October 2, 2007
Metabolic Engineering of Plant L-Ascorbic Acid Biosynthesis: Recent Trends and Applications
By Zhang, Lei Wang, Zinan; Xia, Yang; Kai, Guoyin; Et al
ABSTRACT Vitamin C (L-ascorbic acid; AsA) is the major soluble antioxidant found in plants and is also an essential component of human nutrition. Although numerous biotechnological methods have been exploited to increase its yield, pressures such as commercial competition and environmental concerns make it urgent to find a new way for industrial production of plant-derived AsA. Engineering plant AsA has now become feasible because of our increased understanding of its biosynthetic pathway. Several possible strategies could be followed to increase AsA production, such as overcoming the rate limiting steps in the biosynthetic pathway, promoting recycling, and reducing catabolism. For these purposes, genes of plant, microbial and animal origins have been successfully used. Several examples will be given to illustrate these various approaches. The existing and potential achievements in increasing AsA production would provide the opportunity for enhancing nutritional quality and stress tolerance of crop plants. KEYWORDS ascorbic acid, biosynthesis, metabolism, metabolic engineering, genetic modification, plantAbbreviation: 2-CP, 2-cysteine peroxiredoxin; ADLT, aldono- lactonase (EC 220.127.116.11); ALO, D-arabinono-1, 4-iactone oxidase; AO, Ascorbate oxidase (EC 18.104.22.168); APX, Ascorbate peroxidase (ECI. 11. 1.11); AsA. L-ascorbic acid; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase (EC 22.214.171.124); GaIUR, D-galacturonate reductase (EC 126.96.36.199); GDPME,GDP-D-mannose-3, 5-epimerase (EC 188.8.131.52); GDPMPPase,GDP-D-mannosepyrophosphorylase (EC 184.108.40.206); GR, glutathione reductase; GSH, glutathione; GSSG, Oxidized glutathione; HK, hexokinase (EC 220.127.116.11); L-GaIDH, L -galactose dehydrogenase; L-GalPPase, L-galactose 1-P phosphatase; L-GLDH, L- galactono-1, 4-lactone dehydrogenase (EC 18.104.22.168); MDHA, monodehydroascorbate; MDHAR, monodehydroascorbate reductase (EC 22.214.171.124); ME, methylesterase; Ml, myo-inositol; MIOX, homozygous myo- inositol oxygenase; PGI, phosphoglucose isomerase (EC 126.96.36.199); PMI, phosphomannose isomerase (EC 188.8.131.52); PMM, phosphomannose mutase (EC 184.108.40.206); RNAi, RNA interfering; VHb, Vttreoscttta hemoglobin.
This research was financially supported by the National Natural Science Foundation of China (20572130. 30600807), National Basic Research Program of China (973 Program, 2007CB 108805), Shanghai Natural Science Foundation (04ZR14051) and Shanghai Rising-Star Program (06QB14010).
Vitamin C (defined as L-ascorbic acid and its oxidation product dehydroascorbate) is one of the best known plant metabolites and possesses significant biochemical functions as an antioxidant, enzyme co-factor, and electron donor and acceptor in electron transport.43 This compound also plays an important role in plant growth and metabolism. It is involved in cell division, expansion and elongation.17 Together with flavonoids, polyphenolics and water insoluble compounds such as alpha-tocopherol (vitamin E), AsA contributes to the overall intake of "free radical scavengers" or "anti-oxidative metabolites" in the human diet. There is now convincing evidence that such metabolites, singly and in combination, benefit human health and well-being, acting as anti- cancer forming agents and protecting against coronary heart disease.28 Comprehensive reviews of functions and biomedical roles for ascorbic acid in higher plants and fungi appeared in 1996.29,73 AsA is synthesized by all higher plants, nearly all higher animals2 and by a number of yeasts.30 However, humans and a few other animal species have lost the capacity to synthesize the compound because they have a nonfunctional gene for L-gulono-1, 4-lactone oxidase, which acts in the last step of AsA synthesis in animals.55 Since AsA cannot be stored in the body, plant-derived AsA must be obtained from thetary sources regularly.
Because of its proposed functions and high values as an essential micronutrient, AsA is of great economic significance. The current world production of AsA is estimated at 80,000 tons per annum with a global market value in excess of US$ 600 million and an annual growth rate of 3-4%.7,11 At present, the majority of commercially available AsA is synthesized from D-glucose via the seven-step Reichstein process.28 Although the yield of AsA can reach up to ~50%,7 there are obvious economical and environmental disadvantages associated with this technology. Attempts have been made to exploit microalgae for direct production of AsA from inexpensive feedstocks.19,66,67,72 However, at present these methods do not provide a commercially viable alternative for AsA bio- manufacturing.
Much knowledge has been obtained recently on the AsA biosynthetic pathway in higher plants; the intermediates and enzymes involved have been well characterized.27,74,81 This, together with the rapid advancements in biotechnology such as genetic transformation of plants and other organisms, has opened up the possibilities of using genetic engineering approaches to achieve efficient AsA production in plants.
In this review we endeavor to provide an update on some of the recent advances and discuss various aspects of metabolic engineering of plant AsA, with a focus on illustrative examples from this broad field of research that may serve to indicate trends and, perhaps, suggest future directions.
