Enhanced biosynthesis and characterization of surfactin isoforms with engineered Bacillus subtilis through promoter replacement and Vitreoscilla hemoglobin co-expression

[Display omitted] •An engineered Bacillus subtilis with high surfactin titer 10.2 g/L was constructed.•Vitreoscilla hemoglobin co-expression successfully enhanced surfactin biosynthesis.•Surfactin isoform C15 showed the best surface activity of the surfactin products. The surfactin produced by Bacillus subtilis has great potential in enhanced oil recovery, agriculture and bio-mediation. Its further applications, however, are restrained by its low producing titer and the limited information about its isoforms. Using a strong IPTG-inducible promoter Pg3, the native promoter of surfactin synthase in wild-type B. subtilis THY-15 was replaced, which gave engineered THY-15/Pg3-srfA. In a shaking flask, the surfactin titer of THY-15/Pg3-srfA reached 8.2 g/L, significantly higher than 1.2 g/L of THY-15. In a 5-L fermenter, however, the surfactin titer of THY-15/Pg3-srfA shrank to 5.6 g/L, which was later confirmed to be related to the low dissolved oxygen limit during fermentation. A Vitreoscilla hemoglobin (VHb) gene was then introduced into the engineered strain, obtaining a novel THY-15/Pg3-srfA(VHb). Through the successful expression of VHb, the surfactin titer of THY-15/Pg3-srfA(VHb) increased to 10.2 g/L in the flask and 8.6 g/L in the fermenter, for increases of 24% and 51%, respectively, compared with the non-VHb engineered strain. The contents, structures and surface activity of the surfactin isoforms produced by the engineered strains were further highlighted. Surfactin C15-isoform with 14.8 mg/L Critical Micelle Concentration, which can reduce surface tension of water to 27.1 mN/m (25 °C), is the most preferred.