[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-554] Exploring the Potential of CPP–PNA Conjugates for Translational Regulation of Secondary Metabolism in Streptomyces

○Ryuya Furukawa¹, Saruul Erdenebat¹, Tetsushi Mori¹ (1. Tokyo University of Agriculture and Technology (Japan))

Keywords:

Cell-penetrating peptides,Streptomycetes,Functional characterization

Streptomycetes are ubiquitous soil-dwelling bacteria that play essential roles in nutrient cycling through organic matter decomposition and significantly contribute to ecosystem health. Beyond their ecological importance, they are prolific producers of bioactive secondary metabolites, including antibiotics, antitumor agents, and immunosuppressants, making them highly valuable for drug development. Their genomes typically contain 20–50 biosynthetic gene clusters (BGCs); however, nearly 90% remain silent or poorly expressed under standard laboratory conditions, representing a vast, largely untapped resource of novel bioactive compounds. Although various genetic engineering approaches have been developed for actinomycetes, these methods are often complex, require host strain modification, and remain highly strain-dependent, limiting broader applicability. To address these challenges, we developed a rapid and broadly applicable strategy using cell-penetrating peptides (CPPs) conjugated with peptide nucleic acids (PNA) for nucleic acid manipulation and translation control, eliminating the need for direct genetic modification. First, we evaluated CPP permeability in Streptomyces avermitilis using FAM-labeled CPPs, identifying cationic CPPs as most effective for cellular uptake. Subsequently, CPP-PNA probes were designed and synthesized to regulate protein translation. Growth inhibition assays targeting housekeeping genes demonstrated that optimized CPP-PNA probes achieved specific translational inhibition without observable cytotoxic effects, highlighting the precision and safety of this approach. To validate CPP-PNA applications in secondary metabolite research, antiSMASH analysis identified four pigment-related BGCs responsible for two melanin types, flaviolin, and carotenoids from the genome sequence of S. avermitilis. CPP-PNA probes targeting genes within these clusters are currently under synthesis to directly modulate and study secondary metabolic pathways at the translational level. This approach aims to facilitate activation and characterization of cryptic BGCs, potentially accelerating novel natural product discovery and improving production efficiency in industrial strains. This strategy provides a flexible, non-invasive alternative to conventional genome editing, enabling rapid functional gene analysis without permanent genetic alterations. It also offers temporal control of gene expression, allowing investigation of dynamic regulatory mechanisms in secondary metabolism. The established platform demonstrates strong potential for widespread application in native Streptomycetes strains and may extend to other actinomycetes. This approach also has the potential to facilitate activation of cryptic biosynthetic pathways while providing new opportunities for advancing drug discovery, biotechnology, and microbial ecology research.