WO2002103001A1

WO2002103001A1 - Gdp-mannose-3',5'-epimerase and methods of use thereof

Info

Publication number: WO2002103001A1
Application number: PCT/EP2002/006891
Authority: WO
Inventors: Beata Wolucka
Original assignee: Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
Priority date: 2001-06-15
Filing date: 2002-06-14
Publication date: 2002-12-27

Abstract

The identification of a new key enzyme in vitamin C synthesis, and its use to modulate vitamin C synthesis in eukaryotic cells are disclosed. Specifically, the isolation of a multimeric GDP-mannose-3',5'-epimerase, the identification, cloning and expression of a nucleic acid sequence encoding GDP-mannose-3',5'-epimerase, methods of producing GDP-mannose-3',5'-epimerase, transgenic plants and microorganisms that express the GDP-mannose-3',5'-epimerase, and methods of production of ascorbic acid using the GDP-mannose-3',5'-epimerase are disclosed.

Description

GDP- ANNOSE-3',5'-EPIMERASE AND METHODS OF USE THEREOF

Field of the Invention

The present invention relates to ascorbic acid (vitamin C) synthesis in eukaryotic cells, and preferably plant cells. The invention relates to the identification of a new key enzyme in vitamin C synthesis, and its use to modulate vitamin C synthesis in eukaryotic cells. Specifically, this invention relates to the isolation of a multimeric GDP-mannose-3',5'-epimerase, to the identification, cloning and expression of a nucleic acid sequence encoding GDP-mannose-3',5'- epimerase, to methods of producing GDP-mannose-3',5'-epimerase, to transgenic plants and microorganisms that express the GDP-mannose-3',5'-epimerase, and to methods of production of ascorbic acid using the GDP-mannose-3',5'-epimerase.

Background of the Invention Vitamin C or ascorbic acid (also referred to herein as L-ascorbic acid, L-AA or AA) is an important metabolite for most living organisms, is present in millimolar quantity, and is well known for its antioxidant properties. Ascorbic acid is synthesized in all higher plants and in almost all higher animals except humans, other primates, guinea pigs and some birds (Burns, J.J. 1957, Nature 180:533; Chatterjee, 1973, Science 182:1271-1272; Chaudhuri et al., 1969, Science 164:435-436). Opinions differ about the presence of ascorbic acid in microorganisms and several ~ reports suggest that ascorbic acid analogues, rather than ascorbic acid itself are present in microorganisms (Takahashi et al., 1976, Agric. Biol. Chem. 40:121-129; Leung et al., 1985, Plant Sci. 38:65-69; Nick et al., 1986, Plant Sci. 46:181-187).

The biosynthesis of ascorbic acid follows different pathways in the animal and plant kingdom. In animals, D-glucose is the primary precursor in the biosynthesis of ascorbic acid and the last step of the biosynthetic pathway is catalyzed by a microsomal enzyme, L-gulono-γ-lactone oxidase which oxidises L-gulono-γ-lactone to ascorbic acid. This enzyme has been isolated and characterized from rat, goat and chicken (Nishikimi et al., 1976, Arch. Biochem. Biophys. 175:427- 435; Kiuchi et al., 1982, Biochemistry 21 :5076-5082).

Despite the importance of ascorbic acid in plants, its biosynthesis in plants is not completely understood and it is one of the few primary plant metabolic pathways that are still not completely elucidated. A biosynthetic pathway from D-galactose proceeding via L-galactono-γ-lactone has been proposed as long ago as 1954 by Isherwood et al. (1954, Biochem. J. 56:1-15) and Mapson et al. (1954, Biochem. J. 56:21-281954), based on the initial studies of the oxidation of L- galactono-γ-lactone to ascorbic acid by the enzyme L-galactono-γ-lactone dehydrogenase. L- galactono-γ-lactone dehydrogenase activity has been described in plants such as peas, cabbage, cauliflower florets, potato and sweet potato roots. Recently, the isolation and cloning of a gene encoding cauliflower L-galactono-γ-lactone dehydrogenase has been described (østergaard et al., 1997, J. Biol. Chem. 272:30009-30016). Loewus (1988, The Biochemistry of Plants, Vol. 14, pp. 85-107, Academic Press, New York) has proposed an alternative pathway in which ascorbic acid is synthesized from D-glucose via L-sorbosone. The presence of an enzyme able to convert L- sorbosone to ascorbic acid with concomitant reduction of NADP was demonstrated in bean and spinach leaves (Loewus et al., 1990, Plant Physiol. 94:1492-1495; Saito et al., 1990, Plant Physiol. 94:1496-1500). Conceivably, these distinct routes may be present in different subcellular compartments or in different plant species.

Wheeler et al. (1998, Nature 393:365-369) (see also PCT Publication Nos. WO 99/33995 and WO 01/72974 to Ascorbex Limited) found that D-mannose and L-galactose are efficient precursors for ascorbic acid synthesis. These authors identified the enzyme L-galactose dehydrogenase from pea and Arabidopsis thaliana, and proposed an ascorbic acid biosynthetic pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-γ-lactone. The L-galactose dehydrogenase enzyme was also cloned and expressed in plants. Wheeler et al. suggest that interconversion of GDP-D-mannose into GDP-L-galactose may be carried out by a GDP-mannose-3',5'-epimerase. However, although they showed some L-galactose formation from GDP-D-mannose, the existence of an epimerase activity was not clearly demonstrated, as the enzyme itself was not isolated and the intermediates have not been identified. Moreover, at least two other steps are needed to explain the L-galactose formation, and the relative importance of those steps in the transformation is unclear.

PCT Publication WO 99/64618 to BioTechnical Resources, also disclosed a proposed ascorbic acid biosynthetic pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose-1- phosphate, L-galactose, and L-galactono-γ-lactone. WO 99/64618 disclosed several mutants of the microalgae Prototheca and demonstrated a correlation between an increase in GDP-D- mannose:GDP-L-galactose epimerase (i.e., GDP-mannose-3',5'-epimerase) activity and an increase in ascorbic acid production in these mutants. However, like PCT Publication Nos. WO 99/33995 and WO 01/72974, WO 99/64618 did not purify or isolate an epimerase, or describe the structural characteristics of GDP-D-mannose:GDP-L-galactose epimerase.

Apart from the publication by Wheeler et al., WO 99/33995, WO 01/72974 and WO 99/64618, GDP-mannose-3',5'-epimerase activity was reported in snail (Goudsmit and Neufield, 1967), in the green alga Chlorella pyrenoidosa and in the plant Linum usitatissimum (Barber, 1971 , Arch. Biochem. Biophys. 147:619-623). However, none of these enzymes have ever been characterized or purified and the corresponding genes are unknown.

PCT Publication WO 98/50558 to Vlaams Interuniversitair Instituut voor Biotechnologie discloses the isolation and cloning of a gene encoding L-galactono-γ-lactone dehydrogenase, which is the final enzyme in the ascorbic acid pathway described above, as well as its expression in plants. PCT Publication WO 99/33995 and WO 01/72974, both to Ascorbex Limited, disclose the isolation and cloning of a gene encoding of a plant L-galactose dehydrogenase, which is the second to last enzyme in the ascorbic acid pathway described above, as well as expression of this enzyme in plants. However, overexpression of either of these enzymes alone has not been shown lead to a significant increase in ascorbic acid, as those steps in the biosynthesis do not appear to be rate limiting.

Several attempts have been made to create genetically modified plants with an increased ascorbic acid content. The primary function of ascorbic acid is to act as a reducing agent. Ascorbic acid is also important as a cofactor for certain enzymatic reactions, including the production of collagen in vertebrates. Since humans are completely dependent upon ingested food for the acquisition of ascorbic acid, it is desirable to increase the vitamin C content of plants and fruits. Moreover, owing to its reducing activity, ascorbic acid plays a role in the protection of plants and animals against environmental stresses including, as non-limiting examples, cold, heat, drought and oxidative stress. Ascorbic acid-overproducing strains that are less stress-sensitive or even stress-resistant can play an important role in the economy and agriculture of the world. Therefore, it is desirable to be able to increase the production of ascorbic acid in plants and microorganisms.

Summary of the Invention

One embodiment of the present invention relates to an isolated, multimeric GDP-mannose- 3',5'-epimerase. Preferably, the epimerase is isolated from a plant, such as, but not limited to, Arabidopsis thaliana, Oryza sativa, Lycopersicon esculentum, Zea mays, Mesembryanthemum crystallinum, Glycine max, Solanu tuberosum, Medicago truncatula, Sorghum bicolor, Triticum aestivum, Hordeum vulgare and Lotus japonicus. In one aspect, the epimerase is a dimer, which may have a molecular weight of between about 80 kDa and about 90 kDa. Such an epimerase may also have the following characteristics: (a) a K'_eq for the formation of GDP-L-galactose of about 0.15; (b) a K_m value for GDP-mannose of about 4.4 μM; and (c) a K, value for GDP of about 0.7 μM. In one aspect of the invention, at least one monomer of the multimeric GDP-mannose-3',5'- epimerase includes an amino acid sequence selected from: (a) an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein the amino acid sequence has GDP-mannose- 3',5'-epimerase activity; or (b) a fragment of an amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity. In another aspect, at least one monomer of the multimeric GDP-mannose-3',5'-epimerase includes an amino acid sequence that is at least about 80% identical to SEQ ID NO:2, and in another aspect, an amino acid sequence that is at least about 90% identical to SEQ ID NO:2, and in another aspect, an amino acid sequence selected from: SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46.

Such a multimeric GDP-mannose-3',5'-epimerase as described herein can be used to modulate the synthesis of a product in a cell, the product selected from: GDP-L-galactose, L- galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone. Such an epimerase can also be used to modulate ascorbic acid synthesis in a cell, including, but not limited to, a eukaryotic cell

(e.g., a plant cell) or a prokaryotic cell.

One embodiment of the present invention relates to an isolated GDP-mannose-3',5'- epimerase monomer which includes an amino acid sequence that is at least about 70% identical and less than 100% identical to SEQ ID NO:2, wherein the amino acid sequence has GDP- mannose-3',5'-epimerase activity. In one aspect, the monomer comprises an amino acid sequence selected from: SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54.

Yet another embodiment of the present invention relates to an isolated nucleic acid molecule which includes a nucleic acid sequence selected from: (a) a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical and less than 100% identical to SEQ ID NO:2, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; (b) a nucleic acid sequence encoding a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity; (c) a nucleic acid sequence that is a probe or primer that hybridizes under high stringency conditions to a nucleic acid sequence of (a) or (b); or (d) a nucleic acid sequence that is a complement of any of the nucleic acid sequences of (a)-(c). In one aspect, the nucleic acid sequence encodes an amino acid sequence that is at least about 80% identical and less than 100% identical to SEQ ID NO:2, and in another aspect, the nucleic acid sequence encodes an amino acid sequence that is at least about 90% identical and less than 100% identical to SEQ ID NO:2, and in another aspect, the nucleic acid sequence encodes an amino acid sequence that is less than about 95% identical to SEQ ID NO:2, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity. In one aspect, the nucleic acid sequence is at least about 70% identical and less than 100% identical to SEQ ID NO:1. In yet another aspect, the nucleic acid sequence is a fragment of a nucleic acid sequence selected from: SEQ ID NO:1 , SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO.31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:53, wherein the fragment encodes an amino acid sequence that has GDP-mannose- 3',5'-epimerase activity. Another embodiment of the present invention relates to a recombinant nucleic acid molecule comprising an expression vector and a nucleic acid molecule as described above, operatively linked to at least one transcription control sequence.

Yet another embodiment of the present invention relates to a recombinant nucleic acid molecule comprising an expression vector and a nucleic acid sequence operatively linked to at least one transcription control sequence, wherein the nucleic acid sequence is selected from: (a) a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; and (b) a nucleic acid sequence encoding a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity.

Another embodiment of the present invention relates to a recombinant host cell transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence that encodes any of the amino acid sequences for a protein having GDP-mannose-3',5'-epimerase activity described above. The host cell can include, but is not limited to, a eukaryotic cell (e.g., a yeast or a plant cell) or a prokaryotic cell. In one aspect, expression of the recombinant nucleic acid molecule by the host cell is sufficient to increase the synthesis of a product in the host cell, the product selected from: GDP-L-galactose, L-galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone. In another aspect, expression of the recombinant nucleic acid molecule by the host cell is sufficient to increase ascorbic acid production in the host cell. Yet another embodiment of the present invention relates to a transgenic plant or part of a plant having one or more cells comprising a nucleic acid sequence that encodes any of the amino acid sequences for a protein having GDP-mannose-3',5'-epimerase activity described above. In one aspect, the plant or part of a plant has increased synthesis of a product as compared to a non- transgenic plant, the product selected from: GDP-L-galactose, L-galactose-1 -phosphate, L- galactose, and L-galactono-γ-lactone. In another aspect, the transgenic plant or part of a plant has increased production of ascorbic acid as compared to a non-transgenic plant.

Another embodiment of the present invention relates to a method to increase ascorbic acid synthesis in a host cell. The method includes growing a host cell that is transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one subunit of a multimeric GDP-mannose-3',5'-epimerase, wherein the nucleic acid sequence is operatively linked to a transcription control sequence. In one aspect, the transcription control sequence comprises a native GDP-mannose-3',5'-epimerase promoter located upstream of the nucleic acid sequence encoding the at least one subunit of a multimeric GDP-mannose-3',5'- epimerase. In another aspect, the transcription control sequence comprises a non-native promoter located upstream of the nucleic acid sequence encoding the at least one subunit of a multimeric GDP-mannose-3',5'-epimerase. The GDP-mannose-3',5'-epimerase can include any of the GDP- mannose-3',5'-epimerases described above. The host cell can include eukaryotic or prokaryotic cells.

Another embodiment of the present invention relates to a method to increase ascorbic acid synthesis in a cell comprising a multimeric GDP-mannose-3',5'-epimerase, comprising introducing into the genome of the cell a non-native promoter upstream of a gene encoding the at least one subunit of a multimeric GDP-mannose-3',5'-epimerase. In one aspect, the non-native promoter is a plant promoter. The gene comprises a nucleic acid sequence encoding any of the GDP-mannose- 3',5'~epimerases described above. The cell is preferably a eukaryotic cell, such as a plant cell. Another embodiment of the present invention relates to a method to increase ascorbic acid synthesis in a cell comprising a multimeric GDP-mannose-3'5'-epimerase, comprising genetically modifying the cell to increase the activity of the GDP-mannose-3',5'-epimerase in the cell. In one aspect, the genetic modification comprises expressing a recombinant GDP-mannose-3',5'- epimerase promoter in the cell upstream of the gene encoding the at least one subunit of a multimeric GDP-mannose-3',5'-epimerase, wherein expression of the recombinant promoter increases the expression of GDP-mannose-3',5'-epimerase by the cell. In another aspect, the genetic modification comprises a modification of GDP-mannose-3',5'-epimerase in the cell which increases the expression or activity of the GDP-mannose-3',5'-epimerase in the cell. The gene comprises a nucleic acid sequence encoding any of the GDP-mannose-3',5'-epimerases described above. The cell is preferably a eukaryotic cell, such as a plant cell. One aspect of the invention relates to a plant which is obtainable according to this method, followed by regeneration of the plant cell produced thereby.

Yet another embodiment of the present invention relates to an isolated antibody that selectively binds to a multimeric GDP-mannose-3',5'-epimerase. In one aspect, the multimeric GDP-mannose-3',5'-epimerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 and SEQ ID NO:54.

Yet another embodiment of the present invention relates to a recombinant nucleic acid molecule comprising an expression vector and a nucleic acid molecule comprising: (a) a first nucleic acid sequence encoding an amino acid sequence selected from: (i) an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; and (ii) a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity; and (b) at least one additional nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein the enzyme is selected from: phosphomannose isomerase, phosphomannomutase, GDP- D-mannose pyrophosphorylase, GDP-L-galactose pyrophosphorylase, L-galactose-1 -P- phosphatase, L-galactose dehydrogenase, or L-galactono-γ-lactone dehydrogenase. Another embodiment of the present invention relates to a recombinant host cell transformed with at least two recombinant nucleic acid molecules comprising: (a) a first recombinant nucleic acid molecule comprising a nucleic acid sequence selected from: (i) a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein the amino acid sequence has GDP-mannose-S'.δ'-epimerase activity; and (ii) a nucleic acid sequence encoding a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose- 3',5'-epimerase activity; and (b) at least one additional recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein the enzyme is selected from: phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-L-galactose pyrophosphorylase, L-galactose-1 -P-phosphatase, L-galactose dehydrogenase, or L-galactono-γ- lactone dehydrogenase. In one aspect, the first and the at least one additional recombinant nucleic acid molecules are contained within a single recombinant vector. In this aspect of the invention, the recombinant vector can be dicistronic. The host cell can include eukaryotic (e.g., plant or yeast) or prokaryotic cells. In a preferred embodiment, the host cell has increased synthesis of a product in the host cell as compared to a non-transformed host cell, the product selected from: GDP-L-galactose, L-galactose-1 -phosphate, L-galactose, or L-galactono-γ-lactone. In another preferred embodiment, the host cell has increased production of ascorbic acid as compared to a non-transformed host cell. Another embodiment of the present invention relates to a transgenic plant or part of a plant having one or more cells comprising at least two recombinant nucleic acid molecules comprising: (a) a first recombinant nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: (i) a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein the amino acid sequence has GDP-mannose- 3',5'-epimerase activity; and (ii) a nucleic acid sequence encoding a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity; and (b) at least one additional recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein the enzyme is selected from: phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP- L-galactose pyrophosphorylase, L-galactose-1 -P-phosphatase, L-galactose dehydrogenase, or L- galactono-γ-lactone dehydrogenase.

Another embodiment of the invention includes the use of a multimeric GDP-mannose-3',5'- epimerase as described above for in vitro synthesis of a product selected from: GDP-L-galactose, L-galactose-1 -phosphate, L-galactose, or L-galactono-γ-lactone. Yet another embodiment of the invention relates to the use of a multimeric GDP-mannose-

3',5'-epimerase as described above, or a nucleic acid sequence encoding at least one subunit of the multimeric GDP-mannose-3',5'-epimerase, as a selectable marker in eukaryotic cells. Another embodiment of the invention relates to the use of a multimeric GDP-mannose-3',5'- epimerase as described above to increase the reductive capacity of a eukaryotic cell. In one aspect, the cell is a plant cell or a yeast cell.

Brief Description of the Drawings

Fig. 1 is a schematic representation of the proposed de novo pathway for the synthesis of L-ascorbic acid from D-mannose in plants. Enzymes: (1) hexokinase; (2) phosphomannomutase; (3) GDP-Man pyrophosphorylase; (4) GDP-mannose-3',5'-epimerase; (5) L-galactose dehydrogenase; (6) L-galactono-1 ,4-lactone dehydrogenase. Fig.2 is a line graph illustrating the correlation between L-ascorbic acid (L-AA) content and

GDP-mannose-3',5'-epimerase activity during the growth of A. thaliana cell suspension. Total L-AA content of cells and epimerase specific activity of the corresponding 55 % to 70 % ammonium sulphate fractions were determined, as described in the Examples.

Fig. 3 is a graph showing the purification of GDP-mannose- 3',5'~epimerase from A. thaliana suspension's cells. (A) DEAE-Sepharose FPLC of the ammonium sulphate fraction. The enzyme was eluted with a linear gradient of NaCl from 0 to 200 mM, as described in Material and Methods. (B) Sephacryl S-200 gel filtration chromatography of epimerase fractions from DEAE- Sepharose. (C) Hydroxylapatite FPLC of epimerase fractions from Sephacryl gel filtration. The proteins were eluted with a linear gradient of 2 to 500 mM potassium phosphate (pH 7.2), as described in the Examples.

Fig. 4 is a digitized image of an SDS-PAGE of the partially purified GDP-mannose-3',5'- epimerase. Proteins were visualized by Coomassie Blue staining. Left lane: molecular mass standards. Right lane: the NAD-eluted fraction from Blue-Sepharose. Proteins were identified by mass spectrometry of in-gel tryptic digests as follows: Band 1 , betaine aldehyde dehydrogenase; Band 2, glutathione synthase; Bands 3 and 4 (apparent molecular masses of 46 kDa and 43 kDa, respectively), GDP-mannose-3',5'-epimerase.

