Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) in Escherichia coli

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dc.contributor.authorLee, SangYupko
dc.contributor.authorYIM, KSko
dc.contributor.authorChang, Ho Namko
dc.contributor.authorChang, YongKeunko
dc.date.accessioned2010-11-16T06:42:54Z-
dc.date.available2010-11-16T06:42:54Z-
dc.date.created2012-02-06-
dc.date.created2012-02-06-
dc.date.created2012-02-06-
dc.date.created2012-02-06-
dc.date.issued1994-02-
dc.identifier.citationJOURNAL OF BIOTECHNOLOGY, v.32, no.2, pp.203 - 211-
dc.identifier.issn0168-1656-
dc.identifier.urihttp://hdl.handle.net/10203/20042-
dc.description.abstractPlasmids containing the Alcaligenes eutrophus poly(3-hydroxybutyric acid) (PHB) biosynthetic genes were constructed for the production of PHB in Escherichia coli and plasmid stability was investigated by repeated subculturing without antibiotic pressure. Both pSYL101 (high copy) and pSYL102 (medium copy) were unstable during the subcultures. Higher instability was observed when cells were accumulating PHB. Segregational instability was aggravated by the faster growth of plasmid-free cells and by appearance of non-dividing cells harboring large amount of PHB during the fed-batch culture. Two derivatives, pSYL103 and pSYL104, were then developed by cloning the parB locus of plasmid R1 into pSYL102 and pSYL101, respectively. They showed 100% stability even during PHB synthesis and accumulation over 110 generations. All four plasmids were structurally stable. The final cell mass, PHB concentration, and PHB per dry cell weight (P/X, w/w, %) of 10 1.4 g l-1, 81.2 g l-1, and 80.1%, respectively, were obtained in 39 h by high cell density culture of XL1-Blue (pSYL104). The final PHB concentration was lower using XL1-Blue (pSYL103), which suggested that high gene dosage was required for the synthesis and accumulation of PHB to a high concentration in E. coli.-
dc.description.sponsorshipWe would like to thank Dr. Steinbuchel and Dr. Gerdes for supplying the plasmids pSK2665 and pKG1022, respectively. We also thank Yoo Kyung Lee and Beom Soo Kim for technical assistance. Part of this work was presented at the Engineering Foundation Conference 'Recombinant DNA Technology II' held at Palm Coast, FL, USA, 1993. This work was supported by Korea Science and Engineering Foundation.en
dc.languageEnglish-
dc.language.isoen_USen
dc.publisherELSEVIER SCIENCE BV-
dc.titleConstruction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) in Escherichia coli-
dc.typeArticle-
dc.identifier.wosidA1994ND08700011-
dc.identifier.scopusid2-s2.0-0028213524-
dc.type.rimsART-
dc.citation.volume32-
dc.citation.issue2-
dc.citation.beginningpage203-
dc.citation.endingpage211-
dc.citation.publicationnameJOURNAL OF BIOTECHNOLOGY-
dc.embargo.liftdate9999-12-31-
dc.embargo.terms9999-12-31-
dc.contributor.localauthorLee, SangYup-
dc.contributor.localauthorChang, Ho Nam-
dc.contributor.localauthorChang, YongKeun-
dc.contributor.nonIdAuthorYIM, KS-
dc.type.journalArticleArticle-
dc.subject.keywordAuthorPOLY(3-HYDROXYBUTYRIC ACID)-
dc.subject.keywordAuthorPHB BIOSYNTHETIC GENE-
dc.subject.keywordAuthorPLASMID STABILITY-
dc.subject.keywordAuthorSTABLE PLASMID-
dc.subject.keywordAuthorHIGH CELL DENSITY CULTURE-
dc.subject.keywordAuthorESCHERICHIA-COLI-
dc.subject.keywordPlusPOLY-BETA-HYDROXYBUTYRATE-
dc.subject.keywordPlusALCALIGENES-EUTROPHUS H16-
dc.subject.keywordPlusFED-BATCH CULTURE-
dc.subject.keywordPlusDENSITY CULTIVATION-
dc.subject.keywordPlusCOPY NUMBER-
dc.subject.keywordPlusIDENTIFICATION-
dc.subject.keywordPlusMETABOLISM-
dc.subject.keywordPlusCLONING-
dc.subject.keywordPlusPHB-
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