2) United States Patent Porro et al. US006630330B1 US 6,630,330 BL Oct. 7, 2003 a0) Patent No.: (45) Date of Patent: (54) ASCORBIC ACID PRODUCTION FROM YI (75) Inventors: Danilo Porro, Erba (11); Michael Sauer, Mider (AT) (73) Assignee: Biopolo S.C.a.R.L., Milan (11) (+). Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35, USC. 1540) by 0 days. (21) Appl. No.: 09/630,983 22) Filed! 2, 2000 (51) In. cl? C12P 17/00; C12P 7162 (52) US. Ch 435/117; 435/126; 435/135; 435/137; 435/254.21; 549/315 (58) Field of Search 435/126, 135, 435/137, 254.21, 117; 549/315 (56) References Cited U.S. PATENT DOCUMENTS 59 A 6/1986. Roland eta. 016,068 A 4/1990 Roland eta. 2002001297 AL * 1/2002 Berry et al 485/136 FOREIGN PATENT DOCUMENTS wo wossi1745 41985 wo WORSSISSS 11/1998 wo wo9933005 7/1909, wo wosm61618 12/1999 wo womn's4502 72000 OTHER PUBLICATIONS Berendsen A Glimpse of the Holy Grail Oct, 23, 1998 Vol 282 Science pp. 642-643.* EMBL/GenBank/DDBJ Entry O81884.* Darnis etal. Cloning, sequencing and functional expression of a eDNA encoding porcine pancreatic precarhoxypepti= dase AL. Feb, 1999, European Journal of Biochemistry 259: 719-725." Wheeler et al. The biosynthetic pathway of vitamin C in higher plants. May 1998. Nature 393:365-369.* US. patent application Publication No. 2002/0076771 (pub lished Jun. 20, 2002). PCT/GROLOS485 International S 2002). Onolii etal, “Influence of L-Galactonic Acid y-Lactone on Ascorbate Production in Some Yeasts,” Antonie van Leew- wenhoek 71:227-280 (1997). v, arch Report (Jul Krasnov et al, “Expression of Rat Gene far _-Gilono-y-Lactone Oxidase, the Key Enzyme of ,-AScoe- bie Acid Biosyahesis, in Guinea Pig Cells and in Teleost Fish Rainbow Trout (Oncorhynchus mykis)” Biockinica et Biophysica Acta 1381:241-248 (1998). Kanagasundaram etal "Isolation and Charaterizaton of the Gene Encoding Gluconolactonase from Zymomonas mob lis," Biochinica et Biophysiea Acta 1171: 198-200 (1992) Koshizaka et al., J. Biol. Chem, 263:1619-1621 (1998). Huh etal, Mot Microb. 30:895-903 (1998) Huh etal, Eur J. Biochem, 225:1073-1079 (1994) Kim etal, Biochim. Biophys, Acta 1297:1-8 (1996). Kim etal, Biochim. Biophys. Acta 1429:29-39 (1998) Dumbeava et al, Biodhim, Biophys. Acta 926:331-338 as87) Nick et al., Plant Science 46:181-187 (1986). Lee etal, App. En. Microb. 65:4685-4687 (1099) Oxtergaard etal, J. Biol. Chem. 272:30009-30016 (1997) ‘Hancock et al., FEMS Microbiol. Lett, 186:245~250 (2000). Spickett et al, Free Rad, Biol. Med, 28:183-192 (2000) Daruwala ota, FEBS Let, 460:480-484 (1995) * cited by examiner Primary Examiner—David Sura Assistant Examiner—David A. Lambertson (7) Auorney, Agent, or Firm—Williams, Morgan & Amerson, P.C 67 Herein is disclosed a method of generating ascorbic acid from yeast. In one embodiment, the yeast is a Zygosaceha- romyces spp. or a Kluyveromyces spp. growing in a medium ‘comprising an ascorbic acid precursor. In a second embodi- ‘ment the Yeast is a recombinant yeast geowing ia a medium comprising an ascorbic acid precursor. Preferably the recombinant yeast is iransformed with a coding region encoding an enzyme selected from L-galactose dehydroge- nase (LGDH), L-galactono-1,4-lactone dehydrogenase (AGD), D-arabinose dehydrogenase (ARA), D-arabinono- 1 4-lactone oxidase (ALO) or L-gulono-1,4-lactone oxidase (RGLO). The ascorbic acid precursor is preferably D-glucose, L-galactose, L-galactono-1,4-lactone, of L-gulono-1;4-lactone. In another preferred embodiment the ascorbic acid is accumulated in the medium at levels greater than background, Preferably, the yield of the conversion of the precursor to ascorbic acid is preferably at least about 35% ABSTRACT 15 Claims, 7 Drawing SheetsU.S. Patent Oct. 7, 2003 Sheet 1 of 7 US 6,630,330 BL Fig. 1 D.Gtucose LAscorbateUS 6,630,330 B1 Sheet 2 of 7 Oct. 7, 2003 U.S. Patent Fig.2 | See [4]US 6,630,330 B1 Sheet 3 of 7 Oct. 7, 2003 U.S. Patent Fig. 3 ean © 8 tt 2 gy e¢ 6 6 S$ 6 6 8 G {iGO st Gul] yueqD pIoy ofoosy sepMosesUy | aUS 6,630,330 B1 Sheet 4 of 7 Oct. 7, 2003 U.S. Patent Fig. 4 «yee 22°73 o4 02 ® 3 [-€0 b+ Bui) suaques plow syquoosy seingooryyyUS 6,630,330 B1 Sheet 5 of 7 Oct. 7, 2003 U.S. Patent ob eo ££ 2 » © & & SS & eu; u) pray aIquoosy [b+ 6ui} anosg eaming | Ct nnnmnnnnneninnennnonennnnnnsnumanaaU.S. Patent Oct. 7, 2003 Sheet 6 of 7 US 6,630,330 BLU.S. Patent Oct. 7, 2003 Sheet 7 of 7 US 6,630,330 BL [x GRFIeUW © GRF18U ALO ‘4 GRF18U ALO+LGDH Ascorbic Acid in the | time th]US 6,630,330 BL 1 ASCORBIC ACID PRODUCTION FROM. YEAST BACKGROUND OF THE INVENTION 1, Field of the Invention ‘The present invention relates generally to the field of ascorbic acid production, Mote particularly, it relates 10 a process for the production of L-ascorbic acid from yeast, including recombinant yeast 2, Description of Related Art L-aseorbie acid (Vitamin C) is a powerful water-soluble antioxidant that is vital for growth and maintenance of all tissue types in humans. One important role of ascorbie acid is its involvement in the production of collagen, an essential cellular component for connective tissues, muscles, teadons, bones, teeth and skin, Collagen is also required forthe repair ‘of blood vessels, bruises, and broken bones. Ascorbic ac helps regulate blood pressure, contributes to reduced cho- lesterol levels, and aids inthe removal of cholesterol depos its from arterial walls. Ascorbic acd also aids in the metabo- lization of folic acid, regulates the uptake of iron, and is required for the conversion of the amino acids L-tyrosine and L-phenylalanine into noradrenaline. The conversion of lryptophan into seratonin, the neurobormone responsible for sleep, pain control, and well-being, also requires adequate supplies of ascorbic acid, ‘Adeficiency of L-ascorbic acid can impair the production Of collagen and lead to joint pain, anemia, nervousness and retarded growth. Other eflects are reduced immune response and inereased susceptibility to infections, The most extreme form of ascorbic acid deficiency is scurvy, a condition ‘evidenced by swelling ofthe joints, bleeding gums, and the hhemorthaging of capillaries below the surface ofthe skia. If left untreated, scurvy is fatal Although intestines easily absorb ascorbie acid, it is exereied 10 the urine within two fo four hours of ingestion ‘Therefore it cannot be store in the body. L-ascorbie acid is produced in all higher plants and in the liver or kidney of ‘most higher animals, but not humans, bats, some birds and a variety of fishes. Therefore, humans must have access to Sullicient amounts of ascorbic acid from adequate dietary ‘sources or supplements in order to maintain optimal health Food sources of ascorbic acid include citrus fruits, potatoes, peppers, green leafy vegetables, tomatoes, and berries. Ascorbic acid is also commercially available as a ‘supplement in forms such as pills tablets, powders, wafers, and sycups. L-Ascorbie acid is approved for use as a dietary supple- ‘ment and chemical preservative by the U.S, Food and Deug Administration and is on the FDA’s list of substances aenerally recognized as safe. L-Ascorbie acid may be used in soft drinks as an antioxidant for flavor ingredients, in meat and meat-containing products, for curing and pickling, in flour to improve baking quality, in beer asa stabilizer, in fats and oils as an antioxidant, and in a wide variety of foods for ascorbic acid enrichment. L-Ascorbic acid may also find use in stain removers, hair-care products, plasties manufacture, photography, and water treatment. ‘The enzymes of the biosynthetic pathways leading 10 ascorbic acid have not been identified yet to completion, Current understanding of the physiological pathways. in planis and animals is shown in FIG. 1 In animals, D-glucose serves asthe frst precursor and the last step is catalyzed by a microsomal L-gulono-1,4-lactone 20 os ss ss 6s 2 oxidase. The enzyme has been isolated and characterized from different sources. The gene from rat has been cloned and sequenced (Koshizaka Tet al, 1998, J. Biol. Chem 263, 1619-1621.) ‘Two diserete pathways have been reported for ascorbie acid synthesis in plants. In one pathway, L-ascorbie acid is synthesized from D-glucose via L-sosbosone (Loews M. W. ct al, 1990, Plant. Physiol. 94, 1492-1495), Current evidence’ suggests that the main physiological pathway proceed from D-glucose via L-galactose and L-galactono- 1 4-lactone to Laascorbie acid (Wheeler G. L. etal. 1998, Nature, 393, 365-369,). The last two steps are catalyzed by the enzymes L-galactose dehydrogenase and T-galactono- Iyttactone dehydrogenase. Also in this case, the last cazyme has been isolated and characterized, and the gene from Brassica oleracea has been cloned and sequenced (@stergaard J. et al. 1997, J. Biol. Chem., 272, 30009-30016), For use a5 a dietary supplement, ascorbie acid ean be isolated from natural sources or synthesized chemically by the oxidation of L-sorbese asin variations of the Reichstein process (US. Pat, No. 2,265,121, Tt remains desirable to have methods for the production of ascorhie acid by convenient processes. Two main require- rents in the production of ascorbic acid are thatthe syn- thesis should be enantioselective, because only the LL-tnantiomer of ascorbic aed i biologically active, and that the environment of the final steps of the process should be ‘non-oxidative, because ascorbic acid is very easly oxidized ‘One possible approach is the production of L-aseorbic acid from microorganisms. Microorganisms can be easily grown on an industrial scale. Although the produetion of ascorbic acid from microorganisms and fungi has been reported in the past, recent evidenee proves that L-ascorbie acid analogues, and not L-ascorbic acid, are found (Hub W. K etal, 198, Mol, Microbiol. 30,4, 895-908)HHancock R D. et al, 2000, FEMS Microbiol. Let. 186, 245-250) (Dambrava V. A. etal. 1987, BBA 926, 331-338\Nick J. A. et al, 1986, Plant Science, 46, 181-187). In yeasts (Candida and ‘Saccharomyces species), the production of eryth- roascorbie acd has been reported (Huh W. K. et al, 1994, Eur J. Biochem, 225, 1073-1079\Huh W. K. et al, 1998, Mol. Microbiol. 30, 4, 895-903). In such yeasts, a physi ological pathway has been proposed proceeding from D-glucose via D-arabinose and D-arabinono-1,4-actone to cxythroascorbie acid (Kim S. T. et al, 1996, BBA, 1297, 1-8). The enzymes D-arabinose dehydrogenase and Daarabinono-1,4lactone oxidase from Candida albicans as well as S. cerevisiae have been characterized. Interestingly, Legalactose and L-galactono-1-lactone are substrates for these activities in vito. In vivo production of L-ascorbie acid has been obtained by feeding L-galactono-L,4-lactone to wildtype Candida cells (Intemational Patent Application W08S/01745) Recently it has been shown that wild-type S. cerevisiae cells accumulated intracellularly L-aseorbie acid when incubated with L-galactose, L-galaciono-1,4-lacone, of L-gulono-1,4- lactone (Hancock et a, 2000, FEMS Microbiol. Lett. 186, 245-250Spickett C. M. et al, 2000, Free Rad. Biol. Med 28, 183-192), Wild-type Candida cells incubated with L-galactono- lactone accumulate L-ascorbic acid in the medium, siagesting that this yeast has a biological mechanism forthe release of the intracellular accumulated L-ascorbie acid; indeed, L-ascotbie acid is a complex molecule and itis scientifically reasonable that its accumulation in the mediumUS 6,630,330 BL 3 is ot related to a simple diflusion process, but should depend on facilitated or active transport. This conclusion is ‘supported by the identification and characterization of L-ascorbie acid transporters in higher eukaryotic (mammalian) cells (Daruwala R. et al., 1999, FEBS Letters. 