Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described. These plants accumulated more than 4% of their fresh weight (approximately 40% of their dry weight) in the form of PHB in leaf chloroplasts. These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and sugar alcohols.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCLPHAs) are synthesized in bacteria by using intermediates of the ␤-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the ␤-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids (1-3). Polyhydroxybutyrate (PHB) is the most well-characterized PHA. High molecular weight PHB is found as intracellular inclusions in a wide variety of bacteria (2). In Alcaligenes eutrophus, PHB accumulates up to 80% of the dry weight (dwt), with inclusions being typically 0.2-1 m in diameter. Small quantities of PHB oligomers of approximately 150 monomer units also are found associated with membranes of bacteria and eukaryotes, where they form channels permeable to calcium (4). High molecular weight PHAs have the properties of thermoplastics and elastomers. Numerous bacteria and fungi can hydrolyze PHAs to monomers and oligomers, which are metabolized as a carbon source. PHAs thus have attracted attention as a potential source of renewable and biodegradable plastics and elastomers. PHB is a highly crystalline polymer with rather poor physical properties, being relatively stiff and brittle (5). In contrast, PHA copolymers containing monomer units ranging from three to five carbons for short-chain-length PHA or 6-14 carbons for medium-chain-length PHA (MCL-PHA) are less crystalline and more flexible polymers (5).PHB has been produced in Arabidopsis thaliana and cotton expressing the A. eutrophus PHB biosynthetic enzymes (6-8).In Arabidopsis expressing the PHB pathway in the plastids, leaves accumulated up to 14% PHB per g of dwt (7). High-level synthesis of PHB in plants opened the possibility of using agricultural crops as suitable systems for the production of PHAs on ...

SummaryTransgenic plants producing peroxisomal polyhydroxyalkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon¯ow through the b-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the b-oxidation of these fatty acids. Tween±laurate was a stronger inducer of b-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween±oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced¯ow of capric acid towards b-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esteri®ed unusual fatty acids, resulting in changes in the activity of the b-oxidation cycle as well as removal and degradation of these unusual fatty acids through b-oxidation. Such enhanced ow of fatty acids through b-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon¯ow through boxidation as well as to analyse the degradation pathway of unusual fatty acids.