WO2004000233A2

WO2004000233A2 - Algal gdp-mannose-3',5'-epimerases and methods of use thereof

Info

Publication number: WO2004000233A2
Application number: PCT/US2003/019951
Authority: WO
Inventors: Thomas W. Mcmullin; Susan Shuyun Peng
Original assignee: Arkion Life Sciences Llc
Priority date: 2002-06-25
Filing date: 2003-06-25
Publication date: 2003-12-31
Also published as: AU2003249364A8; AU2003249364A1; WO2004000233A3

Abstract

GDP-mannose-3',5'-epimerases from algae have been identified, isolated, purified, and/or cloned. Disclosed herein are algal GDP-mannose-3',5'-epimerases from any algae, including those of the genera Chlamydomonas, Prototheca, and Chlorella. Also disclosed herein are nucleic acid and amino acid sequences for algal GDP-mannose-3',5'-epimerases and the use thereof to produce genetically modified host cells, as well as in methods to produce L-galactose, ascorbic acid, or intermediate products in the ascorbic acid biosynthetic pathway.

Description

ALGAL GDP-MANNOSE-3',5'-EPIMERASES AND METHODS OF USE THEREOF

Field of the Invention This invention generally relates to ascorbic acid (vitamin C) synthesis in eukaryotic cells, and preferably plant cells. The invention relates to the identification of algal GDP-mannose-S'jS'- epimerases; to the characterization of purified algal GDP-mannose-3',5'-epimerases; to the identification, cloning and expression of a nucleic acid sequence encoding an algal GDP-mannose-3',5'- epimerase; to methods of producing an algal GDP-mannose-3',5'-epimerase; to transgenic plants and microorganisms that express the algal GDP-mannose-3',5'-epimerase; and to methods of production of ascorbic acid using the algal GDP-mannose-3',5'-epimerase.

Background of the Invention Nearly all forms of life, both plant and animal, either synthesize ascorbic acid (vitamin C) (also referred to herein as L-ascorbic acid, ascorbic acid, L -AA or AA) or require it as a nutrient. Ascorbic acid was first identified to be useful as a dietary supplement for humans and animals for the prevention of scurvy. Ascorbic acid, however, also affects human physiological functions such as the adsorption of iron, cold tolerance, the maintenance of the adrenal cortex, wound healing, the synthesis of polysaccharides and collagen, the formation of cartilage, dentine, bone and teeth, the maintenance of capillaries, and is useful as an antioxidant.

For use as a dietary supplement, ascorbic acid can be isolated from natural sources, such as rosehips, synthesized chemically through the oxidation of L-sorbose, or produced by the oxidative fermentation of calcium D-gluconate by Acetobacter suboxidans. Considine, "Ascorbic Acid," Van Nostrand's Scientific Encyclopedia, Vol. 1, pp. 237-238, (1989). Ascorbic acid (predominantly intracellular) has also been obtained through the fermentation of strains of the microalga, Chlorella pyrenoidosa. See U.S. Patent No. 5,001,059 by Skatrud, which is assigned to the assignee of the present application.

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). It is believed that ascorbic acid is produced inside the chloroplasts of photosynthetic microorganisms and functions to neutralize energetic electrons produced during photosynthesis. Accordingly, ascorbic acid production is known in photosynthetic organisms as a protective mechanism.

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 oxidizes 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 and algae, its biosynthesis in these organisms is not completely understood. 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 dArabidopsis thaliana, and proposed an ascorbic acidbiosynthetic 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.

Recently, an alternative plant pathway in ripe strawberry fruit based on D-galacturonic acid (derived from sources such as pectin from cell wall breakdown) reduction by the GalUR gene product to L-galactonic acid, a precursor to L-galactono- γ-lactone and L-ascorbic acid was described (Agius et al., 2003, Nature Biotechnology 21: 177-181). Overexpression of GalUR in Arabidopsis thaliana enhanced vitamin C content by two- to threefold.

Recycling of oxidized forms of L-ascorbic acid has recently been shown to increase the L- ascorbic acid levels in plants. This can be achieved by overexpression of a dehydroascorbate reductase (DHAR) cDNA encoded by a plant gene (Chen, 2003, Proc. Natl. Acad. Sci. 100:3525-3530). Expression was shown to increase L-ascorbic acid by two- and four-fold in tobacco and maize, respectively. While this strategy does not increase the amount of L-ascorbic acid being made by plants, it does effectively increase the concentration of the reduced form of ascorbic acid in cells.

PCT Publication WO 99/64618 to Bio-Technical Resources, also disclosed a proposed ascorbic acid biosynthetic pathway in plants and photosynthetic microalgae 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 definitive 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.

One investigator 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 (Wolucka et al., Proceedings of the National Academy of Sciences, 2001, 98: 14843- 14848). On the basis of the purified protein, the sequence was determined, and the epimerase was cloned and expressed recombinantly in an E. coli host cell. The Arabidopsis thaliana GDP-mannose- 3',5'-epimerase sequence 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 Arabidopsis 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 Dl 8. 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.

To the present inventors' knowledge, no GDP-mannose-3 ',5'epimerases from algae have been identified prior to the present invention.

Since products and processes which improve the ability to biosynthetically produce ascorbic acid are desirable and beneficial for the improvement of human health, 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 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 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, 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; and (c) a nucleic acid sequence that is fully complementary to any of the nucleic acid sequences of (a) or (b). In preferred embodiments, the nucleic acid sequence encodes an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21. In another embodiment, the nucleic acid sequence is less than about 100% identical to any of the nucleic acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. In one embodiment, the nucleic acid sequence is at least about 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:20, and less than 100% identical to any of the nucleic acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. In other preferred embodiments, the nucleic acid sequence encodes a protein comprising an amino acid sequence of SEQ ID NO:21, or the nucleic acid sequence is SEQ ID NO:20. In one aspect, the nucleic acid sequence encodes a biologically active fragment of SEQ ID NO: 10 or SEQ ID NO:21 , wherein the fragment has GDP-mannose-3',5'-epimerase activity.

Another embodiment of the present invention relates to an oligonucleotide probe or primer that hybridizes under high stringency conditions to a nucleic acid sequence comprising SEQ ID NO:20 or the complement thereof. Yet another embodiment of the present invention relates to a recombinant nucleic acid molecule comprising a nucleic acid sequence operatively linked to at least one expression control sequence, the nucleic acid sequence being selected from the group consisting of: (a) a nucleic acid sequence that encodes an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO :21, wherein the amino acid sequence has GDP-mannose-3 ',5 '-epimerase activity; or (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 as described above. The host cell can include a eukaryotic cell (e.g., a yeast or a plant cell) or a prokaryotic cell. In one aspect, the 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 the group consisting of 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 genetically modified plant or part thereof, wherein the plant has been genetically modified to recombinantly express a GDP-mannose- 3',5'-epimerase or biologically active fragment thereof, wherein the GDP-mannose-3',5'-epimerase comprises an amino acid sequence selected from the group consisting of: (a) an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3',5'- epimerase activity; or (b) an amino acid sequence that is a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity.

Yet another embodiment of the present invention relates to an isolated algal GDP-mannose- 3 ',5 '-epimerase having characteristics comprising: (a) a monomeric molecular weight of between about 40kD and about 50kD; (b) an optimum pH of from about 7 to about 8.5; and (c) a K,,, of between about 7 μM and 50 μM for GDP-D-mannose. The isolated algal GDP-mannose-3',5'-epimerase has been purified by at least 2-fold as compared to a crude extract having GDP-mannose-3 ',5 '-epimerase activity andhas GDP-mannose-3',5'-epimerase enzymatic activity. The epimerase can be isolated from an algal genus including, but not limited to, Chlamydomonas, Prototheca, Chlorella, Platimonas, Euglena, Scenedesmus, Pterocladia, Porphyridium, Ochromonas, and Cyclotella, with Chlamydomonas, Prototheca, and Chlorella being particularly preferred.

Preferably, an isolated algal epimerase of the invention forms a dimer. In one aspect, the epimerase has a V_max of between about 22.4 nmol min^"1 mg^"1 and about 4.8 nmol min^"1 mg^'1 for GDP-D- mannose. In one aspect, the epimerase has characteristics comprising: (a) a monomeric molecular weight of about 43kD; (b) an optimum pH of about 7.2; and (c) a K_m of at least about 30 μM for GDP- D-mannose. Such an epimerase can be isolated from Prototheca. In one aspect, the epimerase has been isolated by detergent solubilization. In another embodiment, the epimerase has been isolated by hydrophobic interaction chromatography. In another embodiment, the epimerase has been isolated by anion exchange chromatography. In another embodiment, the epimerase has been isolated by size exclusion chromatography. Preferably, the epimerase is of a purity to appear as a single band on an SDS-PAGE gel, or of a purity to elute from a chromatography column and appear as a single band on an SDS-PAGE gel.

An isolated algal epimerase can also comprise an amino acid sequence selected from the group consisting of: (a) an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21 , wherein the amino acid sequence has GDP-mannose-3 ',5 '-epimerase activity; and (b) an amino acid sequence that is a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity. In other embodiments, the epimerase comprises any of the amino acid sequences set forth above (as encoded by nucleic acid sequences of the invention). In one aspect, the epimerase is bound to a solid support. Another embodiment of the invention relates to a method to produce L-galactose. The method includes the steps of: (a) contacting the isolated algal GDP-mannose-3 ',5'-epimerase as described herein with GDP-D-mannose under conditions that result in the production of GDP-L-galactose; and (b) converting the GDP-L-galactose to L-galactose. The step of converting can be performed by a method including, but not limited to, hydrolysis of the GDP-L-galactose under acid conditions, or enzymatic treatment of the GDP-L-galactose to produce L-galactose. In one aspect, the method can also include a step of purifying the L-galactose.

Another embodiment of the invention relates to a method for producing L-galactose, comprising: (a) growing a host cell that is transformed with a recombinant nucleic acid molecule encoding a GDP-mannose-3 ',5'-epimerase as previously described herein, to produce GDP-L-galactose; (b) converting the GDP-L-galactose to L-galactose; and (c) recovering the GDP-L-galactose or the L- galactose from the host cell. In one aspect, step (c) comprises purifying the GDP-L-galactose or the L-galactose from a crude extract of microbial host cells. In another aspect, the GDP-L-galactose is recovered in step (c) and wherein step (b) of converting is performed after step (c) of recovering. In another aspect, step (b) comprises producing a crude extract of host cells comprising GDP-L-galactose and converting the GDP-L-galactose to L-galactose, followed by recovering the L-galactose.

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 a recombinant nucleic acid molecule encoding a GDP-mannose-3 ',5 '-epimerase to increase ascorbic acid synthesis by the host cell, wherein the recombinant nucleic acid molecule comprises a nucleic acid sequence operatively linked to at least one expression control sequence, the nucleic acid sequence being any of the nucleic acid sequences encoding a GDP-mannose-3',5'-epimerase described herein. The host cell can include a eukaryotic cell (e.g., a plant cell, an algal cell or a yeast cell), or a prokaryotic cell. Yet another embodiment of the invention relates to a method to increase ascorbic acid synthesis in an algal cell comprising a GDP-mannose-3 ',5'-epimerase, comprising introducing into the genome of the cell a non-native promoter upstream of a gene encoding the GDP-mannose-3',5'- epimerase, wherein the epimerase comprises an amino acid sequence selected from the group consisting of: (a) an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21 , wherein the amino acid sequence has GDP-mannose-3 ',5'-eρimerase activity; or (b) an amino acid sequence that is 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 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 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; or (ii) an amino acid sequence that is 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 the group consisting of phosphomannose isomerase, phosphomarmomutase, GDP-D-mannose pyrophosphorylase, GDP-L- galactose pyrophosphorylase, L-galactose- 1-P-phosphatase, L-galactose dehydrogenase, and 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 the group consisting of: (i) a nucleic acid sequence that encodes an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21 , wherein the amino acid sequence has GDP-mannose-3 ',5'-epimerase activity; (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 the group consisting of phosphomannose isomerase, phosphomarmomutase, GDP-D- mannose pyrophosphorylase, GDP-L-galactose pyrophosphorylase, L-galactose- 1-P-phosphatase, L- galactose dehydrogenase, and L-galactono-γ-lactone dehydrogenase.

Yet another embodiment of the present invention relates to a genetically modified host cell, wherein the host cell comprises at least one genetic modification to increase the activity of a GDP- mannose-3',5'-epimerase in the host cell, wherein the GDP-mannose-3',5'-epimerase comprises an amino acid sequence selected from the group consisting of: (a) an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; and (b) an amino acid sequence that is a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity. In one aspect, the host cell has been modified by transformation with a recombinant nucleic acid molecule encoding the GDP-mannose-3',5'-epimerase. In another aspect, the host cell is an algal cell that has been genetically modified by the introduction into the genome of the algal cell of a non-native promoter upstream of a gene encoding the GDP- mannose-3 ',5 '-epimerase.

Brief Description of the Drawings FIG. 1 is a schematic drawing of the pETChlam.C1.6 plasmid. FIG. 2 is a schematic drawing of the pETChlam.Cl.10 plasmid.

FIG. 3 shows the molecular mass of functional (native) recombinant Chlamydomonas epimerase.

FIG. 4 shows the pH dependence of the recombinant Chlamydomonas epimerase. FIG. 5. shows the time course of the epimerization of GDP-D-mannose to GDP-L-galactose by recombinant Chlamydomonas GDP-mannose-3',5'-epimerase.

