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GENETIC MODIFICATION OF PRIMARY METABOLISM | Biopolymers
GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers 477 Products: Epicuticular Waxes; Wax Pathways. Seed Development: Germination. Water Relations of Plants: Uptake, Loss and Control.
Further Reading Ackman RG (1990) Canola fatty acids – An ideal mixture for health, nutrition, and food use. In: Shahidi F (ed.) Canola and Rapeseed – Production, Chemistry, Nutrition, and Processing Technology, pp. 81–98. New York: Van Nostrand Reinhold. Gunstone FD (2001) Oilseed crops with modified fatty acid composition. Journal of Oleo Science 50: 3–13. Hildebrand DF, Rao SS, and Hatanaka T (2002) Redirecting lipid metabolism in plants. In: Kuo TM and Gardner HW (eds) Lipid Biotechnology, pp. 57–84. New York: Marcel Dekker. Kinney AJ (2002) Perspectives on the production of industrial oils in genetically engineered oilseeds. In: Kuo TM and Gardner HW (eds) Lipid Biotechnology, pp. 85–93. New York: Marcel Dekker. Miege C and Marechal E (1999) 1,2-sn-Diacylglycerol in plant cells: Product, substrate and regulator. Plant Physiology and Biochemistry 37: 795–808. Murphy DJ (2002) Biotechnology and the improvement of oil crops – genes, dreams and realities. Phytochemistry Reviews 1: 67–77. Schultz DJ and Ohlrogge JB (2002) Metabolic engineering of fatty acid biosynthesis. In: Kuo TM and Gardner HW (eds) Lipid Biotechnology, pp. 1–25. New York: Marcel Dekker. Slabas AR, Hanley Z, Schierer T, et al. (2001) Acyltransferases and their role in the biosynthesis of lipids – opportunities for new oils. Journal of Plant Physiology 158: 505–513. Thelen JJ and Ohlrogge JB (2002) Metabolic engineering of fatty acid biosynthesis in plants. Metabolic Engineering 4: 12–21. Topfer R, Martini N, and Schell J (1995) Modification of plant lipid synthesis. Science 268: 681–686. Voelker T and Kinney AJ (2001) Variations in the biosynthesis of seed-storage lipids. Annual Review of Plant Physiology and Plant Molecular Biology 52: 335–361.
Biopolymers Y Poirier, University of Lausanne, Lausanne, Switzerland Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction Plants naturally synthesize a variety of polymers that have been used by mankind as a source of useful
biomaterials. For example, cellulose, the main constituent of plant cell wall and the most abundant polymer on earth, has been used for several thousand years as a source of fibers for various fabrics. Similarly, rubber extracted from the bark of the tree Hevea brasiliensis, has been a major source of elastomers until the development of similar synthetic polymers. In the last century, the usefulness of plant polymers as biomaterials has been expanded through the chemical modification of the natural polymers. For example, a number of plastics have been made by substituting the hydroxyl groups present on the glucose moiety of cellulose with larger groups, such as nitrate or acetate, giving rise to materials such as cellulose acetate, a clear plastic used in consumer products such as toothbrush handles and combs. Similarly, starch has been used in the manufacture of plastics by either using it in blends with synthetic polymers or as the main constituent in biodegradable plastics. The advent of transformation and expression of foreign genes in plants has created the possibility of expanding the usefulness of plants to include the synthesis of a range of biomolecules. In view of the capacity of certain crops to produce a large quantity of organic raw material at low cost, such as oils and starch, it is of interest to explore the possibility of using transgenic plants as efficient vectors for the synthesis of biopolymers. Such plant based biopolymers could replace, in part, the synthetic plastics and elastomers produced from petroleum, offering the advantage of renewability and sustainability. Furthermore, being natural products, biopolymers are usually biodegradable and can thus contribute to alleviate problems associated with the management of plastic waste. In this article, the emphasis will be on the use of transgenic plants for the synthesis of two novel classes of industrially useful polymers, namely protein based polymers made from natural or artificial genes, and polyhydroxyalkanoates, a family of bacterial polyesters having the properties of biodegradable plastics and elastomers.
Protein Based Polymers A number of proteins with interesting plastic or adhesive properties are synthesized in nature. For example, marine mussels (Mytilus spp.) can produce strong protein based adhesives that are effective in aqueous environments, allowing these organisms to stick to many surfaces. Several proteins composed of blocks of amino acids repeated extensively form tough and flexible fibers that could find applications in many areas of material science, including in the medical field. Such proteins include silk, collagen,
478 GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers
Silks represent a broad class of polymers that can be loosely defined as externally spun fibrous protein secretions. Commercial silk used in the textile industry is produced from the domesticated worm Bombyx mori. Spiders also produce a variety of silks that are used in the construction of webs or as draglines. The different types of spider silks produced have impressive properties, ranging from lycra-like elastic fibers to kevlar-like superfibers. The exceptional material properties of spider silk have made it an early target for its synthesis in larger quantities in a more amenable production system. Cloning of the spidoin proteins forming the dragline of the spider Nephila clavipes enabled the exploration of their synthesis in Escherichia coli. However, expression of a synthetic gene coding for a protein containing highly repeated blocks of amino acids found in spidoins led only to low production of a mixture of proteins of variable sizes. This heterogeneity in the proteins synthesized was due to premature termination of translation. In contrast, expression of a similar gene construct in the yeast Pichia pastoris, as well as in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum), resulted in the synthesis of a higher amount (between 2 and 10% of total proteins) of a full length protein, indicating that, at least in these eukaryotes, the repetitive nature of the gene construct was not an obstacle to the synthesis of these unusual proteins. Several synthetic genes containing various combinations of the repetitive peptides found in the
Synthesis of Artificial Protein Polymers
Elastin is a very strong natural fiber found in ligaments and arterial walls. Analysis of the primary structure of all mammalian elastins has shown the presence of a repeated block of amino acids with the sequence GVGVP. Synthetic proteins made from multiple repeats of this sequence display elastic properties. These protein polymers are also biodegradable as well as biocompatible, being nontoxic and naturally resorbed by animal tissues. Polymers based on the GVGVP sequence have been shown to prevent postsurgical adhesions and scars in rats. Other medical applications of similar bioelastics include tissue reconstruction, wound covering, and programed drug delivery. Expression in E. coli of a synthetic protein made of 121 repeats of the GVGVP peptide was shown to result in accumulation of polymer inclusion bodies that occupied up to 90% of the cell volume. However, expression of the same synthetic gene in the fungus Aspergillus nidulans, as well as in tobacco, only led to low polymer accumulation despite adequate accumulation of mRNA. It is thought that efficient expression in eukaryotes of such proteins made of repeated blocks of peptides is limited by the availability of certain amino acids or tRNAs. Thus, accumulation of high levels of artificial protein polymers in plants may be dependent on additional genetic engineering, in particular, modifications in the amino acid biosynthetic pathways and/or tRNA pools.
Polyhydroxyalkanoates Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids synthesized by a wide variety of bacteria (Figure 1). The best-characterized PHA is polyhydroxybutyrate (PHB), a homopolymer of 3-hydroxybutyrate. Although initially described by M. Lemoigne in 1926, it was not until 1962 that the value of PHB as a thermoplastic was recognized in a US patent, and not until 1982 when the first PHA produced by bacterial fermentation was
R
O − −
Synthesis of Silk Proteins
N. clavipes spidoin protein have been expressed in tobacco, allowing an assessment of the link between protein structure and physical properties.
−
and elastin. Plant cell walls also contain proteins that are partially composed of repeated blocks of amino acids, such as extensin, the glycine rich proteins, and the proline rich proteins. The mechanical properties of the pure form of these natural plant proteins have not been well studied. The ability to synthesize protein polymers from genes allows the precise determination of both molecular mass and amino acid sequence, making it possible, in principle, to achieve a degree of control over physical properties and functionality that is outside the scope of chemical polymerization technologies. The development and use of these protein based biomaterials are, in many cases, limited by the difficulties in producing sufficient quantities of the material to establish the structure/function relationships and design applications, as well as difficulties in the economical large-scale production of polymers for commercialization. It is in this respect that plants have been explored as a potential production vector for protein based biopolymers.
