Enhanced Cultivation of Actinomycetota Strains from Millipedes (Diplopoda) Using a Helper Strain-Assisted Method

The limited cultivability of Actinomycetota strains restricts the exploration of their novel antibiotics, highlighting the need for improved isolation techniques. This study employed a helper strain-assisted cultivation method which utilizes culture supernatants from helper strains to isolate diverse members of the Actinomycetota from millipedes and compared its efficacy with a standard method. Using a preliminary dual-layer solid media assay and subsequent confirmation experiments, eight helper strains (M3, M9, M13, N3, N4, N6, N8, and N9) were identified, whose supernatants promoted the growth of Actinomycetota and other microbes. Application of this method to millipede samples established a novel cultivation strategy based on co-cultivation with helper strains. The new method enabled the isolation of 233 bacterial species in total, of which 143 were species of the phylum Actinomycetota, including 49 novel species. In contrast, the standard method yielded only 42 total bacterial species and 29 species of Actinomycetota, with merely 8 novel species. Comparative diversity analysis revealed that the helper strain-assisted method yielded Actinomycetota strains from 85 genera, which was 3.5 times higher than the standard method. This demonstrates that the helper strain-assisted approach is a highly effective strategy for accessing diverse and novel microbial majority. Among the isolated Actinomycetota strains, 75 strains predicted to have high biosynthetic gene clusters (BGCs) numbers or expected to be novel species were screened for antibacterial activity. Fourteen strains (17%) exhibited inhibitory effects against at least one indicator bacterium. One novel strain, Streptomyces sp. N8-31, was selected for whole-genome sequencing. AntiSMASH analysis predicted 40 biosynthetic gene clusters in N8-31, with 60% showing less than 70% similarity to known clusters; among these, 20 clusters showed less than 50% similarity. These findings indicate that strain N8-31 is a rich reservoir of novel genetic resources, and its broad-spectrum antibacterial activity is likely linked to these unique secondary metabolite gene clusters. Critically, this study confirms that helper strain-assisted cultivation is a powerful tool for unlocking the hidden biosynthetic potential of previously inaccessible Actinomycetota.