Module and individual domain deletions of NRPS to produce plipastatin derivatives in Bacillus subtilis

Abstract

BackgroundPlipastatin, an antifungal lipopeptide, is synthesized by a non-ribosomal peptide synthetase (NRPS) in Bacillus subtilis. However, little information is available on the combinatorial biosynthesis strategies applied in plipastatin biosynthetic pathway. In this study, we applied module or individual domain deletion strategies to engineer the plipastatin biosynthetic pathway, and investigated the effect of deletions on the plipastatin assembly line, as well as revealed the synthetic patterns of novel lipopeptides.ResultsModule deletion inactivated the entire enzyme complex, whereas individual domain (A/T domain) deletion within module 7 truncated the assembly line, resulting in truncated linear hexapeptides (C16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ala/Val). Interestingly, within the module 6 catalytic unit, the effect of thiolation domain deletion differed from that of adenylation deletion. Absence of the T6-domain resulted in a nonproductive strain, whereas deletion of the A6-domain resulted in multiple assembly lines via module-skipping mechanism, generating three novel types of plipastatin derivatives, pentapeptides (C16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu), hexapeptides (C16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ile), and octapeptides (C16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Gln-Tyr-Ile).ConclusionsNotably, a unique module-skipping process occurred following deletion of the A6-domain, which has not been previously reported for engineered NRPS systems. This finding provides new insight into the lipopeptides engineering. It is of significant importance for combinatorial approaches and should be taken into consideration in engineering non-ribosomal peptide biosynthetic pathways for generating novel lipopeptides.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0929-4) contains supplementary material, which is available to authorized users.

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“…The remaining modules, which are adjacent to the deletion, were recombined at different highly conserved sequence motifs that are characteristic of amino acid-incorporating modules of peptide synthetases. Gao et al (2018) stated that the entire deletion of an NRPS module, which was a producer of plipastatin, caused the inactivation of the enzyme. However, the authors observed that individual domain deletion (A and T domains) leads to the creation of three novel plipastatin derivatives.…”

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