− (− O − CH − CH2 − C −)n − R = methyl 3-hydroxybutyrate R = ethyl 3-hydroxyvalerate Figure 1 Chemical structure of 3-hydroxyacid monomers constituting PHA. The monomer can range from 3 to 16 carbons in length, depending on the size of the pendant R group.
GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers 479
commercialized. Since then, there has been a growing interest in producing and commercializing PHA as a family of biodegradable thermoplastics and elastomers. Synthesis of PHA in Bacteria
PHAs represent a large group of bacterial polyesters, which can include over 150 different hydroxyacids. Although the majority of PHAs are composed of R-3-hydroxyacids ranging from 3 to 16 carbons in length, some polymers also contain monomers of 4-hydroxy and 5-hydroxyacids. Furthermore, a diversity of functional groups have been found on the monomers forming PHAs, including unsaturated groups, as well as methyl, halogenated, cyano, and phenoxy groups, just to name a few. The majority of bacteria synthesizing PHAs can be broadly subdivided into two groups. One group produces shortchain-length PHAs (SCL-PHAs) with monomers ranging from 3 to 5 carbons in length, while a distinct group synthesizes medium-chain-length PHAs (MCL-PHAs) with monomers from 6 to 16 carbons. This classification is, however, not strict as a few bacteria have been shown to produce ‘‘hybrid’’ PHAs with monomers ranging from 4 to 10 carbons or higher. PHAs accumulate in bacteria as a highmolecular-weight polymer forming intracellular granules of 0.2–0.3 mm in diameter. Typically, PHAs accumulate to a significant proportion of the cell dry weight when bacteria are grown in a media that is limited in a nutrient essential for growth (typically nitrogen or phosphorus), but with an abundant supply of carbon (e.g., glucose). Under these conditions, bacteria convert the extracellular carbon into an intracellular storage form, namely PHA. When the limiting nutrient is resupplied, intracellular PHA is degraded and the resulting carbon is used for growth. PHAs have been shown to occur in nearly 100 genera of bacteria, encompassing Gram-positive and Gramnegative species, as well as in some archea. The large diversity of monomers found in PHA translates into a wide spectrum of physical properties. The homopolymer PHB is a relatively stiff and brittle plastic with limited applications. In contrast, PHA copolymers composed primarily of 3-hydroxybutyrate with a fraction of longer chain monomers, such as 3-hydroxypentanoate, 3-hydroxyhexanoate, or 3-hydroxyoctanoate, are more flexible and tougher plastics. These PHAs can be used in a wide variety of consumer products, such as containers and bottles, as well as water resistant films. PHAs made of longer monomers, such as MCL-PHAs, are typically elastomers and sticky materials. Numerous bacteria and some fungi have been found to degrade extracellular PHA and metabolize
the breakdown products to CO2 and water. Degradation of extracellular PHA is made possible by the secretion of PHA depolymerases, enzymes catalyzing the hydrolysis of PHAs to monomers and oligomers. PHAs are thus biodegradable polymers, and products made from them can be degraded completely in a variety of environments. Degradation is particularly rapid in the microbe rich environment of a compost (typically 3–9 months for a bottle made of PHB). PHAs can also be degraded, albeit slowly, in animal tissues. Since the breakdown products are 3-hydroxyacids, which are naturally found in animals, PHAs are biocompatible, making them useful for some medical applications, such as implants, gauzes, and resorbable suture filaments. The main limitation in using bacterial PHAs as a source of biodegradable polymers is their production cost. Synthesis of PHAs in native or recombinant bacteria using cheap carbon sources, such as glucose or sucrose, has been estimated to cost a minimum of 2–4 US$/kg, making them 5–10 times more expensive than petroleum based polymers, such as polypropylene. Thus, although bacterial PHAs could be used in high-value applications where the cost of the material is small compared to the value of the final product, such as in medical products, bacterial PHA cannot compete in price with synthetic plastics for high-volume, low-value consumer products, such as packaging material and disposable items. It is in this context that agriculture has been regarded as a promising alternative for the production of PHA on a large scale and at low cost. In comparison to bacterial or yeast fermentation, crop plants are capable of producing very large amounts of a number of useful chemicals at low cost. For example, starch and lipids are two of the most industrially useful and versatile products harvested from crop plants. Starch is used extensively in the food industry and as a source of fermentation substrate for the production of a wide variety of chemicals, including polylactic acid. Several million tonnes of cornstarch is produced annually in the USA, with a market value of approximately US$0.25/kg, making it one of the cheapest commodity products. Plant lipids are also utilized by the food and nonfood industries. Nonfood industrial uses of lipids include the manufacture of soaps and detergents, paints, varnishes, lubricants, adhesives, and plastics, as well as biodiesel. The USA produces approximately 10 million tonnes of vegetable oil annually, with a market value of approximately US$0.50/kg. In view of the high productivity of crop plants, it was reasoned that if PHA could be synthesized in plants at a level comparable to reserve lipids in oilseed plants, i.e., approximately 10–40% dry weight, several million tonnes of PHA could
480 GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers
potentially be produced at a cost comparable to vegetable oil, making it competitive with petroleum derived plastics.
wt). Growth of these transgenic plants was however compromised, being severely stunted, although seeds could still be produced. It is thought that synthesis of PHB from acetyl CoA present in the cytoplasm leads to a limitation in the accumulation of isoprenoids and flavonoids, which are also synthesized from cytoplasmic acetyl CoA, resulting in a growth defect. PHB in transgenic plants accumulated in the form of intracellular granules with a size and appearance identical to bacterial PHA granules. Analysis of the chemical structure and physical properties of the polymer confirmed that PHA synthesized in plants was similar to the bacterial polymer. Similar low amounts of PHB synthesized in the cytoplasm of plants have also been demonstrated in tobacco, oilseed rape (Brassica napus), and cotton (Gossypium spp.) fiber cells (Table 1). In plants, biosynthesis of fatty acids from acetyl CoA occurs in the plastid. The plastid is therefore a site of higher flux of carbon through acetyl CoA compared to the cytoplasm. This flux is particularly
Synthesis of PHB in Plants
The first PHA produced in plants was the homopolymer PHB. In bacteria, PHB is synthesized from the condensation of two acetyl CoA by a 3-ketothiolase to form acetoacetyl CoA, which is subsequently reduced to R-3-hydroxybutyryl CoA by an acetoacetyl CoA reductase (Figure 2A). The R-3-hydroxybutyryl CoA is finally polymerized by the PHA synthase to form PHB. Expression of the PHB biosynthetic pathway in plants was first demonstrated in 1992 in the plant Arabidopsis thaliana. Constitutive expression of the acetoacetyl CoA reductase and PHA synthase from the bacterium Ralstonia eutropha into the cytoplasm of transgenic A. thaliana plant cells led to the production of PHB up to 0.1% of the shoot dry weight (dry
(B)
(A) Acetyl CoA
Acyl CoA
Acetyl CoA Malonyl CoA
Acetoacetyl CoA (phaB)
(phaB)
(phaCRe ) Isoprenoids
2-trans -enoyl CoA S-3OH-acyl CoA
Acetoacetyl CoA
3-Hydroxybutyryl CoA
Flavonoids
3-Ketoacyl CoA
(phaA)
R-3OH-acyl CoA
R-3-Hydroxybutyryl CoA
(phaC1Pa)
(phaCRe )
PHB
MCL-PHA
PHB
(phaC1Ac ) SCL-PHA
(C) Threonine (ilvA) 2-Ketobutyrate
Propionyl CoA PDC
Acetyl CoA (phaA)
(btkB) 3-Ketovaleryl CoA
Acetoacetyl CoA (phaB)
(phaB) R-3-Hydroxyvaleryl CoA
R-3-Hydroxybutyryl CoA
(phaCRe) Isoleucine
P(HB-HV)
(phaCRe) PHB
Figure 2 Modification of plant metabolic pathways for the synthesis of PHAs. (A) Synthesis of PHB from acetyl CoA in the cytoplasm; (B) synthesis of PHB, SCL-PHA, and MCL-PHAs in the peroxisome; and (C) synthesis of PHB and P(HB-HV) copolymer in the plastid. The pathways created or enhanced by the expression of transgenes are highlighted in bold, while endogenous plant pathways are in normal type. The various transgenes expressed in plants are indicated in parentheses. The phaA, phaB, and phaCRe genes encode a 3-ketothiolase, an acetoacetyl CoA reductase, and a PHA synthase from R. eutropha, respectively. The btkB gene encodes a second 3-ketothiolase isolated from R. eutropha, which shows high affinity for both propionyl CoA and acetyl CoA. The ilvA gene encodes a threonine deaminase from E. coli. The phaC1Pa and phaCAc genes encode PHA synthases from P. aeruginosa and A. caviae, respectively. While the PHA synthase from R. eutropha can polymerize 3-hydroxyacyl CoAs ranging from 3 to 5 carbons, the PHA synthases from A. caviae and P. aeruginosa can polymerize 3-hydroxyacyl CoAs ranging from 3 to 7 carbons and 6 to 16 carbons, respectively. PDC refers to the endogenous plant pyruvate dehydrogenase complex.
GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers 481 Table 1 Summary of transgenic plants producing PHAs Subcellular compartment
Species
Tissue
PHA type
PHA quantity (%dry wt)
Cytoplasm
A. thaliana Oilseed rape Tobacco Cotton
Shoot Shoot Shoot Fiber
PHB PHB PHB PHB
Plastid
A. thaliana Oilseed rape Cotton A. thaliana Oilseed rape
Shoot Seed Fiber Shoot Seed
PHB PHB PHB P(HB-HV) P(HB-HV)
40 8 0.05 0.8 2.3
Peroxisome
A. thaliana
Whole plants
MCL-PHA
0.6
0.1 0.1 0.01 0.3
Figure 3 Accumulation of PHB granules in chloroplasts of transgenic A. thaliana. Granules are seen as agglomerations of electronlucent granules in the stroma of the chloroplast. Scale bar ¼ 1 mm.
enhanced in the seed of oil-accumulating plants, such as A. thaliana, where up to 40% of the seed dry weight is triacylglycerides. It was therefore hypothesized that the larger flux of acetyl CoA in the plastid may lead to a significant increase in polymer production in transgenic plants expressing the PHB pathway in chloroplasts. Constitutive expression of the PHB biosynthetic pathway in the chloroplasts of leaves of A. thaliana led to polymer accumulation up to 10–40% of the leaf dry weight. These experiments were done by modifying the R. eutropha enzymes 3-ketothiolase, acetoacetyl CoA reductase, and PHB
synthase for plastid targeting by fusing the transit peptide of the small subunit of ribulose bisphosphate carboxylase to the N-terminal end of the proteins (Figure 2C). Transmission electron micrographs revealed that the plastids contained numerous inclusions typical of PHB granules (Figure 3). The accumulation of PHB in leaves was found to increase over the life span of the plant, with fully expanded presenescing leaves typically showing ten times more PHB than younger expanding leaves of the same plant. Plants producing approximately 5–10% dry wt PHB showed little signs of reduced growth,
482 GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers
although chlorosis of leaves was observed, indicating that some aspect of chloroplast function was affected. In plants accumulating higher levels of PHB, a defect in growth was observed, being severe at 40% dry wt and accompanied by sterility. These experiments indicated that although the plastid was a much better site for the synthesis of PHB than the cytoplasm, accumulation of high amounts of PHB in such an important organelle as the chloroplast has its limits. In contrast to the chloroplast, the leukoplast of developing seeds is not involved in photosynthesis and its metabolism is simpler, being devoted mainly to the synthesis of reserve lipids and protein in the case of oilseed crops. Restriction of the expression of the PHB biosynthetic pathway to the leukoplast of developing seeds of oilseed rape resulted in accumulation of PHB up to 8% of the mature seed dry weight. These transgenic seeds had a normal size, appearance, and germination frequency. Furthermore, since the transgenes were not expressed in the vegetative tissues, growth of the plants was normal. Acetyl CoA, the building block of PHB, is found not only in the cytoplasm and plastid, but also in the mitochondria and peroxisomes, being implicated in these organelles in the tricarboxylic acid and b-oxidation cycles, respectively. Although no conclusive demonstration of PHB in plant mitochondria has been reported, synthesis of up 2% dry wt PHB was reported in transgenic black Mexican sweetcorn (Zea mays L. cv. Black Mexican Sweet) suspension cell cultures expressing the PHB biosynthetic pathway in the peroxisomes (Figure 2B). Further experiments are needed to explore the potential advantages and disadvantages of synthesizing PHB in the peroxisomes of plants growing in the field.
Synthesis of PHA Copolymers in Plants Since PHB is a polymer with relatively poor physical properties, being too stiff and brittle for use in most commodity products, successful production of PHA in crops depends on the synthesis of polymers having better physical properties. In the late 1970s, the copolymer poly(hydroxybutyrate-hydroxyvalerate) (P(HB-HV)) was identified as a valuable polymer with sufficient flexibility and impact resistance for its use as a commodity plastic. This co-polymer, marketed under the trade name BiopolTM, was first synthesized by bacterial fermentation using R. eutropha growing in media supplemented with propionate in order to create an intracellular pool of propionyl CoA that could be condensed to acetyl CoA to form 3-ketovaleryl CoA.
In order to synthesize P(HB-HV) in the plastids of plants, propionyl CoA was generated by modifying the branched amino acid biosynthetic pathway (Figure 2C). An E. coli threonine deaminase modified for plastid targeting was overexpressed in plants, leading to the accumulation of 2-ketobutyrate, which can be converted, albeit at low efficiency, to propionyl CoA by the plant pyruvate dehydrogenase complex. A novel 3-ketothiolase from R. eutropha showing high affinity for both propionyl CoA and acetyl CoA was also targeted to the plastids along with an acetoacetyl CoA reductase and a PHA synthase. Expression of all four genes in A. thaliana under the control of the constitutive CaMV 35S promoter resulted in the accumulation of P(HB-HV) of between 0.2% and 0.8% dry wt in shoots, with a 4–17 mol% HV content in the polymer. Seed specific expression of the same pathway in the leukoplast of oilseed rape embryos resulted in PHA accumulation up to 2.3% dry wt in seeds, with up to 6.5 mol% HV. Although these levels of P(HB-HV) copolymer production are lower than for PHB, synthesis of BiopolTM in plants is an important milestone on the route to commercialization of PHA-producing crops. Synthesis of the copolymer MCL-PHAs in several pseudomonads was shown to be mediated by two distinct pathways. In one pathway, the intermediate 3-hydroxyacyl-acyl carrier protein (ACP) of the fatty acid biosynthetic pathway is first converted to 3-hydroxyacyl CoAs by a transacylase before being polymerized by the PHA synthase. The creation of this pathway in the plastids of plant cells has not been successfully demonstrated to date. In the second pathway, the 3-hydroxyacyl CoA intermediates generated by degradation of fatty acids through the b-oxidation cycle are utilized by the PHA synthase to form MCL-PHAs (Figure 2B). Synthesis of MCLPHAs using intermediates of b-oxidation has been successfully demonstrated in A. thaliana expressing a PHA synthase from Pseudomonas aeruginosa modified at the C-terminal end by the addition of a tripeptide allowing targeting of the protein to the peroxisomes. MCL-PHA production in peroxisomes was, however, low, reaching a maximum of 0.4% dry wt in 7-day-old germinating seedlings. The monomer composition of the polymer was complex, including saturated and unsaturated monomers ranging from 6 to 16 carbons. Growth of these transgenic plants in liquid media supplemented with various fatty acids resulted in a significant increase in MCL-PHA containing monomers derived from the b-oxidation of the external fatty acids. For example, addition of either tridecanoic acid, tridecenoic acid (C13:1, D12), or 8-methyl-nonanoic acid resulted in the production of PHA containing mainly saturated
GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers 483
