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

Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany¹

Author for correspondence: Bernd H. A. Rehm. Tel: +49 251 833 9848. Fax: +49 251 833 8388. e-mail: rehm{at}uni-muenster.de

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 5·5x10⁵ 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 ß-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.

Keywords: polyhydroxyalkanoate, PHA synthase, PHA depolymerase

Abbreviations: CDW, cell dry weight; GC–MS, gas chromatography–mass spectrometry; GPC, gel permeation chromatography; PHA, polyhydroxyalkanoate; PHB, polyhydroxybutyrate

The GenBank accession numbers for the phaC and phaZ sequences reported in this paper are AY007313 and AF311864, respectively.

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