Evaluation of an oxygen-dependent self-inducible surfactin synthesis in B. subtilis by substitution of native promoter P by anaerobically active P and P.

A novel approach targeting self-inducible surfactin synthesis under oxygen-limited conditions is presented. Because both the nitrate (NarGHI) and nitrite (NasDE) reductase are highly expressed during anaerobic growth of B. subtilis, the native promoter P of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters P and P to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native P expression. As hypothesized, activity of P and P increased with lower oxygen levels and surfactin was not produced by P::P as well as P::P mutant strains under conditions with highest oxygen availability. P showed expressions similar to P at lowest oxygen availability, while maximum value of P was more than 5.5-fold higher. Although the promoter exchange P::P resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying P::P reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall Y of 1.007 g/g. This value also surpassed the Y of the reference strain JABs24 at highest and moderate oxygen availability. Bioreactor cultivations illustrated that significant cell lysis occurred when the process of "anaerobization" was performed too fast. However, processes with a constantly low agitation and aeration rate showed promising potential for process improvement, especially by employing the strain carrying P::P promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter P represents a promising tool for anaerobic-inducible bioprocesses in Bacillus.