efficient blue-white screening based gene inactivation system for Streptomyces
Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, usin...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2015-02, Vol.99 (4), p.1923-1933 |
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| container_title | Applied microbiology and biotechnology |
| container_volume | 99 |
| creator | Li, Pengwei Li, Jine Guo, Zhengyan Tang, Wei Han, Jianshan Meng, Xiangxi Hao, Tingting Zhu, Yaxin Zhang, Lixin Chen, Yihua |
| description | Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86. |
| doi_str_mv | 10.1007/s00253-014-6369-0 |
| format | Article |
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A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-014-6369-0</identifier><identifier>PMID: 25666782</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer-Verlag</publisher><subject>Analysis ; Antibiotic resistance ; Antibiotics ; Bacteria ; bioactive properties ; Biological activity ; Biomedical and Life Sciences ; Biosynthesis ; Biotechnology ; Crosses, Genetic ; DNA, Bacterial - chemistry ; DNA, Bacterial - genetics ; Drug resistance ; Gene Silencing ; Genes ; Genes, Bacterial ; Genes, Reporter ; Genetic aspects ; genetic engineering ; Genetic screening ; Genetic Testing - methods ; Genetic Vectors ; Genetics, Microbial - methods ; Inactivation ; Laboratories ; Life Sciences ; Metabolites ; Methods ; Methods and Protocols ; Microbial Genetics and Genomics ; Microbiology ; Molecular Biology - methods ; Molecular Sequence Data ; Morphology ; mutants ; Piperidones - metabolism ; Plasmids ; Proteins ; Recombination, Genetic ; screening ; Secondary metabolites ; Sequence Analysis, DNA ; Signal transduction ; Streptomyces ; Streptomyces - genetics ; Streptomyces - isolation & purification ; Streptomyces lavendulae ; Studies ; Suicide</subject><ispartof>Applied microbiology and biotechnology, 2015-02, Vol.99 (4), p.1923-1933</ispartof><rights>Springer-Verlag Berlin Heidelberg 2015</rights><rights>COPYRIGHT 2015 Springer</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c600t-830541bb3ff2ad8d3d59301bd6c8037e1c9c9c14ba3f5c3353257b9141c928073</citedby><cites>FETCH-LOGICAL-c600t-830541bb3ff2ad8d3d59301bd6c8037e1c9c9c14ba3f5c3353257b9141c928073</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00253-014-6369-0$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-014-6369-0$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,777,781,27905,27906,41469,42538,51300</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25666782$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Pengwei</creatorcontrib><creatorcontrib>Li, Jine</creatorcontrib><creatorcontrib>Guo, Zhengyan</creatorcontrib><creatorcontrib>Tang, Wei</creatorcontrib><creatorcontrib>Han, Jianshan</creatorcontrib><creatorcontrib>Meng, Xiangxi</creatorcontrib><creatorcontrib>Hao, Tingting</creatorcontrib><creatorcontrib>Zhu, Yaxin</creatorcontrib><creatorcontrib>Zhang, Lixin</creatorcontrib><creatorcontrib>Chen, Yihua</creatorcontrib><title>efficient blue-white screening based gene inactivation system for Streptomyces</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86.</description><subject>Analysis</subject><subject>Antibiotic resistance</subject><subject>Antibiotics</subject><subject>Bacteria</subject><subject>bioactive properties</subject><subject>Biological activity</subject><subject>Biomedical and Life Sciences</subject><subject>Biosynthesis</subject><subject>Biotechnology</subject><subject>Crosses, Genetic</subject><subject>DNA, Bacterial - chemistry</subject><subject>DNA, Bacterial - genetics</subject><subject>Drug resistance</subject><subject>Gene Silencing</subject><subject>Genes</subject><subject>Genes, Bacterial</subject><subject>Genes, Reporter</subject><subject>Genetic aspects</subject><subject>genetic engineering</subject><subject>Genetic screening</subject><subject>Genetic Testing - methods</subject><subject>Genetic Vectors</subject><subject>Genetics, Microbial - methods</subject><subject>Inactivation</subject><subject>Laboratories</subject><subject>Life Sciences</subject><subject>Metabolites</subject><subject>Methods</subject><subject>Methods and Protocols</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Molecular Biology - methods</subject><subject>Molecular Sequence Data</subject><subject>Morphology</subject><subject>mutants</subject><subject>Piperidones - metabolism</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Recombination, Genetic</subject><subject>screening</subject><subject>Secondary metabolites</subject><subject>Sequence Analysis, DNA</subject><subject>Signal transduction</subject><subject>Streptomyces</subject><subject>Streptomyces - genetics</subject><subject>Streptomyces - isolation & purification</subject><subject>Streptomyces