Isolation and identification of a new lasso peptide cattlecin from Streptomyces cattleya based on genome mining

Actinomycetes strains isolated from different habitats in Switzerland were investigated for production of antibacterial and antitumoral compounds. Based on partial 16S rRNA gene sequences, the isolated strains were identified to genus level. Streptomyces as the largest genus of Actinobacteriawas isolated the most frequently. A screening assay using the OmniLog instrument was established to facilitate the detection of active compounds from actinomycetes. Extracts prepared from the cultivated strains able to inhibit Staphylococcus aureusand Escherichia coliwere further analysed by HPLC and MALDI-TOF MS to identify the produced antibiotics. In this study, the bioactive compound echinomycin was identified from two isolated Streptomycesstrains. Natural compounds similar to TPU-0037-C, azalomycin F4a 2-ethylpentyl ester, a derivative of bafilomycin A1, milbemycin-α8 and dihydropicromycin were detected from different isolated Streptomyces strains. Milbemycin-α8 showed cytotoxic activity against HT-29 colon cancer cells. The rare actinomycete,Micromonospora sp. Stup16_C148 produced a compound that matches with the antibiotic bottromycin A2. The draft genome sequence from Actinokineospora strain B136.1 was determined using Illumina and nanopore-based technologies. The isolated strain was not able to produce antibacterial compounds under standard cultivation conditions. The antiSMASH bioinformatics analyses of the genome from strain B136.1 identified biosynthetic gene clusters with identity values between 4% to 90% to known gene clusters encoding antibiotics. The combinations of cultivation conditions, screening assays, analytical methods and genome mining are important tools to characterize strains of actinomycetes for the identification of their potential to produce natural compounds with antimicrobial activity.

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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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Heterologous production of new lasso peptide koreensin based on genome mining

The Journal of Antibiotics ◽

10.1038/s41429-020-00363-5 ◽

2020 ◽

Vol 74 (1) ◽

pp. 42-50

Author(s):

Hiroki Fuwa ◽

Hikaru Hemmi ◽

Issara Kaweewan ◽

Ikko Kozaki ◽

Hiroyuki Honda ◽

...

Keyword(s):

Genome Mining ◽

Heterologous Production ◽

Lasso Peptide

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A new genome-mining tool redefines the lasso peptide biosynthetic landscape

Nature Chemical Biology ◽

10.1038/nchembio.2319 ◽

2017 ◽

Vol 13 (5) ◽

pp. 470-478 ◽

Cited By ~ 153

Author(s):

Jonathan I Tietz ◽

Christopher J Schwalen ◽

Parth S Patel ◽

Tucker Maxson ◽

Patricia M Blair ◽

...

Keyword(s):

Genome Mining ◽

Lasso Peptide ◽

Mining Tool

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Isolation and structure determination of a new lasso peptide specialicin based on genome mining

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2018.11.007 ◽

2018 ◽

Vol 26 (23-24) ◽

pp. 6050-6055 ◽

Cited By ~ 9

Author(s):

Issara Kaweewan ◽

Hikaru Hemmi ◽

Hisayuki Komaki ◽

Shigeyoshi Harada ◽

Shinya Kodani

Keyword(s):

Structure Determination ◽

Genome Mining ◽

Lasso Peptide

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Precursor-centric genome-mining approach for lasso peptide discovery

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1208978109 ◽

2012 ◽

Vol 109 (38) ◽

pp. 15223-15228 ◽

Cited By ~ 98

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.

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Albusnodin: an acetylated lasso peptide fromStreptomyces albus

Chemical Communications ◽

10.1039/c7cc08620b ◽

2018 ◽

Vol 54 (11) ◽

pp. 1339-1342 ◽

Cited By ~ 27

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.

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Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance

10.1101/2020.11.25.399105 ◽

2020 ◽

Author(s):

Yuanyuan Si ◽

Ashley M. Kretsch ◽

Laura M. Daigh ◽

Mark J. Burk ◽

Douglas A. Mitchell

Keyword(s):

Genome Mining ◽

Efficient Production ◽

Surface Receptors ◽

Lasso Peptide ◽

Anticancer Properties ◽

Lasso Peptides ◽

Isolation And Characterization ◽

Precursor Peptide ◽

A Cell ◽

Substrate Tolerance

AbstractLasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like structure. Owing to a rigid topology, lasso peptides are unusually stable towards heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. Known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent RiPP class; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address the longstanding challenges associated with lasso peptide production. We report the successful formation of a diverse array of lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants in order to evaluate the substrate tolerance of the biosynthetic pathway. We show that the lasso-forming cyclase from the fusilassin pathway can produce millions of sequence-diverse lasso peptides via CFB with an extraordinary level of sequence permissiveness within the ring region of the lasso peptide. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.

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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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Isolation and Identification of Pentalenolactone Analogs from Streptomyces sp. NRRL S-4

Molecules ◽

10.3390/molecules26237377 ◽

2021 ◽

Vol 26 (23) ◽

pp. 7377

Author(s):

Huanhuan Li ◽

Hongji Li ◽

Shuo Chen ◽

Wenhui Wu ◽

Peng Sun

Keyword(s):

Density Functional ◽

Genome Mining ◽

Biosynthetic Gene Cluster ◽

Antimicrobial Activities ◽

Gram Negative Bacteria ◽

Genomic Information ◽

Isolation And Identification ◽

Terpene Synthases ◽

Functional Theory ◽

Streptomyces Sp

Terpene synthases are widely distributed in Actinobacteria. Genome sequencing of Streptomyces sp. NRRL S-4 uncovered a biosynthetic gene cluster (BGC) that putatively synthesizes pentalenolactone type terpenes. Guided by genomic information, the S-4 strain was chemically investigated, resulting in the isolation of two new sesquiterpenoids, 1-deoxy-8α-hydroxypentalenic acid (1) and 1-deoxy-9β-hydroxy-11-oxopentalenic acid (2), as shunt metabolites of the pentalenolactone (3) biosynthesis pathway. Their structures and absolute configurations were elucidated by analyses of HRESIMS and NMR spectroscopic data as well as time-dependent density functional theory/electronic circular dichroism (TDDFT/ECD) calculations. Compounds 1 and 2 exhibited moderate antimicrobial activities against Gram-positive and Gram-negative bacteria. These results confirmed that the pentalenolactone pathway was functional in this organism and will facilitate efforts for exploring Actinobacteria using further genome mining strategies.

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