Radically Expanding the Landscape of Noncanonical Amino Acids in RiPPs

Abstract

Natural products provide a rich source of therapeutics and inspiration for synthetic and medicinal chemists alike. 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, our lab previously generated a network of about 600 uncharacterized streptococcal ribosomally synthesized and post- translationally modified peptide (RiPP) natural products that are quorum sensing-regulated and tailored by radical S-adenosylmethionine (RaS) enzymes. RaS enzymes are a superfamily of metalloenzymes present in all domains of life that utilize a [4Fe-4S] cluster and S- adenosylmethionine (SAM) to catalyze versatile and intricate chemical reactions. From this network of streptococcal RaS-RiPPs, 16 distinct subfamilies emerged revealing a diverse landscape of unchartered enzymatic reactions. The most complex of these is the GRC subfamily, named after a conserved motif in the precursor peptide and found in Streptococcus pneumoniae, an opportunistic pathogen and the causative agent of bacterial pneumonia. In this study, using a heterologous co-expression in vivo platform, we have elucidated the modifications installed by the grc biosynthetic enzymes, including a ThiF-like adenylyltransferase/cyclase that generates a 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 di- dehydrohistidine in RiPP biosynthesis, respectively. The modifications reported expand the landscape of noncanonical amino acids in RiPP natural product biosynthesis and motivate downstream biocatalytic applications of the corresponding enzymes. In addition, we present our efforts towards utilizing a different metalloenzyme, a cytochrome P450, in order to generate a combinatorial library of vancomycin-inspired scaffolds.