Evaluation of an oxygen‐dependent self‐inducible surfactin synthesis in B. subtilis by substitution of native promoter PsrfA by anaerobically active PnarG and PnasD

Abstract

Abstract 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 srfA of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters P narG and P nasD to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native P srfA expression. As hypothesized, activity of P narG and P nasD increased with lower oxygen levels and surfactin was not produced by P srfA ::P narG as well as P srfA ::P nasD mutant strains under conditions with highest oxygen availability. P narG showed expressions similar to P srfA at lowest oxygen availability, while maximum value of P nasD was more than 5.5-fold higher. Although the promoter exchange P srfA ::P narG resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying P srfA ::P nasD reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall Y P/X of 1.007 g/g. This value also surpassed the Y P/X 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 srfA ::P nasD promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter P nasD represents a promising tool for anaerobic-inducible bioprocesses in Bacillus .

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