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Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(beta-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760-12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a beta-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(beta-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation.

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase.

This paper describes the biosynthesis and properties of bacterial poly(hydroxyalkanoate)s (PHA) with predominantly hydroxyl end groups. Hydroxy termination is achieved by the addition of low molecular weight diols to the culture. It is found that low molecular weight diols of various structures can be easily incorporated as chain ends, when used during fermentation, by a variety of microorganisms. Incorporation of a chiral diol does not appear to be stereospecific; both (R)- and (S)-1,2-propanediols were incorporated into the polymer. Moreover, both primary and secondary hydroxyl groups of 1,2-propanediol were found to have reacted. It was found that an increase in the hydroxy termination in P(3HB) leads to an increase in the thermal stability, most likely by prolonging the condensation reaction and delaying the degradation reaction.

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