odd-chain, unsaturated odd-chain, or branchedchain 3-hydroxyacid monomers, respectively. These studies demonstrated that the plant b-oxidation pathway was capable of generating a large spectrum of monomers from fatty acids, which can be included in MCL-PHAs. In a further extension of the study of MCL-PHA synthesis in plant peroxisomes, it has been shown that an endogenous control over the flux of substrates toward b-oxidation and PHA synthesis could be achieved through the combined expression of a peroxisomal PHA synthase with an acyl ACP thioesterase. Expression of a plastidial caproyl ACP thioesterase from Cuphea lanceolata in A. thaliana was used to channel decanoic acids toward peroxisomal b-oxidation, resulting in a 8-fold increase in synthesis of MCL-PHA in mature leaves, with the polymer being composed of approximately 40 mol% 3-hydroxydecanoic acid, 32 mol% 3-hydroxyoctanoic acid, and 4 mol% 3-hydroxyhexanoic acid. This strategy initially applied to the synthesis of MCL-PHAs in vegetative green tissues was further extended to the synthesis of PHA in developing seeds of A. thaliana. In dry seeds, a maximal amount of 0.1% dry wt was detected. From these studies, a working model was established whereby enzymes and genes involved in the synthesis of unusual fatty acids in plants can be used to modulate the quantity and quality of substrates channeled toward MCLPHAs. The absolute amount of MCL-PHA accumulating in mature shoots or seeds of transgenic plants expressing both thioesterase and PHA synthase remains relatively low (p0.6% dry wt) compared to PHB synthesis in plastids (Table 1). The reasons for this difference could be multiple, including differential activity of bacterial enzymes in various plant subcellular compartments, and the relative ability of the created and endogenous metabolic pathways to compete for the same substrates (acetyl CoA for PHB and 3-hydroxyacyl CoAs for MCLPHAs). Synthesis of a small amount (0.04% dry wt) of SCL-PHA containing 3-hydroxyhexanoic, 3-hydroxyvaleric, and 3-hydroxybutyric acids has also been reported in A. thaliana expressing the PHA synthase from the bacterium A. caviae in the peroxisomes. Whereas the 3-hydroxyhexanoic acid and 3-hydroxybutyric acid monomers are derived from the b-oxidation of fatty acids, the source of the 3-hydroxyvaleric acid is unknown.
polymer as a thermoplastic or elastomer, a novel inventive use of PHA in plants was demonstrated by the expression of the PHB biosynthetic pathway in the cytoplasm of cotton fiber cells. In this system, the polymer is produced in plants only to change the physical properties of the fiber. Accumulation of 0.3% dry wt PHB in the cytoplasm of the fiber was shown to be sufficient to significantly decrease the rate of heat uptake and cooling of the fiber, resulting in a higher heat capacity and an improvement in the fiber insulating properties. Interestingly, in contrast to the large increase in accumulation of PHB in leaves of A. thaliana expressing the pathway in the chloroplast, expression of the PHB pathway in the plastid of the cotton fiber resulted in a lower amount of polymer, indicating a difference either in the number or metabolic activity of the plastids in fiber cells compared to leaves. It is also tempting to speculate whether the properties of wood, rubber, or starch could also be positively modified through the coaccumulation of PHAs.
Conclusions A spectrum of PHAs ranging from the stiff PHB to the more flexible P(HB-HV) plastics and MCL-PHA elastomers have now been successfully synthesized in plants. The challenge for the future is to succeed in high-level production (X15% dry wt) of a limited number of useful PHAs without a decrease in crop yield. Although quite complex, this task appears to be feasible. Initial experiments with expression of protein based polymers in plants indicate that, like PHAs, engineering of various metabolic pathways is also likely to be necessary before high-level accumulation of polymer is accomplished. For both protein based polymers and PHAs, synthesis of polymers for the modification of the properties of plant products, such as fibers, are attractive alternatives to the extraction of polymers for industrial uses. PHAs and protein based polymers are only two out of a large group of polymers produced in nature that have interesting properties. In the future, novel industrially useful polymers could perhaps also be developed through the in situ modification of natural plant compounds. See also: Genetic Modification of Primary Metabolism: Proteins. Primary Products: Proteins.
Further Reading PHA to Modify the Properties of Plant Fibers
Although the synthesis of PHA in plants is mainly focused on the extraction and utilization of the
Daniell H and Guda C (1997) Biopolymer production in microorganisms and plants. Chemistry and Industry 14: 555–558.
484 GENETIC MODIFICATION OF PRIMARY METABOLISM / Photosynthesis Hinman MB, Jones JA, and Lewis RV (2000) Synthetic spider silk: a modular fiber. Trends in Biotechnology 18: 374–379. Mittendorf V, Robertson EJ, Leech RM, et al. (1998) Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid b-oxidation. Proceedings of the National Academy of Sciences USA 95: 13397–13402. Mittendorf V, Bongcam V, Allenbach L, et al. (1999) Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through b-oxidation. Plant Journal 20: 45–55. Nawrath C, Poirier Y, and Somerville C (1995) Plant polymer for biodegradable plastics: cellulose, starch and polyhydroxyalkanoates. Molecular Breeding 1: 105–122. Poirier Y (2002) Polyhydroxyalkanoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism. Progress in Lipid Research 41: 131–155. Poirier Y, Nawrath C, and Somerville C (1995) Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants. Biotechnology 13: 142–150. Steinbu¨chel A (1991) Polyhydroxyalkanoic acids. In: Byrom D (ed.) Novel Biomaterials from Biological Sources, pp. 123–213. New York: MacMillan. Steinbu¨chel A and Fu¨chtenbusch B (1998) Bacterial and other biological systems for polyester production. Trends in Biotechnology 16: 419–427. Urry DW, McPherson DT, Xu J, et al. (1996) Protein-based polymeric materials: synthesis and properties. In: Salamone JC (ed.) The Polymeric Materials Encyclopedia: Synthesis, Properties and Applications, pp. 7263–7279. Florida: CRC Press. Yamamoto H (1995) Marine adhesive proteins and some biotechnological applications. Biotechnology and Genetic Engineering Reviews 13: 133–165.
Photosynthesis M J Paul and D W Lawlor, Rothamsted Research, Harpenden, UK Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction Photosynthetic organisms use the physical energy of light to form reduced compounds (‘‘reductants’’ ferredoxin and NADPH), and the energy source adenosine triphosphate (ATP): these are required for the synthesis of organic products, primarily sugars and amino acids, from inorganic starting materials, carbon dioxide (CO2), and nitrate (NO3 ) and sulfate (SO24 ) ions. This article considers the general mechanisms of photosynthetic CO2 assim-
ilation for plants with the C3 type of assimilation (Figure 1), with brief comments on the C4 type, and the feasibility of altering them by genetic modification to increase the photosynthetic rate (A). This depends on environmental conditions, particularly light, CO2 concentration and temperature, and on many genetically determined subprocesses. These may be separated into: (1) light reactions, including light capture, electron transport (et), and synthesis of reduced pyridine nucleotide (NADPH) and adenosine triphosphate (ATP), and (2) CO2 assimilation by the enzyme ribulose bisphosphate (RuBP) carboxylase/oxygenase (rubisco) in which RuBP, synthesized by the photosynthetic carbon reduction (PCR) cycle, is carboxylated (CO2 added) forming sugar phosphates. These are metabolized to sucrose. In addition, reaction of RuBP with oxygen (O2), a process called oxygenation, and also catalyzed by rubisco, results in photorespiration (PR), a major inefficiency in C3 photosynthesis. Regulation of these subprocesses is complex, with extensive interactions which determine the response to environmental conditions, and also to use of assimilates by the plant for growth and respiration. The effects on photosynthetic rate of genetically modifying amounts, activities and characteristics of enzymes and other components involved in carbon assimilation are outlined, and the interaction with environmental conditions discussed.
