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Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-  co-3-hydroxyvalerate) copolymer production


Poly(hydroxyalkanoates) are natural polymers with thermoplastic properties. One polymer of this class with commercial applicability, poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) can be produced by bacterial fermentation, but the process is not economically competitive with polymer production from petrochemicals. Poly(hydroxyalkanoate) production in green plants promises much lower costs, but producing copolymer with the appropriate monomer composition is problematic. In this study, we have engineered Arabidopsis and Brassica to produce PHBV in leaves and seeds, respectively, by redirecting the metabolic flow of intermediates from fatty acid and amino acid biosynthesis. We present a pathway for the biosynthesis of PHBV in plant plastids, and also report copolymer production, metabolic intermediate analyses, and pathway dynamics.

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Figure 1: A pathway designed to produce PHBV in the plastids of plants.
Figure 2: Concentrations of selected 2-keto acids and amino acids in control plants and in Arabidopsis expressing threonine deaminase.
Figure 3: 13C Nuclear magnetic resonance spectra demonstrating PHBV copolymer production in transgenic Arabidopsis.
Figure 4

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We gratefully acknowledge the excellent technical assistance of Debra Broyles, Laura Casagrande, Kathleen Gonzalez, Catharine Gunter, Brad La Vallee, Susan Lowry, Debbie Mahadeo, Robert Stock, and Gregory Thorne.

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Correspondence to Kenneth J. Gruys.

Supplementary information

Alternatives for Generation of Propionyl-CoA in planta

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be produced in plants by using the Pyruvate Dehydrogenase Complex (PDC) for converting 2-ketobutyrate to propionyl-CoA. However, under in vivo conditions in transformed plants expressing the ilvA gene, PDC performs this reaction inefficiently, and production of copolymer to high internal concentrations in planta will likely require a supplementary route for conversion of 2-ketobutyrate to propionyl-CoA. There are ways to bypass PDC or supplement its activity, but all require additional transgenes. These routes include modifying the PDC to more readily accept 2-ketobutyrate1, 2, expression of an alternative enzyme system capable of forming propionyl-CoA from 2-ketobutyrate3, or co-expression of a 2-ketoacid oxidase with a propionyl-CoA synthetase or acyl-CoA transferase1, 4.

Propionyl-CoA can also be generated by other routes, although none presents a straightforward alternative to the threonine-derived pathway (See Figure 1). For instance, propionyl-CoA may be generated from acetyl-CoA using a 5-step pathway, part of which is involved in propionyl-CoA degradation in plants5-9. Conversion of acrylyl-CoA to propionyl-CoA is potentially problematic, but an appropriate enzyme may be available from Chroroflexus aurantiacus5. Propionyl-CoA can also be derived from succinyl-CoA using a pathway present in both Rhodococcus ruber and Nocardia corallina10, 11. This pathway is initiated by methylmalonyl-CoA mutase, an enzyme that requires vitamin B12 as a cofactor. However, vitamin B12 is not synthesized in plants9. Rhodococcus and Nocardia also produce minor amounts of 3-hydroxyvaleryl-CoA via a different, uncharacterized route10, 11 that may link to enzymes for degradation of valine and isoleucine. These pathways might also be engineered in plants, but a large number of genes are required.

Figure 1. Potential routes to produce propionyl-CoA in Plants. (GIF 35.5 KB)

Many alternative pathways have the potential to produce propionyl-CoA in plants. However, production of propionyl-CoA from threonine provides the most direct route.

Several other amino acids can be used to produce propionyl-CoA. Methionine, like threonine, generates 2-ketobutyrate during catabolism. This conversion can be performed by a multi-enzyme pathway that also produces cysteine12, or in a single step by L-methionine ?-lyase in a reaction that also produces ammonia and methanethiol13. In either case, supplementation of PDC activity would still be required to efficiently produce propionyl-CoA. Another pathway, present in Clostridium propionicum, converts alanine to propionyl-CoA via lactic acid, lactyl-CoA and acrylyl-CoA14, 15. However, none of the required genes have been cloned, and some of the necessary enzymes are oxygen sensitive16, 17. ß-alanine is another potential starting metabolite for the production of propionyl-CoA18-20. ß-alanine normally plays a critical role as a precursor to Coenzyme-A and acyl carrier protein. However, little is known about the concentration and compartmentalization of ß-alanine in plants, and propionyl-CoA may actually be required for its synthesis.

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Slater, S., Mitsky, T., Houmiel, K. et al. Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-  co-3-hydroxyvalerate) copolymer production. Nat Biotechnol 17, 1011–1016 (1999).

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