Lanthionine Rings Research Articles

Discovery, biosynthesis, and characterization of a lanthipeptide from Bacillus subtilis EH11 with a unique lanthionine ring pattern

Discovery, biosynthesis, and characterization of a lanthipeptide from Bacillus subtilis EH11 with a unique lanthionine ring pattern

Dual targeting of the class V lanthipeptide antibiotic cacaoidin

Antibiotic resistance is reaching alarming levels, demanding for the discovery and development of antibiotics with novel chemistry and mechanisms of action. The recently discovered antibiotic cacaoidin combines the characteristic lanthionine residue of lanthipeptides and the linaridin-specific N-terminal dimethylation in an unprecedented N-dimethyl lanthionine ring, being therefore designated as the first class V lanthipeptide (lanthidin). Further notable features include the high D-amino acid content and a unique disaccharide substitution attached to the tyrosine residue. Cacaoidin shows antimicrobial activity against gram-positive pathogens and was shown to interfere with peptidoglycan biosynthesis. Initial investigations indicated an interaction with the peptidoglycan precursor lipid IIPGN as described for several lanthipeptides. Using a combination of biochemical and molecular interaction studies we provide evidence that cacaoidin is the first natural product demonstrated to exhibit a dual mode of action combining binding to lipid IIPGN and direct inhibition of cell wall transglycosylases.

The structure of MadC from Clostridium maddingley reveals new insights into class I lanthipeptide cyclases.

The rapid emergence of microbial multi-resistance against antibiotics has led to intense search for alternatives. One of these alternatives are ribosomally synthesized and post-translationally modified peptides (RiPPs), especially lantibiotics. They are active in a low nanomolar range and their high stability is due to the presence of characteristic (methyl-) lanthionine rings, which makes them promising candidates as bacteriocides. However, innate resistance against lantibiotics exists in nature, emphasizing the need for artificial or tailor-made lantibiotics. Obviously, such an approach requires an in-depth mechanistic understanding of the modification enzymes, which catalyze the formation of (methyl-)lanthionine rings. Here, we determined the structure of a class I cyclase (MadC), involved in the modification of maddinglicin (MadA) via X-ray crystallography at a resolution of 1.7 Å, revealing new insights about the structural composition of the catalytical site. These structural features and substrate binding were analyzed by mutational analyses of the leader peptide as well as of the cyclase, shedding light into the mode of action of MadC.

Investigating the Specificity of the Dehydration and Cyclization Reactions in Engineered Lanthipeptides by Synechococcal SyncM.

ProcM-like enzymes are class II promiscuous lanthipeptide synthetases that are an attractive tool in synthetic biology for producing lanthipeptides with biotechnological or clinically desired properties. SyncM is a recently described modification enzyme from this family used to develop a versatile expression platform for engineering lanthipeptides. Most remarkably, SyncM can modify up to 79 SyncA substrates in a single strain. Six SyncAs were previously characterized from this pool of substrates. They showed particular characteristics, such as the presence of one or two lanthionine rings, different flanking residues influencing ring formation, and different ring directions, demonstrating the relaxed specificity of SyncM toward its precursor peptides. To gain a deeper understanding of the potential of SyncM as a biosynthetic tool, we further explored the enzyme's capabilities and limits in dehydration and ring formation. We used different SyncA scaffolds for peptide engineering, including changes in the ring's directionality (relative position of Ser/Thr to Cys in the peptide) and size. We further aimed to rationally design mimetics of cyclic antimicrobials and introduce macrocycles in prochlorosin-related and nonrelated substrates. This study highlights the largest lanthionine ring with 15 amino acids (ring-forming residues included) described to date. Taking advantage of the amino acid substrate tolerance of SyncM, we designed the first single-SyncA-based antimicrobial. The insights gained from this work will aid future bioengineering studies. Additionally, it broadens SyncM's application scope for introducing macrocycles in other bioactive molecules.

Class V Lanthipeptide Cyclase Directs the Biosynthesis of a Stapled Peptide Natural Product.

Lanthipeptides are a class of cyclic peptides characterized by the presence of one or more lanthionine (Lan) or methyllanthionine (MeLan) thioether rings. These cross-links are produced by α,β-unsaturation of Ser or Thr residues in peptide substrates by dehydration, followed by a Michael-type conjugate addition of Cys residues onto the dehydroamino acids. Lanthipeptides may be broadly classified into at least five different classes, and the biosynthesis of classes I-IV lanthipeptides requires catalysis by LanC cyclases that control both the site-specificity and the stereochemistry of the conjugate addition. In contrast, there are no current examples of LanCs that occur in class V biosynthetic clusters, despite the presence of lanthionine rings in these compounds. In this work, bioinformatics-guided co-occurrence analysis identifies more than 240 putative class V lanthipeptide clusters that contain a LanC cyclase. Reconstitution studies demonstrate that the cyclase-catalyzed product is notably distinct from the product formed spontaneously. Stereochemical analysis shows that the cyclase diverts the final product to a configuration that is distinct from one that is energetically favored. Structural characterization of the final product by multi-dimensional NMR spectroscopy reveals that it forms a helical stapled peptide. Mutational analysis identified a plausible order for cyclization and suggests that enzymatic rerouting to the final structure is largely directed by the construction of the first lanthionine ring. These studies show that lanthipeptide cyclases are needed for the biosynthesis of some constrained peptides, the formations of which would otherwise be energetically unfavored.