4460, 480484). However, L-ascosbate transporters have not been described among the yeast genera, Nevertheless, while Candida cells growing in media containing L-galactono- 1,4-lactone accumulate L-ascorbic acid in the medium, accumulation in the medium of L-ascorbie acid from wild type S. cerevisiae cells has, surprisingly, never been described ‘A desirable method for the large-scale production of ascorhie acid comprises the use of genetically engineered microorganisms (ic., recombinant microorganisms). Both prokaryotic and eukaryotic microorganisms are today easily and successfully used for the production of heterologous proteins as well as for the production of heterologous metabolites, Among. prokaryotes, Escherichia coli and Bacillus subiilis are often used. Among eukaryotes, the yeasts S. cerevisiae and Kluyveromyces lactis are often used Despite the great suecess of these hosts, only one example has been described for the production of L-ascorbic acid by transformed microbial cells. Since only eukaryotic cells are natural L-ascotbic acid producers, itis even more surprising that only a prokaryotic transformed microbial host has been described to lead to the intracellular accumulation of L-ascorbie acid. Lee et al. (Appl. Environment. Mierobiol., 1999, 65, 4685-4687), showed that the cloning of the S' cerevisiae gene encoding D-arabinono-1,4-lactone oxidase into E. col allows the produetion of L-ascorbie acid from F. coli incubated with L-galactono- 1,4-lactone. Accumulation ‘of L-ascorbie acid was observed only at the intracellular level No experimental data have been described in the literature about the production of L-ascorbic acid from tansfoemed eukaryotic: microorganisms. @stergaard et al. cloned the zene encoding L-yalactono-1,4-lacione dehydrogenase from ‘cauliflower in the yeast S, cerevisiae (J. Biol. Chem., 1997, 272, 48, 30009-30016). While, in vitro, the authors Found L-galactono-1 4-lactone dehydrogenase activity in the yeast cell extract (cytochrome c assay, see Ostergaard et al), n0 production of L-aseorbic acid was proven ia vivo. Berry et al., International Patent Appin. WO 99/64618 discuss the potential use of the plant biosynthetic pathway of | ascorhic acid; special emphasis is given to the activity ‘catalyzing the conversion of GDP-D-mannose to GDP-L- galactose. However, characterization of the enzyme cala- Iyzing this step has aot been presented in detail. An over- ‘expressed £. coli homologue turned out to be inactive. ‘Smirnoff et al., WO 99/33995, discuss the use of L-galactose dehydrogenase for production of ascorbic acid The enzyme was purified from pea seedlings and the N-terminal protein sequence was determined, The complete ‘sequence is not known and has not yet been reported, The L-galactose dehydrogenase enzyme partial sequence was 72% identical to amino acids 5-22 of an unidentified puta- tive coding sequence from Arabidopsis thaliana, accession no. 3549669, Roland et al., US. Pat, Nos. 4,595,659 and 4,916,068, discuss the use of non-recombinant Candida steains 10 convert L-galactonic substrates to L-ascorbic acid. Roland et al. described the responsible enzyme as L-galactono-1,4- lactone oxidase, Kumar, WO 00/34502, discusses the production of L-ascorbic acid in Candida blankii and Cryptococcus 20 os ss 4s ss 6s 4 dimennae yeast capable of using 2-keto-L-gulonic acid as 4 sole carbon source in the production. Kumar specifically excludes the production from yeast by a pathway involving L-galactonolactone oxidase or by conversion of L-galactonie precursors, Tl remains desirable to have methods for the production of | ascorbic acid by a convenient fermentation process, SUMMARY OF THE INVENTION In one embodiment, this invention relates to a method of generating ascorbic acid, comprising () culturing a Kluyveromyces spp. or & Zygosaccharomyees spp. yeast in 4 medium comprising an ascorbic acid precursor, thereby forming ascorbic acid, and (i) isolating the ascorbic acid Ina second embodiment, the present invention relates 4 method of generating ascorbic acid, comprising i) eultue ing a recombinant yeast in a medium comprising an ascorbic acid precursor, thereby forming ascorbic acd, and (i) iso- lating the aseorbie acid. Preferably, the recombinant yeast accumulates ascorbic acid in the medium at a level greater than the background. Alko preferably, the recombinant yeast produces ascorbic acid at a yield greater than about 35% from the precursor. In third embodiment, the present invention relates to a method of stabilizing aseorbie aid in a medium, comprising culturing a yeast in the medium. ‘The present invention provides methods for the produc- tioa of ascorbic acid by a convenient fermentation process. DESCRIPTION OF THE DRAWINGS FIG. 1 provides a schematic representation of the current understanding of the physiological biosynthetic pathways leading from D-glucose to L-ascorbic acid in plants or animals, respectively. The following enzymes ae involved! A, L-galactono-1,4-lactone dehydrogenase (1.3.2.3), B, galactose dehydrogenase, C, sugar phosphatase (3.1.3.23, pulative), D, hydrolase (putative), E, GDP-mannose-3,5- epimerase (5.1.3.18), F, mannose-I-phosphate guanylyl- transferase (2.7.7.22), G, phosphomannomutase (5.4.28), H, mannose-6-phosphate isomerase (5.3.1.8), 1, glucose-6- phosphate isomerase (5.3.1.9), J; hexokinase (2.7.1.1); 1; L-gulono-1,4-lactone oxidase (1.1.38); 2; aldonolactonase G.1.1.17} 2a, ghucurono lactone reductase (1.11.20) 3; Deghicuronate reductase (1.1.1.19}; 3a, wronolactonase G.1.1.19) or spontaneous, 4; D-glucurona kinase (2.7.1.43); 5; slucuronate-L-phosphate uridylyltransferase (2.7.7.44); 6; LUDP-D-glucose dehydmgenase (1.11.22); 7, UTP-ghucose- L-phophate uridylyttransferase (2.7.7.9); 8, phosphogluco- mutase (5.42.2), 9, hexokinase (2.7.1.1). However, it has to bee stressed that in the scope of the present invention to produce L-ascorbie acid, the enzymes useful are not limited to the enzymes of the physiological pathways, FIGS. 2A-B shows the stability of ascombic acid under culture conditions. Ascorbic acid was added to mineral medium (2% glucose, 0.67% YNB) and incubated under standard culture conditions for 7 days. The flask of panel A. ‘was inoculated a time 0 with non-transformed S. cerevisiae GREISU to an initial OD” of 0.05, whereas the flask of panel B was kept sterile Samples were taken a the indicated times and the ascorbie acid concentration was determined. Although the ascorbic acid was stable in this medium when rowing yeast was present, it was completely degraded within 7 days in sterile medium. FIG. 3 shows the endogenous ability of yeasts to convert the precursors L-galactono-1,4-lactone (Gal) or L-gulono-US 6,630,330 BL 5 1 4-lactone (Gul) to ascorbic acid. Non-transformed yeast cxlls (S. cerevisiae GRFISU, W3031B and Z, baili) were sgrovn on mineral medium (2% glucose, 0.67% YNB) in the presence of 100 mM L-galactono-1,4-lactone or L-gulono- 1 4-lactone, respectively, for 72 br initial OD" was 0.05); + signifies that no precursor was added. While ascorbic acid was accumulated within the eell, no ascorbic acid could be detected in the culture broth FIG, 4 shows the endogenous ability of yeasts to convert galactose to ascorbic acid. Non-transformed S. cerevisiae (GREI8U and W303 1 B), Z bai and K. lactis were grown ‘on mineral mesium (2% glucose, 0.67% YNB) starting from an OD" of 0.05 overnight. Then, 250 mg 1” L-galactose were added and the cultures were kept under standard conditions for another 24 hr before the determination of ascorbic acid, All ofthese strains accumulated ascorbic acid intracellulary while no ascorbic acid was measurable in the ‘culture broth (It is believed the high background in K. lactis is due to erythroascorbie acid, naturally present inthis yeast species at higher concentrations than seen in S. cerevisiae). FIG. $ shows the conversion of L-galactono-1 4-lactone to ascorbic acid by recombinant yeasts. S. cerevisiae GRFISU t (contol), oF transformed with AGD or ALO, respectively, were grown on mineral medium (2% glucose, (0.67% YNB) starting from an OD of 0.05 in the presence ‘of 50 mM L-galactono-1,4-lactone (Gal) for 72hr. While the ‘control cells did not accumulate ascorbie acid inthe culture ‘medium, cells transformed with AGD or ALO unexpectedly accumulated considerable amounts (ie. greater than back- aground levels) of ascorbic acid in the culture medium. No ascorbic acid was detected in cultures without the addition of L-galactono-1,4-lactone (marked), FIG. 6 shows the conversion of L-galactose to ascorbic acid by recombinant yeasts. S. cerevisiae GRFISU wt (Control), transformed with LGDH; AGD; ALO; AGD and LGDH; ALO and LGDH; or ARA and ALO, respectively, ‘were grown on mineral medium (2% glucose, 0.67% YNB) starting from an OD®®? of 0.05 over night. Then 250 mg I~" Legalaciose were added and the cultures were kept under standard conditions for another 24 hr before the determina- tion of ascorbic acid. The conteol cells or cells transformed with only LGDH did not accumulate ascorbic acid in the culture medium, Cells transformed with LGDH and either AGD or ALO, as well as cells transformed with ARA and ‘ALO, accumulate considerable amounts (ie. greater than background levels) of ascorbic acid in the medium, FIG. 7 shows the conversion of L-galactase to ascorbic acid in a high cell density culture of recombinant yeast. S ‘cerevisiae GRFISU wt (conteol) or transformed with ALO, ‘or LGDH and ALO, respectively, were grown on mineral medium (2% glucose, 0.67% YNB) starting from an OD ‘0f 0.05 over night. At time 0 the cells were concentrated 10 times and 250 mg 1-* Legalactose were added and the ‘cultures were kept under standard conditions for 6 days. At the times indicated samples were taken and the aseorbie acid ‘concentration in the culture broth was measured. While the ‘control cells did not accumulate ascorbie acid inthe culture medium, cells transformed with ALO alone or ALO and LGDH accumulated considerable amounts (ie. greater than background levels) of ascorbic acid in the medium. DESCRIPTION OF ILLUSTRATIVE, EMBODIMENTS, In one embodiment, this invention relates to a method of ‘generating ascorbic acid, comprising (i) culturing Kluyveromyces spp. or a Zygosaccharomyces spp. yeast in os ss 4s ss 6s 6 2 medium comprising an ascorbic acid precursor, thereby forming ascorbic acid, and (ii) isolating the ascorbic acid ‘This method is based on the scientific observation that wild-type yeast of the genus Kluyveromyces or Zygosac- charomyees are capable of generating L-ascorbie acid when cultured on a medium containing an ascorbic acid pathway precursor. Preferably, the yeast is Z. bul ot K. lactis, More preferably, the yeast is Z. bailii ATCC 60483 or K lactis PM6-7A, ‘The medium in which the yeast is cultured can be any medium known in the art to be suitable for this purpose Cilturing techniques and media are well known in the art ‘Typically, but itis not limited to, culturing is performed by aqueous fermentation in an appropriate vessel. Examples for typical vessel for yeast fermentation comprise 4 shake flask or a bioreactor ‘The meclium comprises any component required for the srowth of the yeast and one or more precursors for the production of ascorbie acid. Components for growth of the ‘yeast and precursors forthe production of ascorbic acid! may ‘or may be not identical ‘The medium comprises a carbon source, such as ghucose or other earbohydrates (such as sucrose, fructose, lactose, Degalactose, or hydrolysates of vegetable matter, among others). Typically, the medium also comprises a nitrogen source, either organic or inorganic, and optionally the medium may also comprise components such as amino acids; purines; pyrimidines; com steep liquor; yeast extract; protein hydrolysates; water-soluble. vitamins, such as B complex vitamins; or inorganic salts such as chlorides, hydrochlorides, phosphates, or sulfates of Ca, Mg, Na, K, Fe, Ni, Co, Cu, Ma, Mo, or Zn, among others. Further components known to one of ordinary skill inthe art to be useful in_yeast culturing or fermentation can also be included. The medium may or may be not buffered. ‘The medium also comprises an ascorbic acid precursor. ‘The ascorbic acid precursor is any compound that, in the yeast, can be converted, either directly or through interme- diate'steps, into L-ascorbic acid. Ascorbic acid precursors include, but are not limited to D-glucose; trehalose; fructose; D-glucose-6-P; D-glucose-1-P; UDP-D-glucose; UDP- slucuronie acid; D-alucuronie acid-1-P; D-glucuronic acids D-glucurono lactone; L-gulonic acid; D-fructose-6-P; D-mannose-6-P; D-mannose-I-P; GDP-D-mannose; GDP- L-galactose; L-galactose-1-P; L-galactose; L-gulono-1,4- lactone; or L-galactono-I.4-Iactone, Preferably, the ascorbic acid precursor is selected from D-glucose: L-galactose; L-galactono-1,4-lactone; ot L-gulono-1,4-lactone. Two of mote ascorbic acid precursors can also be used. During the course of the fermentation, the ascorbic acid precursor is internalized by the yeast and converted, through ‘one or more steps, into L-ascorbie acid. The L-aseorbie acid so produced can be contained within the yeast, or can be accumulated in the medium at greater