Recent patents on vitamin C: opportunities for crop improvement and single-step biological manufacture.

Vitamin C is an essential human nutrient obtained primarily from plant foods. Despite the necessity of the vitamin for human health and the essential role that it plays in plant stress resistance, it is only in the last decade that an understanding of its biosynthesis in plants has emerged. These fundamental discoveries present for the first time the opportunity to manipulate crop plants for improved human nutrition and enhanced agronomic performance and are discussed in relation to recent patents protecting aspects of vitamin C synthesis and degradation in plants. Vitamin C has a wide range of medicinal and industrial uses resulting in an annual market exceeding US$600 million. In recent years, there has been strong competition in vitamin C markets as a result of emerging Chinese producers and one response has been increased investment in research and development for the biological manufacture of the vitamin. Knowledge of the plant biosynthetic pathway has provided novel opportunities for the synthesis of vitamin C in yeasts, although successes have been limited to date. A more promising route is the direct synthesis of vitamin C in acetic acid bacteria and significant research and development has gone into the genetic improvement of appropriate strains.

Related papers:

title: Biotechnological approaches for L-ascorbic acid production.
abstract: Over the past decade there has been increasing pressure to develop alternatives to the Reichstein process, a largely chemical synthesis by which the vast majority of world vitamin C (L-ascorbic acid, L-AA) is produced. The pressures include increasing environmental concerns and legislation, and the need to increase process efficiency and reduce capital costs. The development of efficient fermentation processes in the past ten years has also represented a catalyst for change. Here, we describe the development of biotechnological alternatives for the synthesis of Reichstein intermediates by industrial microorganisms. The recent elucidation of the plant biosynthetic pathway represents new opportunities not only for the direct synthesis of L-AA by fermentation but also for the production of human crop plants and animal fodder with enhanced nutritional value. We discuss the potential for these developments in the light of recent findings concerning L-AA biosynthesis in plants.

title: The biosynthetic pathway of vitamin C in higher plants
abstract: Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown—one of the few primary plant metabolic pathways forwhich this is the case. L-ascorbate is abundant in plants (found at concentrations of 1–5 mM in leaves and 25 mM in chloroplasts,) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose anascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

title: The use of microorganisms in L-ascorbic acid production.
abstract: L-Ascorbic acid has been industrially produced for around 70 years. Over the past two decades, several innovative bioconversion systems have been proposed in order to simplify the long time market-dominating Reichstein method, a largely chemical synthesis by which still a considerable part of L-ascorbic acid is produced. Here, we describe the current state of biotechnological alternatives using bacteria, yeasts, and microalgae. We also discuss the potential for direct production of l-ascorbic acid exploiting novel bacterial pathways. The advantages of these novel approaches competing with current chemical and biotechnological processes are outlined.

title: The use of micro-organisms for L-ascorbic acid production: current status and future perspectives
abstract: L-Ascorbic acid (L-AA) has been industrially produced for around 60 years in a primarily chemical process utilising D-glucose (D-glc) as starting material. Current world production is estimated at approximately 80,000 tonnes per annum with a worldwide market in excess of U.S. $600 million. We present a brief overview of research geared to exploiting micro-organisms for the industrial production of vitamin C, with emphasis on recent approaches using genetically engineered bacterial strains. We also discuss the potential for direct production of L-AA exploiting novel biochemical pathways with particular reference to yeast fermentations. The potential advantages of these novel approaches over current chemical and biotechnological processes are outlined.

title: Biosynthesis and Catabolism of L-Ascorbic Acid in Plants
abstract: L-Ascorbic acid (AsA) is a vital antioxidant compound that plays a critical role in the cellular metabolism of plants and animals. Research on plant AsA metabolism experienced a significant resurgence after 1998 following the identification of AsA-deficient Arabidopsis mutants and the elucidation of a biosynthetic pathway accepted by the overwhelming majority of the plant science community. The identification and cloning of novel biosynthetic genes and the ensuing metabolic engineering of plant AsA content has however revealed a more complex picture. Additional biosynthetic routes have been identified and unexpected biochemical phenotypes were observed upon expression of animal AsA biosynthetic genes. The isolation of novel AsA conjugates from plant tissues and the evidence for long distance transport of AsA in plants have provided additional facets to its functionality. Although some progress has been made regarding the impact of AsA recycling on pool size, we still do not have a clear picture of the biochemistry of AsA degradation. This communication comprehensively reviews new developments in the AsA metabolic system and prompts directions for future research.

title: Progress in manipulating ascorbic acid biosynthesis and accumulation in plants
abstract: l-Ascorbic acid (vitamin C) is synthesized from hexose sugars. It is an antioxidant and redox buffer, as well as an enzyme cofactor, so it has multiple roles in metabolism and in plant responses to abiotic stresses and pathogens. Plant-derived ascorbate also provides the major source of vitamin C in the human diet. An understanding of how ascorbate metabolism is controlled should provide a basis for engineering or otherwise manipulating its accumulation. Biochemical and molecular genetic evidence supports synthesis from GDP-d-mannose via l-galactose (d-Man/l-Gal pathway) as a significant source of ascorbate. More recently, evidence for pathways via uronic acids has been obtained: overexpression of myo-inositol oxygenase, d-galacturonate reductase and l-gulono-1,4-lactone oxidase all increase leaf ascorbate concentration. Interestingly, this has proved more effective in pathway engineering than overexpressing various d-Man/l-Gal pathway genes. Ascorbate oxidation generates the potentially unstable dehydroascorbate, and the overexpression of glutathione-dependent dehydroascorbate reductase has resulted in increased ascorbate. Ascorbate is catabolized to products such as oxalate, l-threonate and l-tartrate. The enzymes involved have not been identified, so catabolism is not yet amenable to manipulation. In the examples of pathway engineering so far, the increase in ascorbate has been modest on an absolute or proportional basis. Therefore, a deeper understanding of ascorbate metabolism is needed to achieve larger increases. Identifying genes that control ascorbate accumulation by techniques such as analysis of quantitative trait loci (QTL) or activation tagging may hold promise, particularly if regulatory genes can be identified.

title: Metabolic Engineering of Plant L-Ascorbic Acid Biosynthesis: Recent Trends and Applications
abstract: (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.