Modular Use of the Uniquely Small Ring A of Mersacidin Generates the Smallest Ribosomally Produced Lanthipeptide.

Mersacidin is an antimicrobial class II lanthipeptide. Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), characterized by intramolecular lanthionine rings. These rings give lanthipeptides their bioactive structure and stability. RiPPs are produced from a gene cluster that encodes a precursor peptide and its dedicated unique modification enzymes. The field of RiPP engineering aims to recombine modification enzymes from different RiPPs to modify new substrates, resulting in new-to-nature molecules with novel or improved functionality. The enzyme MrsM from the mersacidin gene cluster installs the four lanthionine rings of mersacidin, including the uniquely small ring A. By applying MrsM in RiPP engineering, this ring could be installed in linear peptides to achieve stabilization by a very small lanthionine or to create small lanthionine-stabilized modules for chemical modification. However, the formation of unique intramolecular structures like that of mersacidin’s ring A can be very stringent. Here, the formation of ring A of mersacidin is characterized by mutagenesis. A range of truncated mersacidin variants was made to identify the smallest possible construct in which this ring could still be formed. Additionally, mutants were created to study the flexibility of ring A formation. It was found that although the formation of ring A is stringent, it can be formed in a core peptide as small as five amino acids. The truncated mersacidin core peptide CTFAL is the smallest ribosomally produced lanthipeptide reported to date, and it has exciting prospects as a new module for application in RiPP engineering.

Mutational Studies of the Mersacidin Leader Reveal the Function of Its Unique Two-Step Leader Processing Mechanism.

The class II lanthipeptide mersacidin, a ribosomally synthesized and post-translationally modified peptide (RiPP), displays unique intramolecular structures, including a very small lanthionine ring. When applied in the growing field of RiPP engineering, these can add unique features to new-to-nature compounds with novel properties. Recently, a heterologous expression system for mersacidin in Escherichia coli was developed to add its modification enzymes to the RiPP engineering toolbox and further explore mersacidin biosynthesis and leader-processing. The dedicated mersacidin transporter and leader protease MrsT was shown to cleave the leader peptide only partially upon export, transporting GDMEAA-mersacidin out of the cell. The extracellular Bacillus amyloliquefaciens protease AprE was shown to release active mersacidin in a second leader-processing step after transport. The conserved LanT cleavage site in the mersacidin leader is present in many other class II lanthipeptides. In contrast to mersacidin, the leader of these peptides is fully processed in one step. This difference with mersacidin leader-processing raises fundamentally interesting questions about the specifics of mersacidin modification and processing, which is also crucial for its application in RiPP engineering. Here, mutational studies of the mersacidin leader–core interface were performed to answer these questions. Results showed the GDMEAA sequence is crucial for both mersacidin modification and leader processing, revealing a unique leader layout in which a LanM recognition site is positioned downstream of the conserved leader-protease LanT cleavage site. Moreover, by identifying residues and regions that are crucial for mersacidin-type modifications, the wider application of mersacidin modifications in RiPP engineering has been enabled.

Heterologous expression of a cryptic gene cluster from a marine proteobacterium Thalassomonas actiniarum affords new lanthipeptides thalassomonasins A and B.

The aim of this study was to utilize a cryptic biosynthetic gene cluster (BGC) of a marine proteobacterium Thalassomonas actiniarum for production of new lanthipeptides by heterologous expression system. Based on genome mining, a new BGC of class I lanthipeptide was found in the genome sequence of a marine proteobacterium T. actiniarum. Molecular cloning was performed to construct an expression vector derived from commercially available plasmid pET-41a(+). Heterologous production of new lanthipeptides named thalassomonasins A and B was performed using the host Escherichia coli BL21(DE3) harbouring the expression vector. The structure of thalassomonasin A was determined by the interpretation of NMR and MS data. As a result, thalassomonasin A was determined to be a lanthipeptide with three units of lanthionine. The bridging pattern of the lanthionine rings in thalassomonasin A was determined by interpretation of NOESY data. The structure of thalassomonasin B was proposed by MS/MS experiment. We succeeded in heterologous production of new class I lanthipeptides using a BGC of a marine proteobacterium T. actiniarum. To the best of our knowledge, this is the first report of heterologous production of lanthipeptides derived from proteobacterial origin. There are many cryptic biosynthetic gene clusters (BCGs) of this class of lanthipeptides in proteobacterial genomes. This study may lead to the production of new lanthipeptides by utilizing the BCGs.