than background levels. ‘Appreferred medium comprises glucose, YNB, and atleast fone of L-galactono-1,4-lactone; L-gulono-t 4-lactone; or L-galactose. Alter culturing has progressed for a sufficient length of time to produce a desired concentration of L-ascorbic acid in the yeast, the culture medium, or both, the L-ascorbie acid is isolated. “Isolated,” as used herein to refer to ascorbic acid, means being brought to a state of greater purty by separation of ascorbic acid from at least one non-ascorbie acid component of the yeast or the medium. Preferably, the isolated ascorbic acid is at least about 95% pure, more preferably at least about 99% pureUS 6,630,330 BL 7 ‘To isolate L-ascorbie acid from the yeast, the first step of isolation, after the yeast is separated from the medium, typically is lysing of the yeast by chemical or enzymatic treatment, treatment with glass beads, sonication, freeze! thaw cycling, or other known techniques. L-ascorbic acid ‘can be purified from the membrane, protein, and nucleic acid fractions of the yeast lysate by appropriate techniques, such as centrifugation, filtration, microfiltration, ultrafiltration, ‘nanofiltration, liquid-liquid extraction, crystallization, enzy- matic treatment with nuclease or protease, or ‘chromatography, among others. ‘To isolate L-aseorbic acid accumulated in the medium, the isolation comprises purifying the ascorbic acid from’ the medium. Purification can be performed by known techniques, such as the use of an ion exchange resin, activated carbon, microfiltration, ultrafiltration, nanofiltration, liquid-liquid extraction, erystallization, ot ‘chromatography, among others. L-ascorbie acid ean he isolated from both the yeast and the medium, If the yeast accumulates L-ascorbic acid in the medium uring the culturing step, preferably the concentration of L-ascorbie acid is stabilized or allowed to increase. In a second embodiment, the present invention relates to ‘a method of generating ascorbic acid, comprising () cultur- ing a recombinant yeast in a medium comprising an ascorbie acid precursor, thereby forming ascorbic acid, and (ii) iso- lating the ascorbic acial A“recombinant” yeast is @ yeast that contains @ nucleic acid sequence not naturally occurring in the yeast or an additional copy of copies of an endogenous nucleic acid ‘sequence, wherein the nucleic acid sequence is introduced into the yeast or an ancestor cell thereof by human action Recombinant DNA techaiques are well-known, such as in ‘Sambrook etal, Molecular Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory Press, which provides fur- ther information regarding various techniques known in the art and discussed herein. In this embodiment, a coding region of the homologous and/or heterologous gene is isolated from an organism, which possesses the gene. The ‘organism can be a bacteritim, a prokaryote, a eukaryote, a microorganism, a fungus, a plant, or an animal Genetic material comprising the coding region can be ‘extracted from cells of the organism by any known tech- nique. Thereafter, the coding region can be isolated by any appropriate technique. In one known technique, the coding region is isolated by, frst, preparing a genomic DNA library ‘ora cDNA library, and second, identifying the coding region in the genomic DNA library or cDNA library, such as by probing the library with a labeled nucleotide probe selected to be or presumed to be at least partially homologous with the coding region, determining whether expression of the ‘coding region imparts a detectable phenotype 10 a library microorganism comprising the coding region, or ampli the desired sequence by PCR. Other known techniques for isolating the coding region can also be used. ‘The recombinant yeast can be selected from any known, sgenus and species af yeast. Yeasts are described by N. J. W. Kreger-van Rij, “The Yeasts,” Vol. 1 of Biology of Yeasts, Ch. 2, AH. Rose and J. S. Harrison, Eds. Academic Press, London, 1987. For example, the yeast genus can be Saccharomyces, Zygosaccharomyces, Candida, Hansenula, Kluyveromyces, Debaromyces, Nadsonia, Lipomyces, Torvlopsis, Kloeckera, Pichia, Schizosaccharomyces, ‘Trigonopsis, Brettanomyces, Cryptococeus, Trichosporon, Aureobasidium, Phailia, Rhodotorula, Yarrowia, or ss 4s ss 6s 8 Schwanniomyees, among others. Saccharomyces, Zygosaccharomyces, Kluyveromyces spp. are preferred More preferably the yeasts are S. cerevisiae, Z. bait and K lactis. Even more preferably, the yeas! is S. cerevisiae strain GREISU or W3031B, Z. bailii ATCC 60483, of K. lactis PMO-7A. Preferably, recombinant yeast ofthe present invention is not able to produce L-ascorbic acid from 2-keto-L-gulonic ad. Preferably, the recombinant yeast comprises at least one coding region encoding an enzyme associated with the conversion of a carbon source 10 ascorbate In a preferred embodiment of the present invention, the coding region introduced into the recombinant yeast encodes an enzyme selected from L-galactose dehydrogenase (LGDH), L-zalactono-t,4-lactone dehydrogenase (AGD), Dearabinose dehydrogenase (ARA), D-arabinono-1,4- lactone oxidase (ALO), L-gulono-I,4-lactone oxidase (RGLO). In one mote prefered embexdiment, the coding region of L-galactose dehydrogenase (GDI), L-galactono-1.4- lactone dehydrogenase (AGD), D-arabinose dehydrogenase (ARA), D-arabinono-1.4-lactone oxidase (ALO), L-gulono- 1 4-lacione oxidase (RGILO) at isolated from A. thaliana or cerevisiae or Rattus norvegicus. I should be noted thatthe term “isolated,” as used herein in reference to a ncleic acid sequence, refers to the ultimate source, not the immediate source, of the coding region. ‘Tha is, a coding region is “isolated” from an organism if it encodes a protein sequence substantially identical to that of the same protein purified from cells of the organism. In even more preferted embodiments, the coding regions encoding LGDH and AGD ae isolated from A. thaliana the coding regions encoding ‘ALO and ARA are isolated from S. cerevisiae, and the coding region encoding RGLO is isolated from R. nonvegi- In another more preferred! embodiment, the amino acid sequence of the LGDH enzyme has at least about 70%, more preferably about 80%, and most preferably about 90% similarity with SEQ ID'NO=1; the amino acid sequence of| the AGD enzyme has at least about 70%, more preferably about 80%, and most preferably about 90% similarity with SEQ ID NO:1 or SEQ ID NO:3; the amino acid sequence of the ARA enzyme has at least about 70%, more preferably about 80%, and most preferably about 90% similarity with SEQ ID NO:20; the amino acid sequence of the ALO enzyme has at least about 70%, more preferably about 80%, and most preferably about 90% similarity with SEQ ID 1NO:S or SEQ ID NO-7; the amino acid sequence of the RGLO enzyme bas at least shout 70%, more preferably about 80%, and most preferably about 904% similarity with SEQ ID NO:9; wherein “similarity” is determined by a sequence alignment performed using the CLUSTAL pro- grim In another more preferred! embodiment, the amino acid sequence of the LGDH enzyme has at least about 70%, more preferably about 80%, and most preferably about 90% identity with SEQ ID NO: 11; the amino acid sequence of the AGD enzyme has at east about 70%, more preferably about 80%, and most preferably about 90% identity with SEQ ID NO:1 or SEQ ID NO33; the amino acid sequence of the ARA enzyme has atleast about 70%, more preferably about 80%, and most preferably about 90% identity with SEQ ID 1NO:20; the amino acid sequence of the ALO enzyme has at least about 70%, more preferably about 804%, and most preferably about 90% identity with SEQ ID NO:S or SEQ IDUS 6,630,330 BL 9 NO the amino acid sequence of the RGLO enzyme bas at least about 70%, more preferably about 80%, and most preferably about 90% identity with SEQ ID NO:9; wherein “identity” is determined by a sequence alignment performed using the CLUSTAL program, In another more preferred embodiment, the coding gion encoding the LGDH enzyme has at leas about 70%, mote preferably about 80%, and most preferably about 90% ‘entity with SEQ ID NO 12; the coding region encoding he AAGD enzyme as a least about 70, more preferably about 806%, and most preferably about 90% identity with SEQ ID NO 2 or SEQ ID NO 45 the coding region encoding the ARA, enzyme has a east about 70%, more preferably about 80%, aixd most preferably about 90% identity with SEO 1D NO 21; the coding region encoding the ALO cazyme has atleast about 720%, more preferably about 80%, and most preferably about 909 identity with SEQ TD NO 6 or SEQ ID NO 8; the ‘coding region encoding the RGLO enzyme has at least about 70¢, more preferably about 80%, and most preferably about 9048 identity with SEQ ID NO 10, wherein “identity” is determined by a sequence alignment performed using the CLUSTAL progeam. In another prefered embodiment, wherein the enzyme is ARA, the enzyme comprises motif I and motif TT of the aldo-keto reductase (AKR) superfamily, specifically the fumino- acid. sequences GXRXXDXAXXXXXEXXXG (SEO ID NO:13) and GXXN (SEQ ID NO:26), respectively (Kim $. T-et al. 1998, BBA, 1429, 29-30), Tn a more preferred embodiment, the recombinant yeast further comprises at least one coding region encoding an enayme associated with the conversion of a earbon source to Legalactore Preferably, a coding region encoding a desired enzyme is incorporated into the yeast in such a manner that the desired ‘enzyme is produced in the yeast and is substantially fune- tional. Such a yeast may be referred to herein as being “functionally ansformed.” Once the casing region has been isolated, it ean be prepared for transformation into and expression in the yeast Useful inthe present iavention, At minimum, this involves the insertion ofthe coding region nto a vector and operable linkage to a promoter found on the veetor and active in the target organism (ie, inthe present invention, a yeas). Any vector (integrative, chromosomal or episomal) canbe used ‘Any promoter active in the target host (homologous or heterologous, constitutive, inducible or represible) can be used, Such insertion often involves the use of restriction endonucleases to “open up” the vector ata desired point ‘where operable linkage tothe promoter is possible, followed by ligation of the coding region into the desied point. If desired, before insertion into the vector, the coding rexion ‘ean be prepared for use in the target organism. This can involve allering the codons used in the coding region 10 more fully match the codon use of the larget organism; changing sequences in the coding region that eould impair the transcription oF translation of the coding region or the stability of an mRNA transcript of the coding region; or adding or removing portions encoding signaling peptides (egions of the protein encoded by the coding region that iret the protein to specific locations (ean organelle, the membrane of the cell of an organelle, or extracellular secretion), among other possible preparations known inthe art. In one embodiment of the present invention, the L-ailactono-1,4lactone dehydrogenase protein (AGD) ‘comprises a sigoaling peptide and the coding region encox- ing the L-galactono-1 ‘lactone dehydrogenase also encodes 0 as os ss 4s 50 ss 6s 10 the signaling peptide. In another embodiment ofthe present invention, the L-galactono-1,4-lactone dehydrogenase pro- tein (AGD) does not comprise a signaling peptide and the coding region encoding the L-galactono-1,4-lactone dehy- rogenase also does not encode the signaling. peptide Specifically, the AGD sequence given in SEQ ID NO:1 comprises a signaling peptide of amino acids 1-100, and the AGD sequence given in SEQ ID NO:2 comprises a signaling peptide of amino acids 1-90. As one of skill in the art will recognize, deletion of a nucieie acid sequenee encoding a signaling peptide from a longer mucleic acid. sequence encoding desired enzyme may require the addition of an in-frame ATG codon to allow for proper initiation of trans- lation of the desired enzyme. Regardless whether the coding region is modified, when the coding region is inserted into the vector, it is operably linked to promoter active in the yeast. A promoter, as known, is a DNA sequence that can direet the transcription of a nearby coding region. As already described, the pro- ‘moter canbe constitutive, inducible orrepressibl. Inducible promoters can be induced by the addition to the