Fig. 5 is a nano-ESI MS/MS spectrum of the m/z 785.9 [M + 2H]⁺² peptide ion derived from in-gel tryptic digestion of the 46 and 43 kDa bands of GDP-mannose-3',5'-epimerase as shown in Fig. 4. Fig. 6 shows the amino acid sequence of a epimerase/dehydratase-like protein of A. thaliana (SEQ ID NO:2) corresponding to a monomeric subunit of the purified GDP-mannose-3',5'- epimerase. The shadowed region corresponds to the m/z 785.9 peptide sequence obtained by the nano-electrospray tandem mass spectrometry of the in-gel tryptic digest of the 46 kDa and 43 kDa epimerase bands (see Fig 5). Peptide ions corresponding to the underlined regions were observed by the matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (see Table 2).

Fig. 7 shows the determination of molecular mass of GDP-mannose- 3',5'-epimerase. (A)

Molecular mass of the native enzyme determined by Sephacryl S-200 gel filtration chromatography; where k_av = (V₈|_Ution B V_eXciusion) (V_totai B V_exC|_USion)^"1, and the molecular standards were: thyroglobulin (670 kDa), bovine γ-globulin (γ-GLO, 158 kDa), chicken ovalbumin (OVO, 44 kDa), equine myoglobin (MYO, 17 kDa) and vitamin B₁₂ (VitB₁₂, 1.35 kDa). (B) Molecular mass of the enzyme subunit (band 3; Fig. 4) determined by SDS-PAGE. The molecular mass standards were: β- galactosidase (β-GAL, 116.3 kDa), phosphorylase b (PHO, 97.4 kDa), bovine serum albumin (BSA,' 66.3 kDa), glutamic dehydrogenase (GDH, 55.4 kDa), lactate dehydrogenase (LDH, 36.5 kDa), carbonic anhydrase (CAN, 31 kDa).

Fig. 8 is a graph showing inhibition of GDP-mannose-3',5'-epimerase by GDP. Double reciprocal plots are shown and each line represents a fixed GDP concentration: circles 0 μM, triangles 2 μM, squares 4 μM. V is pmol of GDP-L-Gal produced per min. GDP-Man concentrations were from 1.1 to 5.5 μM. Inset: Secondary plot of slope versus GDP concentration was used to determine the K, value for GDP.

Fig. 9 shows the inhibition of GDP-mannose-3',5'-epimerase by purine nucleoside- pyrophospho-hexoses. Incubations contained GDP-[¹⁴C]Man (3.4 μM), various amounts of either GDP-D-glucose, GDP-L-fucose or ADP-D-glucose, and an aliquot of the hydroxylapatite fraction of epimerase.

Fig. 10 illustrates the effect of pH (A) and temperature (B) on GDP-mannose-3',5¹- epimerase activity.

- Detailed Description of the Invention

The present invention generally relates to the purification, isolation, cloning and sequencing of a multimeric GDP-mannose-3',5'-epimerase and to uses of the epimerase to increase the production of ascorbic acid and/or precursor products in the ascorbic acid synthesis pathway in both eukaryotic and prokaryotic host cells. Surprisingly, the present inventor was able to isolate and purify a GDP-mannose-3',5'-epimerase from a crude extract of Arabidopsis cells, and has now demonstrated its role as a homodimeric enzyme in ascorbic acid biosynthesis. On the basis of the purified protein, the sequence was determined. The present inventor has also cloned and expressed the recombinant epimerase in an E. coli host cell and showed that epimerase activity could be detected in the ammonium sulfate fraction of the induced host cells (these data are also described in a publication by the present inventor subsequent to the priority date of the present application in Wolucka et al., Proceedings of the National Academy of Sciences, 2001 , 98:14843- 14848, which is incorporated herein by reference in its entirety). The sequence identified by the present inventor was previously presented as part of an Arabidopsis thaliana BAC clone (EMBO Accession No. AF272706). A nucleic acid sequence within the BAC sequence was identified through the Arabadopsis genome sequencing project as encoding a hypothetical protein having epimerase/dehydratase homology, with a similarity to the Arabidopsis thaliana dTDP-glucose 4-6- dehydratase homologue D18. However, this protein was not identified in the EMBO database as being a GDP-mannose-3',5'-epimerase, or as having any function in an ascorbic acid biosynthetic pathway.

A European application (EP1033405) published September 6, 2000 also contains the sequence for the GDP-mannose-3',5'-epimerase, in addition to multiple other proteins from Arabidopsis thaliana, rice, soybean, and other plants, as part of a large sequencing project in which numerous "Sequence Determined DNA Fragments" were sequenced and then included in a large patent application. However, this document describes the sequence only as encoding a putative protein, without disclosing any possible link with GDP-mannose-3',5'-epimerase activity or ascorbic acid biosynthetic activity, clearly indicating that the function of the enzyme was not known and cannot be simply deduced from its sequence.

The purification, identification, cloning and expression of the GDP-mannose-3',5'-epimerase from the ascorbic acid biosynthetic pathway is surprising and completely unpredictable from a disclosure of a putative protein with homology to the generic class of epimerases. Indeed, the GDP-mannose-3',5'-epimerase is a unique epimerase which catalyzes the conversion of GDP-D- mannose into GDP-L-galactose using a unique double epimerization of the hexosyl residue. Therefore, identification of a generic homology to epimerases is not predictive of the function of a GDP-mannose-3',5'-epimerase. The present inventor is believed to be the first investigator to have specifically identified, purified, sequenced and cloned a GDP-mannose-3', 5'-epimerase gene in the ascorbic acid biosynthetic pathway. Moreover, to the best of the present inventor's knowledge, until the present invention, the Arabidopsis thaliana GDP-mannose 3', 5'-epimerase is believed to be the only GDP-mannose 3', 5'-epimerase to have been purified and specifically identified by function, as well as sequenced and cloned.

One embodiment of the present invention relates to an isolated, multimeric GDP-mannose- 3',5'-epimerase. According to the present invention, a GDP-mannose-3',5'-epimerase is defined as an enzyme which catalyzes the conversion of GDP-D-mannose (substrate) into GDP-L-galactose (product) using a unique double epimerization of the hexosyl residue. The GDP-mannose-3',5' - epimerase catalyzes the first dedicated step in the vitamin C (ascorbic acid) synthesis pathway in plant and microbial cells (see Wheeler etal., 1998, Nature 393:365-369; PCT Publication Nos. WO 99/33995 and WO 01/72974 to Ascorbex Limited; and PCT Publication WO 99/64618 to BioTechnical Resources). The present inventor has discovered that the native GDP-mannose- 3',5'-epimerase exists as a multimer (e.g., is multimeric) and more specifically, as a dimer. The dimer from Arabidopsis thaliana has a molecular weight of between about 80 kDa and about 90 kDa. The multimeric GDP-mannose-3',5'-epimerase from Arabidopsis thaliana also has the following characteristics: (a) a K'_eq for the formation of GDP-L-galactose of about 0.15; (b) a K_m value for GDP-mannose of about 4.4 μM; and (c) a Kj value for GDP of about 0.7 μM. The detailed structural and biochemical characteristics of a purified and isolated multimeric GDP-mannose-3',5'- epimerase are described in detail in Examples 1-3. Both the native multimeric GDP-mannose-3',5'- epimerase and biologically active monomers of the GDP-mannose-3',5'-epimerase, as well as homologues and particularly fragments thereof, are intended to be encompassed by the present invention.

According to the present invention, a GDP-mannose-3',5'-epimerase is a protein that has GDP-mannose-3',5'-epimerase biological activity, including full-length proteins, fusion proteins, or any homologue of a naturally occurring GDP-mannose-3',5'-epimerase (including natural allelic variants, fragments, related GDP-mannose-3',5'-epimerases from different organisms and synthetically or artificially derived variants). A homologue of a GDP-mannose-3',5'-epimerase includes proteins which differ from a given naturally occurring GDP~mannose-3',5'-epimerase in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol). One preferred homologue is a biologically active fragment of a naturally occurring GDP-mannose-3',5'-epimerase. Other preferred homologues of naturally occurring GDP-mannose~3',5'-epimerases are described in detail below.

An isolated protein, such as an isolated GDP-mannose-3',5'-epimerase, according to the present invention, is a protein that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. Both purified and recombinant produced GDP-mannose-3',5'-epimerases are described in the Examples section. As such, "isolated" does not reflect the extent to which the protein has been purified. Preferably, an isolated GDP-mannose-3',5'-epimerase of the present invention is produced recombinantly. In addition, and by way of example, an "Arabidopsis thaliana GDP-mannose-3',5'- epimerase" refers to a GDP-mannose-3',5'-epimerase (including a homologue of a naturally occurring GDP-mannose-3',5'-epimerase) from Arabidopsis thaliana or to a GDP-mannose-3',5'- epimerase that has been otherwise produced from the knowledge of the structure (e.g., sequence) and perhaps the function of a naturally occurring GDP-mannose-3',5'-epimerase from Arabidopsis thaliana. In other words, an Arabidopsis thaliana GDP-mannose-3',5'-epimerase includes any GDP-mannose-3',5'-epimerase that has substantially similar structure and function of a naturally occurring GDP-mannose-3',5'-epimerase from Arabidopsis thaliana or that is a biologically active (i.e., has biological activity) homologue of a naturally occurring GDP-mannose-3',5'-epimerasefrom Arabidopsis thaliana as described in detail herein. As such, an Arabidopsis thaliana GDP- mannose-3',5'-epimerase can include purified, partially purified, recombinant, mutated/modified and synthetic proteins. This discussion applies similarly to GDP-mannose-3',5'-epimerases from other plants as disclosed herein. According to the present invention, an isolated multimeric GDP-mannose-3',5'-epimerase, or a biologically active subunit (e.g., a GDP-mannose-3',5'-epimerase monomer), homologue or fragment thereof, has GDP-mannose-3',5'-epimerase activity (i.e., biological activity). In general, the biological activity or biological action of a protein refers to any function(s) exhibited or performed by the protein that is ascribed to the naturally occurring form of the protein as measured or observed in vivo (i.e., in the natural physiological environment of the protein) or in vitro (i.e., under laboratory conditions). The biological activity of GDP-mannose-3',5'-epimerase includes the ability to catalyze the conversion of GDP-D-mannose into GDP-L-galactose using the double epimerization referenced above. Modifications of a protein, such as in a homologue or mimetic (discussed below), may result in proteins having the same biological activity as the naturally occurring protein, or in proteins having decreased or increased biological activity as compared to the naturally occurring protein. Modifications which result in a decrease in expression or a decrease in the activity of the protein, can be referred to as inactivation (complete or partial), down- regulation, or decreased action of a protein. Similarly, modifications which result in an increase in expression or an increase in the activity of the protein, can be referred to as amplification, overproduction, activation, enhancement, up-regulation or increased action of a protein. A functional subunit, homologue, or fragment of a GDP-mannose-3',5'-epimerase is preferably capable of performing substantially the same (e.g., at least qualitatively the same) biological function of the native GDP-mannose-3',5'-epimerase protein (i.e., has biological activity). A preferred multimeric GDP-mannose-3',5'-epimerase is isolated from a plant or derived from a GDP-mannose-3',5'-epimerase from a plant (e.g., a homologue or modified sequence as described below). In one embodiment, the GDP-mannose-3',5'-epimerase is from a plant that includes, but is not limited to, Arabadopsis thaliana, Oryza sativa, Lycopersicon esculentum, Zea mays, Mesembryanthemum crystallinum, Glycine max, Solanum tuberosum, Medicago truncatula, Sorghum bicolor, Tήticum aestivum, Hordeum vulgare or Lotus japonicus.

With regard to the GDP-mannose-3',5'-epimerase of the present invention, it is preferred that modifications in GDP-mannose-3',5'-epimerase homologues, when the homologues are modified forms of a naturally occurring GDP-mannose-3',5'-epimerase, do not substantially change or at least do not substantially decrease, the basic biological activity of the epimerase as compared to the naturally occurring protein. Increased biological activity (e.g., increased enzyme activity), may be desirable in a GDP-mannose-3',5'-epimerase homologue. GDP-mannose-3',5'-epimerase homologues may have differences in characteristics other than the functional, or enzymatic, activity of the protein as compared to the naturally occurring form, such as a decreased sensitivity to inhibition by certain compounds as compared to the naturally occurring protein. As discussed above, a protein that has "GDP-mannose-3',5'-epimerase biological activity" or that is referred to as a "GDP-mannose-3',5'-epimerase" refers to a protein that catalyzes the conversion of GDP-D-mannose to GDP-L-galactose using a unique double epimerization of the hexosyl residue. An isolated GDP-mannose-3',5'-epimerase of the present invention, including full- length proteins, truncated proteins, fusion proteins and homologues, can be identified in a straightforward manner by the proteins' ability to catalyze the above-identified conversion. GDP-mannose- 3',5'-epimerase biological activity can be evaluated by one of skill in the art by any suitable in vitro or in vivo assay for enzyme activity. Assays for the specific evaluation and measurement of GDP- mannose-3',5'-epimerase biological activity are described herein. One assay, described in detail in Wolucka et al., 2001 , Anal. Biochem. 294:161-168, incorporated herein by reference in its entirety, uses an HPLC radio-method using GDP-D-[U-¹⁴C] mannose as substrate. This method is described in detail in the Examples section (Examples 1-3). Another assay for GDP-mannose- 3', 5' -epimerase activity, described in Example 5, is a thin layer chromatography method using ¹⁴C labeled GDP-D-mannose as a substrate. Either assay, or any other assay which specifically measures GDP-mannose-3',5'-epimerase activity, is suitable for the detection and measurement of GDP-mannose-3',5'-epimerase biological activity according to the present invention.

Several highly homologous GDP-mannose-3',5'-epimerases from different plants are specifically identified herein. As discussed above, the present inventor purified, cloned and identified what is believed to be the only GDP-mannose-3',5'-epimerase at the time of the invention, if not to date, to have been purified and identified by specific function, as well as sequenced and cloned. This GDP-mannose-3',5'-epimerase was isolated from Arabidopsis thaliana and has an amino acid sequence represented herein by SEQ ID NO:2. SEQ ID NO:2 is encoded by a nucleic acid sequence of SEQ ID NO:1. Once the identity of a verified GDP- mannose-3',5'-epimerase was discovered, this sequence was used to identify several other sequences of previously unknown function which are highly homologous to the Arabidopsis GDP- mannose-3',5'-epimerase and which are believed to be additional GDP-mannose-3',5¹- epimerases from other plants. These epimerases are described in Example 4 and their homology to the Arabidopsis GDP-mannose-3',5'-epimerase at the nucleic acid and amino acid level is shown in Table 5. All of the sequences described in Example 4/Table 5 have at least 80% identity, and most have about 90% identity, to the amino acid sequence of the Arabidopsis thaliana GDP-mannose-3',5'-epimerase (SEQ ID NO:2) and therefore, each is representative of a homologue of the Arabidopsis thaliana GDP-mannose-3',5'-epimerase according to the present invention.

In one embodiment, a GDP-mannose-3',5'-epimerase of the present invention has an amino acid sequence that is at least about 70% identical to an amino acid sequence of selected from the group of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, over the full length of any of such sequences, wherein the protein is a GDP-mannose-3',5'-epimerase (i.e., has GDP-mannose-3',5'-epimerase biological activity). In a preferred embodiment, amino acid sequence identity is determined with reference to SEQ ID NO:2. In another embodiment, a GDP-mannose-3',5'-epimerase of the present invention has an amino acid sequence that is at least about 75% identical, and even more preferably at least about 80% identical, and even more preferably at least about 85% identical, and even more preferably at least about 90% identical and even more preferably at least about 95% identical, and even more preferably at least about 96% identical, and even more preferably at least about 97% identical, and even more preferably at least about 98% identical, and even more preferably at least about 99% identical to any of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, and most preferably to SEQ ID NO:2, over the full length of any of such sequences, wherein the protein has GDP-mannose-3',5'-epimerase biological activity. It is noted that each of SEQ ID NOs:26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46 is at least about 90% identical to SEQ ID NO:2 (e.g., see Table 5).

In another embodiment, a GDP-mannose-3', 5'-epimerase of the present invention has an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, and most preferably to SEQ ID NO:2, over at least 50 amino acids of any of such sequences. More preferably, a GDP- mannose-3', 5'-epimerase of the present invention has an amino acid sequence that is at least about 75% identical, and more preferably at least about 80% identical, and more preferably at least about 85% identical, and more preferably at least about 90% identical and more preferably at least about 95% identical, and more preferably at least about 96% identical, and more preferably at least about 97%o identical, and more preferably at least about 98% identical, and more preferably at least about 99% identical to any of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, and most preferably to SEQ ID NO:2, over at least 75 amino acids, and more preferably 100 amino acids, and more preferably 125, and more preferably 150, and more preferably 175, and more preferably 200, and more preferably 225, and more preferably 250, and more preferably 275, and more preferably 300, and more preferably 325, and more preferably 350 amino acids of any of the above-identified sequences. In a most preferred embodiment, such a protein has GDP-mannose-3',5'-epimerase biological activity.

In one embodiment of the present invention, a GDP-mannose-3', 5'-epimerase according to the present invention has an amino acid sequence that is less than about 100% identical to SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, and particularly, to SEQ ID NO:2. In another aspect of the invention, a GDP-mannose- 3',5'-epimerase according to the present invention has an amino acid sequence that is less than about 99% identical to any of the above-identified amino acid sequences, and in another about 99% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than is less than 98% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 97% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 96% identical to any of the above- identified amino acid sequences, and in another embodiment, is less than 95% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 94% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 93% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 92% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 91% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 90% identical to any of the above-identified amino acid sequences.

As used herein, unless otherwise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1 ) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches and blastn for nucleic acid searches with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S.F., Madden, T.L., Schaaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position-Specific Iterated BLAST. It is noted that due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences might be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a "profile" search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position- specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs.

Two specific sequences can be aligned to one another using BLAST 2 sequence as described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST 2 sequence alignment is performed using the standard default parameters as follows:

For blastn, using 0 BLOSUM62 matrix: Reward for match = 1

Penalty for mismatch = -2

Open gap (5) and extension gap (2) penalties gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties gap x_dropoff (50) expect (10) word size (3) filter (on).

A GDP-mannose-3',5'-epimerase of the present invention can also include proteins having an amino acid sequence comprising at least 30 contiguous amino acid residues of any of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, and most preferably SEQ ID NO:2, (i.e., 30 contiguous amino acid residues having 100% identity with 30 contiguous amino acids of any of the above-identified sequences. In a preferred embodiment, a GDP-mannose-3', 5'-epimerase of the present invention includes proteins having amino acid sequences comprising at least 50, and more preferably at least 75, and more preferably at least 100, and more preferably at least 115, and more preferably at least 130, and more preferably at least 150, and more preferably at least 200, and more preferably, at least 250, and more preferably, at least 300, and more preferably, at least 350 contiguous amino acid residues of any of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, and most preferably SEQ ID NO:2. In one embodiment, such a protein has GDP-mannose-3',5'-epimerase biological activity.

According to the present invention, the term "contiguous" or "consecutive", with regard to nucleic acid or amino acid sequences described herein, means to be connected in an unbroken sequence. For example, for a first sequence to comprise 30 contiguous (or consecutive) amino acids of a second sequence, means that the first sequence includes an unbroken sequence of 30 amino acid residues that is 100% identical to an unbroken sequence of 30 amino acid residues in the second sequence. Similarly, for a first sequence to have "100% identity" with a second sequence means that the first sequence exactly matches the second sequence with no gaps between nucleotides or amino acids.