2. A PROPOSED RATIONAL SCHEME FOR AsA BIOSYNTHESIS IN PLANTS
The biosynthesis pathway of AsA in animals has been well established. D-glucose is converted to AsA dirough D-glucuronic acid, L-gulonic acid, and Lgulono-1, 4-lactone.8 Given the important biochemical and physiological roles of AsA and its abundance in all plants tested, it is surprising that its biosynthetic pathway in plants has remained enigmatic and been a subject of controversy for many years. However, significant progress has recendy been made toward understanding AsA biosynthesis in plants.74 Wheeler et al.iX proposed a plant AsA biosynthetic pathway that does not predict inversion of the carbon skeleton of glucose, with D-mannose and L- galactose as key intermediates (Figure 1). The first step in the biosynthetic pathway of plant AsA is the formation, at the level of sugar nucleotide, of L-galactosyl residues, catalyzed by a previously described GDP-D-mannose-3', 5'-epimerase (GDPME) activity.4,5 By using combined conventional biochemical and mass spectrometry methods, Wolucka et al.82 obtained a highly purified preparation of GDP-D-mannose 3', 5'-epimerase from an Arabidopsis thaliana cell suspension. The steps involved in the synthesis of L- galactose from GDP-L-galactose in plants were not fully characterized until recendy. Conklin etal.13 characterized a low AsA- content mutant (vtc4-1) of Arabidopsis and presented evidences for an in vivo role for L-GaI 1-P phosphatase (Gal PPase) in plant AsA biosynthesis. L-galactono-1, 4-lactone can be produced from L- galactose by a newly identified L-galactose dehydrogenase (GaLDH) activity. This reaction proceeds widiout carbon skeleton inversion. L-galactono-1, 4-lactone can then be converted to AsA by the well- documented L-galactono-1, 4-lactone dehydrogenase enzyme (GLDH).53,59,63 However, until now the mechanism of the sugar release from its nucleotide-bound form (GDP-L-galactose) was not well understood. This pathway proceeds via GDP-D-mannose and GDP-L- galactose, as indicated by radio-labeling data.47 Conklin et al.14 had taken a complementary genetic approach to unraveling AsA biosynthesis in higher plants by isolation and analysis of a collection of AsA-deficient Arabidopsis mutants (vtc), which gave valuable in vivo support for the proposed scheme. A fascinating implication of identification of this pathway is diat it fills a major gap in plant carbohydrate metabolism and it should allow engineering of plants for increased AsA production, dius increasing their nutritional value and stress tolerance.
Isherwood32 proposed an alternative pathway in plants indicating D-galacturonic acid as a metabolic precursor of L-galactono-1, 4- lactone (Figure 1). Radiotracer and biochemical evidence, including data demonstrating the enzymatic reduction of derivatives of D- galacturonic acid to L-galactonic acid by plant extracts, supports this alternative padiway. This pathway could constitute a carbon salvage mechanism in certain organs after the breakdown of cell walls, such as occurs during fruit ripening,12,17 and in photosynthesizing plant cells, where the L-galactose padiway prevails and contributes to maintain the cell's oxidation-reduction state.21
FIGURE 1 Generalized scheme of AsA biosynthetic network in plants. Two potential branch pathways operating in plants, the Man pathway and alternative galacturonate pathway to L-galactono-1,4- lactone, are shown. Plants synthesize AsA from D-glucose via a complex ten-step pathway involving phosphorylated sugar intermediates and sugar nucleotides. Oxidation of AsA produces monodehydroascorbate (MDHA) by the enzymes ascorbate peroxidase (APX) and ascorbate oxidase (AO), which is converted to AsA by MDHAR or dlsproportionates nonenzymatically to AsA and dehydroascorbate (DHA). DHA spontaneously hydrolyzes to 2, 3-diketogulonic acid unless salvaged by DHAR, which uses GSH as the reductant. Oxidized glutathione (GSSG) is reduced by glutathione reductase (GR).
Enzymatic ascorbate loss would probably involve ascorbate oxidase (AO) and ascorbate peroxidase (APX). AO is a copper-containing blue oxidase that catalyzes the aerobic oxidation of AsA to monodehydroascorbate and functions as an AsA free radical producer.20,27,37,40,65 In concert with other enzymes, APX is considered to be a key antioxidant enzyme in plants and may function as a scavenger of hydrogen peroxide produced by basal metabolism.1,33,52 AsA then enters the ascorbate-glutathione cycle75 or the AsadaHalliwell pathway71 for AsA regeneration (Figure 1). Finally, AsA catabolism occurs with tartrate and oxalate as major products.75 Although recent progress has revealed biosynthetic pathways for AsA, the degradative pathways remain unclear. In some plants (Vitaceae), AsA is degraded via L-idonate to L-threarate (L- tartrate), with the latter arising from carbons 1-4 of AsA.2 In most plants, AsA degradation can occur via dehydroascorbate, yielding oxalate83 plus L-threonate, with the latter from carbons 3-6 of ascorbate.2,31,68 The metabolic steps between AsA and oxalate/ L- threonate and their subcellular location are still unknown.
3. METABOLIC ENGINEERING OF PLANTS FOR ENHANCED PRODUCTION OF AsA
None of the traditional methods for producing vitamin C has a sufficiently high production level to allow for commercialization of the process. Our recent understanding of the AsA biosynthetic pathways in plants has made metabolic engineering a promising approach for manipulating the content of AsA in plants. So far most of the genes known to encode enzymes in this pathway have been cloned using classical and molecular approaches. Alteration in AsA and substrate accumulation can be achieved by several strategies based on regulating the expression of the key enzymes (Table 1). The details are discussed below.
TABLE 1 The Development of Biotechnological Approaches for L- Ascorbic Acid Production
3.1 Promoting AsA Biosynthesis
To improve the yield of plant AsA, one general approach has been utilized to increase the carbon flux towards AsA by changing the expression of biosynthetic gene(s), thereby overcoming specific rate- limiting steps in the pathway, or by blocking competitive pathways.
L-galactose is an important precursor and only used in the synthesis of AsA in plants, so engineering Lgalactose biosynthesis should not disturb the overall carbohydrate metabolism. L-galactose is also an extremely rare sugar, so the isolation of genes involved in the production of free L-galactose and overexpression of these key enzymes might provide useful tools for extending the metabolic capacity.
