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Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression.

ABSTRACT: Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

SUBMITTER: McMahon MD

PROVIDER: S-EPMC3346366 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression.

McMahon Matthew D MD Guan Changhui C Handelsman Jo J Thomas Michael G MG

Applied and environmental microbiology 20120316 10

Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologica ...[more]

PMID: 22427497

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Project description:BACKGROUND:Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS:In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION:The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

Dataset Information

Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression.

Publications

Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression.

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