New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Rapid and Selective Chemical Editing of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs) via CuII -Catalyzed ß-Borylation of Dehydroamino Acids.

We report the fast and selective chemical editing of ribosomally synthesized and post-translationally modified peptides (RiPPs) by ß-borylation of dehydroalanine (Dha) residues. The thiopeptide thiostrepton was modified efficiently using CuII -catalysis under mild conditions and 1D/2D NMR of the purified product showed site-selective borylation of the terminal Dha residues. Using similar conditions, the thiopeptide nosiheptide, lanthipeptide nisin Z, and protein SUMO_G98Dha were also modified efficiently. Borylated thiostrepton showed an up to 84-fold increase in water solubility, and minimum inhibitory concentration (MIC) assays showed that antimicrobial activity was maintained in thiostrepton and nosiheptide. The introduced boronic-acid functionalities were shown to be valuable handles for chemical mutagenesis and in a reversible click reaction with triols for the pH-controlled labeling of RiPPs.

BAGEL4: a user-friendly web server to thoroughly mine RiPPs and bacteriocins.

Interest in secondary metabolites such as RiPPs (ribosomally synthesized and posttranslationally modified peptides) is increasing worldwide. To facilitate the research in this field we have updated our mining web server. BAGEL4 is faster than its predecessor and is now fully independent from ORF-calling. Gene clusters of interest are discovered using the core-peptide database and/or through HMM motifs that are present in associated context genes. The databases used for mining have been updated and extended with literature references and links to UniProt and NCBI. Additionally, we have included automated promoter and terminator prediction and the option to upload RNA expression data, which can be displayed along with the identified clusters. Further improvements include the annotation of the context genes, which is now based on a fast blast against the prokaryote part of the UniRef90 database, and the improved web-BLAST feature that dynamically loads structural data such as internal cross-linking from UniProt. Overall BAGEL4 provides the user with more information through a user-friendly web-interface which simplifies data evaluation. BAGEL4 is freely accessible at http://bagel4.molgenrug.nl.

Selective Modification of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs) through Diels-Alder Cycloadditions on Dehydroalanine Residues.

We report the late-stage chemical modification of ribosomally synthesized and post-translationally modified peptides (RIPPs) by Diels-Alder cycloadditions to naturally occurring dehydroalanines. The tail region of the thiopeptide thiostrepton could be modified selectively and efficiently under microwave heating and transition-metal-free conditions. The Diels-Alder adducts were isolated and the different site- and endo/exo isomers were identified by 1D/2D 1 H NMR. Via efficient modification of the thiopeptide nosiheptide and the lanthipeptide nisin Z the generality of the method was established. Minimum inhibitory concentration (MIC) assays of the purified thiostrepton Diels-Alder products against thiostrepton-susceptible strains displayed high activities comparable to that of native thiostrepton. These Diels-Alder products were also subjected successfully to inverse-electron-demand Diels-Alder reactions with a variety of functionalized tetrazines, demonstrating the utility of this method for labeling of RiPPs.

Major gene-regulatory mechanisms operating in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis.

Post-translationally modified peptides commonly display antimicrobial activity, but can also aid the development of bacterial colonies, giving a competitive advantage in the ecological niche. The production of post-translationally modified peptides by bacteria is a complex and energetically costly process that is strictly orchestrated in the cell. The onset of peptide production is linked to the different enzymes that take part during maturation, the transporters and the immunity determinants (if required). Thus, the population can make optimal use of available resources and obtain the benefits of production at an advantageous moment during growth, avoiding toxicity to itself. The timing and level of expression of the different operons is controlled by diverse (complex) regulatory pathways in response to environmental changes, stress or master regulators during specific growth transition phases. In this review, we highlight the basic principles and mechanisms of regulation of expression of post-translationally modified peptides and the relationship with the overall culture developmental processes and/or cellular differentiation. We also discuss the biotechnological consequences derived from the understanding of regulatory networks involved in the biosynthesis of these natural products.

Guiding antibiotics towards their target using bacteriophage proteins.

