Abstract
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 Actinosynnema pretiosum ATCC 31280. An excessive mycelial fragmentation of A. pretiosum ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene APASM_4178 was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the APASM_4178 deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of APASM_4178 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, APASM_4178 was overexpressed in salinomycin-producing Streptomyces albus BK 3-25 and validamycin-producing S. hygroscopicus 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.
Highlights
The filamentous actinomycetes, with Streptomyces spp. as the major genus, are well known for their rich and diverse secondary metabolites, many of which have been developed into drugs and agents for plant protection
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
The inoculated mycelia or mycelia germinated from spores start to form pellets, following which programmed cell death (PCD) occurs at the center, and new multinucleated mycelia develop inside or from the edge of the pellets to carry out antibiotic production [2]
Summary
The filamentous actinomycetes, with Streptomyces spp. as the major genus, are well known for their rich and diverse secondary metabolites, many of which have been developed into drugs (e.g., erythromycin, vancomycin) and agents for plant protection (e.g., avermectin, validamycin). For industrial production of antibiotics in large scale, actinomycetes are usually subjected to submerged fermentation in liquid cultures. The inoculated mycelia or mycelia germinated from spores start to form pellets, following which programmed cell death (PCD) occurs at the center, and new multinucleated mycelia develop inside or from the edge of the pellets to carry out antibiotic production [2]. Classical strategies for morphology engineering, including adjustments of pH, temperature, medium composition, aeration, and agitation, have been implemented for the production of lipstatin, ε-poly-L-lysine, rapamycin, etc. [8,9,10] These manipulations usually affect large pellets and have limited effect on small pellets [11]
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