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    Biotechnological approaches for the production ofpolyhydroxyalkanoates in microorganisms and plants A review

    Pornpa Suriyamongkol a ,b , Randall Weselake b , Suresh Narine b,Maurice Moloney c , Saleh Shah a ,

    a Plant Biotechnology Unit, Alberta Research Council, Vegreville, Alberta, Canada T9C 1T4b Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2P5

    cDepartment of Biological Sciences, University of Calgary, Calgary, Canada T2N 1N4

    Received 14 August 2006; received in revised form 23 November 2006; accepted 23 November 2006Available online 30 November 2006

    Abstract

    The increasing effect of non-degradable plastic wastes is a growing concern. Polyhydroxyalkanoates (PHAs), macromolecule-polyesters naturally produced by many species of microorganisms, are being considered as a replacement for conventional plastics.Unlike petroleum-derived plastics that take several decades to degrade, PHAs can be completely bio-degraded within a year by avariety of microorganisms. This biodegradation results in carbon dioxide and water, which return to the environment. Attemptsbased on various methods have been undertaken for mass production of PHAs. Promising strategies involve genetic engineering ofmicroorganisms and plants to introduce production pathways. This challenge requires the expression of several genes along withoptimization of PHA synthesis in the host. Although excellent progress has been made in recombinant hosts, the barriers toobtaining high quantities of PHA at low cost still remain to be solved. The commercially viable production of PHA in crops,however, appears to be a realistic goal for the future. 2006 Elsevier Inc. All rights reserved.

    Keywords:Polyhydroxyalkanoates; PHA; Polyhydroxybutyrate; PHB; Bioplastics; Microorganisms; E. coli; Yeast; Transgenic plants

    Contents

    1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1492. Monomer composition and physical properties of PHAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

    Biotechnology Advances 25 (2007) 148175www.elsevier.com/locate/biotechadv

    Abbreviations:ACP, acyl carrier protein; CaMV35S, cauliflower mosaic virus 35S; CoA, coenzyme A; DGAT, diacylglycerol acyltransferase;dwt, dry weight; ER, endoplasmic reticulum;fad, fatty acid desaturase; HA, 3-hydroxyalkanoate; HB, 3-hydroxybutyrate; 4HB, 4-hydroxybutyrate;HD, 3-hydroxydecanoate; HH, 3-hydroxyhexanoate; HO, 3-hydroxyoctanoate; HV, 3-hydroxyvalerate; lcl-, long-chain-length; mcl-, medium-chain-length; PCR, polymerase chain reaction; PHA, polyhydroxyalkanoate; PhaA, -ketothiolase; PhaB, acetoacetyl-CoA reductase; PhaC, PHAsynthase; PHB, poly(3-hydroxybutyrate); P(HB-HH), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); P(HB-HV), poly(3-hydroxybutyrate-co-3-hydroxyvalerate); PDC, pyruvate dehydrogenase complex; scl-, short-chain-length. Corresponding author. Fax: +1 780 632 8612.

    E-mail address:[email protected](S. Shah).

    0734-9750/$ - see front matter 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.biotechadv.2006.11.007

    mailto:[email protected]://dx.doi.org/10.1016/j.biotechadv.2006.11.007http://dx.doi.org/10.1016/j.biotechadv.2006.11.007mailto:[email protected]
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    3. PHA synthesis in microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513.1. Genes and enzymes involved in PHA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513.2. PHA production in recombinantEscherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1523.3. Fatty acid-oxidation and PHA production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1543.4. Engineering of PHA synthases to enhance and change polymer synthesis . . . . . . . . . . . . . . . . . . . 1553.5. Production of PHA copolymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

    4. PHA production in eukaryotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575. PHA synthesis in transgenic plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

    5.1. Cytosolic PHA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1585.2. PHA synthesis in plastids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1605.3. PHA synthesis in peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1635.4. Modulating the quantity and monomer composition of PHA in transgenic plants . . . . . . . . . . . . . . . 1655.5. Barriers to increasing PHA production in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

    6. PHA extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1687. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

    1. Introduction

    Growth in the human population has led to theaccumulation of huge amounts of non-degradable wastematerials across our planet. The accumulation of plasticwastes has become a major concern in terms of theenvironment (Guillet, 2002; Derraik, 2002; Thompsonet al., 2004). Conventional plastics not only take manydecades to be decomposed in nature, but also produce

    toxins during the process of degradation. For thisreason, there is special interest in producing plasticsfrom materials that can be readily eliminated from ourbiosphere in an environmentally friendly fashion(Gross and Kalra, 2002). The allure of bioplastic isalso linked to diminishing petrochemical reserves. Theindustrialized world is currently highly dependent onfossil fuels as a source of energy for industrial processesand for the production of structural materials. Fossilfuels are, however, a finite resource and currentevidence suggests, based on recent usage trends and

    the rate of discovery, that utilization rates will outstripdiscovery from about 2010 (Zagar, 2000). This is aglobal problem as our economy is still very oil-dependent. The world currently consumes approximate-ly 140 million tons of plastics per annum. Processing ofthese plastics uses approximately 150 million tons offossil fuels, which are difficult to substitute. All carbon-based structural materials (e.g., plastics, foams, coating,and adhesives) owe their properties to long arrays ofcarboncarbon bonds. The challenge to the world iswhether we can substitute the source of these longcarbon arrays from a non-sustainable source with asustainable renewable one.

    Bioplastics are natural biopolymers that are synthe-sized and catabolized by various organisms (Jendrossekand Handrick, 2002; Kim and Rhee, 2003; Tokiwa andCalabia, 2004; Kragelund et al., 2005; Akar et al., 2006)and these materials do not cause toxic effects in the hostand have certain advantages over petroleum-derivedplastic (Steinbchel and Fchtenbusch, 1998; Angelovaand Hunkeler, 1999; Zinn et al., 2001; Williams andMartin, 2002; Reddy et al., 2003; Chen and Wu, 2005a,

    b; Steinbchel, 2005). These biopolymers accumulateas storage materials in microbial cells under stressconditions (Barnard and Sander, 1989; Sudesh et al.,2000; Chen et al., 2001; Kadouri et al., 2005; Berlangaet al., 2006). The most widely produced microbialbioplastics are polyhydroxyalkanoates (PHAs) and theirderivatives (Madison and Huisman, 1999; Witholt andKessler, 2002; Kim and Lenz, 2001). Beijerinck firstobserved lucent granules of PHA in bacterial cells in1888 (reported inChowdhury, 1963). The compositionof PHAs was first described by Lemoigne as an

    unknown material in the form of a homopolyester ofthe 3-hydroxybutyric acids, called polyhydroxybutyrate(PHB) (Lemoigne, 1926, 1927). During the following30 years, interest in this unknown material wasnegligible. The first report on functions of PHBappeared in 1958 by Macrae and Wilkinson (Macraeand Wilkinson, 1958). They reported the rapid biode-gradability of PHB produced by Bacillus megaterium,by B. cereus and B. megaterium itself. From here on,the interest in PHB increased dramatically. In thefollowing years, research on PHB and other forms ofPHAs included investigations with other microorgan-isms and the potential use of these biopolymers was

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    realized (Braunegg et al., 1998; Volova, 2004; Scott,2005; Noda et al., 2005; Pandey et al., 2005; Ren et al.,2005). The current review begins with a discussion ofthe chemical structure and properties of PHAs. This isfollowed by an examination of PHA synthesis in micro-

    organisms. Finally, numerous strategies are presentedshowing how genes encoding PHA synthases have beenused to produce PHA in plants. Market opportunities ofPHA polymers have been discussed inPlatt (2006).

    2. Monomer composition and physical properties of

    PHAs

    PHAs are composed of 3-hydroxy fatty acid mono-mers, which form linear, head-to-tail polyester (Fig. 1).PHA is typically produced as a polymer of 103 to 104

    monomers, which accumulate as inclusions of 0.2

    0.5 m in diameter. These inclusions or granules aresynthesized and stored by both gram-positive and gram-negative bacteria without hazardous effects to the hosts(Luengo et al., 2003). PHA accumulation occurs whenthe cells experience a nutrient imbalance such as excesscarbon with limited nitrogen, phosphorus or oxygen(Anderson and Dawes, 1990; Steinbchel, 1991;Steinbchel and Fchtenbusch, 1998). The bacteriastore the excess nutrients intracellularly by forminginsoluble biopolymers from soluble molecules. Thebiopolymers become mobilized when conditions for

    normal growth return. The structure, physio-chemicalproperties, monomer composition and the number and

    size of the granules vary depending on the organism(Anderson and Dawes, 1990; Ha and Cho, 2002). Of allthe characterized PHAs, alkyl groups, which occupy theRconfiguration at the C-3, vary from one carbon (C1) toover 14 carbons (C14) in length. PHAs can be sub-

    divided into three broad classes according to the size ofcomprising monomers. PHAs containing up to C5monomers are classified as short chain length PHAs(scl-PHA). PHAs with C6C14 and NC14 monomersare classified as medium chain length (mcl-PHA) andlong chain length (lcl-PHA) PHAs, respectively (Madi-son and Huisman, 1999). scl-PHAs have propertiesclose to conventional plastics while the mcl-PHAs areregarded as elastomers and rubbers. There are alsoreports on functional modification of the monomers toimprove the properties of the resulting bioplastic, such

    as the introduction of unsaturated and halogenatedbranched chains. As well, heteropolymers can beformed by polymerization between more than onekind of monomer. PHB is the most common type ofscl-PHA and this homopolymer of 3-hydroxybutyricacid has been studied most extensively. Copolymers ofPHA can be formed containing 3-hydroxybutyrate(HB), 3-hydroxyvalerate (HV), 3-hydroxyhexanoate(HH) or 4-hydroxybutyrate (4HB) monomers. Most ofthe microbes synthesize either scl-PHAs containingprimarily 3HB units or mcl-PHAs containing 3-hydro-xyoctanoate (HO) and 3-hydroxydecanoate (HD) as the

    major monomers (Anderson and Dawes, 1990; Stein-bchel, 1991; Steinbchel and Schlegel, 1991; Lee,1996). Bacteria synthesize a wide range of PHAs andapproximately 150 different constituents of PHAs havebeen identified (Steinbchel and Valentin, 1995).