The Need for Improved Photosynthesis Photosynthesis is fundamental to all biological activity on the planet and underpins all agricultural production of crops, and thereby also animals, for the world’s human population of over 6 billion. Selection breeding has increased yield potential of many crops but has not increased the potential rate of CO2 assimilation per unit leaf area (Amax) nor potential biomass production, rather it has increased harvest index (yield of required product/total biomass ratio). Improved agronomic practices, such as increasing fertilization and irrigation and pest and disease control, have maximized the actual rates of CO2 assimilation per unit leaf area (A) and the leaf area index (leaf area per unit area of ground surface) to the point where biomass and yields in intensive agriculture are approaching their maximum. To increase yields further, Amax and A must be increased. A major aim of current studies of photosynthesis is to understand the mechanisms and to manipulate them and so to increase Amax; another aim is to control the types and amounts of products formed.
Further Reading Ackman RG (1990) Canola fatty acids – An ideal mixture for health, nutrition, and food use. In: Shahidi F (ed.) Canola and Rapeseed – Production, Chemistry, Nutrition, and Processing Technology, pp. 81–98. New York: Van Nostrand Reinhold. Gunstone FD (2001) Oilseed crops with modified fatty acid composition. Journal of Oleo Science 50: 3–13. Hildebrand DF, Rao SS, and Hatanaka T (2002) Redirecting lipid metabolism in plants. In: Kuo TM and Gardner HW (eds) Lipid Biotechnology, pp. 57–84. New York: Marcel Dekker. Kinney AJ (2002) Perspectives on the production of industrial oils in genetically engineered oilseeds. In: Kuo TM and Gardner HW (eds) Lipid Biotechnology, pp. 85–93. New York: Marcel Dekker. Miege C and Marechal E (1999) 1,2-sn-Diacylglycerol in plant cells: Product, substrate and regulator. Plant Physiology and Biochemistry 37: 795–808. Murphy DJ (2002) Biotechnology and the improvement of oil crops – genes, dreams and realities. Phytochemistry Reviews 1: 67–77. Schultz DJ and Ohlrogge JB (2002) Metabolic engineering of fatty acid biosynthesis. In: Kuo TM and Gardner HW (eds) Lipid Biotechnology, pp. 1–25. New York: Marcel Dekker. Slabas AR, Hanley Z, Schierer T, et al. (2001) Acyltransferases and their role in the biosynthesis of lipids – opportunities for new oils. Journal of Plant Physiology 158: 505–513. Thelen JJ and Ohlrogge JB (2002) Metabolic engineering of fatty acid biosynthesis in plants. Metabolic Engineering 4: 12–21. Topfer R, Martini N, and Schell J (1995) Modification of plant lipid synthesis. Science 268: 681–686. Voelker T and Kinney AJ (2001) Variations in the biosynthesis of seed-storage lipids. Annual Review of Plant Physiology and Plant Molecular Biology 52: 335–361.
Biopolymers Y Poirier, University of Lausanne, Lausanne, Switzerland Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction Plants naturally synthesize a variety of polymers that have been used by mankind as a source of useful
biomaterials. For example, cellulose, the main constituent of plant cell wall and the most abundant polymer on earth, has been used for several thousand years as a source of fibers for various fabrics. Similarly, rubber extracted from the bark of the tree Hevea brasiliensis, has been a major source of elastomers until the development of similar synthetic polymers. In the last century, the usefulness of plant polymers as biomaterials has been expanded through the chemical modification of the natural polymers. For example, a number of plastics have been made by substituting the hydroxyl groups present on the glucose moiety of cellulose with larger groups, such as nitrate or acetate, giving rise to materials such as cellulose acetate, a clear plastic used in consumer products such as toothbrush handles and combs. Similarly, starch has been used in the manufacture of plastics by either using it in blends with synthetic polymers or as the main constituent in biodegradable plastics. The advent of transformation and expression of foreign genes in plants has created the possibility of expanding the usefulness of plants to include the synthesis of a range of biomolecules. In view of the capacity of certain crops to produce a large quantity of organic raw material at low cost, such as oils and starch, it is of interest to explore the possibility of using transgenic plants as efficient vectors for the synthesis of biopolymers. Such plant based biopolymers could replace, in part, the synthetic plastics and elastomers produced from petroleum, offering the advantage of renewability and sustainability. Furthermore, being natural products, biopolymers are usually biodegradable and can thus contribute to alleviate problems associated with the management of plastic waste. In this article, the emphasis will be on the use of transgenic plants for the synthesis of two novel classes of industrially useful polymers, namely protein based polymers made from natural or artificial genes, and polyhydroxyalkanoates, a family of bacterial polyesters having the properties of biodegradable plastics and elastomers.
Protein Based Polymers A number of proteins with interesting plastic or adhesive properties are synthesized in nature. For example, marine mussels (Mytilus spp.) can produce strong protein based adhesives that are effective in aqueous environments, allowing these organisms to stick to many surfaces. Several proteins composed of blocks of amino acids repeated extensively form tough and flexible fibers that could find applications in many areas of material science, including in the medical field. Such proteins include silk, collagen,
478 GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers
Silks represent a broad class of polymers that can be loosely defined as externally spun fibrous protein secretions. Commercial silk used in the textile industry is produced from the domesticated worm Bombyx mori. Spiders also produce a variety of silks that are used in the construction of webs or as draglines. The different types of spider silks produced have impressive properties, ranging from lycra-like elastic fibers to kevlar-like superfibers. The exceptional material properties of spider silk have made it an early target for its synthesis in larger quantities in a more amenable production system. Cloning of the spidoin proteins forming the dragline of the spider Nephila clavipes enabled the exploration of their synthesis in Escherichia coli. However, expression of a synthetic gene coding for a protein containing highly repeated blocks of amino acids found in spidoins led only to low production of a mixture of proteins of variable sizes. This heterogeneity in the proteins synthesized was due to premature termination of translation. In contrast, expression of a similar gene construct in the yeast Pichia pastoris, as well as in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum), resulted in the synthesis of a higher amount (between 2 and 10% of total proteins) of a full length protein, indicating that, at least in these eukaryotes, the repetitive nature of the gene construct was not an obstacle to the synthesis of these unusual proteins. Several synthetic genes containing various combinations of the repetitive peptides found in the
Synthesis of Artificial Protein Polymers
Elastin is a very strong natural fiber found in ligaments and arterial walls. Analysis of the primary structure of all mammalian elastins has shown the presence of a repeated block of amino acids with the sequence GVGVP. Synthetic proteins made from multiple repeats of this sequence display elastic properties. These protein polymers are also biodegradable as well as biocompatible, being nontoxic and naturally resorbed by animal tissues. Polymers based on the GVGVP sequence have been shown to prevent postsurgical adhesions and scars in rats. Other medical applications of similar bioelastics include tissue reconstruction, wound covering, and programed drug delivery. Expression in E. coli of a synthetic protein made of 121 repeats of the GVGVP peptide was shown to result in accumulation of polymer inclusion bodies that occupied up to 90% of the cell volume. However, expression of the same synthetic gene in the fungus Aspergillus nidulans, as well as in tobacco, only led to low polymer accumulation despite adequate accumulation of mRNA. It is thought that efficient expression in eukaryotes of such proteins made of repeated blocks of peptides is limited by the availability of certain amino acids or tRNAs. Thus, accumulation of high levels of artificial protein polymers in plants may be dependent on additional genetic engineering, in particular, modifications in the amino acid biosynthetic pathways and/or tRNA pools.
Polyhydroxyalkanoates Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids synthesized by a wide variety of bacteria (Figure 1). The best-characterized PHA is polyhydroxybutyrate (PHB), a homopolymer of 3-hydroxybutyrate. Although initially described by M. Lemoigne in 1926, it was not until 1962 that the value of PHB as a thermoplastic was recognized in a US patent, and not until 1982 when the first PHA produced by bacterial fermentation was
R
O − −
Synthesis of Silk Proteins
N. clavipes spidoin protein have been expressed in tobacco, allowing an assessment of the link between protein structure and physical properties.
