Regulation of Surfactin Biosynthesis Pathways in Bacillus sp.: Regulatory Network Reconstruction and Metabolic Engineering Perspectives

Surfactin is an amphiphilic lipopeptide, composed of a cyclic heptapeptide linked to a β-hydroxy fatty acid. This structural configuration gives rise to its remarkable biosurfactant properties. The synthesis is performed by a non-ribosomal peptide synthesis mechanism that involves surfactin synthetase, a multi-enzyme complex encoded by the srfA operon. The expression of this operon and surfactin synthetase activity are regulated by quorum sensing pathways such as Rap-Phr and ComXQPA controlling natural competence, YcxA and YerP efflux pumps, sporulation phosphorelay and degradative enzymes as well as surfactin-regulated pathways. The objective of this manuscript is to review well-studied regulatory metabolic pathways of Bacillus subtilis that control surfactin biosynthesis and to construct a global metabolic regulatory network in order to design the most efficient metabolic engineering strategies to improve surfactin production while maintaining strain stability. The surfactin synthesis regulatory network was built using KEGG pathway maps for two-component systems, quorum sensing, bacterial secretion, and ABC transporters. Most genes and interactions were based on Bacillus subtilis subsp. subtilis str. 168. Gene details were obtained from the NCBI database. The network map was created manually using Canva’s flowchart tool. Modifications in amino acid and fatty acid precursor pathways did not have a significant impact on surfactin yield despite an increase in amino acid availability. The modification of genes involved in quorum sensing pathways has been associated with the regulation of natural competence development and cell cycle progression. For example, srfA promoter replacement and comA overexpression resulted in a significant enhancement of srfA operon expression and surfactin production. The overexpression of efflux pumps ycxA and yerP resulted in enhanced surfactin secretion and increased self-resistance to surfactin. In contrast the deletion of sporulation genes resulted in an increase in surfactin yield per biomass; however, it caused inhibition of growth. Knockout of biofilm-related genes and competing lipopeptide pathways resulted in an increase in surfactin yield. A hypothesis of triple-mutant strain combining comA overexpression, ycxA and yerP enhanced efflux transport, and kinC deletion was proposed based on the constructed regulatory metabolic network that theoretically can improve surfactin yield and strain stability; however, further validation is required to assess potential unintended effects.