title: Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase
abstract: L-Ascorbic acid (vitamin C) in fruits and vegetables is an essential component of human nutrition. Surprisingly, only limited information is available about the pathway(s) leading to its biosynthesis in plants. Here, we report the isolation and characterization of GalUR, a gene from strawberry that encodes an NADPH-dependent D-galacturonate reductase. We provide evidence that the biosynthesis of L-ascorbic acid in strawberry fruit occurs through D-galacturonic acid, a principal component of cell wall pectins. Expression of GalUR correlated with changing ascorbic acid content in strawberry fruit during ripening and with variations in ascorbic acid content in fruit of different species of the genus Fragaria. Reduced pectin solubilization in cell walls of transgenic strawberry fruit with decreased expression of an endogenous pectate lyase gene resulted in lower ascorbic acid content. Overexpression of GalUR in Arabidopsis thaliana enhanced vitamin C content two- to threefold, demonstrating the feasibility of engineering increased vitamin C levels in plants using this gene.

title: myo-Inositol Oxygenase Offers a Possible Entry Point into Plant Ascorbate Biosynthesis1
abstract: Two biosynthetic pathways for ascorbate (l-ascorbic acid [AsA]; vitamin C) in plants are presently known, the mannose/l-galactose pathway and an l-GalUA pathway. Here, we present molecular and biochemical evidence for a possible biosynthetic route using myo-inositol (MI) as the initial substrate. A MI oxygenase (MIOX) gene was identified in chromosome 4 (miox4) of Arabidopsis ecotype Columbia, and its enzymatic activity was confirmed in bacterially expressed recombinant protein. Miox4 was primarily expressed in flowers and leaves of wild-type Arabidopsis plants, tissues with a high concentration of AsA. Ascorbate levels increased 2- to 3-fold in homozygous Arabidopsis lines overexpressing the miox4 open reading frame, thus suggesting the role of MI in AsA biosynthesis and the potential for using this gene for the agronomic and nutritional enhancement of crops.

title: GDP-Mannose 3′,5′-Epimerase Forms GDP-L-gulose, a Putative Intermediate for the de Novo Biosynthesis of Vitamin C in Plants*
abstract: Despite its importance for agriculture, bioindustry, and nutrition, the fundamental process of l-ascorbic acid (vitamin C) biosynthesis in plants is not completely elucidated, and little is known about its regulation. The recently identified GDP-Man 3′,5′-epimerase catalyzes a reversible epimerization of GDP-d-mannose that precedes the committed step in the biosynthesis of vitamin C, resulting in the hydrolysis of the highly energetic glycosyl-pyrophosphoryl linkage. Here, we characterize the native and recombinant GDP-Man 3′,5′-epimerase of Arabidopsis thaliana. GDP and GDP-d-glucose are potent competitive inhibitors of the enzyme, whereas GDP-l-fucose gives a complex type of inhibition. The epimerase contains a modified version of the NAD binding motif and is inhibited by NAD(P)H and stimulated by NAD(P)+. A feedback inhibition of vitamin C biosynthesis is observed apparently at the level of GDP-Man 3′,5′-epimerase. The epimerase catalyzes at least two distinct epimerization reactions and releases, besides the well known GDP-l-galactose, a novel intermediate: GDP-l-gulose. The yield of the epimerization varies and seems to depend on the molecular form of the enzyme. Both recombinant and native enzymes co-purified with a Hsp70 heat-shock protein (Escherichia coli DnaK and A. thaliana Hsc70.3, respectively). We speculate, therefore, that the Hsp70 molecular chaperones might be involved in folding and/or regulation of the epimerase. In summary, the plant epimerase undergoes a complex regulation and could control the carbon flux into the vitamin C pathway in response to the redox state of the cell, stress conditions, and GDP-sugar demand for the cell wall/glycoprotein biosynthesis. Exogenous l-gulose and l-gulono-1,4-lactone serve as direct precursors of l-ascorbic acid in plant cells. We propose an l-gulose pathway for the de novo biosynthesis of vitamin C in plants.

title: Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability.
abstract: Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L-galactose to L-galactose 1-P. We show that a GDP-L-galactose phosphorylase, encoded by the Arabidopsis thaliana VTC2 gene, catalyses this step in the ascorbate biosynthetic pathway. Furthermore, a homologue of VTC2, At5g55120, encodes a second GDP-L-galactose phosphorylase with similar properties to VTC2. Two At5g55120 T-DNA insertion mutants (vtc5-1 and vtc5-2) have 80% of the wild-type ascorbate level. Double mutants were produced by crossing the loss-of-function vtc2-1 mutant with each of the two vtc5 alleles. These show growth arrest immediately upon germination and the cotyledons subsequently bleach. Normal growth was restored by supplementation with ascorbate or L-galactose, indicating that both enzymes are necessary for ascorbate generation. vtc2-1 leaves contain more mannose 6-P than wild-type. We conclude that the GDP-mannose pathway is the only significant source of ascorbate in A. thaliana seedlings, and that ascorbate is essential for seedling growth. A. thaliana leaves accumulate more ascorbate after acclimatization to high light intensity. VTC2 expression and GDP-L-galactose phosphorylase activity rapidly increase on transfer to high light, but the activity of other enzymes in the GDP-mannose pathway is little affected. VTC2 and At5g55120 (VTC5) expression also peak in at the beginning of the light cycle and are controlled by the circadian clock. The GDP-L-galactose phosphorylase step may therefore play an important role in controlling ascorbate biosynthesis.

title: L-tartaric acid synthesis from vitamin C in higher plants.
abstract: The biosynthetic pathway of L-tartaric acid, the form most commonly encountered in nature, and its catabolic ties to vitamin C, remain a challenge to plant scientists. Vitamin C and L-tartaric acid are plant-derived metabolites with intrinsic human value. In contrast to most fruits during development, grapes accumulate L-tartaric acid, which remains within the berry throughout ripening. Berry taste and the organoleptic properties and aging potential of wines are intimately linked to levels of L-tartaric acid present in the fruit, and those added during vinification. Elucidation of the reactions relating L-tartaric acid to vitamin C catabolism in the Vitaceae showed that they proceed via the oxidation of L-idonic acid, the proposed rate-limiting step in the pathway. Here we report the use of transcript and metabolite profiling to identify candidate cDNAs from genes expressed at developmental times and in tissues appropriate for L-tartaric acid biosynthesis in grape berries. Enzymological analyses of one candidate confirmed its activity in the proposed rate-limiting step of the direct pathway from vitamin C to tartaric acid in higher plants. Surveying organic acid content in Vitis and related genera, we have identified a non-tartrate-forming species in which this gene is deleted. This species accumulates in excess of three times the levels of vitamin C than comparably ripe berries of tartrate-accumulating species, suggesting that modulation of tartaric acid biosynthesis may provide a rational basis for the production of grapes rich in vitamin C.