medium of an appropriate inducer molecule, which will be determined by the idcatty of the promoter. Repressible promoters can be repressed by the addition to the medium of an appropriate repressor molecule, which will be determined by te identity ofthe promoter. Constitutive promoters are preferred, 2s the use of an inducer or repressor molecule is not required. A preferred constitutive promoter is the S. cerevisiae triose- phosphateisomerase (TPI) promoter ‘The vector comprising the costing region operably linked to the promoter can be a plasmid, a cosmid, or a yeast artificial chromosome, among others known in the art to be appropriate for use in yeast genera. In addition tothe coding region operably linked to the promoter, the vector can also comprise other genetic elements. For example, if the vector isnot expected to integrate into the yeast genome, the vector desirably comprises an origin of replication, which allows the vector to be passed on to progeny cells of a yeast comprising the vector. If integeation of the vector into the yeast genome is desired, the vector can comprise sequences homologous to sequences found in the yeast yenome, ark! can also comprise coding regions that can facilitate integra- tioa. To determine which yeast cells are transformed, the vector preferably comprises a sclectable marker or screcn- able marker which imparts a phenotype to the yeast that distinguishes it from untransformed yeast, e.g. it survives on 1 medium comprising an antibiotic fatal to untransformed yeast or it metabolizes a component of the medium into a product thatthe untransformed yeast does not, among other phenotypes. In addition, the vector may comprise other genetic elements, such as restriction endonuclease sites and others typically found in vectors. ‘After the vector is prepared, with the coding region ‘operably linked to the promoter, the yeast is transformed withthe vector (ie. the vector is introduced into at least one of the cells of a yeast population). Techniques for yeast transformation are well established, and include electroporation, microprojectile bombardment, and the LiAcissDNA/PEG method, among others, Yeast cells, which are transformed, can then be detected by the use of a scteenable or selectable marker on the vector. It should be noted that the phrase “transformed yeast” has essentially the same meaning as “recombinant yeast,” as defined above ‘The transformed yeast can be one that received the vector in «transformation technique, or can be a progeny of such a yeas ‘Alter a recombinant yeast has been obtained, the yeast is cultured in a medium. The medium is as described above,US 6,630,330 BL u ‘A preferred medium comprises glucose, YNB, and L-galactono-1,4-lactone. Preferred recombinant yeasts ‘which can be cultured in this medium include S. cerevisiae strain GREISU yeast bearing a S. cerevisiae TPL promoter ‘operably linked to a coding region encoding A. thaliana [-galaciono-1 4-lactone dehydrogenase (AGD); and S. cer- ‘esisiae strain GRFISU yeast bearing a S. cerevisiae TPL promoter operably linked to a coding region encoding 5: ‘cerevisiae D-arabinono-L,4-lactone oxidase (ALO). ‘Another preferred medium comprises glucose, YNB and L-gulono-1,4-lactone, One particularly preferred recombi- nant yeast which can be cultured in this medium include S. ‘cerevisiae strain GRFI8U bearing a S. cerevisiae TPL pro- moter operably linked to a coding region encoding R. rnonvegicus L-gulono-1,4-lactone oxidase (RGLO). ‘Another preferred medium comprises glucose, YNB and Legalactose. One particularly preferred transformed yeast ‘which ean be cultured in this medium is S. cerevisiae Strain GRFISU yeast beating (i) a S. cerevisiae TPL promoter ‘operably Tinked to coding region encoding A. thaliana L-galactono-1-lactone dehydrogenase (AGD) and (ji) a ‘TPL promoter operably linked to a costing region encoding A. thaliana L-galactose: dehydrogenase (LGDH). A second particularly preferred transformed yeast which can be cul- tured in this medium is S. cerevisiae strain GRFISU yeast ‘comprising (i) a TPI promoter operably linked to a coding region encoding S, cerevisiae D-arabinono-1,4-lactone oxi ‘dase (ALO) and (ii) a TPL promoter operably linked 10 a ‘coding region encoding A, thaliana L-galactose dehydrose~ nase (LGDH). A third particularly preferred transformed yeast which can be cultured in this medium is S. cerevisiae Strain GRFISU yeast comprising (i) a TPL promoter oper- ably linked 10 a coding region encoding S. cerevisiae Darabinono-1,4-lactone oxidase (ALO) and (il) « TPI pro- moter operably linked 10 a coding region encoding S ‘cerevisiae D-arabinose dehydrogenase (ARA), ‘As described for non-recombinant yeast, above, during the course ofthe fermentation, the ascorbic acid precursor is ‘converted, through one or more steps, into L-sscorbie acid While the non-recombinant yeast cells (described above) incubated in similar media typically donot accumulate ascorbic acid above background levels in the medium, surprisingly the particularly prefered recombinant stains herein described are able to accumulate considerable amounts of L-ascorbic acid above background levels. The ‘only exception relates to a yeast transformed with only LGDH, which does not accumulate L-ascorbie acid above background levels, that indicates the LGDH expression is not the limiting factor. The data taken together indicate that the conversion of L-galactono-1 4-laetone to ascorbic acid is the limiting factor in the pathway leading from L-galactose to ascorbie acid ‘Therefore, in a preferred embodiment, the recombinant yeast accumulates L-ascorbie acid in the medium above background levels. Isolation of the ascorbic acid from the media is as described above. Yields of ascorbic acid of greater than about 35% have been observed, as will be described in the Examples below. Therefore, in a further preferred cemboxtiment, the recombinant yeast produce ascorbic acid with a yield higher than 35% of the precursor. The term jeld” refers to the amount of ascorbic acid (molar as well as weightivolume) produced divided by the amount of precursor consumed (molar as well as weight/volume) mul- plied by 100. ‘The following definitions are provided in order t0 aid those skilled inthe art in understanding the detailed descrip- tion of the present invention 0 as os ss 4s ss 6s 12 ‘The term “accumulation of ascorbic acid above back- ground levels" refers to the accumulation of ascorbic acid above the undetectable levels as determined using the pro- cedures described herein “Ascorbic acid” as well as “ascorbate” as used herein, refers to L-ascorbie acid “Ascorbic acid precursor” is a compound that can be converted by a yeast of the present invention, either directly or through one or more intermediates, into L-ascorbic acid, “Amplification” refers to increasing the number of copies of a desired nucleic acid molecule orto increase the activity of an enzyme, by whatsoever means. “Codon” refers to a sequence of three mucleotides that specify a particular amino acid, “DNA ligase” refers to an enzyme that covalently joins two pieces of double-stranded DNA. “Electroporation” refers to a method of introducing for- eign DNA into cells that uses a brief, high voliage DC charge to permeabilize the host cells, causing them to take up extra-chromosomal DNA. ““Endonuclease” refers to an enzyme that hydrolyzes double stranded DNA at internal locations. Enzyme 1.13.37, D-arabinono-1,4-lactone oxidase, refers to a protein’ that catalyzes ‘the conversion of D-arabinono-1,-lactone+0, to D-erythroascorbate+H1.0. ‘The same enzyme due to broadness of substrate range catalyses the conversion of L-galactono-1,¢-lactone+0, to ascorbic acid+H.O.. Exroneously the same enzyme is referred to as L-galactono-1,4-lactone oxidase (enzyme 1.1.3.24) (see Huh, W. Ket al, 1998, Mol. Microbiol. 30, 4, 1895-903) Enzyme 1.3.2.3, L-galactono-1,4-lactone dehydrogenase, refers to a protein that catalyzes the conversion of L-galactono-1,4-lactone+2 ferrieytochrome C to L-ascorbic aacid42 ferrocytochrome C. Enzyme 1.1.38, L-gulono-1,4-Laetone oxidase, refers to a protein that catalyzes the oxidation of L-gulono-1,4-lactone to L-xylo-hexulonolactone which spoatancously isomerizes to L-ascorbie acid, Other enzymes of interest, and their classification aumbers, are as follows: ‘Glucne-@Piomerse Mannow-@P womerse Phorphomannomaee Matose--P punyllianetere GDP-Maasos 4-epimense Sugar phosphaise Galactose debydiogenaie T-Galacono i lstone dehydrogenase Desaonone kine Phosphoplucomutse UTP-Glucowe 1 undlyl tamer [UDP-D-Gionse debydrogensee UUbe.Givctonate &eimerase lucuroate--P uriylyltansense B-chicworotie DeGhuronsteredctase AAlonolctonsse {-Gulooo-1tactone oxide Uroaolastonse ‘Glucronoacone reductase activity T-Gulactonoi lactone piers GalctaronateP uidylyesnserne ‘Gaicturonokinase Hexuonste(Dgalaturnate) reductase )US 6,630,330 BL 13 Sea Myoinon 1? monophosphate sagas Mycizontal oxygenase aoe DGalactoknnse ar UnPHexose 1 udyyiasterse Bhnt0 UDP-Glucoe epimeme S133) Soe sythase 2uhas Fracokinae ara 7) Chssiteation sumer aot avaable la dnabase, ‘The term “expression” refers to the transcription of a gene to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product, is, a peptide, polypeptide, or protein ‘The phrase “functionally linked” or “operably linked” refers 10 a promoter or promoter region and a coding or structural sequence in such an orientation and distance that transcription of the coding or structural sequence may be directed by the promoter or promoter region ‘The term “gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protcin, or RNA molecule, and regions flanking the coding Sequence involved in the regulation of expression. ‘The term “genome” encompasses both the chromosomes and plasmids within a host cell. Encoding DNAS of the present invention introduced into host cells can therefore be «ither chromosomally integrated or plasmid-localized. “Heterologous DNA” refers to DNA from a source dif ferent than that of the recipient cell “Homologous DNA” refers to DNA from the same source a that of the recipient cel. “Hybridization” refers tothe ability ofa strand of nucleic acid to join with a complementary sieand via base pairing. Hybridization occurs when complementary sequences in the two micleic acid strands bind to one another. ‘The term “medium” refers to the chemical environment of the yeast comprising any component required for the growth fof the yeast or the recombinant yeast and one or mote precursors for the production of ascorbic acid. Components for growth of the yeast and precursors for the production of ascorbic acid may or may be not identical “Open reading frame (ORF)” refers to a region of DNA or RNA encoding a peptide, polypeptide, or protein, “Plasmid” refers t0 a circular, extra chromasomal, repi- ceatable piece of DNA. “Polymerase chain reaction (PCR)” refers to an enzymatic technique w ereate multiple copies of one sequence of rucleie acid. Copies of DNA sequence are prepared by shuttling a DNA polymerase between two amplimers. The basis of this amplification metbod is multiple eycles of temperature changes to denature, then re-anneal amplimers, followed by extension to synthesize new DNA strands in the region located between the flanking amplimers. ‘The term “promoter” or “promoter region” refers to @ DNA sequence, usually found upstream (5') to a coding ‘sequence, that controls expression of the coding sequence by controlling procuction of messenger RNA (mRNA) by pro- ‘viding the reogoition site for RNA polymerase and/or other factors necessary for start of transcription atthe correct site ‘A“recombinant cell” or “transformed cel” is a cell that ‘contains a nucleic acid sequence not naturally occurting in the cell or an additional copy or copies of an endogenous 0 as os ss 4s ss 6s 14 aucleic acid sequence, wherein the nucleic acid sequence is introduced into the cell of an aneesior thereof by human action, ‘The term “recombinant vector” or “recombinant DNA or RNA construct” refers to any agent such as a plasmid, ccosini, virus, autonomously replicating sequence, phaze, ot linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence, derived from any source, capable ‘of genomic integration or autonomous replication, compris- ing a nucleic acid molecule in which one or more Sequences hhave been linked in a functionally operative manner. Such recombinant constructs or vectors are capable of