Polyhydroxyalkanoates

A wide variety of bacteria produce polyhydroxyalkanoates (PHAs) as a carbon reserve and electron sink (for a review, see van Beilen and Poirier 2008). These PHAs consist of 3-hydroxy fatty acids with a chain length of 4-16 carbons and have wide-ranging potential for applications such as the formation of plastic bags, fibres and films. Besides their CO2 neutral production, PHA products can be decomposed, which is desirable for the environmental friendly dispersal of disposable items as well as for some medical products which otherwise have to be removed from the body. Poly-3-hydroxybutyrate (PHB) is the most widespread and best characterized PHA found in bacteria like Ralstonia eutropha (for a review, see van Beilen and Poirier 2008). In contrast to cyanophycin synthesis, three enzymes are necessary for PHB synthesis. The first enzyme, p-ketothiolase, catalyses the reversible condensation of two acetyl-CoA moieties to form acetoacetyl-CoA. The acetoacetyl-CoA reductase in turn reduces acetoacetyl-CoA to R-(-)-3-hydroxybutyryl-CoA, which is subsequently polymerized through PHA synthase to form PHB. As an alternative to petrochemicals, PHA production was established in plants, first in Arabidopsis thaliana by the expression of the PHB synthase in the cytoplasm leading to a maximum of 0.1% PHB present in the cytoplasm, nucleus or vacuoles (van Beilen and Poirier 2008). However, the plants showed strong growth retardation and reduced seed production. PHB synthesis in the cytoplasm of tobacco (0.01%), cotton (0.3%) and oilseed rape (0.1%; John and Keller 1996; Nakashita et al. 1999; Poirier et al. 1992) showed similar plant damage. The deleterious effects of PHB production in the cytoplasm of plants might be caused by the diversion of acetyl-CoA and acetoacetyl-CoA away from the endogenous flavonoid and iso-prenoid pathways, which are responsible for the synthesis of a range of plant hormones and sterols (van Beilen and Poirier 2008). Due to their high metabolic flow of acetyl-CoA, chloroplasts might provide a more suitable production platform, although p-ketothiolase is not present. Therefore the required enzymes -including p-ketothiolase - were targeted to plastids, using signal sequences for plastid import. The highest PHB accumulation was observed in Arabidopsis, with a maximum of 14% of dry weight in leaves without significant effects on plant growth but visible leaf chlorosis (Nawrath et al. 1994). In seeds of oil rape up to 8% dry weight PHB accumulation was detected in leucoplasts after the transfer of all three genes (Houmiel et al. 1999), a strategy leading to even 30-40% of dry weight in leaves of A. thaliana. Nevertheless, in contrast to the intact canola seeds, these plants were heavily reduced in growth and did not produce any seeds. Slightly reduced amounts were detected in corn leaves (6% dry weight), sugar cane leaves (2% dry weight) and sugar beet hairy roots (5% dry weight), whereas expression of the PHB pathway in plastids of alfalfa and tobacco led to only low amounts (<0.5% dry weight; Arai et al. 2001; Saruul et al. 2002. Since nucleus-encoded proteins are expressed to a lesser extent than those encoded by plastidic genes, it was supposed that the direct expression of the PHB pathway in the plastid genome might increase the PHB yield without increasing the deleterious effects. Nevertheless, in tobacco this strategy only leads to relatively low amounts up to 1.7% dry weight, accompanied by reduced growth and male sterility (Arai et al. 2001; Bohmert et al. 2002; Lossl et al. 2003).

In order to improve the physical properties of PHB, extensive efforts have made to synthesise co-polymers with better properties like poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] and medium-chain-length PHA (mclPHA). P(HB-co-HV) is produced by the inclusion of 3-hydroxyvalerate in PHB, which is less stiff and tougher than PHB and also easier to process, making it to a good target for commercial application (Noda et al. 2005). Coexpression of a threonine deaminase from E. coli along with the three PHB biosynthetic proteins in plastids led to P(HB-co-HV) accumulation up to 2.3% dry weight in seeds of oil rape and 1.6% dry weight in A. thaliana (Slater et al. 1999; Valentin et al. 1999). However there is a constriction providing 3-hydroxyvaleryl-CoA to the PHA synthase, which is caused by the inefficiency of the pyruvate dehydrogenase complex in converting 2-ketobutyrate to propionyl CoA (for a review, see van Beilen and Poirier 2008).

MclPHA are described as elastomers and their physical properties depend on the monomer composition (for a review, see van Beilen and Poirier 2008) In A. thaliana mclPHA monomers were produced up to 0.4% dry weight in seedlings and consisted of 40-50 mol% of C12 and longer monomers. The production of mclPHAs with longer-chain monomers by using the conversion of the fatty acid biosynthetic intermediate 3-hydroxyacyl-ACP into 3-hydroxyacyl-CoA led to only low amounts (below 0.03% dry weight) of mclPHA in plastids of potato leaves (Romano et al. 2005).

The most useful PHA would be a polymer containing primarily 3-hydroxybuty-rate with a fraction of longer-chain monomers of C6 and higher. In terms of PHA quantity, plastids are the best location for PHA synthesis. However, the synthesis, regulation of precursors (like acetyl-CoA, propionyl-CoA or 3-hydroxyacyl-ACP) and the efficacy to channel them towards PHA without deleterious effects on plant growth needs more investigation (for a review, see van Beilen and Poirier 2008).

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