FIG. 6 is a Lineweaver-Burk plot of purified native recombinant Chlamydomonas epimerase. FIG. 7 shows the inhibitory effect of GDP-L-fucose on recombinant Chlamydomonas epimerase activity.

FIG. 8 shows formation of GDP-L-galactose as a function of reaction time. FIG. 9 shows the stability of purified Prototheca epimerase.

FIG. 10 shows epimerase assay progress curves for the formation of GDP-L-galactose from GDP-D-mannose by Prototheca cell-free extracts.

FIG. 11 shows a Lineweaver-Burk plot of partially-purified native Prototheca epimerase.

Detailed Description of the Invention

This invention generally relates to the discovery of a nucleic acid sequence from a microalga (alga) that encodes GDP-mannose-3',5'-epimerase from the L-ascorbic acid biosynthetic pathway, as well as the GDP-mannose-3',5'-epimerase encoded thereby. This invention also relates to methods that can be used to isolate other microalgal (algal) nucleic acid sequences that exhibit sequence similarity to the GDP-mannose-3 ',5'-epimerase gene and protein described herein. This invention also describes methods to demonstrate GDP-mannose-3 ',5'-epimerase enzyme activity of the proteins expressed from those genes. This invention also relates to fragments and homologues of any of the algal GDP- mannose-3',5'-epimerase-encoding nucleic acid sequences or proteins described herein or identified using the methods disclosed herein, and to the use of any of these nucleic acid sequences to produce a GDP-mannose 3',5'-epimerase, to produce recombinant nucleic acid molecules comprising such sequences, to produce genetically modified microorganisms and plants, and to methods of producing L-galactose, L-ascorbic acid, or intermediate products in the ascorbic acid biosynthetic pathway using such genetically modified microorganisms and plants. Genetically modified microorganisms and plants can include organisms that comprise modifications to an endogenous GDP-mannose 3 ',5'- epimerase according to the present invention, and/or organisms that have been transfected with a recombinant nucleic acid molecule encoding a GDP-mannose-3 ',5'-epimerase according to the present invention. This invention also relates to isolated GDP-mannose-3',5'-epimerases, including substantially purified GDP-mannose-3',5'-epimerases. The invention includes, but is not limited to, epimerases from the algal strains: Chlamydomonas reinhardtii dangerard ATCC 18798 (wild type [WT]) mi Prototheca moriformis ATCC 75669 (WT) (American Type Culture Collection (ATCC; Manassas, VA); Prototheca moriformis ATCCPTA111 (mutant strain from Bio-Technical Resources (BTR) collection); Chlorella pyrenoidosaXJTEX 1613 (WT) andUTEX 166-4 (mutant), both from the University of Texas at Austin UTEX collection.

The GDP-mannose-3',5'-epimerase catalyzes the first dedicated step in the vitamin C synthesis pathway in plant and microbial cells. As set forth in pending U.S. Application Serial No. 09/318,271, filed May 25, 1999, entitled "Vitamin C Production in Microorganisms and Plants" (incorporated herein by reference in its entirety), the GDP-mannose 3' ,5'-epimerase is an important step in the ascorbic acid biosynthetic process and therefore is an important target for genetic modification. 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.

To attempt to identify GDP-mannose-3 ',5 'epimerases from algae, the present inventors first identified three NCBI sequence database entries (SEQ ID NOs: 1-3; see Table 2) that corresponded to the Arabidopsis thaliana gene that had recently been shown to encode a GDP-mannose-3 ',5 '-epimerase (Wolucka et al., Proceedings of the National Academy of Sciences, 2001, 98: 14843-14848). Scanning public DNA sequence databases with these sequences identified sequences of high homology at both the DNA and protein levels in the Chlamydomonas EST database. None of these EST cDNA sequences were associated with a deduced encoded protein, nor with a proposed function, according to the database records. Moreover, none of these sequences, upon examination by the present inventors, appeared to encode a full-length protein (Table 2).

The present inventors assembled a full-length Chlamydomonas sequence from the five overlapping EST sequence entries in the Chlamydomonas Genome Project database and represented herein by SEQ ID NOs:4-8. Alignment of this assembled DNA sequence (represented herein by SEQ ID NO:9) with the Arabidopsis gene showed 63.2% identity over the entire lengths of their open reading frames. The Chlamydomonas sequence has a 51 bp extension at its 3'-end as compared to the Arabidopsis sequence. SEQ ID NO:9 encodes a deduced amino acid sequence of SEQ ID NO: 10.

Using this sequence information, the present inventors cloned and sequenced a Chlamydomonas GDP-mannose-3 ',5 '-epimerase from a Chlamydomonas cDNA library. The cloned sequence has notable differences from that of the sequence that was assembled from the Chlamydomonas Genomic EST Sequence Database (i.e., from SEQ ID NO:9). The nucleic acid sequence encoding a Chlamydomonas GDP-mannose-3 ',5 '-epimerase cloned by the present inventors is represented herein in by SEQ ID NO:20. The amino acid sequence encoded by such nucleic acid sequence is represented herein in SEQ ID NO:21. As discussed in the Examples, the cloned sequence is about 30bp shorter than the assembled contig of SEQ ID NO:9, and additionally includes seven nucleic acid differences that resulted in three amino acid differences between SEQ ID NO: 10 and SEQ ID NO:21 (see Table 5). Therefore, the present invention comprises the isolated nucleic acid sequences encoding the

GDP-mannose-3 ',5 '-epimerase from Chlamydomonas reinhardtii, as well as fragments andhomologues of such sequence. Also included in the present invention are isolated GDP-mannose-3 ',5'-epimerases including epimerases from the algal strains Chlamydomonas reinhardtii dangerard ATCC 18798, Prototheca moriformis ATCC 75669, Prototheca moriformis ATCCPTA111, Chlorella pyrenoidosa UTEX 1613, and Chlorella pyrenoidosa UTEX 166-4. In particular, the present inventors have purified to substantial homogeneity the GDP-mannose-3',5'-epimerase from a Prototheca strain, and have characterized the purified enzyme in detail (see Examples). The methods for purification of GDP-mannose-3',5'-epimerases can be used to isolate and purify the GDP-mannose-3',5'-epimerases from other algal species, which are encompassed by the present invention. One embodiment of the present invention relates to an isolated algal 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 et al, 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). Biological activity of a GDP-mannose-3',5'-epimerase according to the present invention is discussed in more detail elsewhere herein and in the Examples. The native GDP-mannose-3',5'- epimerase from the algae described herein exists as a dimer. The dimer from Chlamydomonas has a molecular weight of about 90 kDa (monomer molecular weight of about 43 kDa).

Preferably, the isolated GDP-mannose-3 ',5'-epimerase has been purified by at least 2-fold, and in one aspect, at least 3-fold, and in another aspect, at least 4-fold, and so on, in whole integers (i.e., 5-fold, 6-fold, 7-fold, etc.) up to at least about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, as compared to a crude extract having GDP-mannose-3 ',5 '-epimerase activity.

In one embodiment, the epimerase has been isolated by detergent solubilization. In another embodiment, the epimerase has been isolated by hydrophobic interaction chromatography. In another embodiment, the epimerase has been isolated by anion exchange chromatography. In another embodiment, the epimerase has been isolated by size exclusion chromatography. Preferably, the epimerase is of a purity to appear as a single band on an SDS-PAGE gel, or of a purity to elute from a chromatography column and appear as a single band on an SDS-PAGE gel.

An isolated algal epimerase can be isolated from an alga belonging to a genus including, but not limited to, Chlamydomonas, Prototheca, Chlorella, Platimonas, Euglena, Scenedesmus, Pterocladia, Porphyridium, Ochromonas, and Cyclotella, with Chlamydomonas, Prototheca, and Chlorella being particularly preferred.

An isolated GDP-mannose-3',5'-epimerase of the present invention can be characterized by its molecular weight. Preferably, an isolated GDP-mannose-3 ',5'-epimerase of the present invention has a monomer molecular weight (the molecular weight of a monomer of the GDP-mannose-3',5'- epimerase) of between about 35 kDa and about 55kDa, and more preferably, between about 40 kDa and about 50kDa. For example, the molecular weight of a Chlamydomonas GDP-mannose-3',5'- epimerase of the invention is about 43 kDa (the dimer having a molecular weight of about 90 kDa).

An isolated GDP-mannose-3',5'-epimerase of the present invention can be characterized by its specific activity. A "specific activity" refers to the rate of conversion of GDP-D-mannose to GDP-L- galactose by the enzyme. More specifically, it refers to the number of molecules of GDP-D-mannose converted to GDP-L-galactose per mg of the enzyme per time unit.

Another way to characterize the isolated GDP-mannose-3',5'-epimerase is by its Michaelis- Menten constant (K_m). K_^ is a kinetic (i.e., rate) constant of the enzyme-substrate complex under conditions of the steady state. Preferably, an isolated GDP-mannose-3',5'-epimerase of the present invention has a K_m for GDP-D-mannose of between about 7μM and about 50μM, and more preferably, between about 7.5 μM and about 40μM, and more preferably, between about 7.5 μM and about 30μM, with any K_m for GDP-D-mannose between 7 μM and 50μM being included, in 0.1 μM increments (i.e., 7.1 μM, 7.2 μM, etc.). For example, an isolated GDP-mannose-3 ',5'-epimerase from Chlamydomonas reinhardtii has a K,,, for GDP-D-mannose of at least about 7.6 μM at a temperature of about 20°C. An isolated GDP-mannose-3',5'-epimerase from Prototheca moriformis has aK^ for GDP-D-mannose of at least about 30 μM at a temperature of about 20 °C.

Yet another way to characterize a GDP-mannose-3',5'-epimerase is by a GDP-L-fucose inhibition rate constant (Kj). Specifically, K_; is a dissociation rate of the GDP-L-fucose-enzyme complex. For example, an isolated an isolated GDP-mannose-3',5'-epimerase from Chlamydomonas reinhardtii of the present invention has a Kj of about 55 μM.

Still another way to characterize a GDP-mannose-3 ',5'-epimerase is by its initial velocity (v₀), i.e., initial rate of product formation. The initial velocity (v₀) refers to the initial conversion rate of GDP-D-mannose to GDP-L-galactose by the enzyme. Specifically, it refers to the number of molecules of GDP-D-mannose converted to GDP-L-galactose per mg of the enzyme per time unit.

In one embodiment, the isolated GDP-mannose-3',5'-epimerase of the invention has a V_max of between about 22.4 nmol min^"1 mg^"1 and about 4.8 nmol min^"1 mg^'1 for GDP-D-mannose.

The isolated GDP-mannose-3',5'-epimerase can be further characterized by its optimum pH. The optimum pH refers to the pH at which the GDP-mannose-3',5'-epimerase has a maximum initial velocity. Preferably the optimal pH is between about pH 7 and pH 8.5, and more preferably between about pH 7.2 and pH 8.2, and more preferably, is about pH 7.2

Overall, the GDP-mannose-3',5'-epimerase from Chlamydomonas reinhardtii also has the following characteristics: (a) a K_m value for GDP-mannose of about 7.6 μM; (b) an optimum pH of about 7.2 in Tris and about 8.2 in potassium phosphate buffer solutions; (c) a V_max for GDP-D-mannose of about 22.4 nmol min^"1 mg^"1; (d) a K₍ value for GDP-L-fucose of about 55 μM; (e) is inhibited by metal salts other than Ca²⁺ salts, by L-ascorbic acid and by ADP-D-glucose by 10 to 90%; and (f) is inhibited by 1 mM GDP, GDP-D-glucose or GDP-L-fucose. The GDP-mannose-3',5'-epimerase from Prototheca moriformis described herein, in addition to having a similar molecular weight as the GDP- mannose-3 ',5 '-epimerase from Chlamydomonas reinhardtii, has the following characteristics: (a) aK,,, value for GDP-mannose of about 30 μM; (b) an optimum pH of about 7.2; (c) a V_max for GDP-D- mannose of about 4.8 nmol min^"1 mg^"1; (d) is inhibitedby Ca ⁺. The detailed structural and biochemical characteristics of a purified and isolated GDP-mannose-3',5'-epimerase of the invention are described in detail in the Examples. Both the native dimeric 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). Ahomologue 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., by glycosylation, 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. Anisolated protein, suchasanisolatedGDP-mannose-3',5'-epimerase, accordingtothepresent 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, a " Chlamydomonas reinhardtii 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 Chlamydomonas reinhardtii 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 Chlamydomonas reinhardtii. In other words, a Chlamydomonas reinhardtii 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 Chlamydomonas reinhardtii or that is a biologically active (i.e., has biological activity) homologue of a naturally occurring GDP-mannose-3',5'-epimerase from Chlamydomonas reinhardtii as described in detail herein. As such, a Chlamydomonas reinhardtii 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 algae as disclosed herein.