In another embodiment, a GDP-mannose-3',5'-epimerase of the present invention, including a GDP-mannose-3',5'-epimerase homologue, includes a protein having an amino acid sequence that is sufficiently similar to a naturally occurring GDP-mannose-3',5'-epimerase amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under moderate, high, or very high stringency conditions (described below) to (i.e., with) a nucleic acid molecule encoding the naturally occurring GDP-mannose-3',5'-epimerase (i.e., to the complement of the nucleic acid strand encoding the naturally occurring GDP-mannose-3', 5'- epimerase amino acid sequence). Preferably, a GDP-mannose-3',5'-epimerase is encoded by a nucleic acid sequence that hybridizes under moderate, high or very high stringency conditions to the complement of a nucleic acid sequence that encodes a protein comprising an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, and most preferably SEQ ID NO:2. Even more preferably, a GDP-mannose-3', 5'-epimerase of the present invention is encoded by a nucleic acid sequence that hybridizes under moderate, high or very high stringency conditions to the complement of the coding region of a nucleic acid sequence selected from SEQ ID NO:1 , SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:53, with SEQ ID NO:1 being particularly preferred. Such hybridization conditions are described in detail below. A nucleic acid sequence complement of nucleic acid sequence encoding a GDP-mannose- 3',5'-epimerase of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to the strand which encodes the GDP-mannose-3', 5'-epimerase. It will be appreciated that a double stranded DNA which encodes a given amino acid sequence comprises a single strand DNA and its complementary strand having a sequence that is a complement to the single strand DNA. As such, nucleic acid molecules of the present invention can be either double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes an amino acid sequence of a GDP-mannose-3',5'-epimerase, and/or with the complement of the nucleic acid sequence that encodes any of such amino acid sequences. Methods to deduce a complementary sequence are known to those skilled in the art. It should be noted that since amino acid sequencing and nucleic acid sequencing technologies are not entirely error-free, the sequences presented herein, at best, represent apparent sequences of GDP-mannose-3', 5'- epimerases of the present invention.

A particularly preferred protein of the present invention comprises an isolated multimeric GDP-mannose-3',5'-epimerase comprising an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, or a subunit (e.g., monomers) or fragment of such sequence that has GDP-mannose-3', 5'- epimerase biological activity. GDP-mannose-3', 5'-epimerase homologues can, in one embodiment, be the result of natural allelic variation or natural mutation. GDP-mannose-3', 5'-epimerase homologues can also be naturally occurring GDP-mannose-3',5'-epimerases from different organisms with at least 70% identity to one another at the nucleic acid or amino acid level as described herein. GDP-mannose- 3',5'-epimerase homologues of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. A naturally occurring allelic variant of a nucleic acid encoding a given GDP-mannose-3',5' -epimerase is a gene that occurs at essentially the same locus (or loci) in the genome as the gene which encodes the given GDP-mannose-3¹, 5'-epimerase, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Natural allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. One class of allelic variants can encode the same protein but have different nucleic acid sequences due to the degeneracy of the genetic code. Allelic variants can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art.

GDP-mannose-3',5'-epimerase proteins of the present invention also include expression products of gene fusions (for example, used to overexpress soluble, active forms of the recombinant protein), of mutagenized genes (such as genes having codon modifications to enhance gene transcription and translation), and of truncated genes (such as genes having membrane binding domains removed to generate soluble forms of a membrane protein, or genes having signal sequences removed which are poorly tolerated in a particular recombinant host).

The minimum size of a protein and/or homologue of the present invention is, in one aspect, a size sufficient to have GDP-mannose-3', 5'-epimerase biological activity. In another embodiment, a protein of the present invention is at least 30 amino acids long, and more preferably, at least about 50, and more preferably at least 75, and more preferably at least 100, and more preferably at least 115, and more preferably at least 130, and more preferably at least 150, and more preferably at least 200, and more preferably, at least 250, and more preferably, at least 300, and more preferably, at least 350 amino acids long. There is no limit, other than a practical limit, on the maximum size of such a protein in that the protein can include a portion of a GDP-mannose-3',5'- epimerase protein or a full-length GDP-mannose-3', 5'-epimerase, plus additional sequence (e.g., a fusion protein sequence), if desired.

The present invention also includes a fusion protein that includes a GDP-mannose-3', 5'- epimerase-containing domain (i.e., an amino acid sequence for a GDP-mannose-3',5'-epimerase according to the present invention) attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; provide other desirable biological activity; and/or assist with the purification of a GDP-mannose-3',5'-epimerase (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, solubility, biological activity; and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the GDP-mannose-3',5'-epimerase-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a GDP-mannose-3', 5'-epimerase. Fusion proteins are preferably produced by culturing a recombinant cell transfected with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a GDP-mannose- 3',5'-epimerase-containing domain.

The present invention also includes a mimetic of a GDP-mannose-3',5'-epimerase. As used herein, the term "mimetic" is used to refer to any peptide or non-peptide compound that is able to mimic the biological action of a naturally occurring peptide, often because the mimetic has a basic structure that mimics the basic structure of the naturally occurring peptide and/or has the salient biological properties of the naturally occurring peptide. Mimetics can include, but are not limited to: peptides that have substantial modifications from the prototype such as no side chain similarity with the naturally occurring peptide (such modifications, for example, may decrease its susceptibility to degradation); anti-idiotypic and/or catalytic antibodies, or fragments thereof; non- proteinaceous portions of an isolated protein (e.g., carbohydrate structures); or synthetic or natural organic molecules, including nucleic acids and drugs identified through combinatorial chemistry, for example.

Such mimetics can be designed, selected and/or otherwise identified using a variety of methods known in the art. Various methods of drug design, useful to design mimetics or other therapeutic compounds useful in the present invention are disclosed in Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., which is incorporated herein by reference in its entirety. A GDP-mannose-3', 5'-epimerase mimetic can be obtained, for example, from molecular diversity strategies (a combination of related strategies allowing the rapid construction of large, chemically diverse molecule libraries), libraries of natural or synthetic compounds, in particular from chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the similar building blocks) or by rational, directed or random drug design. See for example, Maulik et al., supra.

In a molecular diversity strategy, large compound libraries are synthesized, for example, from peptides, oligonucleotides, carbohydrates and/or synthetic organic molecules, using biological, enzymatic and/or chemical approaches. The critical parameters in developing a molecular diversity strategy include subunit diversity, molecular size, and library diversity. The general goal of screening such libraries is to utilize sequential application of combinatorial selection to obtain high-affinity ligands for a desired target, and then to optimize the lead molecules by either random or directed design strategies. Methods of molecular diversity are described in detail in Maulik, et al., ibid.

Maulik et al. also disclose, for example, methods of directed design, in which the user directs the process of creating novel molecules from a fragment library of appropriately selected fragments; random design, in which the user uses a genetic or other algorithm to randomly mutate fragments and their combinations while simultaneously applying a selection criterion to evaluate the fitness of candidate ligands; and a grid-based approach in which the user calculates the interaction energy between three dimensional receptor structures and small fragment probes, followed by linking together of favorable probe sites. One embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence that encodes a GDP-mannose-3', 5'-epimerase of the present invention, as well as nucleic acid sequences fully complementary thereto. A nucleic acid molecule encoding a GDP-mannose-3', 5'-epimerase of the present invention includes a nucleic acid molecule encoding any of the GDP-mannose-3', 5'-epimerase proteins, including homologues, discussed above. More particularly, one embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a protein having an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, over the full length of any of such sequences, wherein the protein is a GDP-mannose-3¹, 5'- epimerase (i.e., has GDP-mannose-3', 5' -epimerase biological activity). More preferably, an isolated nucleic acid molecule of the present invention comprises a nucleic acid sequence encoding an amino acid sequence that is at least about 75% identical, and even more preferably at least about 80% identical, and even more preferably at least about 85% identical, and even more preferably at least about 90% identical and even more preferably at least about 95% identical, and even more preferably at least about 96% identical, and even more preferably at least about 97% identical, and even more preferably at least about 98% identical, and even more preferably at least about 99% identical to any of the above-identified amino acid sequences, with SEQ ID NO:2 being particularly preferred. Preferably, the encoded protein has GDP-mannose-3', 5'-epimerase biological activity.

In yet another embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleic acid sequence encoding an amino acid sequence that has any of the above- referenced percent identities to any of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54 over at least 50 amino acids, and more preferably 100, and more preferably 25, and more preferably 150, and more preferably 175, and more preferably 200, and more preferably 225, and more preferably 250, and more preferably 275, and more preferably 300, and more preferably 325, and more preferably 350 amino acids of any of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54. Preferably, the protein has GDP-mannose-3',5'-epimerase biological activity. Percent identity is determined using BLAST 2.0 Basic BLAST default parameters, as described above.

In one embodiment of the present invention, a nucleic acid molecule according to the present invention comprises a nucleic acid sequence that encodes an amino acid sequence that is less than about 100% identical to SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, and particularly, to SEQ ID NO:2. In another aspect of the invention, a nucleic acid molecule comprises a nucleic acid sequence that encodes an amino acid sequence that is less than about 99% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than is less than 98% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 97% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 96% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 95% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 94% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 93% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 92% identical to any of the above-identified amino acid sequences, and in another embodiment, is less than 91 % identical to any of the above- identified amino acid sequences, and in another embodiment, is less than 90% identical to any of the above-identified amino acid sequences. In one embodiment, nucleic acid molecules encoding a GDP-mannose-3', 5'-epimerase of the present invention include isolated nucleic acid molecules that hybridize under moderate stringency conditions, and even more preferably under high stringency conditions, and even more preferably under very high stringency conditions with the complement of a nucleic acid sequence encoding a naturally occurring GDP-mannose-3', 5'-epimerase. Preferably, an isolated nucleic acid molecule encoding a GDP-mannose-3',5'-epimerase of the present invention comprises a nucleic acid sequence that hybridizes under moderate or high stringency conditions to the complement of a nucleic acid sequence that encodes a protein comprising an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54. In one embodiment, an isolated nucleic acid molecule comprises a nucleic acid sequence that hybridizes under moderate, high or very high stringency conditions to the complement of the coding region of a nucleic acid sequence represented by SEQ ID NO:1 , SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO.31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:53, with SEQ ID NO:1 being particularly preferred.

As used herein, hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31 -9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or less mismatch of nucleotides). High stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 80%) nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or less mismatch of nucleotides). Very high stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 90% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides). As discussed above, one of skill in the art can use the formulae in Meinkoth et al., ibid, to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary, depending on whether DNA.RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10°C less than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na⁺) at a temperature of between about 20°C and about 35°C (lower stringency), more preferably, between about 28°C and about 40°C (more stringent), and even more preferably, between about 35°C and about 45°C (even more stringent), with appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na⁺) at a temperature of between about 30°C and about 45°C, more preferably, between about 38°C and about 50°C, and even more preferably, between about 45°C and about 55°C, with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%. Alternatively, T_m can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions can include a combination of salt and temperature conditions that are approximately 20-25°C below the calculated T_m of a particular hybrid, and wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20°C below the calculated T_m of the particular hybrid. One example of hybridization conditions suitable for use with DNA.DNA hybrids includes a 2-24 hour hybridization in 6X SSC (50% formamide) at about 42°C, followed by washing steps that include one or more washes at room temperature in about 2X SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37°C in about 0.1X-0.5X SSC, followed by at least one wash at about 68°C in about 0.1X-0.5X SSC).

In another embodiment, nucleic acid molecules encompassed by the present invention include isolated nucleic acid molecules comprising a nucleic acid sequence having at least about 12 contiguous nucleotides of a nucleic acid sequence selected from SEQ ID NO: 1 , SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:53, and preferably at least about 15 contiguous nucleotides, and more preferably at least about 18 contiguous nucleotides, and more preferably at least about 21 contiguous nucleotides, and more preferably at least about 24 contiguous nucleotides, and so on, in increments of whole integers (e.g., 25, 26, 27, 28), up to the full length of a coding region of a nucleic acid sequence selected from SEQ ID NO:1 , SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:53. In one embodiment, such a nucleic acid sequence can be used as a probe or primer to identify and/or clone other nucleic acid sequences encoding GDP-mannose-3', 5'-epimerases. In another embodiment, the present invention includes an isolated nucleic acid molecules comprising a nucleic acid sequence encoding a protein having an amino acid sequence comprising at least 30 contiguous amino acid residues of any of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, (i.e., 30 contiguous amino acid residues having 100% identity with 30 contiguous amino acids of any of such amino acid sequences). In a preferred embodiment, an isolated nucleic acid molecule comprises a nucleic acid sequence encoding a protein having an amino acid sequence comprising at least 50, and more preferably at least 75, and more preferably at least 100, and more preferably at least 115, and more preferably at least 130, and more preferably at least 150, and more preferably at least 200, and more preferably, at least 250, and more preferably, at least 300, and more preferably, at least 350 contiguous amino acid residues of any of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54. Such a protein preferably has GDP- mannose-3', 5'-epimerase biological activity.

Particularly preferred nucleic acid molecules of the present invention comprise nucleic acid sequences encoding SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:54, or fragments of such sequences that encode a GDP- mannose-3',5'-epimerase having biological activity. Particularly preferred nucleic acid molecules of the present invention comprise SEQ ID NO:1, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:53, or the coding regions of such molecules, or fragments of such sequences that encode a GDP-mannose-3',5'-epimerase having biological activity.

In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic acid molecule is found in nature. As such, "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified, but indicates that the molecule does not include an entire genome or an entire chromosome in which the nucleic acid molecule is found in nature. An isolated nucleic acid molecule can include a gene, such as a GDP-mannose-3',5'-epimerase gene described herein (e.g., EMBO Accession No. AF272706 contains a complete GDP-mannose-3',5'- epimerase gene, including regulatory sequences). An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the same chromosome. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., are heterologous sequences). Isolated nucleic acid molecules can include DNA, RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA). Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein.

Preferably, an isolated nucleic acid molecule of the present invention is produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications provide the desired effect on protein biological activity. Allelic variants and protein homologues (e.g., proteins encoded by nucleic acid homologues) have been discussed in detail above.

A nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid and/or by hybridization with a wild-type gene. Similarly, the minimum size of a nucleic acid molecule of the present invention is a size sufficient to encode a protein having the desired biological activity, or sufficient to form a probe or oligonucleotide primer that is capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding the natural protein (e.g., under moderate, high or very high stringency conditions, and preferably under very high stringency conditions). As such, the size of a nucleic acid molecule of the present invention can be dependent on nucleic acid composition and percent homology or identity between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions perse (e.g., temperature, salt concentration, and formamide concentration). The minimal size of a nucleic acid molecule that is used as an oligonucleotide primer or as a probe is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 18 bases in length if they are AT-rich. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule of the present invention, in that the nucleic acid molecule can include a portion of a protein-encoding sequence (e.g., a GDP-mannose-3', 5'-epimerase-encoding sequence) or a nucleic acid sequence encoding a full-length protein. Any of the above-described GDP-mannose-3',5'-epimerase, including homologues, can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal end of the GDP-mannose-3',5'-epimerase protein. Such a protein can be referred to as "consisting essentially of a given GDP-mannose-3',5'-epimerase amino acid sequence. According to the present invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the GDP- mannose-3', 5'-epimerase sequence or which would not be encoded by the nucleotides that flank the naturally occurring GDP-mannose-3',5'-epimerase nucleic acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the GDP-mannose-3',5' -epimerase is derived. Similarly, the phrase "consisting essentially of, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a GDP-mannose-3',5'-epimerase (including fragments/homologues) that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the GDP-mannose-3', 5'-epimerase. The nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the GDP-mannose-3',5'-epimerase coding sequence as it occurs in the natural gene. Another embodiment of the present invention includes a recombinant nucleic acid molecule comprising a recombinant vector and a nucleic acid sequence encoding a GDP-mannose-3', 5'- epimerase, or a biologically active subunit or homologue (including a fragment) thereof, as previously described herein. Such nucleic acid sequences are described in detail above. According to the present invention, a recombinant vector is an engineered (i.e., artificially produced) nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice and/or for introducing such a nucleic acid sequence into a host cell. The recombinant vector is therefore suitable for use in cloning, sequencing, and/or otherwise manipulating the nucleic acid sequence of choice, such as by expressing and/or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences including nucleic acid sequences that are not naturally found adjacent to nucleic acid sequence to be delivered, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) which are naturally found adjacent to nucleic acid molecules of the present invention (discussed in detail below). The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a plasmid. The vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome of the recombinant host cell. The entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule encoding a GDP-mannose-3¹, 5'-epimerase, subunit, or homologue thereof. The integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome.

A recombinant vector of the present invention typically contains at least one selectable marker. Selection markers typically allow transformed cells to be recovered by negative selection (i.e., inhibiting growth of cells that do not contain the selection marker) or by screening for a product encoded by the selection marker. The most commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptll) gene, isolated from Tn5, which, when placed under the control of plant regulatory signals, confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3'-adenyl transferase, and the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet. 210:86 (1987), Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil. Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-eno/pyruvylshikimate-3- phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shah et al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643 (1990).

Commonly used genes for screening presumptively transformed cells include β- glucuronidase (GUS), β-galactosidase, luciferase, and chloramphenicol acetyltransferase.

Jefferson, R.A., Plant Mol. Biol. Rep. 5:387 (1987)., Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. USA 84:131 (1987), De Block et al., EMBO J. 3:1681 (1984), green fluorescent protein (GFP) (Chalfie et al., Science 263:802 (1994), Haseloff et al., TIG 11 :328-329 (1995) and PCT application WO 97/41228). Another approach to the identification of relatively rare transformation events has been use of a gene that encodes a dominant constitutive regulator of the Zea mays anthocyanin pigmentation pathway. Ludwig et al., Science 247:449 (1990).

Suitable selection markers for use in prokaryotes and eukaryotes other than plants are also well known. See, e.g., PCT WO 96/23898 and PCT WO 97/42320. For instance, resistance to antibiotics (ampicillin, kanamycin, tetracyline, chloramphenicol, neomycin or hygromycin) may be used as the selection marker.

As used herein, the phrase "recombinant nucleic acid molecule" is used primarily to refer to a recombinant vector into which has been ligated the nucleic acid sequence to be cloned, manipulated, transformed into the host cell (i.e., the insert). "DNA construct" can be used interchangeably with "recombinant nucleic acid molecule" in some embodiments and is further defined herein to be a constructed (non-naturally occurring) DNA molecules useful for introducing

DNA into host cells, and the term includes chimeric genes, expression cassettes, and vectors.

In one embodiment, a recombinant vector of the present invention is an expression vector. As used herein, the phrase "expression vector" is used to refer to a vector that is suitable for production of an encoded product (e.g., a protein of interest). In this embodiment, a nucleic acid sequence encoding the product to be produced is inserted into the recombinant vector to produce a recombinant nucleic acid molecule. The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operatively links the nucleic acid sequence to regulatory sequences in the vector (e.g., a promoter) which enable the transcription and translation of the nucleic acid sequence within the recombinant host cell. Typically, a recombinant vector includes at least one nucleic acid molecule of the present invention (e.g., a nucleic acid molecule comprising a nucleic acid sequence encoding a GDP- mannose-3',5'-epimerase) operatively linked to one or more transcription control sequences to form a recombinant nucleic acid molecule. As used herein, the phrase "recombinant molecule" or "recombinant nucleic acid molecule" primarily refers to a nucleic acid molecule or nucleic acid sequence operatively linked to a transcription control sequence, but can be used interchangeably with the phrase "nucleic acid molecule", when such nucleic acid molecule is a recombinant molecule as discussed herein. According to the present invention, the phrase "operatively linked" refers to linking a nucleic acid molecule to a transcription control sequence (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in a manner such that proteins encoded by the nucleic acid sequence can be expressed when transfected (i.e., transformed, transduced, transfected, conjugated or conduced) into a host cell. Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989).