    PHAs extracted from bacterial cells have propertiessimilar to conventional plastics, such as polypropylene(Byrom, 1987). PHAs can be degraded at a high rate (39 months) by many microorganisms into carbon dioxideand water using their own secreted PHA depolymerases(Jendrossek, 2001). They can be produced from

    renewable resources, are recyclable, and are considerednatural materials. These properties make PHAs appro-priate for substitution to petrochemical thermoplastics(Poirier, 1999a). The large diversity of monomers foundin PHAs provides a wide spectrum of polymers withvarying physical properties. The homopolymer PHB is arelatively stiff and brittle bioplastic, which is of limiteduse. PHAs made of longer monomers, such as mcl-PHAs, are typically elastomers and sticky materials,which can also be modified to make rubbers. PHAcopolymers composed of primarily HB with a fractionof longer chain monomers, such as HV, HH or HO, aremore flexible and tougher plastics. They can be used in a

    Fig. 1. Chemical structure of PHAs. The pendantR groups (shadedboxes) vary in chain length from one carbon (C1) to over 14 carbons

    (C14). Structures shown here are PHB (R =methyl), PHV (R =ethyl),and PHH (R =propyl).

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    wide variety of products including containers, bottles,razors and materials for food packaging. The latex ofPHAs can be used to produce a water-resistant layer forpaper, film or cardboard (Hocking and Marchessault,1994). Manufacturers in the U.S. used PHB and co-

    polymer P(HBHV) as water-proof films on the back ofdiaper sheets (Martini et al., 1989). This copolymer P(HBHV), with flexibility and impact resistance, wasmarketed under the trade name Biopolby ICI/Zenecaand later by Monsanto till 1995. Moreover, PHAs arealso used to produce fiber materials, such as non-wovenfabrics. PHAs with long side chain hydroxyacids havebeen used in pressure-sensitive adhesive formulations(Yalpani, 1993). In addition to their biodegradability,many PHAs are also biocompatible. Their breakdownproducts are 3-hydroxyacids, which are naturally found

    in animals. These PHAs can be very useful in manymedical applications, such as implants, gauzes, suturefilaments, osteosynthetic materials, and a matrixmaterial for slow release drugs and in vitro cell cultures(Zinn et al., 2001; Sudesh, 2004; Chen, 2005; Chen andWu, 2005a,b; Sudesh and Doi, 2005).

    3. PHA synthesis in microorganisms

    3.1. Genes and enzymes involved in PHA synthesis

    Many species of bacteria, which are members of the

    family Halobactericeae of the Archaea, synthesizePHAs. The list of such microorganisms is growingand currently contains more than 300 organisms(Anderson and Dawes, 1990; Steinbchel and Valentin,1995; Braunegg et al., 1998; Madison and Huisman,1999; Zinn et al., 2001; Ciesielski et al., 2006; Berlangaet al., 2006). The chemical diversity of PHAs is large; ofwhich the most well-known and widely produced formis PHB (Hankermeyer and Tjeerdema, 1999; Kim andLenz, 2001). The synthesis of PHB is considered thesimplest biosynthetic pathway. The process involves

    three enzymes and their encoding genes (Fig. 2)(Schbert et al., 1988, 1991; Peoples and Sinskey,1989a,b; Madison and Huisman, 1999; Steinbchel,2001; Steinbchel and Hein, 2001; Reddy et al., 2003).phaAgene encodes-ketothiolase, the first enzyme forthe condensation of two acetyl-CoA molecules to form

    Fig. 2. PHB and P(HBHV) biosynthetic pathways inR. eutropha. phaAand bktBencode-ketothiolase and 3-ketothiolase, enzymes involved information of acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively.phaBencodes acetoacetyl-CoA reductase, which reduces both acetoacetyl-CoA

    and 3-ketovaleryl-CoA to form (R)-3-hydroxybutyryl-CoA and (R)-3-hydroxyvaleryl-CoA, respectively.phaCencodes PHA synthase, which is thelast enzyme responsible for polymerization of the monomers (adapted fromPoirier, 2002).

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    acetoacetyl-CoA. The next step is the reduction ofacetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA cata-lyzed by the acetoacetyl-CoA reductase (Steinbcheland Schlegel, 1991). The enzyme is encoded by thephaBgene and is NADPH-dependent. The last reaction

    is the polymerization of (R)-3-hydroxybutyryl-CoAmonomers catalyzed by PHA synthase, which is en-coded by thephaCgene (Rehm, 2003; Stubbe and Tian,2003). PHA synthase in Ralstonia eutropha, formerlyknown as Alcaligenes eutrophus, reacts with a narrowrange of substrates, with chain length of C3C5 andprefers C4-substrates (Steinbchel and Schlegel, 1991).Therefore, PHAs obtained by this pathway containshort-chain-length monomers. All three enzymes forPHB synthesis are located in the cytosol of the cellwhere PHB accumulation takes place (Anderson et al.,

    1990). Apart from PHB, bacteria also synthesize a widerange of other PHAs (Kim and Lenz, 2001; Luengoet al., 2003). A number of PHAs with different C3 to C5monomers have been produced in several bacteria in-cludingR. eutrophathrough alterations in the type andrelative quantity of the carbon sources in the growthmedia (Steinbchel and Schlegel, 1991; Dias et al.,2006). For example, addition of propionic acid or vale-ric acid in glucose media leads to the production of arandom copolymer composed of HB and HV [P(HBHV)]. In this pathway, the condensation of propionyl-CoA with acetyl-CoA is mediated by a distinct

    ketothiolase (3-ketothiolase, bktB; Slater et al., 1998)(Fig. 2). Reduction of 3-ketovaleryl-CoA to (R)-3-hydroxyvaleryl-CoA and subsequent polymerization toform P(HBHV) are catalyzed by the same enzymesinvolved in PHB synthesis, namely acetoacetyl-CoAreductase and PHA synthase (Poirier, 2002) (Fig. 2).PHA synthases isolated from different bacteria are cap-able of using a wide range of hydroxyacyl-CoA thio-esters as substrates. All known PHA synthases can beclassified into four classes according to their substratespecificities and their subunit compositions (Rehm and

    Steinbchel, 2002; Hai et al., 2004).Genes encoding key enzymes involved in PHA syn-thesis have been cloned from several natural producersof the biopolymer. The firstphaAgene was cloned fromZoogloea ramigera using anti-thiolase antibodies(Peoples et al., 1987). Later it was found that thephaB gene in this species, and in Paracoccus deni-trificans and Rhizobium meliloti, existed in the sameoperon, while the phaCgene was in a different operon(Fig. 3b,Tombolini et al., 1995; Yabutani et al., 1995;Lee et al., 1996; Ueda et al., 1996). In R. eutropha,Acinetobacter spp., Alcaligenes latus and Pseudomo-nas acidophila, the phagenes form a phaCABoperon,

    although the three genes are not in the same sequence inthese species (Fig. 3a). In some cases the genome carriesmore than one copy of the operon (Peoples and Sinskey,1989a,b; Schembri et al., 1994; Umeda et al., 1998). Insome species including Chromatium vinosum, Thiocys-

    tis violacea, Thiocapsa pfennigiiand Synechocystissp.PCC 6803, the PHA synthase consists of two sub-units,PhaC and PhaE (Fig. 3c). This type III synthase mainlycatalyzes the synthesis of scl-PHAs, but also catalyzesthe polymerization of scl- and mcl-monomers (Heinet al., 1998; Liebergesell and Steinbchel, 1992, 1993;Steinbchel and Hein, 2001).

    Additional genes encode other enzymes thatindirectly contribute to PHA synthesis. The PHAsynthase gene (phaC) inAeromonas caviaeis flankedby phaJ, which encodes enoyl-CoA hydratase

    (Fig. 3d). This enzyme catalyzes (R)-specific hydra-tion of 2-enoyl-CoA for supplying (R)-3-hydroxyacyl-CoA monomer units for PHA synthesis through thefatty acid -oxidation pathway (Fukui and Doi, 1997,Fukui et al., 1998). Unlike R. eutropha, Burkholderiacaryophylli, P. oleovoransand P. aeruginosaare ableto form mcl-PHAs. The phaC1ZC2D operon in theseorganisms contains two phaCgenes separated by thephaZgene (Fig. 3e), which encodes a PHA depoly-merase (Huisman et al., 1991; Hang et al., 2002). Therole of PhaD remains unclear although it seems to berequired for PHA formation (Klinke et al., 2000). PHA

    synthesis in P. oleovorans and P. aeruginosa directlyutilizes intermediates from the fatty acid -oxidationpathway to form larger molecules of 3-hydroxyacyl-CoA (Lageveen et al., 1988). The PHA synthase ofP. oleovoranscan also catalyze the polymerization of awider range of monomers, which results in highermolecular weight polymers with better elastic proper-ties. Several microorganisms also carry an additionalcluster (phaF I) located downstream from thephaC1ZC2D operon (Fig. 3e,f, Nishikawa et al.,2002). PhaI participates in the formation and stabili-

    zation of the granules, while PhaF is involved in thestabilization of the granules and acts as a regulator(Prieto et al., 1999).

    3.2. PHA production in recombinant Escherichia coli

    In recent years, a combination of genetic engineeringand molecular microbiology techniques has beenapplied to enhance PHA production in microorganisms.Several mutants with phenotypes in PHA synthesis werecharacterized in order to develop optimal recombinanthost strains. Over-expression ofphagenes in the naturalPHA producer, however, resulted in little difference in

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    polymer accumulation. Natural producers, such asR. eutropha, are well adapted to PHA accumulation intheir cells. R. eutropha can store up to 90% of its dryweight (dwt) in PHA granules. Most natural producers,however, take a long time to grow during fermentationand extraction of polymers from their cells is difficult.Therefore, these PHA producers are not suitable forindustrial production of the biopolymer. On the otherhand, althoughE. colidoes not naturally produce PHA,this bacterium is considered to be appropriate host forgenerating higher yields of the biopolymer because ofits fast growth and the ease with which it can be lysed(Li et al., 2006). pha genes were first introduced into

    E. coliin 1988 by Slater et al. and Schbert et al. PHBgranules were formed in recombinantE. colihost cells(Slater et al., 1988). Even after extensive attempts atmaximizing PHB production in non-PHB producingmicroorganisms, the PHB accumulation level was not ashigh as what could be obtained with the naturalproducers of the biopolymer. One of the major obstaclesin producing PHB in recombinant organisms isassociated with the instability of the introduced phagenes. Loss of the plasmid due to metabolic load oftenlimits high yields of the biopolymer (Lee et al., 1994c;Madison and Huisman, 1999). Other parameters havebeen adjusted to enhance PHB production including

    Fig. 3.phagene operons in different microorganisms. (a) A completephaCABoperon ofR. eutropha; (b) interrupted locus ofZ. ramigera; (c) locuswith two polymerase subunits,phaCandphaE, ofC. vinosum; (d)phaCJoperon ofA. caviaefor copolymer P(HBHH) formation; (e)phaC1ZC2Doperon for mcl-PHA formation in P. oleovorans with two phaC genes (C1 and C2); (f) pha locus with depolymerase (phaZ) between two

    polymerase subunits inP. aureofaciensand phaFgene situated downstream ofphaC1ZC2Doperon.