GDP-mannose pyrophosphorylase (GDPM PPase) catalyzes the synthesis of GDP-D-mannose, which represents the first committed step in the formation of all guanosine-containing sugar nucleotides found in plants that are precursors for cell wall biosynthesis and, probably more importantly, the synthesis of AsA.39 Reduction of AsA in the vtc-mutant of Arabidopsis is caused by a point mutation in the gene encoding GDPM PPase, leading to a reduced activity of the enzyme.14 The antisense system represents a powerful tool for analyzing key steps in metabolic pathways, as it allows the selection of lines with different levels of reduction in enzyme activity. Keller et al.39 constructed an antisense vector that carried a sequence from the GDPM PPase gene from S. tuberosum, and introduced it into potato. They found that the transgenic plants contain reduced AsA and mannose content, resulting in developmental changes during senescence.
Berry etal.6 reported a correlation between GDPME activity and AsA content in randomly mutagenized strains of the unicellular alga Prototheca and suggested that up-regulation of this step could allow the production of algae and plants with enhanced AsA content. However, until now there are no reports of microalgae or plants with enhanced AsA content as a result of overexpression of this gene.
Although the significantly lower AsA and perturbed L-galactose metabolism in vtc4-l indicate that Gal PPase plays a role in plant AsA biosynthesis, the presence of AsA in the T-DNA insertion mutant suggests there is a bypass to this enzyme or that other pathways also contribute to AsA biosynthesis.13 Nevertheless, the potential of engineering this gene for enhancing AsA production should still be exploited.
Biosynthesis of AsA in strawberry fruit occurs through D- galacturonic acid and the NADPHdependent D-galacturonate reductase (GaIUR) is a key enzyme in this pathway. Expression of GaIUR correlated with changing AsA content in strawberry fruit during ripening and with variations in AsA content in fruit of different species of the genus Fragaria. Overexpression of strawberry GaIUR in Arabidopsis resulted in twoto three-folds increase in AsA content.21 Since the substrate of GaIUR D-galacturonic acid, is an abundant component of the cell walls of all plants and constitutes a potentially universal substrate for the synthesis of AsA, the result from transgenic Arabidopsis suggested a general feasibility of using GaIUR overexpression to increase vitamin C content in plants.
The function of GaIDH in AsA biosynthesis was investigated by overexpression in tobacco and antisense suppression in Arabidopsis.26 In tobacco the highest expressing lines had a 3.5- fold increase in extractable GaIDH activity, but this did not lead to an increase in leaf AsA concentration. Arabidopsis lines transformed with an antisense GaIDH construct contained 30% of the wild-type GaIDH activity. L-galactose pool size increased in antisense transformants with low GaIDH activity, and L-galactose concentration was negatively correlated with AsA. Cotransformation of yeast cells or Z. bailii with Arabidopsis GaIDH and GLDH or S. cerevisiae endogenous D-arabinono-l,4-lactone oxidase (ALO) successfully boosted the second-last and last steps converting L- galactose into AsA.70
We suggest that the mere overexpression of the GaIDH gene is ineffective. High concentrations of Lgalactose and other cofactors such as NADPH are necessary for GaIDH producing a marked effect.
The conversion of L-galactono-1, 4-lactone into AsA seems to be the best characterized step in plant AsA biosynthesis and this step is catalyzed by GLDH, an enzyme located on the inner mitochondrial membrane.15 Although GLDH is present in higher plants, a direct correlation between GLDH expression and AsA level has yet to be confirmed. All observations suggest that regulation of both AsA biosynthesis and turnover may be sensitive to the endogenous pool size.15 Oba et al.58 detected a stimulation of GLDH activity followed by an increase in AsA level in sliced potato tubers exposed to air, and Tabata et al.77 obtained AsA-deficient tobacco cells by antisensing GLDH. However, both wild-type and vtcl plants were able to oxidize L-galactono-1, 4-lactone to AsA, making it unlikely that the AsA deficiency in vtcl is caused by a defect in GLDH activity.15 Additionally, in spite of the higher GLDH activity in turning strawberry, the end amount of total AsA after substrate infiltration was close to the average content usually found in small green fruit,16 which could be the result of the maintenance of AsA at physiological levels by the contribution of several other enzymes, including those involved in AsA oxidation/reduction. Considering the limitations of the in vitro assay, kinetic regulation of GLDH cannot be discarded, since it has been described in potatoes.78 Regardless of the metabolic route, the majority of plant AsA seems to be produced by the conversion of l-galactono-1, 4-lactone through GLDH activity. In this respect, GLDH plays an essential role in AsA synthesis and, as a consequence, its activity is essential to the metabolism of the plant cell. Transformation of plants with antisense or RNA interference (RNAi) constructs will allow us to downregulate the expression of GLDH and study its role in plant AsA biosynthesis. Furthermore, the manipulation of GLDH through genetic modification may give us the opportunity to generate crops containing an increased level of vitamin C.
3.2 Inhibiting AsA Degradation or Reducing Catabolism
Because catabolism of AsA direcdy affects its accumulation and thus yield, blocking of catabolism would be of significant value for engineering high AsA content. This can be achieved by knocking out an enzymatic step in the consumption pathway or reducing the level of the corresponding mRNA via antisense, cosuppression or RNAi technologies, or by overexpression of an antibody against the enzymes.79
AO and APX play important roles in ascorbate metabolism. AO may function apoplastically as an ascorbate oxidizer in the process of cell elongation.38'60 APX reduces hydrogen peroxide to water, with ascorbate as an electron donor, and helps plants survive oxidative stress.50 Transgenic plants expressing antisense cytosolic APX showed increased susceptibility to ozone injury62 and were hyper responsive to pathogen attack.50 In the absence of Arabidopsis APX1, the entire chloroplastic H202-scavenging system collapses in response to a moderate level of light stress.18
The activities of AO and APX were negatively correlated with ascorbate content38- 46 and we propose that inhibiting their activities would reduce degradation of AsA and ultimately increase the AsA content. However, until now few attempts have been made to engineer this degradation pathway, and direct evidence for the feasibility of the strategy has not been reported. Further studies using transgenic plants in which AO or APX expression is down- regulated by antisense or RNAi constructs are required to examine the effect of reduced AsA degradation or catabolism on AsA accumulation. However, the potential problems associated with downregulation of AO and/or APX, such as reduced plant oxidative stress tolerance, must be considered.
