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

Abstract

Abstract 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.