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Mining the Metagenome for Natural Product Discovery · PDF fileMining the Metagenome for...
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Mining the Metagenome for Natural Product Discovery Matthew McMahon 1 , Jo Handelsman 2 and Michael Thomas 1 1. Univeristy of Wisconsin - Madison Department of Bacteriology 2. Yale University Department of Molecular, Cellular, and Developmental Biology Abstract Streptomyces lividans is a well-studied member of the phylum Actinobacteria, the source of over two-thirds of naturally derived antibiotics in current use. Well-developed and extensive genetic tools are available for use with S. lividans, which makes it a favorable host for expression of natural product gene clusters. To enable heterologous expression in S. lividans, we constructed a suitable vector to allow conjugation of cosmid metagenomic libraries from Escherichia coli to S. lividans. We also deleted gene clusters responsible for production of two antibiotics native to S. lividans to enhance detection of heterologous antibiotic production associated with metagenomic DNA. We constructed two metagenomic libraries of soil organisms and developed protocols for efficiently conjugating these libraries into S. lividans. The screening of 23,600 clones revealed antimicrobial activity, hemolytic activity, quorum sensing induction and several distinct pigment-producing clones. One active clone that warranted further analysis produces a melanin-like molecule that has antimicrobial activity against Bacillus subtilis. Clones with reproducible phenotypes are analyzed further to identify genes required for natural product biosynthesis as well as structures and biological activities of associated molecules. Streptomycetes as Heterologous Hosts While metagenomic techniques have been used for about a decade to access the metagenome, the success of identifying functional clones has been limited by the heterologous host used. Most functional metagenomic studies, which involve screening clones for expressed activities, use either Escherichia coli or related γ-proteobacteria as hosts. Although screening metagenomic libraries in E. coli has led to a number of discoveries, alternative hosts may contain precursors, sigma factors, rare tRNAs, or cofactors required for natural product biosynthesis that are not present in E. coli (1). This research uses Streptomyces lividans, a well-studied member of the phylum Actinobacteria, as an alternative host for expression of metagenomic libraries (2). Actinobacteria are the source of over two-thirds of naturally derived antibiotics in current use, as well as many other pharmaceuticals, which makes them a good choice for expression of natural product gene clusters (3). Metagenomic Libraries AKAIM1: 10,500 clones in vector pMM436 with an average insert size of 35 kb was constructed from the genomic DNA of 14 cultured Alaskan isolates shown to have antimicrobial activity. AKM2: a library of 70,000 clones in vector pMM436 with an average insert size of 35 kb was constructed with DNA extracted from the uncultured microbial community of Alaskan soil. Vector pMM436 Cosmid vector that can conjugate into S. lividans, integrates into a stable region of the S. lividans chromosome using site-specific recombination at attachment site attP, and can be removed by digesting chromosomal DNA with PacI. Integration is important for screening because it allows the insert to be stably maintained within the S. lividans chromosome without continuous antibiotic selection, which would limit the tester organisms that could be used. Contact Information: 6225 Microbial Sciences Building 1550 Linden Drive Madison, WI 53706 Phone: (608) 263-9075 Email: [email protected] Host Streptomyces lividans ΔActΔRed Streptomyces lividans was mutated for its ability to produce two naturally occurring antibiotics, actinorhodin and prodigiosin. The deletion of these two biosynthesis gene clusters removed the background antibiotic activity that would obscure new antibiotics in metagenomic clones. Furthermore, since metabolites commonly used to generate antibiotics are no longer funneled into making these two endogenous antibiotics, more metabolic flux can occur through newly introduced antibiotic biosynthesis pathways. Vector Integration Restriction Digest / Insert Retrieval Vector Reconstruction/ Phenotype Testing Functional Screens Patched and replica-printed the metagenomic library clones in S. lividans Δact Δred onto screening media. Overlayed with appropriete host shown below. Patched S. aureus Gram + P. aeruginosa Gram - METREX Quorum sensing Blood agar Hemolytic activity CVO26 Quorum sensing No Assay ΔActΔRed Active Clones Hemolytic Rapid Sporulators Dark Spore Pigment Brown Pigment/ Antimicrobial Brown/ Antimicrobial Clone Analysis Confirm phenotype Sequence analysis Identify required genes Purify active compound