Vectors for transferring recombinant sequences into eukaryotic cells are known to the person skilled in the art and include, but are not limited to self-replicating vectors, integrative vectors, artificial chromosomes, Agrobactehum based transformation vectors and viral vector systems such as retroviral vectors, adenoviral vectors or lentiviral vectors. Transcription control sequences are sequences which control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in a host cell useful in the present invention. The transcription control sequences includes a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the chosen host cell or organism. The promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic. The promoter may be a native promoter (i.e., the promoter that naturally occurs within the GDP-mannose-3',5'-epimerase gene and regulates transcription thereof) or a non-native promoter (i.e., any promoter other than the promoter that naturally occurs within the GDP-mannose-3',5'-epimerase gene, including other promoters that naturally occur within the chosen host cell). Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds, Nucleic Acids Res., 15, 2343-61 (1987). Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts, et al., Proc. Natl Acad. Sci. USA, 76, 760-4 (1979). Many suitable promoters for use in prokaryotes and eukaryotes are well known in the art. For instance, suitable constitutive promoters for use in plants include, but are not limited to: the promoters from plant viruses, such as the 35S promoter from cauliflower mosaic virus (Odell et al., Nature 313:810-812 (1985), the full length transcript promoter with duplicated enhancer domains from peanut chlorotic streak caulimovirus (Maiti and Shepherd, BBRC 244:440-444 (1998)), promoters of Chlorella virus methyltransferase genes (U.S. Patent No. 5,563,328), and the full-length transcript promoter from figwort mosaic virus (U.S. Patent No. 5,378,619); the promoters from such genes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990)), ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)), pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)), MAS (Velten et al., EMBO J. 3:2723-2730 (1984)), maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231:276-285 (1992) and Atanassova et al., Plant Jouma/2(3):291 -300 (1992)), βrass/ca napus ALS3 (PCT application WO 97/41228); and promoters of various Agrobacterium genes (see U.S. Patents Nos. 4,771 ,002, 5,102,796, 5,182,200, 5,428,147).

Suitable inducible promoters for use in plants include, but are not limited to: the promoter from the ACE1 system which responds to copper (Mett et al. PNAS 90:4567-4571 (1993)); the promoter of the maize In2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen. Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)), and the promoter of the Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genet. 227:229-237 (1991). A particularly preferred inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter of this type is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. USA 88:10421 (1991). Other inducible promoters for use in plants are described in EP 332104, PCT WO 93/21334 and PCT WO 97/06269.

Suitable promoters for use in bacteria include, but are not limited to, the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gene, the Bacillus pumilus xylosidase gene, the phage lambda P and PL promoters, and the Escherichia colilac, trp and tac promoters. See PCT WO 96/23898 and PCT WO 97/42320.

Suitable promoters for use in yeast host cells include, but are not limited to, promoters from yeast glycolytic genes, promoters from alcohol dehydrogenase genes, the TPI1 promoter, and the ADH2-4c promoter. See, e.g., PCT WO 96/23898. Finally, promoters composed of portions of other promoters and partially or totally synthetic promoters can be used. See, e.g., Ni et al., Plant J., 7:661-676 (1995)and PCT WO 95/14098 describing such promoters for use in plants.

The promoter may include, or be modified to include, one or more enhancer elements. Preferably, the promoter will include a plurality of enhancer elements. Promoters containing enhancer elements provide for higher levels of transcription as compared to promoters which do not include them. Suitable enhancer elements for use in plants include the 35S enhancer element from cauliflower mosaic virus (U.S. Patents Nos. 5,106,739 and 5,164,316) and the enhancer element from figwort mosaic virus (Maiti et al., Transgenic Res., 6, 143-156 (1997)). Othersuitable enhancers for use in other cells are known. See PCT WO 96/23898 and Enhancers And Eukaryotic Expression (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1983).

Recombinant nucleic acid molecules of the present invention, which can be either DNA or RNA, can also contain additional regulatory sequences, such as translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell. In one embodiment, a recombinant molecule of the present invention, including those which are integrated into the host cell chromosome, also contains secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed protein to be secreted from the cell that produces the protein. Suitable signal segments include a signal segment that is naturally associated with the protein to be expressed or any heterologous signal segment capable of directing the secretion of the protein according to the present invention. In another embodiment, a recombinant molecule of the present invention comprises a leader sequence to enable an expressed protein to be delivered to and inserted into the membrane of a host cell. Suitable leader sequences include a leader sequence that is naturally associated with the protein, or any heterologous leader sequence capable of directing the delivery and insertion of the protein to the membrane of a cell. For efficient expression, the coding sequences are preferably also operatively linked to a 3' untranslated sequence. The 3' untranslated sequence contains transcription and/or translation termination sequences. The 3' untranslated regions can be obtained from the flanking regions of genes from bacterial, plant or other eukaryotic cells. For use in prokaryotes, the 3' untranslated region will include a transcription termination sequence. For use in plants and other eukaryotes, the 3' untranslated region will include a transcription termination sequence and a polyadenylation sequence. Suitable 3' untranslated sequences for use in plants include those of the cauliflower mosaic virus 35S gene, the phaseolin seed storage protein gene, the pea ribulose biphosphate carboxylase small subunit E9 gene, the soybean 7S storage protein genes, the octopine synthase gene, and the nopaline synthase gene. In plants and other eukaryotes, a 5' untranslated sequence is typically also employed. The

5' untranslated sequence is the portion of an mRNA which extends from the 5' CAP site to the translation initiation codon. This region of the mRNA is necessary for translation initiation in eukaryotes and plays a role in the regulation of gene expression. Suitable 5' untranslated regions for use in plants include those of alfalfa mosaic virus, cucumber mosaic virus coat protein gene, and tobacco mosaic virus.

It will be appreciated by one skilled in the art that use of recombinant DNA technologies can improve control of expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within the host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Additionally, the promoter sequence might be genetically engineered to improve the level of expression as compared to the native promoter. Recombinant techniques useful for controlling the expression of nucleic acid molecules include, but are not limited to, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgamo sequences), modification of nucleic acid molecules to correspond to the codon usage of the host cell, and deletion of sequences that destabilize transcripts.

In one embodiment of the present invention, a recombinant nucleic acid molecule comprises an expression vector and a nucleic acid molecule comprising a first nucleic acid sequence encoding a GDP-mannose-3', 5'-epimerase as previously described herein (including subunits and homologues) and at least one additional nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein the enzyme is, in a preferred aspect, another enzyme in the ascorbic acid biosynthetic pathway. Such an enzyme can include: phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP- L-galactose pyrophosphorylase, L-galactose-1 -P-phosphatase, L-galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase. The nucleic acid sequences encoding each of phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, L- galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase from at least one organism are known in the art. For example, for Arabidopsis thaliana, the nucleic acid and amino acid sequences for phosphomannose isomerase are disclosed in GenBank Accession Nos. NC_ 003070 and NP_176878, respectively, as well as in Privalle, 2002, Ann. NY. Acad. Sci. 129- 138); the amino acid sequence for phosphomannomutase is disclosed in GenBank Accession No. O80840 and in Lin et al., 1999, Nature 402:761-768); the amino acid sequence for GDP-D- mannose pyrophosphorylase is disclosed in GenBank Accession No. NP_181507; the nucleic acid and amino acid sequence for L-galactose dehydrogenase is disclosed in PCT Publication Nos. WO 99/33995 and WO 01/72974, supra, and the nucleic acid and amino acid sequence for L- galactono- γ -lactone dehydrogenase is disclosed in PCT Publication WO 98/50558, supra. In one aspect, the recombinant nucleic acid molecule includes one additional nucleic acid sequence, and in another aspect, at least two additional nucleic acid sequences, and in another aspect, at least three additional nucleic acid sequences, and in another aspect, at least four additional nucleic acid sequences encoding any of the above-referenced enzymes. The additional sequences are not required to be isolated from or derived from the same organism as the GDP-mannose-3', 5'- epimerase.

One or more recombinant molecules of the present invention can be used to produce an encoded product (e.g., GDP-mannose-3', 5'-epimerase) of the present invention. In one embodiment, an encoded product is produced by expressing a nucleic acid molecule as described herein under conditions effective to produce the protein. A preferred method to produce an encoded protein is by transfecting (transforming) a host cell with one or more recombinant molecules to form a recombinant host cell. Suitable host cells to transfect include, but are not limited to, any prokaryotic or eukaryotic cell that can be transfected, with bacterial, fungal (e.g., yeast), algal and plant cells being particularly preferred. Host cells can be either untransfected cells or cells that are already transfected with at least one other recombinant nucleic acid molecule. According to the present invention, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" can be used interchangeably with the term "transfection" when such term is used to refer to the introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and yeast, or into plant cells. In microbial systems and plant systems, the term "transformation" is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism or plant and is essentially synonymous with the term "transfection". Therefore, transfection techniques include, but are not limited to, transformation, particle bombardment, electroporation, microinjection, chemical treatment of cells, lipofection, adsorption, infection (e.g., Agrobacterium mediated transformation and virus mediated transformation) and protoplast fusion (protoplast transformation).

Methods of transforming prokaryotic and eukaryotic host cells are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989); PCT WO 96/23898 and PCT WO 97/42320. For instance, numerous methods for plant transformation have been developed, including biological and physical transformation protocols. See, for example, Miki et al., "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition, vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-119. The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C.I., Crit Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for y4grojbacter/t//77-mediated gene transfer are provided by numerous references, including Gruber et al., supra, Miki et al., supra, Moloney et al., Plant Cell Reports 8:238 (1989), and U.S. Patents Nos. 4,940,838 and 5,464,763. A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds sufficient to penetrate plant cell walls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J.C., Trends Biotech. 6:299 (1988), Sanford, J.C., Physiol. Plant 79:206 (1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively, liposome or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNA into protoplasts using CaCI₂ precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet 199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982). Electroporation of protoplasts and whole cells and tissues have also been described. Donn et al., In Abstracts of Vllth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); DΗalluin etal., PlantCell 4: 1495-1505 (1992) and Spencer et al., PlantMol. Biol. 24:51-61 (1994). Accordingly, it is the object of the present invention to create genetically modified host cells, and particularly, genetically modified plants or microorganisms, that have increased production of intermediates within the ascorbic acid pathway and/or that contain an increased content of ascorbic acid, relative to non-modified (i.e., non-transformed or wild-type) plants or microorganisms. According to the present invention, a genetically modified microorganism or plant includes a microorganism or plant that has been modified using recombinant technology and/or classical mutagenesis techniques. According to the present invention, genetic modifications that result in an increase in gene expression or function (the preferred embodiment) can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene. For example, a genetic modification in a gene encoding GDP-mannose-3', 5'-epimerase which results in an increase in the function of the GDP-mannose-3', 5'-epimerase, can be the result of an increased expression of the GDP-mannose-3', 5'-epimerase, an enhanced activity of the GDP-mannose-3',5'-epimerase, or an inhibition of a mechanism that normally inhibits the expression or activity of the GDP-mannose-3', 5' -epimerase. Genetic modifications which result in a decrease in gene expression, in the function of the gene, or in the function of the gene product (i.e., the protein encoded by the gene) can be referred to as inactivation (complete or partial), deletion, interruption, blockage, silencing or down-regulation of a gene. For example, a genetic modification in a gene encoding GDP-mannose-3', 5'-epimerase which results in a decrease in the function of the GDP-mannose-3¹, 5'-epimerase, can be the result of a complete deletion of the gene (i.e., the gene does not exist, and therefore the protein does not exist), a mutation in the gene which results in incomplete or no translation of the protein (e.g., the protein is not expressed), a mutation in the gene or genome which results in silencing of a gene, or a mutation in the gene which decreases or abolishes the natural function of the protein (e.g., a protein is expressed which has decreased or no enzymatic activity).

The genetic modification of a microorganism or plant to provide increased expression and/or activity of a GDP-mannose-3', 5'-epimerase according to the present invention preferably affects the activity of an ascorbic acid biosynthetic pathway expressed by the microorganism or plant, whether the ascorbic acid biosynthetic pathway is endogenous and genetically modified, endogenous with the introduction of one or more recombinant nucleic acid molecules into the organism, or provided completely by recombinant technology. According to the present invention, to "affect the activity of an ascorbic acid biosynthetic pathway" includes any genetic modification that causes any detectable or measurable change or modification in the ascorbic acid biosynthetic pathway expressed by the organism as compared to in the absence of the genetic modification. A detectable change or modification in the ascorbic acid biosynthetic pathway can include, but is not limited to, a detectable change in the production of at least one product in the ascorbic acid biosynthetic pathway including the immediate product of the GDP-mannose-3',5'-epimerase (i.e., GDP-L-galactose), as well as products lying downstream of the GDP-mannose-3',5'-epimerase (e.g., L-galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone), or a detectable change in the production of ascorbic acid by the microorganism or plant.

It should be noted that reference to increasing the activity of a GDP-mannose-3',5'- epimerase refers to any genetic modification in the organism containing the GDP-mannose-3', 5'- epimerase (or into which the GDP-mannose-3', 5'-epimerase is to be introduced) which results in increased functionality of the GDP-mannose-3',5'-epimerase, and can include higher activity of the GDP-mannose-3¹, 5'-epimerase (e.g., specific activity or in vivo enzymatic activity), reduced inhibition or degradation of the GDP-mannose-3¹, 5'-epimerase, and overexpression of the GDP- mannose-3', 5'-epimerase. For example, gene copy number can be increased, expression levels can be increased by use of a non-native promoter that gives higher levels of expression than that of the native promoter (i.e., the GDP-mannose-S'.δ'-epimerase promoter), or a gene can be altered by genetic engineering or classical mutagenesis to increase the activity of the encoded GDP- mannose-3',5'-epimerase. Similarly, reference to decreasing the activity of a GDP-mannose-3', 5'-epimerase refers to any genetic modification in the organism containing such GDP-mannose-3', 5'-epimerase (or into which the GDP-mannose-3',5'-epimerase is to be introduced) which results in decreased functionality of the GDP-mannose-3', 5'-epimerase, and includes decreased activity of the GDP- mannose-3',5'-epimerase, increased inhibition or degradation of the GDP-mannose-3', 5'- epimerase and a reduction or elimination of expression of the GDP-mannose-3', δ'-epimerase. For example, the activity of a GDP-mannose-S'.δ'-epimerase of the present invention can be decreased by blocking or reducing the production of the GDP-mannose-S'.δ'-epimerase, "knocking out" the gene or portion thereof encoding the GDP-mannose-3', 5'-epimerase, reducing GDP- mannose-3',5'-epimerase activity, or inhibiting the activity of the GDP-mannose-3', 5'-epimerase. Blocking or reducing the production of a GDP-mannose-3', 5'-epimerase can include placing the gene encoding the GDP-mannose-3¹, ^-epimerase under the control of a promoter that requires the presence of an inducing compound in the growth medium. By establishing conditions such that the inducer becomes depleted from the medium, the expression of the gene encoding the GDP- mannose-3',5'-epimerase (and therefore, of protein synthesis) could be turned off. Blocking or reducing the activity of GDP-mannose-3', 5'-epimerase could also include using an excision technology approach similar to that described in U.S. Patent No.4,743,546, incorporated herein by reference. To use this approach, the gene encoding the protein of interest is cloned between specific genetic sequences that allow specific, controlled excision of the gene from the genome. Excision could be prompted by, for example, a shift in the cultivation temperature of the culture, as in U.S. Patent No. 4,743,546, or by some other physical or nutritional signal.

In one embodiment of the present invention, a genetic modification includes a modification of a nucleic acid sequence encoding a GDP-mannose-3', 5'-epimerase as described herein. Such a modification can be to an endogenous GDP-mannose-S'.^-epimerase, whereby a microorganism or plant that naturally contains such a system is genetically modified by, for example, classical mutagenesis and selection techniques and/or molecular genetic techniques, include genetic engineering techniques. Genetic engineering techniques can include, for example, using a targeting recombinant vector to delete a portion of an endogenous gene, orto replace a portion of an endogenous gene with a heterologous sequence, such as an improved GDP-mannose-S^δ'- epimerase or a different promoter that increases the expression of the endogenous GDP- mannose-3', 5'-epimerase.

For example, a non-native promoter can be introduced upstream of at least one gene encoding a subunit of a GDP-mannose-3¹, 5'-epimerase. Preferably the 5' upstream sequence of a endogenous gene encoding a subunit of a multimeric GDP-mannose-3',5'-epimerase is replaced by a constitutive promoter or a promoter with optimal expression under the growth conditions used. This method is especially useful when said endogenous gene is not active or is not sufficiently active under the growth conditions used. In another aspect of this embodiment of the invention, the genetic modification can include the introduction of a recombinant nucleic acid molecule encoding a GDP-mannose-3',5'-epimerase, including a subunit or homologue thereof, into a host. The host can include: (1) a host cell that does not express an ascorbic acid biosynthetic pathway, wherein all functional enzymes of an ascorbic acid biosynthetic pathway are introduced into the host cell, including a recombinant nucleic acid molecule encoding a GDP-mannose-3¹, 5'-epimerase; or (2) the preferred and most typical embodiment, a host cell that expresses an ascorbic acid biosynthetic pathway, wherein the introduced recombinant nucleic acid molecule encodes a GDP-mannose-3',5'-epimerase alone or together with at least one, and as many as three or four, recombinant nucleic acid molecules encoding other enzymes in the ascorbic acid biosynthetic pathway or regulatory sequences that enhance the expression and/or activity of other enzymes in the ascorbic acid biosynthetic pathway. The present invention intends to encompass any genetically modified organism (e.g., microorganism or plant), wherein the organism comprises at least one modification to increase the expression and/or activity of a GDP-mannose-3', 5'-epimerase according to the present invention. Genetic modification of a microorganism can be accomplished using classical strain development and/or molecular genetic techniques. Such techniques known in the art and are generally disclosed for microorganisms, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press. The reference Sambrook et al., ibid., is incorporated by reference herein in its entirety. A genetically modified microorganism can include a microorganism in which nucleic acid molecules have been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect within the microorganism.

As used herein, a genetically modified plant can include any genetically modified plant including higher plants and particularly, any consumable plants or plants useful for producing ascorbic acid. Such a genetically modified plant has a genome which is modified (i.e., mutated or changed) from its normal (i.e., wild-type or naturally occurring) form such that the desired result is achieved (i.e., increased or modified GDP-mannose-3', 5'-epimerase activity and, in some embodiments, production of a desired product using the ascorbic acid biosynthetic pathway). Genetic modification of a plant can be accomplished using classical strain development and/or molecular genetic techniques. Methods for producing a transgenic plant, wherein a recombinant nucleic acid molecule encoding a desired amino acid sequence is incorporated into the genome of the plant, are known in the art and are discussed below.

One embodiment of the present invention relates to a recombinant host cell transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a GDP- mannose-3',5'-epimerase according to the present invention. Nucleic acid sequences encoding GDP-mannose-3¹, 5'-epimerases and the proteins encoded by such sequences have been described in detail above and all such nucleic acid sequences and proteins are encompassed by the present invention (e.g., multimeric GDP-mannose-3',5'-epimerases and biologically active subunits and homologues (including fragments) thereof). Preferred host cells to transform with a recombinant nucleic acid molecule of the present invention include any prokaryotic or eukaryotic host cell. Preferred prokaryotic cells include bacterial cells. Preferred eukaryotic host cells include fungal cells (preferably yeast cells), algal cells (preferably algal cells having an ascorbic acid biosynthetic pathway, such as microalgae of the genera Prototheca or Chlorella), and higher plant cells. Preferably, the host cell is an acid-tolerant host cell. Acid-tolerant yeast and bacteria are also known in the art. All known species of the microalga, Prototheca, produce L-ascorbic acid. Production of ascorbic acid by microalgae of the genera Prototheca and Chlorella is described in detail in U.S. Patent No. 5,792,631, issued August 11, 1998, and in U.S. Patent No. 5,900,370, issued May 4, 1999, both of which are incorporated herein by reference in their entirety. Preferred bacteria for use in the present invention include, but are not limited to, Azotobacter, Pseudomonas, and Escherichia, although acid-tolerant bacteria are more preferred, such as, but not limited to, lactic acid bacteria. Preferred fungi for use in the present invention include yeast, and more preferably, yeast of the genus Saccharomyces.

