Targeted knockout and plasmid-based transfer of NRPS/PKS for improving lipopeptide iturin A synthesis.

The nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) assembly lines are a large enzymatic machinery that facilitates the transfer and synthesis of intermediates between multimodular megasynthases through complex protein-protein interactions. Although NRPS/PKS systems display a highly sophisticated biosynthetic pathway, a similar strategy for the holistic editing of these gene clusters has not been described. In this study, practical gene-editing tools were developed for long gene clusters, allowing the efficient knockout of entire NRPS/PKS gene clusters. In addition, a strategy for the genome cleavage and plasmid capture of NRPS/PKS genes was developed. Using iturin A synthesis as a case study, the competing lipopeptide biosynthesis gene cluster was knocked out, and the NRPS/PKS gene cluster responsible for its production were transferred into a plasmid. The engineered strain exhibited significantly enhanced expression of iturin A NRPS/PKS genes and increased production of iturin A. This approach facilitates the optimization of NRPS/PKS systems and other long-gene cluster-derived natural product biosynthesis.