title: Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-l-threonate
abstract: Increasing the l-ascorbate (vitamin C) content of crops could in principle involve promoting its biosynthesis or inhibiting its degradation. Recent progress has revealed biosynthetic pathways for ascorbate, but the degradative pathways remain unclear. The elucidation of such pathways could promote an understanding of the roles of ascorbate in plants, and especially of the intriguing positive correlation between growth rate and ascorbate oxidase (or its products). In some plants (Vitaceae), ascorbate is degraded via l-idonate to l-threarate (l-tartrate), with the latter arising from carbons 1–4 of ascorbate. In most plants, however (including Vitaceae), ascorbate degradation can occur via dehydroascorbate, yielding oxalate plus l-threonate, with the latter from carbons 3–6 of ascorbate. The metabolic steps between ascorbate and oxalate/l-threonate, and their subcellular location, were unknown. Here we show that this pathway operates extracellularly in cultured Rosa cells, proceeds via several novel intermediates including 4-O-oxalyl-l-threonate, and involves at least one new enzyme activity. The pathway can also operate non-enzymatically, potentially accounting for vitamin losses during cooking. Several steps in the pathway may generate peroxide; this may contribute to the role of ascorbate as a pro-oxidant that is potentially capable of loosening the plant cell wall and/or triggering an oxidative burst.

title: Tracer studies on ascorbic acid formation in plants
abstract: Tracer studies in which D -glucose-1- 14 C, -2- 14 C or -6- 14 C; D -galactose-1- 14 C; D -glucuronic acid-6- 14 C; D -glucuronolactone-1- 14 C or -6- 14 C; D -galacturonic acid-1- 14 C; D -galacturonate-U- 14 C methyl ester; myo -inositol-2 14 C; or D -glucose-1-t or -6-t are administered to intact plant tissues, usually to the detached ripening strawberry or the detached parsley leaf, reveal the existence of two processes of L -ascorbic acid formation in higher plants. In one process, D -glucose and related sugars are utilized as their hexose phosphates. Oxidation proceeds at carbon 1 of the sugar to form the carboxyl group at carbon 1 of L -ascorbic acid. Except for partial equilibration of the hexose phosphate with triose and pentose phosphates, the six carbon chain is utilized without cleavage. Carbon 5 is epimerized from the D - to the L - configuration during the conversion. Carbon 6 remains reduced throughout the process. The other process of L -ascorbic acid formation proceeds from exogenously supplied D -glucuronolactone or D -galacturonate methyl ester. Here, the carboxyl group at carbon 6 of the uronic acid derivative is conserved, becoming carbon 1 of L -ascorbic acid. The aldehydic group of the uronic acid derivative is reduced to a primary alcohol corresponding to carbon 6 in the L -ascorbic acid. There is no cleavage of the carbon chain during the conversion. D -Glucuronic acid supplied exogenously or generated enzymatically within the plant tissue from myo -inositol is not utilized for L -ascorbic acid synthesis. The physiological significance of the two processes is discussed.

title: d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves.
abstract: d-[6-(14)C]Glucosone that had been prepared enzymically from d-[6-(14)C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, (14)C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-(14)C]glucose underwent considerable redistribution during AA formation, whereas (14)C from [6-(14)C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-(14)C]sorbosone was found to be equivalent to [6-(14)C]glucose as a source of (14)C for AA. In the presence of 0.1% d-glucosone, conversion of [6-(14)C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.

title: Antisense suppression of l-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l-galactose synthesis.
abstract: l-Galactose dehydrogenase (l-GalDH), a novel enzyme that oxidizes l-Gal to l-galactono-1,4-lactone (l-GalL), has been purified from pea seedlings and cloned from Arabidopsis thaliana. l-GalL is a proposed substrate for ascorbate biosynthesis in plants, therefore the function of l-GalDH in ascorbate biosynthesis was investigated by overexpression in tobacco and antisense suppression in A. thaliana. In tobacco the highest expressing lines had a 3.5-fold increase in extractable activity, but this did not increase leaf ascorbate concentration. Arabidopsis thaliana, transformed with an antisense l-GalDH construct, produced lines with 30% of wild-type activity. These had lower leaf ascorbate concentration when grown under high light conditions. l-Gal pool size increased in antisense transformants with low l-GalDH activity, and l-Gal concentration was negatively correlated with ascorbate. The results provide direct evidence for a role of l-GalDH in ascorbate biosynthesis. Ascorbate pool size in A. thaliana is increased by acclimation to high light, but l-GalDH expression was not affected. l-Gal accumulation was higher in antisense plants acclimated to high light, indicating that the capacity to synthesize l-Gal from GDP-mannose is increased. Because the only known function of l-GalL is ascorbate synthesis, these antisense plants provide an opportunity to investigate ascorbate function with minimal effects on carbohydrate metabolism. Measurements of other antioxidants revealed an increase in ascorbate- and pyrogallol-dependent peroxidase activity in low-ascorbate lines. As ascorbate is the major hydrogen peroxide-scavenging antioxidant in plants, this could indicate a compensatory mechanism for controlling hydrogen peroxide concentration.

title: Partial purification and identification of GDP-mannose 3",5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway
abstract: The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, of l-galactosyl residues, catalyzed by a largely unknown GDP-d-mannose 3",5"-epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-d-mannose 3",5"-epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a blast search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP-d-mannose 5"-epimerase. The plant GDP-d-mannose 3",5"-epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase/reductase family. The enzyme was cloned and expressed in Escherichia coli cells.

title: An Arabidopsis Purple Acid Phosphatase with Phytase Activity Increases Foliar Ascorbate1[OA]
abstract: Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activation-tagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.

