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

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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Identification of the Novobiocin Biosynthetic Gene Cluster of Streptomyces spheroides NCIB 11891

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.5.1214-1222.2000 ◽

2000 ◽

Vol 44 (5) ◽

pp. 1214-1222 ◽

Cited By ~ 133

Author(s):

Marion Steffensky ◽

Agnes Mühlenweg ◽

Zhao-Xin Wang ◽

Shu-Ming Li ◽

Lutz Heide

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Biosynthetic Gene Cluster ◽

Streptomyces Lividans ◽

Open Reading Frames ◽

Biosynthetic Gene ◽

Insertional Inactivation ◽

Key Enzyme ◽

Novobiocin Resistance ◽

Reading Frames

ABSTRACT The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Double-stranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB r, and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4,6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, inStreptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.

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Genome Mining and Metabolic Profiling Uncover Polycyclic Tetramate Macrolactams from Streptomyces koyangensis SCSIO 5802

Marine Drugs ◽

10.3390/md19080440 ◽

2021 ◽

Vol 19 (8) ◽

pp. 440

Author(s):

Wenjuan Ding ◽

Jiajia Tu ◽

Huaran Zhang ◽

Xiaoyi Wei ◽

Jianhua Ju ◽

...

Keyword(s):

Natural Products ◽

Gene Cluster ◽

Metabolic Profiling ◽

Biosynthetic Pathway ◽

Genome Mining ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Bioactive Metabolites ◽

Bioinformatics Analyses ◽

Reading Frames

We have previously shown deep-sea-derived Streptomyces koyangensis SCSIO 5802 to produce two types of active secondary metabolites, abyssomicins and candicidins. Here, we report the complete genome sequence of S. koyangensis SCSIO 5802 employing bioinformatics to highlight its potential to produce at least 21 categories of natural products. In order to mine novel natural products, the production of two polycyclic tetramate macrolactams (PTMs), the known 10-epi-HSAF (1) and a new compound, koyanamide A (2), was stimulated via inactivation of the abyssomicin and candicidin biosynthetic machineries. Detailed bioinformatics analyses revealed a PKS/NRPS gene cluster, containing 6 open reading frames (ORFs) and spanning ~16 kb of contiguous genomic DNA, as the putative PTM biosynthetic gene cluster (BGC) (termed herein sko). We furthermore demonstrate, via gene disruption experiments, that the sko cluster encodes the biosynthesis of 10-epi-HSAF and koyanamide A. Finally, we propose a plausible biosynthetic pathway to 10-epi-HSAF and koyanamide A. In total, this study demonstrates an effective approach to cryptic BGC activation enabling the discovery of new bioactive metabolites; genome mining and metabolic profiling methods play key roles in this strategy.

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Discovery, Bioactivity Evaluation, Biosynthetic Gene Cluster Identification, and Heterologous Expression of Novel Albofungin Derivatives

Frontiers in Microbiology ◽

10.3389/fmicb.2021.635268 ◽

2021 ◽

Vol 12 ◽

Author(s):

Weiyi She ◽

Wenkang Ye ◽

Aifang Cheng ◽

Xin Liu ◽

Jianwei Tang ◽

...

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Pathogenic Bacteria ◽

Streptomyces Coelicolor ◽

Draft Genome ◽

2D Nmr ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Terminal Deoxynucleotidyl Transferase ◽

Chemical Structures

The crude extract of Streptomyces chrestomyceticus exhibited strong and broad activities against most “ESKAPE pathogens.” We conducted a comprehensive chemical investigation for secondary metabolites from the S. chrestomyceticus strain and identified two novel albofungin (alb) derivatives, i.e., albofungins A (1) and B (2), along with two known compounds, i.e., albofungin (3) and chloroalbofungin (4). The chemical structures of the novel compounds were elucidated using HRMS, 1D and 2D NMR, and electronic circular dichroism spectroscopy. The draft genome of S. chrestomyceticus was sequenced, and a 72 kb albofungin (alb) gene cluster with 72 open reading frames encoding type II polyketide synthases (PKSs), regulators, and transporters, and tailoring enzymes were identified using bioinformatics analysis. The alb gene cluster was confirmed using the heterologous expression in Streptomyces coelicolor, which successfully produced the compounds 3 and 4. Furthermore, compounds 1–4 displayed remarkable activities against Gram-positive bacteria and antitumor activities toward various cancer cells. Notably, compounds 1 and 3 showed potent activities against Gram-negative pathogenic bacteria. The terminal deoxynucleotidyl transferase (dUTP) nick-end labeling and flow cytometry analysis verified that compound 1 inhibited cancer cell proliferation by inducing cellular apoptosis. These results indicated that albofungins might be potential candidates for the development of antibiotics and antitumor drugs.