3.3 Enhancing Ascorbate Recycling
Oxidation of AsA produces the short-lived radical monodehydroascorbate (MDHA), which is converted to AsA by MDHA reductase (MDHAR) or disproportionates nonenzymatically to AsA and dehydroascorbate (DHA) (Figure 1). DHA undergoes irreversible hydrolysis to 2, 3-diketogulonic acid80 or is recycled to AsA by dehydroascorbate reductase (DHAR), which uses glutathione (GSH) as the reductant56,74 (Figure 1). Thus, DHAR allows the plant to recycle DHA, thereby recapturing AsA before it is lost. Chen et al.9 did excellent work to confirm that overexpression of DHAR in plants would increase the level of ascorbic acid through improved ascorbate recycling. A DHAR cDNA from wheat was isolated and expressed in tobacco and maize, where DHAR expression was increased up to 32- and 100-fold, respectively. The increase in DHAR expression increased foliar and kernel endogenous ascorbic acid levels 2- to 4-fold and significantly increased the ascorbate redox state in both tobacco and maize. Increasing the level of AsA through enhanced recycling provided greater protection against oxidative damage10 and at the same time did not exhibit any deleterious effects such as growth impairment, chlorosis, necrosis, or premature senescence. While these results suggest that the vitamin C content in plants can be elevated by increasing the expression of enzymes responsible for recycling ascorbate, so far the potential value of engineering the MDHAR- catalyzed AsA recycling pathway has not been examined.
3.4 Engineering an Alternative pathway by Introduction of Exogenous Novel Gene(s)
To over-express an enzyme, a sense gene is needed. This gene can be a gene from the same plant, from another plant species, or from another organism. It has been shown that entire metabolic pathways not present in an organism can be introduced efficiently by means of molecular genetics.64 Although plants and animals use different biosynthetic pathways to produce AsA, it is possible to metabolically engineer plants to supplement their production of AsA using one or more components from the animal pathway. Transgenic tobacco and lettuce plants expressing a rat cDNA encoding L-gulono- y -lactone oxidase, which was involved in the final step in the animal AsA biosynthetic pathway, accumulated up to seven times more ascorbic acid than untransformed plants.34 Although the specific biochemical step(s) leading to this increase remain to be elucidated in the transgenic plants, manipulation of a biosynthetic pathway by expressing a novel heterologous gene may prove to be a useful approach for nutritional engineering. Use of heterologous genes could also have the advantage of minimizing the problems associated with gene silencing and co-suppression because of the sequence divergence between the introduced genes and the host genes. It should be noted, however, that plants genetically modified with genes from rats or other animals might not be appealing to consumers.
By expressing a human-derived DHAR gene in tobacco chloroplasts, transgenic plants showed a 2.29 times higher DHAR activity and a 1.43 times higher glutathione reductase activity than the non- transgenic control.41 Although the total AsA content was not significantly affected, the ratio of AsA/DHA was increased from 0.21 to 0.48. As a result, it enhanced stresstolerance of the transgenic plants.41
Lorence et al.AS presented a possible biosynthetic route using >>ryo-inositol (MI) as the initial substrate and they believed that wyo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Ascorbate levels increased 2- to 3-fold in homozygous MI oxygenase (MIOX) transgenic Arabidopsis lines. So MI can also be used as a precursor for AsA for the agronomic and nutritional enhancement of crops.
3.5 Switching the Sub-Cellular Localization of Compartment Enzymes
In mapping biosynthetic pathways, compartmentalization of metabolites and enzymes should be studied as this plays a major role in the regulation of metabolite pathways.79 Compartmentalization of desired compounds or targeted expression of key enzymes is one strategy that can be used to enhance the production of desired pharmaceuticals through genetic engineering.61 By specific localization of the introduced sesquiterpene synthase to the mitochondria, Kappers etal.36 obtained transgenic Arabidopsis plants emitting two new isoprenoids. The biosynthesis of plant AsA requires at least five compartments-apoplast, cytosol, vacuole, chloroplasts and mitochondrion.38 AsA de novo synthesized in cytosol is transported to the apoplast and oxidized. It accumulates in source leaf phloem and is transported to sink tissues in plants.22 The last downstream enzyme GLDH is an integral protein of the inner mitochondrial membrane15 and it has been assumed to date that the supply of direct precursor L-galactono-1, 4-lactone to the mitochondrion is the major limitation on AsA production and accumulation in plants. In our laboratory we are progressing towards making plants transformed with GaIDH fused with a bona fide mitochondrial targeting signal. By switching specific subcellular localization sequences, one could efficiently deliver GaIDH expression into mitochondrion and specifically increase the concentration of L-galactono-1, 4-lactone, the very important precursor of AsA in this compartment.
