Lanthionine-containing peptides (lanthipeptides) are a family of ribosomally synthesized and posttranslationally modified peptides containing (methyl)lanthionine residues. Here we present a phylogenomic study of the four currently known classes of lanthipeptide synthetases (LanB and LanC for class I, LanM for class II, LanKC for class III, and LanL for class IV). Although they possess very similar cyclase domains, class II-IV synthetases have evolved independently, and LanB and LanC enzymes appear to not always have coevolved. LanM enzymes from various phyla that have three cysteines ligated to a zinc ion (as opposed to the more common CysCys-His ligand set) cluster together. Most importantly, the phylogenomic data suggest that for some scaffolds, the ring topology of the final lanthipeptides may be determined in part by the sequence of the precursor peptides and not just by the biosynthetic enzymes. This notion was supported by studies with two chimeric peptides, suggesting that the nisin and prochlorosin biosynthetic enzymes can produce the correct ring topologies of epilancin 15X and lacticin 481, respectively. These results highlight the potential of lanthipeptide synthetases for bioengineering and combinatorial biosynthesis. Our study also demonstrates unexplored areas of sequence space that may be fruitful for genome mining. molecular evolution | natural products | phylogeny | posttranslational modification | lantibiotics

Phosphinothricin-tripeptide (PTT, phosphinothricyl-alanyl-alanine) is a natural product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 21705 1 and Streptomyces viridochromogenes DSM 40736 2 . PTT has attracted widespread interest due to its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see 3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of heretofore undescribed catalyses, including a highly unusual reaction for carbon bond cleavage. In sum, we define a more complex pathway for early PTT biosynthesis that includes biochemically unprecedented and chemically interesting steps. KeywordsStreptomyces viridochromogenes; phosphinothricin; biosynthesis; bialaphos; phosphonate metabolism * Corresponding Author: Department of Microbiology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, Urbana, IL 61801, Phone: 217-244-1943, Fax: 217-244-6697, metcalf@uiuc.edu. # Present Address: Shanghai Chemspec Corp., No. 3 Lan 1273. Accession codesSequence data from the PTT biosynthetic gene cluster that was used in designing the experiments reported here was derived solely from previously available sequence (GenBank accession number AY632421). Sequence data generated in this study that corrects apparent sequencing errors in S. hygroscopicus PTT biosynthetic genes (found in GenBank accessions AB029917.1 and D37878.1) were deposited in GenBank under accession numbers EF486265 and EF486266 respectively. The bioactive portion of the PTT (also known as bialaphos, 1) tripeptide is phosphinothricin (PT, 2). PT is the only known naturally occurring compound to incorporate a direct carbon to phosphorus to carbon (C-P-C) bond motif. PTT has become a model for the biosynthesis of phosphonic acid antibiotics based on numerous studies 3 , leading to a pathway that is regarded as mostly solved. It should be noted, however, that many of the PTT non-producing mutants analyzed to define the pathway were generated by random chemical mutagenesis and some mutations were not rigorously mapped to individual genes. Accordingly, the resulting biosynthetic model is based upon a patchwork of chemical, enzymatic, and genetic data. Further, though many PTT biosynthetic genes were previously cloned and sequenced, the full gene cluster was not completely sequenced from either producer until very recently, when the cluster from S. viridochromogenes was isolated and characterized 7,8 . Sequence analysis of the cluster revealed that only about half of the genes identified had rigorous genetic or biochemical data establishing the...

Ribosomally synthesized and post-translationally modified peptides are a rapidly expanding class of natural products. They are typically biosynthesized by modification of a C-terminal segment of the precursor peptide (the core peptide). The precursor peptide also contains an N-terminal leader peptide that is required to guide the biosynthetic enzymes. For bioengineering purposes, the leader peptide is beneficial because it allows promiscuous activity of the biosynthetic enzymes with respect to modification of the core peptide sequence. However, the leader peptide also presents drawbacks as it needs to be present on the core peptide and then removed in a later step. We show that fusing the leader peptide for the lantibiotic lacticin 481 to its biosynthetic enzyme LctM allows the protein to act on core peptides without a leader peptide. We illustrate the use of this methodology for preparation of improved lacticin 481 analogues containing non-proteinogenic amino acids.

In recent years, the number of known peptide natural products that are synthesized via the ribosomal pathway has rapidly grown. Taking advantage of sequence homology among genes encoding precursor peptides or biosynthetic proteins, in silico mining of genomes combined with molecular biology approaches has guided the discovery of a large number of new ribosomal natural products, including lantipeptides, cyanobactins, linear thiazole/oxazole-containing peptides, microviridins, lasso peptides, amatoxins, cyclotides, and conopeptides. In this review, we describe the strategies used for the identification of these ribosomally-synthesized and posttranslationally modified peptides (RiPPs) and the structures of newly identified compounds. The increasing number of chemical entities and their remarkable structural and functional diversity may lead to novel pharmaceutical applications.

SUMMARY Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides. The recently discovered lantibiotic epilancin 15X produced by Staphylococcus epidermidis 15X154 contains an unusual N-terminal lactate group. To understand its biosynthesis, the epilancin 15X biosynthetic gene cluster was identified. The N-terminal lactate is produced by dehydration of a Ser residue in the first position of the core peptide by ElxB, followed by proteolytic removal of the leader peptide by ElxP, and hydrolysis of the resulting new N-terminal dehydroalanine. The pyruvate group thus formed is reduced to lactate by an NADPH dependent oxidoreductase designated ElxO. The enzymatic activity of ElxB, ElxP, and ElxO were investigated in vitro or in vivo and the importance of the N-terminal modification for peptide stability against bacterial aminopeptidases was assessed.

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for “stable isotope labeling of phosphonates in extract” (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S‐adenosyl methionine. Mass‐guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.

The final step in lanthipeptide biosynthesis involves the proteolytic removal of an N-terminal leader peptide. In the class I lanthipeptide epilancin 15X, this step is performed by the subtilisin-like serine peptidase ElxP. Bioinformatic, kinetic, and mass spectrometric analysis revealed that ElxP recognizes the stretch of amino acids DLNPQS located near the proteolytic cleavage site of its substrate, ElxA. When the ElxP recognition motif was inserted into the noncognate lanthipeptide precursor NisA, ElxP was able to proteolytically remove the leader peptide from NisA. Proteolytic removal of the leader peptide by ElxP during the biosynthesis of epilancin 15X exposes an N-terminal dehydroalanine on the core peptide of ElxA that hydrolyzes to a pyruvyl group. The short-chain dehydrogenase ElxO reduces the pyruvyl group to a lactyl moiety in the final step of epilancin 15X maturation. Using synthetic peptides, we also investigated the substrate specificity of ElxO and determined the 1.85 Å resolution X-ray crystal structure of the enzyme.

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for “stable isotope labeling of phosphonates in extract” (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S‐adenosyl methionine. Mass‐guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.