Recombinant production of the lantibiotic nisin using Corynebacterium glutamicum in a two-step process

BackgroundThe bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products.ResultsWe established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin.ConclusionsWe demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.

Characterization of Leader Processing Shows That Partially Processed Mersacidin Is Activated by AprE After Export.

The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing.

Functional Expression and Characterization of the Highly Promiscuous Lanthipeptide Synthetase SyncM, Enabling the Production of Lanthipeptides with a Broad Range of Ring Topologies.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of lanthionine rings that provide stability and functionality. Genome mining techniques have shown their huge diversity and potential for the discovery of novel active molecules. However, in many cases, they are not easily produced under laboratory conditions. The heterologous expression of these molecules using well-characterized lanthipeptide biosynthetic enzymes is rising as an alternative system for the design and production of new lanthipeptides with biotechnological or clinical properties. Nevertheless, the substrate-enzyme specificity limits the complete modification of the desired peptides and hence, their full stability and/or biological activity. New low substrate-selective biosynthetic enzymes are therefore necessary for the heterologous production of new-to-nature peptides. Here, we have identified, cloned, and heterologously expressed in Lactococcus lactis the most promiscuous lanthipeptide synthetase described to date, i.e., SyncM from the marine cyanobacteria Synechococcus MITS9509. We have characterized the functionality of SyncM by the successful expression of 15 out of 18 different SyncA substrates, subsequently determining the dehydration and cyclization processes in six representatives of them. This characterization highlights the very relaxed substrate specificity of SyncM toward its precursors and the ability to catalyze the formation of exceptionally large rings in a variety of topologies. Our results suggest that SyncM could be an attractive enzyme to design and produce a wide variety of new-to-nature lanthipeptides with a broad range of ring topologies.

Heterologous Expression of Mersacidin in Escherichia coli Elucidates the Mode of Leader Processing.

The lanthipeptide mersacidin is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by Bacillus amyloliquefaciens. It has antimicrobial activity against a range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, giving it potential therapeutic relevance. The structure and bioactivity of mersacidin are derived from a unique combination of lanthionine ring structures, which makes mersacidin also interesting from a lantibiotic-engineering point of view. Until now, mersacidin and its derivatives have exclusively been produced in Bacillus strains and purified from the supernatant in their bioactive form. However, to fully exploit its potential in lanthipeptide-engineering, mersacidin would have to be expressed in a standardized expression system and obtained in its inactive prepeptide form. In such a system, the mersacidin biosynthetic enzymes could be employed to create novel peptides, enhanced by the recent advancements in RiPP engineering, while the leader peptide prevents activity against the expression host. This system would however need a means of postpurification in vitro leader processing to activate the obtained precursor peptides. While mersacidin’s native leader processing mechanism has not been confirmed, the bifunctional transporter MrsT and extracellular Bacillus proteases have been suggested to be responsible. Here, a modular system is presented for the heterologous expression of mersacidin in Escherichia coli, which was successfully used to produce and purify inactive premersacidin. The purified product was used to determine the cleavage site of MrsT. Additionally, it was concluded from antimicrobial activity tests that in a second processing step mersacidin is activated by specific extracellular proteases from Bacillus amyloliquefaciens.

Importance of the leader peptide sequence on the lanthipeptide secretion level.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides. Their precursor peptide comprises of an N-terminal leader peptide and a C-terminal core peptide. Here, the leader peptide is crucial for enzyme recognition especially for the modification enzymes and acts furthermore as a secretion signal for the lanthipeptide exporter. The core peptide is the target site for the posttranslational modifications and contains dehydrated amino acids and lanthionine rings. Nisin produced by the Gram-positive bacterium Lactococcus lactis is one of the best-studied lanthipeptides and used as a model system to study their modification and secretion processes. Nisin is secreted as a precursor peptide. Here, we present an in vivo secretion analysis of NisT in the absence of the modification machinery allowing the secretion of leader peptide mutants and their impact solely on the secretion activity of NisT. Additionally, we created leader peptide hybrids to provide new insights, how the secretion is effected by unnatural leader peptides. The focus on the secretion activity of the transporter alone enabled us to determine the recognition site of NisT within the leader peptide of nisin.