Determine structure of active molecule(s) Sequence Analysis Generated small-insert library for sequencing of brown clone. Sequence revealed genes responsible for melanin production. In Eukaryotes these genes have been shown to produce a melanin precursor with antimicrobial activity (4). Chemical Analysis Partially purified pigment from brown clone run through DOWEX cation exchange resin with most pigment in the 1.0 M ammonium hydroxide eluent. Sample was run through HPLC with 0-100% acetonitrile gradient and fractions were collected every minute. Fractions tested against B. subtilis. Conclusion New methods of uncovering previously inaccessible natural product diversity are needed to combat increased frequency of infection resistance to antibiotics. The tools and protocols we have developed for screening metagenomic libraries in the alternative host S. lividans were used to screen 23,600 clones and uncovered antimicrobial activity, hemolytic activity, quorum sensing induction, alterations in host phenotype and several distinct pigment-producing clones. Our future research will focus on the sequence analysis and determination of the active molecules structure for four individual clones. One of these clones produces a melanin-like molecule that has antimicrobial activity against Bacillus subtilis. This work may present an opportunity to rejuvenate the process of drug discovery from soil microorganisms, and our research will evaluate and enhance this exciting strategy. Acknowledgments We would like to thank Christine Mlot and Lynn Williamson for previous work on the 14 Alaskan isolates and Changhui Guran, Rosivette Santiago and Erik Ransom for technical assistance. References 1. Riesenfeld, C. S., P. D. Schloss, and J. Handelsman. 2004. Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38:525-552. 2. Challis, G. L. and D. A. Hopwood. 2003. Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc. Natl. Acad. Sci. U. S. A. 100 Suppl 2:14555-14561. 3. Bentley et. al. 2002. Complete genome sequence of the model actinomycete Streptomycescoelicolor A3(2). Nature 417:141-147. 4. Zhao, P., J. Li, Y. Wang, H. Jiang. 2007. Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase. Insect Biochem. Mol. Biol. 37: 952-959.
Transcript of Mining the Metagenome for Natural Product Discovery · PDF fileMining the Metagenome for...
Mining the Metagenome for Natural Product DiscoveryMatthew McMahon1, Jo Handelsman2 and Michael Thomas1
1. Univeristy of Wisconsin - Madison Department of Bacteriology 2. Yale University Department of Molecular, Cellular, and Developmental Biology
AbstractStreptomyces lividans is a well-studied member of the phylum Actinobacteria, the source of over two-thirds of naturally derived antibiotics in current use. Well-developed and extensive genetic tools are available for use with S. lividans, which makes it a favorable host for expression of natural product gene clusters. To enable heterologous expression in S. lividans, we constructed a suitable vector to allow conjugation of cosmid metagenomic libraries from Escherichia coli to S. lividans. We also deleted gene clusters responsible for production of two antibiotics native to S. lividans to enhance detection of heterologous antibiotic production associated with metagenomic DNA. We constructed two metagenomic libraries of soil organisms and developed protocols for efficiently conjugating these libraries into S. lividans. The screening of 23,600 clones revealed antimicrobial activity, hemolytic activity, quorum sensing induction and several distinct pigment-producing clones. One active clone that warranted further analysis produces a melanin-like molecule that has antimicrobial activity against Bacillus subtilis. Clones with reproducible phenotypes are analyzed further to identify genes required for natural product biosynthesis as well as structures and biological activities of associated molecules.
Streptomycetes as Heterologous HostsWhile metagenomic techniques have been used for about a decade to access the metagenome, the success of identifying functional clones has been limited by the heterologous host used. Most functional metagenomic studies, which involve screening clones for expressed activities, use either Escherichia coli or related γ-proteobacteria as hosts. Although screening metagenomic libraries in E. coli has led to a number of discoveries, alternative hosts may contain precursors, sigma factors, rare tRNAs, or cofactors required for natural product biosynthesis that are not present in E. coli (1). This research uses Streptomyces lividans, a well-studied member of the phylum Actinobacteria, as an alternative host for expression of metagenomic libraries (2). Actinobacteria are the source of over two-thirds of naturally derived antibiotics in current use, as well as many other pharmaceuticals, which makes them a good choice for expression of natural product gene clusters (3).
