Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum

Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, the...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Microbial cell factories 2016-01, Vol.15 (12), p.12, Article 12
Hauptverfasser: Son, Yeon Jeong, Ryu, Ae Jin, Li, Ling, Han, Nam Soo, Jeong, Ki Jun
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page
container_issue 12
container_start_page 12
container_title Microbial cell factories
container_volume 15
creator Son, Yeon Jeong
Ryu, Ae Jin
Li, Ling
Han, Nam Soo
Jeong, Ki Jun
description Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α-amylase. The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.
doi_str_mv 10.1186/s12934-015-0400-8
format Article
fullrecord <record><control><sourceid>gale_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_4714500</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A441818640</galeid><sourcerecordid>A441818640</sourcerecordid><originalsourceid>FETCH-LOGICAL-c528t-71b011df16c874d5d1aa343b3c0999eea664085345be9fa027b40ff900434dfd3</originalsourceid><addsrcrecordid>eNptUk1rHSEUldLSpGl_QDdF6KqLSXV0RmdTCOlX4EGhH2tx9PqeYUan6oTk39fHS9M8KHfhxXvO4X4chF5Tck6p7N9n2g6MN4R2DeGENPIJOqVcdE0ru-Hpo_wEvcj5mhAqpGDP0Unbi17U_BT5j3ADU1xmCAVHhzXe-e2uMXG5w8uk8-wtdjFhCDsdDFi8pGhXU3wMe3gCE-fRB13ZtVLAh4x9wBtYTQwxl2iw8SXBOr9Ez5yeMry6f8_Qr8-ffl5-bTbfvlxdXmwa07WyNIKOhFLraG-k4LazVGvG2cgMGYYBQPc9J7JjvBthcJq0YuTEuYEQzrh1lp2hDwfdZR1nsKYOlvSkluRnne5U1F4dV4LfqW28UVxQ3hFSBd7eC6T4e4Vc1HVcU6g9KyoEJ7Q2Iv6htnoC5YOLVczMPht1wTmV9T58r3X-H1QNC7OvGwLn6_8R4d0RoWIK3JatXnNWVz--H2PpAWtSzDmBexiSErU3iDoYRFWDqL1BlKycN4-388D46wj2B9TptkM</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1774010997</pqid></control><display><type>article</type><title>Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum</title><source>Springer Open Access</source><source>MEDLINE</source><source>DOAJ Directory of Open Access Journals</source><source>PubMed Central Open Access</source><source>EZB-FREE-00999 freely available EZB journals</source><source>PubMed Central</source><source>SpringerLink Journals - AutoHoldings</source><creator>Son, Yeon Jeong ; Ryu, Ae Jin ; Li, Ling ; Han, Nam Soo ; Jeong, Ki Jun</creator><creatorcontrib>Son, Yeon Jeong ; Ryu, Ae Jin ; Li, Ling ; Han, Nam Soo ; Jeong, Ki Jun</creatorcontrib><description>Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α-amylase. The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.</description><identifier>ISSN: 1475-2859</identifier><identifier>EISSN: 1475-2859</identifier><identifier>DOI: 10.1186/s12934-015-0400-8</identifier><identifier>PMID: 26767787</identifier><language>eng</language><publisher>England: BioMed Central Ltd</publisher><subject>Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Glutathione transferase ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; Leuconostoc - genetics ; Leuconostoc - metabolism ; Plasmids - genetics ; Polymerase Chain Reaction ; Recombinant proteins ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism</subject><ispartof>Microbial cell factories, 2016-01, Vol.15 (12), p.12, Article 12</ispartof><rights>COPYRIGHT 2016 BioMed Central Ltd.</rights><rights>Copyright BioMed Central 2016</rights><rights>Son et al. 2016</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c528t-71b011df16c874d5d1aa343b3c0999eea664085345be9fa027b40ff900434dfd3</citedby><cites>FETCH-LOGICAL-c528t-71b011df16c874d5d1aa343b3c0999eea664085345be9fa027b40ff900434dfd3</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/PMC4714500/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714500/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26767787$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Son, Yeon Jeong</creatorcontrib><creatorcontrib>Ryu, Ae Jin</creatorcontrib><creatorcontrib>Li, Ling</creatorcontrib><creatorcontrib>Han, Nam Soo</creatorcontrib><creatorcontrib>Jeong, Ki Jun</creatorcontrib><title>Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum</title><title>Microbial cell factories</title><addtitle>Microb Cell Fact</addtitle><description>Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α-amylase. The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.