MULTIPLE BETA-KETOTHIOLASES MEDIATE POLY(BETA-HYDROXYALKANOATE) COPOLYMER SYNTHESIS IN RALSTONIA-EUTROPHA

Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental li mitation. The type of polymer produced depends on the carbon sources a vailable, the flexibility of the organism's intermediary metabolism, a nd the substrate specificity of the PHA biosynthetic enzymes, Ralstoni a eutropha produces both the homopolymer poly-beta-hydroxybutyrate (PH B) and, when provided with the appropriate substrate, the copolymer po ly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV). A required st ep in production of the hydroxyalerate moiety of PHBV is the condensat ion of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form beta-k etovaleryl-CoA. This activity has generally been attributed to the bet a-ketothiolase encoded by R. eutropha phbA. However, we have determine d that PhbA does not significantly contribute to catalyzing this conde nsation reaction. Here we report the cloning and genetic analysis of b ktB, which encodes a beta-ketothiolase from R. eutropha that is capabl e of forming beta-ketovaleryl-CoA. Genetic analyses determined that Bk tB is the primary condensation enzyme leading to production of beta-hy droxyvalerate derived from propionyl-CoA, We also report an additional beta-ketothiolase, designated BMC, that probably serves as a secondar y route toward beta-hydroxyvalerate production.