(437d) A Transcription Factor Decoy Strategy for Activation of Streptomyces Polyketide and Non-Ribosomal Peptide Gene Clusters

Discovery of novel antibiotics remains an important task for modern biotechnology, especially with the emergence and prevalence of antibiotic resistance. As a rich source of pharmaceutical agents, microbial natural products are synthesized by the biosynthetic gene clusters (BGCs) encoded in genomes. Recent advances in genomics and bioinformatics revealed the existence of a vast number of uncharacterized BGCs. However, those BGCs are mostly transcriptionally silent under conventional conditions due to tight regulation in native hosts. Although many methods have been developed to fully exploit this largely untapped reservoir of natural products, their success rates are relatively low and few of them can be carried out in a high-throughput manner. Here we developed a transcription factor decoy (TFD) strategy to activate the polyketide and non-ribosomal peptide BGCs from Streptomycetes in a targeted and high-throughput manner. This strategy consists of design and construction of TFDs or a library of TFDs encoding the regulatory DNA elements from the target BGCs in a Streptomyces strain using a multicopy plasmid, followed by screening for highly expressed TFD recombinants based on a reporter gene. As a proof-of-concept, we applied TFDs to activate two silent pigment BGCs for actinorhodin and undecylprodigiosin, respectively, in Streptomyces lividans 66 and 4 out of 10 BGCs in Streptomyces albus and Streptomyces sp. NRRL F-5635. In addition, we applied a library of TFDs to activate 10 BGCs in Streptomyces griseofuscus NRRL B-5429 and Streptomyces sp. NRRL F-4335 in a high throughput manner. We are currently screening the resulting libraries and characterizing one of the new products from Streptomyces sp. NRRL F-4335. Due to its simplicity and ease, this strategy can be readily scaled up for discovery of hundreds of novel natural products from Streptomycetes.