scholarly journals Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification

2021 ◽  
Author(s):  
Li Cao ◽  
Moshe Beiser ◽  
Joseph D Koos ◽  
Margarita Orlova ◽  
Hader E Elashal ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Genome Mining ◽  
Gene Clusters ◽  
Post Translational Modification ◽  
Protein Repair ◽  
Lasso Peptide ◽  
Lasso Peptides ◽  
Modified Peptides ◽  
Asparagine Deamidation

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

scholarly journals Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool

2018 ◽  
Author(s):  
Javier Santos-Aberturas ◽  
Govind Chandra ◽  
Luca Frattaruolo ◽  
Rodney Lacret ◽  
Thu H. Pham ◽  
...  
Keyword(s):  
Genome Mining ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
Bioactive Molecules ◽  
Post Translational Modification ◽  
Biosynthetic Gene Clusters ◽  
New Family ◽  
The Family ◽  
Modified Peptides ◽  
Mining Tool

ABSTRACTThe rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, and it has been proposed that a YcaO-TfuA pair of proteins also catalyzes their formation. Potential biosynthetic gene clusters encoding YcaO and TfuA protein pairs are common in Actinobacteria but the chemical diversity generated by these pathways is almost completely unexplored. A RiPPER analysis reveals a highly diverse landscape of precursor peptides encoded in previously undescribed gene clusters that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new family of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

scholarly journals Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34T Yields a Novel Lasso Peptide, Leepeptin

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Juan Pablo Gomez-Escribano ◽  
Jean Franco Castro ◽  
Valeria Razmilic ◽  
Scott A. Jarmusch ◽  
Gerhard Saalbach ◽  
...  
Keyword(s):  
Natural Products ◽  
Heterologous Expression ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Bioinformatic Analysis ◽  
Growth Media ◽  
Content Type ◽  
Lasso Peptide ◽  
Lasso Peptides ◽  
New Family

ABSTRACT Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides. IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

scholarly journals Precursor-centric genome-mining approach for lasso peptide discovery

2012 ◽  
Vol 109 (38) ◽  
pp. 15223-15228 ◽  
Author(s):  
Mikhail O. Maksimov ◽  
István Pelczer ◽  
A. James Link
Keyword(s):  
Solution Structure ◽  
Caulobacter Crescentus ◽  
Genome Mining ◽  
Gene Clusters ◽  
Bacterial Phyla ◽  
Lasso Peptide ◽  
Lasso Peptides ◽  
Genes Encoding ◽  
Peptide Gene ◽  
Conserved Amino Acids

Lasso peptides are a class of ribosomally synthesized posttranslationally modified natural products found in bacteria. Currently known lasso peptides have a diverse set of pharmacologically relevant activities, including inhibition of bacterial growth, receptor antagonism, and enzyme inhibition. The biosynthesis of lasso peptides is specified by a cluster of three genes encoding a precursor protein and two enzymes. Here we develop a unique genome-mining algorithm to identify lasso peptide gene clusters in prokaryotes. Our approach involves pattern matching to a small number of conserved amino acids in precursor proteins, and thus allows for a more global survey of lasso peptide gene clusters than does homology-based genome mining. Of more than 3,000 currently sequenced prokaryotic genomes, we found 76 organisms that are putative lasso peptide producers. These organisms span nine bacterial phyla and an archaeal phylum. To provide validation of the genome-mining method, we focused on a single lasso peptide predicted to be produced by the freshwater bacterium Asticcacaulis excentricus. Heterologous expression of an engineered, minimal gene cluster in Escherichia coli led to the production of a unique lasso peptide, astexin-1. At 23 aa, astexin-1 is the largest lasso peptide isolated to date. It is also highly polar, in contrast to many lasso peptides that are primarily hydrophobic. Astexin-1 has modest antimicrobial activity against its phylogenetic relative Caulobacter crescentus. The solution structure of astexin-1 was determined revealing a unique topology that is stabilized by hydrogen bonding between segments of the peptide.

scholarly journals Albusnodin: an acetylated lasso peptide fromStreptomyces albus

2018 ◽  
Vol 54 (11) ◽  
pp. 1339-1342 ◽  
Author(s):  
Chuhan Zong ◽  
Wai Ling Cheung-Lee ◽  
Hader E. Elashal ◽  
Monika Raj ◽  
A. James Link
Keyword(s):  
Heterologous Expression ◽  
Genome Mining ◽  
Post Translational Modification ◽  
Lasso Peptide

Genome mining and heterologous expression has revealed a new lasso peptide, albusnodin, with an obligate acetyllysine post-translational modification.

scholarly journals Reactivity-based screening for citrulline-containing natural products reveals a family of bacterial peptidyl arginine deiminases