According to the present invention, an isolated GDP-mannose-3',5'-epimerase, including 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 GDP-mannose-3',5'-epimerase is isolated from an alga or derived from a GDP- mannose-3',5'-epimerase from an alga (e.g., a recombinant epimerase, or a homologue or modified sequence as described below). In one embodiment, the GDP-mannose-3',5'-epimerase is from an alga that includes, but is not limited to, Chlamydomonas, Prototheca, Chlorella, Platimonas, Euglena, Scenedesmus, Pterocladia, Porphyridium, Ochromonas, and Cyclotella, with GDP-mannose-3',5'- epimerases from Chlamydomonas, Prototheca, or Chlorella being particularly preferred. Several different algal species that are known to produce ascorbic acid and thus are likely to have a GDP- mannose-3 ',5'-epimerase according to the invention are shown in Table 1. TABLE 1

L-Ascorbic Acid

Organism (mg g^"1 dew cells)

Chlamydomonas reinhardtii 2.0

Prototheca moriformis 0.69 to 4.8

Prototheca zopfi 0.73 to 2.4

Prototheca chlorelloides 2.4

Protothecakruegeri 2.25

Protothecawickerhamii 0.74

Prototheca ulmea 0.63 to 1.20

Chlorella pyrenoidosa 0.53 to 2.0

C. vulgaris 1.2 to 15.0

Platimonas viridis 0.54

Euglena gracilis 0.20

Scenedesmus acutus 1.7

Scenedesmus obliquus 2.0

Pterocladia capillaceae 0.79

Porphyridium cruentum 1.7

Ochromonas danica 0.83 to 2.1

Cyclotella cryptica 0.80

Methods that can be used to isolate algal genes that encode, or are believed to encode, an algal GDP-mannose-3 ',5'-epimerase gene are encompassed by the present invention. Also encompassed by the present invention are methods to demonstrate GDP-mannose-3',5'-epimerase enzyme activity of the proteins expressed from those genes. A sequence from the microalga Chlamydomonas has already been identified as discussed above by searching the Chlamydomonas Genomic EST sequence database, and methods that can be used to isolate this gene are described below. Due to the high degree of sequence conservation observed among the GDP-mannose-3 ',5'-epimerase genes fromArabidopsis and Chlamydomonas, methods based on a hybridization approach are also provided below that describe the isolation of epimerase genes from other algae, including Prototheca and Chlorella. Other algae can be considered as well (Table 1). Isolated GDP-mannose-3 ',5'-epimerase genes from any of the algae listed in Table 1 are contemplated to be part of the present invention.

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) maybe 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 straight-forward 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 (see Examples). 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. Another assay for GDP- mannose-3',5'-epimerase activity is a thin layer chromatography method using ¹⁴C labeled GDP-D- mannose as a substrate (e.g., see Example 8). 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.

As discussed above, the present inventors have purified, cloned and identified what is believed to be the only algal GDP-mannose-3',5'-epimerase at the time of the invention to have been purified to substantial homogeneity and identified by specific function, as well as sequenced and cloned. This GDP-mannose-3',5'-epimerase was isolated from Chlamydomonas reinhardtii and has an amino acid sequence represented herein by SEQ ID NO:21. SEQ ID NO:21 is encoded by a nucleic acid sequence of SEQ ID NO:20. A second amino acid sequence for a GDP-mannose-3 ',5 '-epimerase from Chlamydomonas reinhardtii is represented herein by SEQ ID NO: 10. SEQ ID NO: 10 is the deduced amino acid sequence encoded by the cDNA sequence assembled from five Chlamydomonas reinhardtii ESTs of unknown function that is represented herein by SEQ ID NO:9. Although the cloned sequence represented by SEQ ID NO:20 (encoding SEQ ID NO:21) is believed to be the more accurate representation of the GDP-mannose-3',5'-epimerase from Chlamydomonas reinhardtii, SEQ ID NO:9 is also likely to encode a functional GDP-mannose-3',5'-epimerase, and therefore is encompassed by the present invention. Indeed, SEQ ID NO:9 is a homologue of SEQ ID NO:20 and SEQ ID NO: 10 is a homologue of SEQ ID NO:21. In addition, the present inventors have purified to substantial homogeneity a second algal GDP-mannose-3',5'-epimerase from Prototheca. Once the identity of a verified algal GDP-mannose-3',5'-epimerase was discovered, this sequence can be used to identify and clone other algal GDP-mannose-3',5'-epimerase sequences as discussed above.

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: 10 and SEQ ID NO:21, over the full length of either 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 : 21. 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, or any percent identity between 70% and 99%, in whole integers (i.e., 71%, 72%, etc.), to either of SEQ ID NO:10 or SEQ ID NO:21, preferably over the full length of either of such sequences.

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: 10 or SEQ ID NO:21, and most preferably to SEQ ID NO: 10, 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% identical, and more preferably at least about 98% identical, and more preferably at least about 99%o identical, or any percent identity between 70% and 99%, in whole integers (i.e., 71%, 72%, etc.), to either of SEQ ID NO: 10 or SEQ ID NO:21, and most preferably to SEQ ID NO:21, 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, andmore preferably 250, and more preferably 275, and more preferably 300, and more preferably 325, and more preferably 350 amino acids of either 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 either one or both of SEQ ID NO: 10 and SEQ ID NO:21. 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 either of the above-identified amino acid sequences, and in another embodiment, is less than is less than 98% identical to either of the above-identified amino acid sequences, and in another embodiment, is less than 97% identical to either of the above-identified amino acid sequences, and in another embodiment, is less than 96% identical to either of the above-identified amino acid sequences, and in another embodiment, is less than 95% identical to either of the above-identified amino acid sequences, and in another embodiment, is less than 94% identical to either of the above-identified amino acid sequences, and in another embodiment, is less than 93% identical to either of the above- identified amino acid sequences, and in another embodiment, is less than 92% identical to either of the above-identified amino acid sequences, and in another embodiment, is less than 91 % identical to either of the above-identified amino acid sequences, and in another embodiment, is less than 90% identical to either of the above-identified amino acid sequences. In one embodiment, a GDP-mannose-3',5'- epimerase according to the present invention has an amino acid sequence that is less than 100% identical, or less than 99% identical, and so on in whole integer increments, down to less than about 90% identical, to an amino acid sequence encoded by any one or more of the nucleic acid sequence represented by SEQ ID NO:4-8.

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 either one of SEQ ID NO: 10 or SEQ ID NO:21 , and most preferably SEQ ID NO:21 , (i.e., 30 contiguous amino acid residues having 100% identity with 30 contiguous amino acids of either 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 either of SEQ ID NO: 10 or SEQ ID NO:21, and most preferably SEQ ID NO:21. 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 : 10 or SEQ ID NO :21 , and most preferably SEQ ID NO:21. 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:9 or SEQ ID NO:20, with SEQ ID NO:9 being particularly preferred. Such hybridization conditions are described in detail below.

Anucleic 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.

As used herein, reference to hybridization conditions refers 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 ^CC, 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).

A particularly preferred protein of the present invention comprises an isolated GDP-mannose- 3',5'-epimerase comprising an amino acid sequence selected from SEQ ID NO: 10 or SEQ ID NO:21, or a fragment of such sequence that has GDP-mannose-3',5'-epimerase biological activity. GDP- mannose-3',5'-epimerases comprising SEQ ID NO: 10 or SEQ ID NO:21 can include monomers, dimers, or multimers comprising such sequences.

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-3',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 (e.g., a second enzyme function); 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 canbejoinedto 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.

In one embodiment of the present invention, any of the amino acid sequences described herein 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 ends of the specified amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of the specified 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 specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino 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 given amino acid sequence 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 specified amino acid sequence 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 specified amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.

Another embodiment of the present invention relates to a composition comprising at least about 500 ng, and preferably at least about 1 μg, and more preferably at least about 5 μg, and more preferably at least about 10 μg, and more preferably at least about 25 μg, and more preferably at least about 50 μg, and more preferably at least about 75 μg, and more preferably at least about 100 μg, and more preferably at least about 250 μg, and more preferably at least about 500 μg, and more preferably at least about 750 μg, and more preferably at least about 1 mg, and more preferably at least about 5 mg, of an isolated GDP-mannose-3',5'-epimerase comprising any of the GDP-mannose-3',5'-epimerases or homologues thereof discussed herein. Such a composition of the present invention can include any carrier with which the protein is associated by virtue of the protein preparation method, a protein purification method, or a preparation of the protein for use in any method according to the present invention.

Further embodiments of the present invention include nucleic acid molecules that encode a GDP-mannose-3',5'-epimerase. A nucleic acid molecule of the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence encoding any of the isolated GDP-mannose-3',5'-epimerases, including a GDP-mannose-3',5'-epimerase homologue, described above. In a preferred embodiment a nucleic molecule of the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence represented by SEQ ID NO:9, SEQ ID NO:20, or fragments or homologues thereof encoding a GDP-mannose-3 ',5'-epimerase or portion thereof (e.g., fragment thereof) useful in the invention, or encompassing useful oligonucleotides and complementary nucleic acid sequences.

More particularly, one embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a protein comprising an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from SEQ ID NO: 10 or SEQ ID NO:21 , over the full length of either of such sequences, wherein the protein is a GDP-mannose-3 ',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, or any percent identity between 70% and 99%, in whole integers (i.e., 71%, 72%, etc.), to either of the above- identified amino acid sequences, with SEQ ID NO:21 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 either of SEQ ID NO: 10 or SEQ ID NO:21 over at least 50 amino acids, and more preferably 100, 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 either of SEQ ID NO: 10 or SEQ IDNO:21. Preferably, theproteinhas 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: 10 or SEQ ID NO:21, or to the amino acid sequences encoded by any one or more of SEQ ID NOs:4-8. 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 : 10 or SEQ ID NO:21. 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:9 or SEQ ID NO:20, with SEQ ID NO: 20 being particularly preferred.

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:9 or SEQ ID NO:20, 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:9 or SEQ ID NO:20. 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 fragment of any of the GDP-mannose-3 ',5'-epimerase proteins described above. 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: 10 or SEQ ID NO:21, or fragments of such sequences that encode a GDP-mannose-3',5'-epimerase having biological activity. Particularly preferred nucleic acidmolecules of the present invention comprise SEQ ID NO: 9 and SEQ ID NO: 20, 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. 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 acidmolecules 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.

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 per se (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 GDP-mannose-3',5'-epimerase encoding sequence, a nucleic acid sequence encoding a full-length GDP-mannose-3',5'-epimerase (including a GDP-mannose-3',5'- epimerase gene), including any length fragment between about 20 and 1155 nucleotides, in whole integers (e.g., 20, 21, 22, 23, 24, 25 1118, 1155 nucleotides), or multiple genes, or portions thereof. 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 (e.g., monomer) 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-3',5'-epimerase 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 (nptIT) 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-e«o/pyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic CellMol. 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 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 expression control sequences (including transcription and translation 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 an expression control sequence 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, Agrobacterium 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

Journal 2(3):29l- 00 (1992)), Brassica napus A S3 (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 ACEl 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 TnlO (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_R and P_L promoters, and the Escherichia coli lac, 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 35 S 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)). Other suitable 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 acidmolecules 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-Dalgarno 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, phosphomarmomutase, 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, phosphomarmomutase, 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.

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" mMethods 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 rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, CL, Crit. Rev. Plant. Sci. 10: 1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-medi&ted 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 CaCL, precipitation, polyvinyl alcohol or poly-L-omithine 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. Dorrn et al., In Abstracts of Vllth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24:51-61 (1994). Accordingly, it is the obj ect 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-3 ',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-3',5'- epimerase (e.g., specific activity or in vivo enzymatic activity), reduced inhibition or degradation of the GDP-mannose-3 ',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 anon-native promoter that gives higher levels of expression than that of the native promoter (i.e., the GDP- mannose-3',5'-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',5'-epimerase. For example, the activity of a GDP-mannose-3 ',5'- epimerase of the present invention can be decreased by blocking or reducing the production of the GDP-mannose-3',5'-epimerase, "knocking out" the gene orportion 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-3',5'-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-3',5'-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, or to replace a portion of an endogenous gene with a heterologous sequence, such as an improved GDP-mannose-3',5'-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-3',5'-epimerase. Preferably the 5' upstream sequence of a endogenous gene encoding a 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-3',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 aplant canbe 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- 3',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., 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 Chlamydomonas, 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 (i.e., Escherichia coli), although acid-tolerant bacteria are more preferred in some aspects, 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, Candida, Hansenula, Pichia, Kluveromyces, and Phaffia.

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 genetically modified plants (transgenic plants) or parts of such plants that are transformed with a recombinant nucleic acid molecule encoding a GDP-mannose-3',5'-epimerase of the present invention (including 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 apreferred embodiment, expression of the recombinant nucleic acidmolecule by the host cell or transgenic plant is sufficient to increase the synthesis of GDP-L-galactose by the host cell, which is useful in a method to produce L-galactose (discussed below). In another 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 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 homologues thereof); 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, phosphomarmomutase, 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 acidmolecule comprising a nucleic acidsequence encodingaGDP-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 a 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.

A related embodiment of the present invention relates to a method to increase ascorbic acid synthesis in an algal host cell comprising an endogenous GDP-mannose-3 ',5 '-epimerase as described herein, comprising introducing into the genome of the cell a non-native promoter upstream of a gene encoding the at least one monomer of the GDP-mannose-3',5'-epimerase. Such a non-native promoter can include, but is not limited to, other algal promoters or promoters that can be used in algal cells. 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 GDP-mannose-3 '5 '-epimerase of the invention, 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 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-3',5'-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',5'-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',5'-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 algal GDP-mannose-3',5'-epimerases as described previously herein, including the epimerase having an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:21. 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 imrnunoassay), 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.

