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

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.