Genome-Modified Bacillus subtilis 1A751

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

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