title: Biological synthesis of ascorbic acid: the conversion of derivatives of D-galacturonic acid into L-ascorbic acid by plant extracts.
abstract: Broquist, H. P., Kohler, A. R., Hutchison, D. J. & Burchenal,  (1953).  Brown, E. G., Goodwin, T. W. & Pendlington, S. (1955).  Flaks, J. G. & Gladner, J. A. (1955). In Amino Acid Metabolism, p. 743. Ed. by McElroy, W. D. & Glass, B. Baltimore: Johns Hopkins Press. Fosse, M. R. (1914).  Paris, 158, 1076. Goodwin, T. W. & Pendlington, S. (1954).  F. (1945). U.S. Pat., 2,367,644. Klungs0yr, L. K. (1954). Acta chem. 8cand. 8, 723. Kuhn, R. & Rudy, H. (1934). Ber. dtsch. chem. Gae. 66,1956. Maclaren,  (1952).  (1945). U.S. Pat., 2,367,646. McNutt, W. S. (1954).  Plaut, G. W. E. (1954).  208, 513.

title: Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence
abstract: Ascorbic acid is a well-known antioxidant and cellular reductant with an intimate and complex role in the response of plants to ozone. It is clear from a number of studies that sensitivity to ozone is correlated with total ascorbic acid levels, and that a first line of defence against the reactive oxygen species generated in the apoplastic space by ozone is ascorbic acid. For activity, ascorbic acid must be in the fully reduced state. Therefore, both the rate of ascorbic acid synthesis and recycling via dehydroascorbate and monodehydroascorbate reductases are critical in the maintenance of a high ascorbic acid redox state. Active transport of ascorbic acid across the plasma membrane is necessary to achieve reduction of oxidized ascorbic acid by cytoplasm-localized reductases. It has been known for some time that the chlorotic lesions produced by exposure to ozone are not unlike lesions produced by the hypersensitive response to avirulent pathogen attack. Surprisingly, activation of a defence gene-signalling network by both ozone and pathogens is influenced by the level of ascorbic acid. Indeed, in addition to acting simply as an antioxidant in the apoplastic space, ascorbic acid appears to be involved in a complex phytohormone-mediated signalling network that ties together ozone and pathogen responses and influences the onset of senescence.

title: Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant.
abstract: The Arabidopsis thaliana ascorbate-deficient vtc-1 mutant has only 30% ascorbate contents of the wild type (WT). This ascorbate-deficient mutant was used here to study the physiological roles of ascorbate under salt stress in vivo. Salt stress resulted in a more significant decrease in CO2 assimilatory capacity in the vtc-1 mutant than in the WT. Photosystem II function in the Arabidopsis vtc-1 mutant also showed an increased sensitivity to salt stress. Oxidative stress, indicated by the hydrogen peroxide content, increased more dramatically in the vtc-1 mutant than in the WT under salt stress. To clarify the reason for the increased oxidative stress in the vtc-1 mutant, the contents of small antioxidant compounds and the activities of several antioxidant enzymes in the ascorbate-glutathione cycle were measured. Despite an elevated glutathione pool in the vtc-1 mutant, the ascorbate contents and the reduced form of ascorbate decreased very rapidly under salt stress. These results showed that the activities of MDAR and DHAR were lower in the vtc-1 mutant than in the WT under salt stress. Thus, low intrinsic ascorbate and an impaired ascorbate-glutathione cycle in the vtc-1 mutant under salt stress probably induced a dramatic decrease in the reduced form of ascorbate, which resulted in both enhanced ROS contents and decreased NPQ in the vtc-1 mutant.

title: Ascorbate-Deficient Mutants of Arabidopsis Grow in High Light Despite Chronic Photooxidative Stress1
abstract: Acclimation to changing environments, such as increases in light intensity, is necessary, especially for the survival of sedentary organisms like plants. To learn more about the importance of ascorbate in the acclimation of plants to high light (HL), vtc2, an ascorbate-deficient mutant of Arabidopsis, and the double mutants vtc2npq4 and vtc2npq1 were tested for growth in low light and HL and compared with the wild type. The vtc2 mutant has only 10% to 30% of wild-type levels of ascorbate, vtc2npq4 has lower ascorbate levels and lacks non-photochemical quenching of chlorophyll fluorescence (NPQ) because of the absence of the photosystem II protein PsbS, and vtc2npq1 is NPQ deficient and also lacks zeaxanthin in HL but has PsbS. All three genotypes were able to grow in HL and had wild-type levels of Lhcb1, cytochrome f, PsaF, and 2-cysteine peroxiredoxin. However, the mutants had lower electron transport and oxygen evolution rates and lower quantum efficiency of PSII compared with the wild type, implying that they experienced chronic photooxidative stress. The mutants lacking NPQ in addition to ascorbate were only slightly more affected than vtc2. All three mutants had higher glutathione levels than the wild type in HL, suggesting a possible compensation for the lower ascorbate content. These results demonstrate the importance of ascorbate for the long-term acclimation of plants to HL.

title: The Timing of Senescence and Response to Pathogens Is Altered in the Ascorbate-Deficient Arabidopsis Mutant vitamin c-11
abstract: The ozone-sensitive Arabidopsis mutant vitamin c-1 (vtc1) is deficient in l-ascorbic acid (AsA) due to a mutation in GDP-Man pyrophosphorylase (Conklin et al., 1999), an enzyme involved in the AsA biosynthetic pathway (Smirnoff et al., 2001). In this study, the physiology of this AsA deficiency was initially investigated in response to biotic (virulent pathogens) stress and subsequently with regards to the onset of senescence. Infection with either virulent Pseudomonas syringae or Peronospora parasitica resulted in largely reduced bacterial and hyphal growth in the vtc1 mutant in comparison to the wild type. When vitamin c-2 (vtc2), another AsA-deficient mutant, was challenged with P. parasitica, growth of the fungus was also reduced, indicating that the two AsA-deficient mutants are more resistant to these pathogens. Induction of pathogenesis-related proteins PR-1 and PR-5 is significantly higher in vtc1 than in the wild type when challenged with virulent P. syringae. In addition, the vtc1 mutant exhibits elevated levels of some senescence-associated gene (SAG) transcripts as well as heightened salicylic acid levels. Presumably, therefore, low AsA is causing vtc1 to enter at least some stage(s) of senescence prematurely with an accompanying increase in salicylic acid levels that results in a faster induction of defense responses.