introducing 45 regulatory sequence or promoter region and a DNA sequence for a selected gene product into a cell in such a ‘manner that the DNA sequence is transcribed into a func- tional mRNA, which may or may not be translated and therefore expressed “Restriction enzyme” refers to an enzyme that recognizes 4 specific sequence of nucleotides in double stranded DNA and cleaves both strands; also called a restriction endonu- lease. Cleavage typically occurs within the restriction site or close to “Sclectable marker” refers to a nucleic aci whose expression confers a phenotype fac cation of cells containing the nucleic acid sequence. Select- able markers include those, which confer resistance to toxie chemicals (€.g, ampicillin, kanamycin) or complement a nutritional deficieney (cg, uracil, histidine, leucine) “Screenable marker” refers to a nucleic acid sequence whose expression imparts a visually distinguishing charac- teristic (eg, color changes, fluorescence, “Transcription” refers to the process of producing an RNA copy from a DNA template “Transformation” refers to & process of introducing an exogenous nucleic acid sequence (eg, a vector, plasmid, oF recombinant nucleic acid molecule) into a cell in which that exogenous nucleic acid is incorporated into a chromosome or is capable of autonomous replication. A cell that has uncengone transformation, or a descendant of such a eel, is “transformed” of “eecombinant." If the exogenous aucleie acid comprises coding region encoding a desired protein, and the desired protein i proxiuced inthe transformed yeast and is substantially functional, such a transformed yeast is “functionally transformed.” “Translation” refers to the production of protein from messenger RNA. The term “yield” refers to the amount of ascorbie acid produced (molar or weightvolume) divided by the amount bf precursor consumed (molar of Weight volume) multiplied by 100. “Unit” of enzyme refers to the enzymatic activity and indicates the amount of mieromoles of subsirate converted per mg of total cell proteins per minute “Vector” refers to @ DNA or RNA molecule (such as 2 plasmid, cosmid, bacteriophage, yeast artificial chromosome, oF vis, among others) that carries nucleic acid sequences into a host cell. The vector ora portion of it can be inserted into the genome of the host cell LIST OF ABBREVIATIONS. Ase L-ascorbie acid (vitamin C) ‘AGD L-galactono-1,4-lactone dehydrogenase (without sig- naling peptide, from A. thaliana)US 6,630,330 BL 15 ALO D-arabinono-1.4-lactone oxidase from S. cerevisiae ARA D-arabinose dehydrogenase [rom S. cerevisiae Gal L-galactono-1,4-lactone Gul L-gulono-1,4-lactone LGDH L-galactose dehydrogenase from A. thaliana RGLO L-gulono-1,4-lactone oxidase fiom R. norvegicus TCA trichloro acetic acid ‘TPL triosephosphateisomerase EXAMPLES. ‘The following examples are included to demonstrate preferred embodiments of the invention. It should be appre~ ciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well ia the practice Of the inveation, and thus can be considered to con: preferred modes for its practice. However, those of ski the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments Which ace disclosed and still obtain a like or similar result ‘without departing from the spirit and scope ofthe invention, MATERIALS AND METHODS 1, Determination of Ascorbic Acid Ascorbic acid was determined speetrophotometrcally fol- lowing a method after Sullivan etal, (1955, Assoc. Off. Agr Chem., 38, 2, 514-518). 135 sl of sample were mixed in a ‘cuvette with 40 ai of HyPO, (85%). Then 675 sl cue Bipyridyl (0.5%) and 135 yl FeCl, (1%) were added. After 10min the absorbance at 525 nm was measured. The identity ‘of the ascorbie acid was confirmed by HPLC (Tracer Extra~ sil Column C8, 5M, 15x0.46 em, Teknokroma, $. Coop. C. Lida. #'TR-016077; Eluent: 5 mM cetyltrimethylammonium bromide, 50 mM KHsPO, in 95/5 H,0/Acetonitrile; Flow rate: 1 ml min, Detection UV @254 nm) with pure L-ascorbie acid (Aldrich, A9,200-2) as standard 2, Determination of Protein Concentration Protein concentrations were determined following Low- 1y's method (Lowry O. H. et al, 1951, J. Biol. Chem. 193, 265-275), using the Bio-Rad DC Protein Assay Kit Il (Cat Nr. 500-0112) with BSA as standard 3. Amplification of Specific Gene Sequences “To amplify specific gene sequences, PfuTurbo DNA poly- merase (Stratagene #600252) was used on a GeneAmp PCR ‘Sysicm 9700 (PE Appl. Biosystems, Inc.) Standard condi- tions used were: 400 uM aNTP, 0.5 4M primers, 0.5 mM MgCl. (in ation to the buffer), and 3.75 U Pfu per 100 jl reaction. ‘The sequences of the genes used have been publicly reported via Genbank, as follows: Gene Genbank ssesion (0) ‘SEQ ID No: hop) ALDOGSS (Gene =o TITIAN) 2 [AGD bomolog fom 2070680 4 Basin Ato ‘vanss0, aBono4oL 5s Roto mas36 to RA, ‘¥15134, 236018 (ORF YBRI408) 21 ‘The following program was used for amplification of AGD: 0 20 os ss 4s ss 6s 16 ees ‘The following program was used for amplification of ALO: wee see sre } soe we aminaos eth re 2 ‘The following program was used for amplification of ARA: OG eC at eyes me re ‘The following program was used for amplification of LGDH wee gee see } some Re tminaos Be thn Fé 2 ‘The following program was used for amplification of RGLO: oe we ee sy me 4a Byles me 5 oa ve 2 ‘Template DNA for AGD an! LGDH: 50 ng. plasmid DNA library pFL61 Arabicopsis (ATCC #77500 (Minet M. ct al, 1992, Plant J,, 2, 417-422). Template DNA for RGLO: 0.5 ng rat liver marathon-ready cDNA library (Clontech #7471-1). Template DNA for ALO and ARA: 50 rng genomic DNA from S. cerevisiae GRFISU, extracted using a standard method, PCR products were blunt end cloned into the EcoRV site of pSTBlue-1 using the perfectly blunt cloning kit from Novagen Inc. (#70191-4),US 6,630,330 BL 17 ‘Oligonuclenies we Gene anplied SEQIDNO: 1 cangagqoctnatgtongtacpece SEQID NO: 1S: ngggcsetiangegtgtggnerctgge AOD (plan) SEOID NO: 16: tsaeestengepetinstos: SEQIDNO: 17; iggastsagertzapscassggg —RGLO (at) SEQID NO: 18: teccattglasetce SEQ ID NO: 19: aaggncsgtoggrnscte ALO (yes) SEQID NO: 22: sgaewusstgegetgsge SEQIDNO: 25: tagutgatggtccasigg GDH (past SEQID NO: 26: stgleuctsgisgetenace SEQID NO: 2: taslactsnaugloaagiigaie ARA (ea) 4, Plasmid) Construction ‘The naming convention used herein is that pSTBIue-1 containing, for example, AGD ia sense direction regarding its multiple cloning site (MCS) was designated pSTB AGD- 1. In further example, pSTBIue-1 containing AGD in antisense direction regarding its MCS was designated pSTB AGD-2, and s0 on. Inserts were cloned using the pYX series (RED Systems, Inc) below. Standard procedures were employed for all ‘cloning purposes (Sambrook J etal, Molecular Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory Press). 18 (2% glucose, 0.67% YNB) and incubated the solution in shake flasks shaking at 30° C. FIG. 2 shows the respective results. In sterile medium, ascorbic acid is rapidly degraded (See panel B), whereas it is completely stable if growing yeast is present (see panel A). This result shows that culturing yeast in a medium is a method of stabilizing ascorbic acid. 2. Ascorbie Acid Produetion From Non-transformed Yeasts ‘According to the literature, wild-type (wt) yeast com- prises a D-arabinono-1-lactone oxidase activity with a broad substrate specificity (Huh W. K. et al, 1994, Eur. J Biochem. 225, 1073-1079). Such activity has been demon- strated in vitro, To determine whether the substrates or the product could cross the cell membrane, we incubated three different yeast strains (S. cerevisiae GRFISU and W3031 B, as well as Z. bailii) with L-galactono-1,4-lactone (the last precursor of the plant biosynthetic pathway leading to ascorbie acid) or L-gulono-1,4-lactone (the last precursor of the animal metabolic pathway). As shown in FIG. 3, both of the substances can be internalized into the yeast cell and can bbe converted to ascorbic acid, No ascorbic acid was acc- mulated in the culture broth (not shown) but significant amounts were measured in whole cell extracts. ‘The next prior precursor in the plant pathway is L-galactose. FIG. 4 shows the results of incubations of yeast as PSTBAGDA Boot pYND? PLAGD PSTB Lott Boot pyxn22 PELGDH, PSTB ALO: Boot pyrene PLALO PSTBARA? Soc blunt Bani pYXO22 EcoRI bon! Bal pH ARA PSTBRGLO- Noll Hunt Kpol blunt pYNO!2— PooRE bt pLRGLO 5. Yeast Cultivation and Examiaation: ‘Yeast strains used were S. cerevisiae GRFISU (Brambilla, L. et al, 1999, FEMS Microb. Lett. 171, 133-140), W3031B, Z. bailii ATCC 60483, and K. lactis PM6-7A (Wésolowski-Louvel, M. et al, 1992, Yeast 8, 711719). All Strains were cultivated in shake flasks ia minimal medium (0.67% wiv YNB (Dito Laboratories, Detroit, Mich. #919- 15), 2% wiv glucose, addition ofthe appropriate amino acids, for adenine or uracil, respectively, 10 50 jag 1!) under Standard conditions (Shaking at 30° C.) The initial optical density at 660 nm was about 0.05. For incubation with L-galactose the cells were grown over night, then 250 mg. 1-? of L-galactose were added and the cells were incubated for 24 he. For incubation with substrates ‘ther than L-galactose, the cells were grown in presence of 50 mM or 100 mM of the respective substrates for 72 hr. Cells were recovered by centrifugation at 4000 rpm for 5 min at 4° C,, washed once with cold distilled HO, and treated as follows: for determination of intracellular ascorbie acid, cells were resuspended in about 3 times the pellet volume of cold 10% TCA, vortexed vigorously, kept on ice for about 20 min then the supernatant was cleared from the cell debris by centrifugation. 6. Yeast Transformation: Transformation of yeast cells was done following the Standard LiAcis-DNA/PEG method (Gietz, R. D. and Schiestl, R-H., 1996, Transforming Yeast with DNA, Meth- ‘ods in Mol, and Cell, Biol). Transformed yeast are being deposited with ATCC, catalog numbers not yet assigned, EXPERIMENTAL RESULTS 1. Stability of L-ascorbie Acid ‘To determine the stability of ascorbic acid under culture conditions, we added ascorbie acid to our standard medium 5 cells with this substate.S, cerevisiae Z. bail, and K. lactis fe able to produce ascorbic acid fom this compound, but also inthis ease sscorbie acid is accumulated o a significant amount inside ofthe ell (FIG. 4), but the conceatraton in the culture medium remains under the deteetion iit (aot shown). 3. Ascorbie Acid Production and Accumulation in the ‘Medium From Transformed Yeasis We cloned the homologous genes of D-arabinono-1,4- lactone oxidase (ALO) and D-arabinose. dehydrogenase (ARA), as well asthe heterologous 4. thaliana genes for L-galactono-1,4-lactone dehydrogenase (AGD) and Legalactose dehydrogenase (GDH), These genes were cloned into available yeast expression vectors like outlined in materials and methods. In short, the plasmids are inte- arative and the TPI promoter, a naturally sitong and consti- {utive promoter ofS. cerevisiae, drives the expression of the genes in question. Upon incubation ofS. cerevisiae GRF1SU transformed with AGD or ALO with L-galactono-1,4- lactone, the cells not only accumulated ascorbie aeid intra cellularly (not shown), but also, surprisingly, accumulated considerable amounts of ascorbic acid into the culture broth (FIG. §). This was also true forthe same transformed cells incubated with L-galactose (FIG. 6). Cotransformation of LLegalactose dehydrogenase or D-arabinose dehydeogenase significantly increased the ability of the respective yeast sirain to convert L-galactose to aseorhie acid (FIG. 6). FIG. 7 shows data of a high-density culture converting Legalactose to ascorbic acid. The respective yeast strains were grown overnight in standard minimal medium. The next day, the cells were aseptically centrifuged and the pellet ‘was resuspended in Yio ofthe supernatant to concentrate the cells 10 times. Then, 250 mg I”? of L-galactose were added 4s 50 ss 6sUS 6,630,330 BL 19 and the cultures were incubated under standard conditions for 6 days. After 6 days the strain transformed with ALO and LGDH accumulated over 70 mg ascorbic acid per liter ‘culture medium. 