An Environmental DNA‐Derived Type II Polyketide Biosynthetic Pathway Encodes the Biosynthesis of the Pentacyclic Polyketide Erdacin

King, Ryan W.; Bauer, John D.; Brady, Sean F.

doi:10.1002/anie.200901209

Cited by 46 publications

(33 citation statements)

References 29 publications

(44 reference statements)

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“…To identify type II PKS gene clusters in these libraries, cosmid DNA isolated from each library was used as the template in PCR reactions with degenerate primers designed to amplify full-length KS β genes (10,(14)(15)(16). Amplicons of the correct predicted size (1.5 kb) were gel purified, sequenced, and compared to deposited KS β genes from cultured bacteria (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Feng

Kallifidas²,

Brady³

2011

Proc. Natl. Acad. Sci. U.S.A.

135

149

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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-moleculedirected 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. D espite the historical success of bacterial natural products as lead structures for the development of small molecule therapeutics and the continued need for new antimicrobials and chemotherapeutics, large screening programs have deemphasized the use of microbial extracts over the past two decades. The reason most frequently cited for this decline is the persistent rediscovery of known metabolites (1-3). Most environmental bacteria remain recalcitrant to standard culture methods (4-6), and the difficulties associated with growing these organisms prohibit their use as new sources of bioactive small molecules. Although it is not yet possible to easily culture the majority of environmental bacteria, it is possible to extract microbial DNA directly from environmental samples (environmental DNA, eDNA) and to clone this DNA into cultured bacteria where it can be functionally characterized. This general approach has been termed metagenomics (7). The application of metagenomics to the study of bacterial secondary metabolism is particularly appealing in light of the fact that the genes required for the biosynthesis of a natural product are typically clustered on a bacterial chromosome. The heterologous expression of natural product gene clusters ...

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Section: Resultsmentioning

confidence: 99%

“…4), were found to encode the biosynthesis of the pentacyclic metabolite erdacin (5) and previously uncharacterized fluostatin (6) derivatives, respectively (14,15). Of the five eDNA-derived type II PKS systems that have now been functionally characterized in heterologous hosts (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Feng

Kallifidas²,

Brady³

2011

Proc. Natl. Acad. Sci. U.S.A.

135

149

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“…To compare the biosynthetic potentials of different soil microbiomes, environmental DNA (eDNA) extracted directly from three geographically distinct arid soils collected in the American Southwest (the Sonoran Desert of Arizona [AZ], the Anza-Borrego region of the Sonoran Desert of California [AB], and the Great Basin Desert of Utah [UT]) was used to construct three independent eDNA cosmid libraries. Each library contains in excess of 350 gigabases of DNA (ϳ100,000 bacterial genome equivalents) and is predicted to provide sufficient sequence coverage to capture the major constituents of the respective soil metagenome (2,3,6,16,17,21,22). The enormous size of these cloned soil metagenomes makes it difficult to shotgun sequence to a depth that would provide statistically relevant comparisons of differences in secondary metabolite genes.…”

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confidence: 99%

Natural Product Biosynthetic Gene Diversity in Geographically Distinct Soil Microbiomes

Reddy

Kallifidas

Kim

et al. 2012

Appl Environ Microbiol

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The number of bacterial species estimated to exist on Earth has increased dramatically in recent years. This newly recognized species diversity has raised the possibility that bacterial natural product biosynthetic diversity has also been significantly underestimated by previous culture-based studies. Here, we compare 454-pyrosequenced nonribosomal peptide adenylation domain, type I polyketide ketosynthase domain, and type II polyketide ketosynthase alpha gene fragments amplified from cosmid libraries constructed using DNA isolated from three different arid soils. While 16S rRNA gene sequence analysis indicates these cloned metagenomes contain DNA from similar distributions of major bacterial phyla, we found that they contain almost completely distinct collections of secondary metabolite biosynthetic gene sequences. When grouped at 85% identity, only 1.5% of the adenylation domain, 1.2% of the ketosynthase, and 9.3% of the ketosynthase alpha sequence clusters contained sequences from all three metagenomes. Although there is unlikely to be a simple correlation between biosynthetic gene sequence diversity and the diversity of metabolites encoded by the gene clusters in which these genes reside, our analysis further suggests that sequences in one soil metagenome are so distantly related to sequences in another metagenome that they are, in many cases, likely to arise from functionally distinct gene clusters. The marked differences observed among collections of biosynthetic genes found in even ecologically similar environments suggest that prokaryotic natural product biosynthesis diversity is, like bacterial species diversity, potentially much larger than appreciated from culture-based studies.

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“…PCR screening of a soil metagenomic library with degenerate primers (type II PKS), followed by the expression of biosynthetic genes in S. lividans and S. albus resulted in the novel natural products erdacin (Fig. 2) (King et al 2009) and utahmycins A and B (Bauer et al 2010), which exhibit novel carbon skeletons. Another soil metagenomic library was screened with primers specific for type I PKS genes (Courtois et al 2003).…”

Section: Sequence-based Approach Of Metagenomic Library Screeningmentioning

confidence: 99%

Bioprospecting microbial metagenome for natural products

Nováková

Farkašovský

2013

Biologia

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Mining of natural sources for new secondary metabolites has a successful history, which is reflected by the fact that over 50% of all drugs, currently on the market, are derived from natural products. Bacteria are one of the most important sources of bioactive natural products destined for drug discovery. However, less than 1% of the microorganisms observed in different habitats have been cultivated and characterized. To explore the genomic and functional diversity of the vast majority of the microbial world, novel methods were introduced, which are based on analysis of a DNA isolated from environmental communities. Metagenomics represents a strategy offering access to the genetic information present in uncultured bacteria by screening of libraries constructed from DNA isolated from different habitats. Functional-and sequence-driven screens are the major approaches employed to mine metagenomic libraries. This review aims to highlight discoveries in this area and discusses the possible future directions of the field.

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An Environmental DNA‐Derived Type II Polyketide Biosynthetic Pathway Encodes the Biosynthesis of the Pentacyclic Polyketide Erdacin

Cited by 46 publications

References 29 publications

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

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

Natural Product Biosynthetic Gene Diversity in Geographically Distinct Soil Microbiomes

Bioprospecting microbial metagenome for natural products

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