Polyhydroxyalkanoates (PHAs) are biodegradable polyesters synthesized by a wide variety of microorganisms. Newly isolated Ralstonia eutropha strain A-0.4 screened from soil in Thailand could produce high content of PHAs. Thus, the PHA biosynthesis genes of this strain were cloned, characterized and expressed in Escherichia coli for achieving higher productivity. The PHA biosynthesis genes were analyzed by PCR and gene walking techniques to obtain the intact genes. Three major open reading frames (ORFs) were obtained and found to be 1,770, 1,182 and 741 bp, respectively. Sequence analysis showed that they were closely related to the PHA synthase (phaC), beta-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB) genes of R. eutropha H16. The recombinant E. coli strain harboring phaCAB operons of R. eutropha strain A-04 was constructed and expressed under the control of the arabinose-inducible araBAD promoter (pBAD/TOPO[superscript ®] ThioFusion[superscript TM] vector). In flask cultivation, the PHB production of recombinant E. coli was investigated by varying types of media, concentrations of inducer (L-arabinose) and initial inoculum sizes. The obtained results showed that the cultivation using a low inoculum size and LB medium containing 50 µg/ml carbinicillin supplemented with 1% L-arabinose gave 6.11 g/l of dry cell weight and 5.7 g/l of PHB amount at 24 h. It is indicated that the expression of these genes in E. coli results in a high level of 93.3% (w/w) of PHB content at 24 h of cultivation comparing with the wild type R. eutropha strain A-04 accumulated 78% (w/w) of PHB content at 60 h. Thus, the ability of the recombinant E. coli to express the phaCAB operons leading to the production of PHB with superior characteristic in rapid growth and PHB production
