Expanded natural product diversity revealed by analysis of lanthipeptide-like gene clusters in actinobacteria

Abstract

Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Lanthipeptides are widely distributed in taxonomically distant species, and their currently known biosynthetic systems and biological activities are diverse. Building on the recent natural product gene cluster family (GCF) project, we report here large-scale analysis of lanthipeptide-like biosynthetic gene clusters from Actinobacteria. Our analysis suggests that lanthipeptide biosynthetic pathways, and by extrapolation the natural products themselves, are much more diverse than currently appreciated and contain many different posttranslational modifications. Furthermore, lanthionine synthetases are much more diverse in sequence and domain topology than currently characterized systems, and they are used by the biosynthetic machineries for natural products other than lanthipeptides. The gene cluster families described here significantly expand the chemical diversity and biosynthetic repertoire of lanthionine-related natural products. Biosynthesis of these novel natural products likely involves unusual and unprecedented biochemistries, as illustrated by several examples discussed in this study. In addition, class IV lanthipeptide gene clusters are shown not to be silent, setting the stage to investigate their biological activities.

FIG 1

Biosynthesis of lanthipeptides. (A) The mechanism of (methyl)lanthionine formation. (B) The four currently known classes of lanthipeptide synthetases. Xn represents a peptide linker. The conserved zinc-binding motifs are highlighted by the purple lines in the cyclase domains. SpaB_C is an elimination domain present as a stand-alone protein in thiopeptide biosynthesis and as the C-terminal domain of LanB enzymes in lanthipeptide biosynthesis.

FIG 2

An ML tree of LanCs and LanC-like domains. In the outer ring, the four different classes of enzymes are depicted by the color defined in the legend, whereas the different GCFs are shown in the inner ring with random colors. The GCF numbers are defined in reference and can be found at http://www.igb.illinois.edu/labs/metcalf/gcf/lant.html. PKS, polyketide synthetase; NRPS, nonribosomal peptide synthetase; TOMM, thiazole/oxazole-modified microcins.

FIG 3

GCFs that likely produce two-component lanthipeptides. (A) A representative gene cluster of GCF146 and the sequence logos of the precursor peptide substrates. The predicted LanKC substrate/enzyme pair is shown in blue, and the predicted LanM substrate/enzyme pair is shown in red. The strictly conserved CxSxxS motif of the putative LanKC substrate and the GG/A leader peptide cleavage site of the putative LanM substrate are highlighted by red boxes. (B) A representative gene cluster of GCF30 that likely encodes a haloduracin-like system. The gene clusters appear in two different types, and homologous enzymes are linked by blue dashed lines. (C) A representative gene cluster of GCF46 that may encode class III two-component lanthipeptides. (D) A representative gene cluster of GCF140 that may encode three precursor peptides and a LanKC. The putative functions of the gene products are shown by colors. The logos of the precursor peptides in GCF30, GCF46, and GCF140 are shown in Fig. S2 to S4, respectively, in the supplemental material. The boundaries of each gene cluster are predicted based on the conserved genes within each GCF and are not clearly defined. RS, radical SAM; OR, oxidoreductase.

FIG 4

Association of genes encoding lanthipeptide synthetases with PKS and NRPS genes. (A) A representative gene cluster of GCF133 that likely encodes lanthipeptide synthetase-PKS hybrid systems. (B) A representative gene cluster of GCF26 that likely encodes lanthipeptide synthetase-NRPS hybrid systems, and a logo depicting the precursor peptide sequences. The empty sites in the leader peptide region are a consequence of high sequence divergence (i.e., no conserved residues). The sequence alignment of GCF26 precursor peptides is shown in Fig. S5 in the supplemental material. The GG leader peptide cleavage site and the conserved Cys residue in the putative core region are highlighted by a red box and a red asterisk, respectively. The putative functions of gene products are shown by colors. MT, malonyl transferase; ACP, acyl carrier protein; KS, ketosynthase; KR, ketoreductase; DH, dehydratase/enoyl-CoA hydratase; AL, ATP-dependent ligase; RNR, ribonucleotide reductase-like (these RNR-like proteins appear to contain the ligands for a dinuclear metal cluster but not a tyrosine for radical formation); sLanB, small LanB. The boundaries of each gene cluster are predicted based on the conserved genes within each GCF and are not clearly defined.

FIG 5

Putative LanC-containing lanthipeptide systems without LanB enzymes. (A) A representative gene cluster and the precursor peptide sequence logo of GCF125. (B) A representative gene cluster and the precursor peptide sequence logo of GCF114. The putative functions of gene products are shown by colors. The boundaries of each gene cluster are predicted based on the conserved genes within each GCF and are not clearly defined.

FIG 6

A representative gene cluster of GCF127 that likely encodes lanthipeptide-TOMM hybrid systems. The putative functions of gene products are shown by colors. The boundaries of each gene cluster are predicted based on the conserved genes within each GCF and are not clearly defined. CD, cyclodehydratase; DHG, dehydrogenase.

FIG 7

O-Methyltransferases in class I lanthipeptide biosynthesis. (A) Representative gene clusters of GCFs that encode class I lanthipeptide systems with a putative O-methyltransferase. (B) Sequence similarity network analysis of the putative O-methyltransferases in panel A.

FIG 8

Production of venezuelins in Actinobacteria. (A) Representative gene clusters of GCF147. (B) A logo depicting the GCF147 precursor peptide sequences. (C) MALDI-TOF MS analysis of venezuelin produced by Streptomyces katrae ISP5550. MALDI-TOF MS analysis of venezuelins produced by Streptomyces lavendulae subsp. lavendulae NRRL B-2508 and Streptomyces sp. NRRL B-2375 are shown in Fig. S8 in the supplemental material. (D) ESI-MS-MS analysis of venezuelin produced by S. katrae ISP5550. The lack of fragmentation is consistent with a globular, overlapping ring topology. (E) Chiral GC-MS analysis of hydrolyzed and derivatized venezuelin residues, which revealed the presence of dl-lanthionine and dl-methyllanthionine. Trace i, sample showing the presence of dl-lanthionine; trace ii, sample spiked with dl-lanthionine standard; trace iii, sample showing the presence of dl-methyllanthionine; trace iv, sample spiked with dl-methyllanthionine standard.