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

Abstract

AbstractBackgroundPlipastatin is a potentBacillusantimicrobial 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. subtilissynthesizes plipastatin by a non-ribosomal peptide synthetase encoded by theppsABCDEoperon. In this study,B. subtilis3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.ResultsThe 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain,sfpgene is inactive. Accordingly, the first step was an integration of a repairedsfpversion in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expresseddegQversion fromB. subtilisDSM10Tstrain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls theppsABSDEoperon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pvegled to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaireddegQexpression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatorycomSwhich is located withinsrfABand 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.ConclusionsThis study provides evidence thatdegQstimulates 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-producerB. subtilisstrain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production byB. subtilisplipastatin mono-producer strains.