Evaluation of an oxygen\u2010dependent self\u2010inducible surfactin synthesis in B. subtilis by substitution of native promoter PsrfA by anaerobically active PnarG and PnasD

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

doi:10.1186/s13568-021-01218-4

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.

Highlights

The cyclic lipopeptide surfactin synthesized by Bacillus subtilis displays promising characteristics in a variety of industrial sectors (Geissler et al 2019a) due to its excellent surface-active properties and antimicrobial activities (Falardeau et al 2013; Li et al 2019)
Influence of different oxygen availabilities on nitrogen metabolism Varying filling volumes in shake flasks are associated with different oxygen availabilities for cells
Low filling volumes result in highest oxygen availability (Heyman et al 2019; Schiefelbein et al 2013) and aerobic growth is predominant

Summary

Introduction

The cyclic lipopeptide surfactin synthesized by Bacillus subtilis displays promising characteristics in a variety of industrial sectors (Geissler et al 2019a) due to its excellent surface-active properties and antimicrobial activities (Falardeau et al 2013; Li et al 2019). Yeh et al (2006) reported that up to a certain high level of aeration and stirrer speed surfactin synthesis was improved in carrier-assisted cultivation due to improved oxygen transfer rate and mass transfer efficiency In contrast to these circumstances, other studies described that an enhanced surfactin production rate was reached in oxygen-limited conditions (Davis et al 1999; Kim et al 1997). Still, promising and high values with regard to product per biomass yields were obtained (Geissler et al 2019b; Willenbacher et al 2015) Another aspect is the negative impact of nitrite as well as acetate on anaerobic cell growth. The latter one increased drastically throughout nitrate respiration of B. subtilis and as such is an interesting candidate for strain engineering (Hoffmann et al 2020)

Methods

Results

Conclusion