Schroeder, William A. (Brooklyn Park, MN, US)
Hicks, Paula M. (Eden Prairie, MN, US)
Mcfarlan, Sara C. (St. Paul, MN, US)
Abraham, Timothy W. (Wayzata, MN, US)

Plaque It! Sponsored by: Flash of Genius

10/472317

02/05/2008

03/19/2002

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Cargill, Incorporated (Wayzata, MN, US)

435/137

435/488, 435/471, 435/25, 435/455, 435/69.100, 435/132, 435/483, 435/155

C12N9/02; C12N15/00; C12N15/74; C12N15/75; C12P7/00; C12P21/06; C12Q21/06

435/254.11, 435/455, 435/471, 435/254.2, 435/183, 435/325, 435/91.1, 435/320.1, 435/252.3

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Prouty, Rebecca E.

Raghu, Ganapathirama

Klarquist Sparkman, LLP

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. §371 that claims the benefit of PCT/US02/08404, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional application Ser. No. 60/277,148, filed Mar. 19, 2001.

What is claimed is:

1. A method of oxidizing myo-inositol to glucuronic acid, said method comprising: transforming an isolated cell with a nucleic acid molecule encoding a polypeptide having a sequence at least 95 percent identical to SEQ ID NO 19, wherein the polypeptide has myo-inositol oxygenase activity, and wherein said isolated cell expresses said polypeptide; extracting said polypeptide from the isolated cells; and contacting said polypeptide with myo-inositol such that the myo-inositol is oxidized to glucuronic acid.

2. The method of claim 1, wherein said isolated cell is a prokaryotic cell.

3. The method of claim 2, wherein said prokaryotic cell is selected from the group consisting of Eseherichia, Pseudomonas, Bacillus, Lactobacillus, Lactococcus, and Corynebacterium cells.

4. The method of claim 1, wherein said isolated cell is a eukaryotic cell.

5. The method of claim 4, wherein said eukaryotic cell is selected from the group consisting of yeast, fungi, insect, and mammalian cells.

6. The method of claim 1, wherein said isolated cell is selected from the group consisting of Saccharomyces, Pichia, Aspergillus, Cryptococcus, Schwanniomyces, Schizosaccharomyces, Spodoptera, Cricetulus, and Homo sapiens cells.

7. The method of claim 1, wherein said nucleic acid molecule is integrated into the genome of said isolated cell.

8. The method of claim 1, wherein said polypeptide comprises an amino acid sequence at least 99 percent identical to the amino acid sequence of SEQ ID NO: 19.

9. The method of claim 1, wherein said polypeptide comprises an amino acid sequence at least 98 percent identical to the amino acid sequence of SEQ ID NO: 19.

10. The method of claim 1, wherein said isolated cell produces L-ascorbic acid or glucaric acid.

11. The method of claim 1, wherein said isolated cell comprises glucuronate reductase activity.

12. The method of claim 1, wherein said isolated cell comprises 1,4-lactone hydroxyacyihydrolase activity, D-glucono-1,5-lactone lactonohydrolase activity, or uronolactonase activity.

13. The method of claim 1, wherein said isolated cell comprises gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidase activity, hexose oxidase activity, or gulono-γ-lactone dehydrogenase activity.

14. The method of claim 1, wherein said isolated cell comprises phosphatase activity.

15. The method of claim 1, wherein said isolated cell comprises phytase activity.

16. The method of claim 1, wherein said isolated cell lacks L-gulonate 3-dehydrogenase activity.

17. The method of claim 1, wherein said isolated cell comprises myo-inositol oxygenase activity with a specific activity greater than 40 mg glucuronic acid per gram dry cell weight per hour.

18. The method of claim 1, wherein isolated said cell comprises myo-inositol oxygenase activity such that an extract from 1×10⁶ cells comprises a specific activity greater than 150 μg glucuronic acid formed per 10 mg total protein per 10 minutes, wherein each of said 1×10⁶ cells is said cell or a progeny of said cell.

19. The method of claim 1, wherein said polypeptide lacks an N-terminal polyhistidine tag.

20. The method of claim 1, wherein said polypeptide lacks glutathione-S-transferase sequence.

21. The method of claim 1, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:19.

22. The method of claim 1, wherein said polypeptide consists of the amino acid sequence of SEQ ID NO:19.

23. A method of oxidizing myo-inositol to glucuronic acid, comprising: transforming an isolated cell which produces myo-inositol with a nucleic acid molecule encoding a polypeptide having a sequence at least 95 percent identical to SEQ ID NO 19, wherein the polypeptide has myo-inositol oxygenase activity, wherein said isolated cell expresses said polypeptide, and wherein culturing said transformed cell to express said polypeptide and oxidize said myo-inositol into glucuronic acid.

24. A method of oxidizing myo-inositol to glucuronic acid, said method comprising: transforming an isolated cell with at least one nucleic acid molecule, wherein said at least one nucleic acid molecule encodes at least two polypeptides, a first polypeptide having myo-inositol monophosphatase activity (E.C. 3.1.3.25) or having myo-inositol-1 phosphate synthase activity (E.C. 5.5.1.4), and a second polypeptide comprising at least 95% sequence identity to SEQ ID NO: 19, wherein the second polypeptide comprises myo-inositol oxygenase activity (E.C. 1.13.99.1), and wherein said isolated cell comprises myo-inositol-1-phosphtase, myo-inositol, and glucuronic acid, thereby permitting oxidation of myo-inositol to glucuronic acid.

25. The method of claim 24, wherein the polypeptide having myo-inositol monophosphatase activity is encoded by SEQ ID NO: 73.

26. The method of claim 24, wherein the polypeptide having myo-inositol-1 phosphate synthase activity is encoded by SEQ ID NO: 74.

BACKGROUND

1. Technical Field

The invention relates to methods and materials involved in producing organic compounds such as organic acids.

2. Background Information

Ascorbic acid (vitamin C) has many important nutritional uses. In fact, ascorbic acid is an essential nutrient to humans, and must be obtained from diet to prevent vitamin C deficiencies such as scurvy. In addition, some medical practitioners claim that ascorbic acid has the potential to prevent and treat the common cold, flu, and cancer. Thus, diet supplements containing ascorbic acid are widely used.

Ascorbic acid also has many important industrial uses. For example, ascorbic acid can be used in meat processing, nutritional supplements, and animal foods. In fact, several industrial manufactures can produce 10,000 metric tons annually of ascorbic acid and related ascorbic acid compounds such as calcium ascorbate and sodium ascorbate.

The “Reichstein” method is a commonly used method for producing ascorbic acid from D-glucose or a D-glucose precursor such as corn syrup. This method involves six discrete chemical steps as well as a fermentation step. For example, one of the chemical steps involves converting 2-keto-L-gulonic acid into ascorbic acid by treating the 2-keto-L-gulonic acid with acid at a temperature greater than 60° C.