Novel therapeutic strategies against difficult-to-treat bacterial infections are desperately needed, and the faster and cheaper way to get them might be by repurposing existing antibiotics. Nanodelivery systems enhance the efficacy of antibiotics by guiding them to their targets, increasing the local concentration at the site of infection. While recently described nanodelivery systems are promising, they are generally not easy to adapt to different targets, and lack biocompatibility or specificity. Here, nanodelivery systems are created that source their targeting proteins from bacteriophages. Bacteriophage receptor-binding proteins and cell-wall binding domains are conjugated to nanoparticles, for the targeted delivery of rifampicin, imipenem, and ampicillin against bacterial pathogens. They show excellent specificity against their targets, and accumulate at the site of infection to deliver their antibiotic payload. Moreover, the nanodelivery systems suppress pathogen infections more effectively than 16 to 32-fold higher doses of free antibiotics. This study demonstrates that bacteriophage sourced targeting proteins are promising candidates to guide nanodelivery systems. Their specificity, availability, and biocompatibility make them great options to guide the antibiotic nanodelivery systems that are desperately needed to combat difficult-to-treat infections.

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.

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.

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.

Semisynthetic Macrocyclic Lipo-lanthipeptides Display Antimicrobial Activity Against Bacterial Pathogens.

A large number of antimicrobial peptides depend on intramolecular disulfide bonds for their biological activity. However, the relative instability of disulfide bonds has limited the potential of some of these peptides to be developed into therapeutics. Conversely, peptides containing intramolecular (methyl)lanthionine-based bonds, lanthipeptides, are highly stable under a broader range of biological and physical conditions. Here, the class-II lanthipeptide synthetase CinM, from the cinnamycin gene cluster, was employed to create methyllanthionine stabilized analogues of disulfide-bond-containing antimicrobial peptides. The resulting analogues were subsequently modified in vitro by adding lipid tails of variable lengths through chemical addition. Finally, the created compounds were characterized by MIC tests against several relevant pathogens, killing assays, membrane permeability assays, and hemolysis assays. It was found that CinM could successfully install methyllanthionine bonds at the intended positions of the analogues and that the lipidated macrocyclic core peptides have bactericidal activity against tested Gram-positive and Gram-negative pathogenic bacteria. Additionally, fluorescence microscopy assays revealed that the lipidated compounds disrupt the bacterial membrane and lyse bacterial cells, hinting toward a potential mode of action. Notably, the semisynthesized macrocyclic lipo-lanthipeptides show low hemolytic activity. These results show that the methods developed here extend the toolbox for novel antimicrobial development and might enable the further development of novel compounds with killing activity against relevant pathogenic bacteria.

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.

Synthesis and Characterization of Heterodimers and Fluorescent Nisin Species by Incorporation of Methionine Analogues and Subsequent Click Chemistry.

Noncanonical amino acids form a highly diverse pool of building blocks that can render unique physicochemical properties to peptides and proteins. Here, four methionine analogues with unsaturated and varying side chain lengths were successfully incorporated at four different positions in nisin in Lactococcus lactis through force feeding. This approach allows for residue-specific incorporation of methionine analogues into nisin to expand their structural diversity and alter their activity profiles. Moreover, the insertion of methionine analogues with biorthogonal chemical reactivity, e.g., azidohomoalanine and homopropargylglycine, provides the opportunity for chemical coupling to functional moieties and fluorescent probes as well as for intermolecular coupling of nisin variants. All resulting nisin conjugates retained antimicrobial activity, which substantiates the potential of this method as a tool to further study its localization and mode of action.

Conjugation of Synthetic Polyproline Moietes to Lipid II Binding Fragments of Nisin Yields Active and Stable Antimicrobials.

Coupling functional moieties to lantibiotics offers exciting opportunities to produce novel derivatives with desirable properties enabling new functions and applications. Here, five different synthetic hydrophobic polyproline peptides were conjugated to either nisin AB (the first two rings of nisin) or nisin ABC (the first three rings of nisin) by using click chemistry. The antimicrobial activity of nisin ABC + O6K3 against Enterococcus faecium decreased 8-fold compared to full-length nisin, but its activity was 16-fold better than nisin ABC, suggesting that modifying nisin ABC is a promising strategy to generate semi-synthetic nisin hybrids. In addition, the resulting nisin hybrids are not prone to degradation at the C-terminus, which has been observed for nisin as it can be degraded by nisinase or other proteolytic enzymes. This methodology allows for getting more insight into the possibility of creating semi-synthetic nisin hybrids that maintain antimicrobial activity, in particular when synthetic and non-proteinaceous moieties are used. The success of this approach in creating viable nisin hybrids encourages further exploring the use of different modules, e.g., glycans, lipids, active peptide moieties, and other antimicrobial moieties.