Impact of the nisin modification machinery on the transport kinetics of NisT

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. One of the best-studied examples is nisin produced by Lactococcus lactis. Nisin is synthesized as a precursor peptide comprising of an N-terminal leader peptide and a C-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABC transporter NisT that secretes nisin in a proposed channeling mechanism. Here, we present an in vivo secretion analysis of this process in the presence and absence of the nisin maturation machinery, consisting of the dehydratase NisB and the cyclase NisC. Our determined apparent secretion rates of NisT show how NisB and NisC modulate the transport kinetics of NisA. Additional in vitro studies of the detergent-solubilized NisT revealed how these enzymes and the substrates again influence the activity of transporter. In summary, this study highlights the pivotal role of NisB for NisT in the secretion process.

Frontispiece: Cacaoidin, First Member of the New Lanthidin RiPP Family

Natural Products In their Communication on page 12654, F. J. Ortiz-López et al. describe a novel glycosylated lantibiotic, cacaoidin, which inaugurates a new RiPP family characterized by the co-occurrence of lanthionine rings and N-terminus dimethylation.

Cacaoidin, First Member of the New Lanthidin RiPP Family

AbstractLantibiotics are ribosomally synthesized and post‐translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d‐amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N‐dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram‐positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.

Cacaoidin, First Member of the New Lanthidin RiPP Family

Lantibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d-amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N-dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram-positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.

A Structural View on the Maturation of Lanthipeptides.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides, which display diverse bioactivities (e.g., antifungal, antimicrobial, and antiviral). One characteristic of these lanthipeptides is the presence of thioether bonds, which are termed (methyl-) lanthionine rings. These modifications are installed by corresponding modification enzymes in a two-step modality. First, serine and threonine residues are dehydrated followed by a subsequent catalyzed cyclization reaction, in which the dehydrated serine and threonine residues are undergoing a Michael-type addition with cysteine residues. The dedicated enzymes are encoded by one or two genes and the classification of lanthipeptides is pending on this. The modification steps form the basis of distinguishing the different classes of lanthipeptides and furthermore reflect also important mechanistic differences. Here, we will summarize recent insights into the mechanisms and the structures of the participating enzymes, focusing on the two core modification steps – dehydration and cyclization.

High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB.

Microbial lanthipeptides are formed by a two-step enzymatic introduction of (methyl)lanthionine rings. A dehydratase catalyzes the dehydration of serine and threonine residues, yielding dehydroalanine and dehydrobutyrine, respectively. Cyclase-catalyzed coupling of the formed dehydroresidues to cysteines forms (methyl)lanthionine rings in a peptide. Lanthipeptide biosynthetic systems allow discovery of target-specific, lanthionine-stabilized therapeutic peptides. However, the substrate specificity of existing modification enzymes impose limitations on installing lanthionines in non-natural substrates. The goal of the present study was to obtain a lanthipeptide dehydratase with the capacity to dehydrate substrates that are unsuitable for the nisin dehydratase NisB. We report high-throughput screening for tailored specificity of intracellular, genetically encoded NisB dehydratases. The principle is based on the screening of bacterially displayed lanthionine-constrained streptavidin ligands, which have a much higher affinity for streptavidin than linear ligands. The designed NisC-cyclizable high-affinity ligands can be formed via mutant NisB-catalyzed dehydration but less effectively via wild-type NisB activity. In Lactococcus lactis, a cell surface display precursor was designed comprising DSHPQFC. The Asp residue preceding the serine in this sequence disfavors its dehydration by wild-type NisB. The cell surface display vector was coexpressed with a mutant NisB library and NisTC. Subsequently, mutant NisB-containing bacteria that display cyclized strep ligands on the cell surface were selected via panning rounds with streptavidin-coupled magnetic beads. In this way, a NisB variant with a tailored capacity of dehydration was obtained, which was further evaluated with respect to its capacity to dehydrate nisin mutants. These results demonstrate a powerful method for selecting lanthipeptide modification enzymes with adapted substrate specificity.

Roseocin, a novel two-component lantibiotic from an actinomycete.

Lantibiotics are lanthionine ring containing natural products that belong to the class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Recent expansion in the availability of microbial genome data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic, roseocin, was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Posttranslationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in Escherichia coli, and were in vitro reconstituted to their bioactive form by exploiting commercial proteases like endoproteinase GluC, and proteinase K. The two peptides displayed synergistic antimicrobial activity against Gram-positive bacteria including the WHO high-priority pathogens, MRSA and VRE. Structural characterization confirmed the installation of four (methyl)lanthionine rings with an indispensable disulfide bond in the α-peptide, and six (methyl)lanthionine rings in the β-peptide, by a single promiscuous lanthionine synthetase, RosM. Roseocin is the first two-component lantibiotic from a non-Firmicute, with extensive lanthionine bridging.