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.