Figure 3
Improved biosynthesis of sublancin in B. subtilis 1A747 [SPβc, prototroph, derivative of B. subtilis 168 (trpC2)] based on three strong heterologous promoters. (a) The absorbances of B. subtilis cell cultures at OD595 of, harbouring P glv , P43, and PluxS, induced by maltose (●) and not induced by maltose (□), and not harbouring those three promoters also treated by maltose (∆). (b) Real–time PCR analysis of the transcription amounts of sunA (●), sunI (○), sunT (▲), bdbA (∆), sunS (■), and bdbB (□) of B. subtilis 1A747 harbouring P glv , P43, and PluxS induced by maltose, compared with the control harbouring native promoters also treated with maltose. (c) Cumulative sublancin production in B. subtilis culture supernatant harbouring those three strong promoters induced by maltose (●) and not harbouring those promoters also induced by maltose (□), the maximum production of 628 mg sublancin was obtained from 1 L recombinant bacteria culture supernatant at 36 h after fermentation. (d) Tricine–SDS–PAGE analysis (a) and western blot analysis (b) of total extracellular proteins from B. subtilis 1A747 induced by maltose, harbouring those three strong promoters (lane 1) or not (lane 2). Bands of sublancin indicated by arrow presented in (a) were confirmed by western blotting (b). Marker lane, broad range protein marker (#P7702, New England Biolabs, USA).