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    increased carbon supply, changes in fermentationtemperature, changes in the number of plasmid copiesand choice of bacterial strains (Kim et al., 1992; Leeet al., 1994a,b,c, 1995; Nikel et al., 2006). Growth of therecombinant cells was impaired in many of these

    studies, especially in nutrient-rich medium (Lee, 1994;Wang and Lee, 1997). Recombinant E. coli culturedunder optimal conditions has been shown to accumulatePHB up to 85% of the cell dwt. PHB formed in theseE. coli, however, were of higher molecular weight thanPHB produced by natural producers (Zhang et al., 1994;Kusaka et al., 1997). The molecular mass of the PHBproduced in E. coli cells depended strongly on culturecondition. In higher glucose concentration (20 g/l),37 C and pH 6.0, cells produced PHB with highestmolecular mass value (20 MDa). It has been suggested

    that a chain-transfer agent is generated in E. colicellsduring the accumulation of PHB (Kusaka et al., 1997).

    3.3. Fatty acid-oxidation and PHA production

    The catabolism of fatty acids represents one of themost common pathways to supply hydroxyalkanoate(HA) monomer substrates for PHA synthesis (Sudeshet al., 2000). Intermediates generated by the degrada-tion of alkanoic or fatty acids via -oxidation (Fad

    pathway) can provide hydroxyalkanoyl-CoA (HA-CoA) substrate for mcl-PHAs. This pathway is foundin several bacteria, such as P. oleovoransand P. fragii,which can synthesize mcl-PHA from alkanoic acids orfatty acids. In these bacteria, the monomer composi-

    tion of PHAs produced is directly related to thesubstrate used for growth and usually has monomersthat are 2n(n0) carbons shorter than the substrates(Lageveen et al., 1988). Although wild type E. colicannot accumulate PHA, its fatty acid metabolicpathways have been utilized to provide mcl-HA-CoAprecursors for PHA accumulation in engineered E. colicells. The intermediates of fatty acid -oxidationpathways including enoyl-CoA, 3-ketoacyl-CoA and(S)-3-hydroxyacyl-CoA, can serve as precursors ofmcl-(R)-3-hydroxyacyl-CoA, which is used directly in

    mcl-PHA synthesis (Fig. 4). Therefore, it was possibleto synthesize mcl-PHA in E. coli cells, which aredefective in the-oxidation pathway (fadAand fadBmutants, accumulate -oxidation intermediates), byintroducing the Pseudomonas mcl-PHA synthase(phaC) gene (Langenbach et al., 1997; Qi et al.,1997, 1998; Ren et al., 2000; Park et al., 2002). Inthese defective E. coli, co-expression of other genes,along with PHA synthase, were necessary to enhanceconversion of -oxidation intermediates to (R)-3-

    Fig. 4. Fatty acid-oxidation pathway ofE. coli.RecombinantE. coliwith defectivefadAandfadBuses intermediates of-oxidation, enoyl-CoA,3-ketoacyl-CoA and (S)-3-hydroxyacyl-CoA, as major substrates for mcl-PHA synthesis. Co-expression of mcl-PHA synthase with enoyl-CoAhydratase (encoded by paaF, paaG, ydbU, maoC, yfcXfrom E. coli; phaJfrom P. aeruginosa, P. putidaand A. caviae) or 3-ketoacyl-CoA/ACP

    reductase (encoded by fabGfrom P. aeruginosaand E. coli; rhlGfrom P. aeruginosa) can enhance PHA production by increasing -oxidationintermediate pools (adapted fromPark and Lee, 2003).

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    hydroxyacyl-CoA, immediate precursor of mcl-PHA(Fig. 4). Examples of these genes, which were co-expressed with PHA synthase for enhanced produc-tion, include paaF, paaG, ydbU from E. coli (Parkand Lee, 2004), phaJ from P. aeruginosa, P. putida

    and A. caviae (Fukui et al., 1999; Park et al., 2001;Fiedler et al., 2002; Tsuge et al., 2002, 2003), maoCfrom E. coli (Park and Lee, 2003), yfcXfrom E. coli(Snell et al., 2002), fabG from P. aeruginosa andE. coli (Taguchi et al., 1999; Ren et al., 2000; Parket al., 2002) and rhlGfrom P. aeruginosa (Campos-Garcia et al., 1998; Park et al., 2002). Co-expression ofother genes of the -oxidation pathway can signifi-cantly enhance PHA production in recombinantE. coli. For example, acyl-CoA dehydrogenase ofE. coli, encoded by yafH and fadE (Campbell and

    Cronan, 2002), catalyzes the dehydration of acyl-CoAto enoyl-CoA (Fig. 4) and is considered the rate-limiting step of -oxidation (Qi et al., 1998). Co-expression of yafH, along with phaC and phaJ,enhanced the supply of enoyl-CoA. With the enhancedprecursor supply, the recombinantE. coliaccumulatedfour times more scl-mcl-PHA, P(HBHH) than cellsexpressing only phaCand phaJ(Lu et al., 2003). Co-expression of the E. coli yafHgene also increased HHcontent of the P(HBHH) copolymer produced inrecombinant Aeromonas hydrophila(Lu et al., 2004).When-oxidation was inhibited by sodium acrylate in

    natural producers of scl-PHBR. eutrophaand grown insodium octanoate as a carbon source, the intermediatesof-oxidation pathway were channeled into mcl-PHAscomposed of HH, HO and HB (Green et al., 2002).

    A second route of mcl-PHA synthesis in bacteria isthrough the use of intermediates of fatty acid de novo

    biosynthesis (Fab pathway) that provides HA-CoAmonomers (Fig. 5). In contrast to P. oleovorans andP. fragii (which use -oxidation intermediates),P. aeruginosa and P. putida produce mcl-PHAs whengrown on unrelated substrates, such as glucose. This is

    becauseP. oleovoransandP. fragiiuse fatty acid as carbonsource through-oxidation pathway to produce 3-hydro-xyacyl-CoA, substrate of mcl-PHA synthase (Fig. 4). Onthe other hand, the fatty acid biosynthesis is the main routefor 3-hydroxyacyl-CoA synthesis in P. aeruginosa and

    P. putidaduring growth on a carbon source which ismetabolized to acetyl-CoA, like carbohydrate, acetate orethanol (Fig. 5). The gene phaG, encoding an enzymelinking fatty acid de novobiosynthesis and PHA synthesis,has been cloned from P. putida (Rehm et al., 1998;Hoffmann et al., 2000a,b). The product of this gene

    catalyzes the conversion of (R)-3-hydroxyacyl-ACP,intermediate of fatty acid biosynthesis pathway, to itscorresponding CoA derivative (Fig. 5). Therefore, expres-sion ofphaGinE. coligave the recombinant bacteria thenovel capacity to produce free mcl-fatty acid precursors(3HD) (Zheng et al., 2004) and its expression in

    P. oleovorans and P. fragii produced mcl-PHA (Fiedleret al., 2000; Hoffmann et al., 2000b), from carbon sourcesnon-related to 3HD structure, such as glucose and fructose.The only time PhaG, along with PhaC, was able to producemcl-PHA inE. coli waswhenfattyacid de novobiosynthesiswas partially inhibited by triclosan (Rehm et al., 2001).

    3.4. Engineering of PHA synthases to enhance and

    change polymer synthesis

    A number of studies have focused on engineeringPHA synthases in attempts to enhance activity and/or

    Fig. 5. Fatty acidde novo biosynthesis (Fab pathway). P. aeruginosaand P. putidause (R)-3-hydroxyacyl-CoA monomers from Fab pathway toproduce mcl-PHAs through expression of PhaG and PhaC when grown on carbon sources which is metabolized to acetyl-CoA, like carbohydrate.

    PhaG acts as a link between Fab pathway and mcl-PHA synthesis by catalyzing (R)-3-hydroxyacyl-ACP, intermediate of Fab pathway, to (R)-3-hydroxyacyl-CoA, substrate for mcl-PHA.

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    alter substrate specificity for customized production ofPHA copolymers in recombinant hosts. Takase et al.(2003) modified the phaC1 gene of Pseudomonas sp.through error-prone PCR mutagenesis and site-specificsaturation mutagenesis. The gene product ofphaC1is a

    type II synthase and, unlike type I enzyme, has a verylow specificity for HB units. The engineered enzymessubstantially enhanced (400 times) PHB synthesis inrecombinantE. coli.Amara et al. (2002)also reportedenhanced activity of PHA synthase from A. punctatamodified through genetic engineering. Kichise et al.(2002) achieved enhanced accumulation and changedmonomer composition in recombinantE. colithrough amodification of the phaCgene from A. punctata. Thisalteration led to a 6-fold enhancement in accumulationof P(HBHH). The PhaJ enzyme from A. caviae

    generates (R)-3-hydroxyacyl-CoAs with chain lengthof 4 to 6 carbon atoms from the fatty acid -oxidationpathway for PHA synthesis (Fig. 4).Tsuge et al. (2003)examined the structure of this enzyme through X-raycrystallography and identified Ser-62, Leu-65 and Val-130 as amino acid residues that define the depth andwidth of the acyl-chain-binding pocket. By changingthese residues through site-directed mutagenesis of thephaJ gene, they created mutants that showed signifi-cantly higher specificities towards octenoyl-CoA (C8)than the wild-type enzyme. When the modified genewas introduced into E. colialong with phaC1ofPseu-domonassp. there was an increased incorporation of HO(C8) and HD (C10) into biopolymer.

