Application of a microdroplet array platform for high-throughput analysis of growth and natural product biosynthesis of Streptomyces

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Abstract Streptomycetes are prolific producers of natural products, accounting for the majority of clinically used antibiotics and other bioactive compounds. Their remarkable biosynthetic diversity and ecological versatility are reflected in the large number of biosynthetic gene clusters (BGCs) within their genomes. However, many of these BGCs remain inactive under standard laboratory conditions, highlighting the need for high-throughput cultivation approaches to prioritise conditions that reveal strain- and medium-dependent metabolite production. Here, we present a microdroplet array platform that enables high-throughput cultivation and screening of Streptomyces . The array consists of 10,450 stationary nanolitre droplets, fixed onto a glass slide, that can be monitored for growth, morphology, bioactivity, and metabolite production. Streptomyces strains exhibited robust and reproducible growth while broadly preserving key strain-specific morphological features. Antimicrobial activity was assessed through nano-injection of Bacillus subtilis , revealing strain- and medium-specific antimicrobial activity. Strong inhibition was observed for Streptomyces sp. MBT27 when grown in TSB and MMMP media, with additional bioactivity detected for several strains when grown in MMMP. Single-droplet MALDI-TOF MS analysis of MBT27 grown in minimal medium droplets detected actinomycin X2, linking droplet-based phenotypes to chemical output. Follow-up cultivation under identical conditions in bulk cultures confirmed both antimicrobial activity and metabolite production by bioassays and LC-MS. These results demonstrate that the microdroplet array provides a high-throughput platform to monitor condition-dependent growth, morphology, and secondary metabolism. The system represents a promising upstream prioritisation tool for comparative cultivation studies and functional bioactivity screening in Streptomyces .

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