Several other manufacturing processes containing at least one chemical step are also used to produce ascorbic acid. Specifically, ascorbic acid has been produced using methods that chemically convert D-glucose into L-sorbitol prior to a fermentation step, methods that chemically convert 2-keto-L-gulonic acid into ascorbic acid after a fermentation step, and methods that chemically convert D-glucose into L-sorbitol prior to a fermentation step in addition to chemically converting 2-keto-L-gulonic acid into ascorbic acid after a fermentation step.

SUMMARY

The present invention relates generally to methods and materials for producing organic compounds such as myo-inositol, glucuronic acid, glucaric acid, and ascorbic acid. Specifically, the invention provides cells (e.g., bacterial, fungal, and insect cells), methods for culturing cells, isolated nucleic acid molecules, and methods and materials for producing various organic compounds. The invention is based on the discovery that cells can be genetically manipulated such that they have the ability to produce a desired organic product. For example, the cells provided herein can produce ascorbic acid. It will be understood that the terms “ascorbate,” “ascorbic acid,” “L-ascorbate,” “L-ascorbic acid,” and “vitamin C” can be used interchangeably to refer to L-ascorbic acid. It also will be understood that the term “glucaric acid” as used herein refers to glucaric acid, glucaro-1,4-lactone, and glucaro-6,3-lactone since these three compounds freely interconvert when in solution.

The invention also is based on the discovery of efficient metabolic pathways that utilize glucose and/or phytic acid to produce ascorbic acid. Specifically, ascorbic acid can be produced from glucose and/or phytic acid using a metabolic pathway that can convert myo-inositol into glucuronate. In general, such pathways require less enzymatic steps than the native metabolic pathways used by plants and animals to produce ascorbic acid from glucose. Any method that can efficiently produce ascorbic acid from a carbon source such as glucose or phytic acid would be useful for large-scale production efforts. In addition, the methods and materials provided herein can be used to produce organic compounds without the need of chemical steps such as an acid treatment at high temperature (e.g., a temperature greater than 60° C.).

In general, one aspect of the invention features a method of providing a cell with a polypeptide having myo-inositol oxygenase activity. The method includes introducing a nucleic acid molecule into the cell, where the nucleic acid molecule encodes the polypeptide, and where the cell expresses the polypeptide. The cell can be a prokaryotic cell (e.g., a Pseudomonas, Bacillus, Lactobacillus, Lactococcus , or Corynebacterium cell). The cell can be a eukaryotic cell (e.g., a yeast, fungi, insect, or mammalian cell). The cell can be a Saccharomyces, Pichia, Aspergillus, Cryptococcus, Schwanniomyces, Schizosaccharomyces, Spodoptera, Cricetulus , or Homo sapiens cell. The nucleic acid molecule can be integrated into the genome of the cell. The polypeptide can contain an amino acid sequence at least about 50 percent identical (e.g., at least about 55, 60, 70, 75, 80, or 90 percent identical) to the sequence set forth in SEQ ID NO:12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. The polypeptide can contain an amino acid sequence at least about 70 percent identical to the sequence set forth in SEQ ID NO:12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. The cell can produce L-ascorbic acid. The cell can have glucuronate reductase activity. The cell can have 1,4-lactone hydroxyacylhydrolase activity, D-glucono-1,5-lactone lactonohydrolase activity, and/or uronolactonase activity. The cell can have gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidase activity, and/or gulono-γ-lactone dehydrogenase activity. The cell can have phosphatase activity and/or phytase activity. The cell can lack L-gulonate 3-dehydrogenase activity. The cell can contain myo-inositol oxygenase activity with a specific activity greater than 40 mg glucuronic acid per gram dry cell weight per hour. The cell can contain myo-inositol oxygenase activity such that an extract from 1×10 ⁶ cells contains a specific activity greater than 150 μg glucuronic acid formed per 10 mg total protein per 10 minutes, where each of the 1×10 ⁶ cells is the cell or a progeny of the cell. The nucleic acid molecule can contain a promoter that is lactose unresponsive. The polypeptide can lack an N-terminal polyhistidine tag. The polypeptide can lack a glutathione-S-transferase sequence.

In another embodiment, the invention features methods of producing glucaric acid as well as cells capable of producing glucaric acid. These methods involve converting myo-inositol to glucuronic acid and converting glucuronic acid to glucaric acid. The substrates (e.g., myo-inositol and glucuronic acid) can be converted to their respective products using polypeptides or chemical conversions. A “chemical conversion” as used herein refers to the changing of a substrate to a product without the aid of a polypeptide having enzymatic activity. Moreover, these methods can be practiced in vivo, in vitro, or by using combinations of in vitro and in vivo steps. When polypeptides are used to convert glucuronic acid to glucaric acid, the polypeptides can have either aldehyde dehydrogenase activity, hexose oxidase activity, or aldehyde oxidase activity. When polypeptides are used to convert myo-inositol to glucuronic acid, the polypeptides can have myo-inositol oxygenase activity.

