Cloning, molecular analysis, and expression of the polyhydroxyalkanoic acid synthase (phaC) gene from Chromobacterium violaceum.

The polyhydroxyalkanoic acid synthase gene from Chromobacterium violaceum (phaCCv) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaCCv and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaACv). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes and Ralstonia eutropha (formerly Alcaligenes eutrophus), harboring phaCCv, accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C. violaceum PHA synthase. Neither recombinant E. coli nor recombinant Pseudomonas putida harboring phaCCv accumulated significant levels of PHA. Sequence analysis of the phaCCv product shows homology with several PHA synthases, most notably a 48% identity with that of Alcaligenes latus (GenBank accession no. AAD10274). Polyhydroxyalkanoic acids (PHAs) are carbon and energy reserve polymers produced in some bacteria when carbon sources are plentiful and another nutrient, such as nitrogen, phosphate, oxygen, or sulfur, becomes limiting. PHAs composed of monomeric units ranging from 3 to 14 carbons exist in nature. When the carbon source is exhausted, PHA is utilized by the bacterium (1, 29, 32). Some PHA polyesters have physical properties similar to those of polypropylene, making them a source of biodegradable plastic from renewable resources. Polymers of various compositions are produced, depending on the substrate specificity of the PHA synthase and the carbon source on which the bacterium is grown, as well as the metabolic pathways involved in the utilization of the carbon source. While homopolymers composed of 3-hydroxybutyric acid (3HB) are very brittle, mixtures possessing longer carbon backbones result in a more flexible polymer and, hence, a more marketable plastic. PHAs have potential applications in medicine and dentistry (1, 29), and a polymer composed of 3HB and 3-hydroxyvaleric acid (3HV) has been marketed under the trademark name BIO-POL (26). Another PHA with attractive physical properties is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), but at the present time there are only a few reports of bacteria that accumulate a copolymer composed entirely of 3HB and 3-hydroxyhexanoic acid (3HC) (4, 7, 14). PHA production is best understood in Ralstonia eutropha (formerly Alcaligenes eutrophus [16, 17, 35]; for reviews see references 1 and 29). Poly(3-hydroxybutyrate) is synthesized from acetyl-coenzyme A (CoA) in a three-step pathway. The first reaction involves a PHA-specific 3-ketothiolase, encoded by phaARe, that condenses two acetyl-CoA molecules into acetoacetyl-CoA. The second reaction, which is the reduction of acetoacetyl-CoA to D-(2)-3-hydroxybutyryl-CoA, is catalyzed