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

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 ofB. subtilis, the native promoter PsrfAof the surfactin operon in strainB. subtilisJABs24 was replaced by promoters PnarGand PnasDto induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native PsrfAexpression. As hypothesized, activity of PnarGand PnasDincreased with lower oxygen levels and surfactin was not produced by PsrfA::PnarGas well as PsrfA::PnasDmutant strains under conditions with highest oxygen availability. PnarGshowed expressions similar to PsrfAat lowest oxygen availability, while maximum value of PnasDwas more than 5.5-fold higher. Although the promoter exchange PsrfA::PnarGresulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying PsrfA::PnasDreached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overallYP/Xof 1.007 g/g. This value also surpassed theYP/Xof 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 PsrfA::PnasDpromoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter PnasDrepresents a promising tool for anaerobic-inducible bioprocesses inBacillus.