Browse
Submit Data
Databases
API
Help

Dataset Information

13 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Fluostatins produced by the heterologous expression of a TAR reassembled environmental DNA derived type II PKS gene cluster.

ABSTRACT: Culture independent approaches for accessing small molecules produced by uncultured bacteria are often hampered by the inability to easily clone environmental DNA (eDNA) fragments large enough to capture intact biosynthetic gene clusters that can be used in heterologous expression studies. Here we show that homology screening of eDNA megalibraries for clones containing natural product biosynthetic genes, coupled with transformation-assisted recombination (TAR) in yeast, can be used to access large, functionally intact, natural product gene clusters from the environment. The eDNA derived gene cluster reported here was functionally reconstructed from two overlapping cosmid clones using TAR. The isolation and structure elucidation of three new fluostatins (F, G, and H) produced by this TAR reconstructed gene cluster is described.

SUBMITTER: Feng Z

PROVIDER: S-EPMC2930618 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Fluostatins produced by the heterologous expression of a TAR reassembled environmental DNA derived type II PKS gene cluster.

Feng Zhiyang Z Kim Jeff H JH Brady Sean F SF

Journal of the American Chemical Society 20100901 34

Culture independent approaches for accessing small molecules produced by uncultured bacteria are often hampered by the inability to easily clone environmental DNA (eDNA) fragments large enough to capture intact biosynthetic gene clusters that can be used in heterologous expression studies. Here we show that homology screening of eDNA megalibraries for clones containing natural product biosynthetic genes, coupled with transformation-assisted recombination (TAR) in yeast, can be used to access lar ...[more]

PMID: 20690632

Similar Datasets

Loseolamycins: A Group of New Bioactive Alkylresorcinols Produced after Heterologous Expression of a Type III PKS from Micromonospora endolithica.

Project description:Natural products are a valuable source of biologically active compounds with potential applications in medicine and agriculture. Unprecedented scaffold diversity of natural products and biocatalysts from their biosynthetic pathways are of fundamental importance. Heterologous expression and refactoring of natural product biosynthetic pathways are generally regarded as a promising approach to discover new secondary metabolites of microbial origin. Here, we present the identification of a new group of alkylresorcinols after transcriptional activation and heterologous expression of the type III polyketide synthase of Micromonospora endolithica. The most abundant compounds loseolamycins A1 and A2 have been purified and their structures were elucidated by NMR. Loseolamycins contain an unusual branched hydroxylated aliphatic chain which is provided by the host metabolism and is incorporated as a starter fatty acid unit. The isolated loseolamycins show activity against gram-positive bacteria and inhibit the growth of the monocot weed Agrostis stolonifera in a germination assay. The biosynthetic pathway leading to the production of loseolamycins is proposed in this paper.

| S-EPMC7587189 | biostudies-literature

Duplication of a Pks gene cluster and subsequent functional diversification facilitate environmental adaptation in Metarhizium species.

Project description:The ecological importance of the duplication and diversification of gene clusters that synthesize secondary metabolites in fungi remains poorly understood. Here, we demonstrated that the duplication and subsequent diversification of a gene cluster produced two polyketide synthase gene clusters in the cosmopolitan fungal genus Metarhizium. Diversification occurred in the promoter regions and the exon-intron structures of the two Pks paralogs (Pks1 and Pks2). These two Pks genes have distinct expression patterns, with Pks1 highly expressed during conidiation and Pks2 highly expressed during infection. Different upstream signaling pathways were found to regulate the two Pks genes. Pks1 is positively regulated by Hog1-MAPK, Slt2-MAPK and Mr-OPY2, while Pks2 is positively regulated by Fus3-MAPK and negatively regulated by Mr-OPY2. Pks1 and Pks2 have been subjected to positive selection and synthesize different secondary metabolites. PKS1 is involved in synthesis of an anthraquinone derivative, and contributes to conidial pigmentation, which plays an important role in fungal tolerance to UV radiation and extreme temperatures. Disruption of the Pks2 gene delayed formation of infectious structures and increased the time taken to kill insects, indicating that Pks2 contributes to pathogenesis. Thus, the duplication of a Pks gene cluster and its subsequent functional diversification has increased the adaptive flexibility of Metarhizium species.

| S-EPMC6042797 | biostudies-literature

Activation of a plasmid-situated type III PKS gene cluster by deletion of a wbl gene in deepsea-derived Streptomyces somaliensis SCSIO ZH66.

