Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids

Burkholderia sp. accumulates polyhydroxyalkanoates (PHAs) containing 3-hydr oxybutyrate and 3-hydroxy-4-pentenoic acid when grown on mineral media unde r limited phosphate or nitrogen, and using sucrose or gluconate as a carbon and energy source. Solvent fractionation and NMR spectroscopic characteriz ation of these polyesters revealed the simultaneous accumulation of two hom opolyesters rather than a co-polyester with random sequence distribution of the monomers [Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbuchel A, Tran M, Asrar J (1999) Macromolecules 32: 7389-7395]. To understand the genetic requirements for such unusual polyester accumulation, we probed to tal genomic DNA from Burkholderia sp. by Southern hybridization experiments using phaC-specific probes. These experiments indicated the presence of mo re than one PHA synthase gene within the genome of Burkholderia sp. However , when total genomic DNA from Burkholderia sp, was used to complement a PHA -negative mutant of Ralstonia eutropha for PHA accumulation, only one PHA s ynthase gene was obtained resembling the R. eutropha type of PHA synthases, based on amino acid sequence similarity. In addition to the PHA synthase g ene, based on high sequence homology, genes encoding a beta-ketothiolase an d acetoacetyl-CoA reductase were identified in a gene cluster with the PHA synthase gene. The arrangement of the three genes is quite similar to the R . eutropha poly-beta-hydroxybutyrate biosynthesis operon.