Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in .

KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate--3-hydroxyvalerate) copolymer. Genomic DNA of was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous probe from was identified. In vivo expression in KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus . In vivo expression of the putative PHA synthase gene () in a recombinant strain was investigated by using glucose and decanoate as substrates. (, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an mutant () grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaC, suggesting that the putative PHA synthase of utilizes mainly short-chain-length PHA precursors as a substrate.