Harnessing Genomics to Decode the Chemistry and Biology of Orphan Polyketide Natural Products
Over half of FDA-approved drugs are derived from natural products, highlighting their importance in medicine. Assembly line polyketide synthases (PKSs) are key enzymes involved in the production of structurally diverse natural products that possess significant therapeutic potential as antibiotics, antifungals, and antitumor agents. Motivated by the abundance of recent genomic data, we mined and cataloged 8,799 PKS clusters, 95% of which are orphan clusters with uncharacterized products, underscoring the untapped potential of genomics-driven discovery. From this catalog, we selected two clusters for structural and biological characterization – one implicated in bacterial pathogenesis and another producing a novel antibacterial compound.
The genomes of 40 Nocardia strains, many associated with life-threatening infections, encode the NOCAP (NOCardiosis-Associated Polyketide) synthase, a highly conserved assembly line PKS. We structurally elucidated the fully decorated glycolipid product by heterologous expression of the 3 MDa synthase in E. coli and spectroscopic analysis of the purified products. The glycolipid features a fully substituted benzaldehyde headgroup, a polyfunctional tail, and an O-linked disaccharide. In both E. coli and Nocardia spp., we observed the extracellular secretion of the glycolipid, leading us to propose a model where two dedicated membrane proteins install the second sugar substituent after the monoglycosyl intermediate is exported across the bacterial cell membrane. These findings set the stage for exploring the evolutionary benefit of NOCAP in Nocardia pathogenesis.
A separate biosynthetic gene cluster that produces a putative antibacterial natural product was identified from the genome of Vibrio ruber DSM 16370 based on its colocalization with a gene encoding an aminoacyl-tRNA synthetase homolog. Biological assays confirmed that the pathway produced an antibacterial metabolite and that the co-localized aminoacyl-tRNA synthetase gene functions as a self-resistance mechanism. The structures of four closely related compounds of mixed polyketide – non-ribosomal peptide origin were elucidated. Sequence analysis, mutagenesis and stable isotope feeding studies led to decoding of a plausible biosynthetic pathway for these natural products. The primary products, a trio of interconverting isomers, designated vibriomycins A, B and B’, were found to be unusual allosteric inhibitors of bacterial threonyl-tRNA synthetases. Our study highlights the feasibility of discovering new anti-infective chemotypes via genome mining.