3.6 Promoting Energy Metabolism to Optimize Metabolites Productivity
Improving plant productivity can be achieved by elevating growth and yield. One of the major determinants of crop growth is photosynthetic carbon metabolism.25- 51,76 To date few molecular approaches aimed at improving the efficiency of this pathway have proven successful. Those strategies that did work generally involved expression of cyanobacterial enzymes in higher plants, which resulted in increased photosynthetic rates and growth under normal and limiting carbon dioxide concentrations, respectively.44,51
Antisense inhibition of the mitochondrial malate dehydrogenase in transgenic tomato enhanced photosynthetic activity and increased the capacity to use L-galactono-lactone, the terminal precursor of ascorbate biosynthesis, as a respiratory substrate. Accumulation of carbohydrates and redox-related compounds such as ascorbate was also markedly elevated in the transgenics.57
Oxygen is often a limiting substrate in secondary metabolite biosynthesis in plant cell cultures. One explanation for this effect is that oxygen might get involved in complex enzymatic pathways into biologically active secondary metabolites. Limitation of the dissolved oxygen supplied during cultivation can decrease the activity of certain enzyme, i.e. oxygenases, required for the processing of the pathway intermediates into their final forms, resulting in the accumulation of intermediates as the primary products.24
Bacterial hemoglobin, acting as an additional oxygen source, increases the intracellular concentration of effective dissolved oxygen under aerobic conditions, and the increase in the dissolved- oxygen tension shifts the relative activities of terminal oxidases, which subsequently causes an enhancement in protonpumping efficiency and ATP production.35 Therefore, for some oxygen-involved metabolisms or energy consuming processes, the overexpression of some bacterial hemoglobins, i.e. Vitreoscitta hemoglobin (Vhb), which can facilitate oxygen diffusion and improve energy metabolism, appears to be a prospective metiiod for metabolite productivity enhancement.23
As the biosynthesis of AsA in plants has been shown to be an oxygen-required process74 and respiration can control AsA syndiesis,49 we believe that improved oxygen diffusion and energy metabolism could change flux distribution through the different branches of the metabolic network in the hosts, and heterologous expression of bacterial hemoglobin to increase the concentration of the effective dissolved oxygen might boost this pathway. This new route is currentiy being actively pursued in our laboratory.
4. CONCLUSIONS AND THE FUTURE
Metabolic engineering is a new approach to the understanding and utilization of metabolic processes. Advances in understanding plant biochemical pathways and availability of gene transfer techniques in plants have led to a growing interest in using this technique to redirect metabolic fluxes in plants for industrial purposes. AsA is the major contributor to the value of nutrition both in plants and humans, and is also an important factor associated with stress tolerance in agronomic species.49 The information gained in recent years about the AsA biosynthesis pathway in plants provides a solid foundation for modern breeding aimed at changing AsA content in crop plants. The examples discussed in this review illustrate the enormous potential of using genetic engineering to achieve high yield of AsA.
Aldiough a substantial increase in productivity is feasible when a rate-limiting enzyme is targeted,42 in most biosyndietic pathways for secondary metabolites rate-limiting may be associated with multiple steps. Directed transgenic manipulation of one enzyme often renders subsequent reactions more rate-limiting, and thus the effect of single-enzyme overexpression may be dampened. In general, it can be concluded that manipulation of a single gene is of limited value. Overcoming one limiting step automatically means that one meets the next one. Strategies should include fortification of multiple steps by up- or down-regulating multiple biosynthetic genes69,84 or the combination of more than one of the strategies mentioned above. Efficient linking and transfer of multiple genes to engineer whole plant AsA biosynthesis pathways is currendy being actively pursued in our laboratory. By using a multigene assembly and transformation vector system, which consists of a transformation-competent artificial chromosome (TAC)-based acceptor vector together with two donor vectors,45 we can study the functions and interactions of multiple key genes associated witli plant AsA metabolic pathways. To exploit the full potential of metabolic engineering, it is necessary to gain detailed knowledge on plant AsA metabolism at the level of intermediates, enzymes and genes. On the physiological aspect of the pathway, the fluxes through the pathway are controlled to a greater extent at the level of enzymes and intracellular and intercellular transport. Fluxes are not only determined by gene expression, but also by post-translational regulation of enzyme activity and enzyme and metabolite compartmentalization and transport. For example, 2- cysteine peroxiredoxin (2-CP) forms an integral part of the antioxidant network of chloroplasts and is functionally interconnected with other defense systems. Suppression of 2-CP specifically enhances the activities and expression of enzymes associated with ascorbate metabolism, possibly due to the increased oxidation state of the leaf ascorbate pool.3 Classic biochemistry will continue to play an important role in determining cellular regulatory features and in measuring flux and intermediate concentrations to provide essential metabolic context. In addition, developmental processes, stress responses and environmental conditions are all known to markedly affect the AsA content of individual species. A better understanding of the mechanisms, internal and external to plants, that control AsA accumulation could lead to novel strategies for increasing the AsA content of crops. Unraveling the plant AsA metabolic pathway with this level of understanding is the challenging way to successful applications in fields such as molecular farming, health foods, functional foods, and plant resistance.
 Asada, K. 1992. Ascorbate peroxidase: a hydrogen peroxide- scavenging enzyme in plants. Physiol. Plant. 85: 235-241.
 Asard, H., May, J. M., and Smirnoff, N. 2004. Vitamin C Function and Biochemistry in Animals and Plants. Bios Scientific Publishers, London.
 Baier, M., Noctor, G., Foyer, C. H., and Karl-Josef Dietz, K. J. 2000. Antisense suppression of 2-cysteine peroxiredoxin in arabidopsis specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism. Plant Physiol. 124: 823-832.
 Barber, G. A. 1971. The synthesis of L-galactose by plant enzyme systems. Aren. Biochem. Biophys. 147: 619-623.
 Barber, G. A. 1979. Observations on the mechanism of the reversible epimerization of GDP-D-mannose to GDP-L-galactose by an enzyme from Chlorella pyrenoidosa. J. Biol. Chem. 254: 7600-7603.
 Berry, A., Running, J., Severson, D. K., and Burlingame, R. P. 1999. Vitamin C production in microorganisms and plants. International Patent Application, W099/64618.
 Boudrant, J. 1990. Microbial processes for ascorbic acid biosynthesis: a review. Enzyme Microb. Technol. 12: 322-329.
 Burns, J. J. 1967. Ascorbic acid. In Metabolic Pathways. Greenberg, D. M., ed. Academic Press, New York, 3rd Ed, Vol. 1.
