Influence of promoters on the production of fengycin in Bacillus spp.

Yaseen, Yazen; Gancel, Frédérique; Drider, Djamel; Béchet, Max; Jacques, Philippe

doi:10.1016/j.resmic.2016.01.008

Cited by 38 publications

(26 citation statements)

References 38 publications

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“…For instance, promoter exchange of native P pps promoter against native P fen from B. subtilis 21332 caused no plipastatin overproduction. In contrast, a tenfold higher plipastatin production was obtained when P fen from strain BBG21 (a spontaneous mutant of B. subtilis ATCC 21332) was integrated [ 37 ]. In the following step, it was observed that the combination of both repair of degQ expression and the P pps promoter exchange (BMV14), had no additional effect on the plipastatin titer compared to BMV11 (constitutive plipastatin producer).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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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 (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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Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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“…BBG235, BBG236 (pnpA mutant strains), BBG201 and BBG258 (wild-type strains) were inoculated in 50 mL of Landy MOPS and cultured at 30 °C, 160 rev min −1 for 16 h, when the maximum fengycin production was observed (Yaseen et al 2016). An equivalent volume of about 2 10 cells was obtained from each point of the kinetics previously defined; these volumes were centrifuged (11,000 g, − 9 °C, 5 min), the supernatant was discarded, and the pellet was stored in 1 ml Ambion RNA later (Thermo Fisher Scientific) at − 20 °C.…”

Section: Rna Extraction and Rt-pcr Experimentsmentioning

confidence: 99%

Polynucleotide phosphorylase is involved in the control of lipopeptide fengycin production in Bacillus subtilis

et al. 2018

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Bacillus subtilis is a wealth source of lipopeptide molecules such as iturins, surfactins and fengycins or plipastatins endowed with a range of biological activities. These molecules, designated secondary metabolites, are synthesized via non-ribosomal peptides synthesis (NRPS) machinery and are most often subjected to a complex regulation with involvement of several regulatory factors. To gain novel insights on mechanism regulating fengycin production, we investigated the effect of the fascinating polynucleotide phosphorylase (PNPase), as well as the effect of lipopeptide surfactin. Compared to the wild type, the production of fengycin in the mutant strains B. subtilis BBG235 and BBG236 altered for PNPase has not only decreased to about 70 and 40%, respectively, but also hampered its antifungal activity towards the plant pathogen Botrytis cinerea. On the other hand, mutant strains BBG231 (srfAA) and BBG232 (srfAC) displayed different levels of fengycin production. BBG231 had registered an important decrease in fengycin production, comparable to that observed for BBG235 or BBG236. This study permitted to establish that the products of pnpA gene (PNPase), and srfAA (surfactin synthetase) are involved in fengycin production.

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“…For instance, promoter exchange of native P pps promoter against native P fen from B. subtilis 21332 caused no plipastatin overproduction. In contrast, a 10-fold higher plipastatin production was obtained when P fen from strain BBG21 (a spontaneous mutant of B. subtilis ATCC 21332) was integrated [37]. In the following step, it was observed that the combination of both repair of degQ expression and the P pps promoter exchange (BMV14), had no signi cant effect on the plipastatin titer compared to BMV11 (constitutive plipastatin producer).…”

Section: Discussionmentioning

confidence: 99%

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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Influence of promoters on the production of fengycin in Bacillus spp.

Cited by 38 publications

References 38 publications

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

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

Polynucleotide phosphorylase is involved in the control of lipopeptide fengycin production in Bacillus subtilis

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

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