    Nomura et al. (2004a,b) altered 3-ketoacyl-ACPsynthase III (FabH) of E. coli and PhaC1 ofPseudo-monas sp. by changing the substrate specificity of theenzymes through site-directed or saturation pointmutagenesis of the encoding genes. These engineeredgenes were then introduced into E. coli, along withphaAand phaB. The cumulative effect of having twomonomer-supplying pathways and genetically engi-neered PHA synthase resulted in accumulation of scl-

    mcl-PHA copolymer from the non-related carbonsource, glucose.

    3.5. Production of PHA copolymers

    scl-PHA homopolymers (C3C5), such as PHB,form stiff crystalline materials, which are brittle andcannot be extended without breakage. This lack offlexibility limits the range of applications of scl-PHAhomopolymers. PHB homopolymer consisting solely ofC4 monomer is difficult to process, because it degradesat a temperature slightly above its melting point (DeKoning, 1995). Polymers consisting of only mcl-PHA

    (C6C14) are semi-crystalline thermoplastic elasto-mers. The mechanical properties of these polymersmay be enhanced by reinforcement. Unlike polymerscomposed solely of either scl- or mcl-monomer units,scl-mcl-PHA copolymers can have a wide range of

    physical properties, depending on the mol% composi-tion of the different monomers in the copolymer(Matsusaki et al., 2000). scl-mcl-copolymers composedof mostly C4 monomers, with a small amount of C6monomer, have properties similar to polypropylene(Abe and Doi, 2002). scl-mcl-PHA copolymer of HBand HH [P(HBHH)] is a tough and flexible material.The copolymer of HB and HV [P(HBHV)] hasreduced crystallinity and melting point, leading toimproved flexibility, strength and easier processing.Therefore, several laboratories have attempted to

    synthesize specific scl-mcl-PHA copolymers in bacteria.phagenes from different natural producers, such as thephaCgene from Pseudomonas spp., were introducedinto E. coli to induce the synthesis of copolymers andmcl-PHA. The phaC1gene from P. oleovoransin fadAand fadB strains accumulated mcl-PHAs when grownon C8 to C18 fatty acids, with yield increases achievedby using inducible promoters (Ren et al., 1996). PHAcopolymers containing HH, HO and HD were producedin the recombinantfadBmutant ofE. coliby introducingphaC1and phaC2genes from P. aeruginosa and Bur-kholderia caryophylli (Langenbach et al., 1997; Qi

    et al., 1997; Hang et al., 2002). E. coli cells were co-transformed with the hbcT gene from Clostridiumkluyveri, which encodes a 4-hydroxybutyric acid-CoAtransferase, and phaCfrom R. eutropha. Up to 20% ofthe cell dwt contained P(4HB) homopolymer when thebacteria were grown in the presence of 4HB. In thepresence of glucose P(4HB) homopolymer was pro-duced, while in the absence of glucose, a P(HB4HB)copolymer accumulated even though phaA and phaBgenes were absent (Hein et al., 1997). Changing glucoseand fatty acid concentration in the medium have also

    been shown to lead to a change in the monomercomposition of PHA copolymer (e.g., Lu et al., 2004).Law et al. (2004)achieved 43% cell dwt P(HBHV)copolymer production in recombinant E. coli throughselection of certain strains of the bacteria. By introduc-ing the succinate degradation pathway from C. kluyveri,along with phagenes from R. eutropha, Valentin andDennis (1997) produced P(HB4HB) directly fromglucose.Nomura et al. (2005)showed that expression of3-ketoacyl-acyl carrier protein reductase (fabG) genesenhanced production of polyhydroxyalkanoate copoly-mer from glucose in recombinant Escherichia coliJM109. Other feeding and fermentation strategies were

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    undertaken to optimize the copolymer production levels(Hong et al., 2000; Ahn et al., 2000; Lee and Choi,2001). However, since E. coliis not a natural producerof PHAs there were difficulties in optimizing the growthof engineeredE. coli(Madison and Huisman, 1999).

    4. PHA production in eukaryotic cells

    The production of bioplastic in bacteria is limited byits high cost compared to the costs associated withpetroleum-derived plastics production. This aspect hasbeen one of the driving forces in exploring eukaryoticsystems, especially crops, as production hosts. Studiesof PHA formation in yeast and insect cells can providevaluable information about how these pathways can beincorporated into plants. Synthesis of PHB has been

    demonstrated in Saccharomyces cerevisiae by expres-sing the PHB synthase gene from R. eutropha (Leafet al., 1996). PHB accumulation, however, was very low(0.5% of cell dwt), possibly because of insufficientendogenous -ketoacyl-CoA-thiolase and acetoacetyl-CoA reductase activities. To improve the yield and tosynthesize copolymers of PHAs, studies have focusedon channeling the intermediates of-oxidation pathwayinto PHA assembly.Poirier et al. (2001) introduced amodified phaC1 gene from P. aeruginosa intoS. cerevisiae. Peroxisomal targeting (PTS1) of thegene product was achieved by developing a construct

    which resulted in the addition of a 34 amino acid stretchfrom the carboxylic end of Brassica napus isocitratelyase. When the recombinant yeast cells were grown inmedia containing fatty acids, they accumulated mcl-PHAs demonstrating that peroxisomal PHA synthaseproduces PHA in the peroxisomes using 3-hydroxyacyl-CoA intermediates of fatty acid oxidation. In contrast toS. cerevisiae,Pichia pastorisgrows vigorously on fattyacids as a carbon source.Poirier et al. (2002)introducedthe above PTS1-modified P. aeruginosa phaC1 geneintoP. pastorisand achieved mcl-PHA synthesis in this

    yeast system with fatty acids in the growth medium. Theyield of PHA in the two described studies with yeastsystems, however, was low, with accumulations lowerthan 1% cell dwt.

    Marchesini et al. (2003)have explored the possibil-ities of changing monomer composition of PHA inrecombinant yeast cells. The investigators demonstratedthat it was possible to alter the PHA monomercomposition of mcl-PHAs produced in yeast from theintermediates of the -oxidation of fatty acids by using amodified form of the peroxisomal multifunctionalenzyme 2 (MFE-2, encoded by the fox2 gene). Theytransformed yeast cells with genes coding for two

    mutant forms of the 3-hydroxyacyl-CoA dehydrogenasedomain of the MFE-2 of S. cerevisiae. The mutantMFE-2(a) retain a broad activity towards short-,medium- and long-chain (R)-3-hydroxyacyl-CoAs,while the mutant MFE-2(b), did not accept short-

    chain (R)-3-hydroxyacyl-CoAs. Expression of MFE-2(b), along with PHA synthase, resulted in a substantialincrease in the proportion of the short-chain 3-hydro-xyacid monomers at the expense of longer monomers.These transformant yeast cells were inefficient at usingshort-chain (R)-3-hydroxyacyl-CoAs generated by the-oxidation cycle, leading to higher levels of theseintermediates available to the PHA synthase. Zhanget al. (2006)engineered the synthesis of PHA polymerscomposed of monomers ranging from 4 to 14 carbonatoms in either the cytosol or the peroxisome of

    S. cerevisiae by harnessing intermediates of fatty acidmetabolism and achieved accumulation of PHA up toapproximately 7% of its cell dry weight.

    Insect cells have also been studied as a model forPHA production in eukaryotes. The phaCgene fromR. eutropha was successfully expressed in cabbagelooper cells and a soluble form of PHB synthase thatcould be rapidly purified was obtained (Williams et al.,1996a). In a separate attempt, Williams et al. (1996b)transfected fall armyworm cells with a modified eukary-otic fatty acid synthase, which did not extend fatty acidsbeyond HB, along with thephaCgene fromR. eutropha.

    PHB production was achieved in the transfected cells,although the yield was very low (b1% of cell dwt).

    5. PHA synthesis in transgenic plants

    PHA production in bacteria and yeast requiresgrowth under sterile condition in a costly fermentationprocess with an external energy source such aselectricity. In contrast, PHA production in plant systemsis considerably less expensive because the system onlyrelies on water, soil nutrients, atmospheric CO2 and

    sunlight. In addition, a plant production system is muchmore environmentally friendly. Plants use photosyn-thetically fixed CO2and water to generate the bioplastic,which after disposal is degraded back to CO2and water.Synthesis of PHAs in crops is also an excellent way ofincreasing the value of the crops (Poirier, 1999b;Somerville and Bonetta, 2001). Since starch and sugarare produced in plants at costs below the cost ofcommodity plastics, it might be possible to producePHA at a similar low cost. Unlike the bacterial cell, theplant cell has different subcellular compartments inwhich PHA synthesis can be metabolically localized. Asmentioned earlier, PHB is synthesized in bacteria from

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    acetyl-CoA. This thioester is present in plant cells in thecytosol, plastids, mitochondria and peroxisomes. There-fore, it should be possible to produce PHB in any ofthese subcellular compartments (Hanley et al., 2000;Moire et al., 2003).

    5.1. Cytosolic PHA synthesis

    Initial work on producing PHB in plants focused oncytosolic expression, which avoids the use of targetingsequences. As well, an endogenous -ketothiolase, thefirst critical enzyme in PHB synthesis, occurs naturallyin the cytosol of higher plants where it takes part in thesynthesis of mevalonate, the precursor to isoprenoids(Fig. 6). Therefore, PHB synthesis in the cytosol shouldrequire only the expression of acetoacetyl-CoA reduc-

    tase and PHB synthase from bacteria.The first attempt to produce bioplastic in transgenicplants was carried out withArabidopsis thaliana, a modelplant with a relatively small genome and short life cyclethat could be easily transformed with Agrobacteriumtumefaciens.In the pioneering work,Poirier et al. (1992)introduced the phaB gene encoding acetoacetyl-CoAreductase and the phaC gene encoding PHB synthasefrom R. eutropha into A. thalianaplant cells under thecontrol of the constitutive cauliflower mosaic virus 35S(CaMV35S) promoter without organelle-specific target-ing signals (Fig. 7a). The reductase and synthase genes

    were first introduced individually in separate transforma-

    tions withArabidopsisplants. Transgenic plants expres-sing both genes were obtained by cross-pollination of thetransgenic plants carrying the individual genes. In thesehybrids, the presence of PHB granules was observed invarious tissues including root, leaf, cotyledon and seed.

