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J Bacteriol, April 1998, p. 1979-1987, Vol. 180, No. 8
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Multiple beta -Ketothiolases Mediate Poly(beta -Hydroxyalkanoate) Copolymer Synthesis in Ralstonia eutropha

Steven Slater,* Kathryn L. Houmiel, Minhtien Tran, Timothy A. Mitsky, Nancy B. Taylor, Stephen R. Padgette, and Kenneth J. Gruys

Sustainable Development and Agricultural Sectors, Monsanto Company, St. Louis, Missouri 63198

Received 22 October 1997/Accepted 16 February 1998

Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism's intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-beta -hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(beta -hydroxybutyrate-co-beta -hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form beta -ketovaleryl-CoA. This activity has generally been attributed to the beta -ketothiolase encoded by R. eutropha phbA. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of bktB, which encodes a beta -ketothiolase from R. eutropha that is capable of forming beta -ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of beta -hydroxyvalerate derived from propionyl-CoA. We also report an additional beta -ketothiolase, designated BktC, that probably serves as a secondary route toward beta -hydroxyvalerate production.

* Corresponding author. Mailing address: Monsanto Company, 700 Chesterfield Parkway North, Mail Zone AA3I, St. Louis, MO 63198. Phone: (314) 737-7266. Fax: (314) 737-6759. E-mail: steven.c.slater{at}monsanto.com.




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