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. 2021 Apr 19;11(1):57.
doi: 10.1186/s13568-021-01218-4.

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

Mareen Hoffmann  1 , Alina Braig  1 , Diana Stephanie Fernandez Cano Luna  1 , Katharina Rief  1 , Philipp Becker  1 , Chantal Treinen  1 , Peter Klausmann  1 , Kambiz Morabbi Heravi  1 , Marius Henkel  1 , Lars Lilge  2 , Rudolf Hausmann  1
Affiliations

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

Mareen Hoffmann et al. AMB Express. .

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

Keywords: Bacillus subtilis; Lipopeptide biosurfactants; Microaerobic; Oxygen; Promoter exchange; Surfactin.

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Conflict of interest statement

The authors declare that they have no conflict of interests.

Figures

Fig. 1
Fig. 1
Cell dry weight (CDW) (g/L) and concentrations of nitrate, nitrite and ammonium (all [mmol/L]) of strain B. subtilis JABs24 cultivated in a mineral salt medium employing 40 g/L glucose, 0.1 mol/L NH4+ and 0.1 mol/L NO3− in baffled shake flasks with three different relative filling volumes of 10%, 50% and 100% representing different oxygen availabilities
Fig. 2
Fig. 2
Time course of Miller units determined by the Miller Assay representing expression of promoters PsrfA, PnarG and PnasD. Data recorded until CDWmax of cultivation of strains B. subtilis KM1016 (PsrfA-lacZ), MG1 (PnarG-lacZ) and MG5 (PnasD-lacZ) in a mineral salt medium employing 40 g/L glucose, 0.1 mol/L NH4+ and 0.1 mol/L NO3− in baffled shake flasks with three different relative filling volumes (Rv) of 10%, 50% and 100% representing different oxygen availabilities
Fig. 3
Fig. 3
Time course of cell dry weight (CDW) [g/L] (a) and surfactin concentration [mg/L] (b) of strains B. subtilis JABs24 (reference), MG12 (PnarG-lacZ; amyE::[PnarG-lacZ, spcR]) and MG14 (PsrfA::PnasD; amyE::[PnasD-lacZ, spcR]) cultivated in a mineral salt medium with 40 g/L glucose, 0.1 mol/L NH4+ and 0.1 mol/L NO3− employing three different relative filling volumes of 10%, 50 and 100% representing different oxygen availabilities
Fig. 4
Fig. 4
Exemplary cultivation results of fed-batch bioreactor cultivations employing strains B. subtilis KM1016 (PsrfA-lacZ) (a, c) and B. subtilis MG14 (PsrfA::PnasD; amyE::[PnasD-lacZ, spcR]) (b, d). Displayed are absolute values of cell dry weight (CDW), glucose, surfactin (all [g]), nitrate, nitrite and ammonium (all [mmol]) (a, b), as well as the time course of OD600, Miller units as determined by the Miller Assay to determine the expression of PsrfA and PnasD, and pO2 [%] (c, d)
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