Reagents for Generation and Analysis of Bioplastic Producing Plants

Polyhydroxyalkanoates (PHAs) are a class of biodegradable polyoxoesters synthesized from acetyl-CoA that naturally accumulate as intracellular granules in a diverse range of bacteria. Biosynthesis of the simplest PHA (PHB, poly-3-hydroxybutyrate) can be accomplished though the action of three enzymes, beta-ketothiolase, acetoacetyl-CoA reductase and PHA polymerase. We constructed plasmids that contain genetic elements for the production of PHB, or the closely related copolymer PHBV (poly-3-hydroxybutyrate-co-3-hydroxyvalerate), in oil palm, which produces abundant levels of acetyl-CoA. These bacterial PHA biosynthetic genes have been engineered to include plant plastid targeting signals, in order to direct biosynthesis of the polymer inside the plastids. In addition, we generated antibodies for the detection of PHA biosynthetic enzymes. The plasmids and antibodies reported in this work should be suitable as tools and reagents for the construction and analysis of PHA-producing oil palm. REAGENTS FOR GENERATION AND ANALYSIS OF BIOPLASTIC PRODUCING PLANTS 57 hydroxyvalerate. The physical properties of PHBV depend on the precise ratio of HB to HV. PHBV is useful for the production of biodegradable plastics, due to its relatively low melting temperature and relatively high flexibility and toughness. The biosynthesis of PHB from acetyl-CoA can be accomplished in the model bacterium Ralstonia eutropha by the action of three enzymes: betaketothiolase (encoded by phaA), acetoacetyl-CoA reductase (encoded by phaB) and PHA polymerase (encoded by phaC) (Peoples and Sinskey, 1989a, b). The pathway for PHB synthesis is diagrammed in Figure 2a. Biosynthesis of PHBV requires the use of an additional gene, bktB, which encodes a thiolase that can act either on acetyl-CoA or propionyl-CoA (Slater et al., 1998) and that can substitute for phaA in PHB biosynthesis. The pathway for PHBV synthesis is diagrammed in Figure 2b. Production of PHA polymer in plants is a goal of the bioplastics industry, since it promises significant cost savings over bacterial fermentation (Snell and Peoples, 2002; Bohlmann, 2006). PHA biosynthesis can be accomplished by the introduction of the bacterial open reading frames with the appropriate plant transcription and translation signals (Snell and Peoples, 2002; Suriyamongkol et al., 2007). Poirier et al. (1992) were the first to report the production of PHB in the cytoplasm and vacuoles of transgenic Arabidopsis; the growth and seed production of these transgenic plants was greatly reduced (Poirier et al., 1992). The group later redirected the heterologous proteins into the plastids; PHB production in shoots was increased to 14% dry weight and furthermore, no obvious deleterious effect was observed (Nawrath et al., 1994). The PHB yield was further increased to 40% dry weight in transgenic Arabidopsis transformed with a single gene construct carrying phaA, phaB, and phaC (Bohmert et al., 2000). However, the plant growth was inversely proportional to the PHB yield and stunted growth and loss of fertility were observed in the high-producing lines. PHA production in transgenic plants has now been reported for several species, including Arabidopsis, Brassica, tobacco, cotton, corn, potato, alfalfa, sugarbeet, flax, sugarcane and switchgrass (Snell and Peoples, 2002). Transgenic rape expressing PHB genes in the seed leucoplast were shown to accumulate PHB up to 8% of the fresh seed weight (Houmiel et al., 1999), the results suggesting that accumulation of PHB in an oilseed system may be better tolerated. Oil palm has the highest productivity of any oil crop, and produces abundant levels of acetyl-CoA, a key precursor in the biosynthesis of both fatty acids O OH R O