PHA Production in Plant Peroxisomes

Abstract

Polyhydroxyalkanoates (PHAs) are a group of bacterial carbon storage polymers which possess diverse plastic-like properties similar to polypropylene and polyethylene. However, unlike their petroleum-derived counterparts, these biorenewable polyesters are fully biodegradable. A wide variety of bacteria synthesise PHAs, with unique monomer units that differ from one another in the R side group attached to the hydroxyalkanoate core. In the Gram-negative bacterium Ralstonia eutropha, polyhydroxybutyrate (PHB), the simplest form of PHA, is produced by a three enzyme pathway. This pathway is driven by the successive activities of three enzymes, a β-ketothiolase, acetoacetyl-CoA reductase and PHA synthase, which are commonly referred to as PhaARe, PhaBRe and PhaCRe. PhaARe condenses two molecules of acetyl-CoA to give acetoacetyl-CoA which is subsequently reduced to hydroxybutyryl-CoA through the action of PhaBRe using one molecule of NADPH. Finally, this hydroxybutyryl-CoA monomer is polymerised to form PHB by PhaCRe. Through expression of such bacterial transgenes, PHB has previously been produced in a number of different plant species. Current research is focused on improving PHA production, including PHB, in the high biomass crop sugarcane (Saccharum sp interspecific hybrids) because this could lead to cost-competitive commercial production of this class of bioplastics. This thesis work is part of a wider research project in plant metabolic engineering where plant cell organelles are being studied to find optimal ways to produce high levels of PHAs and other bioplastic precursors. In this respect, one possibility is to focus on PHA production in plant peroxisomes. In plants, peroxisomes are known to be involved in the catabolism of straight chain fatty acids through the β-oxidation cycle. The peroxisomal fatty acid β-oxidation cycle generates a wealth of substrates for PHA biosynthesis. To further evaluate PHA production in plant peroxisomes, the three enzyme R. eutropha PHA biosynthetic pathway was engineered into peroxisomes in transgenic sugarcane plants. Initially, transient expression analysis of green fluorescent protein (GFP) fusion proteins was used to determine minimal peroxisomal targeting signal type 1 sequences sufficient to target each PHA biosynthetic enzyme. Peroxisomal-targeted PhaARe, PhaBRe and PhaCRe were then constitutively expressed in sugarcane using the maize polyubiquitin promoter. In the resulting transgenic lines, PHB was produced up to 1.6% dry weight in mature leaf tissue, which is on par with previous results when the same R. eutropha PHA pathway under the control of the same promoter was targeted to plastids. PHB biosynthesis at this level was not accompanied by any obvious growth effects. Polymer accumulated throughout most leaf cell types and subcellular localisation studies revealed polymer accumulating in both peroxisomes and vacuoles. Further characterisation of the PHA revealed peroxisomal biosynthesis of both 3-hydroxybutyrate (3HB) and 3-hydroxyvalyrate (3HV) monomers, forming P(HB-co-HV) copolymer. 3HB and 3HV accumulated at levels approximately 95% and 5% of total polymer, respectively. Transgenic Arabidopsis thaliana plants expressing peroxisomal-targeted PhaARe, PhaBRe and PhaCRe were also generated as a model system. Analysis of several independent A. thaliana peroxisomal PHA producing lines revealed PHA accumulation up to 1.8% dry weight in dark grown etiolated seedlings, between 0.1% and 0.16% dry weight in 10-day-old seedlings, and between 0.06% and 0.1% dry weight in 21 day old plants. P(3HB-co-3HV) copolymer was also found in these A. thaliana lines, with 3HB and 3HV monomers present in three-week-old plants in a similar ratio as measured in sugarcane. Peroxisomal PHA biosynthesis in A. thaliana had only a minor effect on plant growth as seen in etiolated hypocotyl elongation where tissue expansion was artificially dependent on peroxisomal fatty acid β-oxidation. Evidence in the literature suggests that substrate availability limits peroxisomal PHA biosynthesis in plants. Therefore several metabolic engineering strategies to overcome substrate limitation and increase peroxisomal PHA production were designed. These strategies focused on either reducing competition for peroxisomal PHA substrates by other metabolic pathways or boosting PHA substrate pools contained in the peroxisomes. Since acetyl-CoA exits peroxisomes via the formation of citrate, endogenous expression of A. thaliana peroxisomal citrate synthase genes were knocked down using artificial microRNA interference technology. A reduction in peroxisomal citrate synthase activity in conjunction with peroxisomal PHA biosynthesis increased total PHB accumulation. Higher level PHB accumulation was observed in A. thaliana peroxisomal PHA producing plants grown in liquid culture supplemented with Tween-20, a source of lauric acid (12:0).This demonstrated that peroxisomal PHA biosynthesis is limited in part by fatty acid flux through the β-oxidation cycle. These same A. thaliana plants were also fed an exogenous supply of cis-10-heptadecanoic acid which dramatically increased the proportion of total polymer comprised of HV monomer. This demonstrated that the biosynthesis of HV monomer is dependent on the presence of suitable substrate molecules, such as those arising from the β-oxidation of odd-chain-length fatty acids. A stable increase in fatty acid flux through the peroxisomal β-oxidation cycle of sugarcane and A. thaliana peroxisomal PHA producing plants was attempted with co-expression of the heterologous thioesterase FatB3 from Cuphea lanceolata. However, no increase in peroxisomal PHA biosynthesis was observed. Overall, this study reveals the potential of peroxisomal PHA biosynthesis in plants. A positive evaluation of select metabolic engineering strategies in A. thaliana warrants further application of similar strategies in sugarcane and other high biomass crops. The continuation of this work may result in peroxisomal PHA biosynthesis reaching commercially viable levels in crop plants, in either isolation or conjunction with PHA biosynthesis in other organelles of the same plant