30 mg 1”? ascorbic acid were accumulated intracellularly (got shown). Taking these two values together corresponds to a conversion of around 40% of the L-galactose added. ‘The following table summarizes the main examples reported in this invention, Example of Yest Examples of Gene oveexpresed ‘Sccereisie mo [Ugutono-i-racone Lgalactow K. tacts m0 gales 2 ba es Ugplstonet tone [Ugutono--acone Lgalactow S.ceribiae GD ae ay galectono stone SS corvisie RA. grlston: nse $corvisiae 1DH cow Aan) Peslectone S connie SDH gran wasn) eaten Eaalcone SGD eas ean). S.corisiae ——ARAALO Legato Sicevsiee ROTO gear wt [gulone4-actone While the compositions and methods and yeast strains of 30 this invention have been described in terms of preferred ‘emboctiments, it will be apparent to those of skill in the art that variations may be applied without departing from the ‘concept, spirit and scope of the invention 3s REFERENCES ‘The following references, to the extent that they provide ‘exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference, [1] Padh H. 1990, Cellular functions of ascorbic acid, Biochem. Cell Biol. 68, 1166-1173, [2] US. Pat, No. 2,265,121 [B] Huh, W. K,, Lee, B. HL, Kim, S.’T, Kim, ¥. R, Rhie, 45 G.E., Back, Y. W,, Hwang, C.S., Lee, S.J., Kang, S (0,, 1998, D-Erythrosscorbie acid is an important anti= ‘oxidant molecule in S. cerevisiae, Mol. Microb. 30, 4, 895~003, [4] Wheeler, G. L., Jones, M.A., Smirnoff, N., 1998, The 5° biosynthetic pathway of vitamin C in higher plants, Nature 393, 365-368 [5] Huh, W. K,, Kim, S. T, Yang, K,S., Seok, ¥. J. Hah, 'Y. C, Kang, S. 0.,” 1994, Characterisation of D-arabinono-I-4-lacione oxidase from Candida albi- cans ATCC 16231, Eur, J. Biochem, 225, 1073-1079 [6] Kim, S. T, Hub, W. K., Kim, J. ¥,, Hwang, S. W. Kang, S. 0., 1996, D-Arabinose dehydrogenase and biosynthesis of erythroascorbic acid in Candida gy albicans, BBA 1297, 1-8 [7] Kim, S."T, Huh, W.K., Lee, B.I., Kang, 8. 0.,1998, D-Arabinose dehydrogenase and its gene from Saccha~ romyces cerevisiae, BBA 1429, 29-39 [8] Roland, J. F,, Cayle, T., Dinwoodie, R. C., Mebnert, D. 65 'W., 1986, Fermentation Production of Ascorbic Acid from L-Galactonie Substrate, U.S. Pat, No, 4,595,659 ss 20 [9] Roland, JF, Cayle, T, Dinwoodie, R.C., Mehnert,D. W,, 1990, Bioconversion Production of Ascorbic Acid with L-Galactono-1,4-Oxidase, US. Pat No. 4,916, 068 [10] Lee, B. H., Huh, W. K., Kim, 8. T, Lee, .S., Kang, 'S.0,, 1999, Bacterial Production of D-Erythroascorbie Acid and L-Ascorbie Acid through Functional Expres- sion of Saccharomyces cerevisiae D-Arabinono-1,4- ve ve ve 1 ve % ve 6 re = t Reed, Rava Lactone Oxidase in Escherichia coli, App.Eav, Microb. 65, 10, 4685-4687 [11] Ostergaard, J, Persiau, G., Davey, M. W., Bauw, Van Montagu, M., 1997, Isolation of a eDNA Coding for L-Galactono-y-Lactone Dehydrogenase, an Enzyme involved in the Biosynthesis of Ascorbie Acid in Plants, J. Biol. Chem, 272, 48, 30009-30016 [12] Bauw, G. J. C., Davey, M. W,, Ostergaard, J., Van Montagu, M. C. E., 1998, Production af Ascorbic Acid in Plants, 1998, International Patent Application, W098 50558 [13] Berry, A., Running, J, Severson, D. K., Burlingame, R. P, 1999, Vitamin C Production in Microorganisms and Plants, International Patent Application, W099) 4618 [14] Smirnoff, N., Wheeler, G., 1999, Plant Galactose Dehydrogenase, International Patent Application, W0099/33095 [15] Hancock, R. D., Galpin, J. R., and Viola, R. 2000, Biosynthesis of L-ascorbic acid (vitamin C) by Saez ‘charomyces cerevisiae. FEMS Microbiol, Lett, 186, 245-250 [16] Nishikimi, M., Noguchi, E., Yagi, K., 1978, Occur- rence in Yeast of L-Galactonolactone Oxidase Which is Similar to a Key Enzyme for Ascorbic Acid Biosyn- thesis in Animals, L-Gulonolactone Oxidase, Arch. Biochem, Biophys. 191, 2, 479-486 [17] Bleeg, H.S., Christensen, F, 1982, Biosynthesis of Ascorbate in Yeast, Purification of L-Galactono-14- lactone Oxidase with Properties Different from Mam- malian L-Gulonolactone Oxidase, Eur. J. Biochem. 127, 391-96 [18] Sullivan, M. X,, Clarke, H.C. N., 1955, A highly specific procedure for ascorbic acid, Assoc. Off. Agr Chem. 38, 2, SSIS [19] Lowry, O. H., Rosebrough, N. J., Farr,A. L., Randall, RJ. 1951, Protein Measurement with the Folin Phenol Reagent, J.Biol. Chem. 193, 265-275US 6,630,330 BL 21 [20] Minet, M., Dufour, M. E., Lacroute, F, 1992, Plant 3,2, 417-422 [21] Sambrook et al., Molecular Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory Press. [22] Gietz, R. D. and Schiest, R. H., 1996, Transforming, ‘Yeast with DNA, Methods in Mol. and Cell. Biol. [23] Kreger-van Rij, N. J. W,, “The Yeasts,” Vol. 1 of Biology of Yeasts, Ch. 2, A. H. Rose and J. S. Harrison, Els, Academic Press, London, 1987. [24] Brambilla, L., Bolzani, D., Compagno, C, Carrera, D., van Dijken, J. P, Pronk, J.T, Ranzi, B. M., 22 Alberghina, L., Porro, D. 1999, NADH reoxidation ‘does not control glycolytic flux during exposure of ing Saccharomyces cerevisiae cultures to ghucose ‘exeess, FEMS Microb. Lett, 171, 133-140 [25] Wésolowski-Louvel, M., Prior, C., Borneeque, D., Fukuhara, H, 1992, Rag-mutations involved in ghicose metabolism in yeast: isolation and genetic character- ization. Yeast 8, 711-719 [26] Kumar, M. 2000 Production of ascorbic acid using yeast, International patent application WO 00/34502 0 SEQUENCE LISTING -<160> NUMBER OP SEQ 1D Nos: 26 «20> 89 10 10 2 2135 ORGANISM: AcabSdopeis thaliana too» sequence: 2 Mot Leu Arg ser Leu Leu Leu Arg Arg Ser Val Gly ‘the teu Ser Pro 20 Ser Ser Ser the tle 28 arg Ser Ser arg Thr Leu 38 cye thr the Gly Gin fH ‘The Lex The Pro 30 Arg Pro Pro Pro Pro 8 Pro Pro Ala the Ala 60 Phe Arg Lys tyr Ala Gly 7% ‘yr Ala Ala Leo Ala 75 ne Ale Thr Tyr Phe Ser 35 Phe Pro Phe Pro cis 0 Aen Als Ala Gin Tle Phe 100 larg Tyr Ala Pro Lea Pro lu Asp 105, Ser Asn trp ns, Ser Gly Thr His ois 120 Val Gin Thr Arg Pro clu 10 Aen Lou Ala Asp Lew 135 ty Ale tes Val Lys 140 tye tye 15 Leu arg Tle Arg 150 Pro Val Gly Ser Gly 155 ie Gly Leu Ser arg Ser Gly Met Vel nen teu Gln Aen Phe Ala Ger Tle Arg Glu Gln Gln nie Phe ‘8 Phe tye tes aan 15 cis ay ser teu sly 30 Pro is cin Ala Gln Ser Gly Val 90 His tye 35 tye Hie no athe val Phe Aen Gin Ser His cla Pro Asn Gly 160 Met Asp. Lye cay Te tleUS 6,630,330 BL continued eu Gly Gly Leu Gly Val Val Als Glu Val Thr Lea Gin Cys Val Als Arg His Glu Leu Val Glu His Thr Tyr Val Ser Asn Leu Gln ole Te Lys tye Aen ile Lye Lye Leu Leu Ser Ala Aen Lys ile val tye Tye 305 310 315 320 Leu Tye Te Pro Tye The Aep Thr Val Val Val Val The cye Aen Pro Val Sex Lye Tep Ser Gly Pro Pro Lye Rep Lye Pro Lys Tyr The The ‘Asp Glu Ala Val Gln tis Val Arg Asp Leu Tyr Arg Glu Ser Tle Val Lye Tye Arg Val Gln Asp Ser Gly Lye Lys Ser Pro Aep Ser Ser Glu Pro Asp Tle Gln Glu Leu Ser Phe Thr Glu Lau Arg Asp Lys Lew Lew ‘Ala Leu Asp Pro Lou Asn Asp Val His val Ala Lys Val Aen Gln Ala 408 ao as clu Ala Glu Phe Typ Lye Lys Ser clu Gly Tyr Arg val Gly Tp ser 0 os Oo ‘op Glu Tle Leu Gly Phe Fry ly Gly Gln Gin ap val ser Glu ue Ser Cys Phe Pro Ala Gly Thr Lou Ala Asn Pro Ser Met Lys Asp Leu 350 455 a0 tu Tyr Te Glu Glu Leu Lys Lys Leu Xe Glu Lys Glu Ala Te Pro 465 70 a5 40 Ala Pro Ala Pro Ie Glu Gln Arg Trp Thr Ala Arg Ser Lys Ser Pro 485 so 395 He Ser Pro Ala Phe Ser Thr Ser Glu Aep Asp Ile Phe Ser Trp Val 500 505 510 Gly Me Te Net Tyr Lew Pro Thr Ala Asp Pro Arg Gin Arg Lys Ap 515 520 525 Ie Me Aep Glu Phe Phe tle Tyr Arg Mle Leu ghe Gin Lys Gin Lew 530 535 540 ‘Arg Tle Leu Ser Aen Aen Met Val Glu Lye Lau Phe Pro Val Ser The ‘the Ala 610 <210> 589 10 82 <213> ORGANISM: Artificial Sequence ‘S20. FEATURE? <2 OTHER INFORMATION: Deecripeton of Artificial Sequencer A. thellana segumice: 2 atgotooggt cacttottct cogacgctec gteggecatt ctctoggaac ectatctcegy 60 totteatcca coatcogtts cteatttteg ccteategta ctctotgeac caccggtcaa 12025 US 6,630,330 BL continued 26 toagaagete getecetatt cattacgete gtacagacta gaatereats ateggtttgt gaceaasese, sooattanag, satagtatea, caggtgatea aaagateegs gaggteacce tegcaagaaa agegggecac gatctotace gacagcasts gocettgate tageanaaet satagteage atgeaagace goncctgete tteagoactt sgeagacccte asttocgta gonactecaa, agaagaagee ctegetetsg agaasagast, actatggect, eteagattag geatgaagce, ageteteeea| eocaatgest, eonagaaaaa cttacacesa, ctaaggacaa, segagagoat, agccagacat, ctotonatss, cagaaggats astagatate etgantacat contagages cogaggatss gocagagaas aagaacteas, 210» 689 10 83 Zlib ORGNITEN: Brassica cleraces - SEQUENCE: 3 atacgeoage attecetgag sesacesgeg leggattcge ‘gatogtgaat tacgatacag eactettongs ageecetona agteactoct eetagetoga escaagacat concagtests accaaagtac tatgnastat acasgascte egttcacgte osegtegas agaatetege ageagagcts gogatggaca ggscatcaca agcottaceg gegetsgee egroctecas| tacgcagcas aaegets aacotegees coogttgget, etagegetta getaggatta, aacteegect acaggtgcta, segaaggsa egtageetty ganctegess eegototees gtegeaacat actacagatg agggtocess vontttacag, gonssagtan tagagtants etecct gets gotegaagta tagattaste gatgaatet, gonagaatan comsatages Met Leu Arg Ser Leu Leu Leu Arg Atg Ser Asn Pro Peo Phe ‘the Pro ala 8 Ser ala ser 30 Pro Pro Leu Arg Thr Leu cys Thr 20 38 “0 lu tye Glu Phe arg Lys Tyr Ala 38 ogotaters ctetggastt senagaagye teaaaterte atotegaage tettgtt 7 egygtetete gectaatoag ggatanage tetagasstg gstecagea attagtegec ceattagaga geagcagatt gettyectes tattgetgag cantegaact tecaagagag guagacttgg agtegteget aacacacata ogtctcaaac ‘onnacaagen tgttaagtac gonatoctgt avon 255 agyctgtaca geatgtocgt actetggtaa gaagtotect, agtegagaga caaactactt atcangetsa ggcagagttt ieeetgag ctttgactgt gancectoge caacectage ‘cages wagga agcaatacea agagesceat tastectscs tanteatgta cotccegaca tocactatag acatttgaca 9 negtee cosagtsaat tectotecas coseatasta Ala arg Sor teu arg Ser Gly Gln Thr teu 30 Pro Pro tle ser ser 3 cay tyr Ala ala teu cs 240 360 70 900 960 1020 1080 ao 1200 1260 1320 1380 1440 1500 1560 1620 174027 US 6,630,330 BL continued 28 Me nen au ‘the vat cay vat cin cin 238 ala Hie he ser nen 4305 cay tye aan cin ay aa nee neg tye a arg cin ne 20 tye ome 280 tye neg Lye neg an 450 Lye arg Phe tye au van tye BS ay ay als cin 215 tes Hie nep van van up a ci ser Hie Bie 100 Phe me cin ay ay ne hr arg 260 cys cis van eye tye ay 20 vat tye ala tye sly tye the Hie ep ala ne op ne 2s oye van si tye 335 ain 408 mp nop ne aie tye va ain alu ay Lye ay me ne cla 230 ois oly ois ne tye 310 ne ala alu aie ale Lye ne vat ne ein ais cin ow ow arg Lys 235 ow ae alu dep the atu 455 ala he cin alu tye ay arg cin 200 vai va ser oy cin 280 tys tye me alu oye aye ye ne mp 105 ‘the atu val ay ne tye aly 265 cts ken ne tye dep atu ne as Phe ne Pre Phe aes val ain Phe ala ay asp 258 Les Les Hie Pro rep 330 Phe ae au ala cor aes ay na ne deg dep ain Ma Me 235 aan ay ra os ne as mp ay na ain ne Phe Phe Pro Phe Pro ™ dep aeg Lye ay 220 Lys ap vat cts Lys 300 the ay Lye ay «a eta als Me ay ue val ne 208 the Pro vat His 265 ap dep tye dep us tye cin the Pro aus 110 ate val tye asp ag ay val as als 270 the Les the val cye rin) aus ag ala tes val Ma ay val nye a cls a me 258 ois ye ser vat nye 35 Hie ue as ay Ma tp cls ie Pro an neg ne cin arg 200 Phe ven ven ome ven 320 nee vat aay aay tye ne me cinUS 6,630,330 BI 29 continued 85 8 5 ep Te Phe Ser Typ Val Gly Tle Tle Met Tyr Leu Peo The Ala Asp 500 505 sto Pro Arg Gln Arg tye Rep Tle Thr Asp Glu Phe Phe His Tye Arg tLe eu The Gln Ala tye Leu Tep ep Gln TYE Ser Ale Tye Glu ile Tep ‘ig tye Tie Glu Tie Pro tye Aep ys lu Glu Lea Glu ale Les Gin uy Aeg tau Acg ys Atg Phe Dro Wal Aep Ala Tye Aen Lys Ale Acg ‘arg Glu Leu Asp Pro Aen Arg Tle Lou Ser Aen Aen Het Val Glo Lye Leu Phe pro Val ser Lye Thr ala 2s 00 tio» sng 20 10 4 SLD tasemts 2064 SIS create! arassica oleracea too» snguences & aereggeae gagetttoge tagctoaggt ttcagatege ctgaactana “ cogatoacte ctecteegce getccascge cogttogctt cgaccscoat ttesecctct 120 cogcacteta tgcacttces gteagacctt gactccagce ectccaccge cgectectec 180 tccacogeeg atttontest cogcetcaga seagsegtte coggatacge 240 agcactogct ctettctoos gegcegensc ttacttetee tteccettee cogagancse 200 canacacaag aaggotoaga tettccsata egetcctete coogaagate tecacacest 160 etotanstgg agtgstacte acgeggtcca gaccaggase tetaaccage cggagactct 420 egeegatcte geagctotes teneggaage tcatsegaay aagancegga tocsaccsst 480 tggstocggt ctetecccss atgggategy ttestctese togggastas tgnatteage 540 goteatggec anggtoctog agstagaten agagaegnag agagtocsts tgcaggctsy 620 agotecsate agagagcage agsttgstgg catesttcny gttggascas atgagacagg 720 tyctagatts cotectateg atgageangt gattageaty angcttgten ctestgctea 720 ccttggtage cttguagttg tegctgaggt cacectocag aacaggagct 900 sseggageac acttacgtet cesccttyga agegeteaag aasagtegct 960 ctctacaat aageatgtcn agtacctyta tattccatat actgacecas togtygttst 1020 tacetgenac cotgtatcas antagastay sgcacctang gacasaccan agtacactac 1080 agaggaggct ttanagoatg toogtgacct gtatagagag agcattgtta agtataggst 1140 ccaqgactct agtangange