title: Ascorbic acid conjugates isolated from the phloem of Cucurbitaceae.
abstract: Analysis of phloem exudates from the fruit of Cucurbitaceae revealed the presence of several compounds with UV-visible absorption spectra identical to that of l-ascorbic acid. In Cucurbita pepo L. (zucchini), the compounds could be isolated from phloem exudates collected from aerial parts of the plant but were not detected in whole tissue homogenates. The compounds isolated from the phloem exudates of C. pepo fruit were eluted from strong anion exchange resin in the same fraction as l-ascorbic acid and were oxidised by ascorbate oxidase (E.C. 1.10.3.3). The major compound purified from C. pepo fruit exudates demonstrated similar redox properties to l-ascorbic acid and synthetic 6-O-glucosyl-l-ascorbic acid (6-GlcAsA) but differed from those of 2-O-glucosyl-l-ascorbic acid (2-GlcAsA) isolated from the fruit of Lycium barbarum L. Parent and fragment ion masses of the compound were consistent with hexosyl-ascorbate in which the hexose moiety was attached to C5 or C6 of AsA. Acid hydrolysis of the major C. pepo compound resulted in the formation of l-ascorbic acid and glucose. The purified compound yielded a proton NMR spectrum that was almost identical to that of synthetic 6-GlcAsA. A series of l-ascorbic acid conjugates have, therefore, been identified in the phloem of Cucurbitaceae and the most abundant conjugate has been identified as 6-GlcAsA. The potential role of such conjugates in the long-distance transport of l-ascorbic acid is discussed.

title: Ascorbate biosynthesis in Arabidopsis cell suspension culture.
abstract: The biosynthesis of L-ascorbic acid (L-AA) in an Arabidopsis (L.) Heynh. cell suspension culture was studied by quantifying the effects of incubation with a range of potential biosynthetic precursors, analogs, and inhibitors on the intracellular levels of reduced and oxidized forms of L-AA. Our results support the recently published biosynthetic pathway of L-AA from L-galactose (G.L. Wheeler, M.A. Jones, N. Smirnoff [1998] ), but suggest that Arabidopsis cell suspension culture simultaneously contains two other routes leading to L-AA. The possible physiological significance of these alternate routes is discussed.

title: Increasing vitamin C content of plants through enhanced ascorbate recycling
abstract: Vitamin C (ascorbic acid) is essential to prevent disease associated with connective tissue (e.g., scurvy), improves cardiovascular and immune cell functions, and is used to regenerate α-tocopherol (vitamin E). In contrast to most animals, humans lack the ability to synthesize ascorbic acid as a result of a mutation in the last enzyme required for ascorbate biosynthesis. Vitamin C, therefore, must be obtained from dietary sources and, because it cannot be stored in the body, it must be obtained regularly. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR). To examine whether 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 ascorbic acid levels 2- to 4-fold and significantly increased the ascorbate redox state in both tobacco and maize. In addition, the level of glutathione, the reductant used by DHAR, also increased, as did its redox state. These results demonstrate that the vitamin C content of plants can be elevated by increasing expression of the enzyme responsible for recycling ascorbate.

title: The Ascorbic Acid Redox State Controls Guard Cell Signaling and Stomatal Movement
abstract: H2O2 serves an important stress signaling function and promotes stomatal closure, whereas ascorbic acid (Asc) is the major antioxidant that scavenges H2O2. Dehydroascorbate reductase (DHAR) catalyzes the reduction of dehydroascorbate (oxidized ascorbate) to Asc and thus contributes to the regulation of the Asc redox state. In this study, we observed that the level of H2O2 and the Asc redox state in guard cells and whole leaves are diurnally regulated such that the former increases during the afternoon, whereas the latter decreases. Plants with an increased guard cell Asc redox state were generated by increasing DHAR expression, and these exhibited a reduction in the level of guard cell H2O2. In addition, a higher percentage of open stomata, an increase in total open stomatal area, increased stomatal conductance, and increased transpiration were observed. Guard cells with an increase in Asc redox state were less responsive to H2O2 or abscisic acid signaling, and the plants exhibited greater water loss under drought conditions, whereas suppressing DHAR expression conferred increased drought tolerance. Our analyses suggest that DHAR serves to maintain a basal level of Asc recycling in guard cells that is insufficient to scavenge the high rate of H2O2 produced in the afternoon, thus resulting in stomatal closure.

title: Synthesis of l-(+)-Tartaric Acid from l-Ascorbic Acid via 5-Keto-d-Gluconic Acid in Grapes.
abstract: 5-Keto-l-idionic acid ( identical with5-keto-d-gluconic acid, d-xylo-5-hexulosonic acid) was found as a metabolic product of l-ascorbic acid in slices of immature grapes, Vitis labrusca L. cv ;Delaware'. Specifically labeled compounds, recognized as metabolic products of l-ascorbic acid in grapes, were fed to young grape tissues to investigate the metabolic pathway from l-ascorbic acid to l-(+)-tartaric acid.Label from dehydro-l-[1-(14)C]ascorbic acid, 2-keto-l-[1-(14)C]idonic acid (l-xylo-2-hexulosonic acid), l-[1-(14)C]idonic acid, or 5-keto-l-[1-(14)C] idonic acid was incorporated into l-(+)-tartaric acid in high yields as it was in the l-[1-(14)C]ascorbic acid experiment. In a double label experiment involving a mixture of l-[1-(14)C]idonic acid and l-[2-(3)H]idonic acid, the (3)H/(14)C ratios of 5-keto-l-idonic acid and l-(+)-tartaric acid synthesized in young grape leaves were almost the same as the value of the l-idonic acid fed. Label from 5-keto-l-[6-(14)C]idonic acid was incorporated into sugars and insoluble residue in the same way as l-[6-(14)C]ascorbic acid was metabolized in grapes.These results provide strong evidence that in grapes l-(+)-tartaric acid is synthesized from the C(4) fragment that corresponds to the C1 to C4 group of the 5-keto-l-idonic acid derived from l-ascorbic acid via 2-keto-l-idonic acid and l-idonic acid.

title: Redox Homeostasis and Antioxidant Signaling: A Metabolic Interface between Stress Perception and Physiological Responses
abstract: Low molecular weight antioxidants, such as ascorbate, glutathione, and tocopherol, are information-rich redox buffers that interact with numerous cellular components. In addition to crucial roles in defense and as enzyme cofactors, cellular antioxidants influence plant growth and development by modulating processes from mitosis and cell elongation to senescence and death (De Pinto and De Gara, 2004; Potters et al., 2004; Tokunaga et al., 2005). Most importantly, antioxidants provide essential information on cellular redox state, and they influence gene expression associated with biotic and abiotic stress responses to maximize defense. Growing evidence suggests a model for redox homeostasis in which the reactive oxygen species (ROS)–antioxidant interaction acts as a metabolic interface for signals derived from metabolism and from the environment. This interface modulates the appropriate induction of acclimation processes or, alternatively, execution of cell death programs.