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Identification and Analysis of the Biosynthetic Gene Cluster Encoding the Thiopeptide Antibiotic Cyclothiazomycin in Streptomyces hygroscopicus 10-22

Applied and Environmental Microbiology ◽

10.1128/aem.01790-09 ◽

2010 ◽

Vol 76 (7) ◽

pp. 2335-2344 ◽

Cited By ~ 43

Author(s):

Jiang Wang ◽

Yi Yu ◽

Kexuan Tang ◽

Wen Liu ◽

Xinyi He ◽

...

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Posttranslational Modifications ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Streptomyces Hygroscopicus ◽

Biosynthetic Gene ◽

Thiopeptide Antibiotics ◽

Reading Frames ◽

Macrocyclic Structure

ABSTRACT Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides.

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Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.1.230-240.2005 ◽

2005 ◽

Vol 49 (1) ◽

pp. 230-240 ◽

Cited By ~ 65

Author(s):

Joshua A. V. Blodgett ◽

Jun Kai Zhang ◽

William W. Metcalf

Keyword(s):

Sequence Analysis ◽

Heterologous Expression ◽

Gene Cluster ◽

Genomic Dna ◽

Biosynthetic Gene Cluster ◽

Fosmid Library ◽

Open Reading Frames ◽

Biosynthetic Gene ◽

Heterologous Host ◽

Reading Frames

ABSTRACT A fosmid library from genomic DNA of Streptomyces viridochromogenes DSM 40736 was constructed and screened for the presence of genes known to be involved in the biosynthesis of phosphinothricin tripeptide (PTT). Eight positives were identified, one of which was able to confer PTT biosynthetic capability upon Streptomyces lividans after integration of the fosmid into the chromosome of this heterologous host. Sequence analysis of the 40,241-bp fosmid insert revealed 29 complete open reading frames (ORFs). Deletion analysis demonstrated that a minimum set of 24 ORFs were required for PTT production in the heterologous host. Sequence analysis revealed that most of these PTT genes have been previously identified in either S. viridochromogenes or S. hygroscopicus (or both), although only 11 out of 24 of these ORFs have experimentally defined functions. Three previously unknown genes within the cluster were identified and are likely to have roles in the stepwise production of phosphonoformate from phosphonoacetaldehyde. This is the first report detailing the entire PTT gene cluster from any producing streptomycete.

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Cloning and Characterization of the Pyrrolomycin Biosynthetic Gene Clusters from Actinosporangium vitaminophilum ATCC 31673 and Streptomyces sp. Strain UC 11065

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01214-06 ◽

2006 ◽

Vol 51 (3) ◽

pp. 946-957 ◽

Cited By ~ 46

Author(s):

Xiujun Zhang ◽

Ronald J. Parry

Keyword(s):

Sequence Analysis ◽

Gene Cluster ◽

Genetic Locus ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Biosynthetic Gene ◽

Genes Encoding ◽

Cloned Gene

ABSTRACT The pyrrolomycins are a family of polyketide antibiotics, some of which contain a nitro group. To gain insight into the nitration mechanism associated with the formation of these antibiotics, the pyrrolomycin biosynthetic gene cluster from Actinosporangium vitaminophilum was cloned. Sequencing of ca. 56 kb of A. vitaminophilum DNA revealed 35 open reading frames (ORFs). Sequence analysis revealed a clear relationship between some of these ORFs and the biosynthetic gene cluster for pyoluteorin, a structurally related antibiotic. Since a gene transfer system could not be devised for A. vitaminophilum, additional proof for the identity of the cloned gene cluster was sought by cloning the pyrrolomycin gene cluster from Streptomyces sp. strain UC 11065, a transformable pyrrolomycin producer. Sequencing of ca. 26 kb of UC 11065 DNA revealed the presence of 17 ORFs, 15 of which exhibit strong similarity to ORFs in the A. vitaminophilum cluster as well as a nearly identical organization. Single-crossover disruption of two genes in the UC 11065 cluster abolished pyrrolomycin production in both cases. These results confirm that the genetic locus cloned from UC 11065 is essential for pyrrolomycin production, and they also confirm that the highly similar locus in A. vitaminophilum encodes pyrrolomycin biosynthetic genes. Sequence analysis revealed that both clusters contain genes encoding the two components of an assimilatory nitrate reductase. This finding suggests that nitrite is required for the formation of the nitrated pyrrolomycins. However, sequence analysis did not provide additional insights into the nitration process, suggesting the operation of a novel nitration mechanism.

