Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes.

In the submerged cultivation of filamentous microbes, including actinomycetes, complex morphology is one of the critical process features for the production of secondary metabolites. Ansamitocin P-3 (AP-3), an antitumor agent, is a secondary metabolite produced by ATCC 31280. An excessive mycelial fragmentation of ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of is specifically regulated by an AdpA-like protein APASM_1021. Moreover, the mycelial fragmentation was alternatively alleviated by the overexpression of subtilisin inhibitor encoded genes, which also led to a 46.50 ± 0.79% yield increase of AP-3. Furthermore, was overexpressed in salinomycin-producing BK 3-25 and validamycin-producing TL01, which resulted in not only dispersed mycelia in both strains, but also a 33.80% yield improvement of salinomycin to 24.07 g/L and a 14.94% yield improvement of validamycin to 21.46 g/L. In conclusion, our work elucidates the involvement of a novel subtilisin-like serine peptidase in morphological differentiation, and modulation of its expression could be an effective strategy for morphology engineering and antibiotic yield improvement in actinomycetes.