Expanding the Landscape of Noncanonical Amino Acids in RiPP Biosynthesis

Advancements in DNA sequencing technologies and bioinformatics have enabled the discovery of new metabolic reactions from overlooked microbial species and metagenomic sequences. Using a bioinformatic co-occurrence strategy, we previously generated a network of ∼600 uncharacterized quorum-sensing-regulated biosynthetic gene clusters that code for ribosomally synthesized and post-translationally modified peptide (RiPP) natural products and are tailored by radical S-adenosylmethionine (RaS) enzymes in streptococci. The most complex of these is the GRC subfamily, named after a conserved motif in the precursor peptide and found exclusively in Streptococcus pneumoniae, the causative agent of bacterial pneumonia. In this study, using both in vivo and in vitro approaches, we have elucidated the modifications installed by the grc biosynthetic enzymes, including a ThiF-like adenylyltransferase/cyclase that generates a C-terminal Glu-to-Cys thiolactone macrocycle, and two RaS enzymes, which selectively epimerize the β-carbon of threonine and desaturate histidine to generate the first instances of l-allo-Thr and didehydrohistidine in RiPP biosynthesis. RaS-RiPPs that have been discovered thus far have stood out for their exotic macrocycles. The product of the grc cluster breaks this trend by generating two noncanonical residues rather than an unusual macrocycle in the peptide substrate. These modifications expand the landscape of nonproteinogenic amino acids in RiPP natural product biosynthesis and motivate downstream biocatalytic applications of the corresponding enzymes.