 Chen, Z., Young, T. E., Ling, J., Chang, S. C, and Gallie, D. R. 2003. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc. Natl. Acad. Sci. USA 100: 3525-3530.
 Chen, Z., and Gallie, D. R. 2005. Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than increasing avoidance. Plant Physiol. 138: 1673- 1689.
 Chotani, G., Dodge, I, Hsu, A., Kumar, M., LaDuca, R., Trimbur, D., Weyler, W., and Sanford, K. 2000. The commercial production of chemicals using pathway engineering. Biochim. Biophys. Acta 1543:434-455.
 Conklin, P. L. 2001 . Recent advances in the role and biosynthesis of ascorbic acid in plants.Plant Cell Environ. 24: 383- 394.
 Conklin, P. L., Gatzek, S., Wheeler, G. L, Dowdle, J., Raymond, M. J., Rolinski, S., Isupov, M., Littlechild, J. A., and Smirnoff, N. 2006. Arabidopsis thaliana VTC4 encodes L-galactose 1 - phosphate phosphatase, a plant ascorbic acid biosynthetic enzyme. J. Biol. Chem. 281: 15662-15670.
 Conklin, P. L, Nprris, S. R., Wheeler, G. L, Williams, E.H., Smirnoff, N., and Last, R. L. 1999. Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc. Natl. Acad. Sci. USA 96: 4198-4203.
 Conklin, P. L, Pallanca, J. E., Last, R. L., and Smirnoff, N. 1997. L-Ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1. Plant Physiol. 115:1 277-1 285.
 Cordenunsi, B. R., Nascimento, J. R. O., Genovese, M. I., and Lajolo, F. M. 2002. Influence of cultivar on quality parameters and chemical composition of strawberry fruits grown in Brazil. J. Agric. Food Chem. 50: 2581-2586.
 Davey, M. W., Van Montagu, M., Inze, D., Sanmartin, M., Kanellis, ?., Smirnoff, N., Benzie, I. J. J., Strain, J. J., Favell, D., Fletcher, J. 2000. Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J. Sci. Food Agric. 80: 825-860.
 Davletova, S., Rizhsky, L., Liang, H. J., Zhong, S. Q., Oliver, D. J., Coutu, J., Shulaev, V, Schlauch, K., and Mittler, R. 2005. Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Want Cell 17: 268-281 .
 Doncheck, J. A., Huss, R. J., Running, J. A., and Skatrud, T. J. 1 996. L-ascorbic acid containing biomass of Chlorella pyrenoidosa. US Patent 5521090.
 Esaka, M., Hattori, T., Fujisawa, K., Sakajo, S., and Asahi, T. 1990. Molecular cloning and nucleotide sequence of full-length cDNA for ascorbate oxidase from cultured pumpkin cells. Eur. J. Biochem. 191:537-541.
 Fernanda, A., Roclo, G. L., Jose, L C, Juan, M. B., and Miguel, A. B., Victoriano, V. 2003. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat. Biotechnol. 21: 177-181.
 Franceschi, V. R., and Tarlyn, N. M. 2002. L-ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants.Plant Physiol. 130: 649-656.
 Frey, A. D., Oberle, B. T., Farres, J., and Kallio, P. T. 2004. Expression of Vitreoscilla haemoglobin in tobacco cell cultures relieves nitrosative stress in vivo and protects from NO in vitro. Plant Biotechnol. J. 2: 221-231.
 Frykman, S. A., Tsuruta, H., Starks, C. M., Regentin, R., Carney, J. R., and Licari, P. J. 2002. Control of secondary metabolite congener distributions via modulation of the dissolved oxygen tension. Biotechnol. Prog. 18: 913-920.
 Galtier, N., Foyer, C. H., Huber, J., Voelker, T. A., and Huber, S. C. 1993. Effects of elevated sucrose phosphate synthase activity on photosynthesis, assimilate partitioning, and growth in tomato (Lycoperscion esculentum var UC82B). Plant Physiol. 101: 535- 543.
 Gatzek, S., Wheeler, G. L., and Smirnoff, N. 2002. Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis. Plant J. 30: 541-553.
 Green, M. A., and Fry, S. C. 2005. Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate. Nature 433: 83-87.
 Hancock, R. D., and Viola, R. 2002. Biotechnological approaches for L-ascorbic acid production. Trends Biotechnol. 20: 299-305.
 Harris, J. R. 1996. Ascorbic Acid: Biochemistry and Biomedical Cell Biology, Subcellular Biochemistry. Plenum Press, New York, Vol. 25.
 Heick, H. M. C, Gra, G. L. A., and Humpers, J. E. C. 1969. The occurrence of ascorbic acid among the yeasts.Can. J. Microbiol. 18: 597-600.
 Helsper, J. P., and Loewus, F. A. 1982. Metabolism of I- Threonic Acid in Rumex x acutus L. and Pelargonium crispum (L.) L'Her. Plant Physiol. 69: 1365-1368.
 Isherwood, F. A. 1954. Synthesis of L-ascorbic acid in plants and animals. Biochem. J. 56: 1-15.
 Ishikawa, T, Yoshimura, K., Sakai, K., Tamoi, M., Takeda, T, and Shigeoka, S. 1998. Molecular characterization and physiological role of a glyoxysome-bound ascorbate peroxidase from spinach. Plant Cell Physiol. 39: 23-34.
 Jain, A. K., and Nessler, C. L. 2000. Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol. Breed. 6: 73-78.
 Kallio, P. T., Kim, D. J., Tsai, P. S., and Bailey, J. E. 1994. Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions.fw: J. Biochem. 219: 201-208.
 Kappers, I. F, Aharoni, A., van Herpen, T. W. J. M., Luckerhoff, L. L. P., Dicke, M., and Bouwmeester, H. J. 2005. Genetic engineering of terpenoid metabolism attracts bodyguards to Arabidopsis. Science 309: 2070-2072.