Project description:Actinomycete genome sequencing has disclosed a large number of cryptic secondary metabolite biosynthetic gene clusters. However, their unavailable or limited expression severely hampered the discovery of bioactive compounds. The whiB-like (wbl) regulatory genes play important roles in morphological differentiation as well as secondary metabolism; and hence the wblA so gene was probed and set as the target to activate cryptic gene clusters in deepsea-derived Streptomyces somaliensis SCSIO ZH66.wblA so from deepsea-derived S. somaliensis SCSIO ZH66 was inactivated, leading to significant changes of secondary metabolites production in the ?wblA so mutant, from which ?-pyrone compound violapyrone B (VLP B) was isolated. Subsequently, the VLP biosynthetic gene cluster was identified and characterized, which consists of a type III polyketide synthase (PKS) gene vioA and a regulatory gene vioB; delightedly, inactivation of vioB led to isolation of another four VLPs analogues, among which one was new and two exhibited improved anti-MRSA (methicillin-resistant Staphylococcus aureus, MRSA) activity than VLP B. Moreover, transcriptional analysis revealed that the expression levels of whi genes (whiD, whiG, whiH and whiI) and wbl genes (wblC, wblE, wblH, wblI and wblK) were repressed by different degrees, suggesting an intertwined regulation mechanism of wblA so in morphological differentiation and secondary metabolism of S. somaliensis SCSIO ZH66.wblA orthologues would be effective targets for activation of cryptic gene clusters in marine-derived Streptomyces strains, notwithstanding the regulation mechanisms might be varied in different strains. Moreover, the availability of the vio gene cluster has enriched the diversity of type III PKSs, providing new opportunities to expand the chemical space of polyketides through biosynthetic engineering.

| S-EPMC4924298 | biostudies-literature

Lysoquinone-TH1, a New Polyphenolic Tridecaketide Produced by Expressing the Lysolipin Minimal PKS II in <i>Streptomyces albus</i>.

Project description:The structural repertoire of bioactive naphthacene quinones is expanded by engineering Streptomyces albus to express the lysolipin minimal polyketide synthase II (PKS II) genes from Streptomyces tendae Tü 4042 (llpD-F) with the corresponding cyclase genes llpCI-CIII. Fermentation of the recombinant strain revealed the two new polyaromatic tridecaketides lysoquinone-TH1 (7, identified) and TH2 (8, postulated structure) as engineered congeners of the dodecaketide lysolipin (1). The chemical structure of 7, a benzo[a]naphthacene-8,13-dione, was elucidated by NMR and HR-MS and confirmed by feeding experiments with [1,2-13C?]-labeled acetate. Lysoquinone-TH1 (7) is a pentangular polyphenol and one example of such rare extended polyaromatic systems of the benz[a]napthacene quinone type produced by the expression of a minimal PKS II in combination with cyclases in an artificial system. While the natural product lysolipin (1) has antimicrobial activity in nM-range, lysoquinone-TH1 (7) showed only minor potency as inhibitor of Gram-positive microorganisms. The bioactivity profiling of lysoquinone-TH1 (7) revealed inhibitory activity towards phosphodiesterase 4 (PDE4), an important target for the treatment in human health like asthma or chronic obstructive pulmonary disease (COPD). These results underline the availability of pentangular polyphenolic structural skeletons from biosynthetic engineering in the search of new chemical entities in drug discovery.

| S-EPMC6164072 | biostudies-literature

Klebsiella pneumoniae pks gene cluster

Project description:Prevalence and characteristics of pks gene cluster harboring Klebsiella pneumoniae from bloodstream infection in China

| PRJEB32094 | ENA

Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites.

Project description:A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts.

| S-EPMC3150919 | biostudies-literature

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

Project description:Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts.

| S-EPMC6701952 | biostudies-literature

Phage p1-derived artificial chromosomes facilitate heterologous expression of the FK506 gene cluster.

Project description:We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L(-1) to 5.5 mg L(-1)) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

| S-EPMC3708917 | biostudies-literature

Oligosaccharides increase the genotoxic effect of colibactin produced by pks+ Escherichia coli strains.

Project description:BackgroundColibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101.MethodsBacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 μM, 25 μM and 125 μM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis.ResultsInulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli. However, the addition of 125 μM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells.ConclusionOur results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli.

| S-EPMC7890614 | biostudies-literature

Molecular cloning and heterologous expression of a biosynthetic gene cluster for the antitubercular agent D-cycloserine produced by Streptomyces lavendulae.

Project description:In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding D-alanyl-D-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, L-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-L-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-L-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the L-arginine derivative, N(G)-hydroxy-L-arginine. The resulting O-ureido-L-serine is then racemized to O-ureido-D-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-D-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein.

| S-EPMC2825969 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data