Genome mining unveils a class of ribosomal peptides with two amino termini

Abstract

The era of inexpensive genome sequencing and improved bioinformatics tools has reenergized the study of natural products, including the ribosomally synthesized and post-translationally modified peptides (RiPPs). In recent years, RiPP discovery has challenged preconceptions about the scope of post-translational modification chemistry, but genome mining of new RiPP classes remains an unsolved challenge. Here, we report a RiPP class defined by an unusual (S)-N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus, which we term the daptides. Nearly 500 daptide biosynthetic gene clusters (BGCs) were identified by analyzing the RiPP Recognition Element (RRE), a common substrate-binding domain found in half of prokaryotic RiPP classes. A representative daptide BGC from Microbacterium paraoxydans DSM 15019 was selected for experimental characterization. Derived from a C-terminal threonine residue, the class-defining Dmp is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. Daptides uniquely harbor two positively charged termini, and thus we suspect this modification could aid in membrane targeting, as corroborated by hemolysis assays. Our studies further show that the oxidative decarboxylation step requires a functionally unannotated accessory protein. Fused to the C-terminus of the accessory protein is an RRE domain, which delivers the unmodified substrate peptide to the oxidative decarboxylase. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional RiPP classes through genome mining.

Biosynthesis of macrocyclic peptides with C-terminal β-amino-α-keto acid groups by three different metalloenzymes

“…4,[38][39][40] Our approach to finding BGCs that encode first-in-class RiPPs starts with bioinformatic searches centered on a prevalent, class-agnostic protein domain termed the RiPP precursor recognition element (RRE). 39,41,42 These domains are found in the majority of prokaryotic RiPP BGCs but have been difficult to identify owing to their small size (80-90 residues), frequent fusion to much larger proteins, and high sequence variability. All known RRE domains contain three alpha helices and a three-stranded beta sheet.…”

Automated, self-resistance gene-guided, and high-throughput genome mining of bioactive natural products fromStreptomyces

“…Conjugation transfer was used to introduce heterologous genes into Streptomyces 34 and the E. coli - Streptomyces di-parental intergenic conjugation method represents a commonly employed strategy in genetic engineering of Streptomyces 35, 36 . However, to our knowledge, no automated workflow for this conjugation process has been reported thus far.…”

Discovery and engineering of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products

“…78,139,140 When combined with other characteristic hallmarks for gene mining, the genetic signatures associated with precursor peptides provide a straightforward approach to exploring RiPP families/subfamilies that were previously overlooked. 6,141 2.2.1 NHLP/Nif11 related RiPPs. The discovery of two specific types of precursor peptides, namely nitrile hydrataserelated leader peptides (NHLP) and Nif11 nitrogen-fixing proteins (N11P), has attracted significant attention in the exploration of new RiPPs classes.…”

“…Recently, all predicted RRE domains from RRE-Finder were subjected to analysis using RODEO to determine their co-occurrence with short open reading frames (ORFs). 6 Among them, one type of fusion protein combining a domain of unknown function (DUF) with an RRE was selected, leading to the identification of 1441 putative precursor peptides from 483 BGCs. The representative BGC encodes a unique product termed daptide (Fig.…”

“…RiPPs have been found in all three kingdoms of life with remarkable structural diversity and wide-ranging biological functions, which arise from the considerable variability in precursor peptide sequences and the evolving catalytic reactions mediated by the modification enzymes. [6][7][8] Compared to the overwhelming demand, the approaches for discovering new RiPPs are currently limited. Historically, the methods of bioassay-guided screening have played a major role in RiPP discovery, but recently this kind of strategy is continuously showing signs of reaching its limits.…”