In another embodiment, the invention features a cell containing an exogenous nucleic acid molecule, where the exogenous nucleic acid molecule encodes a polypeptide having myo-inositol oxygenase activity, and where the cell expresses the polypeptide. The cell can be a prokaryotic cell (e.g., a Pseudomonas, Bacillus, Lactobacillus, Lactococcus , or Corynebacterium cell). The cell can be a eukaryotic cell (e.g., a yeast, fungi, insect, or mammalian cell). The cell can be a Saccharomyces, Pichia, Aspergillus, Cryptococcus, Schwanniomyces, Schizosaccharomyces, Spodoptera, Cricetulus , or Homo sapiens cell. The polypeptide can contain an amino acid sequence at least about 50 percent identical (e.g., at least about 55, 60, 70, 75, 80, or 90 percent identical) to the sequence set forth in SEQ ID NO:12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. The polypeptide can contain an amino acid sequence at least about 70 percent identical to the sequence set forth in SEQ ID NO:12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. The cell can contain a second exogenous nucleic acid molecule, where the second exogenous nucleic acid molecule encodes a second polypeptide, and where the cell expresses the second polypeptide. The second polypeptide can have glucuronate reductase activity. The second polypeptide can contain an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:36. The second polypeptide can have 1,4-lactone hydroxyacylhydrolase activity, D-glucono-1,5-lactone lactonohydrolase activity, or uronolactonase activity. The second polypeptide can contain an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:37 or 38. The second polypeptide can have gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidase activity, or gulono-γ-lactone dehydrogenase activity. The second polypeptide can contain an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:39 or 40. The second polypeptide can have phosphatase activity. The second polypeptide can contain an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:41 or 44. The second polypeptide can have phytase activity. The second polypeptide can contain an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:42 or 43. The cell can contain a second exogenous nucleic acid molecule and a third exogenous nucleic acid molecule, where the second exogenous nucleic acid molecule encodes a second polypeptide, where the third exogenous nucleic acid molecule encodes a third polypeptide, and where the cell expresses the second polypeptide and the third polypeptide. The second polypeptide can have glucuronate reductase activity, 1,4-lactone hydroxyacylhydrolase activity, D-glucono-1,5-lactone lactonohydrolase activity, gulono-γ-lactone oxidase activity, gulono-γ-lactone dehydrogenase activity, uronolactonase activity, galactono-γ-lactone oxidase activity, pyridine nucleotide transhydrogenase activity, phytase, and/or phosphatase activity. The third polypeptide can have glucuronate reductase activity, 1,4-lactone hydroxyacylhydrolase activity, D-glucono-1,5-lactone lactonohydrolase activity, gulono-γ-lactone oxidase activity, gulono-γ-lactone dehydrogenase activity, uronolactonase activity, galactono-γ-lactone oxidase activity, pyridine nucleotide transhydrogenase activity, phytase activity, and/or phosphatase activity. The cell can lack L-gulonate 3-dehydrogenase activity. The cell can have a genetic modification that reduces L-gulonate 3-dehydrogenase activity. The genetic modification can include a nucleic acid deletion in the genome of the cell. The cell can produce ascorbic acid. The cell can have pyridine nucleotide transhydrogenase activity. The cell can have myo-inositol oxygenase activity with a specific activity greater than 40 mg glucuronic acid per gram dry cell weight per hour. The cell can have myo-inositol oxygenase activity such that an extract from 1×10 ⁶ cells comprises a specific activity greater than 150 μg glucuronic acid formed per 10 mg total protein per 10 minutes, where each of the 1×10 ⁶ cells is the cell or a progeny of the cell. The exogenous nucleic acid molecule can contain a promoter that is lactose unresponsive. The polypeptide can lack an N-terminal polyhistidine tag. The polypeptide can lack a glutathione-S-transferase sequence. The exogenous nucleic acid molecule can be integrated into the genome of the cell.

In another aspect, the invention features a method of reducing myo-inositol oxygenase activity in a cell. The method includes genetically modifying the genome of the cell such that the expression of a polypeptide having the myo-inositol oxygenase activity is reduced. The cell can be a eukaryotic cell (e.g., a plant cell). The genetic modification can contain a nucleic acid deletion in the genome of the cell.

Another embodiment of the invention features a cell containing a genetic modification that reduces myo-inositol oxygenase activity. The cell can be a eukaryotic cell (e.g., a plant cell). The genetic modification can include a nucleic acid deletion in the genome of the cell. The cell can lack the myo-inositol oxygenase activity.

Another embodiment of the invention features a cell containing a genetic modification that reduces L-gulonate 3-dehydrogenase activity. The cell can be a eukaryotic cell. The genetic modification can include a nucleic acid deletion in the genome of the cell. The cell can lack the L-gulonate 3-dehydrogenase activity.

Another aspect of the invention features an isolated nucleic acid molecule containing a nucleic acid sequence at least about 50 percent identical to the sequence set forth in SEQ ID NO:1. The isolated nucleic acid molecule can encode a polypeptide having myo-inositol oxygenase activity. The nucleic acid sequence can be as set forth in SEQ ID NO:1.

In another embodiment, the invention features an isolated nucleic acid molecule that encodes a polypeptide having an amino acid sequence at least about 50 percent identical to the sequence set forth in SEQ ID NO:19. The polypeptide can have myo-inositol oxygenase activity. The amino acid sequence can be as set forth in SEQ ID NO:19.

Another aspect of the invention features a method for producing ascorbic acid. The method includes (a) contacting myo-inositol with a first polypeptide having myo-inositol oxygenase activity to form glucuronate, where the first polypeptide is within a cell, (b) contacting the glucuronate with a second polypeptide having glucuronate reductase activity to form gulonate, (c) contacting the gulonate with a third polypeptide to form gulono-γ-lactone, the third polypeptide having 1,4-lactone hydroxyacylhydrolase activity and/or D-glucono-1,5-lactone lactonohydrolase activity, and (d) contacting the gulono-γ-lactone with a fourth polypeptide to form the ascorbic acid, the fourth polypeptide having gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidase activity, and/or gulono-γ-lactone dehydrogenase activity, where at least 10 mg (e.g., at least 20, 30, 40, 50, 100, or more mg) of ascorbic acid is produced per gram dry cell weight per hour.

Another embodiment of the invention features a method for producing ascorbic acid. The method includes (a) contacting myo-inositol with a first polypeptide having myo-inositol oxygenase activity to form glucuronate, where the first polypeptide is within a cell, (b) contacting the glucuronate with a second polypeptide having uronolactonase activity to form glucurono-lactone, (c) contacting the glucurono-lactone with a third polypeptide having glucuronolactone reductase activity to form gulono-γ-lactone, and (d) contacting the gulono-γ-lactone with a fourth polypeptide to form the ascorbic acid, the fourth polypeptide having gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidase activity, and/or gulono-γ-lactone dehydrogenase activity, where at least 10 mg (e.g., at least 20, 30, 40, 50, 100, or more mg) of ascorbic acid is produced per gram dry cell weight per hour.

Another embodiment of the invention features a method for producing ascorbic acid. The method includes (a) contacting myo-inositol with a first polypeptide having myo-inositol oxygenase activity to form glucuronate, where the first polypeptide is extraceilular, (b) contacting the glucuronate with a second polypeptide having glucuronate reductase activity to form gulonate, (c) contacting the gulonate with a third polypeptide to form gulono-γ-lactone, the third polypeptide having 1,4-lactone hydroxyacylhydrolase activity and/or D-glucono-1,5-lactone lactonohydrolase activity, and (d) contacting the gulono-γ-lactone with a fourth polypeptide to form the ascorbic acid, the fourth polypeptide having gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidase activity, and/or gulono-γ-lactone dehydrogenase activity.

Another embodiment of the invention features a method for producing ascorbic acid. The method includes (a) contacting myo-inositol with a first polypeptide having myo-inositol oxygenase activity to form glucuronate, where the first polypeptide is extracellular, (b) contacting the glucuronate with a second polypeptide having uronolactonase activity to form glucurono-lactone, (c) contacting the glucurono-lactone with a third polypeptide having glucuronolactone reductase activity to form gulono-γ-lactone, and (d) contacting the gulono-γ-lactone with a fourth polypeptide to form the ascorbic acid, the fourth polypeptide having gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidase activity, and/or gulono-γ-lactone dehydrogenase activity.