</description><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Glutathione transferase</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>Leuconostoc - genetics</subject><subject>Leuconostoc - metabolism</subject><subject>Plasmids - genetics</subject><subject>Polymerase Chain Reaction</subject><subject>Recombinant proteins</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><issn>1475-2859</issn><issn>1475-2859</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNptUk1rHSEUldLSpGl_QDdF6KqLSXV0RmdTCOlX4EGhH2tx9PqeYUan6oTk39fHS9M8KHfhxXvO4X4chF5Tck6p7N9n2g6MN4R2DeGENPIJOqVcdE0ru-Hpo_wEvcj5mhAqpGDP0Unbi17U_BT5j3ADU1xmCAVHhzXe-e2uMXG5w8uk8-wtdjFhCDsdDFi8pGhXU3wMe3gCE-fRB13ZtVLAh4x9wBtYTQwxl2iw8SXBOr9Ez5yeMry6f8_Qr8-ffl5-bTbfvlxdXmwa07WyNIKOhFLraG-k4LazVGvG2cgMGYYBQPc9J7JjvBthcJq0YuTEuYEQzrh1lp2hDwfdZR1nsKYOlvSkluRnne5U1F4dV4LfqW28UVxQ3hFSBd7eC6T4e4Vc1HVcU6g9KyoEJ7Q2Iv6htnoC5YOLVczMPht1wTmV9T58r3X-H1QNC7OvGwLn6_8R4d0RoWIK3JatXnNWVz--H2PpAWtSzDmBexiSErU3iDoYRFWDqL1BlKycN4-388D46wj2B9TptkM</recordid><startdate>20160115</startdate><enddate>20160115</enddate><creator>Son, Yeon Jeong</creator><creator>Ryu, Ae Jin</creator><creator>Li, Ling</creator><creator>Han, Nam Soo</creator><creator>Jeong, Ki Jun</creator><general>BioMed Central Ltd</general><general>BioMed Central</general><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>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>5PM</scope></search><sort><creationdate>20160115</creationdate><title>Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum</title><author>Son, Yeon Jeong ; Ryu, Ae Jin ; Li, Ling ; Han, Nam Soo ; Jeong, Ki Jun</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c528t-71b011df16c874d5d1aa343b3c0999eea664085345be9fa027b40ff900434dfd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Glutathione transferase</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>Leuconostoc - genetics</topic><topic>Leuconostoc - metabolism</topic><topic>Plasmids - genetics</topic><topic>Polymerase Chain Reaction</topic><topic>Recombinant proteins</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Son, Yeon Jeong</creatorcontrib><creatorcontrib>Ryu, Ae Jin</creatorcontrib><creatorcontrib>Li, Ling</creatorcontrib><creatorcontrib>Han, Nam Soo</creatorcontrib><creatorcontrib>Jeong, Ki Jun</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>ProQuest Health and Medical</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health &amp; Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health &amp; Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>ProQuest Biological Science Journals</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Microbial cell factories</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Son, Yeon Jeong</au><au>Ryu, Ae Jin</au><au>Li, Ling</au><au>Han, Nam Soo</au><au>Jeong, Ki Jun</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum</atitle><jtitle>Microbial cell factories</jtitle><addtitle>Microb Cell Fact</addtitle><date>2016-01-15</date><risdate>2016</risdate><volume>15</volume><issue>12</issue><spage>12</spage><pages>12-</pages><artnum>12</artnum><issn>1475-2859</issn><eissn>1475-2859</eissn><abstract>Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α-amylase. The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.</abstract><cop>England</cop><pub>BioMed Central Ltd</pub><pmid>26767787</pmid><doi>10.1186/s12934-015-0400-8</doi><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 1475-2859
ispartof Microbial cell factories, 2016-01, Vol.15 (12), p.12, Article 12
issn 1475-2859
1475-2859
language eng
recordid cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_4714500
source Springer Open Access; MEDLINE; DOAJ Directory of Open Access Journals; PubMed Central Open Access; EZB-FREE-00999 freely available EZB journals; PubMed Central; SpringerLink Journals - AutoHoldings
subjects Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Glutathione transferase
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Leuconostoc - genetics
Leuconostoc - metabolism
Plasmids - genetics
Polymerase Chain Reaction
Recombinant proteins
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
title Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-07T20%3A22%3A04IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Development%20of%20a%20high-copy%20plasmid%20for%20enhanced%20production%20of%20recombinant%20proteins%20in%20Leuconostoc%20citreum&rft.jtitle=Microbial%20cell%20factories&rft.au=Son,%20Yeon%20Jeong&rft.date=2016-01-15&rft.volume=15&rft.issue=12&rft.spage=12&rft.pages=12-&rft.artnum=12&rft.issn=1475-2859&rft.eissn=1475-2859&rft_id=info:doi/10.1186/s12934-015-0400-8&rft_dat=%3Cgale_pubme%3EA441818640%3C/gale_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1774010997&rft_id=info:pmid/26767787&rft_galeid=A441818640&rfr_iscdi=true