An Integrated Strategy to Reprogram NRPS Initiation Modules for Long-Chain Surfactin Biosynthesis

Engineering of nonribosomal peptide synthetases (NRPSs) is frequently limited by loss of biosynthetic productivity following domain recombination, particularly within the initiation module, where interdomain compatibility plays a critical yet underexplored role. Although substrate selectivity can often be reprogrammed by replacing the starter condensation (Cs) domain, such modifications are typically accompanied by severe reductions in productivity, imposing a major bottleneck in NRPS engineering. To produce long-chain surfactin homologs, we replaced the native Cs domain of the surfactin synthetase SrfAA with a homologous Cs domain from a long-chain lipopeptide-producing NRPS. This enabled incorporation of long-chain fatty acyl substrates but resulted in a substantial decrease in production titer. Systematic analyses suggested that this productivity loss is primarily associated with suboptimal interdomain compatibility within the engineered initiation module rather than intrinsic deficiencies of the substituted Cs domain. By combining targeted mutagenesis of the Cs domain with interdomain engineering and fine-tuning of the carrier (T₁) domain, we achieved recovery of production through combinatorial optimization. Notably, improved productivity was largely achieved without altering acyl-chain specificity, supporting the view that catalytic efficiency can be enhanced independently of substrate selection. These findings suggest that productive reprogramming of NRPS initiation modules requires coordinated optimization of both domain-specific properties and interdomain compatibility.