Glucaric acid, containing two carboxylic acid functional groups, is potentially useful as an acidulent in the food and animal feed industries. Glucaric acid has also been shown to be useful as a chelating agent and can be used as a biodegradable detergent and an additive for cement. Glucaric acid, because it is a potent inhibitor of the enzyme beta-glucuronidase, has also been shown to be valuable as an anti-cancer agent and has been shown to lower serum cholesterol in mammals. Natural sources with particularly high levels of glucaric acid include fruits such as apples and grapefruit and vegetables such as brussel sprouts and broccoli. Because of its metal chelating properties, it can be used as a chelating agent of 99 Tcm for the detection of myocardial infarction and the radio-imaging of tumors. It also is a raw material for the production of polyhydroxylated polymers and as such can be used for the production of fibers, films, and adhesives.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram depicting a seven step metabolic pathway that can produce ascorbic acid from glucose using D-myo-inositol and L-gulonate as intermediates.

FIG. 2 is a diagram depicting a seven step metabolic pathway that can produce ascorbic acid from glucose using D-myo-inositol and D-glucurono-3,6-lactone as intermediates.

FIG. 3 is a diagram depicting an eight step metabolic pathway that can produce ascorbic acid from glucose using UDP-D-glucuronate and L-gulonate as intermediates.

FIG. 4 is a diagram depicting an eight step metabolic pathway that can produce ascorbic acid from glucose using UDP-D-glucuronate and D-glucurono-3,6-lactone as intermediates.

FIG. 5 is a diagram depicting a nine step metabolic pathway that can produce ascorbic acid from glucose using UDP-D-glucuronate and L-gulonate as intermediates.

FIG. 6 is a diagram depicting a nine step metabolic pathway that can produce ascorbic acid from glucose using UDP-D-glucuronate and D-glucurono-3,6-lactone as intermediates.

FIGS. 7A-C shows an alignment of 17 amino acid sequences.

FIG. 8 is a graph plotting μg glucuronate formed per assay versus μg protein per assay for the indicated cell extracts.

FIG. 9 is a sequence listing containing an amino acid sequence from a polypeptide having glucuronate reductase activity. The threonine at position number 2 can be an alanine.

FIG. 10 is a sequence listing containing an amino acid sequence from a polypeptide having D-glucono-1,5-lactone lactonohydrolase activity. The threonine at position number 2 can be an alanine; the valine at position 16 can be an alanine; the methionine at position 17 can be an isoleucine; the glutamic acid at position 34 can be a glutamine; and a valine can be inserted after the valine at position 162.

FIG. 11 is a sequence listing containing an amino acid sequence from a polypeptide having uronolactonase activity. The amino acid residues from position number 2 through position number 20 can be removed to form a mature polypeptide.

FIG. 12 is a sequence listing containing an amino acid sequence from a polypeptide having gulono-γ-lactone oxidase activity. The isoleucine at position number 85 can be an valine, and the glutamine at position 189 can be a histidine.

FIG. 13 is a sequence listing containing an amino acid sequence from a polypeptide having galactono-γ-lactone oxidase activity.

FIG. 14 is a sequence listing containing an amino acid sequence from a polypeptide having acid phosphatase activity.

FIG. 15 is a sequence listing containing an amino acid sequence from a polypeptide having phytase activity.

FIG. 16 is a sequence listing containing an amino acid sequence from a polypeptide having phytase activity.

FIG. 17 is a sequence listing containing an amino acid sequence from a polypeptide having phosphatase activity.

FIG. 18 is a diagram depicting a metabolic pathway that can produce D-glucaric acid from glucose or phytate.

FIG. 19 is a graph plotting μg ascorbic acid per mL per OD600 unit for the indicated samples after a zero, three, or six hour incubation.

FIG. 20 is a graph plotting the concentration of ascorbic acid for the indicated samples after a zero, four, eight, or nineteen hour incubation.

FIG. 21 contains HPLC and mass spectrometry graphs demonstrating the conversion of glucuronic acid into glucaric acid.

DETAILED DESCRIPTION

The invention provides methods and materials related to producing organic compounds such as myo-inositol and ascorbic acid. Specifically, the invention provides cells, methods for culturing cells, isolated nucleic acid molecules, and methods and materials for producing various organic compounds. In addition, the invention provides several metabolic pathways that can be used to produce ascorbic acid.

1. Seven Step Metabolic Pathways

The invention provides several seven step metabolic pathways that can produce ascorbic acid from glucose (FIGS. 1 and 2). As depicted in step one of FIG. 1, D-glucose can be converted into D-glucose-6-phosphate by a polypeptide having either hexokinase activity (EC 2.7.1.1) or glucokinase activity (EC 2.7.1.2). Polypeptides having hexokinase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Homo sapiens, Rattus norvegicus, Saccharomyces cerevisiae, Arabidopsis thaliana , and Aspergillus niger . Polypeptides having glucolinase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Bos taurus, Rattus norvegicus, Mus musculus, Homo sapiens, Saccharomyces cerevisiae, Schistosoma mansoni, Aspergillus nidulans, Schizosaccharomyces pombe, Arabidopsis thaliana, Kluyveromyces lactis, Schwanniomyces occidentalis (also known as Debaryomyces occidentalis ), Plasmodium falciporm, Bacillus subtilis, Aspergillus niger, Staphylococcus xylosus, Brucella abortus, Zymomonas mobilis, Escherichia coli , and Streptomyces coelicolor . For example, nucleic acid that encodes a polypeptide having glucokinase activity can be obtained from Aspergillus niger and can have a sequence as set forth in GenBank® Accession Number X99626.

Alternatively, D-glucose can be converted into D-glucose-6-phosphate by a polypeptide having either polyphosphate:D-glucose 6-phosphotransferase activity (EC 2.7.1.63) or D-glucose-6-phosphate phosphohydrolase activity (EC 3.1.3.9), or extracellular D-glucose can be transported into a cell and converted into D-glucose-6-phosphate by a polypeptide having protein-N(pai)-phosphohistidine-sugar phosphotransferase activity (EC 2.7.1.69). Polypeptides having polyphosphate:D-glucose 6-phosphotransferase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Mycobacterium tuberculosis . Polypeptides having D-glucose-6-phosphate phosphohydrolase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus and Homo sapiens . For example, nucleic acid that encodes a polypeptide having D-glucose-6-phosphate phosphohydrolase activity can be obtained from Rattus norvegicus and can have a sequence as set forth in GenBank® Accession Number U07993. Polypeptides having protein-N(pai)-phosphohistidine-sugar phosphotransferase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Escherichia coli and Bacillus subtilis.

In step two, the resulting D-glucose-6-phosphate can be converted into D-myo-inositol-1-phosphate by a polypeptide having myo-inositol-1-phosphate synthase activity (EC 5.5.1.4). Polypeptides having myo-inositol-1-phosphate synthase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Arabidopsis thaliana, Saccharomyces cerevisiae, Citrus paradisi, Candida albicans , and Spirodela polyrrhiza . For example, nucleic acid that encodes a polypeptide having myo-inositol-1-phosphate synthase activity can be obtained from Saccharomyces cerevisiae and can have a sequence as set forth in GenBank® Accession Number J04453.

