Insights into the availability of surfactin-forming nonribosomal peptide synthetase in Bacillus subtilis

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Bacillus subtilis is capable to produce notable amounts of surfactin, a lipopeptide described as one of the most powerful biosurfactants. Currently, to improve bacterial production, research groups are focused on enhanced provision of precursor molecules as well as adaptations in cultivation parameters and downstream processing. However, the intracellular availability of nonribosomal peptide synthetase (NRPS) subunits for surfactin formation is still unknown. Hence, there is a lack of knowledge about the number of NRPS per cell under respective conditions. Information about this issue will help to calculate a production rate of NRPS enzyme complex during different growth phases and conditions.This knowledge should then be used to adapt cultivation parameters for highest possible NRPS expression combined with enhanced activity for surfactin formation.In this context, a second point addressed in this proposal is the relationship between NRPS expression and availability of precursor molecules for surfactin formation. Several studies showed an increase in surfactin production after improvement of metabolic pathways leading to enhanced availability of precursors. Nevertheless, the open question is about the balance between intracellular concentration of precursor molecules and NRPS enzyme complexes per cell and which factor is limiting during cultivation.It is a reasonable assumption, which is also experimentally supported, that the amount of NRPS translational rate should not be too high so as not to cause too high a metabolic burden. On the other hand, the NRPS complex is needed for biosynthesis of surfactin. Therefore, in order to flexibly optimize the expression level of NRPS for different strains, methods to determine the amount of NRPS per cell must first be established and the actual NRPS protein amount must be determined.