CONSTRUCTION OF PLASMIDS, ESTIMATION OF PLASMID STABILITY, AND USE OF STABLE PLASMIDS FOR THE PRODUCTION OF POLY(3-HYDROXYBUTYRIC ACID) BY RECOMBINANT ESCHERICHIA-COLI

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Plasmids 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.
Publisher
ELSEVIER SCIENCE BV
Issue Date
1994-02
Language
English
Article Type
Article
Citation

JOURNAL OF BIOTECHNOLOGY, v.32, no.2, pp.203 - 211

ISSN
0168-1656
URI
http://hdl.handle.net/10203/20042
Appears in Collection
CBE-Journal Papers(저널논문)
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