Peptide Biosynthetic Gene Clusters Research Articles

Isolation and identification of a new lasso peptide cattlecin from Streptomyces cattleya based on genome mining

Lasso peptides are ribosomally synthesized and posttranslationally modified peptides with diverse biological functions. Recent genome mining has revealed that many species of actinomycetes possibly contain biosynthetic gene clusters of lasso peptides. With genome mining for lasso peptide biosynthesis, we screened several actinomycetes for lasso peptide production using high-performance liquid chromatography and electrospray ionization–mass spectrometry. Consequently, Streptomyces cattleya was identified as a producer of a new lasso peptide named cattlecin. Analysis of amino acid content on cattlecin indicated the presence of four moles each of Asp and His, three moles each of Gly and Tyr, and one mole of Ser. Tandem mass spectrometry (MS/MS) analysis of cattlecin revealed C-terminal sequence of WHHGWYGWWDD. The peptide sequence (SYHWGDYHDWHHGWYGWWDD) was the expected amino acid sequence of cattlecin based on genome mining. As a result of MS/MS analysis, the amine residue of the first Ser was proposed to form a macrolactam ring with the β-carboxyl residue of the ninth Asp. The biosynthetic gene cluster of cattlecin comprised four genes: catA, catC, catB1, and catB2, which is typical of a lasso peptide biosynthetic gene cluster in actinomycetes.

Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA.

Lasso peptides are a member of the superclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Like all RiPPs, lasso peptides are derived from a gene-encoded precursor protein. The biosynthesis of lasso peptides requires two enzymatic activities: proteolytic cleavage between the leader peptide and the core peptide in the precursor protein, accomplished by the B enzymes, and ATP-dependent isopeptide bond formation, accomplished by the C enzymes. In a subset of lasso peptide biosynthetic gene clusters from Gram-positive organisms, the B enzyme is split between two proteins. One such gene cluster is found in the organism Rhodococcus jostii, which produces the antimicrobial lasso peptide lariatin. The B enzyme in R. jostii is split between two open reading frames, larB1 and larB2, both of which are required for lariatin biosynthesis. While the cysteine catalytic triad is found within the LarB2 protein, LarB1 is a PqqD homologue expected to bind to the lariatin precursor LarA based on its structural homology to other RiPP leader peptide binding domains. We show that LarB1 binds to the leader peptide of the lariatin precursor protein LarA with a sub-micromolar affinity. We used photocrosslinking with the noncanonical amino acid p-azidophenylalanine and mass spectrometry to map the interaction of LarA and LarB1. This analysis shows that the LarA leader peptide interacts with a conserved motif within LarB1 and, unexpectedly, the core peptide of LarA also binds to LarB1 in several positions. A Rosetta model built from distance restraints from the photocrosslinking experiments shows that the scissile bond between the leader peptide and core peptide in LarA is in a solvent-exposed loop.

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin

Lasso peptides are a new class of ribosomally synthesized and post-translationally modified peptides and thus far are only isolated from proteo- and actinobacterial sources. Typically, lasso peptide biosynthetic gene clusters encode enzymes for biosynthesis and export but not for tailoring. Here, we describe the isolation of the novel lasso peptide paeninodin from the firmicute Paenibacillus dendritiformis C454 and reveal within its biosynthetic cluster a gene encoding a kinase, which we have characterized as a member of a new class of lasso peptide-tailoring kinases. By employing a wide variety of peptide substrates, it was shown that this novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere. These experiments also reveal that no other recognition motif is needed for efficient enzymatic phosphorylation of the C-terminal serine. Furthermore, through comparison with homologous HPr kinases and subsequent mutational analysis, we confirmed the essential catalytic residues. Our study reveals how lasso peptides are chemically diversified and sets the foundation for rational engineering of these intriguing natural products.

Ustiloxins, fungal cyclic peptides, are ribosomally synthesized in Ustilaginoidea virens.

Ustiloxins A and B are toxic cyclic tetrapeptides, Tyr-Val/Ala-Ile-Gly (Y-V/A-I-G), that were originally identified from Ustilaginoidea virens, a pathogenic fungus affecting rice plants. Contrary to our report that ustiloxin B is ribosomally synthesized in Aspergillus flavus, a recent report suggested that ustiloxins are synthesized by a non-ribosomal peptide synthetase in U.virens. Thus, we analyzed the U.virens genome, to identify the responsible gene cluster. The biosynthetic gene cluster was identified from the genome of U.virens based on homologies to the ribosomal peptide biosynthetic gene cluster for ustiloxin B identified from A.flavus. It contains a gene encoding precursor protein having five Tyr-Val-Ile-Gly and three Tyr-Ala-Ile-Gly motifs for ustiloxins A and B, respectively, strongly indicating that ustiloxins A and B from U.virens are ribosomally synthesized. Accession codes of the U.virens and A.flavus gene clusters in NCBI are BR001221 and BR001206, respectively. Supplementary data are available at Bioinformatics online.

Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry

Lasso peptides are natural products with a unique three dimensional structure resembling a lariat knot. They are from ribosomal origin and are post-translationally modified by two enzymes (B and C), one of which shares little similarity to enzymes outside of lasso peptide biosynthetic gene clusters and as such is a useful target for genome mining. In this study, we demonstrate a B protein-centric genome mining approach through which we were able to identify 102 putative lasso peptide biosynthetic gene clusters from a total of 87 different proteobacterial strains. Ten of these clusters were cloned into the pET41a expression vector, optimized through incorporation of a ribosomal binding site and heterologously expressed in Escherichia coli BL21(DE3). All 12 predicted lasso peptides (namely burhizin, caulonodin I, caulonodin II, caulonodin III, rhodanodin, rubrivinodin, sphingonodin I, sphingonodin II, syanodin I, sphingopyxin I, sphingopyxin II, and zucinodin) were detected by high-resolution Fourier transform mass spectrometry and their proposed primary structure was confirmed through tandem mass spectrometry. High yields (ranging from 0.4 to 5.2 mg/L) were observable for eight of these compounds, while thermostability assays revealed five new representatives of heat labile lasso peptides.

The Genome Sequence of Streptomyces lividans 66 Reveals a Novel tRNA-Dependent Peptide Biosynthetic System within a Metal-Related Genomic Island

The complete genome sequence of the original isolate of the model actinomycete Streptomyces lividans 66, also referred to as 1326, was deciphered after a combination of next-generation sequencing platforms and a hybrid assembly pipeline. Comparative analysis of the genomes of S. lividans 66 and closely related strains, including S. coelicolor M145 and S. lividans TK24, was used to identify strain-specific genes. The genetic diversity identified included a large genomic island with a mosaic structure, present in S. lividans 66 but not in the strain TK24. Sequence analyses showed that this genomic island has an anomalous (G + C) content, suggesting recent acquisition and that it is rich in metal-related genes. Sequences previously linked to a mobile conjugative element, termed plasmid SLP3 and defined here as a 94 kb region, could also be identified within this locus. Transcriptional analysis of the response of S. lividans 66 to copper was used to corroborate a role of this large genomic island, including two SLP3-borne “cryptic” peptide biosynthetic gene clusters, in metal homeostasis. Notably, one of these predicted biosynthetic systems includes an unprecedented nonribosomal peptide synthetase—tRNA-dependent transferase biosynthetic hybrid organization. This observation implies the recruitment of members of the leucyl/phenylalanyl-tRNA-protein transferase family to catalyze peptide bond formation within the biosynthesis of natural products. Thus, the genome sequence of S. lividans 66 not only explains long-standing genetic and phenotypic differences but also opens the door for further in-depth comparative genomic analyses of model Streptomyces strains, as well as for the discovery of novel natural products following genome-mining approaches.

Identification and analysis of the gene cluster involved in biosynthesis of paenibactin, a catecholate siderophore produced by Paenibacillus elgii B69

Bacteria belonging to the genus Paenibacillus are recognized as rich sources of bioactive natural products. To date, there are few characterized siderophores from this genus. Here, through genome analysis, we identified a non-ribosomal peptide biosynthetic gene cluster (pae) responsible for siderophore assembly in Paenibacillus elgii B69. The 12.8 kb gene cluster comprises six open reading frames encoding proteins similar to the components of the bacillibactin biosynthetic machinery and bacillibactin esterase. To examine the product of the pae gene cluster, we cultured P. elgii B69 in iron-deficient medium for siderophore expression. A novel siderophore structurally similar to bacillibactin, designated paenibactin, was purified and characterized. Its structure was determined as a cyclic trimeric lactone of 2,3-dihydroxybenzoyl-alanine-threonine. The involvement of the pae gene cluster in paenibactin biosynthesis was confirmed by the biochemical assay of adenylation domain specificity. Furthermore, we demonstrated that the pae gene cluster evolves from an ancestral bacillibactin biosynthetic gene cluster via sequence and phylogenetic analyses. The structural difference between paenibactin and bacillibactin may stem from a mutation-induced change in the adenylation domain specificity. Based on these findings and published models for bacillibactin, we proposed models for paenibactin biosynthesis, ferric-paenibactin uptake and paenibactin-bounded iron release.

Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile Thermobifida fusca

Bacteria belonging to the order Actinomycetales have proven to be an important source of biologically active and often therapeutically useful natural products. The characterization of orphan biosynthetic gene clusters is an emerging and valuable approach to the discovery of novel small molecules. Analysis of the recently sequenced genome of the thermophilic actinomycete Thermobifida fusca revealed an orphan nonribosomal peptide biosynthetic gene cluster coding for an unknown siderophore natural product. T. fusca is a model organism for the study of thermostable cellulases and is a major degrader of plant cell walls. Here, we report the isolation and structure elucidation of the fuscachelins, siderophore natural products produced by T. fusca. In addition, we report the purification and biochemical characterization of the termination module of the nonribosomal peptide synthetase. Biochemical analysis of adenylation domain specificity supports the assignment of this gene cluster as the producer of the fuscachelin siderophores. The proposed nonribosomal peptide biosynthetic pathway exhibits several atypical features, including a macrocyclizing thioesterase that produces a 10-membered cyclic depsipeptide and a nonlinear assembly line, resulting in the unique heterodimeric architecture of the siderophore natural product.

Identification of a Biosynthetic Gene Cluster and the Six Associated Lipopeptides Involved in Swarming Motility ofPseudomonas syringaepv. tomato DC3000

Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to "mine" the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.