title: Biosynthesis of d-Erythroascorbic Acid by Candida
abstract: and many strains of yeasts.') This paper proves chemically that the analog produced by Candida from various sugars including sucrose, hexose and pentose is D-EAsA. As the results of screening from about 100 strains of yasts, Candida guilliermondii, C. utilis and C. zeylanoi des produced a single ascorbic acid analog in Koji extract or a synthetic sucrose medium . C. utilis IFO 0396 was cultured by shaking in a syn thetic medium composed of 5 % sucrose, 0.5 % NH4NO3 0.5% KH2PO4, 0.05% MgSO4.7H,O, 0,01% CaCl, 2H,O, 0.01 % NaCI and trace amounts of biotin, pH 4.5. After the incubation for 5 days the analog was separated by Amberlite IR-45 column from the broth. The eluate from the resin was reduced by H2S, and con centrated in vacuo. The 2, 4-dinitrophenylhydrazone of this concentrate was purified by adsorption chro matography on silicagel column, and recrystallized from acetone-ethanol mixture. From the Rf values of paper and thin-layer chromatograms and optical rotation analysis, the analog was considered as nEAsA. IR spectra of osazones of L-AsA, D-erythor bic acid (n-EA) and n-EAsA closely resembled to each other, so the identification with this method may not be adequate for the compounds of these kinds. On the other hand, NMR spectrum of osazone of DEAsA in D2O was clearly illustrated in signals that this analog was distinguished from those of L-AsA and DFIG, 1. Paper Chromatogram of Ascorbic Acid Analogs. Developing solvent was methyl ethyl ketone saturated with water and reagent was 2, 6-dichlorophenolindo phenol. a, Candida utilis IFO 0396; b, Candida zeyl anoides IFO 0719; c, Candida guilliermondii IFO 0676; TR, triose reductone.

title: Characterisation of D-arabinono-1,4-lactone oxidase from Candida albicans ATCC 10231.
abstract: D-Erythroascorbic acid was detected from the cell extracts of a dimorphic fungus, Candida albicans. Its concentration in yeast cells grown at 25 degrees C was estimated to be about 0.45 mumol/ml cell water. D-Arabinono-1,4-lactone oxidase, which catalyses the final step in the biosynthesis of D-erythroascorbic acid, was purified 639-fold from the mitochondrial fraction of C. albicans to apparent homogeneity, with an overall yield of 21.2%, by a purification procedure consisting of Triton X-100 solubilisation, ammonium sulphate precipitation, anion-exchange, hydrophobic-interaction, gel-filtration and dye-ligand chromatographies. Gel-filtration chromatography and polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave apparent molecular masses of 110 kDa and 84.4 kDa, respectively. SDS/PAGE showed only one protein band corresponding to a molecular mass of 66.7 kDa. Considering the binding of detergents, the enzyme is suggested to be a single polypeptide. The enzyme showed a typical fluorescence excitation spectrum of a flavin-containing enzyme. The flavin was not released by treatment with SDS, CCl3CO2H or boiling, indicating that it may be covalently bound to the enzyme protein. The enzyme was optimally active at 40 degrees C and at pH 6.1. The enzyme was stable in the range pH 7.5-10. An apparent Km value for D-arabinono-1,4-lactone was 44.1 mM. L-Galactono-1,4-lactone, L-gulono-1,4-lactone and L-xylono-1,4-lactone could also serve as substrates. Competitive inhibition was demonstrated with D-glucono-1,5-lactone, L-arabinono-1,4-lactone, D-galactono-1,4-lactone and D-gulono-1,4-lactone. p-Chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, iodoacetamide and divalent metal ions such as Cd2+, Hg2+, Mn2+ and Zn2+ exhibited inhibitory effects on the enzyme.

title: D-arabinose dehydrogenase and biosynthesis of erythroascorbic acid in Candida albicans.
abstract: D-Arabinose dehydrogenase was purified 2750-fold from the cytosolic fraction of Candida albicans to apparent homogeneity, with an overall yield of 3%, by a purification procedure consisting of ammonium sulfate precipitation and DEAE-Sepharose A-50, Sephacryl S-200, Cibacron blue and phenyl-Sepharose CL-4B chromatographies. Gel-filtration chromatography gave an apparent molecular mass of 41 kDa and SDS-PAGE showed only one protein band corresponding to a molecular mass of 42 kDa, indicating that the enzyme is a single polypeptide. The enzyme was optimally active at pH 8.0 and the pI value of the enzyme was 5.0. The enzyme was relatively stable from pH 4.5 to 7.5. The optimal temperature for the enzyme activity was 30 degrees C. The activity of the enzyme was inhibited by Hg2+, Fe2+, Zn2+, Cu2+, Mg2+, Mn2+, N-ethylmaleimide and p-chloromercuribenzoic acid. The enzyme catalysed the oxidation of D-arabinose, L-fucose, L-xylose and L-galactose, which have the same configurations of hydroxyl groups at C2- and C3-positions, with apparent K(m) values of 29.2, 28.9, 37.1 and 91.3 mM at pH 8.0, respectively, with 50 microM NADP+. The enzyme used NADP+ as a coenzyme. Apparent K(m) value at 60 mM D-arabinose for NADP+ was 44.6 microM. NADPH inhibited the enzyme activity competitively with respect to NADP+ (Ki = 78.6 microM). The amino-terminal sequence of the enzyme was Met-Lys-Leu-Ala-Thr-Glu-Ile-Asp-Phe-X-Leu-Asn-Asn-Gly-. The reaction product was D-arabinono-1,4-lactone, judged from gas-liquid chromatography/mass spectrometry. In C. albicans, D-erythroascorbic acid was formed from D-arabinose by D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase.

