High-Throughput Media Screening for Modulation of Secondary Metabolite Production in Actinomycetes

Abstract New approaches to induce expression of microbial secondary metabolite biosynthetic pathways can accelerate natural product discovery. Here, we present a high-throughput media screening (HTMS) workflow that builds on classical fermentation optimization strategies in which small changes in media composition alter secondary metabolite production. Rather than relying on predefined media recipes, HTMS uses robotic liquid handling to systematically generate and test combinatorial mixtures of commonly used media components, enabling exploration of hundreds of previously untested growth conditions. To validate the approach, we applied HTMS to three actinomycetes: the model organism Streptomyces coelicolor, an unexplored soil isolate Streptomyces GA7, and Amycolatopsis keratiniphila, selected for its encoding of the cryptic keratinimicin glycopeptide family. HTMS yielded three principal findings. First, production of actinorhodin and undecylprodigiosin in S. coelicolor was strongly dependent on specific media formulations. Second, optimized HTMS conditions increased production of target metabolites in Streptomyces GA7 by up to 22-fold. Finally, HTMS identified a small subset of the tested conditions under which keratinimicin A and C1, a previously unreported member of this family, were produced. Together, these results demonstrate that HTMS is a powerful platform for systematically perturbing secondary metabolism in actinomycetes and may accelerate the discovery of cryptic or bioactive natural products.