Cloning of new Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate synthase genes and their use in the development of recombinant microorganisms

Abstract

Pseudomonas sp. MBEL 6-19 was isolated from activated sludge and produced a polyhydroxyalkanoate (PHA) consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA) of $C_6$, $C_8$, $C_{10}$ and $C_{12}$, when sucrose, fructose, gluconate, oleic acid, lauric acid, decanoic acid, octanoic acid and hexanoic acid were fed as carbon source. The PHA produced was blend of homopolymer and copolymer, which could be fractionated with boiling acetone. The acetone-insoluble fraction of the PHA was a homopolymer of 3HB units [P(3HB)], while the acetone-soluble fraction was a copolymer P(3HB-co-3HA) containing both short- and medium-chain-length 3-hydroxyalkanoate (3HB and 3HA) units ranging from $C_4$ to $C_{12}$. The PHA copolymer was shown to be a random copolymer of 3HB and medium-chain-length 3HA units by analysis of the $^{13}C-NMR$ spectrum. PHA biosynthesis genes of Pseudomonas sp. MBEL 6-19 were cloned and sequenced. In the pha locus, a medium-chain-length pha synthase exists alone and two genes encoding PHA synthases ($phaC1_{P_S 619}$ and $phaC2_{P_S 619}$) were flanked by a PHA depolymerase gene ($phaZ_{P_S 619}$).