    Moreover, PHB granules were found in some subcellularcompartments of the cell including the nucleus, vacuolesand cytosol, but not in the chloroplasts and mitochondria.The highest amount of PHB obtained in these plants wasonly 0.1% dwt of leaves. Biopolymer from transformedplants had the size, appearance and structure of thematerial from natural bacterial producers. In a later study,Poirier et al. (1995) produced PHB in cell suspensionculture of transgenic Arabidopsis and found that thesePHB had identical chemical structure and similar thermalproperties as those of bacterial PHB, although the mole-

    cular weight distribution of the plant-produced PHB wasmuch broader than that of bacterial PHB. Strong growthretardation and reduction in seed production wereobservedin transgenic plant lines with high expression of acetoace-tyl-CoA reductase (Poirier et al., 1992; Poirier, 2001). Co-expression of acetoacetyl-CoA reductase with PHBsynthase led to a further reduction in growth compared toplants expressing only the reductase. It was hypothesizedthat the diversion of cytoplasmic acetyl-CoA and acet-oacetyl-CoA away from the endogenous isoprenoid andflavonoid pathways might have depleted the cell ofessential metabolites, thus affecting the growth of the

    transgenic plants. The isoprenoid pathway contributes to

    Fig. 6. Genetically engineered metabolic pathways for PHB synthesis in plant cytoplasm. Dashed arrows indicate expression of transgenes, whileplant native pathways are shown by solid arrows. Enhancement of acetyl-CoA pool by enzyme inhibitors is also shown. Crosses indicate targeted

    sites of enzyme inhibitors. Quizalofop inhibits acetyl-CoA carboxylase (ACCase) of the flavonoid synthetic pathway and mevastatin inhibits 3-hydroxy-3-methylglutaryl-CoA (HMGR) of the mevalonic synthetic pathway.

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    the synthesis of essential plant growth hormones, cytoki-nins, gibberellins and brassinosteroids. Cytoplasmic acetyl-CoA and acetoacetyl-CoA are involved in the synthesis ofsterols, essential components of cell membranes. There-fore, it was likely that small changes in these hormones andsterols severely affected plant growth.

    Similar experiments were carried out in blackMexican sweet maize (Zea mays L.) and in tobacco(Nicotiana tabacum L.). All three pha genes fromR. eutropha were individually introduced under theconstitutive CaMV35S promoter. In transgenic cultures,PHB was obtained up to 0.15% of the cell extract dwt.Transgenic cell cultures showed significant growth

    retardation. Moreover, after weekly sub-culturing over1.5 years, the activity of PHA synthase gene in the cellcultures was lost. This result suggested that thetransgenic lines were genetically unstable (Hahn et al.,1997). The results in transgenic tobacco, carrying phaBfrom R. eutropha and phaC from A. caviae were alsosimilar, with a very low production of PHB occurring atless than 10 g/g of fresh weight (Nakashita et al.,1999). Similar results were obtained by expressingR. eutropha pha genes in the cytosol of potato andBrassica napus. Resultant transgenics exhibited lowexpression levels for the introduced genes, a low yieldof PHB (b0.1% dwt) and stunted growth (Poirier, 2002).

    Fig. 7. Various plant transformation gene constructs. (a) Constructs of phaB and phaC from R. eutropha under constitutive CaMV35S (35S)promoters with Hygromycin (HPT) and Kanamycin (NPTII) resistance; (b) three individual plastid target constructs with each gene attached to thechloroplast transit peptide (CTP) and under CaMV35S promoter; (c) triple plastid target construct with CaMV35S promoter and chloroplast transit

    peptide; (d) polycistronic mRNAphaoperon fromR. eutrophafor chloroplast genomic transformation under prrn promoter; (e) peroxisomal targetconstruct ofphaC1fromP. aeruginosafor mcl-PHA formation with the attachment of DNA segment encoding the last 34 amino acids ofB. napusisocitrate lyase (ICL). Tnos, terminator sequence from nopaline synthase gene; TpsbA, terminator sequence from plastidpsbAgene; RB, right borderof the T-DNA; LB, left border of the T-DNA.

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    Interestingly, over-expression of the bacterial ketothio-lase in plants expressing the reductase and PHAsynthase did not increase PHB yield. Therefore,ketothiolase activity is probably not the limiting factorfor PHB synthesis in the cytoplasm of plants. Instead,

    other factors including the low flux of acetyl-CoA anddifferences in codon usage may represent limitingfactors.

    R. eutropha pha genes were also successfullyexpressed in the cytosol of cotton fibers. Cotton fiberis composed of a thin primary wall (0.4 m) and a thicksecondary wall (810 m). The FbL2A promoter isspecific to fibers and regulates expression duringsecondary cell wall formation (Rinehart et al., 1996).Mature cotton fiber contains 89% cellulose. Thechemical reactivity, thermal characteristics, water ab-

    sorption characteristics and the strength of the fiber areall dependent on fiber composition. In an attempt toimprove the chemical and thermal properties of thefibers by producing PHB in the cell lumen (John andKeller, 1996), the phaB and phaC genes fromR. eutropha were placed under fiber tissue specificpromoters (E6 and FbL2A) and were introduced intocotton genome by particle bombardment. As previouslymentioned -ketothiolase, encoded by the phaA, isubiquitous in the plant cytosol and therefore does notneed to be introduced from R. eutrophato produce PHBin plant cell cytosol. These two promoters initiate

    expression at different stages. E6 has a high activityduring early fiber development whereas FbL2A has ahigher activity in late fiber development. PHB granuleswere found in the cytosol of transgenic plants. The levelof PHB in transgenic fibers increased during early stages(up to 15 days post anthesis), but due to the largeincrease in fiber mass that came with plant development,the PHB level eventually decreased. Transgenic plantsshowed normal growth and morphology. Fibers fromtransgenic plants contained 0.34% PHB, which wassufficient to improve the insulating properties of the

    fiber. The transgenic fibers had higher heat capacity andlower thermal conductivity (John, 1997). The heatuptake of the fiber might have been influenced byinteractions of PHB in the fiber lumen (Chowdhury andJohn, 1998).

    5.2. PHA synthesis in plastids

    Low levels of PHB accumulation in the cytosol(b0.5% dwt) have been associated with a limited supplyof acetyl-CoA. In contrast, the plastid does exhibitrelatively high levels of acetyl-CoA because thisorganelle is the site of fatty acid biosynthesis, which

    relies heavily on an acetyl-CoA input. Plastids canaccumulate a relatively large amount of starch granuleswithout organelle disruption and therefore should beable to accumulate substantial quantities of PHB. Theabsence of an endogenous ketothiolase in plastids,

    however, would necessitate the introduction of bacterialgenes encoding ketothiolase along with acetoacetyl-CoA reductase and PHB synthase.

    To increase PHB production, expression of the threebacterial genes for PHB synthesis was targeted toplastids using gene constructs that included a DNAsegment encoding a chloroplast transit peptide (Nawrathet al., 1994)(Fig. 7b). The genes were integrated into thenuclear genome, while the recombinant enzymes weretranslocated into plastids via the chloroplast transitpeptide (CTP) of the small subunit of ribulose-bispho-

    sphate carboxylase from pea (Coruzzi et al., 1983). PHAproteins encoded by the transgenes were synthesized inthe cytoplasm, and then post-translationally importedthrough the chloroplast membrane by binding with aproteinaceous receptor. Afterward a stromal proteasecatalyzes the cleavage of the transit peptide and leavesthe mature protein free to be functional in the plastids.The transgenes were placed independently under thecontrol of constitutively expressed CaMV35S promoterand were introduced into Arabidopsis. Plants with allthree enzyme activities (obtained by cross-pollinationsof plants expressing individual genes) showed high

    accumulation of PHB granules (Nawrath et al., 1994).PHB inclusions were observed exclusively in plastids,and the size and appearance of the granules were similarto granules produced in bacteria. The quantity of PHBaccumulation increased gradually over time. Themaximum amount of PHB in pre-senescing leaves was10 mg/g fresh weight, which was approximately 14% ofthe dwt. Plastids of these leaves were filled with PHBgranules. There were no major deleterious effects oneither plant growth or fertility, although leaf chlorosiswas observed in plants accumulating more than 3%

    PHB. Transgenic plants expressing only PhaB and PhaCwere not able to produce PHB, which confirmed theabsence of ketothiolase in plastids. Although theproduction of PHB in these plants was about 100-foldgreater than in transgenic lines producing cytosolicPHB, the yield of biopolymer was still lower than thelevel of endogenous starch accumulation (12 mg/g ofthe fresh weight). Since the three genes were individ-ually inserted into multiple loci, they did not strictly co-segregate together. This resulted in a decrease in genecopy and PHB production in later generations.

    A triple gene construct carrying all three genes in onevector for plastidial PHB synthesis was developed in

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    which is involved in isoleucine biosynthesis (Fig. 8)from 2-ketobutyrate. To ease this pressure, the quan-tity of 2-ketobutyrate in plastids was enhanced throughthe expression of ilvA, a threonine deaminase fromE. coli. The gene product of phaA, however, cannotefficiently catalyze the synthesis of 3-ketovaleryl-CoAusing propionyl-CoA. Therefore, in order to pro-duce copolymer containing both HB and HV, bktB

    (fromR. eutropha) was used as a substitute. This geneencodes a novel -ketothiolase having high affinity forboth acetyl-CoA and propionyl-CoA. The E. coli ilvA,the R. eutropha phaB, phaCand bktB genes were allattached to DNA segments encoding plastidial targetsequences and driven by a CaMV35S promoter.Transgenic lines ofArabidopsis produced P(HBHV)copolymer up to 1.6% of the dwt with HV unit fractionof 417 mol% (Slater et al., 1999; Valentin et al., 1999).This was one of the most complex genetic engi-neering experiments performed in plants because theexpression of four genes, modified for targeting to theplastid, was required. The transformation involved

    diversion of carbon from two pathways: acetyl-CoAfrom fatty acid biosynthesis and propionyl-CoA fromamino acid biosynthesis.

