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

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.

Modeling the time course of ComX: towards molecular process control for Bacillus wild-type cultivations

“…For ComX pheromone bioactivity assay, a reporter strain, namely CT2 was engineered. Therefore, a chromosomal integration of P srfA - lacZ fusion was introduced into the amyE locus of BKK31700 (Koo et al 2017 ), carrying a deletion of comX (∆ comX :: kan ), using the plasmid pKAM446 (Hoffmann et al 2021a ). The mutant strains were selected on LB agar plates supplemented with kanamycin (5 µg/mL), spectinomycin (100 µg/mL) or erythromycin (5 µg/mL), respectively.…”

The phylogeny and metabolic potentials of an n-alkane-degrading Venatorbacter bacterium isolated from deep-sea sediment of the Mariana Trench

“…Conversely, all the genomes from the neighboring genera (Oleibacter, Thalassolituus, Oceanobacter, Bacterioplanes, Oleispira, and Bermanella) were predicted to be deficient in denitrification. Considering that the expression of narGHI is typically induced at very low oxygen concentrations (Hoffmann et al, 2021), our results suggested a genus-specific ecological function of Venatorbacter in suboxic or anoxic environments, e.g., the oxygen-minimal zone (OMZ), where ST750PaO-4 was isolated from (BioSample: SAMN15097647), and the deeply buried marine sediments, where C2-1 and MT-18 were isolated from.…”

Deletion of Rap-phosphatases for quorum sensing control in Bacillus and its effect on surfactin production

“…All mutant strains were confirmed for successful erm removal by phenotype verification, PCR (Primer S1464–1465; S1467–1568; S1470–1471; S1560–1561) and Sanger sequencing (Primer S1567–S1574). For strains CT10–CT12, lacZ fusion with P srfA was integrated into the amyE locus, using plasmid pKAM446 as previously described by Hoffmann et al ( 2021 ). Successful integration was checked by phenotype verification and PCR (Primer S1637–S1638) and sanger sequencing confirmed the absence of point mutations (Primer S1639–S1640).…”

“…To address the challenge of interactions between the quorum sensing regulatory network and surfactin production in bioprocessing, there are various approaches. For example, the native P srfA promoter can be exchanged to uncouple surfactin production and the quorum sensing system (Coutte et al 2010 ; Hoffmann et al 2021 ; Willenbacher et al 2016 ). Another option is to incorporate the quorum sensing system for lipopeptide production into the context of molecular process control, for example by modeling the kinetics of quorum sensing molecules (Treinen et al 2021 ).…”