A preferred plant cell to transform according to the present invention is preferably a plant suitable for consumption by animals, including humans, but can include any higher plant in which it may be beneficial to increase the production of ascorbic acid or of an enzyme within the ascorbic acid pathway (discussed in detail below). In particular, cells from crop plants (including peas, soybeans, potatoes, tomatoes, corn, sorghum, rice, wheat, barley, other small grains, legumes, lettuce, melons, other fruits and similar plants) are desirable host cells for use in the present invention, as well as the plants or parts of plants (i.e., transgenic plants) obtainable by transformation of such host cells. Specifically, cells from any dicotyledonous or monocotyledonous plant can be transformed with the recombinant nucleic acid molecules of the present invention. One embodiment of the present invention relates to transgenic plants or parts of such plants that are transformed with a recombinant nucleic acid molecule encoding a GDP-mannose- 3',5'-epimerase (including subunits and homologues thereof). The genetically modified or transgenic plant is not limited to a plant variety, and preferably has increased ascorbic acid synthesis compared with a non-transformed control. The transgenic plant is typically obtainable by regenerating a recombinant plant cell produced according to the invention. Methods for regenerating plant cells into plants are well known to the person skilled in the art. "Plant parts" include seeds, pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, explants, etc.

In a preferred embodiment, expression of the recombinant nucleic acid molecule by the host cell or transgenic plant is sufficient to increase the synthesis of a product of the ascorbic acid biosynthetic pathway in the host cell or transgenic plant. Such a product can include the immediate product of the GDP-mannose-3',5'-epimerase (i.e., GDP-L-galactose), as well as products lying downstream of the GDP-mannose-3', 5'-epimerase (e.g., L-galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone). In a particularly preferred embodiment, expression of the recombinant nucleic acid molecule by the host cell or transgenic plant is sufficient to increase ascorbic acid production in the host cell or transgenic plant. Methods to measure ascorbic acid production are described in Examples 1-3 and are known in the art. For example, PCT Publication Nos. WO 99/33995, WO 01/72974 and WO 99/64618, supra, each of which is incorporated herein by reference in its entirety, describe methods of measuring ascorbic acid production, as well as measuring the production of intermediate products in the ascorbic acid biosynthetic pathway.

In one embodiment of the invention, a recombinant host cell is transformed with at least two recombinant nucleic acid molecules comprising: (a) a first recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a GDP-mannose-3', 5'-epimerase according to the present invention (including subunits and homologues thereof); (b) at least one additional recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein the enzyme is selected from: phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP- L-galactose pyrophosphorylase, L-galactose-1 -P-phosphatase, L-galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase. These enzymes in the ascorbic acid biosynthetic pathway have been described above. In one aspect, the host cell is transformed with one additional recombinant nucleic acid molecule and in another aspect, with two additional recombinant nucleic acid molecules and in another aspect, with three additional recombinant nucleic acid molecules and in another aspect, with at least four additional recombinant nucleic acid molecules as set forth above. In one embodiment, the each of the recombinant nucleic acid molecules is contained within a single recombinant vector. For example, the vector can be a dicistronic vector. Also included in the present invention are transgenic plants or parts of such plants which have been transformed with these additional recombinant nucleic acid molecules. The additional sequences are not required to be isolated from or derived from the same organism as the GDP-mannose-3',5'- epimerase.

Another embodiment of the present invention relates to a method to increase ascorbic acid synthesis in a host cell, comprising growing a host cell that is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one subunit of a multimeric GDP-mannose-3',5'-epimerase, wherein the nucleic acid sequence is operatively linked to a transcription control sequence. Recombinant nucleic acid molecules comprising a nucleic acid sequence encoding at least one subunit of a multimeric GDP-mannose- 3',5'-epimerase (including homologues thereof) have been described in detail above, as have recombinant host cells transformed with such recombinant nucleic acid molecules. Such recombinant nucleic acid molecules can comprise native or non-native promoters and other regulatory or selection sequences as discussed above. In one aspect of this embodiment, the host cell can also be transformed with at least one additional recombinant nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein the enzyme is, in a preferred aspect, another enzyme in the ascorbic acid biosynthetic pathway. Such an enzyme can include: phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-L-galactose pyrophosphorylase, L-galactose-1 -P-phosphatase, L-galactose dehydrogenase, and L-galactono^y- lactone dehydrogenase, which have been discussed above. The additional recombinant nucleic acid sequence(s) can be introduced into the host cell as part of the same recombinant nucleic acid molecule containing the GDP-mannose-3¹, δ^epimerase or as separate recombinant nucleic acid molecules. The host cell can be transformed with one, two, three, four or more additional recombinant nucleic acid molecules. The additional enzymes are not required to be isolated from or derived from the same organism as the GDP-mannose-3¹, 5'-epimerase.

A related embodiment of the present invention relates to a method to increase ascorbic acid synthesis in a cell comprising a multimeric GDP-mannose-3',5'-epimerase, comprising introducing into the genome of the cell a non-native promoter upstream of a gene encoding the at least one subunit of a multimeric GDP-mannose-3', 5'-epimerase. Such a non-native promoter can include, but is not limited to, other plant promoters. Genetic modification of host cells has been discussed in detail above. In other embodiments, other enzymes in the ascorbic acid biosynthetic pathway (e.g., phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-L-galactose pyrophosphorylase, L-galactose-1 -P-phosphatase, L- galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase) can be modified by introduction of a non-native promoter upstream of the gene encoding such enzymes.

Yet another related embodiment of the present invention relates to a method to increase ascorbic acid synthesis in a cell comprising a multimeric GDP-mannose-3'5'-epimerase, comprising genetically modifying the cell to increase the activity of the GDP-mannose-3', 5'-epimerase in the cell. Such a genetic modification can include, in one aspect, expressing a recombinant GDP- mannose-3', 5'-epimerase promoter in the cell upstream of the gene encoding the at least one subunit of a multimeric GDP-mannose-3', 5'-epimerase, wherein expression of the recombinant promoter increases the expression of GDP-mannose-3',5'-epimerase by the cell. In another aspect, such a genetic modification can include a modification to the endogenous GDP-mannose- S^δ'-epimerase that increases the activity of the epimerase. Again, in one aspect, other enzymes in the ascorbic acid biosynthetic pathway can be genetically modified in a similar manner to further increase ascorbic acid production in the host cell.

One aspect of these embodiments of the invention comprises growing a transgenic plant or plant part, or a culture of recombinant plant cells as described above, under conditions effective to increase ascorbic acid synthesis in the cells or plant. Another aspect of these embodiments of the invention comprises culturing a culture containing any of the recombinant host cells described above, wherein the host cell is a microbial cell, under conditions effective to increase ascorbic acid synthesis in the host cell.

In these methods of the present invention, a genetically modified microorganism as described in detail above is cultured or grown in a suitable medium, under conditions effective to produce ascorbic acid. An appropriate, or effective, medium refers to any medium in which a genetically modified microorganism of the present invention, when cultured, is capable of producing the desired product (e.g., ascorbic acid). Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources. Such a medium can also include appropriate salts, minerals, metals and other nutrients. Microorganisms of the present invention can be cultured in conventional fermentation bioreactors. The microorganisms can be cultured by any fermentation process which includes, but is not limited to, batch, fed-batch, cell recycle, and continuous fermentation. Preferred growth conditions for potential host microorganisms according to the present invention are well known in the art. The genetically modified microorganisms of the present invention are engineered to produce increased ascorbic acid through the modified activity of the GDP-mannose-3',δ'-epimerase according to the present invention, alone or in combination with other genetic modifications that the microbes may contain.

Ascorbic acid produced by the genetically modified microorganism can be recovered from the fermentation medium using conventional separation and purification techniques. For example, the fermentation medium can be filtered or centrifuged to remove microorganisms, cell debris and other particulate matter, and the ascorbic acid product can be recovered from the cell-free supernatant by conventional methods, such as, for example, ion exchange, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization and crystallization.

Intracellular ascorbic acid produced in accordance with the present invention can also be recovered and used in a variety of applications. For example, cells from the microorganisms can be lysed and the ascorbic acid which is released can be recovered by a variety of known techniques. Alternatively, intracellular ascorbic acid can be recovered by washing the cells to extract the ascorbic acid, such as through diafiltration.

A genetically modified plant is cultured in a fermentation medium or grown in a suitable medium such as soil. An appropriate, or effective, fermentation medium for recombinant plant cells is known in the art and generally includes similar components as for a suitable medium for the culture of microbial cells (e.g., assimilable carbon, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients). A suitable growth medium for higher plants includes any growth medium for plants, including, but not limited to, soil, sand, any other particulate media that support root growth (e.g. vermiculite, perlite, etc.) or Hydroponic culture, as well as suitable light, water and nutritional supplements which optimize the growth of the higher plant. The genetically modified plants of the present invention are engineered to produce increased ascorbic acid through the modified activity of the GDP-mannose-3',δ'-epimerase according to the present invention, alone or in combination with other genetic modifications that the plants may contain. Again, ascorbic acid produced by the plant may be recovered through purification processes which extract the compound from the plant. In a preferred embodiment, the ascorbic acid is recovered by harvesting the plant. In this embodiment, the plant can be consumed in its natural state or further processed into consumable products.

Any of the above-described methods can also be used to produce any intermediate product in the ascorbic acid biosynthetic pathway as discussed above.

Another embodiment of the present invention relates to an isolated antibody or antigen binding fragment that selectively binds to any of the multimeric GDP-mannose-3',δ'-epimerases as described previously herein, including SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 and SEQ ID NO:54. According to the present invention, the phrase "selectively binds to" refers to the ability of an antibody, antigen binding fragment or binding partner to preferentially bind to specified proteins (e.g., GDP-mannose-3',5'-epimerase). More specifically, the phrase "selectively binds" refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc. Isolated antibodies of the present invention can include serum containing such antibodies, or antibodies that have been purified to varying degrees. Whole antibodies of the present invention can be polyclonal or monoclonal. Alternatively, functional equivalents of whole antibodies, such as antigen binding fragments in which one or more antibody domains are truncated or absent (e.g., Fv, Fab, Fab', or F(ab)₂ fragments), as well as genetically-engineered antibodies or antigen binding fragments thereof, including single chain antibodies, camelid antibodies and derivatives, or antibodies that can bind to more than one epitope (e.g., bi-specific antibodies), or antibodies that can bind to one or more different antigens (e.g., bi- or multi-specific antibodies), may also be employed in the invention.

Generally, in the production of an antibody, a suitable experimental animal, such as, for example, but not limited to, a rabbit, a sheep, a hamster, a guinea pig, a mouse, a rat, or a chicken, is exposed to an antigen against which an antibody is desired. Typically, an animal is immunized with an effective amount of antigen that is injected into the animal. An effective amount of antigen refers to an amount needed to induce antibody production by the animal. The animal's immune system is then allowed to respond over a pre-determined period of time. The immunization process can be repeated until the immune system is found to be producing antibodies to the antigen. In order to obtain polyclonal antibodies specific for the antigen, serum is collected from the animal that contains the desired antibodies (or in the case of a chicken, antibody can be collected from the eggs). Such serum is useful as a reagent. Polyclonal antibodies can be further purified from the serum (or eggs) by, for example, treating the serum with ammonium sulfate.

Monoclonal antibodies may be produced according to the methodology of Kohler and Milstein (Nature 256:495-497, 1975). For example, B lymphocytes are recovered from the spleen (or any suitable tissue) of an immunized animal and then fused with myeloma cells to obtain a population of hybridoma cells capable of continual growth in suitable culture medium. Hybridomas producing the desired antibody are selected by testing the ability of the antibody produced by the hybridoma to bind to the desired antigen.

GDP-L-galactose is an intermediate product in the ascorbic acid biosynthesis, but can be an interesting compound on its own. Modulation of the production of this product as used herein can refer to the increase as well as the decrease of the synthesis or the product, and can be realized by any method known to the person skilled in the art, including, but not limited to adaptation of the promoter region of at least one gene encoding a subunit of a multimeric GDP- mannose-3', 5'-epimerase, recombinant expression of a GDP-mannose-3',5'-epimerase, or the use of antisense RNA.

Accordingly, a further aspect of the invention is the use of a multimeric GDP-mannose-3',5'- epimerase according to the invention for the in vitro synthesis of a compound selected from GDP- L-galactose, L-galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone, and most preferably, GDP-L-galactose. Indeed, GDP-L-galactose may be directly obtained from GDP-D- mannose, by enzymatic treatment with the enzyme according to the invention, or it may be obtained from other precursors that may be transformed directly or indirectly into GDP-D-mannose, which then can be transformed in GDP-L-galactose by the GDP-mannose-3',δ'-epimerase according to the invention. GDP-L-galactose may be used, amongst others, in the study of glycosylation of proteins. Still a further aspect of the invention is the use of a multimeric GDP-mannose-3',5¹- epimerase according to the invention as a selectable marker in eukaryotic cells. A preferred embodiment is the use as a selectable marker, whereby said eukaryotic cell is a yeast cell or a plant cell. Indeed, overexpression of GDP-mannose-3¹, δ'-epimerase results in tolerance against the toxic thiol oxidizing drug diamide. By protection against thiol oxidation and by increase of the ascorbic acid synthesis, overexpression of GDP-mannose-3',δ'-epimerase leads to an increase of the reductive capacity of the cell. Therefore, another aspect of the invention is the use of a multimeric GDP-mannose-3', δ'- epimerase to increase the reductive capacity of an eukaryotic cell. Preferably, said eukaryotic cell is a plant cell or a yeast cell. Increase of the reductive capacity, as used here, is expressed as an increase of the diamide tolerance of the transformed cell, compared with a non-transformed control.

The following examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention.

Examples The following Materials and Methods were used in Examples 1-3.

Reagents

Guanosine diphospho-D-[U-¹⁴C]mannose (specific activity 296 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). All compounds were of analytical grade. Guanosine diphospho-D-mannose, guanosine diphospho-L-fucose, adenosine diphospho-D- glucose, guanosine di- and mono-phosphates, L-ascorbic acid, dithiothreitol, guanosine 5'- diphosphate immobilized on cross-linked 4 % beaded agarose and Heparin-agarose resin were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO). NAD, NADH, NADP and NADPH were from Boehringer Mannheim GmbH (Mannheim, Germany).

DEAE-Sepharose Fast Flow, Sephacryl S-200 HR and Blue Sepharose CL-6B were from Amersham Pharmacia Biotech (Uppsala, Sweden). Hydroxylapatite Bio-Gel HTP was purchased from Bio-Rad Laboratories (Hercules, CA). HPLC-grade acetonitrile and methanol were from LabScan (Dublin, Ireland). Mark12 wide range protein standard was from Novex (San Diego, CA). Cells

Arabidopsis thaliana (L.) Heynh. ecotype Columbia cell suspension was grown on a rotary shaker (120 rpm) under continuous white light at 26°C in a Murashige and Skoog basal salts with minimal organics medium (Sigma-Aldrich Chemicals) supplemented with 3 % sucrose, 20 mM β- morpholino-ethanosulfonate, 2.7 μM naphthaleneacetic acid, 0.2 μM kinetin, and pH adjusted to 6.14. For subculturing, 80 ml aliquots from the mother cell suspension were transferred into 1 I Erlenmeyer flasks containing 320 ml of medium, every 7 days. For harvesting, 7-day-old subcultures were diluted 5 times up to 2.41 with a fresh medium and grown for subsequent 4 days in 6 liter Erlenmeyer flasks. Cells were collected by filtration on a fritted glass funnel, weighed and either used immediately for enzyme extraction or frozen in liquid nitrogen and stored at -80°C. Protein determination

Protein concentration was determined by the method of Bradford (1976, Anal. Biochem. 72:248-254) using bovine serum albumin as standard. Ascorbic acid determination L-Ascorbic acid content of Arabidopsis suspension=s cells was measured by reverse-phase HPLC with on-line UV detection at 243 nm, as described by Wolucka et al., 2001 , Anal. Biochem. 294:161-168, incorporated herein by reference in its entirety. GDP-mannose-3', 5'-epimerase assay The GDP-mannose-S'.δ'-epimerase activity was determined by the HPLC radio-method of

Wolucka et al., 2001 , ibid., using GDP-D-[U-¹⁴C] mannose as substrate. Briefly, the assay mixture consisted of 0.02 μCi of GDP-D-[U-¹⁴C] mannose (68 pmoles), 1 mM EDTA, 50 mM Tris-HCI buffer pH 7.7 and a GDP-mannose-3¹, δ'-epimerase preparation in a total volume of 20 μl. Samples were incubated at 26°C for 10 min. The reaction was stopped by adding 20 μl of cold 3 % metaphosphoric acid on ice. Samples were centrifuged for 3 min at 14000 rpm and supernatants

(30 μl) were immediately analyzed by HPLC with an on-line radioactivity detector. The amount of the reaction product formed was calculated by integration of the area of the 5.9 min GDP-L-

, [¹ C]galactose peak for measurement of GDP-mannose- 3',5'-epimerase activity. One unit of GDP- mannose-3',δ'-epimerase corresponds to an amount of enzyme which produces at 26°C 1 pmole of GDP-L-Gal in 1 min.

Purification of the GDP-mannose-3', 5'-epimerase

Step 1: Preparation of crude extract. 250 g (wet weight) of 4-days-old Arabidopsis suspension cells were ground with liquid nitrogen to a fine powder in a pre-cooled mortar and extracted with 2 volumes of 0.1 M Tris-HCI buffer pH 7.7 containing 5 mM DTT, 1 mM EDTA, 0.6 M sucrose, 1 mM phenylmethylsulphofluoride (PMSF) and 1 % (w/v) polyvinylpolypyrrolidone (buffer A). After centrifugation at 12000 rpm for 20 min at 4°C, the supernatant was removed and used as the crude extract.

Step 2: Ammonium sulphate precipitation. Solid ammonium sulphate (Sigma-Aldrich) was added to the crude enzyme extract up to 65 % of saturation, with a gentle mixing, on ice. After standing on ice for 30 min, the solution was centrifuged at 14 000 rpm for 30 min at 4°C. The ammonium sulphate saturation of the obtained supernatant was increased to 70 %, and the solution was centrifuged again, as described above. The resulting supernatant was discarded and the precipitate was dissolved in δO mM Tris-HCI buffer (pH 7.7) containing 1 mM EDTA, 0.5 mM DTT, 1 mM PMSF and 20 % glycerol (buffer B). The resulting 55 % to 70 % ammonium sulphate fraction was desalted on prepacked NAP-25 columns containing Sephadex G-25, according to the manufacturer's instructions (Amersham Pharmacia Biotech).

Step 3: DEAE-Sepharose anion-exchange FPLC. Fast protein liquid chromatography was performed on a FPLC system from Amersham Pharmacia Biotech. The 55 % to 70 % ammonium sulphate fraction was loaded on a DEAE-Sepharose column (1.2 x 18 cm) equilibrated with buffer B at a flow rate of 2 ml/min. After washing with buffer B, the epimerase activity was eluted with a 200-ml linear gradient from 0 to 200 mM NaCl in buffer B. 5-ml fractions were collected. Fractions containing epimerase activity were pooled and concentrated to 4 ml by ultrafiltration using a 10000 MW cut-off Vivacell (70 ml) concentrator (Sartorius, Stonehouse, UK).

Step 4: Sephacryl S-200 gel filtration FPLC. The concentrated pooled fractions from DEAE-Sepharose were applied to a Sephacryl S-200 column (1.6 x 94 cm) equilibrated with buffer B, at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected and the active fractions were pooled.

Step 5: Hydroxylapatite FPLC. Bio-Gel HT was suspended in 2 mM potassium phosphate buffer (pH 7.2) containing 0.5 mM DTT, 1 mM EDTA, 1 mM PMSF and 20 % glycerol (buffer C), and the slurry was used to pack a 1.2 x 9 cm column, which was then equilibrated with buffer C. The pooled gel-filtration fractions were applied onto the column at a flow rate of 0.5 ml/min and the column was washed with buffer C until the UV absorption at 280 nm was close to zero. The elution was carried out with 100 ml of a linear gradient from 2 to 500 mM potassium phosphate in buffer C and 2-ml fractions were collected. The active fractions were pooled and concentrated to 0.5 ml by using a Viva-Spin 4 ml concentrator with 10000 MW cut-off membrane (Vivascience, Binbrook, U.K.).

Step 6: Heparin-agarose cation-exchange chromatography. The concentrated pooled fractions from hydroxylapatite step were applied on a 3-ml Heparin-agarose column equilibrated with buffer B. The column was washed with 3 column volumes of buffer B and, subsequently, eluted with 3 column volumes of 0.5 M NaCl in buffer B. 2-ml fractions were collected. The epimerase activity was found in the non-adsorbed material; no enzyme activity could be detected in the salt eluate.

