High-level surfactin production by Bacillus subtilis through integrated metabolic engineering and bioprocess optimization.

Abstract

Surfactin is a cyclic lipopeptide biosurfactant produced by Bacillus species and has considerable potential as a sustainable alternative to petroleum-derived surfactants. However, efficient surfactin production remains constrained by the limited productivity of native strains, inadequate precursor supply and suboptimal fermentation conditions. In this study, Bacillus subtilis ATCC 6051a, which harbors an intact endogenous sfp gene, was used as the strain background to develop a high-producing surfactin production system through integrated strain engineering and fermentation optimization. Building on a previously established T7 RNA polymerase expression platform, deletion of codY and spoIIAC together with mutation of abrB generated strain 164LP4, which produced 286mg/L surfactin. Subsequent enhancement of comA, sfp and srfA expression further improved surfactin biosynthesis, with T7 promoter-driven expression of the srfA operon in strain 164LP7-1 increasing the titer to 1.75g/L. Optimization of the cultivation conditions further increased surfactin production to 4.69g/L, while combined supplementation with L-leucine and sodium glutamate markedly increased the titer to 13.62g/L. Further engineering of fatty acid precursor metabolism and secretion-related processes, including enhanced cypC and secA expression, yielded strain 164LP15, which reached a shake-flask surfactin titer of 19.22g/L. In a 5-L fed-batch bioreactor, 164LP15 produced 52.32g/L surfactin within 27h, corresponding to an average productivity of 1.94g/L/h. The integration of multi-level strain engineering with fermentation optimization thus established B. subtilis ATCC 6051a as an effective platform for high-level surfactin production and provides a practical framework for the development of efficient microbial surfactin production systems.