Synthesis of medium-chain-length polyhydroxyalkanoates inArabidopsis thalianausing intermediates of peroxisomal fatty acid β-oxidation

Abstract

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 ...

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs

“…The yields in the two transgenic lines analysed, were 0.017 mg and 0.26 mg of polymer/g of cell dry weight (Table 1). Transgenic A. thaliana expressing the mcl-PHA-polymerase in the peroxisomes accumulated similar amounts of mcl-PHAs in leaves (0.2 mg/g of cell dry weight) and 20 times more polymer (4 mg/g of cell dry weight) in germinating seedlings (Mittendorf et al 1998). The monomer composition of the polymer accumulated in the two transgenic potato lines analysed in the present report is shown in Table 1 and consisted of 3-(R)-hydroxyalkanoates with carbon chains ranging from C-6 to C-12.…”

“…The same was observed in mcl-PHAs accumulated in natural or recombinant prokaryotes (Table 1). In contrast, when b-oxidation intermediates were directed into mcl-PHA biosynthesis in natural hosts (De Waard et al 1993), recombinant yeast (Poirier et al 2001 or recombinant A. thaliana (Mittendorf et al 1998(Mittendorf et al , 1999Allenbach and Poirier 2000), the monomer composition also included unsaturated monomers.…”

“…Until recently, knowledge of mcl-PHA biosynthesis was incomplete, which hampered the identification of the minimum gene-set required for its production in recombinant hosts. It has been shown that the PHApolymerase alone was able to sustain mcl-PHA synthesis deriving precursors from b-oxidation in Escherichia coli (Langenbach et al 1997), Saccharomyces cerevisiae (Poirier et al 2001), Pichia pastoris and in the model plant Arabidopsis thaliana (Mittendorf et al 1998). However, b-oxidation in plants is predominantly active during a limited part of the plant life-cycle (i.e., seed germination and senescence).…”

Production of renewable polymers from crop plants

“…Various approaches were tested to produce mclPHAs in plants (Figure 4). Using the 3-hydroxyacyl-CoA intermediates generated in the peroxisome by the fatty acid b-oxidation pathway, a mclPHA consisting of C6-C16 monomers was produced to a level of 0.4% dry weight in A. thaliana seedlings (Mittendorf et al, 1998). These polymers consisted of more than 40-50 mol% C12 and longer monomers, and also contained a significant amount of unsaturated monomers.…”

Start a Research on Biopolymer Polyhydroxyalkanoate (PHA): A Review

“…Conversely, coupling GC to mass spectrometry detector (GC-MS) ensures more reliable detection, identity confirmation and quantification of PHA monomers, as well as enables tentative identification of novel PHA monomers in the absence of analytical standards [133]. Figure 4 shows representative GC-MS chromatograms of PHA monomers [37]. A recent study by Tan et al [132] showed that PHA monomers with carbon number between 4 and 16 have strong linear correlations with their respective retention times and response factors (R 2 ≥ 0.987) under GC-MS, which enabled the retention time and response factor of other PHA monomers to be predicted.…”