Abstract |
Caulobacter crescentus was investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Polyhydroxyalkanoate (PHA) accumulation contributing to approximately 18% of the cell dry weight was obtained in the presence of glucose. Gas chromatography-mass spectrometry and gel permeation chromatography of the purified PHA showed that this polyester was solely composed of 3-hydroxybutyrate and had a weight average molar mass of 5.5 x 10(5) g mol(-1) and a polydispersity of 1.6. An ORF encoding a conserved, hypothetical protein which shared approximately 47% identity with the PHB synthase from Azorhizobium caulinodans was identified within the complete C. crescentus genomic sequence. This putative C. crescentus PHB synthase gene, phaC, consisted of a 2019 nt stretch of DNA (encoding 673 aa residues), which encoded a PHB synthase with a molecular mass of approximately 73 kDa. This is currently the largest PHA synthase identified. The phaC coding region was subcloned into vector pBBR1-JO2 under lac promoter control. The resulting plasmid, pQQ4, mediated PHB accumulation in the mutant Ralstonia eutropha PHB(-)4 and recombinant Escherichia coli JM109(pBHR69), which produced the beta-ketothiolase and acetoacetyl-CoA reductase from R. eutropha, contributing to approximately 62% and 6% of cell dry weight, respectively. Functional expression of the coding region of phaC was confirmed by immunoblotting and in vitro PHB synthase activity.
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Authors | Q Qi, B H Rehm |
Journal | Microbiology (Reading, England)
(Microbiology (Reading))
Vol. 147
Issue Pt 12
Pg. 3353-8
(Dec 2001)
ISSN: 1350-0872 [Print] England |
PMID | 11739767
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Hydroxybutyrates
- Polyesters
- Recombinant Proteins
- poly-beta-hydroxybutyrate
- Acyltransferases
- poly-beta-hydroxybutyrate polymerase
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Topics |
- Acyltransferases
(genetics, metabolism)
- Caulobacter crescentus
(enzymology, genetics)
- Cupriavidus necator
(genetics, metabolism)
- Escherichia coli
(genetics, metabolism)
- Gene Expression
- Genes, Bacterial
- Hydroxybutyrates
(metabolism)
- Molecular Sequence Data
- Polyesters
(metabolism)
- Recombinant Proteins
(metabolism)
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