PNNL

Abstract

Advances in the prediction of biosynthetic gene clusters (BGCs) from genomic and metagenomic data have made the large-scale identification of candidate secondary metabolite biosynthesis pathways routine. However, most predicted BGCs cannot be successfully expressed in the limited range of expression hosts currently available, which prevents the identification and characterization of their respective products. Here we show that parallelized use of a phylogenetically and physiologically diverse panel of purpose-engineered chassis strains offers a versatile strategy for the rapid activation of BGCs. We domesticated an ensemble of 25 diverse ?-proteobacteria from 11 different genera by integrating a unified landing pad that enables efficient introduction of large BGCs into their genome. We deployed this chassis-independent recombinase-assisted genome engineering (CRAGE) strategy to express several previously posited but experimentally elusive non-ribosomal peptide synthase (NRPS) and NRPSpolyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens. Successful activation of 6 BGCs identified 21 products, including several with likely roles in host-microbe interactions. Taken together, we illustrate how the regulatory and physiological diversity naturally present across bacteria can be harnessed for the exploration of novel bioproducts and provide a sizable panel of domesticated chassis strains to enable this strategy.