Precursor-centric genome-mining approach for lasso peptide discovery

Abstract

Lasso peptides are a class of ribosomally synthesized posttranslationally modified natural products found in bacteria. Currently known lasso peptides have a diverse set of pharmacologically relevant activities, including inhibition of bacterial growth, receptor antagonism, and enzyme inhibition. The biosynthesis of lasso peptides is specified by a cluster of three genes encoding a precursor protein and two enzymes. Here we develop a unique genome-mining algorithm to identify lasso peptide gene clusters in prokaryotes. Our approach involves pattern matching to a small number of conserved amino acids in precursor proteins, and thus allows for a more global survey of lasso peptide gene clusters than does homology-based genome mining. Of more than 3,000 currently sequenced prokaryotic genomes, we found 76 organisms that are putative lasso peptide producers. These organisms span nine bacterial phyla and an archaeal phylum. To provide validation of the genome-mining method, we focused on a single lasso peptide predicted to be produced by the freshwater bacterium Asticcacaulis excentricus. Heterologous expression of an engineered, minimal gene cluster in Escherichia coli led to the production of a unique lasso peptide, astexin-1. At 23 aa, astexin-1 is the largest lasso peptide isolated to date. It is also highly polar, in contrast to many lasso peptides that are primarily hydrophobic. Astexin-1 has modest antimicrobial activity against its phylogenetic relative Caulobacter crescentus. The solution structure of astexin-1 was determined revealing a unique topology that is stabilized by hydrogen bonding between segments of the peptide.bioinformatics | protein NMR

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin

“…2b). In contrast to the lasso peptides that were previously predicted in firmicutes (all of which bear an N-terminal Gly) (25), those predicted here feature an N-terminal Ala, a property only recently observed for a series of lasso peptides from Caulobacter sp. K31 (11).…”

“…Gene Cluster Type in Firmicutes-As only lasso peptides originating from proteo-and actinobacteria have been characterized (3,4,7,25,31), we sought to uncover interesting gene clusters in other bacterial phyla. By employing our previously described B protein-centric genome mining approach (7), a new family of gene clusters predominantly found in Bacillus und Paenibacillus spp.…”

“…Until now, all isolated lasso peptides originate from proteobacterial or actinobacterial sources (3,4,7,31), although biosynthetic clusters from other bacterial phyla have been predicted (25). Here, we describe a family of lasso peptide biosynthetic gene clusters that are found primarily in Paenibacillus and Bacillus spp.…”

“…Interestingly, the studied gene cluster was found to contain not only the conserved genes encoding the precursor peptide, processing enzymes, and ABC transporter, but an additional gene coding for a putative kinase. Such a gene is notably absent from all characterized lasso peptide biosynthetic gene clusters (3,4,7,25,31).…”

“…Generally, all known lasso peptides carry a Gly, Ala, Cys, or Ser residue at position 1 and a ring-forming Glu or Asp residue at positions 7-9 (3,4,7,10,11,21,25). Their respective processing enzymes are highly specific for the nature and location of these residues (6, 9 -11, 16, 28 -30).…”