Metagenomic LibrariesAKAIM1: 10,500 clones in vector pMM436 with an average insert size of 35 kb was constructed from the genomic DNA of 14 cultured Alaskan isolates shown to have antimicrobial activity.AKM2: a library of 70,000 clones in vector pMM436 with an average insert size of 35 kb was constructed with DNA extracted from the uncultured microbial community of Alaskan soil.
Vector pMM436Cosmid vector that can conjugate into S. lividans, integrates into a stable region of the S. lividans chromosome using site-specific recombination at attachment site attP, and can be removed by digesting chromosomal DNA with PacI. Integration is important for screening because it allows the insert to be stably maintained within the S. lividans chromosome without continuous antibiotic selection, which would limit the tester organisms that could be used.
Contact Information:6225 Microbial Sciences Building1550 Linden DriveMadison, WI 53706Phone: (608) 263-9075Email: [email protected]
Host Streptomyces lividans ΔActΔRedStreptomyces lividans was mutated for its ability to produce two naturally occurring antibiotics, actinorhodin and prodigiosin. The deletion of these two biosynthesis gene clusters removed the background antibiotic activity that would obscure new antibiotics in metagenomic clones. Furthermore, since metabolites commonly used to generate antibiotics are no longer funneled into making these two endogenous antibiotics, more metabolic flux can occur through newly introduced antibiotic biosynthesis pathways.
Vector Integration
Restriction Digest / Insert Retrieval
Vector Reconstruction/ Phenotype Testing
Functional ScreensPatched and replica-printed the metagenomic library clones in S. lividansΔactΔred onto screening media. Overlayed with appropriete host shown below.
Patched
S. aureus Gram +
P. aeruginosa Gram -
METREX Quorumsensing
Blood agar Hemolyticactivity
CVO26 Quorumsensing
No Assay
ΔActΔRed
Active ClonesHemolytic Rapid Sporulators
Dark Spore Pigment Brown Pigment/ Antimicrobial
Brown/ Antimicrobial Clone Analysis
Confirmphenotype
Sequenceanalysis
Identifyrequired genes
Purify activecompound
Determine structure of active molecule(s)
Sequence Analysis
Generated small-insert library for sequencing of brown clone. Sequence revealed genes responsible for melanin production. In Eukaryotes these genes have been shown to produce a melanin precursor with antimicrobial activity (4).
Chemical AnalysisPartially purified pigment from brown clone run through DOWEX cation exchange resin with most pigment in the 1.0 M ammonium hydroxide eluent. Sample was run t h rou gh HPL C w i t h 0 - 1 0 0 % a c e t o n i t r i l e gradient and fractions were collected every minute. Fractions tested against B. subtilis.
ConclusionNew methods of uncovering previously inaccessible natural product diversity are needed to combat increased frequency of infection resistance to antibiotics. The tools and protocols we have developed for screening metagenomic libraries in the alternative host S. lividans were used to screen 23,600 clones and uncovered antimicrobial activity, hemolytic activity, quorum sensing induction, alterations in host phenotype and several distinct pigment-producing clones. Our future research will focus on the sequence analysis and determination of the active molecules structure for four individual clones. One of these clones produces a melanin-like molecule that has antimicrobial activity against Bacillus subtilis. This work may present an opportunity to rejuvenate the process of drug discovery from soil microorganisms, and our research will evaluate and enhance this exciting strategy.
AcknowledgmentsWe would like to thank Christine Mlot and Lynn Williamson for previous work on the 14 Alaskan isolates and Changhui Guran, Rosivette Santiago and Erik Ransom for technical assistance.
References1. Riesenfeld, C. S., P. D. Schloss, and J. Handelsman. 2004. Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38:525-552.2. Challis, G. L. and D. A. Hopwood. 2003. Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc. Natl. Acad. Sci. U. S. A. 100 Suppl 2:14555-14561.3. Bentley et. al. 2002. Complete genome sequence of the model actinomycete Streptomycescoelicolor A3(2). Nature 417:141-147.4. Zhao, P., J. Li, Y. Wang, H. Jiang. 2007. Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase. Insect Biochem. Mol. Biol. 37: 952-959.