−
and elastin. Plant cell walls also contain proteins that are partially composed of repeated blocks of amino acids, such as extensin, the glycine rich proteins, and the proline rich proteins. The mechanical properties of the pure form of these natural plant proteins have not been well studied. The ability to synthesize protein polymers from genes allows the precise determination of both molecular mass and amino acid sequence, making it possible, in principle, to achieve a degree of control over physical properties and functionality that is outside the scope of chemical polymerization technologies. The development and use of these protein based biomaterials are, in many cases, limited by the difficulties in producing sufficient quantities of the material to establish the structure/function relationships and design applications, as well as difficulties in the economical large-scale production of polymers for commercialization. It is in this respect that plants have been explored as a potential production vector for protein based biopolymers.
− (− O − CH − CH2 − C −)n − R = methyl 3-hydroxybutyrate R = ethyl 3-hydroxyvalerate Figure 1 Chemical structure of 3-hydroxyacid monomers constituting PHA. The monomer can range from 3 to 16 carbons in length, depending on the size of the pendant R group.
GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers 479
commercialized. Since then, there has been a growing interest in producing and commercializing PHA as a family of biodegradable thermoplastics and elastomers. Synthesis of PHA in Bacteria
PHAs represent a large group of bacterial polyesters, which can include over 150 different hydroxyacids. Although the majority of PHAs are composed of R-3-hydroxyacids ranging from 3 to 16 carbons in length, some polymers also contain monomers of 4-hydroxy and 5-hydroxyacids. Furthermore, a diversity of functional groups have been found on the monomers forming PHAs, including unsaturated groups, as well as methyl, halogenated, cyano, and phenoxy groups, just to name a few. The majority of bacteria synthesizing PHAs can be broadly subdivided into two groups. One group produces shortchain-length PHAs (SCL-PHAs) with monomers ranging from 3 to 5 carbons in length, while a distinct group synthesizes medium-chain-length PHAs (MCL-PHAs) with monomers from 6 to 16 carbons. This classification is, however, not strict as a few bacteria have been shown to produce ‘‘hybrid’’ PHAs with monomers ranging from 4 to 10 carbons or higher. PHAs accumulate in bacteria as a highmolecular-weight polymer forming intracellular granules of 0.2–0.3 mm in diameter. Typically, PHAs accumulate to a significant proportion of the cell dry weight when bacteria are grown in a media that is limited in a nutrient essential for growth (typically nitrogen or phosphorus), but with an abundant supply of carbon (e.g., glucose). Under these conditions, bacteria convert the extracellular carbon into an intracellular storage form, namely PHA. When the limiting nutrient is resupplied, intracellular PHA is degraded and the resulting carbon is used for growth. PHAs have been shown to occur in nearly 100 genera of bacteria, encompassing Gram-positive and Gramnegative species, as well as in some archea. The large diversity of monomers found in PHA translates into a wide spectrum of physical properties. The homopolymer PHB is a relatively stiff and brittle plastic with limited applications. In contrast, PHA copolymers composed primarily of 3-hydroxybutyrate with a fraction of longer chain monomers, such as 3-hydroxypentanoate, 3-hydroxyhexanoate, or 3-hydroxyoctanoate, are more flexible and tougher plastics. These PHAs can be used in a wide variety of consumer products, such as containers and bottles, as well as water resistant films. PHAs made of longer monomers, such as MCL-PHAs, are typically elastomers and sticky materials. Numerous bacteria and some fungi have been found to degrade extracellular PHA and metabolize
the breakdown products to CO2 and water. Degradation of extracellular PHA is made possible by the secretion of PHA depolymerases, enzymes catalyzing the hydrolysis of PHAs to monomers and oligomers. PHAs are thus biodegradable polymers, and products made from them can be degraded completely in a variety of environments. Degradation is particularly rapid in the microbe rich environment of a compost (typically 3–9 months for a bottle made of PHB). PHAs can also be degraded, albeit slowly, in animal tissues. Since the breakdown products are 3-hydroxyacids, which are naturally found in animals, PHAs are biocompatible, making them useful for some medical applications, such as implants, gauzes, and resorbable suture filaments. The main limitation in using bacterial PHAs as a source of biodegradable polymers is their production cost. Synthesis of PHAs in native or recombinant bacteria using cheap carbon sources, such as glucose or sucrose, has been estimated to cost a minimum of 2–4 US$/kg, making them 5–10 times more expensive than petroleum based polymers, such as polypropylene. Thus, although bacterial PHAs could be used in high-value applications where the cost of the material is small compared to the value of the final product, such as in medical products, bacterial PHA cannot compete in price with synthetic plastics for high-volume, low-value consumer products, such as packaging material and disposable items. It is in this context that agriculture has been regarded as a promising alternative for the production of PHA on a large scale and at low cost. In comparison to bacterial or yeast fermentation, crop plants are capable of producing very large amounts of a number of useful chemicals at low cost. For example, starch and lipids are two of the most industrially useful and versatile products harvested from crop plants. Starch is used extensively in the food industry and as a source of fermentation substrate for the production of a wide variety of chemicals, including polylactic acid. Several million tonnes of cornstarch is produced annually in the USA, with a market value of approximately US$0.25/kg, making it one of the cheapest commodity products. Plant lipids are also utilized by the food and nonfood industries. Nonfood industrial uses of lipids include the manufacture of soaps and detergents, paints, varnishes, lubricants, adhesives, and plastics, as well as biodiesel. The USA produces approximately 10 million tonnes of vegetable oil annually, with a market value of approximately US$0.50/kg. In view of the high productivity of crop plants, it was reasoned that if PHA could be synthesized in plants at a level comparable to reserve lipids in oilseed plants, i.e., approximately 10–40% dry weight, several million tonnes of PHA could
480 GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers
potentially be produced at a cost comparable to vegetable oil, making it competitive with petroleum derived plastics.