title: D-arabinose dehydrogenase and its gene from Saccharomyces cerevisiae.
abstract: D-Arabinose dehydrogenase was purified 843-fold from the cytosolic fraction of Saccharomyces cerevisiae with a recovery of 9%. The purified enzyme gave two bands with a molecular mass of 40 and 39 kDa on SDS-PAGE. The native enzyme had a molecular mass of 74 kDa as estimated by Sephacryl S-200 chromatography. Therefore, this enzyme was considered to be a heterodimer. The purified enzyme exhibited maximum activity at pH 10.0 and around 30 degrees C. The enzyme catalysed the oxidation of D-arabinose, L-xylose, L-fucose and L-galactose in the presence of NADP+. The apparent Km values at pH 10.0 with 50 microM NADP+ for D-arabinose, L-xylose, L-fucose, and L-galactose were 161, 24, 98 and 180 mM, respectively. The pH profile of Vmax and kcat/Km showed one ionisable groups around pH 8.3. D-Erythroascorbic acid was formed in vitro from D-arabinose by D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase. The N-terminal amino acid sequence of the heavy subunit was Ser-Thr-Glu-Asn-Ile-Val-Glu-Asn-Met-Leu-His-Pro-Lys-Thr-. The N-terminus of the light subunit was blocked. The obtained peptide sequence was identical to the translational product of an unknown open reading frame, YBR149W, in chromosome II of S. cerevisiae. When compared with the translational product of this open reading frame, the peptide sequence was identical to the amino acid sequences of residues 7 to 20. The first six amino acids of this open reading frame were lost in protein sequence, which may be modified post-translationally. The heavy subunit was composed of 344 amino acid residues and its deduced amino acid sequence contained the motifs I, II, and III of aldo-keto reductase and also leucine zipper motif. This enzyme is the first heterodimeric protein of aldo-keto reductase family. In the deletion mutant of this gene, D-arabinose dehydrogenase activity and D-erythroascorbic acid were not detected.

title: Biosynthesis of L-ascorbic acid (vitamin C) by Saccharomyces cerevisiae.
abstract: Saccharomyces cerevisiae cells incubated with D-glucose (D-Glc), D-galactose or D-mannose (D-Man) synthesised D-erythroascorbic acid (D-EAA) but not L-ascorbic acid (L-AA). Accumulation of D-EAA was observed in cells incubated with D-arabinose (D-Ara) whilst accumulation of L-AA occurred in cells incubated with L-galactose (L-Gal), L-galactono-1,4-lactone and L-gulono-1,4-lactone. When S. cerevisiae cells were incubated with D-[U-(14)C]Glc, D-[U-(14)C]Man or L-[1-(14)C]Gal, incorporation of radioactivity into L-AA was observed only with L-[1-(14)C]Gal. Pre-incubation of yeast cells with D-Ara substantially reduced the incorporation of L-[1-(14)C]Gal into L-AA. Our results indicate that, under appropriate conditions, yeast cells can synthesise L-AA via the pathway naturally used for D-EAA biosynthesis.

title: Biosynthesis of Vitamin C by Yeast Leads to Increased Stress Resistance
abstract: In industrial large scale bio-reactions micro-organisms are generally exposed to a variety of environmental stresses, which might be detrimental for growth and productivity. Reactive oxygen species (ROS) play a key role among the common stress factors–directly-through incomplete reduction of O2 during respiration, or indirectly-caused by other stressing factors. Vitamin C or L-ascorbic acid acts as a scavenger of ROS, thereby potentially protecting cells from harmful oxidative products. While most eukaryotes synthesize ascorbic acid, yeast cells produce erythro-ascorbic acid instead. The actual importance of this antioxidant substance for the yeast is still a subject of scientific debate.  We set out to enable Saccharomyces cerevisiae cells to produce ascorbic acid intracellularly to protect the cells from detrimental effects of environmental stresses. We report for the first time the biosynthesis of L-ascorbic acid from D-glucose by metabolically engineered yeast cells. The amount of L-ascorbic acid produced leads to an improved robustness of the recombinant cells when they are subjected to stress conditions as often met during industrial fermentations. Not only resistance against oxidative agents as H2O2 is increased, but also the tolerance to low pH and weak organic acids at low pH is increased.  This platform provides a new tool whose commercial applications may have a substantial impact on bio-industrial production of Vitamin C. Furthermore, we propose S. cerevisiae cells endogenously producing vitamin C as a cellular model to study the genesis/protection of ROS as well as genotoxicity.

title: The missing step of the l-galactose pathway of ascorbate biosynthesis in plants, an l-galactose guanyltransferase, increases leaf ascorbate content
abstract: The gene for one postulated enzyme that converts GDP-l-galactose to l-galactose-1-phosphate is unknown in the l-galactose pathway of ascorbic acid biosynthesis and a possible candidate identified through map-based cloning is the uncharacterized gene At4g26850. We identified a putative function for At4g26850 using PSI-Blast and motif searching to show it was a member of the histidine triad superfamily, which includes d-galactose uridyltransferase. We cloned and expressed this Arabidopsis gene and the homologous gene from Actinidia chinensis in Escherichia coli and assayed the expressed protein for activities related to converting GDP-l-galactose to l-galactose-1-P. The expressed protein is best described as a GDP-l-galactose-hexose-1-phosphate guanyltransferase (EC 2.7.7.), catalyzing the transfer of GMP from GDP-l-galactose to a hexose-1-P, most likely d-mannose-1-phosphate in vivo. Transient expression of this A. chinensis gene in tobacco leaves resulted in a >3-fold increase in leaf ascorbate as well as a 50-fold increase in GDP-l-galactose-d-mannose-1-phosphate guanyltransferase activity.

title: A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis.
abstract: Ascorbate is a critical compound in plants and animals. Humans are unable to synthesize ascorbate, and their main source of this essential vitamin are plants. However, the pathway of synthesis in plants is yet to be established, and several unknown enzymes are only postulated to exist. We describe a specific L-galactose-1-phosphate (L-gal-1-P) phosphatase that we partially purified from young kiwifruit (Actinidia deliciosa) berries. The enzyme had a native molecular mass of approximately 65 kDa, was completely dependent on Mg2+ for activity and was very specific in its ability to hydrolyze L-gal-1-P. The activity had a pH optimum of 7.0, a K(-M(L-gal-1-P) of 20-40 microM and a Ka(Mg2+) of 0.2 mM. The activity was inhibited by Mg2+ at concentrations >2 mM. The enzyme from Arabidopsis thaliana shoots showed similar properties to the kiwifruit enzyme. The Arabidopsis thaliana enzyme preparation was digested with trypsin, and proteins present were identified by using liquid chromatography-MS. One of 24 proteins present in our preparation was an Arabidopsis thaliana protein, At3g02870, annotated myo-inositol-1-phosphate phosphatase in GenBank, that matched the characteristics of the purified l-gal-1-phosphate phosphatase. We then expressed a kiwifruit homologue of this gene in Escherichia coli and found that it showed 14-fold higher maximum velocity for l-gal-1-P than myo-inositol-1-P. The expressed enzyme showed very similar properties to the enzyme purified from kiwifruit and Arabidopsis, except that its KM(L-gal-1-P) and Ka(Mg2+) were higher in the expressed enzyme. The data are discussed in terms of the pathway to ascorbate biosynthesis in plants.

