Natural products have long been the primary resource as scaffolds for therapeutic drugs, particularly against the threat of antimicrobial resistance. Once dormant, the field of natural product discovery is experiencing a renaissance as advancements in the field of metagenomics has allowed the investigation of a vast, untapped reservoirs of gene clusters. Large environmental DNA (eDNA) segments, previously inaccessible due to lack of cultivability in the host, are now routinely constructed on broad-host vectors and shuttled into heterologous hosts, primarily E. coli and S. lividans. Antimicrobial compounds are discovered through high-throughput screens of eDNA in these host vectors, but are limited by very low ?hit rates?. Heterologous hosts often do not have or poorly express the transcriptional recognition elements, alternative sigma factors, that guide the host RNA polymerase that can activate these biosynthetic gene clusters (BGCs). This proposal outlines the development of a broad-based chassis organism for activation of BGCs from actinobacteria, the organisms responsible for the majority of antimicrobials. Alternative sigma factors most commonly found in actinobacteria will be combinatorially expressed in Pseudomonas putida, an organism that is both genetically tractable like E. coli and has a high-GC content organism with significant tolerance to xenobiotics like S. lividans. Through an interdisciplinary approach combining insights from microbiology, synthetic biology and analytical chemistry, this proposal seeks to provide a system that will increase the ?hit rate? in genome mining, which will be an important tool to the broader scientific community.
Antimicrobial resistance is a rising global threat, and research efforts to discover new antimicrobials have been revitalized by screening natural products encoded in environmental DNA. However, discovery has been hindered by low ?hit? rates as heterologous hosts, primarily Escherichia coli and Streptomyces lividans, lack or poorly express the alternative sigma factors needed to activate these secondary metabolite clusters. This proposal outlines the creation of a chassis that combinatorially expresses alternative sigma factors of actinobacteria, the class of organism responsible for the majority of antimicrobials, as a new platform host for antibacterial and antifungal drug discovery.