wt). Growth of these transgenic plants was however compromised, being severely stunted, although seeds could still be produced. It is thought that synthesis of PHB from acetyl CoA present in the cytoplasm leads to a limitation in the accumulation of isoprenoids and flavonoids, which are also synthesized from cytoplasmic acetyl CoA, resulting in a growth defect. PHB in transgenic plants accumulated in the form of intracellular granules with a size and appearance identical to bacterial PHA granules. Analysis of the chemical structure and physical properties of the polymer confirmed that PHA synthesized in plants was similar to the bacterial polymer. Similar low amounts of PHB synthesized in the cytoplasm of plants have also been demonstrated in tobacco, oilseed rape (Brassica napus), and cotton (Gossypium spp.) fiber cells (Table 1). In plants, biosynthesis of fatty acids from acetyl CoA occurs in the plastid. The plastid is therefore a site of higher flux of carbon through acetyl CoA compared to the cytoplasm. This flux is particularly
Synthesis of PHB in Plants
The first PHA produced in plants was the homopolymer PHB. In bacteria, PHB is synthesized from the condensation of two acetyl CoA by a 3-ketothiolase to form acetoacetyl CoA, which is subsequently reduced to R-3-hydroxybutyryl CoA by an acetoacetyl CoA reductase (Figure 2A). The R-3-hydroxybutyryl CoA is finally polymerized by the PHA synthase to form PHB. Expression of the PHB biosynthetic pathway in plants was first demonstrated in 1992 in the plant Arabidopsis thaliana. Constitutive expression of the acetoacetyl CoA reductase and PHA synthase from the bacterium Ralstonia eutropha into the cytoplasm of transgenic A. thaliana plant cells led to the production of PHB up to 0.1% of the shoot dry weight (dry
(B)
(A) Acetyl CoA
Acyl CoA
Acetyl CoA Malonyl CoA
Acetoacetyl CoA (phaB)
(phaB)
(phaCRe ) Isoprenoids
2-trans -enoyl CoA S-3OH-acyl CoA
Acetoacetyl CoA
3-Hydroxybutyryl CoA
Flavonoids
3-Ketoacyl CoA
(phaA)
R-3OH-acyl CoA
R-3-Hydroxybutyryl CoA
(phaC1Pa)
(phaCRe )
PHB
MCL-PHA
PHB
(phaC1Ac ) SCL-PHA
(C) Threonine (ilvA) 2-Ketobutyrate
Propionyl CoA PDC
Acetyl CoA (phaA)
(btkB) 3-Ketovaleryl CoA
Acetoacetyl CoA (phaB)
(phaB) R-3-Hydroxyvaleryl CoA
R-3-Hydroxybutyryl CoA
(phaCRe) Isoleucine
P(HB-HV)
(phaCRe) PHB
Figure 2 Modification of plant metabolic pathways for the synthesis of PHAs. (A) Synthesis of PHB from acetyl CoA in the cytoplasm; (B) synthesis of PHB, SCL-PHA, and MCL-PHAs in the peroxisome; and (C) synthesis of PHB and P(HB-HV) copolymer in the plastid. The pathways created or enhanced by the expression of transgenes are highlighted in bold, while endogenous plant pathways are in normal type. The various transgenes expressed in plants are indicated in parentheses. The phaA, phaB, and phaCRe genes encode a 3-ketothiolase, an acetoacetyl CoA reductase, and a PHA synthase from R. eutropha, respectively. The btkB gene encodes a second 3-ketothiolase isolated from R. eutropha, which shows high affinity for both propionyl CoA and acetyl CoA. The ilvA gene encodes a threonine deaminase from E. coli. The phaC1Pa and phaCAc genes encode PHA synthases from P. aeruginosa and A. caviae, respectively. While the PHA synthase from R. eutropha can polymerize 3-hydroxyacyl CoAs ranging from 3 to 5 carbons, the PHA synthases from A. caviae and P. aeruginosa can polymerize 3-hydroxyacyl CoAs ranging from 3 to 7 carbons and 6 to 16 carbons, respectively. PDC refers to the endogenous plant pyruvate dehydrogenase complex.
GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers 481 Table 1 Summary of transgenic plants producing PHAs Subcellular compartment
Species
Tissue
PHA type
PHA quantity (%dry wt)
Cytoplasm
A. thaliana Oilseed rape Tobacco Cotton
Shoot Shoot Shoot Fiber
PHB PHB PHB PHB
Plastid
A. thaliana Oilseed rape Cotton A. thaliana Oilseed rape
Shoot Seed Fiber Shoot Seed
PHB PHB PHB P(HB-HV) P(HB-HV)
40 8 0.05 0.8 2.3
Peroxisome
A. thaliana
Whole plants
MCL-PHA
0.6
0.1 0.1 0.01 0.3
Figure 3 Accumulation of PHB granules in chloroplasts of transgenic A. thaliana. Granules are seen as agglomerations of electronlucent granules in the stroma of the chloroplast. Scale bar ¼ 1 mm.
enhanced in the seed of oil-accumulating plants, such as A. thaliana, where up to 40% of the seed dry weight is triacylglycerides. It was therefore hypothesized that the larger flux of acetyl CoA in the plastid may lead to a significant increase in polymer production in transgenic plants expressing the PHB pathway in chloroplasts. Constitutive expression of the PHB biosynthetic pathway in the chloroplasts of leaves of A. thaliana led to polymer accumulation up to 10–40% of the leaf dry weight. These experiments were done by modifying the R. eutropha enzymes 3-ketothiolase, acetoacetyl CoA reductase, and PHB
synthase for plastid targeting by fusing the transit peptide of the small subunit of ribulose bisphosphate carboxylase to the N-terminal end of the proteins (Figure 2C). Transmission electron micrographs revealed that the plastids contained numerous inclusions typical of PHB granules (Figure 3). The accumulation of PHB in leaves was found to increase over the life span of the plant, with fully expanded presenescing leaves typically showing ten times more PHB than younger expanding leaves of the same plant. Plants producing approximately 5–10% dry wt PHB showed little signs of reduced growth,
482 GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers
although chlorosis of leaves was observed, indicating that some aspect of chloroplast function was affected. In plants accumulating higher levels of PHB, a defect in growth was observed, being severe at 40% dry wt and accompanied by sterility. These experiments indicated that although the plastid was a much better site for the synthesis of PHB than the cytoplasm, accumulation of high amounts of PHB in such an important organelle as the chloroplast has its limits. In contrast to the chloroplast, the leukoplast of developing seeds is not involved in photosynthesis and its metabolism is simpler, being devoted mainly to the synthesis of reserve lipids and protein in the case of oilseed crops. Restriction of the expression of the PHB biosynthetic pathway to the leukoplast of developing seeds of oilseed rape resulted in accumulation of PHB up to 8% of the mature seed dry weight. These transgenic seeds had a normal size, appearance, and germination frequency. Furthermore, since the transgenes were not expressed in the vegetative tissues, growth of the plants was normal. Acetyl CoA, the building block of PHB, is found not only in the cytoplasm and plastid, but also in the mitochondria and peroxisomes, being implicated in these organelles in the tricarboxylic acid and b-oxidation cycles, respectively. Although no conclusive demonstration of PHB in plant mitochondria has been reported, synthesis of up 2% dry wt PHB was reported in transgenic black Mexican sweetcorn (Zea mays L. cv. Black Mexican Sweet) suspension cell cultures expressing the PHB biosynthetic pathway in the peroxisomes (Figure 2B). Further experiments are needed to explore the potential advantages and disadvantages of synthesizing PHB in the peroxisomes of plants growing in the field.
Synthesis of PHA Copolymers in Plants Since PHB is a polymer with relatively poor physical properties, being too stiff and brittle for use in most commodity products, successful production of PHA in crops depends on the synthesis of polymers having better physical properties. In the late 1970s, the copolymer poly(hydroxybutyrate-hydroxyvalerate) (P(HB-HV)) was identified as a valuable polymer with sufficient flexibility and impact resistance for its use as a commodity plastic. This co-polymer, marketed under the trade name BiopolTM, was first synthesized by bacterial fermentation using R. eutropha growing in media supplemented with propionate in order to create an intracellular pool of propionyl CoA that could be condensed to acetyl CoA to form 3-ketovaleryl CoA.