GDP-L-galactose is an intermediate product in the ascorbic acid biosynthesis, but can be an interesting compound on its own and is particularly useful herein in a method to produce L-galactose. Modulation of the production of GDP-L-galactose 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 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 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 into GDP-L-galactose by the GDP-mannose-3',5'-epimerase according to the invention. GDP-L-galactose may be used to produce L-galactose, for example by recovering and/or purifying the GDP-L-galactose and then treating the GDP-L-galactose to produce L-galactose, or, more preferably, by converting the GDP-L-galactose to L-galactose directly after fermentation or the enzymatic reaction (i.e., without first purifying or recovering the GDP-L-galactose from the fermentation culture, fermentation broth, or enzymatic reaction medium), followed by recovery and/or purification of the L-galactose. Suitable procedures for converting GDP-L-galactose to L-galactose are known in the art and include hydrolysis of GDP-L-galactose under acid conditions, and preferably mild acid conditions, to produce L- galactose, or enzymatically treating the GDP-L-galactose to produce L-galactose.

In one aspect of this embodiment, an isolated GDP-mannose-3',5'-epimerase according to the invention is contacted with the substrate (GDP-D-mannose) under conditions suitable for enzymatically producing GDP-L-galactose. Suitable conditions are known in the art and are described, for example, in the Examples section. In one aspect, the GDP-mannose-3',5'-epimerase is bound to a solid support, i.e. , an immobilized enzyme. As used herein, a GDP-mannose-3 ',5 '-epimerase bound to a solid support (i.e., an immobilized GDP-mannose-3',5'-epimerase) includes immobilized isolated GDP-mannose-3',5'- epimerase, immobilized cells which contain a GDP-mannose-3',5'-epimerase (including immobilized bacterial, fungal (e.g., yeast), microalgal, insect, plant or mammalian cells), stabilized intact cells and stabilized cell/membrane homogenates. Stabilized intact cells and stabilized cell/membrane homogenates include cells and homogenates from naturally occurring microorganisms expressing GDP-mannose-3 ',5'-epimerase or from genetically modified microorganisms, insect cells or mammalian cells as disclosed elsewhere herein. Thus, although methods for immobilizing GDP-mannose-3',5'- epimerase are discussed below, it will be appreciated that such methods are equally applicable to immobilizing bacterial and other cells and in such an embodiment, the cells can be lysed.

A variety of methods for immobilizing an enzyme are disclosed in Industrial Enzymology 2nd Ed., Godfrey, T. and West, S. Eds., Stockton Press, New York, N.Y., 1996, pp.267-272; Immobilized Enzymes, Chibata, I. Ed., Halsted Press, New York, N.Y., 1978; Enzymes and Immobilized Cells in Biotechnology, Laskin, A. Ed., Benjamin/Cummings Publishing Co., Inc., Menlo Park, California, 1985; and Applied Biochemistry and Bioengineering, Vol. 4, Chibata, I. and Wingard, Jr., L. Eds, Academic Press, New York, N.Y., 1983, which are incorporated herein in their entirety. Briefly, a solid support refers to any solid organic supports, artificial membranes, biopolymer supports, or inorganic supports that can form a bond with GDP-mannose-3',5'-epimerase without significantly effecting the activity of isolated the GDP-mannose-3',5'-epimerase. Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers (e.g., polyacrylamide), stabilized intact whole cells, and stabilized crude whole cell/membrane homogenates. Exemplary biopolymer supports include cellulose, polydextrans (e.g., Sephadex®), agarose, collagen and cliitin. Exemplary inorganic supports include glass beads (porous and nonporous), stainless steel, metal oxides (e.g., porous ceramics such as ZrO₂, TiO₂, Al₂O₃, and NiO) and sand. Preferably, the solid support is selected from the group consisting of stabilized intact cells and/or crude cell homogenates . Preparation of such supports requires a minimum of handling and cost. Additionally, such supports provide excellent stability of the enzyme.

Stabilized intact cells and/or cell/membrane homogenates can be produced, for example, by using bifunctional crosslinkers (e.g., glutaraldehyde) to stabilize cells and cell homogenates. In both the intact cells and the cell membranes, the cell wall and membranes act as immobilizing supports. In such a system, integral membrane proteins are in the "best" lipid membrane environment. Whether starting with intact cells or homogenates, in this system the cells are either no longer "alive" or "metabolizing", or alternatively, are "resting" (i.e., the cells maintain metabolic potential and active GDP-mannose-3 ',5'-epimerase, but under the culture conditions are not growing); in either case, the immobilized cells or membranes serve as biocatalysts.

GDP-mannose-3 ',5'-epimerases can be bound to a solid support by a variety of methods including adsorption, cross-linking (including covalent bonding), and entrapment. Adsoφtion can be through van del Waal' s forces, hydrogen bonding, ionic bonding, or hydrophobic binding. Exemplary solid supports for adsoφtion immobilization include polymeric adsorbents and ion-exchange resins. Solid supports in a bead form are particularly well-suited. The particle size of an adsoφtion solid support can be selected such that the immobilized enzyme is retained in the reactor by a mesh filter while the substrate (e.g., the oil) is allowed to flow through the reactor at a desired rate. With porous particulate supports it is possible to control the adsoφtion process to allow GDP-mannose-3 ',5'- epimerases or bacterial cells to be embedded within the cavity of the particle, thus providing protection without an unacceptable loss of activity. Cross-linking of a GDP-mannose-3 ',5'-epimerase to a solid support involves forming a chemical bond between a solid support and a GDP-mannose-3',5'-epimerase. It will be appreciated that although cross-linking generally involves linking a GDP-mannose-3',5'-epimerase to a solid support using an intermediary compound, it is also possible to achieve a covalent bonding between the enzyme and the solid support directly without the use of an intermediary compound. Cross-linking commonly uses a bifunctional or multifunctional reagent to activate and attach a carboxyl group, amino group, sulfur group, hydroxy group or other functional group of the enzyme to the solid support. The term "activate" refers to a chemical transformation of a functional group which allows a formation of a bond at the functional group. Exemplary amino group activating reagents include water-soluble carbodiimides, glutaraldehyde, cyanogen bromide, N-hydroxysuccinimide esters, triazines, cyanuric chloride, and carbonyl diimidazole. Exemplary carboxyl group activating reagents include water- soluble carbodiimides, and N-ethyl-5-phenylisoxazolium-3-sulfonate. Exemplary tyrosyl group activating reagents include diazonium compounds. And exemplary sulfhydryl group activating reagents include dithiobis-5,5'-(2-nitrobenzoic acid), and glutathione-2-pyridyl disulfide. Systems for covalently linking an enzyme directly to a solid support include Eupergit®, a polymethacrylate bead support available from Rohm Pharma (Darmstadt, Germany), kieselguhl (Macrosorbs), available from Sterling Organics, kaolinite available from English China Clay as "Biofix" supports, silica gels which can be activated by silanization, available from W.R. Grace, and high-density alumina, available from UOP (Des Plains, IL). Entrapment can also be used to immobilize the GDP-mannose-3',5'-epimerase. Entrapment of GDP-mannose-3',5'-epimerase involves formation of, inter alia, gels (using organic or biological polymers), vesicles (including microencapsulation), semipermeable membranes or other matrices. Exemplary materials used for entrapment of an enzyme include collagen, gelatin, agar, cellulose triacetate, alginate, polyacrylamide, polystyrene, polyurethane, epoxy resins, carrageenan, and egg albumin. Some of the polymers, in particular cellulose triacetate, can be used to entrap the enzyme as they are spun into a fiber. Other materials such as polyacrylamide gels can be polymerized in solution to entrap the enzyme. Still other materials such as polyglycol oligomers that are functionalized with polymerizable vinyl end groups can entrap enzymes by forming a cross-linked polymer with UV light illumination in the presence of a photosensitizer. GDP-L-galactose produced by a method of the present invention can be recovered by conventional methods and then further modified, as described above, to produce L-galactose, for example.

Each publication or reference cited or described herein is incoφorated herein by reference in its entirety.

The following examples are provided for the pvnpose of illustration and are not intended to limit the scope of the present invention. EXAMPLES Example 1

This example describes the identification of a GDP-mannose-3',5'-epimerase gene and protein from Chlamydomonas.

Several public DNA sequence databases were scanned for sequences that exhibited similarity to the Arabidopsis thaliana gene that was known to encode GDP-mannose-3',5'-epimerase (three sequence database entries were found for the Arabidopsis thaliana gene, as listed in Table 2; SEQ ID NOs: 1-3). Sequences that showed high similarity at both the DNA and protein level were identified in the Chlamydomonas EST sequence database (SEQ ID NO:s 4 to 8). However, none of these sequences appeared to encode a full-length protein.

TABLE 2

% Identity % Identity

Open Reading to A to A

Organism Database Entry Numbers Frame/Protein thaliana thaliana

DNA Protein

Arabidopsis NCBI NM 122767 1134 bp 100% 100% thaliana AY057660 377 aa AY057694

Chlamydomonas Chlamydomonas AV621729.1 1185 bp 63.2% 73.2% reinhardtii Genome Project AV634490.1 394 aa

AV620290.1

20011023.11431

20011023.1143.2

The full-length Chlamydomonas sequence was assembled from five EST entries in the Chlamydomonas Genome Project sequence database (SEQ ID NOs:4-8). The first four were identified by BLAST search using the Arabidopsis sequence as the query and standard search parameters. A consensus sequence derived from these first four sequences was assembled, and this consensus sequence was used to BLAST search the Chlamydomonas database, also using standard search parameters. This search revealed the fifth cDNA sequence corresponding to the 5'-end of the open reading frame. The fifth sequence was then compiled with the first consensus sequence to generate a second consensus sequence that contains what appears to be the entire open reading frame. The five database entries are AV621729.1 (SEQ ID NO:4), AV634490.1 (SEQ ID NO:5), AV620290.1 (SEQ ID NO:6), 20011023.11431 (SEQ ID NO:7), and 20011023.1143.2 (SEQ ID NO:8). These EST sequences overlap and can be assembled into a consensus sequence that contains an open reading of 1185 base pairs, including putative ATG start and TGA stop codons, represented herein by SEQ ID NO:9. Alignment of this DNA sequence with the Arabidopsis gene showed 63.2% identity over the entire lengths of their open reading frames. The Chlamydomonas open reading frame sequence has a 51 bp extension at its 3'-end as compared to the Arabidopsis sequence. SEQ ID NO:9 encodes a 394 amino acid sequence represented herein by SEQ ID NO: 10. Example 2

This example describes methods for the construction of cDNA from algae. 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 Coφoration

(Madison, WI). 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), ibid. 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, L, Revantos, D., Jaeck, E., and San Degundo, B., Promega Notes Magazine, Number 54, 1995, 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 algae, including Chlamydomonas, Prototheca, and Chlorella. A 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 Coφoration 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.

Since the DNA sequence of the putative epimerase open reading frame from Chlamydomonas is publicly available, oligonucleotide primers can be designed and combined with the cDNA library in PCR reactions to amplify that gene. Alternatively, the Chlamydomonas cDNA can be used directly in PCR reactions to amplify the desired sequences without the need to create a cDNA library. The epimerase genes from other algae such as Prototheca and Chlorella can be obtained by screening their cDNA libraries using a DNA probe derived from a highly conserved region of the

Arabidopsis epimerase gene. Comparison of the Arabidopsis DNA sequence to the putative GDP- mannose-3',5'-epimerase gene from Chlamydomonas revealed several regions where the DNA sequence similarities were reasonably high among the four sequences (greater than 75%) . These regions of DNA can be amplified by PCR using the Arabidopsis gene or Chlamydomonas cDNA library as template.

The amplified DNA will then be purified and used to prepare DNA probes labeled with radioactive or biotinylated nucleotides. This labeling will involve using random primer oligonucleotides and Klenow fragment enzyme. Kits are available fromNew England Biolabs (Beverly, MA) to achieve the labeling of these DNA fragments. Colony lifts of the cDNA libraries can be screened using these probes as described in Maniatis et al (1982) to identify those colonies that contain cDNA clones with sequences similar to the Arabidopsis epimerase gene. Those clones can be rescued by DNA miniprep procedures, and sequenced to determine the length and sequence similarity to the Arabidopsis gene. Those clones that exhibit the highest similarity are tested for expression of GDP-mannose-3',5'-epimerase activity. In the present example, DNA preparations (e.g., cDNA, cDNA libraries synthesized from polyA+ mRNA as described above, and genomic DNA) were made from Chlamydomonas reinhardtii wild type cells. A cDNA library, CC-1690 WT mt+ 21gr, was purchased from the Chlamydomonas Genetic Center (Duke University). Genomic DNA was extracted and purified from fresh cultured cells (Chlamydomonas reinhardtii (WT) ATCC 18798) using a Puregene^® DNA Isolation Kit (Cat. D6000 A; Gentra Systems, Minneapolis, MN). These DNA preparations were used as templates for PCR amplification as described in Example 2 below. Example 3

This example describes PCR amplification of the putative epimerase sequences. PCR can be used to amplify the putative complete open reading frame of the gene encoding

Chlamydomonas GDP-D-mannose-3', 5 '-epimerase. DNA preparations (e.g., cDNA, cDNA libraries, and genomic DNA) were made from Chlamydomonas reinhardtii wild type cells or purchased from the Chlamydomonas Genetic Center (Duke University) as described in Example 2. These DNA preparations were used as templates for PCR amplification of the epimerase open reading frame. Primers (Table 3; SEQ ID NOs: 11 and 12) were designed and synthesized according to the putative full-length epimerase gene resulting from assembly of the DNA sequences identified in public databases into a putative full-length GDP-D-mannose-3, 5 epimerase of Chlamydomonas (SEQ ID NO:9).

