Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)
Synthesis of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) in the Cytosol
As described in a previous section, expression of the R. eutropha acetoacetyl-CoA reductase and PHB synthase in the cytosol of A. thaliana plants led to the accumulation of only 0.1% of the homopolymer PHB (Poirier et al., 1992a). However, expression of the same reductase along with the PHA synthase from A. caviae led to the accumulation of a similar amount of a PHA copolymer containing mostly 3-hydroxybutyrate with 0.2–0.8 mol% of 3-hydroxyvalerate (Matsumoto et al., 2005). The PHA synthase of A. caviae has been previously shown to have unique substrate specificity, being capable of producing a PHA copolymer composed of monomers ranging from 4 to 6 carbons (Fukui and Doi, 1997). Although several potential pathways could provide either the propionyl-CoA or 3-hydroxyvaleryl- CoA thought to be required for the synthesis of P(HB-HV), including amino acid synthesis or degradation, as well as β-oxidation of odd-chain fatty acids (see below for further details), it is not known which of these pathways provides the substrate for copolymer synthesis in the cytosol. Interestingly, use of an in vitro mutated A. caviae PHA synthase having higher catalytic activity led to an approximate fivefold increase in PHA accumulation in the cytoplasm, indicating that the improvement of enzymatic properties though mutagenesis is a valuable approach to increase the amount of PHA produced in plants.Synthesis of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) in the Plastid
Because of the improved properties of P(HB-HV) copolymers over PHB, bacterial production of P(HB-HV), also known under the trade name BiopolTM, has been central to the marketing and commercial production of PHA. It was therefore natural that after the demonstration of high-level PHB synthesis in the plastids, efforts would be focused on the synthesis of PHA copolymers, such as P(HB-HV).Since synthesis of P(HB-HV) in bacteria relies on the production of propionyl- CoA, it was necessary to create an endogenous pool of propionyl-CoA in plants that could be used by the PHA pathway. Furthermore, since the plastid was shown to be the best subcellular compartment for the synthesis of PHB from acetyl-CoA, it was also chosen as the site for P(HB-HV) synthesis from acetyl- CoA and propionyl-CoA. Although several metabolic pathways exist in prokaryotes and eukaryotes that can generate propionyl-CoA, the simplest strategy adopted was the conversion of 2-ketobutyrate to propionyl-CoA by the pyruvate dehydrogenase complex (PDC), an enzyme naturally located in the plastid (Slater et al., 1999). Although PDC normally decarboxylates pyruvate to give acetyl-CoA, the same enzyme can also decarboxylate 2-ketobutyrate, albeit at low efficiency, to give propionyl-CoA. Since 2-ketobutyrate is also found in the plastid as an intermediate in the synthesis of isoleucine from threonine, both the substrate and the enzyme complex required for the generation of propionyl-CoA are present in this organelle. However, since PDC would have to compete for the 2-ketobutyrate with the acetolactate synthase, an enzyme involved in isoleucine biosynthesis, the quantity of 2-ketobutyrate present in the plastid was enhanced through the expression of the E. coli ilvA gene,which encodes a threonine deaminase (Slater et al., 1999).
Expression of the P(HB-HV) pathway in the leucoplast of B. napus seeds has also been achieved by putting the bacterial genes under the control of the seedspecific promoter from the Lesquerella hydroxylase gene. In these experiments, an isoleucine-insensitive mutant of the ilvA gene was coexpressed along with the bktB, phaA, and phaC genes, and all four genes were inserted in a single multigene vector. P(HB-HV) synthesis in the range of 0.7–2.3% dwt was reported with an HV content of 2.3–6.4 mol% (Slater et al., 1999). Interestingly, there was an inverse relationship between the amount of PHA and the proportion of the HV monomer, indicating a ‘‘bottleneck’’ in providing 3-hydroxyvaleryl-CoA to the PHA synthase. This bottleneck is thought to be caused by the inefficiency of the PDC in converting 2-ketobutyrate to propionyl-CoA.