Step 7: GDP-agarose affinity chromatography. The non-adsorbed fraction from the

Heparin-agarose step was applied to a 2-ml column of GDP-agarose equilibrated with buffer B.

The column was washed with three column volumes of buffer B and eluted with three column volumes of either 0.5 M NaCl or 1 mM GDP in buffer B. 2-ml fractions were collected. The material non-adsorbed on GDP-agarose column contained the epimerase activity.

Step 8: Blue-Sepharose chromatography. The material non-adsorbed on GDP-agarose was applied at room temperature to a 3-ml Blue Sepharose CL-6B column equilibrated with buffer B. The column was washed with 3 column volumes of buffer B and, subsequently, eluted with 2 column volumes of 1 mM NAD in buffer B, followed by 3 column volumes of 0.5 M NaCl in buffer B. Fractions of 2 ml were collected. The active fractions from different elution steps (non-retained, 1 mM NAD and 0.5 M NaCl) were pooled separately and concentrated to 30 μl by ultrafiltration using 4-ml Viva-Spin concentrators.

A summary of the purification procedure is presented in Table 1. Determination of enzyme molecular mass

The molecular mass of the native GDP-mannose-3',δ'-epimerase was estimated by gel filtration on a Sephacryl S-200 column (1.6 x 94 cm) in buffer B, as described above, using the

4δ following molecular mass standards: thyroglobulin (670 kDa), bovine γ-globulin (168 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa) and vitamin B ₂ (1.35 kDa). Polyacrylamide gel electrophoresis

Proteins were separated by SDS-PAGE using 12.5 % mini-gels and the buffer system described by Laemmli (1970). Gels were stained with Coomassie Brilliant Blue R-250. Peptide preparation for mass spectrometry

For LCQ nanospray analysis, Coomassie-stained protein bands were excised out of gels, cut in 1 x 1 mm pieces and washed three times with 250 μl of 50 % acetonitrile in 200 mM ammonium bicarbonate (wash buffer) for 10 min per wash on a rocker table. The washings were discarded, the gel pieces were immersed in 100 μl of the wash buffer and 3 μl of 225 mM dithiothreitol were added. The reduction was performed at 50°C for 40 min, after which the DTT solution was replaced by 100 μl of 5δ mM iodoacetamide in 100 mM ammonium bicarbonate, and the samples were incubated for 30 min at room temperature in the dark. The supernatant was removed, the gel pieces were washed for 15 min with 2δ0 μl of the wash buffer and, subsequently, dried in a Speed-Vac concentrator. 1 μl of a 1 mg/ml stock solution of sequencing grade modified trypsin (Promega, Madison, Wl) was added to 69 μl of 40 mM ammonium bicarbonate solution containing 2 M urea, and 20 μl of the so obtained trypsin working solution were added to each sample. Samples were incubated for 16 h at 37°C, and the reaction was stopped by adding 1 μl of formic acid. Supernatants were saved and the obtained peptides were purified by solid-phase extraction on Zip-Tips (Millipore, Bedford, CA) according to the manufacturer procedure, except that a 0.01 % formic acid solution was used, instead of 0.1 % trifluoroacetic acid. Peptides were eluted from Zip-Tip with 6 μl of a methanol-isopropanol-0.01 % formic acid (5: 1: 3; v/v/v) solution, loaded into a nanospray needle (Protana, Odense, Denmark) and analysed immediately by LCQ- MS/MS. For MALDI-TOF analysis, the excised gel pieces were washed three times with water, followed by two washes for 15 min with 100 μl of 50 % acetonitrile in water and a subsequent drying in Speed-Vac centrifugal vacuum concentrator. The dried gel pieces were re-hydrated in 10 μl of 50 mM freshly prepared ammonium bicarbonate containing a total of 0.05 μg of sequencing grade modified trypsin (Promega) for about 10 min, after which the remaining supernatant was removed. δO mM ammonium bicarbonate solution was then added (δO to 100 μl) until the gel pieces were completely submerged, and the digestion proceeded overnight at 37°C. The supernatant containing the generated peptides was removed and acidified by adding 1 μl of formic acid. The peptides were concentrated on a small amount of added Poros 50 R2 beads (Boehringer Mannheim GmbH, Mannheim, Germany) and either stored at -20°C or immediately used for MALDI-MS peptide mass mapping.

Nano-electrospray ionisation tandem mass spectrometry of tryptic peptides Peptide analyses were performed on a Finnigan LCQ quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped with a nano-electrospray source kit. Off-line nanospray ionisation with no sheath gas assistance was carried out using disposable capillary needles (Protana, Odense, Denmark) and a microinjector air pressure device. The LCQ was operated manually in the Tune Plus window, as described by Wilm and Mann (1996). The maximum ion injection time was 500 ms, and up to 1000 ms for less abundant ions. The capillary temperature was hold at 190°C and flow-rates ranged from 20 to 50 nl/min. Peptides were analysed by acquiring first a full mass scan (m/z 400 to 2000), followed by a zoom scan of the selected peptide ion region, and, finally, an MS/MS scan of a selected double-charged ion (typically from 10 to 60 microscans per ion) using a relative collision energy of 15-30 % with an isolation width 2.0-3.0, depending on the m/z value of a fragmented ion. The product ion spectra were interpreted manually. This approach allowed in most cases the unambiguous identification of stretches of amino acids (at least 8 were required for a significant match) if not the complete peptide sequence. The obtained peptide sequences were submitted to database searching for protein identification using NCBI/BLAST tools (e.g., described in Altschul, S.F., Madden, T.L., Schaaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI- BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389- 3402, incorporated herein by reference in its entirety). MALDI-TOF peptide-mass mapping All MALDI-MS measurements were performed on a Bruker Reflex III MALDI-TOF-MS

(Bruker Daltonik GmbH, Bremen, Germany) operating in reflectron mode (Gevaert et al., 1997, Electrophoresis 18:2950-2960). Prior to each analysis, the mass spectrometer was externally linearly calibrated using the calculated masses of two synthetic peptides, spotted on the MALDI target as close as possible to the real sample. Typically 50 to 100 laser shots were accumulated for each measurement. The obtained peptide mass fingerprints were analyzed by using the Mascot algorithm (Electrophoresis, 20(18):3551-67 (1999)) to identify proteins.

Example 1

The following example describes the purification of the GDP-mannose-3¹, δ¹ -epimerase. The GDP-mannose-3', δ'-epimerase was purified from 2δ0 g (fresh weight) of 4-day-old A. thaliana suspension's cells, as described in Materials and Methods above (Table 1). In the preliminary experiments, the level of total L-ascorbic acid (L-ascorbic acid + dehydro-L-ascorbic acid) and the capacity of A. thaliana cells to convert the exogenous ¹⁴C-mannose into the total L- AA pool were both observed to decrease upon aging of the cell culture. Significantly, this decrease correlated with a drop of the specific activity of GDP-mannose-3¹, 5¹ -epimerase (Fig. 2). Since on day 4 of the Arabidopsis cell suspension culture, the capacity of cells to synthesize L-AA, their L- AA content, as well as the specific activity of GDP-mannose-3',5' -epimerase were high, cells were collected at that specific moment. For protein extraction, cells were crushed in liquid nitrogen to avoid any overheating. Homogenization or sonication, both resulted in a dramatic loss of the activity probably because of thermolability of the epimerase (see below). A high osmolarity buffer containing 0.6 M sucrose was used for cell extraction to prevent disruption of cellular organelles, such as mitochondria and chloroplasts. The intact organelles were removed by a subsequent centrifugation, and the obtained crude extract, enriched in the cytoplasmic fraction, contained all the epimerase activity. Omission of sucrose from the extraction buffer resulted in a release of mitochondrial and chloroplastic proteins from broken organelles and, consequently, in an important contamination of the epimerase preparation. This was especially true for the mitochondrial citrate synthase, which, as determined by mass spectrometry, co-purified with the epimerase throughout the whole purification procedure and, because of its abundance, masked the presence of the latter enzyme.

The epimerase activity could not be measured in crude extracts because of the presence of other enzymatic activities utilizing GDP-mannose as substrate, such as GDP-mannose dehydratase and pyrophosphatase (Wolucka et al., 2001 , supra). The hydrolytic activity could be inhibited in the 4-day-old cell extracts by addition of EDTA; however, in older cells, an apparently different, EDTA-insensitive pyrophosphatase activity was present, thus rendering the epimerase determination difficult. GDP-mannose dehydratase activity was separated from the epimerase by the ammonium sulphate fractionation: dehydratase precipitated at 65 % salt saturation, whereas the epimerase was confined to the 5δ % to 70 % ammonium sulphate fraction (Table 1).

The ammonium sulphate fraction was separated further by DEAE-Sepharose anion- exchange FPLC. As shown in Fig. 3A, all epimerase activity bound to DEAE-Sepharose and was eluted as a broad peak (ten fractions) at about 75 mM NaCl. Since the majority of other proteins appeared as sharp peaks (one to three fractions) as judged on the basis of SDS-PAGE analysis of DEAE fractions, the epimerase behavior may suggest the presence of some post-translational modifications within the enzyme molecule as, for example, phosphorylations.

The pooled epimerase fractions from DEAE-Sepharose were concentrated by ultrafiltration and applied to Sephacryl S-200 gel filtration chromatography (Fig. 3B). The epimerase activity eluted in a symmetrical peak corresponding to a 84 kD protein (see below). The gel filtration step resulted, however, in a ten-fold decrease in the enzymatic activity, probably due to the loss of a cofactor from the enzymatic complex. This is consistent with the fact that chromatography of the epimerase on reverse-phase columns, such as Phenyl-Sepharose or RP-Resource, led to a complete loss of the enzymatic activity.

Further enzyme purification was achieved on a hydroxylapatite column (Fig. 3C). The epimerase bound tightly to hydroxylapatite and was eluted at about 40 mM potassium phosphate buffer. The activity of the pooled hydroxylapatite fractions was relatively stable; at this stage the enzyme could be stored for about 3 weeks at 4°C without any significant loss of activity. The two next steps in the epimerase purification, namely a cation-exchange chromatography on Heparin-Agarose, followed by an affinity chromatography on GDP-agarose (Table 1 ) were included to remove some persisting and abundant contaminants identified by nano- ES MS/MS of in-gel digests of the SDS-PAGE epimerase fractions: 10-formyltetrahydrofolate synthetase (Nouretal., 1991 , J. Biol. Chem. 266: 18363-18369) and adenylosuccinate synthetase, respectively. Although GDP is a strong inhibitor of GDP-mannose-3',5'-epimerase (see below), the enzyme did not bind to GDP-Agarose resin, thus suggesting that the GDP-binding site is not located at the surface but rather buried in the epimerase structure.

The non-adsorbed material from GDP-Agarose was applied onto a dye-affinity column of Blue-Sepharose. About 50 % of the epimerase activity of the GDP-agarose non-adsorbed material bound to the Blue-Sepharose column (Table 1). This binding was dependent on temperature; the enzyme bound to the resin at the room temperature, however it was not adsorbed if the chromatography was performed at 4°C. Importantly, re-chromatography of the non-adsorbed epimerase fraction on a Blue-Sepharose column at room temperature led to a complete loss of the activity. Specific elution of Blue-Sepharose with 1 mM NAD resulted in the recovery of a half of the bound epimerase activity (about 25 % of the total activity). The rest of the bound enzyme activity was recovered by a less specific elution with 0.5 M NaCl (Table 1). It is worth noticing that the GDP-mannose-3¹, 5'-epimerase could not be eluted from Blue-Sepharose column with 1 mM GDP- mannose substrate, although the enzyme shows a high affinity for the substrate (K_m = 4.4 μM; see below) and has no requirement for NAD.

The NAD-eluted Blue-Sepharose fraction contained approximately 20 μg of protein in total and showed the highest specific activity of the epimerase (13600 U/mg of protein) (Table 1). However, due to the instability of the enzyme, the obtained values for the purification fold and the enzyme yield were low (Table 1). The Blue-Sepharose fractions (non-adsorbed, NAD- and NaCI- eluate) of the epimerase were extremely unstable: after an overnight period, only 5 % of the original activity could be detected in each of the fractions, thus preventing any further purification steps.

The NAD-eluate from Blue-Sepharose showed four protein bands on the SDS-PAGE (Fig. 4): the slowest-migrating 60 kDa band (band 1), a faint 55 kDa band (band 2) and two discrete bands of 46 and 43 kDa (band 3 and 4, respectively). In-gel tryptic digestion of bands 1 and 2, followed by the MALDI-TOF peptide mass mapping allowed an unambiguous identification of the proteins as betaine aldehyde dehydrogenase (band 1) and glutathione synthase (band 2). However, no significant match could be found for bands 3 and 4. Because the molecular mass of the native epimerase was estimated to be approximately 84 kDa and no protein bands of high molecular mass (> 60 kDa) could be observed on the gel, it was deduced therefore that the enzyme has a dimeric structure with a subunit of about 42 kDa. Thus, the two discrete bands of 46 and 43 kDa, respectively, represent the epimerase subunit(s). Example 2

The following example describes the identification of GDP-mannose-3',5'-epimerase. The 46 and 43 kDa protein bands of the NAD-eluted fraction from Blue-Sepharose (bands 3 and 4; Fig. 4) were subjected to the in-gel tryptic digestion, followed by the nano-electrospray tandem mass spectrometry analysis, as described in Materials and Methods. The MS spectrum of both bands revealed the presence of an abundant doubly charged peptide ion at m/z 786.9, the product ion spectrum of which is shown in Fig. 5. The interpretation of the MS/MS spectrum gave the putative sequence TGAGGFXASHXAR (SEQ ID NO:47), where X denotes either a leucine or an isoleucine and _ denotes an unknown amino acid. Subsequently, all of the 4 possible combinations of the above-mentioned sequence containing either exclusively leucine, isoleucine or permutations of both, were submitted to a database search using BLAST program and different general and Arabidopsis-speclfic protein databases. No perfect match could be found, although some of the submitted combinations showed a significant similarity to several sugar-epimerases. Finally, a search using the DNA database of Arabidopsis revealed that only one of the predicted sequences, namely TGAGGFIASHIAR (SEQ ID NO:48), matches perfectly with a hypothetical

42.8 kDa protein of A. thaliana described as epimerase/dehydratase-like protein (BAC clone having the Accession No. EMBL:AF272706; Fig. δ and 6). In order to confirm further the identity of the GDP-mannose-3', δ'-epimerase, the sequence of the hypothetical 42.8 kDa protein of A. thaliana was subjected to an in-silico tryptic digestion using the MS-Digest algorithm (Clauser K. R., Baker P. R. and Burlingame A. L., "Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.", 1999, Analytical Chemistry 71(14):2871-), and the calculated masses of the theoretical peptides were, subsequently, compared with those observed in the MALDI-TOF analysis of tryptic digests derived from the 46 and 43 kDa protein bands (Fig. 4; Table 2). As shown in Table 2, eight out of nine measured peptide masses for the 46 kDa protein (band 3; Fig. 4) digest corresponded exactly (mass difference < 20 ppm) to the calculated tryptic peptide masses of the hypothetical 42.8 kDa protein of A. thaliana. Similarly, fourteen out of the total sixteen MALDI-TOF peptides derived from the 43 kDa protein (band 4; Fig.4) could be identified in the theoretical digest of the predicted 42.8 kDa epimerase/dehydratase-like protein (Table 2). Interestingly, the m/z 785.9 peptide ion identified by the nano-ES MS/MS (Fig. 5) was also the most abundant ion in the MALDI TOF analysis (m/z 1570.76; Table 2). The tryptic peptides derived from the 46 and 43 kDa proteins and observed in the MALDI-TOF experiment cover 46% of the epimerase sequence (Fig. 6 and Table 2). Obviously, the two discrete epimerase bands of 46 and 43 kDa represent different molecular forms of the same protein. Analysis of the C-terminal peptides observed in the MALDI-TOF of the epimerase bands revealed that the most distal C-terminal peptide present in the 46 kDa band digest (V₃₅₈VGTQAPVQLGSLR₃₇₁, SEQ ID NO:3) was absent from the 43 kDa band-derived sample (Table 2). Instead, the latter showed the presence of two related C-terminal peptides (l33iTYFWIK₃₃ , SEQ ID NO: 12; and l₃₃₁TYFWIKEQIEKEK₃₄₄, SEQ ID NO:20) located more upstream within the protein sequence (Table 2 and Fig. 6). This suggests that the 43 kDa epimerase band represents the product of a proteolytic cleavage at the C-terminus of the 46 kDa form. The loss of about 30 amino acids from the C-terminus would account for an approximately 3 kDa shift in the molecular mass of GDP-mannose-3',5'-epimerase and, consequently, the appearance of the two discrete enzyme bands on the SDS-PAGE (bands 3 and 4; Fig 4).

Therefore, it was concluded that the GDP-mannose-3', δ'-epimerase of A. thaliana is a homodimer composed of two identical 42.8 kDa subunits, the amino acid sequence of which is depicted in Fig. 6, and which is represented herein by SEQ ID NO:2. The nucleic acid sequence encoding SEQ ID NO:2 is represented herein by SEQ ID NO:1. The corresponding gene is located on chromosome 5 of A. thaliana, it contains 5 introns and its GC content is 43.6%. The predicted protein is annotated by the Arabidopsis database as being an epimerase/dehydratase-like protein of 377 amino acids (full length). The polypeptide belongs to alpha-beta structural class and has neither predicted transmembrane domains nor signal peptide sequence. Its calculated molecular mass is 42759 Da and the isoelectric point is 5.85. The GDP-mannose-3',5'-epimerase contains an NAD-dependent epimerase/dehydratase domain (PFAM domain PF01370), it belongs to the UDP-glucose4'-epimerase superfamily and is similar to dTDP-glucose4',6'-dehydratase homolog D18 of A. thaliana.

Example 3

The following example describes the characterization of the GDP-mannose-3', δ'- epimerase.

Enzyme stability. The epimerase activity in crude extracts and the ammonium sulphate fraction was quite stable and could be stored at a frozen state for at least two weeks. Purification resulted in a considerable loss of the enzymatic activity. The most stable epimerase preparations, which could be stored for several weeks at 4°C, were obtained after the hydroxylapatite step.

Molecular mass determination. Based on the mobility of the native enzyme on Sephacryl S- 200 gel filtration column as compared with a number of known proteins, the molecular mass of the native enzyme was estimated to be 84 kDa (Fig. 7A). The apparent molecular mass of the denaturated enzyme subunit was about 46 kDa, as determined by SDS-PAGE (Fig. 7B).

Kinetic properties and enzyme effectors. Enzyme activity was linear with respect to time and enzyme concentration (Wolucka et al., 2001 , supra). The apparent equilibrium constant for the formation of GDP-L-galactose (k₊₁ in reaction 4; Fig. 1), K=eq = [GDP- L-Gal]/ [GDP-D-Man], was 0.1 δ. With GDP-D-mannose as the substrate, the typical Michaelis-Menten kinetics was observed; the K_m value for GDP-mannose was determined from the Lineweaver-Burk plot (Fig. 8) to be 4.4 μM.