The PCR reactions were carried out in a Robocycler Gradient 96 (Stratagene, La Jolla, CA). Upstream and downstream primers (Table 3) were designed according to the DNA sequences identified in databases as putative GDP-mannose-3 ',5'-epimerases from Chlamydomonas. Sequences corresponding to recognition sites for Neo I and Xho I were added to the 5'- and 3 '-ends of the oligonucleotides (indicated by lowercase letters) to facilitate cloning. The PCR reactions were carried out in a Robocycler Gradient 96 (Stratagene, La Jolla, CA) using the parameters listed in Table 4. Under optimized conditions using Pfu and FailSafe polymerases, a product with size of 1.2 kb was amplified from both genomic DNA and the Chlamydomonas cDNA library.

TABLE 3

Oligonucleotides for Amplification of Putative Epimerase Genes

Organism Upstream Primer Downstream Primer

Chlamydomonas aataccatgGCCACCGCCGCAGTTC taatctcgagTCAAATCGGCGCCAGCCCTG

(SEQ ID NO:11) (SEQ ID NO:12) TABLE 4

Conditions for the Amplification of Epimerase Sequences

Number of Cycles Temperature Time

1 94°C 30s

94°C 30s

30 45 to 55°C 30s

72°C 120s

1 72°C 600s

Fidelity of the PCR products was first determined by restriction enzyme digestion. Four enzymes, EcoN I, Mfe I, Neo I and Xho I, were chosen to digest the PCR products. The Chlamydomonas cDNA sequence should cut with EcoN I and Mfe I, based on the putative Chlamydomonas epimerase sequence assembled from the Chlamydomonas Genome Project database EST sequences, at positions 492 and 255 of the assembled putative full-length sequence (SEQ ID NO:9), respectively. Also, neither Neo I nor Xho I sites should exist. The expected restricting map was obtained when PCR products were individually digested with EcoN I, Mfe I and Neo I. However, the PCR product was cut unexpectedly by Xho I, resulting in two fragments with approximate sizes of 980 and 300 bp. Possible explanations for this observation were: 1) a sequencing error exists in the coding sequence in the Chlamydomonas Genome Project EST sequence database (a common occurrence); 2) the PCR product was nonspecifically amplified from the cDNA library; or 3) PCR introduced an error into the amplified product. Based on the result from other restriction enzyme digestions (data not shown), the first explanation seemed to be the most likely.

The PCR product was purified and cloned into the Srfl site of the vector pPCR-ScriptAmp SK(+) (Stratagene, La Jolla, CA) to confirm the sequence of the putative GDP-mannose-3 ',5'-epimerase open reading frame DNA. To determine the presence of the insert, 11 clones, including a negative clone (ligation reaction without PCR product), were analyzed by restriction digestion. Double digestions of the recombinant plasmids with Neo I and BamHl showed that six of them had the in- frame insert. As expected, no PCR product was seen in the double digested plasmid prepared from the control ligation reaction. Two clones containing the insert, Chlam.C1.4 and Chlam.C1.5, were sequenced using the T7 primer. Based on the results of restriction digestion and sequencing, the inserts in the two plasmids were oriented in same direction. Sequence covering the 3 '-end of Chlatnydomonas epimerase (from stop codon to position 663) was obtained. A restriction site for Xho I was found at position 952. Example 4

This example describes construction of vectors with the native form of Chlamydomonas epimerase open reading frame sequence for expression in E. coli. Construction of a vector for overexpression the epimerase under control of the T7 promoter system.

The expression vector, pETChlam.C1.6, was constructed for oveφroduction of the epimerase in its native form. The DNA fragment corresponding to the epimerase coding sequence was released from the pPCR-ScriptAmp SK(+) vector by double digestion with Neo I and BamH I. The nucleotide sequence of this fragment was determined by sequencing (see Example 5). The fragment was purified and then cloned into expression vector pET21d(+) (Novagen, Madison, WI), such that the epimerase open reading frame was placed under control of the T7 promoter, so that it would be transcribed by bacteriophage T7 RNA polymerase. The recombinant plasmid, pETChlam.C1.6 (Fig. 1), was confirmed by DNA sequencing (see Example 5), and then transformed into the E. coli BL21 (DE3) expression host that carries an integrated copy of the gene for T7 RNA polymerase under the control of the IPTG inducible lacUV5 promoter.

Construction of a vector for overexpression the epimerase under control of the T7 promoter system fused to a N-terminal S-tag. The epimerase protein could be produced from expression vectors in either an active or inactive form. Detection of functional expression of the epimerase relies, therefore, on assay of the epimerase activity from extracts of disrupted cells or from enzyme purified from these extracts. To facilitate confirmation of epimerase protein expression and determination of functional activity, the epimerase expression vectors, pETChlam.10 (Fig. 2) and pETChlam.l l, were constructed to oveφroduce N-terminal-S-tag-epimerase fusion proteins. The S-tag provides for simple purification of the enzyme by affinity chromatography.

The S-tag (Raines, 2000, Methods Enzymol. 326:362-76) can be used to purify the fusion protein to homogeneity in a single step . The entire S -tag can be removed by digestion with the protease, thrombin, resulting in a full-length epimerase. The insert was prepared by digesting pETChlam.C1.6 with Neo I and Hind HI. The resulting 1.2kb band was gel purified using the MinElute gel extraction kit from Qiagen (Valencia, CA). The fragment was then ligated to the pET29b(+) vector (Novagen, La Jolla, CA) at the Neo VHind HI sites using the Fast Link DNA ligation from Epicentre (Madison, WI). The ligation reactions were transformed into E. coli cells. Two clones, designated pETChlam.10 and pETChlam.11, were confirmed by restriction enzyme digestion with Neo I and Hind III, and by sequencing with T7 and T7-terminator (T7-ter) primers (see Example 5). Example 5

This example describes determination and analysis of expression vector nucleic acid sequences.

As discussed in Example 4, the epimerase open reading frame PCR product was sequenced before subcloning into the expression vector pET21 d(+) . Differences were found between the putative complete open reading frame of the gene encoding Chlamydomonas GDP-D-mannose-3 ',5'-epimerase assembled from EST sequences in the Chlamydomonas Genomic database (SEQ ID NO:9) and sequences derived by PCR from Chlamydomonas cDNA. To confirm the entire coding sequence for Chlamydomonas epimerase, the expression plasmid pETChlam.6, which was functionally expressed in E. coli (see Example 6), was subjected to a more detailed nucleic acid sequence analysis.

Sequencing with the T7 primer, 656 bp of Chlam.C1.6 nucleic acid sequence corresponding to positions 1 to 605 of the assembled (from EST sequences) putative Chlamydomonas open reading frame was determined (97% similarity of Chlam.C1.6 sequence to the assembled sequence). Sequencing with the T7-ter primer resulted in a sequence of 704 bp with 98% similarity to the assembled sequence from positions 1185 to 590. Two specific sequencing primers, 2003-3 (5'- atgggcttcatccagtccaac-3'; SEQ ID NO:13) and 2003-4 (5'-cttgtcgtcgaaggacatg-3'; SEQ ID NO:14) were designed in order to confirm the coding sequence from positions 319 to 886. Overlapping sequence from additional internal primers (SEQ ID NOs: 15 to 19) provided overlapping nucleic sequence from both strands. Alignment of sequences obtained with these external and internal primers allowed determination of the complete coding sequence for the Chlamydomonas epimerase, which is represented herein by SEQ ID NO:20.

Nucleotide (SEQ ID NO:20) and derived amino acid (SEQ H) NO:21) sequences of the cloned Chlamydomonas epimerase were compared with public database sequences using standard nucleotide BLAST (Blastn) and standard protein BLAST (Blastp) (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, incoφorated herein by reference in its entirety). Two new sequences that showed homology to the cloned Chlamydomonas epimerase gene were identified in genomic sequences of rice (Database AccessionNo. AEO17094) andmaize (Database Accession No. AY104279). Specifically, the cloned Chlamydomonas epimerase is about 66.4% or 64.4% identical to the maize or rice sequences, respectively, and is about 71.4% or 71.2% identical to the proteins encoded by the maize or rice sequences, respectively. TABLE 5

Assembled cDNA position Putative Epimerase Cloned Epimerase

(Database Sequence)

777 to 778 CT TC

795 T G

954 G C

999 G C

1128 T G

1131 G T

1152 to 1154 NGT GTA

The cloned Chlamydomonas epimerase nucleic acid sequence possesses an open reading frame of 1155 bp, represented herein by SEQ ID NO:20, which is 30 bp shorter than the assembled putative epimerase sequence derived from the five EST sequences (1185 bp; SEQ ID NO:s 1 to 8). Therefore, the cloned Chlamydomonas epimerase nucleic acid sequence is about 97% identical to the nucleic acid sequence derived from the EST assembly. SEQ ID NO:20 encodes a Chlamydomonas GDP-mannose- 3',5'-epimerase having the amino acid sequence of SEQ ID NO:21. The calculated molecular mass of this algal epimerase is 43,178 Daltons (62.5% G+C). A alignment analysis of sequences from genes cloned and assembled from database EST epimerase sequences is shown in Table 5. When the cDNA sequence was compared to the assembled Chlamydomonas epimerase sequence from the Chlamydomonas Genomic EST database (SEQ ID NO:9), seven nucleic acid differences in the cloned cDNA (indicated in Table 5 with respect to SEQ ID NO: 20) resulted in three differences in amino acids at positions 376, 384 and 385. The cloned epimerase gene sequence also defines five new restriction enzyme sites (position in parentheses with respect to SEQ ID NO:20): Bpm I (559), Bsm I (773), Nae I (860), NgoMIV (855) mdXho I (952).

Unlike the Arabidopsis epimerase, the Chlamydomonas epimerase did not contain any introns, since the PCR product amplified from Chlamydomonas genomic DNA had the same size as that obtained from the cDNA library. Example 6 This example describes functional expression of recombinant epimerases in E. coli.

E. coli BL21(DE3) was transformed with the epimerase expression plasmid pETChlam.6. Three transformants were cultured individually overnight. For protein expression experiments, 50 ml cultures were inoculated from 1 ml of overnight culture and grown in Luria Broth medium containing ampicillin (100 μg/ml) at 37°C with shaking until the OD₆₀₀ reached approximately 0.6 to 1.0. The cultures were allowed to equilibrate for 30 min with shaking at the induction temperature of either 25 or 37 °C prior to the addition of the IPTG inducer (0.4 mM). The cultures were incubated for an additional period of two hours with shaking. The induced cells were harvested by centrifugation (4,000 x g for 20 min at 4°C) and washed with 50 mM Tris buffer, pH7.0. The cells were either stored at -20 ° C or used immediately to prepare enzyme extracts for assay as described below. As a control, cell extracts were prepared from host strains transformed with empty vector (no insert). Protein expression level was monitored by SDS-PAGE analysis as shown in Figure 3 (lane 1, induced empty vector pET 29b(+); lane 2, uninduced pETChalm.10; lane 3 , 4 and 5, 0.4 mM IPTG induced pETChlam.10 and two clones of pETChlam.6).

All recombinant E. coli strains oveφroducing recombinant epimerase under the control of the T7 promoter produced 43 or 48 kDa peptides (data not shown), which, based on the cloned gene sequence (SEQ ID NO:20), is the expected size of the epimerase peptide and the S-tag-epimerase fusion proteins. Of the proteins that could be observed by SDS-PAGE, the oveφroduced recombinant epimerase protein was in the greatest abundance, typically representing approximately 80 to 90% of the total protein in the cell-free extract. Under the conditions used, 80 to 100 mg of recombinant protein was obtained from one liter of shake flask culture. The expression level was slightly higher at 37°C than at 25°C Both native and S-tag-epimerase fusion proteins were recovered in the soluble protein fraction. Example 7

This example describes purification of Chlamydomonas S-tag-epimerase and release of full- length epimerase by cleavage with thrombin.

Taking advantage of the high solubility of S-tag-epimerase fusion protein, the fusion protein was purified to homogeneity from the soluble fraction using S -protein agarose (Novagen, Madison, WI). The purified fusion protein was subjected to a mild proteolysis by biotinylated thrombin. After cleavage at 4°C overnight plus one hour at room temperature, streptavadin agarose was added to the cleavage reaction to bind the biotinylated thrombin. The agarose mixture was added to a chromatography column and purified Chlamydomonas native epimerase was collected by gravity flow through the column. Cleavage and purity of the released epimerase protein was confirmed by SDS- PAGE and Western blotting (data not shown). Following elution from the S -protein agarose affinity column, protein samples were loaded and separated in 4 to 12% Bis-Tris agarose gels. The gel was electroblotted to a nitrocellulose membrane and probed with anti-S-tag antibody. The blot was visualized with NBT/BCIP (Sigma). The concentration of purified epimerase was determined by SDS- PAGE using BSA as a standard. The Chlamydomonas epimerase, purified to homogeneity, was used to characterize the enzyme. Example 8

This example describes methods to assay epimerase activity in E. coli extracts.

Using cell-free extracts, the oveφroduced proteins were analyzed for functionality, by measuring conversion of GDP-D-mannose to GDP-L-galactose. This was done using whole cell extracts, cell-free extracts, or purified proteins. Cell-free extracts were prepared from induced E. coli transformant cells using the BugBuster^® Protein Extraction Reagent (Novagen, Madison, WI).

