Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach
DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequ...
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creator | Wang, Qiuyan Wu, Huili Wang, Anming Du, Pengfei Pei, Xiaolin Li, Haifeng Yin, Xiaopu Huang, Lifeng Xiong, Xiaolong |
description | DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering. |
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However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M110.139659</identifier><identifier>PMID: 20962349</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Cloning, Molecular ; DNA - genetics ; DNA Family Shuffling ; DNA Primers - genetics ; Enzyme Mutation ; Enzymology ; Escherichia coli - genetics ; Gene Amplification ; Gene Library ; Genetic Engineering - methods ; Genomics ; High Throughput Screening (HTS) ; Homologous Genes ; Lipase ; Lipase - genetics ; Metagenomics - methods ; Molecular Sequence Data ; Phylogeny ; Plasmids - metabolism ; Polymerase Chain Reaction (PCR) ; Polymerase Chain Reaction - methods ; Sequence Homology, Amino Acid ; TMGS-PCR</subject><ispartof>The Journal of biological chemistry, 2010-12, Vol.285 (53), p.41509-41516</ispartof><rights>2010 © 2010 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>2010 by The American Society for Biochemistry and Molecular Biology, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c564t-35a4dbf25c4d97917182ca9dca4f528867c8fad902928c1a5ce3908efb658b863</citedby><cites>FETCH-LOGICAL-c564t-35a4dbf25c4d97917182ca9dca4f528867c8fad902928c1a5ce3908efb658b863</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3009877/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3009877/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20962349$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, Qiuyan</creatorcontrib><creatorcontrib>Wu, Huili</creatorcontrib><creatorcontrib>Wang, Anming</creatorcontrib><creatorcontrib>Du, Pengfei</creatorcontrib><creatorcontrib>Pei, Xiaolin</creatorcontrib><creatorcontrib>Li, Haifeng</creatorcontrib><creatorcontrib>Yin, Xiaopu</creatorcontrib><creatorcontrib>Huang, Lifeng</creatorcontrib><creatorcontrib>Xiong, Xiaolong</creatorcontrib><title>Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.</description><subject>Amino Acid Sequence</subject><subject>Cloning, Molecular</subject><subject>DNA - genetics</subject><subject>DNA Family Shuffling</subject><subject>DNA Primers - genetics</subject><subject>Enzyme Mutation</subject><subject>Enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Gene Amplification</subject><subject>Gene Library</subject><subject>Genetic Engineering - methods</subject><subject>Genomics</subject><subject>High Throughput Screening (HTS)</subject><subject>Homologous Genes</subject><subject>Lipase</subject><subject>Lipase - genetics</subject><subject>Metagenomics - methods</subject><subject>Molecular Sequence Data</subject><subject>Phylogeny</subject><subject>Plasmids - metabolism</subject><subject>Polymerase Chain Reaction (PCR)</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Sequence Homology, Amino Acid</subject><subject>TMGS-PCR</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kUtv1DAQgC0EotvCmRv41lNaP_KwL0irpS1Ibam6rcTNcpzxrqskDnay0vLr8SqlggO-WB5_89B8CH2g5IySKj9_qs3ZDT28uCwL-QotKBE84wX98RotCGE0k6wQR-g4xieSTi7pW3TEiCwZz-UCwV3wcQAzun6Db2DUG-h95wy-6H_tO8Drqba6c-0eX0EPEVsf8JfbJb6cg-vtZG17yK33WONbv4MW363us1pHaPByGILXZvsOvbG6jfD--T5Bj5cXD6uv2fX3q2-r5XVmijIf09g6b2rLCpM3spK0ooIZLRujc1swIcrKCKsbSZhkwlBdGOCSCLB1WYhalPwEfZ7rDlPdQWOgH4Nu1RBcp8Neee3Uvz-926qN3ylOiBRVlQqcPhcI_ucEcVSdiwbaVvfgp6hElVNBSsESeT6TJi0wBrAvXShRBzcquVEHN2p2kzI-_j3cC_9HRgI-zYDVXulNcFE9rhmhnFDJWUVpIuRMQFrizkFQ0TjoDTQuJIeq8e6_7X8DncuoCA</recordid><startdate>20101231</startdate><enddate>20101231</enddate><creator>Wang, Qiuyan</creator><creator>Wu, Huili</creator><creator>Wang, Anming</creator><creator>Du, Pengfei</creator><creator>Pei, Xiaolin</creator><creator>Li, Haifeng</creator><creator>Yin, Xiaopu</creator><creator>Huang, Lifeng</creator><creator>Xiong, Xiaolong</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>20101231</creationdate><title>Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach</title><author>Wang, Qiuyan ; Wu, Huili ; Wang, Anming ; Du, Pengfei ; Pei, Xiaolin ; Li, Haifeng ; Yin, Xiaopu ; Huang, Lifeng ; Xiong, Xiaolong</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c564t-35a4dbf25c4d97917182ca9dca4f528867c8fad902928c1a5ce3908efb658b863</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Amino Acid Sequence</topic><topic>Cloning, Molecular</topic><topic>DNA - genetics</topic><topic>DNA Family Shuffling</topic><topic>DNA Primers - genetics</topic><topic>Enzyme Mutation</topic><topic>Enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Gene Amplification</topic><topic>Gene Library</topic><topic>Genetic Engineering - methods</topic><topic>Genomics</topic><topic>High Throughput Screening (HTS)</topic><topic>Homologous Genes</topic><topic>Lipase</topic><topic>Lipase - genetics</topic><topic>Metagenomics - methods</topic><topic>Molecular Sequence Data</topic><topic>Phylogeny</topic><topic>Plasmids - metabolism</topic><topic>Polymerase Chain Reaction (PCR)</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Sequence Homology, Amino Acid</topic><topic>TMGS-PCR</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Qiuyan</creatorcontrib><creatorcontrib>Wu, Huili</creatorcontrib><creatorcontrib>Wang, Anming</creatorcontrib><creatorcontrib>Du, Pengfei</creatorcontrib><creatorcontrib>Pei, Xiaolin</creatorcontrib><creatorcontrib>Li, Haifeng</creatorcontrib><creatorcontrib>Yin, Xiaopu</creatorcontrib><creatorcontrib>Huang, Lifeng</creatorcontrib><creatorcontrib>Xiong, Xiaolong</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Qiuyan</au><au>Wu, Huili</au><au>Wang, Anming</au><au>Du, Pengfei</au><au>Pei, Xiaolin</au><au>Li, Haifeng</au><au>Yin, Xiaopu</au><au>Huang, Lifeng</au><au>Xiong, Xiaolong</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2010-12-31</date><risdate>2010</risdate><volume>285</volume><issue>53</issue><spage>41509</spage><epage>41516</epage><pages>41509-41516</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. 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subjects | Amino Acid Sequence Cloning, Molecular DNA - genetics DNA Family Shuffling DNA Primers - genetics Enzyme Mutation Enzymology Escherichia coli - genetics Gene Amplification Gene Library Genetic Engineering - methods Genomics High Throughput Screening (HTS) Homologous Genes Lipase Lipase - genetics Metagenomics - methods Molecular Sequence Data Phylogeny Plasmids - metabolism Polymerase Chain Reaction (PCR) Polymerase Chain Reaction - methods Sequence Homology, Amino Acid TMGS-PCR |
title | Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach |
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