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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 P  srfA   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 Δ spo0A abrB*  and Δ spo0A  Δ abrB  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 Δ spo0A 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.

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