61 Guanosine nucleotides, such as GDP, GMP, GDP-D-glucose, GDP-L-fucose, were efficient inhibitors of the GDP-mannose-3',δ'-epimerase (Table 3). Fig. 8 shows a competitive inhibition of the epimerase by GDP. The K, value for GDP was 0.7 μM, as determined from a secondary replot of the slopes of Lineweaver-Burk plots for various inhibitor concentrations (Fig. 8; inset). Comparison of the GDP-mannose-3',δ'-epimerase activity at the presence of other purine nucleoside-pyrophospho-hexoses revealed important differences in the degree of inhibition (Fig. 9). 60% inhibition was observed at the presence of either 10 μM GDP-D-glucose or 70 μM GDP-L- fucose, whereas adenosine-pyrophospho-D-glucose had no inhibitory effect on GDP-mannose epimerase (Fig. 9; Table 3). The effect of the oxidized and reduced forms of nicotinamide adenine dinucleotides was tested. Although the GDP-mannose-3', δ'-epimerase did not require any exogenous NAD⁺ for the activity, the partially purified epimerase fraction from hydroxylapatite column showed some activation at the presence of 1 mM either NAD⁺ or NADP⁺ (145 % and 110 % of the control, respectively) (Table 3). The same level of activation was observed with the NAD⁺ concentrations as low as 1 μM. At the presence of the reduced forms of the nicotinamide adenine dinucleotides, NADH and NADPH, 22 % and 12 % of the inhibition, respectively, were observed (Table 3). This suggests that the epimerase molecule probably contains a firmly bound NAD⁺ cofactor, which may be lost during purification of the enzyme. The activity of, at least some of these cof actor-devoid enzyme molecules could be then restored after binding the exogenous NAD⁺, thus resulting in the observed activation effect. On the other hand, a part of the enzyme molecules with more loosely bound NAD⁺ might be susceptible to NADH inhibition due to an enzyme-bound NAD/exogenous NADH exchange. This would be consistent with the sequence data of the NAD-dependent epimerase/dehydratase-like protein, a member of the UDP-glucose 4-epimerase superfamily (Fig. 6). However, in the case of UDP-glucose 4-epimerase, NADP is not able to replace NAD⁺ in the enzyme-cofactor complex and its reduced form (NADPH), apparently, does not inhibit the enzymatic activity (Gabriel et al., 1975, Enzymology 2:85-135). Therefore, a possibility that the GDP-mannose-3', δ'-epimerase could contain a distinct effector site for nicotinamide adenine dinucleotides allowing to sense the redox state of the cell, cannot be excluded.

Among the tested sugar derivatives, 1 mM lactones such as L-ascorbic acid and L- galactono-1 ,4-lactone, inhibited the partially purified epimerase by about 15 %, whereas 1 mM D- mannose 1 -phosphate had a slightly activating effect (124 % of the control) (Table 3).

Effect of chemical agents. Chelating and reducing agents (EDTA and β-mercaptoethanol, respectively) had no effect on the epimerase activity (Table 3). The enzyme was also relatively resistant to detergents; at the presence of 1 % Triton X-100 more than 50% of the activity could be still detected (Table 3). At the presence of iodoacetamide the epimerase activity was reduced by 30 % (Table 3), which indicates that cysteine or histidine residues could be involved in the enzyme catalysis. Metal ions requirements. GDP-mannose 3', δ'-epimerase showed no requirement for metal ions; however, certain metal ions, such as Zn²⁺, Co²⁺, Ni²⁺ and Fe³⁺, completely abolished the enzyme activity (Table 4). A similar effect was observed with GDP-mannose-3',δ'-epimerase of Chlorella pyrenoidosa (Hebda et al., 1979, Arch. Biochem. Biophys. 194:496-502). pH and temperature optima. The epimerase showed a broad pH optimum between pH 7.0 and 9.0, with a maximum of the activity at pH 7.δ (Fig. 10A). The enzyme proved to be thermolabile; at 37°C, only 20 % of the control activity could be detected (Fig. 10B). Pre-incubation at 40°C for 1 δ min resulted in the complete inactivation of the enzyme. The optimal temperature for the epimerase reaction was 26°C; about 6δ%, 80% and 20% of the control activity could be detected at 1δ-20°C, 30°C and 37°C respectively.

Table 1. Purification of GDP-Mannose-3', δ'-epimerase from A. thaliana cell suspension.

^a B Not determined. B Epimerase activity measured at the presence of 1 mM NAD

63 Table 2. Comparison of peptide ions observed in the matrix-assisted laser desorption/ionisation-time-of-flight analysis of the in-gel tryptic dige the 46 kDa and 43 kDa GDP-mannose S'.δ'-epimerase bands (see: Fig. 4) with those predicted by MS-Digest algorithm (Clauser et al., 1 supra) for the hypothetical epimerase/dehydratase-like protein of A. thaliana (EMBL Accession No.: AF272706). The underlined peptide identified also in the nanoelectrospray tandem mass spectrometry analysis of the epimerase bands (see: Fig. δ).

n 5-

Table 3. Effect of various compounds on GDP-Mannose-3',5'-epimerase activity.

Compound Concentration Relative activity (% of control)

No addition B 100

GDP mM 0

GMP 1 M 0

GDP-D-glucose 1 mM 0

GDP-L-fucose 1 M 37

ADP-D-glucose 1 mM 96

NADH 1 mM 78

NADPH 1 mM 88

NADP 1 mM 110

NAD 1 mM 145

L-ascorbic acid 1 mM 84

L-galactono-γ-lactone 1 mM 85

L-galactose 1 M 94

Man 6-P 1 mM 102

Man 1-P 1 mM 124

EDTA 2 mM 98 β-mercaptoethanol 100 mM 86 iodoacetamide 100 mM 69

Triton X-100 1 % 52

Table 4. Effect of metal ions on GDP-mannose-3',5"-epimerase activity.

Metal ion (5 mM) Relative activity {% of control)

None 100

Mg⁺² 112

Ca⁺² 98

Mn⁺² 77

Zn⁺² 0

Co⁺² 0

Ni⁺² 0

Fe⁺³ 0

Example 4 The following example describes the identification of additional GDP-mannos-3',δ'- epimerases from plants.

Several public DNA sequence databases were scanned for sequences that exhibited similarity to the Arabidopsis thaliana gene now known to code for GDP-mannose-3¹, δ'-epimerase

(three NCBI entries were found for the A. thaliana gene, as listed in Table δ below). Sequences that showed high similarity at both the DNA and protein level were identified in various plant δδ databases. The comparison of sequence identity between each of the identified sequences and the A. Thaliana GDP-mannose-3', δ'-epimerase sequences (SEQ ID NO:1 and SEQ ID NO:2) is shown in Table δ.

To identify a putative GDP-mannose-3'δ¹ -epimerase from rice (Oryza sativa), the National Center for Biotechnology Information (NCBI) database was searched using the BLAST program (Altschul et al., 1997, supra) set on default parameters. The protein sequence (SEQ ID NO:2) encoded by the entire Arabidopsis thaliana GDP-mannose-3¹, δ^epimerase open reading frame sequence (SEQ ID NO:1) was used as the query. Database Accession No. AC016780 was identified from this search as exhibiting a high degree of similarity to the Arabidopsis epimerase protein (AC016780 contains a genomic sequence for Oryza sativa Qaponica cultivar-group), cultivar Nipponbare, clone OSJNBa0061 K21 from chromosome 10). After removal of introns, the Oryza sativa gene comprises a sequence that contains an open reading frame of 1137 basepairs, including an ATG start and TGA stop codons. The nucleic acid sequence encoding the Oryza sativa GDP-mannose-S'δ'-epimerase is represented herein by SEQ ID NO:2δ. Alignment of this DNA sequence with the Arabidopsis sequence (SEQ ID NO:1) showed 79.8% (i.e., about 80%) identity over the entire lengths of their open reading frames. SEQ ID NO:2δ encodes an amino acid sequence of 378 amino acids represented herein by SEQ ID NO:26. SEQ ID NO:26 showed 91% identity to the Arabidopsis thaliana epimerase (SEQ ID NO:2) over the entire length of the sequence. GenBank Accession No. AC016780 was initially submitted to GenBank as Accession No.

6642630 on December 28, 1999, but this submission was only a working draft nucleic acid sequence without annotation. On April δ, 2002, GenBank Accession No. AC016780 was submitted as a fully annotated sequence of 112721 bp, showing open reading frames, intron/exon junctions, peptide sequences derived from the nucleotide sequences, homology analysis, and other annotations. The coding region for the GDP-mannose-3', δ'-epimerase was only listed as a putative epimerase/dehydratase in a broad general term because of its sequence homology to GDP-fuscose synthetase (including GDP-mannose-4-keto-6-D epimerase; GDP-4-keto-6-L- galactose reductase). The sequence also contains the following pfam domains: epimerase NAD dependent epimerase/dehydratase family, e-value=2e^"16; Idh lactate/malate dehydrogenase, NAD binding domain e-value=0.00073. However, neither the specific function of the epimerase (i.e., GDP-mannose-3',δ'-epimerase), nor the association of this epimerase with the ascorbic acid pathway were recognized or proposed in the GenBank submission.

To identify a putative GDP-mannose-3'δ'-epimerase from tomato (Lycopersicon esculentum), The Institute for Genomic Research (TIGR) Tomato EST Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC94184 was identified from this search, and exhibited similarity to the Arabidopsis gene over the much of the Arabidopsis

66 open reading frame. The Lycopersicon esculentum cDNA represented in TC94184 included putative ATG start and TAA stop codons, constituting an open reading frame of 942 basepairs (SEQ ID NO:63), encoding a putative protein of 313 amino acids (SEQ ID NO:δ4). SEQ ID NO:53 is 80.4% identical to the Arabidopsis sequence (SEQ ID NO:1) over the full length of the open reading frame for L. esculentum (which is possibly truncated, as discussed below). SEQ ID NO:64 is 86.6% identical to SEQ ID NO:2 over the entire length of the L esculentum sequence (again, possibly truncated).

Upon further examination of this L. esculentum TIGR sequence, it was determined that there was a possible error in the nucleotide sequence resulting in a frame shift and therefore, it was hypothesized that the TC94184 encoded a truncated protein. To investigate this hypothesis, a single nucleotide (C) was deleted from position 914 of the TC94184 sequence. This deletion causes the open reading frame to be extended and increases the length of the L. esculentum sequence that is homologous to the Arabidopsis thaliana sequence. The protein encoded by this modified nucleic acid sequence is more similar to the Arabidopsis epimerase both in terms of length and in amino acid identity, although this sequence has not been confirmed. Therefore, the nucleic acid sequence encoding the modified Lycopersicon esculentum GDP-mannose-3',δ'- epimerase is represented herein as SEQ ID NO:27, and is an apparent complete open reading frame of about 1131 basepairs. Alignment of SEQ ID NO:27 with the Arabidopsis sequence (SEQ ID NO:1) showed 80.4% identity over the entire lengths of their open reading frames. SEQ ID NO:27 encodes an amino acid sequence of 376 amino acids represented herein as SEQ ID

NO:28. SEQ ID NO:28 is 88.6% identical to SEQ ID NO:2 over the entire length of the sequence.

To identify a putative GDP-mannose-3'δ'-epimerase from corn (Zea Mays), the National

Center for Biotechnology Information (NCBI) database was searched using the BLAST program

(Altschul et al., 997, supra) set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. AY104279 was identified from this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The Zea Mays cDNA included putative ATG start and TAA stop codons, constituting an open reading frame of 1143 basepairs. The nucleic acid sequence encoding the Zea Mays GDP-mannose-3',δ'-epimerase is represented herein as SEQ ID NO:29. Alignment of SEQ ID NO:29 with the Arabidopsis sequence (SEQ ID NO:1) showed 78.7% identity over the entire lengths of their open reading frames. SEQ ID NO:29 encodes an amino acid sequence of 380 amino acids represented herein as SEQ ID NO:30. SEQ ID NO:30 is 89.9% identical to SEQ ID NO:2 over the entire length of the sequence.

To identify a putative GDP-mannose-3'δ'-epimerase from ice plant (Mesembryanthemum crystallinum), The Institute for Genomic Research (TIGR) Ice Plant Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC4342 was identified from

67 this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The M. crystallinum cDNA included putative ATG start and TGA stop codons, constituting an open reading frame of 1134 basepairs. The nucleic acid sequence encoding the M. crystallinum GDP-mannose-3',δ'-epimerase is represented herein as SEQ ID NO:31. Alignment of SEQ ID NO:31 with the Arabidopsis sequence (SEQ ID NO:1) showed 82.3% identity over the entire lengths of their open reading frames. SEQ ID NO:31 encodes an amino acid sequence of 377 amino acids represented herein as SEQ ID NO:32. SEQ ID NO:32 is 91.6% identical to SEQ ID NO:2 over the entire length of the sequence.

To identify a putative GDP-mannose-3'δ'-epimerase from soybean (Glycine max), The Institute for Genomic Research (TIGR) Soybean Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC99477 was identified from this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The Glycine max cDNA included putative ATG start and TGA stop codons, constituting an open reading frame of 1131 basepairs. The nucleic acid sequence encoding the Glycine max GDP-mannose- 3',δ'-epimerase is represented herein as SEQ ID NO:33. Alignment of SEQ ID NO:33 with the Arabidopsis sequence (SEQ ID NO:1) showed 81.6% identity over the entire lengths of their open reading frames. SEQ ID NO:33 encodes an amino acid sequence of 376 amino acids represented herein as SEQ ID NO:34. SEQ ID NO:34 is 92.6% identical to SEQ ID NO:2 over the entire length of the sequence.

To identify a putative GDP-mannose-3'δ'-epimerase from potato (Solanum tuberosum), The Institute for Genomic Research (TIGR) Potato Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC29622 was identified from this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The Solanum tuberosum cDNA included putative ATG start and TAA stop codons, constituting an open reading frame of 1131 basepairs. The nucleic acid sequence encoding the Solanum tuberosum GDP- mannose-3',6¹ -epimerase is represented herein as SEQ ID NO:3δ. Alignment of SEQ ID NO:3δ with the Arabidopsis sequence (SEQ ID NO:1) showed 80% identity over the entire lengths of their open reading frames. SEQ ID NO:3δ encodes an amino acid sequence of 376 amino acids represented herein as SEQ ID NO:36. SEQ ID NO:36 is 89.9% identical to SEQ ID NO:2 over the entire length of the sequence.

To identify a putative GDP-mannose-3'δ'-epimerase from Medicago (Medicago truncatula), The Institute for Genomic Research (TIGR) Medicago Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC43478 was identified from this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The

68 Medicago truncatula cDNA included putative ATG start and TAA stop codons, constituting an open reading frame of 1143 basepairs. The nucleic acid sequence encoding the Medicago truncatula GDP-mannose-S'^-epimerase is represented herein as SEQ ID NO:37. Alignment of SEQ ID NO:37 with the Arabidopsis sequence (SEQ ID NO:1) showed 81.3% identity over the entire lengths of their open reading frames. SEQ ID NO:37 encodes an amino acid sequence of 380 amino acids represented herein as SEQ ID NO:38. SEQ ID NO:38 is 91.3% identical to SEQ ID NO:2 over the entire length of the sequence.

To identify a putative GDP-mannose-3'δ'-epimerase from sorghum (Sorghum bicolor), The Institute for Genomic Research (TIGR) Sorghum Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC34324 was identified from this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The Sorghum bicolor cDNA included putative ATG start and TGA stop codons, constituting an open reading frame of 1143 basepairs. The nucleic acid sequence encoding the Sorghum bicolor GDP- mannose-S'jδ'-epimerase is represented herein as SEQ ID NO:39. Alignment of SEQ ID NO:39 with the Arabidopsis sequence (SEQ ID NO:1) showed 78.8% identity over the entire lengths of their open reading frames. SEQ ID NO:39 encodes an amino acid sequence of 380 amino acids represented herein as SEQ ID NO:40. SEQ ID NO:40 is 90.2% identical to SEQ ID NO:2 over the entire length of the sequence. To identify a putative GDP-mannose-3'δ'-epimerase from wheat (Triticum aestivum), The

Institute for Genomic Research (TIGR) Wheat Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC20664 was identified from this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The Triticum aestivum cDNA included putative ATG start and TAA stop codons, constituting an open reading frame of 1137 basepairs. The nucleic acid sequence encoding the Triticum aestivum GDP- mannose-3',δ'-epimerase is represented herein as SEQ ID NO:41. Alignment of SEQ ID NO:41 with the Arabidopsis sequence (SEQ ID NO:1 ) showed 78% identity over the entire lengths of their open reading frames. SEQ ID NO:41 encodes an amino acid sequence of 378 amino acids represented herein as SEQ ID NO:42. SEQ ID NO:42 is 89.7% identical to SEQ ID NO:2 over the entire length of the sequence.

To identify a putative GDP-mannose-3'δ'-epimerase from barley (Hordeum vulgare), The Institute for Genomic Research (TIGR) Barley Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC16791 was identified from this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The Hordeum vulgare cDNA included putative ATG start and TAA stop codons, constituting an open reading

69 frame of 1137 basepairs. The nucleic acid sequence encoding the Hordeum vulgare GDP- mannose-S'^'-epimerase is represented herein as SEQ ID NO:43. Alignment of SEQ ID NO:43 with the Arabidopsis sequence (SEQ ID NO:1) showed 77.9% identity over the entire lengths of their open reading frames. SEQ ID NO:43 encodes an amino acid sequence of 378 amino acids represented herein as SEQ ID NO:44. SEQ ID NO:44 is 89.4% identical to SEQ ID NO:2 over the entire length of the sequence.

To identify a putative GDP-mannose-3'δ'-epimerase from lotus (Lotus japonicus), The Institute for Genomic Research (TIGR) Lotus japonicus Database was searched using the BLAST program set on default parameters. Again, the entire Arabidopsis epimerase gene sequence was used as the query. The entry having Accession No. TC604 was identified from this search, and exhibited similarity to the Arabidopsis gene over the entire length of the open reading frame. The Lotus japonicus cDNA included a putative ATG start codon, and appears to be a partial reading frame of 1002 basepairs that is estimated to be about 90% complete by comparison to the Arabidopsis open reading frame. The nucleic acid sequence encoding the partial Lotus japonicus GDP-mannose-3', δ'-epimerase is represented herein as SEQ ID NO:4δ. Alignment of SEQ ID NO:4δ with the Arabidopsis sequence (SEQ ID NO: 1 ) showed 82.4% identity over the entire length of the partial open reading frame from the Lotus sequence. SEQ ID NO:4δ encodes an amino acid sequence of 334 amino acids represented herein as SEQ ID NO:46. SEQ ID NO:46 is 92.8% identical to SEQ ID NO:2 over the entire length of the sequence.

Table 5. Identification & Comparison GDP-mannose-3',5' -epimerases from plants

Includes stop codon Example δ

The following example describes the isolation, cloning and expression of additional GDP- mannose-3',δ'-epimerases. Construction of cDNA encoding GDP-mannose-3',5' -epimerase from various plants. mRNA Isolation

Total RNA can be extracted from plant tissue using a variety of methods. For example, general methods for extracting total RNA are described in Maniatis et al (Maniatis, T., Frisch, E.F., and Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor, New York). In addition, kits are available from several companies designed for the isolation of total RNA, including the RNAgents Total RNA Isolation System from Promega Corporation (Madison, Wl). PolyA+ mRNA can then be recovered from the total mRNA using oligo dT-based recovery systems. A method for the recovery of polyA+ mRNA using oligo dT cellulose is described in Maniatis et al (1982). Also, oligo dT-coated paramagnetic particles are available from a variety of vendors to aid in recovery of polyA+ mRNA. Specific descriptions of isolating total RNA and polyA÷ mRNA from plant tissues is given by Murillo et al (Murillo, I., Revantos, D., Jaeck, E., and San Degundo, B., Promega Notes Magazine, Number 64, 1996, pp.02) and J.D. Neill (Promega Notes Magazine, Number 44, Nov. 1993, pp.10) where they describe the use of the Promega RNAgents Total RNA Isolation System and the PolyATtract System to isolate mRNA from maize and tobacco tissues. cDNA Synthesis

Synthesis of cDNA can be achieved using the polyA÷ mRNA isolated from plant of interest (e.g., Oryza sativa). One method to prepare cDNA is described in Maniatis et al. (1982, supra) where the first strand cDNA is synthesized by AMV reverse transcriptase and the second strand is synthesized by the Klenow fragment of DNA polymerase I. Alternatively, several vendors offer kits for the synthesis of cDNA. For example, Invitrogen Corporation offers the Superscript Double- Stranded cDNA Synthesis Kit for preparing DNA from polyA÷ mRNA. The cDNA generated by these methods can be cloned into plasmids or phages to generate cDNA libraries. The cDNA libraries can then be used in PCR reactions to amplify the genes corresponding to the GDP-D- mannose-3,δ-epimerase genes. Alternatively, the cDNA can be used directly in PCR reactions to amplify the desired sequences.