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Identification and Verification of the Prodigiosin Biosynthetic Gene Cluster (BGC) in Pseudoalteromonas rubra S4059

Microbiology Spectrum ◽

10.1128/spectrum.01171-21 ◽

2021 ◽

Author(s):

Xiyan Wang ◽

Thomas Isbrandt ◽

Emil Ørsted Christensen ◽

Jette Melchiorsen ◽

Thomas Ostenfeld Larsen ◽

...

Keyword(s):

Secondary Metabolites ◽

Heterologous Expression ◽

Gene Cluster ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Pseudoalteromonas Rubra

Pigmented Pseudoalteromonas strains are renowned for their production of secondary metabolites, and genome mining has revealed a high number of biosynthetic gene clusters (BGCs) for which the chemistry is unknown. Identification of those BGCs is a prerequisite for linking products to gene clusters and for further exploitation through heterologous expression.

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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 Characterization of the Gene Cluster for Biosynthesis of the Thiopeptide Antibiotic TP-1161

Applied and Environmental Microbiology ◽

10.1128/aem.01442-10 ◽

2010 ◽

Vol 76 (21) ◽

pp. 7093-7101 ◽

Cited By ~ 47

Author(s):

Kerstin Engelhardt ◽

Kristin F. Degnes ◽

Sergey B. Zotchev

Keyword(s):

Gene Cluster ◽

Fermentation Broth ◽

Streptomyces Coelicolor ◽

Draft Genome ◽

Biosynthetic Gene Cluster ◽

Genomic Region ◽

Open Reading Frames ◽

Peptide Sequence ◽

Isolation And Characterization ◽

Targeted Gene Inactivation

ABSTRACT Recently, we isolated a new thiopeptide antibiotic, TP-1161, from the fermentation broth of a marine actinomycete typed as a member of the genus Nocardiopsis. Here we report the identification, isolation, and analysis of the TP-1161 biosynthetic gene cluster from this species. The gene cluster was identified by mining a draft genome sequence using the predicted structural peptide sequence of TP-1161. Functional assignment of a ∼16-kb genomic region revealed 13 open reading frames proposed to constitute the TP-1161 biosynthetic locus. While the typical core set of thiopeptide modification enzymes contains one cyclodehydratase/dehydrogenase pair, paralogous genes predicted to encode additional cyclodehydratases and dehydrogenases were identified. Although attempts at heterologous expression of the TP-1161 gene cluster in Streptomyces coelicolor failed, its identity was confirmed through the targeted gene inactivation in the original host.

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Anacyclamide D8P, a prenylated cyanobactin from a Sphaerospermopsis sp. cyanobacterium

10.26434/chemrxiv.5346739.v1 ◽

2017 ◽

Cited By ~ 1

Author(s):

Joana Martins ◽

Niina Leikoski ◽

Matti Wahlsten ◽

Joana Azevedo ◽

Jorge Antunes ◽

...

Keyword(s):

Gene Cluster ◽

Cyclic Peptides ◽

Biosynthetic Gene Cluster ◽

The Novel ◽

Tyrosine Residue ◽

Biosynthetic Gene ◽

Post Translational Modification ◽

Biological Assays ◽

Mass Spectrometric Data ◽

Spectrometric Data

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. Anacyclamides are macrocyclic cyanobactins with a highly diverse sequence that are common in the genus Anabaena. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, anacyclamide D8P (1), from Sphaerospermopsis sp. LEGE 00249. The anacyclamide biosynthetic gene cluster (acy) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the acy gene cluster in Escherichia coli established the connection between genomic and mass spectrometric data. Unambiguous establishment of the type and site of prenylation required the full structural elucidation of 1 using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound 1 was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium Halomonas aquamarina CECT 5000.

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