Discovery of 5-Chlorotryptophan-Containing Antibiotics through Metabologenomics-Assisted High-Throughput Screening

High Resolution Image Download MS PowerPoint Slide Actinomycetota bacteria have specialized in the biosynthesis of antibacterial natural products (NPs), and extract and fraction libraries made from those strains remain a promising source of NP drug leads. Herein, we present a high-throughput screen (HTS), based on engineered Escherichia coli strains expressing the human (Trm5) or bacterial (TrmD) m 1 G37 tRNA methyltransferase, to discover NPs as novel anti-Gram-negative antibiotic leads. To harness the evolution of NPs with in vivo activity, the cell-based phenotypic HTS was applied to the Actinomycetota extract and fraction library at the Natural Products Discovery Center (NPDC), the Herbert Wertheim UF Scripps Institute for Biomedical Research & Innovation. From a total of 46,031 extracts and 28,739 fractions made from 14,635 strains, extracts from two Actinomycetota species presented reproducible selectivity against the trmD -expressing E. coli strain over the trm5 -expressing counterpart. A shared metabolite was identified as 5-chlorotryptophan, which was correlated to the observed selective inhibitory activities. A metabologenomics analysis indicated 5-chlorotryptophan incorporation into two distinct antibiotic nonribosomal peptide families, longicatenamycins and nonopeptins. Notably, the diketopiperazine-containing heptapeptide nonopeptins display rare chemistry, featuring a 5-nitro-tryptophan moiety that has only been described previously as a biosynthetic shunt product. The most active congener of this new family of NPs, nonopeptin D, exhibits a broad-spectrum antibiotic activity, including against selected Gram-negative pathogens.