    As indicated previously, intermediates of fatty acidbiosynthesis provide HA monomers for the productionof PHA copolymer in some bacteria. 3-hydroxyacyl-ACP-CoA transacylase (PhaG) catalyzes the conversionof (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA

    (Fig. 5). Attempts to produce PHA copolymer in theplastid ofArabidopsis by co-expressing P. aeruginosaPHA synthase andP. putidaPhaG did not lead to PHAaccumulation (V. Mittendorf, unpublished results). It isnot clear why PHA copolymer synthesis using fatty acidbiosynthetic intermediates is difficult to achieve inplastids even though this organelle is a suitable site forPHB and P(HBHV) synthesis.

    The high levels of PHB accumulation (up to 40%dwt) achieved by nuclear expression of three enzymeswith plastid targeting signals suggested that plants mayserve as suitable factories for the mass production ofPHB. The reduction of acetyl-CoA pools in high PHB-

    Fig. 8. Genetically engineered PHB and copolymer P(HBHV) synthetic pathways in plant plastids.ilvAfromE. coliencodes threonine deaminase.phaA, bktB, phaB, andphaCare from R. eutropha. phaAand bktBencode thiolases with different substrate specificities; phaBand phaCencodeacetoacetyl-CoA reductase and PHA synthase, respectively. PDC refers to pyruvate dehydrogenase complex in plant plastids. The cross indicates the

    targeted site of enzyme inhibitor, Quizalofop, which enhances the acetyl-CoA pool in the engineered pathway. Dashed arrows indicate expression oftransgenes, while plant native pathways are shown in solid arrows.

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    producing lines, however, resulted in severe effects onplant growth, fertility and metabolism of sugars whenthe genes were expressed constitutively in all tissues ofthe plant. Therefore, it was anticipated that PHAsynthesis in plastids of specific tissues, such as seeds,

    would have smaller adverse effects on plant growth anddevelopment.Houmiel et al. (1999) transformed Bras-sica napuswith a triplepha R. eutrophagene construct.The CaMV35S promoter was replaced with a seed-specific promoter of Lesquerella fendi fatty acidhydroxylase. When combined with a DNA segmentencoding a chloroplast transit peptide, the expressionsof all transgenes were exclusively targeted to leuco-plasts, which are enriched in acetyl-CoA for fatty acidsynthesis. Transgenic lines carrying the triple constructaccumulated PHB up to 8% of the seed dwt (Houmiel

    et al., 1999; Valentin et al., 1999). PHB production hadno deleterious effects on oil production in the seeds, andthe size and germination rate of the seeds were normal.The size of the leucoplast, however, was larger in PHBproducing seeds, suggesting that leucoplasts can adjusttheir size to accommodate more granules. This exper-iment demonstrated that the seed leucoplast wasprobably a better production system for bioplastic thanleaf chloroplast. The leucoplast appeared more meta-bolically tolerant to the diversion of acetyl-CoA to PHBaccumulation compared to leaf chloroplasts. It will beinteresting to explore the effects of producing very high

    levels of this biopolymer in the developing seed.Synthesis of PHA copolymer was also attempted in

    the B. napus seed leucoplast. The E. coli ilvA, and theR. eutropha phaB,phaCand bktBgenes were all attachedto a plastidial targeting sequence and driven by a seed-specific promoter from the fatty acid hydroxylase gene of

    L. fendi. Transgenic B. napuswith seed-specific P(HBHV) construct produced seeds containing up to 2.3% ofthe dwt copolymer with 6 mol% of HV composition(Slater et al., 1999). There was an inverse relationshipbetween the amounts of PHA to the HV monomer

    composition.Therefore, it was thought that inefficiencyofthe PDC in converting 2-ketobutyrate to propionyl-CoAcould be a reason for low yield. Further analysis of thetransgenic plants revealed that expression of the bacterialilvAresulted in more carbon accumulating in the form ofisoleucine or 2-aminobutarate, instead of the target 2-ketobutarate. Nonetheless, synthesis of Biopol [P(HBHV)] in plants was an important milestone towardscommercial production of bioplastics in plants.

    Accumulation of carbohydrate often takes place inamyloplasts, which are specialized plastids; thereforestorage of carbohydrates in these organelles can poten-tially interfere with PHB accumulation. Sugar beet (Beta

    vulgaris L.) roots, however, store carbohydrates invacuoles, and not amyloplasts. Therefore, in this rootsystem, PHB accumulation in plastidsshould not interferewith the accumulation of carbohydrate. Introduction ofthe three phagenes under the CaMV35S promoter with

    the assistance of the chloroplast transit peptide enabled allthe root tissues of sugar beet to accumulate PHB in theamyloplasts of a hairy roots system grown in culture(Menzel et al., 2003). The transgenic plants accumulatedPHB up to 3.4% of the dwt. Growth retardation in thetransgenic hairy roots occurred in solid and liquid media.The model presented in the hairy root system, however,may not reflect the situation in whole transgenic plant.Nevertheless, this was the first successful demonstrationof PHB production in a carbohydrate-storing crop plant.

    Plastic is often used to improve the mechanical

    properties of fiber-based composites. The incorporationof PHAs into fiber-based composites, however, offers amore environmentally friendly alternative. Recently,Wrbel et al. (2004) generated transgenic flax (LinumusitatissimumL), which produced bioplastic in the stem.The aim of this work was to improve the mechanicalquality of fiber in the plant rather than providing a plantsource of PHB for extraction. A triple construct with three

    phagenes ofR. eutrophawas expressed and targeted toplastids in the stem.phaB andphaCgenes were driven bya CaMV35S promoter, whereasphaA was controlled by a14-3-3 promoter. The 14-3-3 promoter expresses specif-

    ically in stem tissues. PHB inclusions were foundexclusively in the chloroplasts of the stem, and accumu-lated up to a maximum of 4.62g/g of the fresh weight(Wrbel et al., 2004). No growth retardation wasobservedin the transgenic lines. Interestingly, seed production wasenhanced in transgenic lines by a factor of two whencompared to non-transgenic control plants. Furthermore,there were changes in fatty acid composition of the seedoil, which included a decrease in alpha-linolenic acidcontent. This is the most abundant fatty acid in flax seedoil. The transgenic plants also contained less starch in the

    chloroplasts and had lower levels of glucose. Thepresence of PHB in stems improved the elastic propertiesof the fibers. The Young's modulus Evalue, a measureof stem tissues resistance to tensile loads, increased up to2-fold (54.4 MPa) in transgenic plants. This researchdemonstrated the feasibility and potential of producingeffective biocomposites from stem tissue containingrelatively small amounts of PHB.

    5.3. PHA synthesis in peroxisomes

    Peroxisomes are 0.11 m organelles that arebounded by a single lipid bilayer (Brown and Baker,

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    2003). Plant peroxisomes can be categorized asglyoxysomes, leaf peroxisomes and unspecializedperoxisomes. Glyoxysomes are specialized peroxi-somes involved in the -oxidation of fatty acids anddirecting acetyl-CoA to carbohydrate to generate

    soluble carbohydrate to support seedling growth(Weselake, 2005). Since these organelles producehigh levels of acetyl-CoA through -oxidation, theyrepresent a logical subcellular compartment forproduction of PHA. In addition, 3-hydroxyacyl-CoAintermediates in the-oxidation pathway can be used asa precursor in the synthesis of PHAs.Hahn et al. (1999)engineered Black Mexican sweet maize to producePHB by introducing three phagenes fromR. eutropha.The construct was designed to target the gene productsto peroxisomes. For the targeting process, amino acid

    residues RAVARL were added to the carboxy terminusof the proteins. PHB accumulated up to 2% dwt intransformants expressing all three enzymes. They haveproposed an equilibrium effect to explain this unex-pected existence of (R)-3-hydroxybutyryl-CoA in plantperoxisomes.

    While R. eutropha produces PHB, P. aeruginosaproduces mcl-PHAs. P. aeruginosa has a differenttype of PHA synthase (phaC1), which uses 3-hydroxyacyl-CoA intermediates from the -oxidationpathway as the substrates (Fig. 9). mcl-PHAs usually

    have monomers with 2n (n0) carbons shorter thanthe substrates. To make the construct, phaC1 genefrom P. aeruginosa was fused to a DNA segmentencoding the last 34 amino acids ofB. napusisocitratelyase (ICL), in order to target the enzyme to leafperoxisomes and glyoxysomes in cotyledons (Fig. 7e).The construct was first introduced intoArabidopsis. APTS1 sequence, the terminal amino acids of peroxi-somal target sequence, is present in B. napus ICL.Expression of the transgene was driven by theconstitutive CaMV35S promoter. Transgenic plants

    grown in light accumulated mcl-PHAs with C6

    C16monomers in peroxisomes, whereas the biopolymeraccumulation took place in glyoxysomes when growthoccurred in the dark (Mittendorf et al., 1998). Theamount of accumulated mcl-PHAs depended on theactivity level of the -oxidation cycle at different

    Fig. 9. Genetically engineered metabolic pathways of scl- and mcl-PHA formation in plant peroxisomes. Expressions of transgenes are indicated by

    dashed arrows. phaCfrom A. caviae and P. aeruginosa encode PHA synthases that use (R)-3-hydroxyacyl-CoA from fatty acid degradation assubstrate for scl- and mcl-PHA polymerization, respectively.

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    stages of development. The activity of the-oxidationand glyoxylate cycle is high during seed germinationand leaf senescence when fatty acids are converted tocarbohydrates, and the level of activity drops graduallyas the photosynthesis commences in seedlings (Ger-

    hard, 1993). The highest level of PHA was detected in7-day old seedlings with 0.4% of the dwt. PHA levelwas low in leaves (0.02% dwt), but graduallyincreased from 2- to 3-fold during leaf senescence.The PHA inclusions were similar in appearance tobacterial inclusions. The PHA inclusions were alsoobserved in vacuoles, which indicated that PHAscould be transported through single-membrane boundorganelles (both peroxisomes and vacuoles havesingle-membranes). No adverse effects on plantgrowth and seed germination were observed. The

    peroxisomes were substantially larger in size in trans-genic plants and the PHAs accumulated in them werecomposed of 14 different saturated and unsaturatedmonomers of 6 to 16 carbons. These monomers couldbe clearly linked to the corresponding 3-hydroxacyl-CoA generated by the -oxidation of fatty acids.

