High divergence of the precursor peptides in combinatorial lanthipeptide biosynthesis

Abstract

Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and post-translationally modified peptides (RiPPs). These compounds are widely distributed in taxonomically distant species, and their biosynthetic systems and biological activities are diverse. A unique example of lanthipeptide biosynthesis is the prochlorosin synthetase ProcM from the marine cyanobacterium Prochlorococcus MIT9313, which transforms up to 29 different precursor peptides (ProcAs) into a library of lanthipeptides called prochlorosins (Pcns) with highly diverse sequences and ring topologies. Here, we show that many ProcM-like enzymes from a variety of bacteria have the capacity to carry out post-translational modifications on highly diverse precursor peptides, providing new examples of natural combinatorial biosynthesis. We also demonstrate that the leader peptides come from different evolutionary origins, suggesting that the combinatorial biosynthesis is tied to the enzyme and not a specific type of leader peptide. For some precursor peptides encoded in the genomes, the leader peptides apparently have been truncated at the N-termini, and we show that these N-terminally truncated peptides are still substrates of the enzymes. Consistent with this hypothesis, we demonstrate that about two-thirds of the ProcA N-terminal sequence is not essential for ProcM activity. Our results also highlight the potential of exploring this class of natural products by genome mining and bioengineering.

Figure 1

Schematic representation of the biosynthetic pathway of lanthipeptides exemplified by prochlorosin 2.8. A shorthand notation for lanthionine structures is shown in the box. Leader and core peptides are not shown in proportion to their actual lengths.

Figure 2

Genome mining of precursor peptide genes associated with ProcM-like enzymes. (A) Bayesian MCMC phylogram of ProcM-like enzymes (protein sequence) and a summary of the number of their putative LanA substrates. The lacticin 481 synthetase LctM and nukacin synthetase NukM were used as an outgroup for Bayesian MCMC analysis, which is shown as an orange triangle. The detailed Bayesian MCMC tree is shown in Supporting Information Figure 29. The putative lanA genes were categorized into two groups based on whether they are spatially close to their associated lanM genes. For LanAs that lack Cys residues, the substrates are shown as total number of lanAs/the number of lanAs that do not code for Cys. If an enzyme had multiple LanA substrates, then the core peptide sequences were aligned to examine whether these precursor peptides are similar (S, for which the Ser/Thr and Cys residues are aligned well) or diverse (D, for which Ser/Thr and Cys residues did not align well). ProcM from Prochlorococcus MIT9313, NpnM from Nostoc punctiforme PCC 73102, and four LanMs (CyanM1–4) from Cyanothece sp. PCC 7425 are highlighted in red, blue, and green, respectively. CyanM1 is highlighted by an asterisk. Three groups of substrates shown in blue contain leader peptides that share very weak similarities with the N11P family (1 × 10^–4 < e-value < 0.1). NA indicates that the precursors do not belong to any known protein family. (B) Sequence alignment of CyanA1.1–1.3, showing that CyanA1.1 and Cyan1.3 may have been truncated at their N-termini. Alternatively, the open reading frame (ORF) annotation of CyanA1.2 could be incorrect, and its translation start codon may instead be at the light brown arrow, like CyanA1.1 and 1.3. Completely conserved and highly conserved residues in the leader peptides are shown in black and gray boxes, respectively. Ser/Thr and Cys residues in the core peptides are shown in blue and red boxes, respectively. The proteolytic cleavage site is indicated by a green arrow. For detailed information on precursor peptide sequence and the procedures for bioinformatics analysis, see Supporting Information Table 1 and Supporting Information Methods.

Figure 3

Modification of ProcAt.1 by ProcM. (A) MALDI-ToF-MS analysis of ProcAt.1 that was obtained by coexpression with ProcM and treated with endoproteinase Glu-C (trace i) and subsequently derivatized by NEM (trace ii). (B) Sequence of ProcAt.1 modified by ProcM and treated with Glu-C. The ESI-MS/MS fragmentation pattern for the 3-fold dehydrated species is shown (the MS/MS data is presented in Supporting Information Figure 4).

Figure 4

ProcM modification of truncated ProcA2.8 derivatives. (A) Sequence of ProcA2.8 and schematic representation of the truncation variants discussed in this study. The purple arrow shows the physiological proteolytic cleavage site for leader peptide removal. The blue arrow shows the endoprotease Asp-N site that was used in this study to shorten the peptide and allow better analysis of the post-translational modifications in the core peptide. (B) MALDI-ToF MS analysis of ProcA2.8-(31–82) that was obtained either by expressing the peptide alone (trace i) or by coexpression with ProcM (trace ii). (C) MALDI-ToF MS analysis of ProcA-(41–82), presented in the same manner as for ProcA2.8-(31–82) in panel B. (D) ProcA2.8-(31–82) peptides (unmodified and modified) were digested by Asp-N and subsequently treated with NEM. Trace i shows the unmodified peptide before NEM derivatization, and trace ii demonstrates complete NEM derivatization of this peptide. Traces iii and iv show the ProcM-modified peptide before and after NEM treatment, respectively. No derivatization of the modified peptides is observed, strongly suggesting formation of lanthionine rings in the ProcM-modified peptide. (E) MALDI-ToF MS analysis of Asp-N-digested ProcA2.8-(41–82). The data are shown as in panel D. In all of the MS spectral data shown, the signals corresponding to the unmodified and the ProcM-modified peptides are highlighted in yellow and green, respectively, whereas the NEM-derivatized peptides are highlighted in light blue. Part of the nonhighlighted peaks are derived from proteolysis products of the leader peptide and Asp-N.

Figure 5

Lanthipeptide biosynthesis in Cyanothece sp. PCC 7425. (A) Four lanthipeptide biosynthetic systems in Cyanothece sp. PCC 7425, showing the gene clusters of each system and their locations in the genome. (B) Sequence similarity network based on the leader peptide sequence of CyanAs. Each node represents a leader peptide, and each edge (line) indicates a pair of nodes (leader peptides) that have a BlastP e-value more stringent than the cutoff value used (1 × 10^–7). Different biosynthetic systems are depicted by different colors. (C) High level of conservation in the N-terminal leader sequence and hypervariability of the C-terminal core peptide of CyanA3.1–3.12. The GG/GA protease cleavage site for leader peptide removal is marked by a green arrow. For the sequences of CyanA1, CyanA2, and CyanA4, see Supporting Information Table 1.

Figure 6

Coexpression studies of Cys-lacking peptides with LanMs. (A) MALDI-ToF MS analysis of ProcA4.1 that was obtained by coexpression with ProcM. Also shown is the sequence of the ProcA4.1 core (obtained by TEV cleavage of a ProcA4.1 mutant containing an engineered TEV cleavage site just before the predicted core sequence) and the MS/MS fragmentation pattern for the 3-fold dehydrated species. (B) MALDI-ToF MS analysis of NpnA3 that was obtained by coexpression with NpnM in E. coli. Also shown is the sequence of endoproteinase Glu-C cleaved NpnA3 and the MS/MS fragmentation pattern for the 4-fold dehydrated species. (C) MALDI-ToF MS analysis of NpnA6 obtained similarly to that for NpnA3 in panel B. Also presented is the sequence and MS/MS fragmentation pattern for 3-fold dehydrated NpnA6. The MS/MS data for 3-fold dehydrated ProcA4.1, 4-fold dehydrated NpnA3, and 3-fold dehydrated NpnA6 are shown in Supporting Information Figures 24–26, respectively.