Analysis of beta-ketothiolase and acetoacetyl-CoA reductase genes of a methylotrophic bacterium, Paracoccus denitrificans, and their expression in Escherichia coli.

ABSTRACT

The beta-ketothiolase gene (phaA) and acetoacetyl-CoA reductase gene (phaB) were isolated from Paracoccus denitrificans. Nucleotide sequence analysis showed that they encoded proteins of 391 amino acids with a molecular mass of 40,744 Da and of 242 amino acids with a molecular mass of 25,614 Da, respectively. The predicted gene products exhibited high amino acid identities with those from other bacteria 64.4-74.0% for the phaA gene product and 47.6-80.6% for the phaB gene product, respectively. Both genes were co-transcribed in a recombinant Escherichia coli. In addition, promoter activity was detected upstream of the phaA gene. Hence, the two genes are organized as an operon, phaA-phaB, in P. denitrificans. NADH was preferred to NADPH as a cofactor of acetoacetyl-CoA reductase.