 Kato, N., and Esaka, M. 1996. cDNA cloning and gene expression of ascorbate oxidase in tobacco. Plant Mol. Biol. 30: 833- 837.
 Kato, N., and Esaka, M. 1999. Changes in ascorbate oxidase gene expression and ascorbate level in cell division and cell elongation in tobacco cells. Physiol. Plant. 105: 321-329.
 Keller, R., Renz, F. S. A., and Kossmann, J. 1 999. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. Plant J. 19: 131-141.
 Kisu, Y, Harada, Y, Goto, M., and Esaka, M. 1997. Cloning of the pumpkin ascorbate oxidase gene and analysis of cis-acting region involved in induction by auxin. Plant Cell Physiol. 38: 631-637.
 Kwon, S. Y, Choi, S. M., Ahn, Y O., Lee, H. S., Lee, H. B., Park, Y. M., and Kwak, S. S. 2003. Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. J. Plant Physiol. 160: 347-353.
 Leech, M. J., May, K., Hallard, D., Verpoorte, R., de Luca, V. Z., and Christou, P. 1 998. Expression of two consecutive genes of a secondary metabolic pathway in transgenic tobacco: molecular diversity influences levels of expression and product accumulation. Plant Mol. Biol. 38: 765-774.
 Levin, M. 1986. New concepts in the biology and biochemistry of ascorbic acid. N. Engl. J. Med. 314: 892-902.  Lieman- Hurwitz, J., Rachmilevitch, S., Mittler, R., Marcus, Y, and Kaplan, A. 2003. Enhanced photosynthesis and growth of transgenic plants that express ictB, a gene involved in HC03- accumulation in cyanobacteria. Plant Biotechnol. J. 1 : 43-50.
 Lin, L, Liu, Y G., Xu, X. P., and Li, B. J. 2003. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proc. Natl. Acad. Sei. USA 100: 5962- 5967.
 Lin, L., and Varner, J. E. 1 991 . Expression of ascorbic acid oxidase in zucchini squash (Cucurbita pepo L). Plant Physiol. 96:159-165.
 Loewus, F. A., and Kelly, S. 1961 . Identity of L-ascorbic acid formed from D-glucose by the strawberry (Fragaria). Nature 191: 10591061.
 Lorence, A., Chevone, B. I., Mendes, P., and Nessler, C. L. 2004. Myoinositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol. 134: 1200-1205.
 Millar, A. H., Mittova, V, Kiddle, G., Heazlewood, J. L., Bartoli, C. G., Theodoulou, F. L., and Foyer, C. H. 2003. Control of ascorbate synthesis by respiration and its implications for stress responses. Plant Physiol. 133:443-447.
 Mittler, R., Herr, E. H., Orvar, B. L, van Camp, W., Willekens, H., Inze, D., and Ellis, B. E. 1999. Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyper responsive to pathogen infection. Proc. Natl. Acad. Sci. USA 96: 14165-1417.
 Miyagawa, Y, Tamoi, M., and Shigeoka, S. 2001 . Overexpression of a cyanobacterial fructose-1,6-sedoheptulose-1,7- bisphosphatase in tobacco enhances photosynthesis and growth. Nat Biotechnol. 19: 965-969.
 Miyake, C, and Asada, K. 1 992. Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids. Plant Cell Physiol. 33:541-553.
 Mutsuda, M., Ishikawa, T, Takeda, T, and Shigeoka, S. 1995. Subcellular localization and properties of l-galactono-y-lactone dehydrogenase in spinach leaves. Biosci. Biotechnol. Biochem. 59: 1983-1984.
 Nascimento, J. R. O., Higuchi, B. K., Gomez, M. L. P. A., Oshiro, R. A., and Lajolo, F. M. 2005. L-ascorbate biosynthesis in strawberries: l-galactono-1, 4-lactone dehydrogenase expression during fruit development and ripening. Postharvest Biol. Technol. 38: 34-42.
 Nishikimi, M., Fukuyama, R., Minoshima, S., Shimizu, N., and Yagi, K. 1994. Cloning and chromosome mapping of the human nonfunctional gene for L-gulono-lamma-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man. J. Biol. Chem. 269: 13685-13688.
 Noctor, G., and Foyer, C. H. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 249-279.
 Nunes-Nesi, A., Carrari, R, Lytovchenko, A., Smith, A. M. O., Loureiro, M. E., Ratcliffe, R. G., Sweetlove, L. J., and Fernie, A. R. 2005. Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants. Plant Physiol. 1 37: 61 1- 622.
 oba, K., Fukui, M., Imai, Y, lriyama, S., and Nogami, K. 1994. Lgalactono-1, 4-lactone dehydrogenase: partial characterization, induction of activity and role in the synthesis of ascorbic acid in wounded white potato tuber tissue. Plant Cell Physiol. 35: 473-478.
 Oba, K., Ishikawa, S., Nishikawa, M., Mizuno, H., and Yamamoto, T. 1 995. Purification and properties of L-galactono-y- lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots./ Biochem. (Tokyo) 117: 120-124.
 Ohkawa, J., Ohya, T, lto, T, Nozawa, H., Nishi, Y, Okada, N., Yoshida, K., Takano, M., and Shinmyo, A. 1994. Structure of the genomic DNA encoding cucumber ascorbate oxidase and its expression in transgenic plants. Plant Cell Rep. 1 3: 481-488.
 Oksman-Caldentey, K. M., and Inze, D. 2004. Plant cell factories in the post-genomic era: new ways to produce designer secondary metabolites. Trends Plant Sci. 9: 433-440.
 Orvar, B. L, and Ellis, B. E. 1997. Transgenic tobacco plants expression antisense RNA for cytosolic ascorbate peroxidase show increased susceptibility to ozone injure. Plant J. 11:1 297- 1305.
