Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes

• Main Authors: Yuanting Wu, Qianjin Kang, Li-Li Zhang, Linquan Bai
• Format: Article
• Language: English
• Published: MDPI AG 2020-06-01
• Series: Biomolecules
• Subjects: actinomycetes; transcriptome; morphology engineering; mycelial fragmentation; subtilisin
• Online Access: https://www.mdpi.com/2218-273X/10/6/851

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 <i>Actinosynnema pretiosum</i> ATCC 31280. An excessive mycelial fragmentation of <i>A. pretiosum</i> ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene <i>APASM_4178</i> was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the <i>APASM_4178</i> deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of <i>APASM_4178</i> 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, <i>APASM_4178</i> was overexpressed in salinomycin-producing <i>Streptomyces albus</i> BK 3-25 and validamycin-producing <i>S. hygroscopicus</i> 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.