2020 ◽  
Author(s):  
Lonnie A. Harris ◽  
Patricia M. B. Saint-Vincent ◽  
Xiaorui Guo ◽  
Graham A. Hudson ◽  
Douglas A. Mitchell
Keyword(s):  
Natural Products ◽  
Arginine Deiminase ◽  
Post Translational Modification ◽  
Genomic Context ◽  
Lasso Peptide ◽  
Lasso Peptides ◽  
C Terminus ◽  
A Genome ◽  
Modified Peptides ◽  
Phylogenetic Profiling

ABSTRACTRibosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products defined by a genetically encoded precursor peptide that is tailored by associated biosynthetic enzymes to form the mature product. Lasso peptides are a class of RiPP defined by an isopeptide linkage between the N-terminal amine and an internal Asp/Glu residue with the C-terminus threaded through the macrocycle. This unique lariat topology, which provides considerable stability towards heat and proteases, has stimulated interest in lasso peptides as potential therapeutics. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of arginine deimination to yield citrulline. Although a citrulline-containing lasso peptide (i.e., citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for arginine deimination has remained unknown. Herein we describe the use of reactivity-based screening to discriminate bacteria that produce arginine-versus citrulline-bearing citrulassins, culminating in the discovery and characterization of 11 new lasso peptide variants. Phylogenetic profiling identified a distally encoded peptidyl arginine deiminase (PAD) gene ubiquitous to the citrulline-containing variants. Absence of this gene correlated strongly with citrulassin variants only containing arginine (des-citrulassin). Heterologous expression of the PAD in a non-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in arginine deimination. The family of PADs were then bioinformatically surveyed for a deeper understanding of its genomic context and potential role in post-translational modification of RiPPs.

scholarly journals Identification, Cloning and Heterologous Expression of the Gene Cluster Directing RES-701-3, -4 Lasso Peptides Biosynthesis from a Marine Streptomyces Strain

Marine Drugs ◽  
2020 ◽  
Vol 18 (5) ◽  
pp. 238 ◽  
Author(s):  
Daniel Oves-Costales ◽  
Marina Sánchez-Hidalgo ◽  
Jesús Martín ◽  
Olga Genilloud
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Fermentation Broth ◽  
Genome Mining ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Tryptophan Residue ◽  
Post Translational Modification ◽  
Lasso Peptides ◽  
Genes Encoding

RES-701-3 and RES-701-4 are two class II lasso peptides originally identified in the fermentation broth of Streptomyces sp. RE-896, which have been described as selective endothelin type B receptor antagonists. These two lasso peptides only differ in the identity of the C-terminal residue (tryptophan in RES-701-3, 7-hydroxy-tryptophan in RES-701-4), thus raising an intriguing question about the mechanism behind the modification of the tryptophan residue. In this study, we describe the identification of their biosynthetic gene cluster through the genome mining of the marine actinomycete Streptomyces caniferus CA-271066, its cloning and heterologous expression, and show that the seven open reading frames (ORFs) encoded within the gene cluster are sufficient for the biosynthesis of both lasso peptides. We propose that ResE, a protein lacking known putatively conserved domains, is likely to play a key role in the post-translational modification of the C-terminal tryptophan of RES-701-3 that affords RES-701-4. A BLASTP search with the ResE amino acid sequence shows the presence of homologues of this protein in the genomes of eight other Streptomyces strains, which also harbour the genes encoding the RES-701-3, -4 precursor peptide, split-B proteins and ATP-dependent lactam synthetase required for the biosynthesis of these compounds.

scholarly journals Transcription-factor centric genome mining strategy for discovery of diverse unprecedented RiPP gene clusters

2018 ◽  
Author(s):  
Shaozhou Zhu ◽  
Guojun Zheng
Keyword(s):  
Transcription Factor ◽  
Natural Products ◽  
Biological Activity ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Pathways ◽  
Lasso Peptides ◽  
Modified Peptides ◽  
Diverse Biological Activity

ABSTRACTRibosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly emerging group of natural products with diverse biological activity. Most of their biosynthetic mechanisms are well studied and the “genome mining” strategy based on homology has led to the unearthing of many new ribosomal natural products, including lantipeptides, lasso peptides, cyanobactins. These precursor-centric or biosynthetic protein-centric genome mining strategies have encouraged the discovery of RiPPs natural products. However, a limitation of these strategies is that the newly identified natural products are similar to the known products and novel families of RiPP pathways were overlooked by these strategies. In this work, we applied a transcription-factor centric genome mining strategy and diverse unique crosslinked RiPP gene clusters were predicted in several sequenced microorganisms. Our research could significantly expand the category of biosynthetic pathways of RiPP natural products and predict new resources for novel RiPPs.