The enzyme assay mixture (final volume of 50 μl) was composed of cell-free extract (50 μg protein), ¹⁴C-labeled GDP-D-mannose (15 μM) in 50 mM NaPi buffer, pH 7.2 containing 2 mM (each) of EDTA and DTT. The mixture was incubated between 30 min and 15 hours at room temperature. Reactions were stopped by placing the assay mixture in a boiling water bath for 1 min. Trifluoroacetic acid (TFA; 0.5 M final concentration) was added and the mixture was incubated at 100°C for 30 min to hydrolyze the GDP moiety of the sugar nucleotides, releasing free sugars.

Thin Layer Chromatography (TLC) was used to separate the mixture of free sugars. Briefly, aliquots (5 μl) of the hydrolyzed assay mixture were loaded on Silica Gel 60 plates (20x20 cm, EM Science, Gibbstown, NJ). The TLC plates were then developed in acetone-n-butanol-water (8:1:1 [vol/vol/vol]). The amount of radioactivity in resolved free sugars on the developed TLC plate was detected and quantified using a phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). TLC analysis of free sugars from the acid hydrolysis of epimerase assay was performed (data not shown) for: recombinant Arabidopsis native epimerase (pETArab-EP); S-tag Chlamydomonas epimerase (pETChlam.10 and pETChlam.11); empty vector [pET21d (+)]; and recombinant Chlamydomonas native epimerase [pETChlam.6]. ImageQuant software allowed quantification of the ¹⁴C-radioactivity in the L-galactose sugar products. Epimerase activity was expressed as activity units (Units) unless otherwise indicated. One Unit of epimerase activity is defined as one nmole of L-galactose formed per min. Enzyme specific activity is defined as Units mg^"1 protein.

Cell-free extracts from bacterial cells oveφroducing recombinant native or S-tag-epimerase fusion protein converted GDP-D-mannose to GDP-L-galactose, confirming that the Chlamydomonas epimerase was functionally expressed. No significant difference in enzyme activity was observed between cells induced at 37°C or 25°C. Cell-free extracts had an average epimerase specific activity of 2.1 Units mg^"1 protein as compared with a value of 1.7 Units mg^"1 protein for recombinant

Arabidopsis epimerase expressed inE. coli (Arabidopsis recombinant epimerase activity was measured in parallel). Example 9

This example describes properties of recombinant Chlamydomonas epimerase. The molecular mass of purified native active Chlamydomonas

Native PAGE (4 to 12%, Tris-Glycine; Cambrex Coφoration, East Rutherford, NJ) was run at 4°C in lx Tris-Glycine running buffer to determine the Mr of the native epimerase. Mr was determined by comparing the Ry of the Chlamydomonas epimerase with that of the high molecular weight standards (Pharmacia, Piscataway, NJ): thyroglobllin (669 kDa), ferritin (440 kDa), catalase

(232 kDa), lactate dehydrogenase (140 kDa) and BSA (67 kDa). Purified epimerase was eluted from an S -protein affinity column and the native protein was released from the fusion protein by treatment with thrombin. After confirming activity, high molecular weight standards and the purified epimerase were separated in a 4 to 12 % Tris-Glycine agarose gel at 4°C (data not shown). A plot of log Mr vs. the

Ry of the protein relative to that of the molecular weight standards on the gel was used to estimate the

Mr (43 kDa) of the recombinant epimerase. Referring to Fig. 3, the arrow indicates the Ry of native

Chlamydomonas epimerase. As shown in Fig. 3, the Mr of the native Chlamydomonas epimerase was estimated to be 90 kDa, consistent with a native epimerase homodimer of two 43 kDa subunits, similar to the Arabidopsis native epimerase which is a homodimer. pH dependence

The pH dependence of the epimerase activity was determined using 50 mM Tris-HCl and potassium phosphate buffer in the range of 6.0 to 9.0 with 15 μM GDP-D-mannose as substrate. Chlamydomonas epimerase had an optimum pH of 7.2 and 8.2 in Tris and potassium phosphate buffer solutions, respectively (Fig. 4).

Epimerase kinetic properties

The purified recombinant native epimerase was incubated with 15 μM GDP-D-mannose at room temperature. A time course of the reaction showed that the enzyme activity remained linear for up to a 60 min incubation time (Fig. 5).

The initial reaction velocity was strongly dependent on substrate concentration (Fig. 6). Substrate inhibition was observed at concentration greater than 120 μM. Fig. 8 shows a Lineweaver- Burk plot used to derive epimerase K., and V_max values. The apparent K_m and V_max values for GDP-D- mannose were of 7.6 ±0.3 μMand22.4± l nmolmin^"1 g^"1 protein, respectively. Addition of GDP, L-ascorbic acid and or L-galactono-γ-lactone increased the apparent K-, (Table 6).

TABLE 6

Concentration Vmax

Addition Km (μM) (μM) (nmol min^"1 mg^"1)

None - 7.6 20.7

GDP 2 11.9 13.7

4 52.9 33.6

L-ascorbic acid 20 38.5 18.8

40 42.9 18.5

L-galactono-γ-lactone 20 8.8 13

Inhibitory effect of GDP-L-fucose

The effect of GDP-L-fucose on epimerase activity was determined at a fixed concentration of GDP-D-mannose (15 μM). Enzyme activity was dramatically reduced with an increasing concentration of GDP-L-fucose. The inhibition constant, Ki calculated from secondary plots (1/v vs. [S]) was 55 μM

(Fig. 7).

Effect of cofactors and metal ions

The effect of cofactors and metal ions on epimerase activity is shown in Table 7. Of the cofactors and metal ions tested, only NAD had a slight stimulatory effect on enzyme activity. The inclusion of NADP, NADH, DTT, EDTA and Ca^÷÷ (as CaCl₂) in the reaction had no significant effect on the enzyme activity. Other metal salts, L-ascorbic acid and ADP-D-glucose inhibited enzyme activity by 10 to 90%. The addition of 1 mM GDP, GDP-D-glucose or GDP-L-fucose resulted in total loss of epimerase activity.

TABLE 7

Concentration Specific Activity % of Control

Addition (mM) (nmol min^"1 mg^"1) Activity

None 14.2 100

NAD 18.9 129

NADP 14.1 99

EDTA 13.9 98

L-ascorbic acid 12.3 87

NADH 11.9 84

L-galactono-γ-lactone 11.7 82

ADP-D-glucose 11.5 81

DTT 10.5 74

GDP 0 0

GDP-L-fucose 0 0

GDP-D-glucose 0 0

CaCI₂ 5 13.1 92

MgCI₂ 5 8.8 62 Concentration Specific Activity % of Control

Addition

(mM) (nmol min^'1 mg^"1) Activity

MnCI₂ 5 7.7 54 FeCI₃ 5 6.1 43 ZnCI₂ 5 5.4 38 CuCI₂ 5 1.6 11

Example 10

This example describes cell growth and extraction of epimerase and other enzymes from microalgae.

Chlorella pyrenoidosa UTEX 1613 (WT) and UTEX 166-4 (mutant); Prototheca moriformis ATCC 75669 (WT) and ATCCPTAl 11 (mutant) and Chlamydomonas reinhardtii dangerard ATCC 18798 (WT) were analyzed. All algal cells were cultured in ATCC medium #5 (ATCC, Manassas, VA) unless otherwise indicated. Growing cultures of Prototheca moriformis and Chlorella pyrenoidosa were harvested (in 1.0 ml of fresh broth) from slants was used to inoculate 50 ml of fresh broth medium. Cultures were grown heterotrophically at 37 °C with agitation (200 φm). Chlamydomonas reinhardtii was incubated under an illumination of 100 μEinsteins m^"2 s^"1 from cool-white fluorescent tubes at 26°C with a 14/10 h light/dark photoperiod. Cells were harvested by centrifugation at 2,000 x g for 10 min at 4°C when the culture OD₆₀₀ reached 2 to 2.5. Cell pellets were washed once with buffer (50 mM KPi, pH 7.2, 100 mM NaCl, 10% glycerol, 2 mM (each) of EDTA and DTT). The cells were either used immediately to prepare enzyme extracts for activity determination or frozen in liquid nitrogen for storage at -80 °C.

To prepare enzyme extracts, fresh or thawed frozen cells were resuspended in buffer at 4°C and disrupted by: 1) passing through an ice-cold SLM Aminco French Pressure cell at 10,000 psi, or 2) grinding frozen cells in liquid nitrogen to a powder in a mortar and pestle; the broken cell paste was resuspended in ice-cold breakage buffer. Disrupted cell suspensions were then sonicated three times at 4 ° C, 30 seconds per sonication. Debris was removed by a centrifugation at 4,000 x g for 10 min. The supernatant was then centrifuged at 18,000 x g for 60 min. The supernatants were further centrifuged at 45,000 φm at 4 °C for 1 hour. This clarified supernatant was designated the soluble enzyme fraction. The pellet, solubilized with 1% (wt/vol) n-octyl-β-D-glucoside (OG) was designated the insoluble enzyme fraction. Protein concentration was detected by its UV absoφtion at 280 nm or by the Bradford method using bovine serum albumin as standard. Example 11

This example describes methods to assay epimerase activity in algal extracts. The expressed natural or recombinant epimerase proteins were analyzed for the ability to convert GDP-D-mannose to GDP-L-galactose. This can be done with either whole cell extracts, cell- free extracts or purified protein.

Unless otherwise specified, the GDP-D-mannose-3,5-epimerase (EC 5.1.3.18; epimerase) assay was carried out at room temperature in a final volume of 50 μl. In a typical enzyme assay, an appropriate amount (10 to 50 μg) of the enzyme extract was incubated with 0.1 to 1.0 nmol of ¹⁴C- labeled GDP-D-mannose substrate (uniformly in the D-mannosyl moiety [specific activity 52.7 μCi/μmol]; Perkin Life Sciences [Boston, MA]) in assay buffer (50 mM NaPi buffer, pH 7.2, 2 mM [each] of EDTA and DTT). A blank (control) was run under identical conditions in parallel with denatured enzyme (boiled at 100°C for 10 min). After the desired time of incubation (5 to 240 minutes), the reactions were stopped by placing the mixtures in a boiling water bath for 1 minute and then hydrolyzed in 0.5 M TFA at 100°C for 30 min to hydrolyze the GDP moiety of the sugar nucleotides releasing free sugars.

Using conditions established at BioTechnical Resources, Manitowoc, Wisconsin, thin layer chromatography (TLC) was used to separate the mixture of free sugars. Briefly, an aliquot (5 μl) of the hydrolyzed mixture was loaded onto Silica Gel 60 plates impregnated with 0.3 M NaH₂P0₄ and dried overnight at 45 °C. The TLC plates were then developed in a solvent containing acetone-n- butanol-water (8:1: 1 [vol/vol/vol]). The amount of radioactivity in resolved free sugars on the developed TLC plate was detected and quantified using a phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). L-galactose and D-mannose released from the GDP-L- galactose substrate and GDP-D-mannose enzymatic products, respectively, was identifiedby comparing their respective Ry values with standards after visualization with the p-anisaldehyde/H₂S0₄ reagent. Enzyme activity was expressed as activity units unless otherwise indicated. One Unit of epimerase activity is defined as one nmole of L-galactose formed per min. Enzyme specific activity is defined as Units mg^"1 protein. Alternatively, one can use the method described in detail in Wolucka et al., 2001, Anal.

Biochem. 294:161-168, incoφorated herein by reference in its entirety, which uses an HPLC radio- method using GDP-D-[U-¹⁴C] mannose as substrate. Example 12

This example compares activity levels of GDP-D-mannose-3,5-epimerase in microalgae producing different amounts of vitamin C.

GDP-D-mannose-3 ,5-epimerase catalyzes the formation of GDP-L-galactose from GDP-D- mannose and has been proposed as the rate-limiting step for the biosynthesis of L-ascorbic acid in algae (WO 99/64618; Running et al., 2003). Algal strains with enhanced epimerase activity were screened. Strains screened were: Chlorella pyrenoidosa UTEX 1613 (WT) and UTEX 166-4 (mutant); Prototheca moriformis ATCC 75669 (WT) and ATCCPTAl 11 (mutant); and Chlamydomonas reinhardtii dangerard ATCC 18798 (WT).

To compare the level of the epimerase activity in the different alga, cells were cultured in ATCC liquid medium #5 and harvested by centrifugation when the culture OD₆₀₀ reached 2 to 2.5. After washing once with breakage buffer, harvested cells were used immediately for enzyme preparation by grinding in liquid N₂. Freshly-prepared enzyme solution was assayed with 15 μM ¹⁴C- labeled GDP-D-mannose in 100 μl of assay buffer. Following incubation, free sugars were obtained by hydrolyzing the reactions in TFA at 100^CC for 30 minutes. Free sugars were separated by TLC. Separation of the hydrolyzed reaction products (Prototheca WT; Prototheca mutant; Chlorella WT; Chlorella mutant; Chlamydomonas WT; and Prototheca mutant (boiled)) were analyzed by TLC from an assay mixture of crude cell-free extracts incubated at room temperature for 60 min. All soluble cell-free extracts catalyzed formation of L-galactose, released from GDP-L-galactose (data not shown). L-galactose formation was not observed in the control reaction (denatured soluble extract of Prototheca moriformis ATCCPTAl 1 boiled at 100°C for 10 min).