In order to synthesize P(HB-HV) in the plastids of plants, propionyl CoA was generated by modifying the branched amino acid biosynthetic pathway (Figure 2C). An E. coli threonine deaminase modified for plastid targeting was overexpressed in plants, leading to the accumulation of 2-ketobutyrate, which can be converted, albeit at low efficiency, to propionyl CoA by the plant pyruvate dehydrogenase complex. A novel 3-ketothiolase from R. eutropha showing high affinity for both propionyl CoA and acetyl CoA was also targeted to the plastids along with an acetoacetyl CoA reductase and a PHA synthase. Expression of all four genes in A. thaliana under the control of the constitutive CaMV 35S promoter resulted in the accumulation of P(HB-HV) of between 0.2% and 0.8% dry wt in shoots, with a 4–17 mol% HV content in the polymer. Seed specific expression of the same pathway in the leukoplast of oilseed rape embryos resulted in PHA accumulation up to 2.3% dry wt in seeds, with up to 6.5 mol% HV. Although these levels of P(HB-HV) copolymer production are lower than for PHB, synthesis of BiopolTM in plants is an important milestone on the route to commercialization of PHA-producing crops. Synthesis of the copolymer MCL-PHAs in several pseudomonads was shown to be mediated by two distinct pathways. In one pathway, the intermediate 3-hydroxyacyl-acyl carrier protein (ACP) of the fatty acid biosynthetic pathway is first converted to 3-hydroxyacyl CoAs by a transacylase before being polymerized by the PHA synthase. The creation of this pathway in the plastids of plant cells has not been successfully demonstrated to date. In the second pathway, the 3-hydroxyacyl CoA intermediates generated by degradation of fatty acids through the b-oxidation cycle are utilized by the PHA synthase to form MCL-PHAs (Figure 2B). Synthesis of MCLPHAs using intermediates of b-oxidation has been successfully demonstrated in A. thaliana expressing a PHA synthase from Pseudomonas aeruginosa modified at the C-terminal end by the addition of a tripeptide allowing targeting of the protein to the peroxisomes. MCL-PHA production in peroxisomes was, however, low, reaching a maximum of 0.4% dry wt in 7-day-old germinating seedlings. The monomer composition of the polymer was complex, including saturated and unsaturated monomers ranging from 6 to 16 carbons. Growth of these transgenic plants in liquid media supplemented with various fatty acids resulted in a significant increase in MCL-PHA containing monomers derived from the b-oxidation of the external fatty acids. For example, addition of either tridecanoic acid, tridecenoic acid (C13:1, D12), or 8-methyl-nonanoic acid resulted in the production of PHA containing mainly saturated
GENETIC MODIFICATION OF PRIMARY METABOLISM / Biopolymers 483
odd-chain, unsaturated odd-chain, or branchedchain 3-hydroxyacid monomers, respectively. These studies demonstrated that the plant b-oxidation pathway was capable of generating a large spectrum of monomers from fatty acids, which can be included in MCL-PHAs. In a further extension of the study of MCL-PHA synthesis in plant peroxisomes, it has been shown that an endogenous control over the flux of substrates toward b-oxidation and PHA synthesis could be achieved through the combined expression of a peroxisomal PHA synthase with an acyl ACP thioesterase. Expression of a plastidial caproyl ACP thioesterase from Cuphea lanceolata in A. thaliana was used to channel decanoic acids toward peroxisomal b-oxidation, resulting in a 8-fold increase in synthesis of MCL-PHA in mature leaves, with the polymer being composed of approximately 40 mol% 3-hydroxydecanoic acid, 32 mol% 3-hydroxyoctanoic acid, and 4 mol% 3-hydroxyhexanoic acid. This strategy initially applied to the synthesis of MCL-PHAs in vegetative green tissues was further extended to the synthesis of PHA in developing seeds of A. thaliana. In dry seeds, a maximal amount of 0.1% dry wt was detected. From these studies, a working model was established whereby enzymes and genes involved in the synthesis of unusual fatty acids in plants can be used to modulate the quantity and quality of substrates channeled toward MCLPHAs. The absolute amount of MCL-PHA accumulating in mature shoots or seeds of transgenic plants expressing both thioesterase and PHA synthase remains relatively low (p0.6% dry wt) compared to PHB synthesis in plastids (Table 1). The reasons for this difference could be multiple, including differential activity of bacterial enzymes in various plant subcellular compartments, and the relative ability of the created and endogenous metabolic pathways to compete for the same substrates (acetyl CoA for PHB and 3-hydroxyacyl CoAs for MCLPHAs). Synthesis of a small amount (0.04% dry wt) of SCL-PHA containing 3-hydroxyhexanoic, 3-hydroxyvaleric, and 3-hydroxybutyric acids has also been reported in A. thaliana expressing the PHA synthase from the bacterium A. caviae in the peroxisomes. Whereas the 3-hydroxyhexanoic acid and 3-hydroxybutyric acid monomers are derived from the b-oxidation of fatty acids, the source of the 3-hydroxyvaleric acid is unknown.
polymer as a thermoplastic or elastomer, a novel inventive use of PHA in plants was demonstrated by the expression of the PHB biosynthetic pathway in the cytoplasm of cotton fiber cells. In this system, the polymer is produced in plants only to change the physical properties of the fiber. Accumulation of 0.3% dry wt PHB in the cytoplasm of the fiber was shown to be sufficient to significantly decrease the rate of heat uptake and cooling of the fiber, resulting in a higher heat capacity and an improvement in the fiber insulating properties. Interestingly, in contrast to the large increase in accumulation of PHB in leaves of A. thaliana expressing the pathway in the chloroplast, expression of the PHB pathway in the plastid of the cotton fiber resulted in a lower amount of polymer, indicating a difference either in the number or metabolic activity of the plastids in fiber cells compared to leaves. It is also tempting to speculate whether the properties of wood, rubber, or starch could also be positively modified through the coaccumulation of PHAs.
Conclusions A spectrum of PHAs ranging from the stiff PHB to the more flexible P(HB-HV) plastics and MCL-PHA elastomers have now been successfully synthesized in plants. The challenge for the future is to succeed in high-level production (X15% dry wt) of a limited number of useful PHAs without a decrease in crop yield. Although quite complex, this task appears to be feasible. Initial experiments with expression of protein based polymers in plants indicate that, like PHAs, engineering of various metabolic pathways is also likely to be necessary before high-level accumulation of polymer is accomplished. For both protein based polymers and PHAs, synthesis of polymers for the modification of the properties of plant products, such as fibers, are attractive alternatives to the extraction of polymers for industrial uses. PHAs and protein based polymers are only two out of a large group of polymers produced in nature that have interesting properties. In the future, novel industrially useful polymers could perhaps also be developed through the in situ modification of natural plant compounds. See also: Genetic Modification of Primary Metabolism: Proteins. Primary Products: Proteins.
Further Reading PHA to Modify the Properties of Plant Fibers
Although the synthesis of PHA in plants is mainly focused on the extraction and utilization of the
Daniell H and Guda C (1997) Biopolymer production in microorganisms and plants. Chemistry and Industry 14: 555–558.
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Photosynthesis M J Paul and D W Lawlor, Rothamsted Research, Harpenden, UK Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction Photosynthetic organisms use the physical energy of light to form reduced compounds (‘‘reductants’’ ferredoxin and NADPH), and the energy source adenosine triphosphate (ATP): these are required for the synthesis of organic products, primarily sugars and amino acids, from inorganic starting materials, carbon dioxide (CO2), and nitrate (NO3 ) and sulfate (SO24 ) ions. This article considers the general mechanisms of photosynthetic CO2 assim-
ilation for plants with the C3 type of assimilation (Figure 1), with brief comments on the C4 type, and the feasibility of altering them by genetic modification to increase the photosynthetic rate (A). This depends on environmental conditions, particularly light, CO2 concentration and temperature, and on many genetically determined subprocesses. These may be separated into: (1) light reactions, including light capture, electron transport (et), and synthesis of reduced pyridine nucleotide (NADPH) and adenosine triphosphate (ATP), and (2) CO2 assimilation by the enzyme ribulose bisphosphate (RuBP) carboxylase/oxygenase (rubisco) in which RuBP, synthesized by the photosynthetic carbon reduction (PCR) cycle, is carboxylated (CO2 added) forming sugar phosphates. These are metabolized to sucrose. In addition, reaction of RuBP with oxygen (O2), a process called oxygenation, and also catalyzed by rubisco, results in photorespiration (PR), a major inefficiency in C3 photosynthesis. Regulation of these subprocesses is complex, with extensive interactions which determine the response to environmental conditions, and also to use of assimilates by the plant for growth and respiration. The effects on photosynthetic rate of genetically modifying amounts, activities and characteristics of enzymes and other components involved in carbon assimilation are outlined, and the interaction with environmental conditions discussed.
The Need for Improved Photosynthesis Photosynthesis is fundamental to all biological activity on the planet and underpins all agricultural production of crops, and thereby also animals, for the world’s human population of over 6 billion. Selection breeding has increased yield potential of many crops but has not increased the potential rate of CO2 assimilation per unit leaf area (Amax) nor potential biomass production, rather it has increased harvest index (yield of required product/total biomass ratio). Improved agronomic practices, such as increasing fertilization and irrigation and pest and disease control, have maximized the actual rates of CO2 assimilation per unit leaf area (A) and the leaf area index (leaf area per unit area of ground surface) to the point where biomass and yields in intensive agriculture are approaching their maximum. To increase yields further, Amax and A must be increased. A major aim of current studies of photosynthesis is to understand the mechanisms and to manipulate them and so to increase Amax; another aim is to control the types and amounts of products formed.