Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

Vahidinasab, Maliheh; Lilge, Lars; Reinfurt, Aline; Pfannstiel, Jens; Henkel, Marius; Heravi, Kambiz Morabbi; Hausmann, Rudolf

doi:10.1186/s12934-020-01468-0

Cited by 43 publications

(36 citation statements)

References 54 publications

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“…At both 50% and 100% Rv, increased growth rates were obtained with decreasing surfactin titers. Hence, expression and presence of surfactin seems to result in reduced growth rates, which is in accordance with previous studies (Tsuge et al 2001 ; Vahidinasab et al 2020 ). Shake flask cultivations employing different Rv have been shown to be a suitable approach to investigate the activation of the promoters P narG and P nasD .…”

Section: Discussionsupporting

confidence: 92%

“…For promoter exchange studies, strain JABs24 was used to replace the native promoter P srfA markerless by either P nasD or P narG similar to the protocol described by Vahidinasab et al ( 2020 ). Briefly, generated fragments upstream(P srfA )-P narG -downstream(P srfA ) and upstream(P srfA )-P nasD -downstream(P srfA ) were ligated into SmaI digested plasmid pJOE4786.1 resulting in plasmids pRIK2 for P srfA ::P narG and pPB1 for P srfA ::P nasD .…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionsupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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“…Future studies should also look into the effect of the deletions on surfactin synthesis decoupled from quorum sensing regulation. For this purpose, a constitutive promoter should be used to express the srfA operon in the presented mutant strains as has been demonstrated previously in Willenbacher et al [ 37 ] and Vahidinasab et al [ 33 ].…”

Section: Discussionmentioning

confidence: 99%

Influence of B. subtilis 3NA mutations in spo0A and abrB on surfactin production in B. subtilis 168

et al. 2021

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Background Bacillus subtilis is a well-established host for a variety of bioproduction processes, with much interest focused on the production of biosurfactants such as the cyclic lipopeptide surfactin. Surfactin production is tightly intertwined with quorum sensing and regulatory cell differentiation processes. As previous studies have shown, a non-sporulating B. subtilis strain 3NA encoding a functional sfp locus but mutations in the spo0A and abrB loci, called JABs32, exhibits noticeably increased surfactin production capabilities. In this work, the impacts of introducing JABs32 mutations in the genes spo0A, abrB and abh from 3NA into strain KM1016, a surfactin-forming derivative of B. subtilis 168, was investigated. This study aims to show these mutations are responsible for the surfactin producing performance of strain JABs32 in fed-batch bioreactor cultivations. Results Single and double mutant strains of B. subtilis KM1016 were constructed encoding gene deletions of spo0A, abrB and homologous abh. Furthermore, an elongated abrB version, called abrB*, as described for JABs32 was integrated. Single and combinatory mutant strains were analysed in respect of growth behaviour, native PsrfA promoter expression and surfactin production. Deletion of spo0A led to increased growth rates with lowered surfactin titers, while deletion or elongation of abrB resulted in lowered growth rates and high surfactin yields, compared to KM1016. The double mutant strains B. subtilis KM1036 and KM1020 encoding Δspo0A abrB* and Δspo0A ΔabrB were compared to reference strain JABs32, with KM1036 exhibiting similar production parameters and impeded cell growth and surfactin production for KM1020. Bioreactor fed-batch cultivations comparing a Δspo0A abrB* mutant of KM1016, KM681, with JABs32 showed a decrease of 32% in surfactin concentration. Conclusions The genetic differences of B. subtilis KM1016 and JABs32 give rise to new and improved fermentation methods through high cell density processes. Deletion of the spo0A locus was shown to be the reason for higher biomass concentrations. Only in combination with an elongation of abrB was this strain able to reach high surfactin titers of 18.27 g L−1 in fed-batch cultivations. This work shows, that a B. subtilis strain can be turned into a high cell density surfactin production strain by introduction of two mutations.

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“…In the genome of Bacillus spp., bacteria encoding for fengycin biosynthesis also show the ability to produce surfactin (Kim et al, 2010;Yaseen et al, 2018). In this context, regulatory crosstalk of nonribosomal peptide synthetases (NRPSs) is conceivable (Vahidinasab et al, 2020;Yaseen et al, 2018). Surfactin is described as one of the most powerful microbially produced biosurfactants and has great potential to be used in many industrial sectors such as cosmetics, pharmaceuticals, as well as food (Henkel et al, 2017;.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, superordinated stimuli such as quorum sensing and nutrient availability influence lipopeptide biosynthesis. Specifically, several global regulators including Spo0A, AbrB, CodY, and DegU are involved in the control of NRPS expression (Nakano et al, 1992;Serror & Sonenshein, 1996;Sun et al, 2021;Vahidinasab et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains

et al. 2021

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Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B . subtilis strains JABs24 and DSM10 T , surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho‐transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B . subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14‐fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B . subtilis JABs24, a lipopeptide‐producing derivative of strain 168, and B . subtilis wild‐type strain DSM10 T , which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ , a deletion mutant was constructed for DSM10 T , while a natively expressed degQ version was integrated into strain JABs24. This allowed strain‐specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10 T . While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a sixfold increase in plipastatin was observed. In contrast, degQ deletion in DSM10 T increased surfactin titer by threefold but decreased plipastatin production by fivefold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

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Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

Cited by 43 publications

References 54 publications

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

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

Influence of B. subtilis 3NA mutations in spo0A and abrB on surfactin production in B. subtilis 168

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains

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