Polyhydroxybutyrate biosynthesis in Caulobacter crescentus: molecular characterization of the polyhydroxybutyrate synthase.

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.

• 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)

Chemical References

• Hydroxybutyrates
• Polyesters
• Recombinant Proteins
• poly-beta-hydroxybutyrate
• Acyltransferases
• poly-beta-hydroxybutyrate polymerase

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)