Enhancing surfactin production in B. velezensis Bs916 combined cumulative mutagenesis and expression key enzymes.

Surfactin is a lipopeptide which has attracted massive attention due to its versatile bioactive properties, although it has less commercial application due to its low yield in wild strains. The B. velezensis Bs916 has enable commercial production of surfactin due to its outstanding capacity to synthesize lipopeptides and amenable to genetically engineering. In this study, 20 derivatives with high surfactin production were obtained firstly by transposon mutagenesis and knockout techniques, and the surfactin yield of the derivative H5 (△GltB) was increased approximately 7-folds, reaching to 1.48 g/L. The molecular mechanism of high yielding surfactin in △GltB was investigated by the transcriptomic and KEGG pathway analysis. The results indicated that △GltB enhanced its ability to synthesize surfactin mainly by promoting transcription of the srfA gene cluster and inhibiting degradation of some key precursors such as fatty acid. Secondly, we obtained a triple mutant derivative BsC3 by cumulative mutagenesis of the negative genes GltB, RapF, and SerA, and it could increase the surfactin titer by twofold, reaching to 2.98 g/L. Thirdly, we achieved overexpression of two key rate-limiting enzyme genes, YbdT, and srfAD, and the derivative BsC5 which further increased the surfactin titer by 1.3-fold, reaching to 3.79 g/L. Finally, the yield of surfactin by derivatives was significantly increased under the optimal medium, particularly the BsC5 increased the surfactin titer to 8.37 g/L. To the best of our knowledge, this is one of the highest yields that have been reported. Our work may pave way for large scale production of surfactin by B. velezensis Bs916. KEY POINTS: • Elucidation of the molecular mechanism of surfactin high-yielding transposon mutant. • Genetically engineering of B. velezensis Bs916 surfactin titer to 8.37 g/L for large scale preparation.