Exploiting Bacillus subtilis as production platform for antimicrobial compounds using mersacidin as an exemplary product
Final Report Abstract
The production of effective antimicrobial substances is necessary at a time when the development of new antibiotics is limited. For screening novel substances, the microbial cell factory Bacillus subtilis seems to be a promising production organism, which has already been established as a workhorse for industrially and pharmaceutically useful proteins and fine chemicals. To realize strategies for the production of antimicrobial substances such as lantibiotics, mersacidin was identified as a promising representative of peptide antibiotics. In subsequent molecular strain engineering approaches, different B. subtilis strains were applied for the construction of production hosts for heterologous mersacidin bioproduction. In detail, the well-established laboratory B. subtilis strain 168, the high cell-density B. subtilis strain BMV9 and the genome-reduced miniBacillus PG10 were selected. In more detail, while the miniBacillus strain PG10 was not able to produce mersacidin, the B. subtilis lab-strain 168 produced about 5-fold higher mersacidin concentrations compared to the native Bacillus amyloliquefaciens strain BH072. In subsequent studies using B. subtilis strain BMV9 as production host for mersacidin biosynthesis, the molecular regulatory circuit of Spo0A, a master regulator of cell differentiation including sporulation initiation, and the global transcriptional regulator AbrB, which is involved in cell adaptation processes in the transient growth phase, was identified to control the initial stimulation of the mersacidin core gene cluster. In a second downstream regulatory step, the activator MrsR1, encoded in the core gene cluster, acts as a stimulatory element for mersacidin biosynthesis. In adaptive production strain engineering approaches, mersacidin concentrations were achieved, which were 7- (BMV9 strain) and 11-fold (168 strain) higher, respectively, compared to the native production strain. These findings are important to understand the mechanisms linking environmental conditions and microbial responses with respect to the bioproduction of bioactive metabolites including antimicrobials such as mersacidin. This information will also support the construction of production strains for bioactive metabolites with antimicrobial properties. Since the B. subtilis mediated bioproduction was achieved without the mersacidin-specific immune system, stress response signatures of the B. subtilis production strain were determined during the production process, allowing more insights about the detoxification mechanisms stimulated in the absence of the mersacidin-specific immunity system. In final fedbatch bioreactor fermentation processes, an upscaled production of mersacidin was aimed for representing the heterologous production of antimicrobial substances using B. subtilis as a production organism for innovative antibiotics from nature.