In step three, D-myo-inositol-1-phosphate can be converted into D-myo-inositol by a polypeptide having myo-inositol-1 (or 4) monophosphatase activity (EC 3.1.3.25). Polypeptides having myo-inositol-1(or 4) monophosphatase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Lycopersicon esculentum, Xenopus laevis , and Mesembryanthemum crystallinum . For example, nucleic acid that encodes a polypeptide having myo-inositol-1(or 4) monophosphatase activity can be obtained from Homo sapiens and can have a sequence as set forth in GenBank® Accession Number NM _— 005536.

In step four, the resulting D-myo-inositol can be converted into D-glucuronate by a polypeptide having myo-inositol oxygenase activity (EC 1.13.99.1). Polypeptides having myo-inositol oxygenase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus, Sus scrofa, Bos taurus, Cryptococcus neoformans, Schwanniomyces occidentalis, Homo sapiens, Avena sativa, Pinus radiata, Cryptococcus terreus, Arabidopsis thaliana , and Pleurotus ostreatus.

In step five, D-glucuronate can be converted into L-gulonate by a polypeptide having glucuronate reductase activity (EC 1.1.1.19). Polypeptides having glucuronate reductase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus, Sus scrofa , and Bos taurus.

In step six, the resulting L-gulonate can be converted into L-gulono-γ-lactone by a polypeptide having 1,4-lactone hydroxyacylhydrolase activity (EC 3.1.1.25), D-glucono-1,5-lactone lactonohydrolase activity (EC 3.1.1.17), or uronolactonase activity (E.C. 3.1.1.19). Polypeptides having 1,4-lactone hydroxyacylhydrolase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Homo sapiens and Rattus norvegicus . Polypeptides having D-glucono-1,5-lactone lactonohydrolase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Zymomonas mobilis, Escherichia coli, Saccharomyces cerevisiae, Aspergillus niger, Rattus norvegicus, Sus scrofa , and Bos taurus . For example, nucleic acid that encodes a polypeptide having D-glucono-1,5-lactone lactonohydrolase activity can be obtained from Zymomonas mobilis and can have a sequence as set forth in GenBank® Accession Number X67189. Polypeptides having uronolactonase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Fusarium oxysporum, Aryctolagus cuniculas, Cavia parcellus, Canis familiaris, Macaca philippinensis, Rattus norvegicus, Sus scrofa , and Bos taurus . For example, nucleic acid that encodes a polypeptide having uronolactonase activity can be obtained from Fusarium oxysporum and can encode a sequence as set forth in GenBank® Accession Number BAA34218.

In step seven, L-gulono-γ-lactone can be converted into L-ascorbate by a polypeptide having gulono-γ-lactone oxidase activity (EC 1.1.3.8), apolypeptide having galactono-γ-lactone oxidase activity (EC 1.1.3.24), or a polypeptide having gulono-γ-lactone dehydrogenase activity. Polypeptides having gulono-γ-lactone oxidase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus, Tachyglossus aculeatus, Ornithorhynchus anatinus, Perameles nasuta, Isoodon macrourus, Macropus rufogiseus, Thylogale thetis, Limulus polyphemus, Gallus gallus, Rana catesbeiana, Capra hircus , and Mus musculus . For example, nucleic acid that encodes a polypeptide having gulono-γ-lactone oxidase activity can be obtained from rat and can encode a sequence as set forth in GenBank® Accession Number P10867. Polypeptides having galactono-γ-lactone oxidase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Saccharomyces cerevisiae . For example, nucleic acid that encodes a polypeptide having galactono-γ-lactone oxidase activity can be obtained from Saccharomyces cerevisiae and can encode a sequence as set forth in GenBank® Accession Number BAA23804. Polypeptides having gulono-γ-lactone dehydrogenase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Euglena gracilis (See, e.g., U.S. Pat. No. 5,250,428).

The seven step metabolic pathway depicted in FIG. 2 is identical to the pathway depicted in FIG. 1 except that, in step five, D-glucuronate can be converted into D-glucurono-3,6-lactone by a polypeptide having either 1,4-lactone hydroxyacylhydrolase activity (EC 3.1.1.25) or D-glucono-1,5-lactone lactonohydrolase activity (EC 3.1.1.17) or uronolactonase activity (EC 3.1.1.19) and, in step six, the resulting D-glucurono-3,6-lactone can be converted into L-gulono-γ-lactone by a polypeptide having glucuronolactone reductase activity (EC 1.1.1.20). Polypeptides having uronolactonase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Fusarium oxysporum, Oryctolagus cuniculus, Cavia porcellus, Canis familiaris, Macaca philippinensis, Rattus norvegicus, Sus scrofa , and Bos taurus , while polypeptides having glucuronolactone reductase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus . For example, nucleic acid that encodes a polypeptide having uronolactonase activity can be obtained from Fusarium oxysporum and can encode a sequence as set forth in GenBank® Accession Number BAA34218.

2. Eight Step Metabolic Pathways

The invention provides several eight step metabolic pathways that can produce ascorbic acid from glucose (FIGS. 3 and 4). As depicted in step one of FIG. 3, D-glucose can be converted into D-glucose-6-phosphate in the same manner as described in the seven step metabolic pathways depicted in FIGS. 1 and 2. For example, D-glucose can be converted into D-glucose-6-phosphate by a polypeptide having hexokinase activity (EC 2.7.1.1), glucokinase activity (EC 2.7.1.2), polyphosphate:D-glucose 6-phosphotransferase activity (EC 2.7.1.63), or D-glucose-6-phosphate phosphohydrolase activity (EC 3.1.3.9), or extracellular D-glucose can be transported into a cell and converted into D-glucose-6-phosphate by a polypeptide having protein-N(pai)-phosphohistidine-sugar phosphotransferase activity (EC 2.7.1.69).

In step two, the resulting D-glucose-6-phosphate can be converted into D-glucose-1-phosphate by a polypeptide having phosphoglucomutase activity (EC 5.4.2.2). Polypeptides having phosphoglucomutase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitations Arabidopsis thaliana, Homo sapiens, Saccharomyces cerevisiae , and Xanthomonas campestris . For example, nucleic acid that encodes a polypeptide having phosphoglucomutase activity can be obtained from Saccharomyces cerevisiae and can have a sequence as set forth in GenBank® Accession Number X72016.

In step three, D-glucose-1-phosphate can be converted into UDP-D-glucose by a polypeptide having UTP-glucose-phosphate uridylyltransferase activity (EC 2.7.7.9). Polypeptides having UTP-glucose-1-phosphate uridylyltransferase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Bos taurus, Solanum tuberosum, Pseudomonas aeruginosa, Bacillus subtilis , and Escherichia coli . For example, nucleic acid that encodes a polypeptide having UTP-glucose-1-phosphate uridylyltransferase activity can be obtained from Bos taurus and can have a sequence as set forth in GenBank® Accession Number L14019.