ctectyacay tegggageca gacattaccg agettteatt 1200 tacagagttg agagataage tgattgecct agatcctete aatgacstte acgttggaaa 1260 agegaatcan gotgaggetg agttetggan ansatcagen ggatacageg teggstagag 1320 tgntgeaate ctgggettts actstastas tonacagtag gtatcagana cttgttttce 1380 tgotggaact ctogctaaac ctagcatgan agacctegag tacataganc agctaaaga 1440US 6,630,330 BL 3 continued Gregacacas aaagaagens caccagcace trovaceata gageagege® ggacaggecy 1500 agtaagage cotatgagte ctgcatteny cxctgcagey geggacattt tetcatgagt 1560 atetetecas tatagacatt tgueseagye ansatigtan gaccagtatt etgegtetgs 1620 acattgggct aanattgege tacceaagga tasagagge ano actcagaas cgattceegg tggatgeata cascaaagea 1800 cageatecte coaaacaaca eggtggeaaa getettcest 1860 tagtttt tktgetcett guagtacesr ttttggeate ctatasegtt 1920 geatctacas gtgtstgtas gasgagtgaa geegetacat eggtca aaaagtetac 1900 ategagttet actactatee eetetttege agtecccctg aataaatare ctegttgees 2000 559.10 0 5 Sub tener: 526 Sip timer BRT S21 ORGRIISH: Saccharcnyees cerevielae sEgumice: 5 Met Ser Thr Tle Pro Phe Arg Lys Asn Tyr Val Pho Lys Aen Trp Ala 1 5 10 3 ly Me Tyr Ser Als Lys Pro Glu Arg yr Phe Gin Pro Ser Ser Tle 20 28 30 Asp Glu Val Val Glu Leu Val Lys Sor Ala Arg Leu Als lu Lys ser 5 %0 3 Lou Val Thr Val Gly Ser Gly His Sor Pro Ser Aen Met cys Val Thr 50 38 a Asp Glu Trp Leu Val Aen Lou Asp Arg Lou Asp Lys Val Gln Lye Phe 3 0 75 80 Van Giu Tyr Pro Glu Leu His Tyr Ala Asp Val Thr Val Asp Als Gly 35 30 38 Met Aeg Leu tye Gin Leu Asn Glu Phe Leu Gly Ala Lys Gly tyr ser 100 105 110 Ser the Gly Ser lie Gly Ser Ger Pro Bye lie Gly Les tle der gee ‘ein ys Val Aen Leu thr Ze Val Aen Gly tye Gly Glu Les Lys Phe Leu Aep Ala Glu Aen Asp Pro Glu Val Phe Lys Ala Ala Leu Lew Sex ‘val Gly Lys Tle Gly Tle Tle Val Ser Ala The Tle Arg Val Val Peo 10 15 130 Gly Phe Asn Tle Lys Ser Thr Gln Glu Val Tle Thr Phe Glu Aen Lew 135 200 208 eu Lys Gln Trp Asp The Leu Trp Thr Ser Ser Glu Phe tle Arg Val 210 218 220, ‘Tap Tep Tye Pro Tye The Arg Lye cye Val Leu ep Arg Gly Aen Lye BS 230 28 to ‘The The Asp Ala Gln Aen Gly Pro Ala Lys Ser Tep Trp Gly Thr Lys Lou Gly Arg Phe Phe Tyr Glu Thr Lou Leu trp tle Ser Thr Lys TleUS 6,630,330 BL continued tye tye au ep vat 385 a ala Phe ™ Lys 465 Lye ars ala ain atgtetacta coccatttag Ma aay ne ep Hie op. 0 vat me me ala 450 Lye va tye ne 25 tye ay me ove ne 455 Lys asp ci cis ne geasasceag agegceagge atgegestea, gtegaacate cantegaats sancanagty stgetectt etggatgees, gatatcates sangtgatta 260 ay ove ne cia aes ew tyr ao Phe he clu cin 500 nen wscgetacte ateeetges the atu Phe Ma vat Lys 408 arg si Phe op, mp a8 Asp sly ‘52g 10 wo 6 "TYPE: DBA segumice: 6 agetgaaaa, ctgatgaaty ctgagteaca, eegetageat cteastacgt, negates eototgetac ceeeegaaaa Lye ain arg arg Phe 330, va tye a0 tye ne Phe dep ale cys me 25 the Phe me ay 455 ep ay asp ne vat 280 dep me aep ay 40 asp clu nen asp conaceaagt sagettagtt gettgttaac eatgeegat, gegaasase wcttgact agaagtette eaveagggte cotettgaag 265 atu ‘the eve ay ne ‘me cys as ‘the Phe asp va 505 Pro tye ay ‘mae the ain ae val ‘the ats re] Phe ser gtgetes Phe dep Phe alu tye ‘mae 338 ‘the ala a5 tye Lew ots oaategets acegttgatt, etagacagat gteacagees gotagteaty ategteacts aaagcegcet gteccegsct cantgagata, val vat val uu ay 300 ay ala 60 aes Lys ala 265 ain me ne ay 4 ala ay Phe Aen ser 270 en eg val The Phe val arg ser rye val ser cia val tye eye acs tye ne as ie tye 0 tye Pro aly Pro wot ala arg tye as tys cin S10 asp an nee nee Hie ay 400 tp va a0 ne rp ectaggcosg aatttarect laggtegtoga gttagt egygeaatee tectage " eagacaaagt acaaaageet aegoeggtat gaggettese ssttagg ctotatetca getectoace teatoncsst gtaagggosa attgaagtte eactetcage tggaaaaatt ‘costatggnc ttcatcega 2035 US 6,630,330 BL continued stestengag cttgatsats actacagaty cos 2093 stetacgeas ctetattarg Jatgteaceg attetateas santgagast gecetatage gescaggetg coateaacas stactacat taccttctge coogtetatg gonatstatg getcegttag segteas aggcegtttg gctgtaatac atggtageas gaggtaasce govggaccag tgaanaagsa aggtegaag aatggtatss gavccogata atgtattort ‘ecagecaas gatetetace eggatttaae agttts ageattttae ecettggae cogeceatte sagteas acattgggee eactgattac cgaggatttg ‘sea cotagegast tgtcogacta g aie ain 21s ain ‘589 10 807 EENGTH: 526 ‘Bypes ber ORGANISH: Saccharonyces cerevicite -<400> SEQUENCES 7 1 oy Asp Lew Asp vat ne ain vat ay ne caw ven caw au neg an aye 0p ay Phe tontsstssg, aanatotaty atggactstt, gaagtottae gtecacsese actageaasa, ctggataaca, stgacgtest, aantggttta, aagaactece gargacteeg aanaagttce cagtsgscta ‘Thr Te Pro Phe Arg Lys Asn Tyr val 5 10 ‘tyr Ser Ala Lys Pro Glu Arg Tyr Phe 20 25 Ven Val Glu Leu Val Lys Ser Als Arg ‘the Val Gly Ser Gly His Ser Pro ser ‘ep lew Val Aen Lew Asp Arg Low Asp yr Pro Glu Leu tile Tyr Ala Asp Val eu tyr Gln Leu Aen Glu Phe Leu Gly Aen Leu Gly Ser Tle Ser Glu Gln Ser Gly Ser lie Gly Ser Ser Pro Tyr Me Val Asn Leu the Tle Val Asn Gly Lye ‘Ala Glu Asn Asp Pro Glu Val Pho Lys lye Te Gly Te tle val ser ala Thr 1s 10 Asp Tle Lye Ser Thr cin Glu val le 135 200 gtaccaagct ggstagattt egecattane cecatttsts tgetttcnce attegttgat gttoattgya teatterate ceatggaage cegttgetca 9 cae cagtocesgt caccatcosa tegcagattt cataaatge taccattest costeettga aaatactatg caggetcane cactetaget anatgaggaa gatggeatts ‘gganatang aaaggagea watgg tattatagat Pho tye nen Trp ala Gin Pro Sor Ser The 30 bea Als Gls Lys Ser ‘Aen Wet Cys Val The Lys Val Gln Lys Phe ‘The Val Aep Ala Gly Ala Lys Gly aye ser Val Ale Gly Tle Tle Gly Les te ser ser ‘ly Glu Leu Lys Phe Ala Ala Lau tea ser He Arg val val Pro 130 ‘The Phe Glu Asn Lea 208 900 1320 10 1500 1860 ase37 US 6,630,330 BL continued he tye tye au op vat 385 ala Pro Phe uP bye 465 tye eg Ma ome aay Ma aay ne tp op 8 va Pro ome ome Aa 450 tye vat nye ne asp tye ay 385 he oye tes ne tes 435 tye asp au au ne ale Phe ay ove ne clu arg cis tyr ao Phe nen hr alu ain sin Phe the atu Phe ala vat Lys Aen 408 arg si Phe Asp wp 485 ep ay <2io> seq 10 woe Sin mee: S2lb ORGNITEN: Sacchaconyees cerevisiae segumice: 8 Aen tye Lye cin arg arg Phe 330, van Aen es tye 70 tye ne oly alu Phe dep ala cys me 35 the Phe the sly 455 asp ay dep ne me var dep ala 360 asp nen aly het 40 ser asp alu dep ala atu ‘the eve ay ne ‘the Ser cys as ‘the dep vat tye tye ay ‘the ‘the cin ag nen vat ‘the as rr) Phe mp Phe dep Phe alu tye ‘the 338 Lew ‘the ala Lew Hor a5 tye alu cocatgecte ctateccatt tagasagaac tatgtgteca totgeansac cagancgtta ottcoascea agagtgeca ggetagotga anaagetta acatgegeg teactgatga atggettgtt eeegtegaat atectgagte acattatgce ageecaates gteactgees, gatgtoacag =p ne val vat val eta ay Seo the Pro ay ala 460 acs tye a tp ain Phe aes ne aay 4 als ay oty The ‘the tye en eg val The Phe val arg ser yr val ser cla val Tye cye arg tyr He as His tye 0 ys Pro oly Pro Hot Als neg tye a5 Lye cle dep tye ne an nee nee ay Phe 400 Aen rp Hie ven 0 ne tp sctggge cggaatteat aegaggtest cgagttagta getogggeca tectoctagt ‘gateggacaa agtacanaag vegatgocag tatgaggott 120 180 300US 6,630,330 B1 39 continued Taccanttga atgaattcet gggtgogaan ggttactcta tocanaatte aggctetate teagencacn gtattgetgg catentotet actagtagte atgyttccte accttateae gotttgattt cttoreasts ost icttg actattgtta atagtaagag cgasttgaag stettggetg coganaacge cecagaagte ete getg ctttacttes agteas! atoggtates ttgtetetge tactatcagg gttgttcesg gettcaatat tasstecact cangeagtge ctacttttga anscctttts ataccotatg gactteatct daatttetes gautttgats ataccettat gugttotetg gaggagt agasctacag atgeecasas tggtccagee aagtcatget guggtaccea getgagtage etettotacg jctetatt atggatotct acc: sect atgegecatt aaccceastt stggeneagt toyttttces ceggeantac ona stg aguagagere tactygeget gteaacgtea cogattctat cageggatte aatatggact gettgettte acaattegtt aatgentass gotsecetet goa segge teggeagtct tacgencate ggatcattct atugegeagg ctgcoatana onsagaattt tatgtconcg tgectatgga agtoogttge toaaatacts cattacctts tgaacccttg gatactagca agagaacaaa caccagtcoe ggtecogtet atggcaatgt gegcegccea ttcctggata acacaccate ccattgcaga etegetcegt tgganaatgt taccaacage cagttgacgt tgtacataan tcctaccatt tavaggeegt ttggctgtaa tactocaate cataaatggt teacccttte atgatagtag cgggaggtaa gecacattgs gecaagaact toctaggcte aaccactcta gotgctggac cagtga 12 ggatactgat tacgatgact saggatgace btgaaggteg aagaatggta tggegaggat tga ast aagaaaggeg, cangatcecg ataatgtatt cttggcasac aaacagtggg ctatcatann tagtattata gavcctagty agttgtcegs ctagtetets tttgtctces tastototat attttactas wagaatat atatatatat atttatatat agcagtgtga tgtctgttca tgtacattct iateactatt cctagctgcc tatcanagac ttetttttga attagagctt tttagtants atgagaecet Ettttottet cattatectt actatagttt ttttttagen aageeseacy cagtantgat tggtogtate agcasanacg aaacatogge atggcatanc gtagatccta sotscagage agttsttage aatcagatag aaatgtactt egagegatgt atatategca stegte 2 ousacatte geascustgt opetagtage gecstyoata togtttgtet cgecenatce gtettatecs exttgtacty gattgettct ganttatgty gttacacege eetacttttg gossscgeaa astogtagen tostante <2is» sng 20 109 SID Ensen 40 SID cnentran: aateue norvegicus - sequence: 9 et val His Gly Tyr Lye Gly val Gla Phe Gln Asn Trp Ala tye Thr 1 5 19 1S ‘Tye Gly cys Ser Pro Glu Val Tyr Tyr Gla Pro Thr Ser Val cla clu 20 ES 30 val arg Glu Val teu Ala Leu Ala Arg Glu Gln Lys Lys Lys val Lys 38 “0 as 360 1080 1200 1260 1320 1380 1440 1500 1560 1620 1600 170 19604 US 6,630,330 BL continued 42 van Phe au Hie vat ‘the ala ne the ctw 235 ser caw ne ser 305 ‘the 35 Phe ven tye aay 0p. 20 Aen aa Phe Aen Aen 280 oye aye ne oye oye aye oly ne tye ain dep ne the van he Phe 135 tye ven ser tes arg 28 es ay the dep 3s oly Hie ain vat tye me Phe va 180 arg ser nen ew 260 Phe ser aep tye tye ain ne ao alu sly ne ep the Hie ep cin he ne mp 25 ™p Phe Hie wp nep tye Lye a8 tye <210> 589.20 Wo 10 Sup tenors 2120 ain mee: ey we te te 310 va Phe 330, aep alu va Ser ys vat Bie ale ne alu ala cin tyr ais tye ep ser ep ne 295 ay Phe Lye Phe ot alu ay ay vat arg oye tye 200 phe cin asp hr hot 280 Phe aes ay alu tye 40 Ser hen ae va ae val va ues ata arg asp ur yr 265 Lew ‘the arg tye aes atu dep os ep arg ay 30 aa ne the au His val Phe Hie als 250 Les Phe ye dep ao Te va ne ay ain cye ain ‘the 235 ne Pro hen ots tye 315 ne 335 ye ‘the Tala Cys Thr Asp ea vat ay Phe ep mp 230 en ay cvs cvs cys 300 tp mp ay can ay va ue cys cin 208 Phe Lys Phe Lys 25 arg Lye aa vat aa the ala 10 als ye vat 210 tye aa tye Phe Phe rey asp dep 35 ay Hie neg ay cin op 255 oy ois tye tye aye a His as ay. tye Ma ne cin me ser 200 rp ser can 320 aw Hie 400 Lye43 US 6,630,330 BL continued [2iis ORGANTEN: Rattus norvegicus - sequence: 10 ggntcctect gatcactaga atcatggtes ataggtacea aggagtccag ttccaanatt 60 goscasagac ctatggttge agtecagagy tgtactacca geceacctee gtgzaggegy 120, eagagagst getagceetg gecegggage agaagaagea agtgaagets gtoggtgsty 100 geeactegee tteagacatt gectgeacty acggttteat gatecacatg ggcsagatgs 200 acegagttet coaggtggac aeggeqasye agcagatase agtygaagce gatatcetes 300 tagetgecct geacecacag ctygatgage atagcctaye catgtecest ctgsgagcay 360 guctgatgt gacagttget ggtgteatts gatcaggase acatancece gggatcaege 420 acggeatcct ggccacteag gtagtggcce tgaccctgat gecagetgat ggagaagtes 400 tggaacgtte tgagvoanga antgoagatg tgttccagge tgeaeggstg eacotggstt 540 geetgggcat catccteace gteacectye agtgtgtgce teagttteag ettcaggags 600 catccttcce ttegaccete aaagaggtec tegacaacct agacagccac ctgaagagst 660 ctgageactt cogettects eggtttecte acactgagaa egteagcate atctaccaay — 720 acoacaccan canggocece tostotgent ctanctygtt ttgygactat gocatoggst 780 totacctact ggagttcteg ctctggacca geacctacct gecatgecte gtaggctsga 840 toaacogett cttcttctgy atgctgttca actgcaagaa ggagagcage aacctoagts 900 acaagatott cacctacgag tgtegetton agoagoatgt acaagactsg gocatoccta 960 gasegnagec canggagsce ctactggage tanaggcost gctggaggec caccccanag 1020 tagtagecca ctacocegta gagstgogct teaccogagg egatgacatt etgctgage> 1080 cotgettece gagggacags tgctacatga acatoattat gtacaggecs tatgganagy 1140 acgtgecteg gctagactac tgzctggect atgagaccat catgaagang tteggaggea 1200 gaceceacty ggcanaggcs cacaattgea cccagaagga ctttgaggan atgtacceca 1260 covttcacas gttetgtgac atcogtgaga agetggacce cactggaatg ttcttgastt 1320, eqtacctgga gauagtcttc tactaaagea ggagtggasa casuccacce tgaccectea 1380 cacttotgct goccscggay gtotggggag cagagaagtg cotcacaage acastgggea 1440 ctgucctete ctectgacea casaganagg ctgggetety ggcegggtce tetotgectt 1500 atktecetgg gtescacsta asctgeaste ctotcaguag sapugugate ectgetacet 1620 coratetate cagactaagg atgtggttcr tectagatte tetggetcce ccaggtatay 1600 agagatteet yoysectges gttetecate cctettcaga agggagggst ecettsgegs 1740 gagtteggct cagaggtage atgaagcaty ctetgetete tettacett gaaggtectt 1000 eggatgocca gagatgtoty