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

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.

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 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 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 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>

Isolation and characterization of a cell-associated protein of Bacillus pumilus PH-01

2001 ◽  
Vol 56 (3-4) ◽  
pp. 402-405 ◽  
Author(s):  
H.-B. Hong ◽  
Y.-S. Chang ◽  
S.-D. Choi ◽  
I.-H. Nam ◽  
Y.-E. Lee
Keyword(s):  
Bacillus Pumilus ◽  
Isolation And Characterization ◽  
A Cell

scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

scholarly journals A generic cell surface ligand system for studying cell-cell recognition

2019 ◽  
Author(s):  
Eleanor M Denham ◽  
Michael I Barton ◽  
Susannah M Black ◽  
Marcus J Bridge ◽  
Ben de Wet ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Density ◽  
Cell Receptor ◽  
Surface Receptors ◽  
Ligand System ◽  
Ligand Interactions ◽  
A Cell ◽  
Surface Ligand ◽  
Receptor Biology ◽  
Cell Cell

AbstractDose-response experiments are a mainstay of receptor biology studies and can reveal valuable insights into receptor function. Such studies of receptors that bind cell surface ligands are currently limited by the difficulty in manipulating the surface density of ligands at a cell-cell interface. Here we describe a generic cell surface ligand system that allows precise manipulation of cell surface ligand densities over several orders of magnitude. We validate the system for a range of immunoreceptors, including the T cell receptor (TCR), and show that this generic ligand stimulates via the TCR at a similar surface density as its native ligand. This system allows the effect of surface density, valency, dimensions, and affinity of the ligand to be manipulated. It can be readily extended to other receptor-cell surface ligand interactions, and will facilitate investigation into the activation of, and signal integration between, cell surface receptors.

The putative sponge aggregation receptor. Isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats

1998 ◽  
Vol 111 (17) ◽  
pp. 2635-2644 ◽  
Author(s):  
B. Blumbach ◽  
Z. Pancer ◽  
B. Diehl-Seifert ◽  
R. Steffen ◽  
J. Munkner ◽  
...  
Keyword(s):  
Cell Surface ◽  
Scavenger Receptor ◽  
Cell Surface Receptor ◽  
Functional Assay ◽  
Cdna Clones ◽  
The Third ◽  
Short Consensus Repeats ◽  
Isolation And Characterization ◽  
A Cell ◽  
Cell Cell

Porifera (sponges) are the oldest extant metazoan phylum. Dissociated sponge cells serve as a classic system to study processes of cell reaggregation. The reaggregation of dissociated cells is mediated by an extracellularly localized aggregation factor (AF), based on heterophilic interactions of the third order; the AF bridges two cells by ligating a cell-surface-bound aggregation receptor (AR). In the present study we report cloning, expression and immunohistochemical localization of a polypeptide from the marine sponge Geodia cydonium, which very likely represents the AR. The presumed AR gene gives rise to at least three forms of alternatively spliced transcripts of 6.5, 4.9 and 3.9 kb, as detected by northern blotting. Two cDNA clones corresponding to the shorter forms were already reported earlier; here we present an analysis of the largest. All three putative polypeptides feature scavenger receptor cysteine-rich (SRCR) domains. The largest form, SRCR-SCR-Car, is a cell-surface receptor of molecular mass 220 kDa, which is assumed to be the cell-adhesion receptor AR; the second form, SRCR-Re, is also a putative receptor of 166 kDa, while the third form, SRCR-Mo, is a soluble molecule of 129 kDa. The SRCR-SCR-Car molecule consists of fourteen SRCR domains, six short consensus repeats (SCRs), a C-terminal transmembrane domain and a cytoplasmic tail; its fourteenth SRCR domain features an Arg-Gly-Asp tripeptide. To obtain monoclonal antibodies, a 170-amino-acid-long polypeptide that is found in all three forms of the SRCR-containing proteins was expressed in E. coli. In a western blot of sponge cells lysate the monoclonal antibody raised against the recombinant polypeptide recognized two major immuno-reacting polypeptides (220 and 117 kDa) and two minor bands (36 and 32 kDa). The antibody was found to react with antigen(s) predominantly localized on the plasma membranes of cells, especially those of spherulous cells. In a functional assay Fab' fragments of the antibodies suppressed AF-mediated cell-cell reaggregation. Additionally, a recombinant SRCR-soluble fragment effectively inhibited AF-mediated cell-cell reaggregation. We conclude that the 220 kDa SRCR-containing protein of the sponge G. cydonium is very likely the AR.

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 A translocated erythropoietin receptor gene in a human erythroleukemia cell line (TF-1) expresses an abnormal transcript and a truncated protein

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 179-185 ◽  
Author(s):  
JC Winkelmann ◽  
J Ward ◽  
P Mayeux ◽  
C Lacombe ◽  
L Schimmenti ◽  
...  
Keyword(s):  
Cell Line ◽  
Receptor Gene ◽  
Erythropoietin Receptor ◽  
S1 Nuclease ◽  
Surface Receptors ◽  
Considerable Excess ◽  
A Cell ◽  
S1 Nuclease Mapping ◽  
High Level ◽  
Nuclease Mapping

We previously identified a translocation breakpoint in exon 8 of the erythropoietin receptor (EpoR) gene in TF-1 cells, a cell line derived from a human erythroleukemia. To investigate the potential pathogenetic significance of this abnormality, we more precisely mapped the breakpoint within exon 8 and studied the expression of the translocated gene by S1 nuclease mapping of EpoR transcripts and chemical crosslinking of labeled erythropoietin (Epo) to TF-1 cell surface receptors. Transcripts from the abnormal gene were found to be highly expressed in relation to normal EpoR transcripts in TF-1 cells. The breakpoint predicted by S1 mapping of abnormal EpoR transcripts agreed closely with that determined by Southern analysis. Chemical cross- linking of 125I-Epo to TF-1 cells showed an abnormal, low-molecular- weight cross-linked species directly recognized by anti-EpoR antibodies and present in considerable excess over the normal EpoR. Karyotype analysis showed that each of 10 TF-1 cell metaphases had, in addition to multiple other alterations, one chromosome 19 with additional chromosomal material translocated onto the short arm at 19p13.3, the location of the EpoR gene. We conclude that the structurally abnormal EpoR gene in TF-1 cells is highly expressed and produces an abnormal protein. We speculate that the chromosomal material brought into the EpoR locus by translocation is responsible for the high level of expression. We hypothesize that this translocation participated in the evolution of the erythroleukemia from which TF-1 cells were derived.

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