 Ostergaard, J., Persiau, G., Davey, M. W., Bauw, G., and van Montagu, M. 1 997. Isolation of a cDNA coding for L-galactono-gamma- lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants. J. Biol. Chem. 272: 30009-30016.
 Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E., and Khostla, C. 2001. Biosynthesis of complex polyketides in a metabolic engineering stain of E. coli. Science 291: 1790-1792.
 Pignocchi, C, Fletcher, J. M., Wilkinson, J. E., Barnes, J. D., and Foyer, C. H. 2003. The function of ascorbate oxidase in tobacco. Plant Physiol. 132: 1631-1641.
 Running, J. 1999. Process for the production of ascorbic acid with Prototheca. US Patent 5900370.
 Running, J. A., Huss, R. J., and Olson, P. T. 1994. Heterotrophic production of ascorbic acid by microalgae. J. Appi. Phycol. 6: 99-104.
 Saito, K., Ohmoto, J., and Kuriha, N. 1997. Incorporation of ^sup 18^O into oxalic, l-threonic and l-tartaric acids during cleavage of l-ascorbic and 5-keto-d-gluconic acids in plants. Phytochemistryte: 805-809.
 Sato, F., Hashimoto, T., Hachiya, A., Tamura, K., Choi, K. B., Morishige, T, Fujimoto, H., and Yamada, Y. 2001 . Metabolic engineering of plant alkaloid biosynthesis. Proc. Natl. Acad. Sci. USA 98: 367-372.
 Sauer, M., Branduardi, P., Valli, M., and Porro, D. 2004. Production of L-ascorbic acid by metabolically engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii. Appi. Environ. Microbiol. 70: 6086-6091.
 Shimaoka, T., Yokota, A., and Miyake, C. 2000. Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves. Plant Cell Physiol. 41:1110-1118.
 Skatrud, T. J., and Huss, R. J. 1991. L-Ascorbic acid production in microorganisms. US Patent 5001059.
 Smirnoff, N. 1 996. The function and metabolism of ascorbic acid in plants. Ann. Bot. 78: 661-669.
 Smirnoff, N., Conklin, P. L., and Loewus, F. A. 2001 . Biosynthesis of ascorbic acid in plants: a renaissance.Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 437-467.
 Smirnoff, N., and Pallanca, J. E., 1996. Ascorbate metabolism in relation to oxidative stress. Biochem. Soc. Trans. 24: 472-478.
 Sweetlove, L. J., Kossmann, J., Riesmeier, J. W., Trethewey, R. N., and Hill, S. A. 1 998. The control of source to sink carbon flux during tuber development in potato. Plant J. 15: 697-706.
 Tabata, K., Oba, K., Suzuki, K., and Esaka, M. 2001. Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for l-galactono 1 ,4-lactone dehydrogenase.Plant. J. 27: 139-148.
 Tudela, J. A., Hernandez, J. A., Gil, M. A., and Espln, J. C. 2003. L-galactono-gamma-lactone dehydrogenase and vitamin C content in fresh-cut potatoes stored under controlled atmospheres. J, Agric. Food Chem. 51 : 4296-4302.
 Verpoorte, R., and Memelink, J. 2002. Engineering secondary metabolite production in plants. Curr. Opin. Biotechnol. 13: 181- 187.
 Washko, P. W., Welch, R. W., Dhariwal, K. R., Wang, Y, and Levine, M. 1992. Ascorbic acid and dehydroascorbic acid analyses in biological samples.Anal. Biochem. 204: 1-14.
 Wheeler, G. L., Jones, M. A., and Smirnoff, N. 1998. The biosynthetic pathway of vitamin C in higher plants. Nature 393: 365- 369.
 Wolucka, B. A., Persiau, G., Doorsselaere, J. V, Davey, M. W., Demol, H., Vandekerckhove, J., Montagu, M. V, Zabeau, M., and Wout Boerjan, W. 2001. Partial purification and identification of GDPmannose 3',5'-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc. Natl. Acad. Sci. USA 98: 14843- 14848.
 Yang, J. C, and Loewus, F. A. 1975. Metabolic conversion of L-ascorbic-acid to oxalic-acid in oxalate accumulating plants. Plant Physiol. 56: 283-285.
 Zhang, L., Ding, R. X., Chai, Y. R., Bonfill, M., Moyano, E., Oksman-Caldentey, K. M., Xu, T F, Pi, Y, Wang, Z. N., Zhang, H. M., Kai, G. Y, Liao, Z. H., Sun, X. F, and Tang, K. X. 2004. Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures. Proc. Natl. Acad. Sci. USA 101: 6786-6791.
Lei Zhang, Zinan Wang, and Yang Xia
State Key Laboratory of Genetic Engineering,
School of Life Sciences, Fudan-SJTUNottingham Plant Biotechnology R&D
Center, Fudan University, Shanghai, P. R.
China, and Department of Pharmacognosy,
School of Pharmacy, Second Military Medical
University, Shanghai, P. R. China
Laboratory of Plant Bioengineering, College
of Life and Environment, Shanghai Normal
University, Shanghai, P. R. China
Department of Pharmacy, Changzheng
Hospital, Second Military Medical University,
Shanghai, P. R. China
State Key Laboratory of Genetic Engineering,
School of Life Sciences, Fudan-SJTUNottingham Plant Biotechnology R&D Center,
Fudan University, Shanghai, P. R. China
Address correspondence to Professor Kexuan Tang, State Key Laboratory of Genetic Engineering, School of Life Science, Fudan- SJTU -Nottingham Plant Biotechnology R&D Center, Fudan University, Shanghai, 200433, P. R. China. E-mail: kxtang@ fudan.edu.cn or [email protected]
Copyright Taylor & Francis Ltd. Jul-Sep 2007
(c) 2007 Critical Reviews in Biotechnology. Provided by ProQuest Information and Learning. All rights Reserved.