otgstectyg geaagconte atteanacgy gtecaaccty 1860 geettotgte tgecatgges tguccetoge agtgtctett ccagaggtat ttagagtags 1920 actegettes acctottane cagttgetys tecetgigtt tetetecett etecttggey 1980 actactetty gagggggate ceaccatgte cttggettte cotgggtatt gttctectet 2040 tectertcac aaatatgact ceagtttgar tegtggcctt eetggagtgr tecttggegs 2100 accaagatgt tocagetace 2120 21> sag 20 Wo. Sup tenons 319US 6,630,330 BL 45 continued iz SPE: PRT ‘<400> SEQUENCES 11 Met The Lye Tle Glu Leu Arg Ala Leu Gly Aen Thr Gly Leu tys Val Ser Ala Val Gly Phe Gly Ale Ser Pro Leu Gly Ser Vel Phe Gly Pro Val Ala Glu Asp Asp Ala Val Ala The Val Arg Glu Ala Phe Arg Leu Gly Tle Asn Phe Phe Asp The Ser Pro Tyr Tye Gly Gly The Len ser clu Lys Met Leu Gly Lys Gly Leu Lys Ala Leu Gln Val Pro arg ser ‘op Tyr Tle Val Ala Thr Lys cys Gly Arg Tyr Lys Glu Gly Phe ASP 3 3 38 Phe Ser Ala Glu Arg Val Arg Lye Ser Tle Aep Glu Ser Leu Glu Arg 100 108 ne Lou Gln Leu Aep Tye Val Aop Tle Lou ile cys tle Aep Ile Glu Phe 1s 16 aS Gly Ser teu Asp Gln Tle Val Ser Glu Thr tle Pro Ala Les Gln tye 130 1s ue ‘eu tye Gln Glu Gly Lys Thr Arg Phe Tle Gly Tle Thr Gly Leu Pro 145 130 155 160 eu Asp Tle Phe Thr Tyr Val Leu Asp Arg Val Pro Pro Gly Thr Val 165 70 175 ‘op Val Tle Leu Ser Tyr cys Hie Tyr Gly Val Aen Asp Ser Thr Leu 130 185 130 eu Asp Leu Leu Pro Tyr Leu Lys Ser Lys Gly val Gly val Tle ser 135 200 208 Ala Ser Pro Leu Ala Met Gly Lou Lou Thr Glu Gin Gly Pro Pro iu 210 213 220, ‘Trp His Pro Ala Ser Pro Glu Leu Lys Ser Ala Ser Lys Ala Als Val Ala His cys Lys Ser Lys Gly Lys Lys Ile thr Lys Les Ala Les Cin ‘tye Sor leu Als Aan Lys Glu Tle Ser Ser Val Leu Val Gly Net Ser Ser Val Ser Gln Val Glu Glu Aen Val Ala Ala Val The Glu Lev Glu Ser Leu Gly Met Asp Gln Glu Thr Leu Ser Glu Vel Glu Ala Te Lew Gly Pro Val Lye Aen Leu The Trp Pro Ser Gly Tle His Gin nes ates sng 20 W012 SID Ensen 960 SID cnGaMrSN? Acabldopete that sana sequence: 12 avgacgaann cagagettog agetetggey atcacaggye ttaaggttag ogcosttgst 60 treggegect ctecgotogs angtgtette ggtecagteg cosaagatga tgcsgtegec 120 acogtgegeg aggetttceg teteggtate aacttctteg acacctocee gtattatgsa 180US 6,630,330 BL 47 continued ggnacactgt ctgagaasat gottygtaag ggactaaagg ctttgoaagt coctagaagt 240 gactacatty cogeractas gugtggtaga tataaagaag gettegatee cagtgcesay 300 agagtangas agagtattss cgsgagctts gagaggctte agctegstta tettgacsts 360 ctecategee atgacattga gttegggtet cttgatcaga ttgtgagtge aaceatecct 420 getctcage aactyaasca agegggzaag acceggteca ttggeatcac tggtetecey 400 stagatatet coacttatgt cottgatega gtgectecag gyactgtega tgtgatatey 540 teatactgte attacggest = egatteg acgetgergg atttactece ttacttgeay 600 aceasaggts tgustatget aagtgettct cesttagess tapyectect tacagences 660 gotcctecty aatggcaccs egctteccet gagctcaagt ctgeaagcas agesgeagtt 720 aotcactges Jog caagaagate acaaagttag ctctgcasta cagtttagea 700, acaaggaga tetogtogst gttsattgas atgagctoty tetcacaggt agaaganaat 910 gtegeagcag ctacagaget tgeaagtcts gugatggate aagaaacter gtetgagstt 900 gaagctatte togagcctgt aaagaatctg acatggccaa gtggaatcca tcagaactaa 960 <210> sag. 80 13 Sin tenor ie Sin nips bet S21 ORGANISK: Areiscial sequence 20> FEATUR [223 OTHER INFORMATION: Description of Artificial Sequence: motif 1 of Side-keto reductase superfamily 2215 WAME/ RET: VARIANT 22% LocarroN: (2) 22}> OTHER INFORMATION: Xaa ~ any amino acid 221s WAME/ REY: VARIANT S22 LocarTON: (4)..(5) (22> OTHER INPORMATION: Xaa ~ any amino acid 221s WAME/ REY: VARIANT S2m Locazton: (7) (22> OTHER TNPORMATION: Xaa ~ any amino acid 221s WAME/REYs VARIANT 22% Locations (9). (13) (22> OTHER INFORMATION: aa ~ any amino acid (221s WAME/REY VARIANT 22> LoeATION: (15)..(17) any amine acta ‘ SEQUENCES 13 ‘Gly Xan Acg Xen Kan Asp Xae Ala Xaa X Kea Glu Kaa xaa 1 5 10 5 xan Gly io» seq 19 WO 14 <213> ORGANISM: Artificial Sequence <2) OTHER INFORMATION: Deecriptton of Artificial Sequencer Forward PCR Priner for L-galactono-1,4-lactone dehydroger feom A. thaliane < sequence: 14 cangaaggce canangttes gttacgctee 20 seq 10 WO 15 Sun tener 30 Sip timer ona S21 ORGANISH: artificial sequence 5223 OTHER INFORMATION: Description of Artificial sequence: Reverse PCR Priner for L-galactono-1,4-lactone dehydrogerUS 6,630,330 B1 49 50 continued from A. thaliane - sequence: 15 atgagcoctt aagcagtast esseactass 30 210» $89 10 No 16 <213> ORGANISM: Artificial Sequence <22%> OTHER INFORMATION: Deecriptton of Artificial Sequencer Forward PCR Priner fer Lngulono-1ydelactone oxidase from Re norvegicus - sequence: 16 tgaggggtea gagtgatteg tttcca 26 21> sag 20 80.17 [21> ORGANISH: areiscial sequence ‘SS20> FEATURE? [22% OPMER INFORMATION: Description of Artificial Sequencer Reve Priner for L-gulono-1yd-lactone oxidase from Re norvegicus sequence: 17 tggaatontg gtocatggst acasngss 2 <210> sx9 m0 Wo 18 is tenors 22 2izs Types uA S21 ORGANISH: Artificial sequence 220s FEATURE? <22}> OTHER INFORMATION: Description of Artificial Sequence: Porward PCR Priner for D-arabinono-1,4-lactone oxidase fram 5. ‘<400> SEQUENCES 18 ttecaccata tgtotactat oc 2 -210> $89 10 No 18 21 tencras 22 Gli ORGNITEM: Artificial Sequence (22202 FEATURE: [22h oMmER ThromMTrON: Description of Artificial Sequencer Reverse PCR Briner for Drerebinanen1,i-lactone oxidase fram 5 cerevisiae - sequence: 19 aggetecta gteggacasc te 22 20> sE9 19 Wo 20 Sib tenons 344 <213> ORGRITSH: Saccharomyces cerevielae - sEgumice: 20 Met Sex Ser Ser Val Ala Ser Thr Glu Asn le Val Glu Asn Mat Leu 1 5 10 8 Hie Pro Lye Thr Thr Glu Tle Tyr Phe Sor Leu Aen Aen Gly Val Arg 20 28 38 Me Pro Ala Leu Gly Leu Gly Thr Ala Asn Pro His Glu Lys Leu Ala 38 40 8Si US 6,630,330 BL continued 52 Giw The Lye Gin Ala Val Lys Ala Ale tle bys Te Asp The Ala Trp Ala Tyr Glu Thr Glu Pro Me tye Glu Leu Leu Glu Asp Gly Ser Tle Lye Tle The the Lys Val Tep Pro Val Lau Tep Aep Leu Aen Glu Ser Leu Lye Ale Leu Gly Leu Glu Leu Gin His Tep Pro Leu Cys Phe Glu Lys Tle Me Ser Gly Leu Val Lys The Pro Val Asp Asp ‘ye Ala Ala Asp Gly Asp Tyr Leu Glu The Tyr le aye Leu Asp Pro Asn Asp His Arg Val Arg 180 18 ‘Aen Phe Ser Tle Glu Tyr Leu Glu Arg Leu le 135 200 Lye Pro Thr Val Asn Gln Val Glu Thr His Pro 210 2s lu Lew Arg Lys Phe Cys Phe Met His Asp le 235 230 238 Ser Pro Leu Gly Ser Hie Gly Als Pro Asn Lew 285 250, ys Lys Lew Ala Glu Lys Tyr Asn Val The Gly 260 265 Ser Tyr His Tle Arg Gln Gly Thr He Val tle 25 280 Pro Val Arg Te Ser Ser Ser Tle Glu Phe Ala 280 295 iy Leu Gin Glu Leu Aen Aep Phe Gly Glu Lye 305 310 315 ios seq 10 Wo 22 <213> ORGRITSH: Saccharomyces cerevielae - sEquENCES 21 tencaactte gtttactgas aatgetacta gtacataate tonatanaaa tattatagat ogottanana ctogtttatt agcegetct accettacct cegectggan aaattataat tacagagsta .gganag aggsggatat caagcatcts gocttonatt gatanaageg totegattte covcancosa, catagtc gaaaatatge cacteaacaa tggtgttegt atcccagcac tgsstttasg ie Phe neg uu ye Lye Lys ala Lys 220 Lys en Pro Ser 300 ye oly vat ue va val dep ay cin ne cis 208 es ne ap arg 265 Tye Arg oy ote ep Le 35 ep arg Asp Le Pre tye tye The ew cle ay val 190 cye arg Pro Gln ‘the Ale Pro tes 255 es tes 20 Ser tes ‘the tye val Aeg Hie aa aay Lye va tye 20 ven ne Aen Asp 320 actaagtate taactateee gosgattata aatccacca stataaageg agectegta gacteatteg cactatotce tateatgtce tottcagtas sgactacagaa atatactett ‘gncage: cotcacgaas 120 300 360 2053 US 6,630,330 BL continued lagttagctga aacaaacas googtasag ctgcaatcea agetggetac aggcacatty 480 atactgetty ygectacgag acagegecat tegtagatge agecatoany gagttattey 510 jagetggate tatcanangg gagsatottt tontasccac annagtgtas cegttctat 600 goseegaagt goacagatea eegaatgaat cttegaaage ttagyctey gaatacgtey 660 acttgesctt cogetatgte ttgaaaaget taaggaccct aaggggates 720 seagectast getgatgate ctggaaaase astgeatger geegacgsty 780 actatteage, casttggana aaacttacct egatoctane gatcatesty 940 gagagecat cggegtetea eatettcea ttgagtactt gyaacgtte attsaggeat 900 geagagttas gocaacggtg eaccaagtgy aaacteacce teacttacce canstygeas 960 seagasagtt ctgerttatg cacgacstte age atacteacca ttaggteces 1020 atggegcace aaacttgaaa atcccactag got tgcegaaaag tacaatgeca 1080 caggasatge cttgetasct cettaccata ttagacaagy cectatogte atecegagat 1140 covtgaatee agttaggatt eoctogagta tegaattoge atctetgaca aaggatgaat 1200 tecaagagtt gaucgactes ggtgaasaut acceagtgag atteatogat gagceactey 1260 cagceatect tocagagtet actagtaacg gaccaaactt ggacaattta aagtattaag 1320 acaacgactt tattttcact eeatttagte ogcttcteaa tottgtcaaa aacaagatat 1380 gtgtantog coteangtan acaatatgte tetoataogt gattegaagt ttttaagtat 1440 ctgnantaca tacgogegeg tatgoatatg tattagttaa attactogan tgtectttat 1500 atastatte 1509 210» $89 10 No 22 21s teneris 23 Sim nimer ona ‘21> ORGANTSN: Artificial Sequence (2220s FEATURE! 22> OTHER TNPORKATION: Description of Artificial Sequence: Forward PCR Primer for tngalactose dehydrogenase from A. ‘sheliane - sequences 22 atgecgasea tagagcttes age 210» $89 10 No 23 Glib ORGNITEM: Artificial Sequence 2 {22% OPUER INFORMATION: Deecriptton of Artificial Sequencer Reverse PCR Priner for Legelactose dehydrogenase fron A. thaliana - sequence: 23 ttagttctga tggattccac tess <210> sug 10 Wo 24 S21 ORGANISH: areiscial sequence ‘S20. FEATURE? <2 OTHER INFORMATION: Deecriptton of Artificial Sequencer ‘Saccharomyces cerevisiae - segumice: 24 atgtetectt cagtagecte aace 4 2US 6,630,330 BL 55 56 continued ais 529 15 no 25 "TYPE: DNA Primer for cerevisiae abinose dehydrogenase from 8. - sequence: 25 etgtoca ageetggte ‘58 10 wo 26 ORGANISH: ArtiGclal Sequence fldo-kato reductae MAME/eEYs VARIAN LOCATION: (2)--(3) superfamily ah ny amino acid - sEgumces 26 oly Xaa aa Asn (OTHER INFORMATION: Description of Artifical Sequence: Reverse PCR OTHER INFORMATION: Description of Artificial Sequence: motif II of ‘What is claimed is: 1. A metbod of generating ascorbic acid, comprising: a) obtaining a recombinant Saccharomyces yeast strain capable of converting an ascorbic acid precursor into ascorbie ac >) culturing the recombinant yeast in a medium compris ing the ascorbic acid precursor, wherein the recombi nant yeast produces ascorbic acid with a yield greater than about 35% from the ascorbic acid precursor, and 6) isolating the ascorbic acid. 2. The method of claim 1, wherein the yeast is selected from Saccharomyces cerevisiae (S. cerevisiae) strain GRFISU; or S. cerevisiae strain W3031B, 3. The method of claim 1, wherein the ascorbic acid precursor is selected from L-galactono-1,4-lactone; D-glucose; L-gulono-t,4-lactone; or L-galactose. 4. The method of claim 1, wherein the isolating step ‘comprises lysing the yeast. 5. The method of claim 4, wherein the isolating step further comprises centrifugation, filtration, microfiltration, ultrafiltration, nanofiltration, liquid-liquid extraction, ‘crystallization, enzymatic treatment with nuclease or protease, or chromatography. ‘6. The method of claim 1, wherein the recombinant yeast accumulates L-ascorbic acid in the medium at levels gieater than background. x» ss 4s 7. The method of claim 6, wherein the isolating step comprises chromatography, activated carbon, microfitration, ultrafiltration, nanofiltration, liquid-liquid extraction, or crystallization. 8. The method of claim 1, wherein the yeast is function- ally transformed with a coding region encoding L-galactose dehydrogenase (LGDH).. 9. The method of claim 8, wherein the coding region encoding LGDH was isolated ftom Arabidopsis thaliana (A. thaliana). 10. The method of claim 8 wherein the functionally transformed yeast further comprises at least one coding region encoding an enzyme associated with the conversion of a carbon source to L-gulactose. 11. The method of claim 8, wherein the eoding region is linked to a promoter active in the yeast. 12. The method of claim IL, wherein the promoter is the S. cerevisiae triosephosphateisomerase (TPI) promoter. 13. The method of claim 8, wherein the yeast is further functionally transformed with a coding region encoding D-arabinono-1,4-lactone oxidase (ALO). 14. The method of claim 13, wherein the coding region ‘encoding the ALO is linked to a promoter active in the yeast 15. The method of claim 14, wherein the promoter is the 'S. cerevisiae triosephosphateisomerase (TPI) promoter.UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 6,630,330 BL Page 1 of 1 DATED = October 7, 2003 INVENTOR(S) : Danilo Porro and Michael Sauer Itis certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below. Column 56. Line 43, delet -gulactose” and insert -~ L-galactose - Signed and Sealed this Bighteenth Day of November, 2003 JAMES E. ROGAN Director ofthe United States Patent and Trademark Office