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

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 <i>Escherichia coli</i> strains expressing the human (Trm5) or bacterial (TrmD) m¹G37 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 <i>trmD</i>-expressing <i>E. coli</i> strain over the <i>trm5</i>-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.