    PHA synthase utilizes theR-isomer of 3-hydroxyacyl-CoA. The process of -oxidation, however, onlygenerates the S-isomer of 3-hydroxyacyl-CoA (Grahamand Eastmond, 2002). The accumulation of a variety ofmcl-PHAs in transgenic plants suggested that plant sys-tems contained enzymes that could convert the S-isomer

    into the R-isomer. (R)-3-hydroxylacyl-CoA epimeraseand enoyl-CoA hydratase II present in the plant multi-functional protein (MFP) could potentially be involved inthis conversion (Mittendorf et al., 1998; Preisig-Mlleret al., 1994) (Fig. 9). A third route for R-isomer pro-duction could be hydration of 2-cis-enoyl-CoA bythe enoyl-CoA hydratase I activity (Schulz, 1991). Inbacteria, (R)-3-hydroxyacyl-CoA is generated by a3-hydroxyacyl-CoA epimerase, an R-specific enoyl-CoA hydratase II, or a 3-ketoacyl-CoA reductase(Steinbchel, 1991)(Fig. 4). However, yeast only con-

    tains an enoyl-CoA hydratase II (Hiltunen et al., 1992).Early attempts at producing mcl-PHAs (C6C14)resulted in biopolymers that were too elastic. Theycontained a high proportion of monomers larger than 10carbons, and a high proportion of unsaturated mono-mers. Such PHA polymers have a low melting point andbehave like glue at room temperature. Increasing theproportion of shorter chain length monomers (e.g., C4C8) in PHA copolymer can solve this problem. PhaCsynthase from A. caviae, which produces scl-PHAcopolymers with C3C7 hydroxy fatty acids, was in-troduced into Arabidopsisin an attempt to improve thequality of the resulting biopolymer (Arai et al., 2002).

    The expression of transgenes was driven by theCaMV35S promoter. The construct contained a DNAsegment encoding a 10 amino acid stretch from thecarboxy-terminus of spinach glycolate oxidase attachedto direct the enzyme to peroxisomes. Transgenic plants

    produced scl-PHA copolymers P(HBHVHH) con-taining C4C6 monomers with improved properties.This type of scl-PHA copolymer has better commercialpotential because it is more flexible than PHB and lesselastomeric than mcl-PHA. scl-PHA accumulated up to441.7 g/g (0.044%) of the leaf dwt. There was a widerange of scl-PHA accumulation among different organsand stages of development, suggesting different activitylevels of-oxidation throughout the plant. -oxidationlevels are high during seed germination and reducedafter the commencement of photosynthesis. This met-

    abolic difference was reflected in a high level of PHAaccumulation (120 g/g of the dwt) in 7-day oldseedlings and only 2.2g/g in the leaves of 28-day oldplants. The level of PHA increased to 230g/g in thesenescing leaves of 60-day old plants when -oxidationincreased again.

    5.4. Modulating the quantity and monomer composition

    of PHA in transgenic plants

    It may be possible to modulate PHA accumula-tion in the peroxisome by altering the carbon flux to the-oxidation cycle because PHA synthesis draws uponintermediates of fatty acid degradation. Indeed, produc-tionofPHAhasbeenusedasatooltoanalyzecarbonflowthrough the -oxidation cycle (Poirier, 2002). Plastidialacylacyl carrier protein (ACP) hydrolase catalyzes theremoval of the growing fatty acid chain from the fattyacid synthase complex (Ohlrogge and Browse, 1995).Previous studies withB. napusexpressing California bay(Umbellularia california) lauroyl-ACP hydrolaserevealedthat developing seeds accumulating lauric acid intriacylglycerol produced a substantial portion of lauric

    acid that could be recycled through the -oxidationpathway (Eccleston et al., 1996). These results suggestedthat expression of an acyl-ACP hydrolase specific to aparticular fatty acid might be a way of increasing carbonflux towards -oxidation and peroxisomal PHA synthesisderived from that particular fatty acid. A transgenic Ara-bidopsis line co-expressing a acyl-ACP hydrolase fromCuphea lanceolata along with PHA synthase produced8-fold more PHA in plant shoots than transgenic linescarrying PHA synthase alone (Mittendorf et al., 1999).The mcl-PHA in the transgenic plant carrying both geneswas enriched in saturated 3-hydroxyacid monomers con-taining 10 carbons or less (40 mol% 3-hydroxydecanoic

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    acid, 32 mol% hydroxyoctanoic acid and 4 mol%hydroxyhexanoic acid). This result suggested that expres-sion of acyl-ACP hydrolase channeled decanoic acidstowards peroxisomal-oxidation (Fig. 10).Poirier et al.(1999) extended the strategy of synthesizing mcl-PHA

    enriched with specific polymers in vegetative tissues todeveloping seeds. In mature seeds ofArabidopsis, theinvestigators obtained up to 0.1% dwt in PHA copoly-mers. These results demonstrated that manipulation ofgenes involved in the synthesis of unusual fatty acidscould be used to modulate the quantity and monomercomposition of mcl-PHAs. The amount of PHA synthe-sized in peroxisomes (b1% dwt) by co-expressing acyl-ACP hydrolase and PHA synthase was still much lowerthan the amount of PHB that could be synthesized inplastids (40% dwt). Further studies on mcl-PHA synthesis

    inS. cerevisiaeand P. pastorismight eventually provideinsight into this metabolic limitation on PHA productionin plant peroxisomes (Poirier et al., 2001, 2002).

    Placing limitations on triacylglycerol accumulationin developing seeds has also been shown to affect the

    amount of PHA formed in peroxisomes. Poirier et al.(1999)conducted studies on PHA formation using thetag1 mutant of Arabidopsis, which is deficient indiacylglycerol acyltransferase (DGAT) activity. The de-creased incorporation of fatty acids into triacylglycerolresulted in an increased level of free fatty acids in seeds(Fig. 10). It was anticipated that excess fatty acids wouldbe channeled towards -oxidation resulting in an in-creased availability of intermediates for mcl-PHAsynthesis. Indeed, when the peroxisomal-targeted PHAsynthase wasexpressedin this mutant, there was a 10-fold

    Fig. 10. Strategies of synthesizing PHA in peroxisomes with special monomers, using lipid biosynthesis mutant (e.g. DGAT mutant); co-expressingfatty acid biosynthetic genes (e.g. acyl-ACP hydrolase); exogenous feeding of special fatty acids, etc. (adapted from Poirier, 2002).

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    increase in mcl-PHA in the transgenic seeds compared toexpression of the PHA synthase in the wild-type plant(Poirier et al., 1999).

    Another mutant ofArabidopsis, known asfad3/fad7/fad8, was engineered to obtain PHA with specific

    monomers. This triple mutant is deficient in synthesis oftri-unsaturated fatty acids. mcl-PHA produced in thismutant was almost completely deficient in all 3-hydroxyacids derived from the degradation of tri-unsaturated fatty acids (Mittendorf et al., 1999). Theseresults suggested that it is possible to modulate themonomer composition of PHA in engineered plants byaltering the degree of unsaturation of fatty acids.

    Another approach used to alter the monomercomposition of PHA synthesized in transgenic plantsinvolves the exogenous application of specific fatty

    acids (Fig. 10).Mittendorf et al. (1999)grew transgenicArabidopsisplants expressing a peroxisomal-targetedP.aeruginosa PHA synthase gene in liquid mediumsupplemented with various fatty acids. This resulted ina significant increase in mcl-PHA containing monomersderived from the -oxidation of the externally suppliedfatty acids. For example, addition of tridecanoic acid(C13:1 12) resulted in the production of PHAcontaining mainly unsaturated odd-chain monomers.In contrast, adding Tween-20 (polyethylene sorbitanmonolaurate) to the growth medium resulted in theincorporation of even-numbered, saturated C6C12

    monomers derived from Tween-20. The investigatorsreported that Tween-20 induced the activity of MC-acyl-CoA oxidase (ACOX) and LC-ACOX. More recently,Arai et al. (2002) demonstrated that application ofTween-20 also resulted in 4-fold increase in scl-PHA inArabidopsis plants expressing an A. caviae PhaCsynthase in the peroxisomes. This PHA synthasecatalyzes the production of scl-PHA copolymers. Theincreased synthesis of scl-PHA in these Arabidopsisplants was due to incorporation of monomers derivedfrom fatty acid moieties of Tween-20 that have been

    shunted through -oxidation cycle. Thus, it was sug-gested that Tween-20 activated -oxidation of scl-fattyacids and also probably induced SC-ACOX activity.

    Although the yield of PHA in these experiments didnot reach a commercially viable level, the resultsdemonstrated that PHA quantity and monomer compo-sition could be altered in the plant peroxisome bysupplying specific fatty acid substrates to the -ox-idation cycle. As well, these investigations demonstrat-ed that the plant -oxidation cycle is capable ofgenerating a large spectrum of monomers from fattyacids that are not naturally present in plants. Therefore,this method can be utilized in the analysis of the deg-

    radation of an unknown or unusual fatty acid in plants(Daae and Dunnill, 1999; Allenbach and Poirier, 2000;Poirier, 2002; Moire et al., 2004). Applying exogenousfatty acids is not a cost-effective option for PHAproduction in plants. The results of experiments with

    exogenously applied fatty acids, however, can provideus with clues on how we might alter fatty acidproduction in plant systems to generate PHAs of desiredmonomer composition.

    There appears to be a major metabolic advantage toproducing PHA in the peroxisome. Carbon used for PHAsynthesis in the peroxisome is not diverted from anabolicpathways involved in the synthesis of essential com-pounds like fatty acids and amino acids, but instead isderived from catabolic pathways. Therefore, a high levelof PHA synthesis in the peroxisome might not have as an

    adverse effect on plant growth and development as itwould in a transgenic plant system that generated PHAsfrom precursors produced through anabolic pathwayssuch as fatty acid synthesis in the plastid.

