Exploring the metabolic burden of surfactin biosynthesis and the metabolic costs of srfA operon expression in Bacillus subtilis

Abstract The biosynthesis of surfactin, a potent lipopeptide biosurfactant produced by Bacillus subtilis , imposes a significant metabolic burden on the host organism. Understanding the metabolic costs associated with surfactin production, specifically the ATP demand and precursor diversion resulting from the expression of the large srfAA-AD operon ( srfA operon), is crucial for optimizing production strains. In this study, the metabolic burden and growth impacts associated with surfactin biosynthesis in B. subtilis were quantitatively evaluated by comparing a reference surfactin-producing strain (BMV9) with two mutant strains: BMV12, which lacks the srfA operon, and BMV33, which retains the srfA operon but lacks the sfp gene. Our analysis included theoretical calculations of ATP and NADPH + H + requirements for de novo surfactin synthesis, and we measured growth behavior and carbon and nitrogen source consumption. Results indicated that surfactin production significantly reduces biomass yield ( Y X/S ) and specific growth rates due to the metabolic costs of expressing the non-ribosomal peptide synthetase (NRPS) and the diversion of key precursors. Notably, BMV12 exhibited higher growth rates compared to the surfactin-producing strain. Proteome analyses further revealed differential protein abundance in non-surfactin-producing strains, indicating altered metabolic pathways that may relieve the metabolic burden associated with surfactin synthesis. These findings highlight the complex trade-offs between secondary metabolite production and cellular growth, providing a foundation for metabolic engineering strategies aimed at optimizing surfactin yields while minimizing metabolic costs.