Development of Bacillus subtilis as a production platform for bioactive secondary metabolites

Bacillus subtilis is considered a workhorse in biotechnology. In this context, the Bacillus family demonstrates a remarkable biosynthetic potential for the production of bioactive metabolites. Here, bacteriocins and lipopeptides are important representatives involved in intercellular interactions as well as showing promising application potentials due to their structural properties. However, in order to use the potential of bioactive metabolites in B. subtilis, molecular regulatory circuits in production strains have to be studied and adapted by molecular strain engineering and tailored bioproduction processes. In this way, surfactin as a representative of the lipopeptide family and mersacidin as a member of the bacteriocin class have already been successfully introduced into adapted B. subtilis production strains by understanding the associated regulatory networks, followed by rational strain engineering.

In order to understand molecular regulatory networks within the biosynthetic pathways of surfactin and mersacidin, both the domesticated laboratory B. subtilis strain 168 and the sporulation-deficient strain 3NA were selected. Using reporter strains together with the integration of combinatorial gene knockouts, the expression of biosynthetic machineries could be evaluated and the influence of certain regulatory circuits could be studied. In this context, the cell differentiation associated with Spo0A-derived sporulation initiation and the regulation of the global transcriptional regulator AbrB were found to have significant relevance. This regulatory network plays a crucial role in the productivity of engineered B. subtilis production strains, which has been applied in adapted high cell-density fed-batch bioreactor cultivation processes. Targeted strain engineering allowed an increase in the production of surfactin to about 40 g/L, which is a record in scientific literature, whereas a mersacidin titer of about 100 mg/L could be reached, a 10-fold increase compared to the wild-type production strain.

In sum, molecular strain engineering based on understanding molecular regulatory networks provides the opportunity to realize the biosynthetic potential of B. subtilis as a production platform for bioactive metabolites. In addition, the use of tailored fed-batch bioreactor production processes with high cell-density enables the production of the target products mentioned in high quantities to enable future applications.