In step four, the resulting UDP-D-glucose can be converted into UDP-D-glucuronate by a polypeptide having UDP-glucose dehydrogenase activity (EC 1.1.1.22). Polypeptides having UDP-glucose dehydrogenase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Homo sapiens, Bos taurus, Mus musculus, Drosophila melanogaster , and Pseudomonas aeruginosa . For example, nucleic acid that encodes a polypeptide having UDP-glucose dehydrogenase activity can be obtained from Pseudomonas aeruginosa and can have a sequence as set forth in GenBank® Accession Number AJ010734.

In step five, the resulting UDP-D-glucuronate can be converted into D-glucuronate by a polypeptide having UDP-glucuronate β-D-glucuronosyltranferase activity (EC 2.4.1.17). Polypeptides having UDP-glucuronate β-D-glucuronosyltransferase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Homo sapiens, Rattus norvegicus , and Mus musculus . For example, nucleic acid that encodes a polypeptide having UDP-glucuronate β-D-glucuronosyltransferase activity can be obtained from Homo sapiens and can have a sequence as set forth in GenBank® Accession Number NM _— 001072.

In step six, D-glucuronate can be converted into L-gulonate by a polypeptide having glucuronate reductase activity (EC 1.1.1.19). Polypeptides having glucuronate reductase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus, Sus scrofa , and Bos taurus.

In step seven, the resulting L-gulonate can be converted into L-gulono-γ-lactone by a polypeptide having either 1,4-lactone hydroxyacylhydrolase activity (EC 3.1.1.25) or D-glucono-1,5-lactone lactonohydrolase activity (EC 3.1.1.17) or uronolactonase activity (EC 3.1.1.19). Polypeptides having 1,4-lactone hydroxyacylhydrolase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus and Homo sapiens , while polypeptides having D-glucono-1,5-lactone lactonohydrolase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Zymomonas mobilis, Escherichia coli, Saccharomyces cerevisiae, Aspergillus niger, Rattus norvegicus, Sus scrofa , and Bos taurus . For example, nucleic acid that encodes a polypeptide having D-glucono-1,5-lactone lactonohydrolase activity can be obtained from Zymomonas mobilis and can have a sequence as set forth in GenBank® Accession Numbers X67189 and S53050.

In step eight, L-gulono-γ-lactone can be converted into L-ascorbate by a polypeptide having gulono-γ-lactone oxidase activity (EC 1.1.3.8), a polypeptide having galactono-γ-lactone oxidase activity (EC1.1. 1.3.24), or a polypeptide having gulono-γ-lactone dehydrogenase activity. Polypeptides having gulono-γ-lactone oxidase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus, Tachyglossus aculeatus, Ornithorhynchus anatinus, Perameles nasuta, Isoodon macrourus, Macropus rufogiseus, Thylogale thetis, Limulus polyphemus, Gallus gallus, Rana catesbeiana , and Capra hircus . For example, nucleic acid that encodes a polypeptide having gulono-γ-lactone oxidase activity can be obtained from rat and can encode a sequence as set forth in GenBank® Accession Number P10867. Polypeptides having galactono-γ-lactone oxidase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Saccharomtyces cerevisiae . For example, nucleic acid that encodes a polypeptide having galactono-γ-lactone oxidase activity can be obtained from Saccharomyces cerevisiae and can encode a sequence as set forth in GenBank® Accession Number BAA23804. Polypeptides having gulono-γ-lactone dehydrogenase as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Euglena gracilis (See, e.g., U.S. Pat. No. 5,250,428).

The eight step metabolic pathway depicted in FIG. 4 is identical to the pathway depicted in FIG. 3 except that, in step six, D-glucuronate can be converted into D-glucurono-3,6-lactone by a polypeptide having either 1,4-lactone hydroxyacylhydrolase activity (EC 3.1.1.25) or D-glucono-1,5-lactone lactonohydrolase activity (EC 3.1.1.17) or uronolactonase activity (EC 3.1.1.19) and, in step seven, the resulting D-glucurono-3,6-lactone can be converted into L-gulono-γ-lactone by a polypeptide having glucuronolactone reductase activity (EC 1.1.1.20). Polypeptides having uronolactonase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Fusarium oxysporum, Oryctolagus cuniculus, Cavia porcellus, Canis familiaris, Macaca philippinensis, Rattus norvegicus, Sus scrofa , and Bos taurus , while polypeptides having glucuronolactone reductase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Rattus norvegicus.

3. Nine Step Metabolic Pathways

The invention provides several nine step metabolic pathways that can produce ascorbic acid from glucose (FIGS. 5 and 6). The nine step metabolic pathways depicted in FIGS. 5 and 6 are similar to the eight step metabolic pathways depicted in FIGS. 3 and 4, respectively, except that the conversion of UDP-D-glucuronate into D-glucuronate uses a D-glucuronate-1-phosphate intermediate. As depicted in FIGS. 5 and 6, step five involves the conversion of UDP-D-glucuronate into D-glucuronate-1-phosphate by a polypeptide having UTP:1-phospho-α-D-glucuronate uridylyltransferase activity (EC 2.7.7.44). Polypeptides having UTP:1-phospho-α-D-glucuronate uridylyltransferase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Hordeum vulgare and Typha latifolia.

In step six, the resulting D-glucuronate-1-phosphate can be converted into D-glucuronate by a polypeptide having ATP:D-glucuronate 1-phosphotransferase activity (EC 2.7.1.43). Polypeptides having ATP:D-glucuronate 1-phosphotransferase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Vigna radiata, Nicotiana tabacum, Lilium longiflorum, Zea mays , and Glycine max.