The time course of L-galactose formation indicated that the epimerization reaction started upon the addition of enzyme. The amount of the labeled L-galactose product released from GDP-L-galactose increased as incubation time increased (Fig. 8; data points are the mean of triplicate reactions). Although the epimerization reaction continued for 15 hours (900 min), 80 to 95% of the maximum epimerization measured was reached within the first 60 minutes.

The epimerase activity in mutants was greater than in the wild type strains. Cells were grown and harvested as described in Example 10. Cell-free extracts were prepared from three parallel cultures by grinding in liquid N₂. Epimerase was assayed at room temperature for 10 min (Example 11). Enzyme activity is expressed as Units (nmol L-galactose produced min^"1). The level of epimerase activity in Prototheca and Chlorella mutants that produce elevated levels of vitamin C was only slightly higher than that in wild type strains after 10 min incubation (Table 8). However, this difference became significant when incubation time was extended to 60 min or more. Within a reaction period of 60 min, among three wild type algal strains (open symbols), the highest activity was found in Chlamydomonas reinhardtii cells. For the assay incubated 900 min, Prototheca strain PTA- 111 epimerase activity was 2.6-fold higher than the wild type strain ATCC 75669.

TABLE 8

Specific Activity (Units mg^"1)

% of Stains* Sample Average Ave

Prototheca moriformis

0.11 0.11 0.10 0.11 ± 0.01 100 ATCC 75669 (wt) Prototheca moriformis

0.16 0.12 0.13 0.14 ± 0.04 127 ATCCPTA111 Chlamydomonas reinhardtii (wt) 0.17 0.27 0.27 0.24 ± 0.07 218

™ UTE™X 1*6ζ6{37 (wt™) ^d0Sa 0.16 0.22 0.07 0.15 ± 0.08 136

Chlorella pyrenoidosa ^„ ,_{q n} ^„ _{q n} . r_i i_fi + n n7 ι.ι_<

UTEX 166-4 (mutant) ⁹ °-^{09 0 21} °'^{16 ±} °-^{07 145}

Example 13

This example demonstrates Prototheca cells naturally express L-galactose dehydrogenase and L-galactono-γ-lactone dehydrogenase (or homologues). The primary pathway for biosynthesis of L-ascorbic acid in plants suggests the involvement of the pathway intermediate, L-galactono-γ-lactone. Analysis of algal epimerase reaction products showed at least two additional ¹⁴C-labeled compounds besides D-mannose (from substrate) and L- galactose (epimerization product) (data not shown). L-galactono-γ-lactone dehydrogenase, catalyzing conversion of L-galactose to L-galactono-γ-lactone, requires NAD. Addition of 5 mM NAD to the reaction mixture resulted in a substantial loss of L-galactose and concomitant accumulation of both unknown products.

These results indicated clearly that other enzymes involved in either L-ascorbic acid synthesis and/or in diversion of GDP-D-mannose or GDP-L-galactose to other pathway products are present in the extracts . For example, it has been reported that GDP-D-mannose dehydrogenase converts GDP-D- mannose to GDP-L-fucose in bacteria, plants and mammals. If this activity is present in the Prototheca extracts, one of the unknown products could be L-fucose.

To identify the unknown compounds, hydrolyzed reaction products were co-chromatographed with the authentic compounds, L-fucose, L-galactono-γ-lactone and L-ascorbic acid, by TLC. The enzyme activity was assayed using soluble enzyme extracts of the Prototheca moriformis mutant ATCC PTA-111 (data not shown). An aliquot (100 μg) of cell-free enzyme extract was incubated with 50 μM of GDP-D-[U-¹⁴C]-mannose (specific activity 52.7 μCi μmol^"1) at room temperature for 2, 12 and 24 h. After hydrolysis, free sugars were separated and co-chromatographed with a standard L- galactono-γ-lactone, L-ascorbic acid and L-fucose. TLC was developed using acetone-n-butanol-water (8:1:1, [vol/vol/vol]). Air dried TLC plates were autoradiographed at room temperature for 20 hr using Hyperfilm βmax film (Amersham). After autoradiography, the plates were sprayed with hydroxylamine/FeCl₃ then sprayed with hydroxylamine/FeCl₃ in order to visualize L-galactono-γ- lactone and free sugars, followed by spraying with anisaldehyde H₂S0₄ to visualize L-ascorbic acid. Autoradiography of reaction mixtures separated by TLC revealed that one of the unknown products had the same R_y as L-galactono-γ-lactone and other unknown products had the same R_y as L- ascorbic acid (data not shown). This showed that cell-free extracts of Prothotheca were capable of producing L-ascorbic acid from GDP-D-mannose, confirming that not only are the last two enzymes of the plant vitamin C pathway, L-galactose dehydrogenase and L-galactono-γ-lactone dehydrogenase (EC 1.3.2.3), but that the two enzymes immediately downstream of GDP-mannose-3,5-epimerase also naturally exist in Prototheca, as proposed by Wheeler et al. (1998), Berry et al. (1999) and Running et al. (2003). Example 14

This example demonstrates purification of GDP-D-mannose-3,5-epimerase from Prototheca and properties of the purified enzyme. The GDP-mannose-3 ',5 '-epimerase has been partially purified from Chlorella pyrenoidosa

(Hebda et al., 1979) and highly-purified from Arabidopsis thaliana (Wolucka et al., 2001b), but there have been no reports of purification of any GDP-mannose-3 ',5'-epimerase to homogeneity. Purification of algal epimerases to homogeneity was successfully completed by the present inventors as described below. Prototheca moriformis ATCC PTA-111 mutant was selected for enzyme purification, not only because strain PTA-1 l l's epimerase activity was greater than other strains, but also because it was an oveφroducer of L-ascorbic acid.

Fast protein liquid chromatography (FPLC; Pharmacia, Piscataway, NJ) was used to purify the epimerase. Chromatograpny columns and media (Phenyl Sepharose and various ion exchange resins) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Thin layer chromatography plates (Silica Gel 60, 20 x 20 cm) were purchased from EM Science (Gibbstown, NJ). The Silica Gel 60 plates were impregnated with 0.3 M NaPi prior to loading samples. All HPLC grade solvents for chromatography were obtained from Fisher Scientific (Hampton, New Hampshire). Algal epimerase purification

All purification steps were performed at 4°C.

Step 1 : Preparation and Fractionation of crude extract. Purification was started with 150 grams of frozen cells (ATCC PTA-111). Algal cells were resuspended in extract Buffer A (20 mM MOPS, pH7.2, 10 % (vol/vol) glycerol, 2 mM DTT and 5mM EDTA) and lysed by passing through a SLM Aminco French Pressure cell at 18,000 psi, and then sonicated for three times for 30 seconds per sonication. The debris was removed by a centrifugation at 4,000 x g for 10 min. The supernatant was centrifuged again at 18,000 x g for 60 min. The supernatant was defined as crude cell-free extract. The crude extract was further fractionated by centrifugation at 100,000 x g for 1 hour. The clarified supernatant was defined as the soluble fraction. The pellet was solubilized using 1% (wt/vol) OG (n- octyl-β-D-glucoside) and designated as the insoluble fraction.

Step 2: Ammonium Sulfate Precipitation. The soluble fraction was brought to 50% (wt/vol) (NH₄)₂S0₄. After 6 hours, the precipitate was collected by centrifugation at 20,000 x g for 60 min. The resulting precipitate was dissolved in the least possible volume of Buffer A.

Step 3: Hydrophobic Interaction Chromatography (HIC). HIC was performed on a Hiload (10/10) Phenyl Sepharose column. The column was equilibrated with Buffer B (0.5 M (NH₄)₂S0₄ in

Buffer A), and then eluted with an inverse salt gradient, from 0.5 M Buffer B to 0.5 M Buffer B without (NH₄)₂S0₄. Three-ml fractions were collected at a flow rate of 1.5 ml min^"1. Every other fraction was assayed for epimerase activity.

Step 4: Anion Exchange. Epimerase lost its activity during dialysis. Therefore, the samples from these column chromatography steps were desalted with a PD-10 column (Sephadex G-25) and the resulting sample was applied to an anion exchange column (Mono-Q [HR, 5/5]). The column was washed with Buffer A, and then eluted with a linear gradient, 0 to 1.0 M, of sodium chloride or sodium acetate dissolved in Buffer A.

Step 5: Size Exclusion. The active fractions from step 4 were concentrated and loaded onto a Superdex-200 Column, 1.6x55 cm. The column was eluted with Buffer C (Buffer A containing 0.3 M NaCl). Prototheca epimerase characterization

Prototheca cells were homogenized at 18,000 psi using a SLM Aminco French Press and then sonicated 3 times for 30 s per sonication. Fractionation of crude extract was achieved by centrifugation as described above. Soluble and insoluble fractions were tested for epimerase activity (data not shown). The protein concentration of enzyme extracts was first adjusted to 1 mg ml^"1. The assays were done by incubating 25 μg of enzyme extract with 15 μM of ¹⁴C-labeled substrate at room temperature for about 15 hours (overnight). ¹⁴C-GDP-D-mannose was incubated with approximately 100 μg of enzyme prepared with Buffer 1 (50 mM MOPS, pH 7.2 containing 5 mM DTT and 5mM EDTA), Buffer 1 plus lmM phenylmethylsulfonyl fluoride (PMSF), Buffer 1 plus 10% glycerol, Buffer 2 (50 mM Bis-tris, pH 7.2 containing 5 mM DTT and 5mM EDTA) and Buffer 3 (50 KPi, pH 7.2 containing 5 mM DTT and 5mM EDTA). Control reaction was generated with boiled enzyme that was prepared with Buffer 1 (50 mM MOPS, pH 7.2 containing 5 mM DTT and 5mM EDTA).

The results showed that all soluble fractions converted GDP-D-mannose into GDP-L-galactose (data not shown). Insoluble fractions did not convert GDP-D-mannose to GDP-L-galactose (data not shown), or the soluble fraction (data not shown), which was heated at 100°C for 10 min. The epimerase, therefore, was present in the soluble cytoplasmic fraction and was absent from the insoluble membrane fraction.

The soluble fraction (25 ml total volume) was saturated with 50% (wt/vol) (NH₄)₂S0₄ and held for 6 hours at 4°C. Precipitated protein was collected by centrifugation and dissolved in Buffer A. Epimerase activity was efficiently precipitated and retained its activity at 50% (wt/vol) (NH₄)₂S0₄ saturation (data not shown). Radioactive L-galactono-γ-lactone was not detected by TLC after (NH₄)₂S0₄ precipitation, indicating that L-galactose dehydrogenase did not co-precipitate with the epimerase at this (NH₄)₂S0₄ saturation level. The epimerase lost 50% and 80% of its activity after dialysis against pH 7.2 MOPS and Bis-Tris buffer, respectively. Purification of Prototheca epimerase

A summary of enzyme recovery and purification at each step from Prototheca moriformis mutant ATCC PTA-111 is presented in the Table 9.

The first chromatography step in the purification was Hydrophobic Interaction Chromatography (HIC) on phenyl Sepharose. The 50% (wt/vol) (NH₄)₂S0₄-precipitated fraction was applied directly to a Hiload Phenyl Sepharose column (16/10) equilibrated with 0.5M (NH₄)₂S0₄ in Buffer A. All epimerase activity was bound to the gel. Most proteins eluted soon after the gradient was started. Bounded epimerase activity was eluted with 140 to 80 mM (NH₄)₂S0₄. The active fractions contained about 67% and 5.8% of the applied activity and protein, respectively. The active fractions were pooled, concentrated and chromatographed on a Mono-Q column. Epimerase activity eluted as a shaφ peak with 0.3 M NaCl. The recovery of the activity from this chromatography was 17%. The last step of purification was separation on a Superdex-200 gel filtration column. The enzyme activity appeared at the elution volume of 22 to 26 ml. As seen in Table 9, after successive chromatography, a 28-fold purification was achieved. The purity of the enzyme preparation from each step of purification was analyzed by SDS-PAGE; purity was significantly increased. Considerable difficulty was encountered to achieve an active, homogenous epimerase protein preparation because the partially purified enzyme was unstable (see below).

TABLE 9

Total Total Specific

Fold Yield (%)

Fraction Activity Protein Activity Purification (nmol) (mg) (nmol gm^'1) Activity Protein

Crude Extract 217 1087 0.2 1 100 100

50% (wt/vol) (NH₄)₂S0₄ 109 364 0.3 2 60 33

Phenyl Sepharose 63 211 0.3 2 67 5.8 onoQ 34 8 4.2 21 17 0.7

Gel Filtration 6 1 5.5 28 1 0.1

Properties of Prototheca epimerase

To evaluate enzyme stability, extracts were prepared as described in Example 10. Stability of enzyme in crude extracts was monitored at room temperature, 4°C and -20°C by incubating a 50 μg of crude extract protein aliquot from each condition overnight with 150 μM of ¹⁴C-labeled GDP-D- mannose at room temperature. The reactions were stopped by adding an equal volume of 1M TFA followed by boiling at 100° C for 30 min. The Prototheca epimerase activity had a measured half-life of 1 and 3 days at room temperature and 4°C, respectively as shown in Fig. 9. There was a complete loss of enzyme activity after 10 days storage under either condition. The enzyme in crude cell-free extracts or in 50% (wt/vol) (NH₄)₂S0₄ solutions was relatively stable and could be stored at -20 ° C up to three weeks without significant loss of activity. However, when either of these enzyme solutions was diluted 5 to 10 fold and then stored at -20°C, 50% of the activity was lost within two weeks.