PCR amplification of the putative epimerase sequences

One method to obtain the complete open reading frames of genes coding for the Oryza sativa, Lycopersicon esculentum, Zea mays, Mesembryanthemum crystallinum, Glycine max, Solanum tuberosum, Medicago truncatula, Sorghum bicolor, Triticum aestivum, Hordeum vulgare and Lotus japonicus GDP-D-mannose-3,δ-epimerases, as well as other such epimerases identified in other plants, is the method of polymerase chain reaction (PCR). The cDNA or cDNA libraries synthesized from polyA÷ mRNA as described above will be used as a templates. By way of example, upstream and downstream primers (see Table 6) have been designed according to the DNA sequences identified in databases as putative GDP-D-mannose-3,δ epimerases from rice (Oryza sativa) and tomato (Lycopersicon esculentum). Sequences corresponding to recognition sites for Ncol and Xhol have been added to the δ' and 3' ends of the oligonucleotides (indicated by lowercase letters) to facilitate cloning. The PCR reactions will be carried out in a Robocycler Gradient 96 (Stratagene, California) using the parameters listed in Table 7.

Table 6 Oligonucleotides for Amplification of Putative Epimerase Genes

Table 7 PCR Conditions for the Amplification of Epimerase Sequences

Cloning the PCR Amplified Epimerase Genes for Expression in E. coli

PCR products corresponding to the epimerase coding sequences will be purified and cloned into an expression vector such as pET21d(+) (Novagen, Madison, Wl). The recombinant plasmids will be confirmed by DNA sequencing, and then transformed into an E. coli expression host. Using this vector, the epimerase genes will be placed under control of the T7 promoter and will be transcribed by bacteriophage T7 RNA polymerase. The appropriate E. coli host strain is one that carries an integrated copy of the gene for T7 RNA polymerase under the control of the IPTG inducible lacUVδ promoter. Bacterial expression of recombinant epimerases The E. coli strain BL21 (DE3) will be transformed with the plasmids generated above. For protein expression experiments, 60 ml cultures will be inoculated from 2 ml of overnight cultures and grown in Luria Broth medium containing ampicillin (100 μg/ml) at 37°C until OD₆₀o reaches approximately 0.6-1.0. The cultures will be allowed to equilibrate for 30 min with shaking at various inducing temperature (25-37°C) prior to the addition of IPTG (0.1-1.0 mM). The cultures will be incubated for an additional period of 2 - 4 hours. The cells will be harvested by centrifugation, and used for enzyme preparation and assay as described below. As control, cell extracts will be prepared from the host strain transformed with an empty vector (no insert). Protein expression will be monitored by SDS-PAGE analysis. Assay for epimerase activity

The expressed proteins will be analyzed for the ability to convert GDP-D-mannose to GDP- L-galactose. This can be done using either using whole cell extracts or purified proteins. The BugBuster protein extraction reagent (Novagen, Madison, Wl) will be used to disrupt the cells to prepare cell extracts. The assay can be performed using any suitable assay for GDP-mannose- 3', δ'-epimerase activity, such as by using the method of Wolucka et al., 2001 , Anal. Biochem. 294:161-168 (See Examples 1-3) or by using the following method. The enzyme assay mixture will be in a final volume of 50 μl, and will be composed of cell extract or purified protein, 50 μM of ¹⁴C labeled GDP-D-mannose in 50 mM NaPi buffer, pH 7.2 containing 2 mM each of EDTA and DTT. The mixture will be incubated at room temperature for 1 , 4 and 16 hours. The reactions will be stopped by placing in a boiling water bath for 1 minute and then hydrolyzed in 0.5 M TFA at 100°C for 30 min to release the GDP moiety of sugar nucleotides. Thin Layer Chromatography (TLC) will be used to separate the mixture of free sugars. Briefly, aliquots (6 μl) of hydrolyzed mixture will be loaded on Silica gel 60 plates (20x20 cm, EM Science) which are pre-impregnated with 0.3 M NaH₂PO₄. The TLC plates can be then developed in a solvent containing 80% acetone, 10% n-butanol and 10% water. The amounts of radioactivity in different compounds on the developed TLC plate will be detected by the Phosphorimager system (Molecular Dynamics).

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. An isolated, multimeric GDP-mannose-3',5'-epimerase.

2. The multimeric GDP-mannose-3',5'-epimerase of claim 1 , wherein said epimerase is isolated from a plant. 3. The multimeric GDP-mannose-3',5'-epimerase of claim 2, wherein said plant is selected from the group consisting of Arabidopsis thaliana, Oryza sativa, Lycopersicon esculentum, Zea mays, Mesembryanthemum crystallinum, Glycine max, Solanum tuberosum, Medicago truncatula, Sorghum bicolor, Triticum aestivum, Hordeum vulgare and Lotus japonicus. 4. The multimeric GDP-mannose-3',δ'-epimerase according to any one of claims 1 , 2 or 3, wherein said epimerase is a dimer. δ. The multimeric GDP-mannose-3', δ'-epimerase of claim 4, whereby said epimerase dimer has a molecular weight of between about 80 kDa and about 90 kDa. 6. The multimeric GDP-mannose-3', δ'-epimerase according to any one of the preceding claims, wherein at least one monomer of said epimerase comprises an amino acid sequence selected from the group consisting of: a. an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3',δ'-epimerase activity; and b. a fragment of an amino acid sequence of (a), wherein said fragment has GDP- mannose-3', δ'-epimerase activity. 7. The multimeric GDP-mannose-3',δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises an amino acid sequence selected from the group consisting of: a. an amino acid sequence that is at least about 80% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity; and b. a fragment of said amino acid sequence of (a), wherein said fragment has GDP- mannose-3', δ'-epimerase activity.

8. The multimeric GDP-mannose-3', δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises an amino acid sequence selected from the group consisting of: a. an amino acid sequence that is at least about 90% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity; and b. a fragment of said amino acid sequence of (a), wherein said fragment has GDP- mannose-3', δ'-epimerase activity.

9. The multimeric GDP-mannose-3',δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:46.

10. The multimeric GDP-mannose-3',δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:2.

11. The multimeric GDP-mannose-3',δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:26.

12. The multimeric GDP-mannose-3',5'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:28.

13. The multimeric GDP-mannose-3',δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:30. 14. The multimeric GDP-mannose-3', δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:32. 16. The multimeric GDP-mannose-3', δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:34.

16. The multimeric GDP-mannose-3', 5'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:36.

17. The multimeric GDP-mannose-3', 5'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:38.

18. The multimeric GDP-mannose-3',5'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:40. 19. The multimeric GDP-mannose-3', δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:42.

20. The multimeric GDP-mannose-3', δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:44.

21. The multimeric GDP-mannose-3', δ'-epimerase according to any one of the preceding claims, wherein said epimerase comprises SEQ ID NO:46.

22. The multimeric GDP-mannose-3',δ'-epimerase according to any one of claims 1-δ, wherein said epimerase has the following characteristics: a. a K'eq for the formation of GDP-L-galactose of about 0.15; b. a K_m value for GDP-mannose of about 4.4 μM; and c. a Kj value for GDP of about 0.7 μM.

23. The use of a multimeric GDP-mannose-3',5'-epimerase according to any one of the preceding claims to modulate the synthesis of a product in a cell, said product selected from the group consisting of GDP-L-galactose, L-galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone. 24. The use of a multimeric GDP-mannose-3',5'-epimerase according to any one of claims 1 to 22 to modulate ascorbic acid synthesis in a cell. 25. The use according to claim 23 or 24, wherein said cell is a eukaryotic cell.

26. The use according to claim 26, wherein said eukaryotic cell is a plant cell.

27. The use according to claim 23 or 24, wherein said cell is a prokaryotic cell.

28. An isolated GDP-mannose-3', δ'-epimerase monomer comprising an amino acid sequence that is at least about 70% identical and less than 100% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity.

29. The isolated GDP-mannose-3',δ'-epimerase monomer according to Claim 28, wherein said monomer comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 and SEQ ID NO:54.

30. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a. a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical and less than 100% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity; b. a nucleic acid sequence encoding a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3',δ'-epimerase activity; c. a nucleic acid sequence that is a probe or primer that hybridizes under high stringency conditions to a nucleic acid sequence of (a) or (b); and d. a nucleic acid sequence that is a complement of any of the nucleic acid sequences of

(a)-(c).

31. The isolated nucleic acid molecule according to claim 30, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 80% identical and less than 100% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP- mannose-3', δ'-epimerase activity.

32. The isolated nucleic acid molecule according to claim 30, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 90% identical and less than 100% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP- mannose-3', δ'-epimerase activity. 33. The isolated nucleic acid molecule according to claim 30, wherein said nucleic acid sequence encodes an amino acid sequence that is less than about 96% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3',δ'-epimerase activity. 34. The isolated nucleic acid molecule according to claim 30, wherein said nucleic acid sequence is at least about 70% identical and less than 100% identical to SEQ ID NO:1. 3δ. The isolated nucleic acid molecule according to claim 30, wherein said nucleic acid sequence encodes a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3',δ'-epimerase activity.

36. The isolated nucleic acid molecule according to claim 30, wherein said nucleic acid sequence is a fragment of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:53, wherein said fragment encodes an amino acid sequence that has GDP-mannose-3', δ'-epimerase activity.

37. A recombinant nucleic acid molecule comprising an expression vector and a nucleic acid molecule according to any one of claims 30-36, operatively linked to at least one transcription control sequence. 38. A recombinant nucleic acid molecule comprising an expression vector and a nucleic acid sequence operatively linked to at least one transcription control sequence, wherein said nucleic acid sequence is selected from the group consisting of: a. a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose- 3',δ'-epimerase activity; and b. a nucleic acid sequence encoding a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3', δ'-epimerase activity.

39. A recombinant host cell transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a. a nucleic acid sequence that encodes an amino acid sequence that is at least about

70% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose- 3',5'-epimerase activity; and b. a nucleic acid sequence encoding a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3', δ'-epimerase activity. 40. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 80% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity.

41. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 90% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity.

42. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 and SEQ ID NO:64.

43. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:2 or a biologically active fragment thereof. 44. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:26 or a biologically active fragment thereof. 45. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:28 or a biologically active fragment thereof.

46. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:30 or a biologically active fragment thereof.

47. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:32 or a biologically active . fragment thereof.

48. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:34 or a biologically active fragment thereof.

49. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:36 or a biologically active fragment thereof. 50. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:38 or a biologically active fragment thereof.

51. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:40 or a biologically active fragment thereof.

52. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:42 or a biologically active fragment thereof.

53. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:44 or a biologically active fragment thereof.

54. The recombinant host cell according to claim 39, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:46 or a biologically active fragment thereof. δδ. The recombinant host cell according to any one of claims 39-64 which is a eukaryotic cell.

66. The recombinant host cell according to any one of claims 39-64 which is a yeast.

67. The recombinant host cell according to any one of claims 39-54 which is a plant cell.

58. The recombinant host cell according to any one of claims 39-54 which is a prokaryotic cell.

69. The recombinant host cell according to any one of claims 39-64, wherein expression of said recombinant nucleic acid molecule by said host cell is sufficient to increase the synthesis of a product in said host cell, said product selected from the group consisting of GDP-L- galactose, L-galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone.

60. The recombinant host cell according to any one of claims 39-54, wherein expression of said recombinant nucleic acid molecule by said host cell is sufficient to increase ascorbic acid production in said host cell.

61. A transgenic plant or part of a plant having one or more cells comprising a nucleic acid sequence selected from the group consisting of: a. a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose- 3',δ'-epimerase activity; and b. a nucleic acid sequence encoding a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3',δ'-epimerase activity.

62. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 80% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', 5'-epimerase activity.

63. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 90% identical to SEQ ID

NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity.

64. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence that is less than 00% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity. 66. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 and SEQ ID NO:54. 66. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:2 or a biologically active fragment thereof.

67. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:26 or a biologically active fragment thereof.

68. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:28 or a biologically active fragment thereof. 69. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:30 or a biologically active fragment thereof. 70. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:32 or a biologically active fragment thereof.

71. The transgenic plant or part of a plant according to claim 61, wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:34 or a biologically active fragment thereof.

72. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:36 or a biologically active fragment thereof.

73. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO.38 or a biologically active fragment thereof.

74. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:40 or a biologically active fragment thereof. 75. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:42 or a biologically active fragment thereof.

76. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:44 or a biologically active fragment thereof.

77. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes an amino acid sequence represented by SEQ ID NO:46 or a biologically active fragment thereof.

78. The transgenic plant or part of a plant according to claim 61 , wherein said nucleic acid sequence encodes a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3', δ'-epimerase activity.

79. The transgenic plant or part of a plant according to any one of claims 61-78, wherein said plant or part of a plant has increased synthesis of a product as compared to a non- transgenic plant, said product selected from the group consisting of GDP-L-galactose, L- galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone.

80. The transgenic plant or part of a plant according to any one of claims 61-78, wherein said plant or part of a plant has increased .production of ascorbic acid as compared to a non- transgenic plant.

81. A method to increase ascorbic acid synthesis in a host cell, comprising growing a host cell that is transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one subunit of a multimeric GDP-mannose-3',δ'-epimerase, wherein said nucleic acid sequence is operatively linked to a transcription control sequence.

82. The method according to claim 81, wherein said transcription control sequence comprises a native GDP-mannose-3',δ'-epimerase promoter located upstream of said nucleic acid sequence encoding said at least one subunit of a multimeric GDP-mannose-3',δ'- epimerase.

83. The method according to claim 81 , wherein said transcription control sequence comprises a non-native promoter located upstream of said nucleic acid sequence encoding said at least one subunit of a multimeric GDP-mannose-3', δ'-epimerase.

84. The method according to claim 81 , wherein said GDP-mannose-3', δ'-epimerase is isolated from a plant.

86. The method according to claim 81 , wherein said nucleic acid sequence encoding at least one subunit of a multimeric GDP-mannose-3', δ'-epimerase is selected from the group consisting of: a. a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-

3', δ'-epimerase activity; and b. a nucleic acid sequence encoding a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3', δ'-epimerase activity.

86. The method according to claim 81 , wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 80% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity.

87. The method according to claim 81, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 90% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity. 88. The method according to claim 81 , wherein said nucleic acid sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 and SEQ ID NO:54.

89. The method according to claim 81 , wherein said nucleic acid sequence encodes SEQ ID NO:2 or a biologically active fragment thereof.

90. The method according to claim 81 , wherein said nucleic acid sequence encodes a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3',5'- epimerase activity.

91. The method according to any one of claims 81-90, wherein said cell is a prokaryotic cell.

92. A method to increase ascorbic acid synthesis in a cell comprising a multimeric GDP- mannose-3', δ'-epimerase, comprising introducing into the genome of said cell a non-native promoter upstream of a gene encoding said at least one subunit of a multimeric GDP- mannose-3', δ'-epimerase.

93. The method according to claim 92, wherein said non-native promoter is a plant promoter.

94. A method to increase ascorbic acid synthesis in a cell comprising a multimeric GDP- mannose-3'δ'-epimerase, comprising genetically modifying said cell to increase the activity of said GDP-mannose-3',5'-epimerase in said cell.

95. The method according to claim 94, wherein said genetic modification comprises expressing a recombinant GDP-mannose-3', δ'-epimerase promoter in said cell upstream of said gene encoding said at least one subunit of a multimeric GDP-mannose-3', δ'-epimerase, wherein expression of said recombinant promoter increases the expression of GDP-mannose-3', δ'- epimerase by said cell.

96. The method according to any one of claims 92-96, wherein said gene comprises a nucleic acid sequence that is at least about 70% identical to SEQ ID NO:1.

97. The method according to any one of claims 92-96, wherein said gene comprises a nucleic acid sequence that is at least about 80% identical to SEQ ID NO:1. 98. The method according to any one of claims 92-95, wherein said gene comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO:1.

99. The method according to any one of claims 92-95, wherein said gene comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:3δ, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:4δ and SEQ ID NO:δ3.

100. The method according to any one of claims 92-95, wherein said gene comprises SEQ ID NO:1.

101. The method according to claim 94, wherein said genetic modification comprises a modification of GDP-mannose-3', 5'-epimerase in said cell which increases the expression or activity of said GDP-mannose-3', δ'-epimerase in said cell.

102. The method according to any one of claims 81-101 , wherein said cell is a eukaryotic cell.

103. The method according to any one of claims 81-101 , wherein said cell is a plant cell.

104. A plant obtainable according to the method of claim 103, followed by regeneration of said plant cell.

105. An isolated antibody that selectively binds to a multimeric GDP-mannose-3', δ'- epimerase.

106. The isolated antibody of claim 105, wherein said multimeric GDP-mannose-3',5'- epimerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO.34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 and SEQ ID NO:54.

107. A recombinant nucleic acid molecule comprising an expression vector and a nucleic acid molecule comprising: a. a first nucleic acid sequence encoding an amino acid sequence selected from the group consisting of: i. an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', 5' -epimerase activity; and ii. a fragment of said amino acid sequence of (a), wherein said fragment has GDP- mannose-3', δ'-epimerase activity; and b. at least one additional nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein said enzyme is selected from the group consisting of phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-L-galactose pyrophosphorylase, L-galactose-1 -P- phosphatase, L-galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase. 108. A recombinant host cell transformed with at least two recombinant nucleic acid molecules comprising: a. a first recombinant nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i. a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein said amino acid sequence has

GDP-mannose~3',δ'-epimerase activity; and ii. a nucleic acid sequence encoding a fragment of said amino acid sequence of

(a), wherein said fragment has GDP-mannose-3',5'-epimerase activity; and b. at least one additional recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein said enzyme is selected from the group consisting of phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-L- galactose pyrophosphorylase, L-galactose-1 -P-phosphatase, L-galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase. 109. The recombinant host cell according to claim 108, wherein said first and said at least one additional recombinant nucleic acid molecules are contained within a single recombinant vector.

110. The recombinant host cell according to claim 109, wherein said recombinant vector is dicistronic.

111. The recombinant host cell according to any one of claims 108-110, wherein said host cell is a eukaryotic cell. 112. The recombinant host cell according to any one of claims 108-110, wherein said host cell is a plant cell.

113. The recombinant host cell according to any one of claims 108-110, wherein said host cell is a yeast.

114. The recombinant host cell according to any one of claims 108-110, wherein said host cell is a prokaryotic cell.

115. The recombinant host cell any one of claims 108-110, wherein said host cell has increased synthesis of a product in said host cell as compared to a non-transformed host cell, said product selected from the group consisting of GDP-L-galactose, L-galactose-1 - phosphate, L-galactose, and L-galactono-γ-lactone. 116. The recombinant host cell according to any one of claims 108-110, wherein said host cell has increased production of ascorbic acid as compared to a non-transformed host cell.

117. A transgenic plant or part of a plant having one or more cells comprising at least two recombinant nucleic acid molecules comprising: a. a first recombinant nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i. a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical to SEQ ID NO:2, wherein said amino acid sequence has GDP-mannose-3', δ'-epimerase activity; and ii. a nucleic acid sequence encoding a fragment of said amino acid sequence of (a), wherein said fragment has GDP-mannose-3', δ'-epimerase activity; and b. at least one additional recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one enzyme or biologically active fragment thereof, wherein said enzyme is selected from the group consisting of phosphomannose isomerase, phosphomannomutase, GDP-D-mannose pyrophosphorylase, GDP-L- galactose pyrophosphorylase, L-galactose-1 -P-phosphatase, L-galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase.

118. The use of a multimeric GDP-mannose-3', δ'-epimerase according to any one of claims 1-22 for in vitro synthesis of a product selected from the group consisting of GDP-L- galactose, L-galactose-1 -phosphate, L-galactose, and L-galactono-γ-lactone. 119. The use of a multimeric GDP-mannose-3',δ'-epimerase according to any one of claims 1-22 or a nucleic acid sequence encoding at least one subunit of said multimeric GDP- mannose-3',5'-epimerase as a selectable marker in eukaryotic cells.

76

120. The use of a multimeric GDP-mannose-3',5'-epimerase according to any of the claims 1-22 to increase the reductive capacity of a eukaryotic cell.

121. The use of a multimeric GDP-mannose-3',5'-epimerase according to claim 120, whereby said cell is a plant cell.

122. The use of a multimeric GDP-mannose-3',5'-epimerase according to claim 120, whereby said cell is a yeast cell.