Production of a Biodegradable Plastic Polymer, Poly- -Hydroxybutyrate, in Transgenic Alfalfa

ketothiolase, acetoacetyl-CoA reductase, and PHB synthase. These enzymes are encoded by phbA, phbB, Alfalfa (Medicago sativa L.) is primarily used as a source of forage and phbC, respectively (Slater et al., 1988). The monoand animal feed. Improving the economic value of alfalfa could be achieved by developing new uses of this perennial crop. To investigate meric composition of PHA polymers is determined by the potential of alfalfa as a source of industrial materials, we employed the substrate specificities of -ketothiolase and PHB a genetic transformation approach to produce a biodegradable plastic, synthase and by the availability of acetyl-CoA and propoly-hydroxybutyrate (PHB), in the leaves of alfalfa plants. Three pionyl-CoA. In the case of 4and 5-carbon PHA precurgenes from Ralstonia eutropha (formerly Alcaligenes eutrophus ) sors, when -ketothiolase is encoded by phbA, a homoencoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and polymer of PHB is produced. When -ketothiolase is a copolymer of PHB and polyhydroxyvalerate (polyhydroxybutyrateencoded by bktB, a copolymer of PHB/V is produced co-hydroxyvalerate, or PHB/V) (bktB, phbB, phbC) engineered for (Slater et al., 1998). Because the PHB/V copolymer has plastid targeting were introduced into alfalfa by Agrobacterium-medibetter physical properties than PHB, it is preferred for ated transformation. DNA and RNA blot analyses of transgenic plants most commercial applications (Anderson and Dawes, indicated integration and expression of the PHB biosynthetic pathway genes. Polyhydroxybutyrate content in the leaves of transgenic plants 1990). Presently, the bacterially produced polymers are ranged from ≈0.025 to 1.8 g kg 1 dry weight (DW). Agglomerations not cost-competitive with nonbiodegradable plastic of PHB granules 0.2 to 0.4 m in diameter, similar to bacterial PHB, polymers derived from petroleum. By genetically engiwere located in the chloroplasts of transgenic plants, demonstrating neering crop plants to produce PHA, a less expensive that phb gene products were targeted into the plastids of transgenic source of these polymers could become available. alfalfa. Transgenic plants exhibited growth similar to untransformed PHB production in plants was first demonstrated in plants, suggesting that expression of PHB biosynthetic pathway genes transgenic Arabidopsis thaliana (L.) Heynh. (Poirier et at current levels and accumulation of product in the plastids had no al., 1992). Poly-hydroxybutyrate is tolerated by plants deleterious effect on growth and fertility. F1 hybrid progeny, obtained if it is synthesized in chloroplasts and PHB levels ranging from crosses of PHB transgenic plants with elite alfalfa germplasm, between 0.1 and 14% DW have been reported with exhibited leaf PHB levels similar to the transgenic parental line, demonstrating that PHB production in alfalfa is a stable and dominantly minimal effects on plant growth (Nawrath et al., 1994). inherited trait. Although various approaches for producing PHB in plants have been reported, most of these utilize Arabidopsis. Recently, production of PHB/V was achieved in the seed of oilseed rape (Brassica napus L.), demonstraA is one of the most important forage crops ting that it is possible to produce PHA in crop plants because of its high biomass production, good nutri(Slater et al., 1999). tional qualities, and adaptation to a wide range of enviAt present, the synthesis of PHB as a single endronments. Genetic engineering has the potential to improduct in crops is economically challenging. Polyprove the economic value of alfalfa by introducing genes hydroxybutyrate levels of 15% DW are needed for practo improve its adaptability and agronomic characteristical large-scale commercial production (Valentin et al., tics, and to increase its utilization in nontraditional areas 1999). Recently, Arabidopsis plants with PHB levels of such as in phytoremediation and production of indus40% DW were obtained, but the plants were stunted trial raw materials. One attractive approach is to engiand infertile (Bohmert et al., 2000). However, plants neer alfalfa to produce biodegradable plastics as a valueproducing PHA at levels compatible with growth could added component. be used for multiple purposes to increase overall crop Many species of bacteria accumulate polyhydroxyalvalue. Production of bioplastics in alfalfa leaves could kanoates (PHA) as energy storage compounds in rebe combined with use of alfalfa for feed and energy. sponse to nutritional stress (Anderson and Dawes, In such a system, alfalfa leaves could be harvested to 1990). Some PHA polymers are commercially valuable produce PHB, leaf by-product could be processed into as biodegradable plastics. Polyhydroxybutyrate is a a feed, and stems could be used for producing either PHA produced in R. eutropha (Yabuuchi et al., 1995) electricity by gasification or ethanol by fermentation. via a three-enzyme biosynthetic pathway consisting of As a start towards this goal, the objectives of this study P. Saruul, Dep. of Plant Pathology, 495 Borlaug Hall; F. Srienc, Biologwere to genetically modify alfalfa to produce PHB and ical Process Technol. Inst., 240 Gortner Laboratories; D.A. Somers, Dept. of Agronomy and Plant Genetics, 411 Borlaug Hall; D.A. Samac, Abbreviations: DW, dry weight; GC, gas chromatography; GC-MS, USDA-ARS-Plant Science Research Unit and Dep. of Plant Pathology, gas chromatography–mass-spectrometry; 3-HB, 3-hydroxybutyrate; 495 Borlaug Hall, Univ. of Minnesota, St. Paul, MN 55108-6030. Funding 1H-NMR, proton-nuclear magnetic resonance spectrometry; 3-HV, for this project was provided by the Consortium for Plant Biotechnology 3-hydroxyvalerate; NPTII, neomycin phosphotransferase II; PCR, Research no. CR22072-66 and Monsanto Company. Received 12 June polymerase chain reaction; PHA, polyhydroxyalkanoates; PHB, poly2001. *Corresponding author ([email protected]). -hydroxybutyrate; PHB/V, polyhydroxybutyrate-co-hydroxyvalerate; RT-PCR, reverse transcriptase-polymerase chain reaction. Published in Crop Sci. 42:919–927 (2002).