Prototheca epimerase characteristics were determined with the partially-purified enzyme collected from Mono-Q chromatography. The formation of GDP-L-galactose was linear with incubation time and substrate and enzyme concentration. Typical progress curves for the formation of GDP-L- galactose with different amounts of enzyme as function of incubation time are shown in Fig. 10.

The kinetics of epimerase activity was analyzed by measuring initial velocities over a range of reaction times and concentrations of GDP-D-mannose at fixed enzyme concentrations. The Michaelis constant (K_m) value for GDP-D-mannose was determined using a Lineweaver-Burk plot (Fig. 11). The K™ for GDP-D-mannose, calculated from the plot, was 30 μM. The K,,, value was significantly lower for Prototheca as compared to that determined by Hebda et al. (1979) for the Chlorella epimerase (97 μM).

The pH optimum for the enzyme activity was 7.2 when assayed in 50 mM MOPS buffer. The enzyme activity in 50 mM KPi buffer (pH7.2) was about 30% higher than in MOPS buffer. The divalent cation, Ca^, was a very effective inhibitor of epimerase activity. At a concentration of 1 mM, epimerase activity was reduced to 43% of initial; Mg^ had no effect on enzyme activity. Although EDTA, NADH, and NADPH were activators of the epimerase in the fraction resuspended from 50% (NH₄)₂S0₄, Mg⁺⁺ did not stimulate or inhibit activity of other partially purified epimerase samples. Example 15

This example demonstrates detection of Prototheca and Chlorella epimerase genes using reduced stringency hybridization.

Previously, the Prototheca epimerase was partially purified using traditional protein purification techniques from crude cell-free extracts. Attempts to prepare homogeneous Prototheca epimerase were unsuccessful because of considerable loss of enzymatic activity during the purification. An alternative approach to cloning the Prototheca and other algal (e.g., Chlorella) epimerase gene relies on knowledge of the Chlamydomonas epimerase coding sequence. A Southern blot approach was first used to detect GDP-D-mannose-3,5-epimerase in Prototheca moriformis wild type and mutant genomic DNA (ATCC PTA-111) using the Chlamydomonas epimerase cDNA as probe and in a similar fashion, from Chlorella pyrenoidosa WT genomic DNA.

To isolate genomic DNA, cells of Prototheca WT and Prototheca mutants were grown in liquid #5 medium and harvested when OD₆₀₀ reached 2 to 2.5. The cells were ground in liquid nitrogen and genomic DNA was isolated using the DNeasy Plant mini kit from Qiagen (Valencia, CA). The genomic DNA was digested with Sac I, Pst I, and Hinc III.

The probe was prepared from the recombinant plasmid pETChlam.6. The plasmid DNA was double digested with Neo I and BamHΪ. The fragment containing the entire cDNA sequence of the epimerase gene was isolated by agarose electrophoresis (1 % gel) and purified using the Qiagen Mini Kit (Cat#12123; Valencia, CA). The purified full-length cDNA of Chlamydomonas epimerase was labeled with biotin by random-primer labeling using the NEBlot Phototope Kit (New England BioLabs, Cat#N7550S).

Whole genomic DNA was digested individually with one of three restriction enzymes, Sac I, Pst I, and Hinc III. DNA from each reaction mixture was separated by electrophoresis (0.7% or 0.8% agarose gel). The DNA was denatured in the denaturation solution (0.5 M NaOH, 1.5 M NaCl) and blotted onto 0.45 μm nylon membranes (Biodyne A; Gelman Laboratory, Cat.# 09-734-08) using 2x S SC as the transfer buffer. DNA was fixed to nylon membranes by UV cross-linking using a Stratagene 1800 UV cross-linker. After prehybridizing at 42 ° C for 2 to 3 hours, the DNA was then probed with a 1.1 kb Neo 1-BamHl fragment from plasmid pETChlam.6 using various conditions of stringencies.

Southern blots probed with Chlamydomonas epimerase at low stringency ( = -17.5) revealed a single cross-reacting fragment in Sac I-digested DNA (both WT and mutant). The result provided evidence that the gene for GDP-D-mannose-3,5-epimerase was present in the Prototheca moriformis genome (data not shown). Similar Southern blots with Chlorella and Prototheca genomic DNA provided further evidence that the gene for GDP-D-mannose-3 ,5-epimerase was present in Chlorella as well (data not shown). Inverse PCR can be used to clone these genes using methods as described herein for the cloning of the epimerase GDP-D-mannose-3 ,5-epimerase from Chlamydomonas. 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 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 75% identical to an amino acid sequence selected from the group consisting of SEQ ID

NO: 10 and SEQ ID NO:21, 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; and c) a nucleic acid sequence that is fully complementary to any of the nucleic acid sequences of (a) or (b).

2. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid sequence encodes an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity.

3. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid sequence encodes an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21 , wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity.

4. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid sequence is less than about 100% identical to any of the nucleic acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

5. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid sequence is at least about 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:20, and less than 100% identical to any of the nucleic acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

6. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid sequence encodes a protein comprising an amino acid sequence of SEQ ID NO:21.

7. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid sequence is SEQ ID NO:20.

8. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid sequence encodes a biologically active fragment of SEQ ID NO: 10 or SEQ ID NO:21, wherein the fragment has GDP-mannose-3',5'-epimerase activity.

9. An oligonucleotide probe or primer that hybridizes under high stringency conditions to a nucleic acid sequence comprising SEQ ID NO:20 or the complement thereof.

10. A recombinant nucleic acid molecule comprising a nucleic acid sequence operatively linked to at least one expression control sequence, the nucleic acid sequence being selected from the group consisting of: a) a nucleic acid sequence that encodes an amino acid sequence that is at least about 75%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, 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.

11. A recombinant host cell transformed with a recombinant nucleic acid molecule of claim 10.

12. The recombinant host cell according to claim 11 , wherein the host cell is a eukaryotic cell.

13. The recombinant host cell according to claim 11, wherein the host cell is a yeast.

14. The recombinant host cell according to claim 11 , wherein the host cell is a plant cell.

15. The recombinant host cell according to claim 11 , wherein the host cell is a prokaryotic cell.

16. The recombinant host cell according to claim 11, wherein 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 the group consisting of GDP-L-galactose, L-galactose- 1- phosphate, L-galactose, and L-galactono-γ-lactone.

17. The recombinant host cell according to claim 11, wherein expression of the recombinant nucleic acid molecule by the host cell is sufficient to increase ascorbic acid production in the host cell.

18. A genetically modified plant or part thereof, wherein said plant has been genetically modified to recombinantly express a GDP-mannose-3',5'-epimerase or biologically active fragment thereof, wherein the GDP-mannose-3',5'-epimerase comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; and b) an amino acid sequence that is a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity.

19. An isolated algal GDP-mannose-3 ',5 '-epimerase having characteristics comprising: a) a monomeric molecular weight of between about 40kD and about 50kD; b) an optimum pH of from about 7 to about 8.5; and c) a K_^ of between about 7 μM and 50 μM for GDP-D-mannose; wherein the isolated algal GDP-mannose-3',5'-epimerase has been purified by at least 2-fold as compared to a crude extract having GDP-mannose-3',5'-epimerase activity; and wherein the isolated algal GDP-mannose-3',5'-epimerase has GDP-mannose-3',5'-epimerase enzymatic activity.

20. The isolated algal epimerase of Claim 19, wherein the epimerase is isolated from an alga belonging to a genus selected from the group consisting of: Chlamydomonas, Prototheca, Chlorella, Platimonas, Euglena, Scenedesmus, Pterocladia, Porphyridium, Ochromonas, and Cyclotella.

21. The isolated algal epimerase of Claim 19, wherein the epimerase is isolated from an alga belonging to a genus selected from the group consisting of: Chlamydomonas, Prototheca, and Chlorella.

22. The isolated algal epimerase of Claim 19, wherein the epimerase forms a dimer.

23. The isolated algal epimerase of Claim 19, wherein the epimerase has a V_max of between about 22.4 nmol min^"1 mg^"1 and about 4.8 nmol min^"1 mg^"1 for GDP-D-mannose.

24. The isolated algal epimerase of Claim 19, wherein the epimerase has characteristics comprising: a) a monomeric molecular weight of about 43kD; b) an optimum pH of about 7.2; and c) a K,„ of at least about 30 μM for GDP-D-mannose.

25. The isolated algal epimerase of Claim 24, wherein the epimerase is isolated from Prototheca.

26. The isolated algal epimerase of Claim 19, wherein the epimerase has been isolated by detergent solubilization.

27. The isolated algal epimerase of Claim 19, wherein the epimerase has been isolated by hydrophobic interaction chromatography.

28. The isolated algal epimerase of Claim 19, wherein the epimerase has been isolated by anion exchange chromatography.

29. The isolated algal epimerase of Claim 19, wherein the epimerase has been isolated by size exclusion chromatography.

30. The isolated algal epimerase of Claim 19, wherein the epimerase is of a purity to appear as a single band on an SDS-PAGE gel.

31. The isolated algal epimerase of Claim 19, wherein the epimerase is of a purity to elute from a chromatography column and appear as a single band on an SDS-PAGE gel.

32. The isolated algal epimerase of Claim 19, wherein the epimerase comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3 ',5'-epimerase activity; and b) an amino acid sequence that is a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity.

33. The isolated algal epimerase of Claim 19, wherein the epimerase comprises an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:21.

34. The isolated algal epimerase of Claim 19, wherein the epimerase comprises an amino acid sequence of SEQ ID NO:21, or a fragment thereof having GDP-mannose-3',5'-epimerase activity.

35. The isolated algal epimerase of Claim 19, wherein the epimerase is bound to a solid support.

36. A method to produce L-galactose, comprising: a) contacting the isolated algal GDP-mannose-3',5'-epimerase of Claim 19 with GDP-D-mannose under conditions that result in the production of GDP-L-galactose; and b) converting the GDP-L-galactose to L-galactose.

37. The method of Claim 36, wherein the step of converting is performed by hydrolysis of the GDP-L-galactose under acid conditions.

38. The method of Claim 36, wherein the step of converting is performed by enzymatic treatment of the GDP-L-galactose to produce L-galactose.

39. The method of Claim 36, further comprising purifying the L-galactose.

40. A method for producing L-galactose, comprising: a) growing a host cell that is transformed with a recombinant nucleic acid molecule encoding a GDP-mannose-3',5'-epimerase to produce GDP-L-galactose, wherein the recombinant nucleic acid molecule of (a) comprises a nucleic acid sequence operatively linked to at least one expression control sequence, the nucleic acid sequence being selected from the group consisting of: i) a nucleic acid sequence that encodes an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21 , wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; 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; b) converting the GDP-L-galactose to L-galactose; and c) recovering the GDP-L-galactose or the L-galactose from the host cell.

41. The method of Claim 40, wherein step (c) comprises purifying the GDP-L-galactose or the L-galactose from a crude extract of microbial host cells.

42. The method of Claim 40, wherein GDP-L-galactose is recovered in step (c) and wherein step (b) of converting is performed after step (c) of recovering.

43. The method of Claim 40, wherein step (b) comprises producing a crude extract of host cells comprising GDP-L-galactose and converting the GDP-L-galactose to L-galactose, followed by recovering the L-galactose.

44. 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 encoding a GDP-mannose-3',5'- epimerase to increase ascorbic acid synthesis by the host cell, wherein the recombinant nucleic acid molecule comprises a nucleic acid sequence operatively linked to at least one expression control sequence, the nucleic acid sequence being selected from the group consisting of: a) a nucleic acid sequence that encodes an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, 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.

45. The method of Claim 44, wherein the host cell is a eukaryotic cell.

46. The method of Claim 45, wherein the eukaryotic cell is selected from the group consisting of: a plant cell, an algal cell and a yeast cell.

47. The method of Claim 44, wherein the host cell is a prokaryotic cell.

48. A method to increase ascorbic acid synthesis in an algal cell comprising a GDP- mannose-3 ',5'-epimerase, comprising introducing into the genome of the cell a non-native promoter upstream of a gene encoding the GDP-mannose-3',5'-epimerase, wherein the epimerase comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; and b) an amino acid sequence that is a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity.

49. 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 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ

ID NO:21, wherein the amino acid sequence has GDP-mannose-3 ',5'-epimerase activity; and ii) an amino acid sequence that is 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 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.

50. 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 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3 ',5 '-epimerase activity; 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 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.

51. A genetically modified host cell, wherein the host cell comprises at least one genetic modification to increase the activity of a GDP-mannose-3',5'-epimerase in the host cell, wherein the

GDP-mannose-3',5'-epimerase comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:21, wherein the amino acid sequence has GDP-mannose-3',5'-epimerase activity; and b) an amino acid sequence that is a fragment of the amino acid sequence of (a), wherein the fragment has GDP-mannose-3',5'-epimerase activity.

52. The genetically modified host cell of Claim 51, wherein the host cell has been modified by transformation with a recombinant nucleic acid molecule encoding the GDP-mannose- 3',5'-epimerase.

53. The genetically modified host cell of Claim 51 , wherein the host cell is an algal cell that has been genetically modified by the introduction into the genome of the algal cell of a non-native promoter upstream of a gene encoding the GDP-mannose-3',5'-epimerase.