Accessing Underexplored Biosynthetic Potential by Initiation Unit Engineering of Nonribosomal Peptide Synthetases in <i>Proteobacteria</i>

Nonribosomal peptide synthetases (NRPSs) represent a valuable yet underexplored resource for producing bioactive natural products. However, most NRPSs remain silenced potentially due to factors such as dysfunction of the initiation unit. The starter condensation (Cs) domain of the initiation unit catalyzes the lipoinitiation of nonribosomal peptides via the incorporation of an <i>N</i>-terminal fatty acyl chain. The concept of initiation unit engineering introduced herein encompasses the replacement of the native initiation unit of NRPSs with a foreign and well-characterized Cs domain-containing initiation unit to activate the NRPS and optimize its expression. This strategy was employed herein to successfully access three of the six previously silent NRPS pathways in <i>Mycetohabitans rhizoxinica</i> HKI 454, a bacterium of the class β-<i>proteobacteria</i>, resulting in the identification of three classes of lipopeptides. This strategy was then extended to access two NRPS pathways in <i>Pseudomonas syringae</i> (γ-<i>proteobacteria</i>) and obtain novel lipopeptides, thereby establishing a feasible complement to existing genome mining strategies for natural product discovery. Furthermore, change of the initiation regions of biosynthetic pathways of nonlipidated chitinimide (β-<i>proteobacteria</i>) and pseudotetraivprolide (γ-<i>proteobacteria</i>) with heterologous Cs-containing initiation units enabled the successful incorporation of fatty acyl chains into the <i>N</i>-terminus of both peptide backbones, launching a workable approach to create artificial lipopeptides. Overall, this study provides a practical strategy for the rational recovery of silent BGCs and introduction of fatty acyl chains into nonribosomal peptides, at least in <i>Proteobacteria</i>, thereby enriching genome mining and combinatorial biosynthesis approaches for accessing the underexplored biosynthetic potential of NRPSs from various bacteria.