4. Phytic Acid

The invention provides several pathways that can be used to produce myo-inositol or ascorbic acid from phytic acid. For example, phytic acid can be converted into myo-inositol by a polypeptide having phytase activity, by a polypeptide having phosphatase activity (or a collection of polypeptides having different phosphatase activities), or a mixture of polypeptides having phytase activity and polypeptides having phosphatase activity (or a collection of polypeptides having different phosphatase activities). For example, a polypeptide having phytase activity can be used to convert phytic acid into myo-inositol. Polypeptides having phytase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Schwanniomyces occidentalis, Bacillus subtilis, E. coli, Aspergillus terreus, Homo sapiens , and Zea mays . For example, nucleic acid that encodes a polypeptide having phytase activity can be obtained from E. coli and can have a sequence as set forth in GenBank® Accession Number M58708, or can be obtained from Bacillus subtilis and can have a sequence as set forth in GenBank® Accession Number AF298179 or AI277890. Also, polypeptides having phytase activity as well as nucleic acid encoding such polypeptides can be obtained as described in U.S. Pat. No. 5,830,733; 5,840,561; or 5,830,732. In one embodiment, a polypeptide having the sequence set forth in FIG. 16 can be used to convert phytic acid into myo-inositol. Alternatively, a polypeptide mixture having multiple inositol polyphosphate phosphatase activities can be used to convert phytic acid into myo-inositol. Polypeptides having phosphatase activity (e.g., acid phosphatase activity) as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, E. coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans , and Aspergillus niger . For example, nucleic acid that encodes a polypeptide having phosphatase activity can be obtained from E. coli and can encode a sequence as set forth in GenBank® Accession Number P07102. Also, polypeptides having phosphatase activity as well as nucleic acid encoding such polypeptides can be obtained as described in U.S. Pat. No. 5,830,733. A mixture of polypeptides having phytase activity and polypeptides having phosphatase activities can be used to convert phytic acid into inositol as described in U.S. Pat. No. 5,830,733. In addition, phytic acid can be converted into myo-inositol using any chemical technique such as heat or steam treatments.

The resulting myo-inositol can be converted into any other organic compound (e.g., ascorbic acid) using any of the enzymatic steps described herein. For example, myo-inositol can be converted into ascorbic acid using steps four through seven of the seven step metabolic pathway of FIG. 1.

5. Glucaric Acid

The invention provides pathways that can be used to produce glucaric acid (FIG. 18). For example, glucose or phytic acid can be converted into glucuronic acid as described herein. The resulting glucuronic can be converted into glucaric acid by a polypeptide having non-specific hexose oxidase activity (EC 1.1.3.5), by a polypeptide having aldehyde dehydrogenase [NAD(P)] activity (EC 1.2.1.5, EC 1.2.1.3 (NAD), or EC 1.2.1.4 (NADP)), or by a polypeptide having aldehyde oxidase activity (EC 1.2.3.1). For example, a polypeptide having non-specific hexose oxidase activity can be used to convert glucuronic into glucaric acid. Polypeptides having non-specific hexose oxidase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Chondrus crispus, Yersini apestis, Yersinia pseudotuberculosis , and Ralstonia solanacearum . For example, nucleic acid that encodes a polypeptide having non-specific hexose oxidase activity can be obtained from Chondrus crispus and can encode an amino acid sequence as set forth in GenBank® Accession Number AAB49376.1, or can be obtained from Yersinia pestis and can encode an amino acid sequence as set forth in GenBank® Accession Number NP _— 403959.1, or can be obtained from Ralstonia solanacearum and can encode an amino acid sequence as set forth in GenBank® Accession Number NP _— 518171.1. Also, polypeptides having hexose oxidase activity as well as nucleic acid encoding such polypeptides can be obtained as described elsewhere (U.S. Pat. No. 6,251,626 and Sullivan and Ikawa, Biochimica et Biophysica Acta , 309:11-22 (1973)). In addition, glucuronic can be converted into glucaric acid using any chemical technique such as oxidation using molecular oxygen and a catalyst For example, methods similar to those described in U.S. Pat. No. 5,817,870 or Gallezot et al. ( Chem. Ind . ( Dekker ), 62:331-40 (1995)) can be used to convert glucuronic into glucaric acid.

Additionally, a polypeptide having aldehyde dehydrogenase activity can be used to convert glucuronic into glucaric acid. Polypeptides having aldehyde dehydrogenase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Bacillus stearothermophilus (gi:1169292) and Bacillus subtilus (gi:16077316 or NP _— 388129.1). Similarly, a polypeptide having aldehyde oxidase activity can be used to convert glucuronic into glucaric acid. Polypeptides having aldehyde oxidase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Oryza sativa (gi:1844950 or AAL700116.1), Zea mays (BAA23226.1), and Lycopersicon esculentum (AAG22607.1 or AF258810).

6. Nucleic Acid

The invention provides isolated nucleic acid molecules that contain a nucleic acid sequence at least about 50 percent identical (e.g., at least about 55, 65, 70, 75, 80, 85, 90, 95, or 99 percent identical) to the sequence set forth in SEQ ID NO:1. The invention also provides isolated nucleic acid molecules that encode a polypeptide having an amino acid sequence at least about 50 percent identical (e.g., at least about 55, 65, 70, 75, 80, 85, 90, 95, or 99 percent identical) to the sequence set forth in SEQ ID NO:19.

The term “nucleic acid” as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

The term “isolated” as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

The term “isolated” as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.

The term “exogenous” as used herein with reference to nucleic acid and a particular cell refers to any nucleic acid that does not originate from that particular cell as found in nature. Thus, all non-naturally-occurring nucleic acid is considered to be exogenous to a cell once introduced into the cell. It is important to note that non-naturally-occurring nucleic acid can contain nucleic acid sequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a cell once introduced into the cell, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid.

Nucleic acid that is naturally occurring can be exogenous to a particular cell. For example, an entire chromosome isolated from a cell of person X is an exogenous nucleic acid with respect to a cell of person Y once that chromosome is introduced into Y's cell.

The percent identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site or the United States government's National Center for Biotechnology Information web site. Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: −i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); −j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); −p is set to blastn; −o is set to any desired file name (e.g., C:\output.txt); −q is set to −1; −r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq −i c:.backslash.seq1.txt −j c:.backslash.seq2.txt −p blastn −o c:.backslash.output.txt −q −1 −r 2. To compare two amino acid sequences, the options of B12seq are set as follows: −i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); −j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); −p is set to blastp; −o is set to any desired file name (e.g., C:\output.txt-); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq −i c:\seq1.txt −j c:\seq2.txt −p blastp −o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO:1) followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 711 matches when aligned with the sequence set forth in SEQ ID NO:1 is 75 percent identical to the sequence set forth in SEQ ID NO:1 (i.e., 711÷948*100=75).

It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78:18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.

The invention also provides isolated nucleic acid molecules that (1) encode a polypeptide having myo-inositol oxygenase activity and (2) hybridize, under hybridization conditions, to the sense or antisense strand of a nucleic acid having the sequence set forth in SEQ ID NO:1. The hybridization conditions can be moderately or highly stringent hybridization conditions.

For the purpose of this invention, moderately stringent hybridization conditions mean the hybridization is performed at about 42° C. in a hybridization solution containing 25 mM KPO ₄ (pH 7.4), 5×SSC, 5× Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15 ng/mL probe (about 5×10 ⁷ cpm/μg), while the washes are performed at about 50° C. with a wash solution containing 2×SSC and 0.1% sodium dodecyl sulfate.

Highly stringent hybridization conditions mean the hybridization is performed at about 42° C. in a hybridization solution containing 25 mM KPO ₄ (pH 7.4), 5×SSC, 5× Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15 ng/mL probe (about 5×10 ⁷ cpm/μg), while the washes are performed at about 65° C. with a wash solution containing 0.2×SSC and 0.1% sodium dodecyl sulfate.