scholarly journals Bacteriocins of Non-aureus Staphylococci Isolated from Bovine Milk

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Domonique A. Carson ◽  
Herman W. Barkema ◽  
Sohail Naushad ◽  
Jeroen De Buck
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Infections ◽  
Genome Mining ◽  
Bovine Milk ◽  
Gene Clusters ◽  
Antibiotic Use ◽  
Content Type ◽  
Treatment And Prevention ◽  
Whole Genomes

ABSTRACT Non-aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine udder, potentially protect the udder against infection by major mastitis pathogens due to bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bovine Staphylococcus aureus. Furthermore, inhibiting isolates were tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. We determined the presence of bacteriocin clusters in NAS whole genomes using genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and noninhibiting isolates and determined the genetic organization of any identified bacteriocin biosynthetic gene clusters. Forty isolates from 9 species (S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus) inhibited growth of S. aureus in vitro, 23 isolates of which, from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus, also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. A total of 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications. IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus) costs Canadian dairy producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With increasing antibiotic resistance and regulations regarding use, there is impetus to explore bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of bacterial infections. We examined the ability of 441 NAS bacteria from Canadian bovine milk samples to inhibit growth of S. aureus in the laboratory. Overall, 9% inhibited growth of S. aureus and 58% of those also inhibited MRSA. In NAS whole-genome sequences, we identified >21% of NAS as having bacteriocin genes. Our study represents a foundation to further explore NAS bacteriocins for clinical use.

scholarly journals Discovery of the Class I Antimicrobial Lasso Peptide Arcumycin

2021 ◽  
Author(s):  
Lydia Stariha ◽  
Dewey G. McCafferty
Keyword(s):  
Micrococcus Luteus ◽  
Genome Mining ◽  
Antibiotic Activity ◽  
Peptide Sequence ◽  
Class I ◽  
Gram Positive ◽  
Conformationally Constrained ◽  
Lasso Peptide ◽  
Lasso Peptides ◽  
Lipid Ii

<p>Lasso peptides are a structurally diverse superfamily of</p><p>conformationally-constrained peptide natural products, of which a</p><p>subset exhibits broad antimicrobial activity. Although advances in</p><p>bioinformatics have increased our knowledge of strains harboring</p><p>the biosynthetic machinery for lasso peptide production, relating</p><p>peptide sequence to bioactivity remains a continuous challenge.</p><p>Towards this end, a structure-driven genome mining investigation</p><p>of Actinobacteria-produced antimicrobial lasso peptides was</p><p>performed to correlate predicted primary structure with antibiotic</p><p>activity. Bioinformatic evaluation revealed eight putative novel</p><p>class I lasso peptide sequences. This subset is predicted to</p><p>possess antibiotic activity as characterized members of this class</p><p>have both broad spectrum and potent activity against Gram positive</p><p>strains. Fermentation of one of these hits, Streptomyces</p><p>NRRL F-5639, resulted in the production of a novel class I lasso</p><p>peptide, arcumycin, named for the Latin word for bow or arch,</p><p>arcum. Arcumycin exhibited antibiotic activity against Gram positive</p><p>bacteria including <i>Bacillus subtilis</i> (4 μg/mL),</p><p><i>Staphylococcus aureus </i>(8 μg/mL), and <i>Micrococcus luteus</i> (8</p><p>μg/mL). Arcumycin treatment of <i>B. subtilis</i> liaI-β-gal promoter</p><p>fusion reporter strain resulted in upregulation of the system liaRS</p><p>by the promoter liaI, indicating arcumycin interferes with lipid II</p><p>biosynthesis. Cumulatively, the results illustrate the relationship</p><p>between phylogenetically related lasso peptides and their</p><p>bioactivity as validated through the isolation, structural</p><p>determination, and evaluation of bioactivity of the novel class I</p><p>antimicrobial lasso peptide arcumycin.</p>

scholarly journals Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

2016 ◽  
Vol 82 (19) ◽  
pp. 5795-5805 ◽  
Author(s):  
Min Xu ◽  
Yemin Wang ◽  
Zhilong Zhao ◽  
Guixi Gao ◽  
Sheng-Xiong Huang ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Genome Mining ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Functional Screening ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Content Type ◽  
Bac Libraries

ABSTRACTGenome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries inStreptomycesspp. We demonstrate mining from a strain ofStreptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate hostStreptomyces lividansSBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic fromS. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways.IMPORTANCEMicrobial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites fromStreptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including streptothricins, borrelidin, two novel lipopeptides, and one unknown antibiotic fromStreptomyces rocheiSal35. The transfer, expression, and screening of the library were all performed in a high-throughput way, so that this approach is scalable and adaptable to industrial automation for next-generation antibiotic discovery.

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