GENERATING AN ACTINOBACTERIAL CHASSIS FOR ANTIMICROBIAL DISCOVERY

AbstractNatural products have long been the primary resource as scaffolds for therapeutic drugs, particularly againstthe threat of antimicrobial resistance. Once dormant, the field of natural product discovery is experiencinga 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 inaccessibledue to lack of cultivability in the host, are now routinely constructed on broad-host vectors and shuttledinto heterologous hosts, primarily E. coli and S. lividans. Antimicrobial compounds are discovered throughhigh-throughput screens of eDNA in these host vectors, but are limited by very low ?hit rates?. Heterologoushosts 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). Thisproposal outlines the development of a broad-based chassis organism for activation of BGCs fromactinobacteria, the organisms responsible for the majority of antimicrobials. Alternative sigma factors mostcommonly found in actinobacteria will be combinatorially expressed in Pseudomonas putida, an organismthat is both genetically tractable like E. coli and has a high-GC content organism with significant toleranceto xenobiotics like S. lividans. Through an interdisciplinary approach combining insights frommicrobiology, synthetic biology and analytical chemistry, this proposal seeks to provide a system that willincrease the ?hit rate? in genome mining, which will be an important tool to the broader scientificcommunity.