Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community
Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgras...
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| creator | Allgaier, Martin Reddy, Amitha Park, Joshua I Ivanova, Natalia D'haeseleer, Patrik Lowry, Steve Sapra, Rajat Hazen, Terry C Simmons, Blake A VanderGheynst, Jean S Hugenholtz, Philip |
| description | Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme. |
| doi_str_mv | 10.1371/journal.pone.0008812 |
| format | Article |
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Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0008812</identifier><identifier>PMID: 20098679</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Agricultural engineering ; Bacteria ; Biodiesel fuels ; Biofuels ; Bioinformatics ; Biomass ; Biomass energy ; Bioreactors ; Biotechnology/Environmental Microbiology ; Carbohydrates ; Carbon dioxide ; Carboxymethyl cellulose ; Carboxymethylcellulose ; Cell walls ; Cellulase - metabolism ; cellulases ; Cellulose ; Chromatography ; Communities ; Community development ; Composting ; Composts ; Computational Biology/Metagenomics ; Deconstruction ; Depolymerization ; E coli ; Ecologists ; enzyme activity ; Enzymes ; Escherichia coli ; Gene expression ; Genes ; genetic techniques and protocols ; Genetics and Genomics/Gene Discovery ; Genetics and Genomics/Gene Expression ; Genomes ; Glycoside hydrolase ; Glycoside Hydrolases - genetics ; Glycoside Hydrolases - metabolism ; Hydrolase ; Hydrolases ; Inoculum ; Laboratories ; Lignin ; Microbial activity ; microbial genetics ; Microbiology/Microbial Physiology and Metabolism ; Microorganisms ; Molecular Sequence Data ; O-glycoside hydrolases ; Optimization ; Panicum virgatum ; pH effects ; Phylogenetics ; plant residues ; Poaceae - enzymology ; Respiration ; ribosomal RNA ; RNA ; RNA, Ribosomal - genetics ; rRNA ; small-subunit ribosomal RNA-based community profiles ; Soil ; Synthesis ; thermophilic bacteria ; thermophilic microorganisms</subject><ispartof>PloS one, 2010-01, Vol.5 (1), p.e8812-e8812</ispartof><rights>COPYRIGHT 2010 Public Library of Science</rights><rights>2010. This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. 2010</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c840t-5d29e3b2e57665425764d7441999465ad5a7181f49596ffb4e6f7ff6087172723</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2809096/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2809096/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20098679$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/1153436$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Allgaier, Martin</creatorcontrib><creatorcontrib>Reddy, Amitha</creatorcontrib><creatorcontrib>Park, Joshua I</creatorcontrib><creatorcontrib>Ivanova, Natalia</creatorcontrib><creatorcontrib>D'haeseleer, Patrik</creatorcontrib><creatorcontrib>Lowry, Steve</creatorcontrib><creatorcontrib>Sapra, Rajat</creatorcontrib><creatorcontrib>Hazen, Terry C</creatorcontrib><creatorcontrib>Simmons, Blake A</creatorcontrib><creatorcontrib>VanderGheynst, Jean S</creatorcontrib><creatorcontrib>Hugenholtz, Philip</creatorcontrib><creatorcontrib>Joint Bioenergy Institute (JBEI)</creatorcontrib><title>Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.</description><subject>Agricultural engineering</subject><subject>Bacteria</subject><subject>Biodiesel fuels</subject><subject>Biofuels</subject><subject>Bioinformatics</subject><subject>Biomass</subject><subject>Biomass energy</subject><subject>Bioreactors</subject><subject>Biotechnology/Environmental Microbiology</subject><subject>Carbohydrates</subject><subject>Carbon dioxide</subject><subject>Carboxymethyl cellulose</subject><subject>Carboxymethylcellulose</subject><subject>Cell walls</subject><subject>Cellulase - metabolism</subject><subject>cellulases</subject><subject>Cellulose</subject><subject>Chromatography</subject><subject>Communities</subject><subject>Community development</subject><subject>Composting</subject><subject>Composts</subject><subject>Computational Biology/Metagenomics</subject><subject>Deconstruction</subject><subject>Depolymerization</subject><subject>E coli</subject><subject>Ecologists</subject><subject>enzyme activity</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Gene expression</subject><subject>Genes</subject><subject>genetic techniques and protocols</subject><subject>Genetics and Genomics/Gene Discovery</subject><subject>Genetics and Genomics/Gene Expression</subject><subject>Genomes</subject><subject>Glycoside hydrolase</subject><subject>Glycoside Hydrolases - genetics</subject><subject>Glycoside Hydrolases - metabolism</subject><subject>Hydrolase</subject><subject>Hydrolases</subject><subject>Inoculum</subject><subject>Laboratories</subject><subject>Lignin</subject><subject>Microbial activity</subject><subject>microbial genetics</subject><subject>Microbiology/Microbial Physiology and 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titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Allgaier, Martin</au><au>Reddy, Amitha</au><au>Park, Joshua I</au><au>Ivanova, Natalia</au><au>D'haeseleer, Patrik</au><au>Lowry, Steve</au><au>Sapra, Rajat</au><au>Hazen, Terry C</au><au>Simmons, Blake A</au><au>VanderGheynst, Jean S</au><au>Hugenholtz, Philip</au><aucorp>Joint Bioenergy Institute (JBEI)</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2010-01-21</date><risdate>2010</risdate><volume>5</volume><issue>1</issue><spage>e8812</spage><epage>e8812</epage><pages>e8812-e8812</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>20098679</pmid><doi>10.1371/journal.pone.0008812</doi><tpages>e8812</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2010-01, Vol.5 (1), p.e8812-e8812 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1289252110 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Agricultural engineering Bacteria Biodiesel fuels Biofuels Bioinformatics Biomass Biomass energy Bioreactors Biotechnology/Environmental Microbiology Carbohydrates Carbon dioxide Carboxymethyl cellulose Carboxymethylcellulose Cell walls Cellulase - metabolism cellulases Cellulose Chromatography Communities Community development Composting Composts Computational Biology/Metagenomics Deconstruction Depolymerization E coli Ecologists enzyme activity Enzymes Escherichia coli Gene expression Genes genetic techniques and protocols Genetics and Genomics/Gene Discovery Genetics and Genomics/Gene Expression Genomes Glycoside hydrolase Glycoside Hydrolases - genetics Glycoside Hydrolases - metabolism Hydrolase Hydrolases Inoculum Laboratories Lignin Microbial activity microbial genetics Microbiology/Microbial Physiology and Metabolism Microorganisms Molecular Sequence Data O-glycoside hydrolases Optimization Panicum virgatum pH effects Phylogenetics plant residues Poaceae - enzymology Respiration ribosomal RNA RNA RNA, Ribosomal - genetics rRNA small-subunit ribosomal RNA-based community profiles Soil Synthesis thermophilic bacteria thermophilic microorganisms |
| title | Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-08T20%3A10%3A34IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Targeted%20Discovery%20of%20Glycoside%20Hydrolases%20from%20a%20Switchgrass-Adapted%20Compost%20Community&rft.jtitle=PloS%20one&rft.au=Allgaier,%20Martin&rft.aucorp=Joint%20Bioenergy%20Institute%20(JBEI)&rft.date=2010-01-21&rft.volume=5&rft.issue=1&rft.spage=e8812&rft.epage=e8812&rft.pages=e8812-e8812&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0008812&rft_dat=%3Cgale_plos_%3EA473916946%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1289252110&rft_id=info:pmid/20098679&rft_galeid=A473916946&rft_doaj_id=oai_doaj_org_article_f780bc58f12947fab16130642b7dfffc&rfr_iscdi=true |