title: Enzymatic production of L-tagatose and L-fructose from L-sorbose and L-psicose, respectively.
abstract: from Pseudomonas sp. ST-24 catalyzes the epimerization of all d - and l -ketohexoses. d -TE immobilized on Chitopearl beads BCW 2510 was used for the production of l -tagatose and l -fructose from l -sorbose and l -psicose, respectively. The production yield was 20% for l -tagatose from l -sorbose and 65% for l -fructose from l -psicose.

title: Biotechnological Production of Vitamins
abstract: Vitamins are defined as essential micronutrients that are required in trace quantity and cannot be synthesized by mammals. Apart from their in vivo nutritional and physiological roles as growth factors for men, animals, plants and microorganisms, vitamins are now being increasingly introduced as food/feed additives, as medical-therapeutic agents, as health aids, and also as technical aids. Production of vitamins by chemical synthesis, or extraction from their known sources has serious disadvantages. This led to an increased interest in substituting these processes with biotechnological processes. For several of these compounds microbiological and algal processes exist, or are rapidly emerging. Different methods like media optimization, mutation and screening, genetic engineering and biocatalyst conversion have been used for improvement of the production of vitamins. The survey describes the current state of vitamin production by biotechnological processes and their significance, as compared to the existing chemical processes.

title: Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans
abstract: Gluconobacter oxydans is unsurpassed by other organisms in its ability to incompletely oxidize a great variety of carbohydrates, alcohols and related compounds. Furthermore, the organism is used for several biotechnological processes, such as vitamin C production. To further our understanding of its overall metabolism, we sequenced the complete genome of G. oxydans 621H. The chromosome consists of 2,702,173 base pairs and contains 2,432 open reading frames. In addition, five plasmids were identified that comprised 232 open reading frames. The sequence data can be used for metabolic reconstruction of the pathways leading to industrially important products derived from sugars and alcohols. Although the respiratory chain of G. oxydans was found to be rather simple, the organism contains many membrane-bound dehydrogenases that are critical for the incomplete oxidation of biotechnologically important substrates. Moreover, the genome project revealed the unique biochemistry of G. oxydans with respect to the process of incomplete oxidation.

title: The Genus Gluconobacter Oxydans: Comprehensive Overview of Biochemistry and Biotechnological Applications
abstract: The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in “historical” production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.

title: Taxonomic characterization of Ketogulonigenium vulgare gen. nov., sp. nov. and Ketogulonigenium robustum sp. nov., which oxidize L-sorbose to 2-keto-L-gulonic acid.
abstract: Four bacterial strains that oxidize L-sorbose to 2-keto-L-gulonic acid, a key intermediate in the synthesis of vitamin C, were isolated from soils of geographically distinct locations. All were Gram-negative, facultatively anaerobic, chemoheterotrophic rods. Comparative analysis revealed nearly identical 16S rDNA sequences amongst them (99.7-100% identical) and identified them as members of the alpha-subclass of the Proteobacteria. Phylogenetic analysis identified the closest taxonomically defined genus as Roseobacter (92.1-92.8% identical). On the basis of phylogenetic, phenotypic and genotypic analyses, a new genus is proposed, Ketogulonigenium gen. nov. Based upon these analyses, we also propose the reclassification of strain DSM 4025TP, originally identified as Gluconobacter oxydans, to the genus Ketogulonigenium. Two species are proposed: the type species Ketogulonigenium vulgare gen. nov., sp. nov., consisting of strains 62A-12APP, 266-13BPP and the type strain K. vulgare DSM 4025TP, and Ketogulonigenium robustum gen. nov., sp. nov., consisting of the type strain K. robustum X6LTP (= NRRL B-21627 = KCTC 0858BP). The species affiliation of the fifth strain (291-19PP) remains unresolved.

title: Microbial Production of L-Ascorbic Acid from D-Sorbitol, L-Sorbose, L-Gulose, and L-Sorbosone by Ketogulonicigenium vulgare DSM 4025
abstract: Ketogulonicigenium vulgare DSM 4025, known as a 2-keto-L-gulonic acid producing strain from L-sorbose via L-sorbosone, surprisingly produced L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone as the substrate under a growing or resting condition. As the best result, K. vulgare DSM 4025 produced 1.37 g per liter of L-AA from 5.00 g per liter of L-sorbosone during 4 h incubation time at 30 °C under the resting cell condition having 5.70 g per liter of wet cells. The precursor of L-AA formation from D-sorbitol and L-sorbose, except for L-gulose, was thought to be the putative furanose form of L-sorbosone. This is the first time it is reported that bacteria can produce vitamin C via L-sorbosone.

title: Pyrroloquinoline Quinone-Dependent Dehydrogenases from Ketogulonicigenium vulgare Catalyze the Direct Conversion of l-Sorbosone to l-Ascorbic Acid
abstract: A novel enzyme, l-sorbosone dehydrogenase 1 (SNDH1), which directly converts l-sorbosone to l-ascorbic acid (l-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced l-AA, as well as 2-keto-l-gulonic acid (2KGA), from l-sorbosone, suggesting that tautomerization of l-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.

title: Review: genetically modified plants for the promotion of human health
abstract: Plants are attractive biological resources because of their ability to produce a huge variety of chemical compounds, and the familiarity of production in even the most rural settings. Genetic engineering gives plants additional characteristics and value for cultivation and post-harvest. Genetically modified (GM) plants of the “first generation” were conferred with traits beneficial to producers, whereas GM plants in subsequent “generations” are intended to provide beneficial traits for consumers. Golden Rice is a promising example of a GM plant in the second generation, and has overcome a number of obstacles for practical use. Furthermore, consumer-acceptable plants with health-promoting properties that are genetically modified using native genes are being developed. The emerging technology of metabolomics will also support the commercial realization of GM plants by providing comprehensive analyzes of plant biochemical components.