    5.5. Barriers to increasing PHA production in plants

    Production of PHA in transgenic plants has barriersassociated with expression of transgenes and metabolicload on plant growth. The constitutive expression of oneof the PHA synthesis genes (phaA) is considered acrucial obstacle (Poirier et al., 2000; Bohmert et al.,

    2002). Bohmert et al. (2002) demonstrated thatconstitutive expression of phaA was detrimental toplant growth as early as during the transformation steps.Expression of this gene was responsible for drasticreduction of transformation efficiency in potato andtobacco. It was suggested that the toxic effects ofphaAcould result from PHB biosynthesis intermediates, thedepletion of acetyl-CoA pool, or unexpected interac-tions between -ketothiolase with other substrates in theplastids. Preventing the expression of phaA duringtransformation/regeneration procedure by using induc-

    ible promoter allowed the generation of transformants.Use of inducible promoter also resulted in two-foldincrease in PHB production without alteration inphenotype, compared toArabidopsislines constitutivelyexpressing the phaA gene (Bohmert et al., 2000).However, use of inducible promoter in potato andtobacco, although resulted in generation of transfor-mants, the amount of PHB formed was neverthelessrather low (b0.1% dwt). This suggested that what wastrue forArabidopsismight not necessarily true for otherplant species.

    Several approaches were undertaken to improvethe yield of PHB in plants. Acetyl-CoA is not only a

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    substrate in PHB synthesis, but this thioester is a crucialsubstrate in several metabolic pathways includingflavonoid and isoprenoid synthesis in cytoplasm, andfatty acid synthesis in plastids (Figs. 6, 8). Specificenzyme inhibitors were used to suppress these anabolic

    pathways in order to increase the availability of acetyl-CoA for PHB production (Suzuki et al., 2002).Quizalofop (an herbicide) inhibits acetyl-CoA carbox-ylase, which converts acetyl-CoA to malonyl-CoA.Application of Quizalofop was shown to increase PHBproduction in the cytosol by 170% (from 0.035% dwt to0.06% dwt) and in plastids by 150% (from 0.055% dwtto 0.085% dwt). Mevastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMGR), which has arole in the mevalonic acid synthetic pathway in cytosol(Fig. 6). Application of 1 M of mevastatin increased

    PHB production in cytoplasm by 190% (from 0.035%dwt to 0.065% dwt) (Suzuki et al., 2002), andinterestingly, no effect on plant growth was observed.This suggests increasing the availability of acetyl-CoAin plant cells might be a strategy to improve PHByield.

    6. PHA extraction

    The extraction of bioplastic from biomass poses yetanother challenge. There are two common protocolsused for PHA extraction from bacteria. The conventional

    one is based on the solubility of PHA in chloroform andinsolubility in methanol (Kessler et al., 2001). Afterharvest, lipids and other lipophilic components in thebacterial cells are removed by reflux in hot methanolfollowed by solubilization of PHA in warm chloroform.PHA from chloroform solvent can be recovered bysolvent evaporation or precipitation by addition ofmethanol. Although highly purified PHA is obtainedby this method, a large amount of hazardous solvent isneeded to repeat the same process. Thus, this method isnot environmentally friendly and unsuitable for mass

    production of bioplastic (Byrom, 1987). The secondprotocol is designed to avoid the use of organic solvents.Bacterial cells are treated with a cocktail of enzymes(including proteases, nucleases and lysozymes) anddetergents to remove proteins, nucleic acids, and cellwalls, leaving the PHA intact (Byrom, 1987).

    In the large scale production of PHA in crops, theextraction and purification of PHA from biomass is acritical factor for determining the practical feasibility ofthe technology. It is important that PHAs from transgenicplants can be extracted efficiently and easily, much likethe extraction of endogenous compounds, such as starch,sucrose and oil. Unlike extractions of bacteria, which are

    specifically intended for PHA production, there are otheruseful byproducts that can also be extracted fromharvested crops. Any extraction process from planttissue should accommodate extraction of such com-pounds in unmodified form. The conventional methods

    used for extraction of low molecular weight lipids are notapplicable for bioplastic produced in plant cells. Unlikeseparating vegetable oils from oilseeds, PHAs cannot besqueezed from the seeds by applying mechanicalpressure. Solvent extraction is also difficult because theresulting polymer solution is extremely viscous, makingthe solution very difficult to work with. Also, theremoval of solvent from the polymer is a slow anddifficult process, and separations based on sedimentationare extremely slow.

    On a laboratory scale, the extraction of PHA from

    plant tissue has usually relied on the same method usedto extract the polymer from bacteria (i.e., chloroformand methanol). Components of the recovered PHA arethen analyzed using various analytical methods such asgas chromatography/mass spectrometry. There are nopublications available in scientific literature regardinglarge scale PHA extraction from plant tissues. There aremethods, however, based on both solvent and non-solvent procedures issued in the form of patents (Poirier,2001).

    In oilseeds, the oil was recovered separately fromPHA, and the residual seed meal was used as animal feed

    (Noda, 1997, 1998a). The seeds containing both oil andPHA were crushed, and the oil was obtained by acombination of pressing and extraction with a solventthat did not dissolve PHA (e.g., hexane). The defattedcompounds containing PHA were then extracted with asolvent that solubilized PHA, leaving the meal bypro-duct behind that was rich in protein. By increasing thetemperature and pressure, and with the right choice ofPHA copolymer, a range of less hazardous PHA-poorsolvents was used to extract PHA. For example, P(HBHV) became soluble in methanol when the temperature

    was raised to 120 C. After solubilization, PHA wasrecovered by cooling, adding non-solvent and evaporat-ing the solvent (Martin et al., 1997; Noda, 1997, 1998a;Kurdikar et al., 1998, 2000). PHA was also chemicallytransformed into a PHA derivative and separated fromthe mixture using distillation, extraction or chromatog-raphy (Martin et al., 2004). The solvent extractionmethod was further improved by the use of marginalnon-solvents, such as alkanes, alcohols or even oil(Noda, 1998a). Marginal non-solvents (e.g., acetone) donot effectively dissolve PHA by themselves, but becomecompetent when mixed with a PHA solvent. Both oiland PHA were extracted from the seeds by crushing in

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    acetone. After the removal of acetone, oil, which alonecannot dissolve PHA polymers, became an effectivesuspending medium for the precipitating PHA. Horowitzand Brennan (2002)recommended the use of ozone inPHA-containing slurries and suspensions, either in water

    or in solvent, to obtain odor-free PHA having anenhanced level of purity.

    Similar to non-solvent PHA extraction from bacteria,the use of a cocktail of enzymes has also been suggestedfor extraction of PHA from plant tissues (Liddell, 1997).Oil in the crushed seeds containing PHA was firstrecovered by hexane. The defatted meal was then treatedwith enzymes, surfactants,oxidizing agents and detergentsto digest the non-PHA components such as carbohydrates,proteins and nucleic acids. The PHA granules wererecovered by decantation, filtration andcentrifugation. It is

    not clear, however, if this protocol would be realistic andcost effective in an industrial process.Two new methods for PHA extraction have been

    described which were based on wet and dry millingmethods used in the corn industry. One method is basedon air classification, which separates dry solid compo-nents according to weight and/or size (Noda, 1998b).The other method is based on centrifugal fractionation,which separates particles in solution based on size and/or density (Noda, 1999). Because PHA granules are thesmallest particles (0.21 m) present in plant cells,compared to starch grains or protein bodies, this size

    difference can be used to separate them from the otherplant components.

    7. Conclusions and future prospects

    Research into theproductionof PHAs as petrochemicalalternatives for the future has been explored usingbacterial and plant systems. Great advances have beenmade in the protein engineering of PHA synthases to altertheir specificity properties so as to produce PHAs withdesired monomer composition. Bacterial fermentation,

    however, relies on external carbon sources such asglucose. Synthesis of PHA in plants, which relies oncarbon dioxide and light, represents a more cost-effectiveapproach to produce thisbiopolymer in large quantities. Inplant systems, several ingenious approaches have beenused to capture intermediates of carbon catabolism andconvert them into PHA. Different compartments of theplant cell and different tissues of the plant have beenexamined for their suitability in producing and storingPHA granules. A major challenge in producing commer-cially viable levels (N15% dwt) of PHA in plant tissue isbeing able to do this without compromising the normalgrowth and development of the plant. Production of PHA

    in agricultural crops is likely to be economically viable if itcan be produced as a byproduct with some other plantconstituent such as oil or starch. For example, if PHA isproduced in an oilseed crop, the bioplastic can potentiallybe recovered along with the oil fraction leaving the

    remaining meal for use as animal feed. At this point, levelsof up to 8% dwt of the seed are possible withoutdeleterious effects on plant growth (Houmiel et al., 1999).Improving the yield of PHA in plants along with a desiredmonomer composition represents an ongoing challenge(Snell and Peoples, 2002). More efficient methods need tobe developed for multiple-gene transformation. Plastidtransformation might be an alternative for multiple-gene transformation because the plastidial genomes arematernally inherited. Although previous attempts atplastidial transformation resulted in very low accumu-

    lation of PHB, new methods of high-level expressiondesigned for this organelle (De Cosa et al., 2001) holdpromise. Since high levels of PHA disrupt chloroplastfunction, it might be worthwhile attempting to increasethe number of chloroplasts per cell to obtain more PHAper g tissue.

    The cost of mass production of PHAs in bacteria isprohibitive except for certain specialty bioplastics usedin medical applications. The synthesis of PHA inbacteria and subsequent extraction of the biopolymeris estimated to cost approximately 34 US$/kg (Leeet al., 1997). This is 510 times more expensive than

    petroleum based polymers, such as polypropylene(b1 $/kg). In this context, the synthesis of PHA incrop plants can be regarded as a promising alternativefor the large-scale and low cost production of thispolymer. Producing PHA in plants, however, will likelybe more expensive than producing corn starch orsoybean oil, which cost in the range of 0.250.50 US$/kg. Expenses associated with PHA extraction fromplant sources will represent a relatively large componentof the total cost of producing this polymer in the plant.Although a few methods have been developed for large-

    scale extraction of recombinant proteins from plants(Menkhaus et al., 2004), the extraction and processingof PHA from plant tissue still requires considerableresearch.

    Acknowledgement

    The authors thank the Alberta Crop IndustryDevelopment Fund and Alberta Agriculture ResearchInstitute for supporting this research. R. Weselakeacknowledges the support of a Discovery Grant fromthe Natural Sciences and Engineering Research Councilof Canada and the Canada Research Chairs Program.

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