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

Abstract

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.

Improved Production of Fengycin in Bacillus subtilis by Integrated Strain Engineering Strategy

“…The strain will achieve a state of spore dormancy in the absence of nutrients or during stress, which are harmful to the production of secondary metabolites . The nonsporulating B. subtilis facilitates surfactin synthesis in a high-density fermentation process . Therefore, knockout of the sporulation pathway may facilitate the synthesis of secondary metabolites, such as fengycin.…”

Systemically engineering Bacillus amyloliquefaciens for increasing its antifungal activity and green antifungal lipopeptides production

“…Biofilm is considered as a suppressor of lipopeptides (e.g., surfactin) synthesis, so disruption of biofilm formation is favorable for improving lipopeptides production ( Wu et al, 2019 ). Here, we knocked out several genes related to biofilm formation such as comK ( She et al, 2020 ), sigD ( Fan et al, 2016 ), dhbF (a gene of dhb operon for biosynthesis of siderophore) and fur (a repressor of dhb ) ( Oliveira et al, 2017 ; Pi and Helmann 2017 ), abrB and sinR (regulators to suppress biofilm formation) ( Newman et al, 2013 ; Klausmann et al, 2021 ), and sinI (a regulator to promote biofilm formation) ( López et al, 2009 ). Deletion of respective genes resulted in an obvious change of colony morphology and float pellicle, but only deletion of dhbF led to a significant increase of antifungal activity.…”

“…After phosphorylation, Spo0A regulates biosynthesis of many secondary metabolites ( Rahman et al, 2006 ; Sun et al, 2021 ). For example, Klausmann et al (2021) reported that the null-mutant of spo0A could reduce surfactin production in B. subtilis . Consistently, deletion of spo0A led to a very significant reduction of antifungal activity in WH1.…”

“…Major concerns, however, are attributed to large genetic sequence of the operons encoding iturin ( itu , 38-kb) and fengycin ( fen , 30-kb) synthase ( Tosato et al, 1997 ; Wu et al, 2007 ; Cheng et al, 2017 ; Yang et al, 2020 ; Wan et al, 2021 ). Because direct overexpression of itu and fen operons has been challenging, Bacillus species have been engineered to improve iturin and fengycin yield mainly through promoter exchanges of the synthase operon ( Dang et al, 2019 ), strengthening biosynthesis of the substrates such as fatty acids ( He et al, 2021 ; Tan et al, 2022 ), overexpression of genes encoding the regulators ComA, SigA, DegU, DegQ and Spo0A ( Koumoutsi et al, 2007 ; Wang et al, 2015 ; Zhang et al, 2017 ; Klausmann et al, 2021 ; Sun et al, 2021 ), or deletion of the repressors gene such as abrB ( Xu et al, 2020 ). These studies are able to efficiently increase the lipopeptides biosynthesis, and also demonstrate the importance of understanding the biosynthetic metabolism of iturin and fengycin.…”

Transcriptome Analysis of Bacillus amyloliquefaciens Reveals Fructose Addition Effects on Fengycin Synthesis

“…The signal protein AbrB is a lipopeptide transcription inhibitor, and Spo0A is a transcription regulator in the sporulation process. Here, phosphorylated Spo0A plays a constructive role in fengycin production via inhibiting the expression of AbrB [ 28 ]. Recent studies have proved that DegQ increases the production of Fengycin [ 29 ].…”