lavendulae</subject><subject>Studies</subject><subject>Suicide</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</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>eNp9kktv1TAQhSMEopfCD2ADkdjAImX8TLKsKh6VKpC4dG05zji4unEutlO4_x5HKY-LEJqFJc93jmZ8XBRPCZwRgPp1BKCCVUB4JZlsK7hXbAhntAJJ-P1iA6QWVS3a5qR4FOMNAKGNlA-LEyqklHVDN8UHtNYZhz6V3W7G6tsXl7CMJiB654ey0xH7ckCPpfPaJHerk5t8GQ8x4VjaKZTbFHCfpvFgMD4uHli9i_jk7jwtrt---Xzxvrr6-O7y4vyqMhIgVQ0DwUnXMWup7pue9aJlQLpemgZYjcS0uQjvNLPCMCYYFXXXEp4btIGanRYvV999mL7OGJMaXTS422mP0xwVkYLLtgWAjL74C72Z5uDzdAtFZXam7W9q0DtUztspBW0WU3XOCUjOOOeZOvsHlavH0ZnJo3X5_kjw6kiQmYTf06DnGNXl9tMxS1bWhCnGgFbtgxt1OCgCaslbrXmrnLda8lbLcs_ulpu7Eftfip8BZ4CuQMwtP2D4Y_v_uD5fRVZPSg_BRXW9pUBE_kE8P5dkPwDeJrrg</recordid><startdate>20150201</startdate><enddate>20150201</enddate><creator>Li, Pengwei</creator><creator>Li, Jine</creator><creator>Guo, Zhengyan</creator><creator>Tang, Wei</creator><creator>Han, Jianshan</creator><creator>Meng, Xiangxi</creator><creator>Hao, Tingting</creator><creator>Zhu, Yaxin</creator><creator>Zhang, Lixin</creator><creator>Chen, Yihua</creator><general>Springer-Verlag</general><general>Springer Berlin Heidelberg</general><general>Springer</general><general>Springer Nature B.V</general><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>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope></search><sort><creationdate>20150201</creationdate><title>efficient blue-white screening based gene inactivation system for Streptomyces</title><author>Li, Pengwei ; Li, Jine ; Guo, Zhengyan ; Tang, Wei ; Han, Jianshan ; Meng, Xiangxi ; Hao, Tingting ; Zhu, Yaxin ; Zhang, Lixin ; Chen, Yihua</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c600t-830541bb3ff2ad8d3d59301bd6c8037e1c9c9c14ba3f5c3353257b9141c928073</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Analysis</topic><topic>Antibiotic resistance</topic><topic>Antibiotics</topic><topic>Bacteria</topic><topic>bioactive properties</topic><topic>Biological activity</topic><topic>Biomedical and Life Sciences</topic><topic>Biosynthesis</topic><topic>Biotechnology</topic><topic>Crosses, Genetic</topic><topic>DNA, Bacterial - chemistry</topic><topic>DNA, Bacterial - genetics</topic><topic>Drug resistance</topic><topic>Gene Silencing</topic><topic>Genes</topic><topic>Genes, Bacterial</topic><topic>Genes, Reporter</topic><topic>Genetic aspects</topic><topic>genetic engineering</topic><topic>Genetic screening</topic><topic>Genetic Testing - methods</topic><topic>Genetic Vectors</topic><topic>Genetics, Microbial - methods</topic><topic>Inactivation</topic><topic>Laboratories</topic><topic>Life Sciences</topic><topic>Metabolites</topic><topic>Methods</topic><topic>Methods and Protocols</topic><topic>Microbial Genetics and Genomics</topic><topic>Microbiology</topic><topic>Molecular Biology - methods</topic><topic>Molecular Sequence Data</topic><topic>Morphology</topic><topic>mutants</topic><topic>Piperidones - metabolism</topic><topic>Plasmids</topic><topic>Proteins</topic><topic>Recombination, Genetic</topic><topic>screening</topic><topic>Secondary metabolites</topic><topic>Sequence Analysis, DNA</topic><topic>Signal transduction</topic><topic>Streptomyces</topic><topic>Streptomyces - 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Academic</collection><jtitle>Applied microbiology and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Li, Pengwei</au><au>Li, Jine</au><au>Guo, Zhengyan</au><au>Tang, Wei</au><au>Han, Jianshan</au><au>Meng, Xiangxi</au><au>Hao, Tingting</au><au>Zhu, Yaxin</au><au>Zhang, Lixin</au><au>Chen, Yihua</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>efficient blue-white screening based gene inactivation system for Streptomyces</atitle><jtitle>Applied microbiology and biotechnology</jtitle><stitle>Appl Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2015-02-01</date><risdate>2015</risdate><volume>99</volume><issue>4</issue><spage>1923</spage><epage>1933</epage><pages>1923-1933</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><abstract>Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer-Verlag</pub><pmid>25666782</pmid><doi>10.1007/s00253-014-6369-0</doi><tpages>11</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0175-7598 |
| ispartof | Applied microbiology and biotechnology, 2015-02, Vol.99 (4), p.1923-1933 |
| issn | 0175-7598 1432-0614 |
| language | eng |
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| source | MEDLINE; Springer Nature - Complete Springer Journals |
| subjects | Analysis Antibiotic resistance Antibiotics Bacteria bioactive properties Biological activity Biomedical and Life Sciences Biosynthesis Biotechnology Crosses, Genetic DNA, Bacterial - chemistry DNA, Bacterial - genetics Drug resistance Gene Silencing Genes Genes, Bacterial Genes, Reporter Genetic aspects genetic engineering Genetic screening Genetic Testing - methods Genetic Vectors Genetics, Microbial - methods Inactivation Laboratories Life Sciences Metabolites Methods Methods and Protocols Microbial Genetics and Genomics Microbiology Molecular Biology - methods Molecular Sequence Data Morphology mutants Piperidones - metabolism Plasmids Proteins Recombination, Genetic screening Secondary metabolites Sequence Analysis, DNA Signal transduction Streptomyces Streptomyces - genetics Streptomyces - isolation & purification Streptomyces lavendulae Studies Suicide |
| title | efficient blue-white screening based gene inactivation system for Streptomyces |
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