Albusnodin: an acetylated lasso peptide fromStreptomyces albus

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

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

10.1101/2021.05.19.444711 ◽

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.

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

Marine Drugs ◽

10.3390/md18050238 ◽

2020 ◽

Vol 18 (5) ◽

pp. 238 ◽

Cited By ~ 2

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.

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Deciphering a Cyclodipeptide Synthase Pathway Encoding Prenylated Indole Alkaloids in Streptomyces leeuwenhoekii

Applied and Environmental Microbiology ◽

10.1128/aem.02525-20 ◽

2021 ◽

Author(s):

Yuqiao Zhang ◽

Tingting Yao ◽

Yuechen Jiang ◽

Huayue Li ◽

Weicheng Yuan ◽

...

Keyword(s):

Heterologous Expression ◽

Gene Locus ◽

Indole Alkaloids ◽

Genome Mining ◽

Bifunctional Enzyme ◽

Substrate Selectivity ◽

Drug Discovery And Development ◽

Biochemical Assays ◽

Tailoring Enzymes ◽

New Compounds

Cyclodipeptide synthases (CDPSs) catalyse the formation of cyclodipeptides using aminoacylated-tRNAs as substrates and have great potentials in the production of diverse 2,5-diketopiperazines (2,5-DKPs). Genome mining of Streptomyces leeuwenhoekii NRRL B-24963 revealed a two-gene locus saz encoding a CDPS SazA and a unique fused enzyme SazB harboring two domains: phytoene-synthase-like prenyltransferase (PT) and methyltransferase (MT). Heterologous expression of the saz gene(s) in Streptomyces albus J1074 led to the production of four prenylated indole alkaloids, among which streptoazines A-C (3–5) are new compounds. Expression of different gene combinations showed that the SazA catalyzes the formation of cyclo (L-Trp-L-Trp) (cWW, 1), followed by consecutive prenylation and methylation by SazB. Biochemical assays demonstrated that SazB is a bifunctional enzyme, catalyzing sequential C3/C3’-prenylation(s) by SazB-PT and N1/N1’-methylation(s) by SazB-MT. Of note substrate selectivity of SazB-PT and SazB-MT was probed, revealing the stringent specificity of SazB-PT but relative flexibility of SazB-MT. IMPORTANCE Natural products with 2,5-DKP skeleton have long sparked the interest in drug discovery and development. Recent advances in microbial genome sequencing have revealed that the potentials of CDPS-dependent pathways encoding new 2,5-DKPs are underexplored. In this study, we report the genome mining of a new CDPS-containing two-gene operon and activation of this cryptic gene cluster through heterologous expression, leading to the discovery of four indole 2,5-DKP alkaloids. The cWW-synthesizing CDPS SazA and the unusual PT-MT fused enzyme SazB were characterized. Our results expand the repertoire of CDPSs and associated tailoring enzymes, setting the stage for accessing diverse prenylated alkaloids using synthetic biology strategies.

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Discovery of the Class I Antimicrobial Lasso Peptide Arcumycin

10.26434/chemrxiv.14544213 ◽

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

Lasso peptides are a structurally diverse superfamily ofconformationally-constrained peptide natural products, of which asubset exhibits broad antimicrobial activity. Although advances inbioinformatics have increased our knowledge of strains harboringthe biosynthetic machinery for lasso peptide production, relatingpeptide sequence to bioactivity remains a continuous challenge.Towards this end, a structure-driven genome mining investigationof Actinobacteria-produced antimicrobial lasso peptides wasperformed to correlate predicted primary structure with antibioticactivity. Bioinformatic evaluation revealed eight putative novelclass I lasso peptide sequences. This subset is predicted topossess antibiotic activity as characterized members of this classhave both broad spectrum and potent activity against Gram positivestrains. Fermentation of one of these hits, StreptomycesNRRL F-5639, resulted in the production of a novel class I lassopeptide, arcumycin, named for the Latin word for bow or arch,arcum. Arcumycin exhibited antibiotic activity against Gram positivebacteria including Bacillus subtilis (4 μg/mL),Staphylococcus aureus (8 μg/mL), and Micrococcus luteus (8μg/mL). Arcumycin treatment of B. subtilis liaI-β-gal promoterfusion reporter strain resulted in upregulation of the system liaRSby the promoter liaI, indicating arcumycin interferes with lipid IIbiosynthesis. Cumulatively, the results illustrate the relationshipbetween phylogenetically related lasso peptides and theirbioactivity as validated through the isolation, structuraldetermination, and evaluation of bioactivity of the novel class Iantimicrobial lasso peptide arcumycin.

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

10.1101/494286 ◽

2018 ◽

Cited By ~ 3

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01752-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 5

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01383-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 5795-5805 ◽

Cited By ~ 31

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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