Synthetic biology strategy to discover a novel ribosomal peptide, Xenopeptide

Ribosomally-synthesized and post-translationally-modified peptides (RiPPs) are a major class of natural products. It originates from a corresponding biosynthetic gene cluster where the genes for a precursor peptide and several post-translational modification (PTM) enzymes are located. The precursor peptide is translated by a ribosome, recognized on the N-terminal leader peptide and modified on the C-terminal core peptide by PTM enzymes, to finally give a structurally unique mature compound. So far, the RiPPs found by researchers only account for a small part of the family, and there are a large number of compounds remaining to be discovered. The S-adenosylmethionine radical (rSAM) enzyme family is one of the largest enzyme superfamilies, containing more than 22000 members. These enzymes are widely distributed in eukarya, bacteria and archaea and are considered to be one the earliest biocatalysts on earth which perform essential and inseparable functions in cells. As more substantial microbial genomic information becomes available, a large number of RiPPs are excavated as natural products by high-throughput analyses. Genome mining in microorganisms reveals great abundance of biosynthetic gene clusters consisting of co-occurring rSAM enzyme and RiPPs' peptide genes, mostly leading to complex bioactive compounds whether reported or not. Recently, three genera, Xenorhabdus, Yersinia, and Erwinia, are unified with the definition of XYE system, for they all employ a highly conserved rSAM enzyme responsible for modifying its adjacent peptide to create a distinctive C-C or C-O bond in the final RiPPs product. Yet complex compounds synthesized through the rSAM enzyme modifying a precursor peptide in the XYE system have rarely been reported. Through algorithm analysis, we monitored a biosynthetic gene cluster in the Xenorhabdus sp. KJ12.1 comforming a similar construction of that in an XYE system. We fully synthesized the precursor peptide and the rSAM enzyme genes from Xenorhabdus sp. KJ12.1, heterologously expressed the modifying system in Escherichia coli and successfully obtained a novel ribopeptide Xenopeptide. Structure analysis showed that the rSAM enzyme XenB is involved in the formation of two C-C bonds on the side groups of the precursor, one between tryptophan14(W14) and asparagine16 (N16), the other between tryptophan21(W21) and lysine23(K23). Although bioactivity tests proved the compound without a confirmed antimicrobial activity, we can still expect the utilization of the product elsewhere with its chemical complexity. These results indicate a developmental potential of combining computer technology and biological engineering. Our work provides a fundamentally theoretical basis for in-depth genome mining and a practically technical flow for finding modified compounds in microorganisms. The combination of genetic manipulation, biosynthetic characterization and structure determination is expected to further decipher the mechanistic enzymology involved in C-C crosslink formation. This strategy will shed light on the structure-activity relationships involved in the maturation of this subclade of RiPPs. Moreover, thorough biosynthetic engineering of Xenopeptide's precursor peptide and relevant enzymes through rational design and directed mutation will facilitate the development of a variety of Xenopeptide analogs with enhanced bioactivities. This paper will not only disclose the enzymatic underpinnings underlying the biosynthesis of C-C crosslinks, but also point out new directions for structural derivatization of bioactive peptides.