Isolated nucleic acid molecules within the scope of the invention can be obtained using any method including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, PCR can be used to obtain an isolated nucleic acid molecule containing a nucleic acid sequence sharing similarity to the sequence set forth in SEQ ID NO:1. PCR refers to a procedure or technique in which target nucleic acid is amplified in a manner similar to that described in U.S. Pat. No. 4,683,195, and subsequent modifications of the procedure described therein. Generally, sequence information from the ends of the region of interest or beyond are used to design oligonucleotide primers that are identical or similar in sequence to opposite strands of a potential template to be amplified. Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. For example, a nucleic acid sequence can be isolated by PCR amplification from total cellular RNA, total genomic DNA, and cDNA as well as from bacteriophage sequences, plasmid sequences, viral sequences, and the like. When using RNA as a source of template, reverse transcriptase can be used to synthesize complimentary DNA strands.

Isolated nucleic acid molecules within the scope of the invention also can be obtained by mutagenesis. For example, an isolated nucleic acid containing a sequence set forth in SEQ ID NO:1 can be mutated using common molecular cloning techniques (e.g., site-directed mutagenesis). Possible mutations include, without limitation, deletions, insertions, and substitutions, as well as combinations of deletions, insertions, and substitutions.

In addition, nucleic acid and amino acid databases (e.g., Genank®) can be used to obtain an isolated nucleic acid molecule within the scope of the invention. For example, any nucleic acid sequence having some homology to a sequence set forth in SEQ ID NO:1, or any amino acid sequence having some homology to a sequence set forth in SEQ ID NO:19 can be used as a query to search GenBank®.

Further, nucleic acid hybridization techniques can be used to obtain an isolated nucleic acid molecule within the scope of the invention. Briefly, any nucleic acid molecule having some homology to a sequence set forth in SEQ ID NO:1 can be used as a probe to identify a similar nucleic acid by hybridization under conditions of moderate to high stringency. Once identified, the nucleic acid molecule then can be purified, sequenced, and analyzed to determine whether it is within the scope of the invention as described herein.

Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, which hybridizes to a probe. The probe can be labeled with a biotin, digoxygenin, an enzyme, or a radioisotope such as ³² P. The DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory, Plainview, N.Y. Typically, a probe is at least about 20 nucleotides in length. For example, a probe corresponding to a 20-nucleotide sequence set forth in SEQ ID NO:1 can be used to identify an identical or similar nucleic acid. In addition, probes longer or shorter than 20 nucleotides can be used.

7. Genetically Modified Cells

The invention provides genetically modified cells (e.g., cells containing an exogenous nucleic acid molecule). Such cells can be used to produce an organic compound such as ascorbic acid, glucuronic acid, and glucaric acid. The cells can be eukaryotic or prokaryotic. For example, genetically modified cells of the invention can be mammalian cells (e.g., human, murine, and bovine cells), plant cells (e.g., corn, wheat, rice, and soybean cells), fungal cells (e.g., yeast cells), or bacterial cells (e.g., E. coli cells). A cell of the invention also can be a microorganism. The term “microorganism” as used herein refers to all microscopic organisms including, without limitation, bacteria, algae, fungi, and protozoa Thus, E. coli, S. cerevisiae, Kluyveromyces lactis, A. niger, Cr. terreus, Sch. occidentalis , and Sz. pombe are considered microorganisms.

Typically, a cell of the invention is genetically modified such that a particular organic compound is produced. Such cells can contain one or more exogenous nucleic acid molecules that encode polypeptides having enzymatic activity. For example, a microorganism can contain exogenous nucleic acid that encodes a polypeptide having myo-inositol oxygenase activity. In this case, D-myo-inositol can be converted into D-glucuronate which can lead to the production of ascorbic acid. It is noted that a cell can be given an exogenous nucleic acid molecule that encodes a polypeptide having an enzymatic activity that catalyzes the production of a compound not normally produced by that cell. Alternatively, a cell can be given an exogenous nucleic acid molecule that encodes a polypeptide having an enzymatic activity that catalyzes the production of a compound that is normally produced by that cell. In this case, the genetically modified cell can produce more of the compound, or can produce the compound more efficiently, than a similar cell not having the genetic modification.

A polypeptide having a particular enzymatic activity can be a polypeptide that is either naturally occurring or non-naturally occurring. A naturally occurring polypeptide is any polypeptide having an amino acid sequence as found in nature, including wild-type and polymorphic polypeptides. Such naturally occurring polypeptides can be obtained from any species including, without limitation, mammalian, fugal, and bacterial species. A non-naturally occurring polypeptide is any polypeptide having an amino acid sequence that is not found in nature. Thus, a non-naturally occurring polypeptide can be a mutated version of a naturally occurring polypeptide or an engineered polypeptide. For example, a non-naturally occurring polypeptide having myo-inositol oxygenase activity can be a mutated version of a naturally occurring polypeptide having myo-inositol oxygenase activity that retains at least some myo-inositol oxygenase activity. A polypeptide can be mutated by, for example, sequence additions, deletions, and/or substitutions using standard methods such as site-directed mutagenesis of the corresponding nucleic acid coding sequence.

The invention provides genetically modified cells that can be used to perform one or more steps of a metabolic pathway described herein. For example, an individual microorganism can contain an exogenous nucleic acid molecule such that each of the polypeptides necessary to perform all seven steps of a seven step metabolic pathway is expressed. It is important to note that such cells can contain any number of exogenous nucleic acid molecules. For example, a particular cell can contain seven exogenous nucleic acid molecules with each one encoding one of the seven polypeptides necessary to perform a seven step metabolic pathway, or a particular cell can endogenously produce polypeptides necessary to perform the first six of the seven steps of a seven step metabolic pathway while containing an exogenous nucleic acid molecule that encodes a polypeptide necessary to perform the seventh step. It is noted that a cell containing an exogenous nucleic acid molecule that encodes a polypeptide having a particular activity can also endogenously express a polypeptide having a similar activity. In such cases, providing a cell with an exogenous nucleic acid molecule that encodes a polypeptide having an activity similar to an endogenously expressed polypeptide is expected to provide that cell with enhanced activity as compared to a similar cell lacking the exogenous nucleic acid molecule. It also is noted that a cell can contain an exogenous nucleic acid molecule that encodes a polypeptide having pyridine nucleotide transhydrogenase activity. Such a polypeptide can be used to generate NADPH within a cell by catalyzing a chemical reaction (e.g., NADH+NADP→NAD+NADPH). Any source can be used to obtain a polypeptide having pyridine nucleotide transhydrogenase activity or a nucleic acid encoding such a polypeptide. For example, nucleic acid encoding a polypeptide having pyridine nucleotide transhydrogenase activity can be obtained as described elsewhere (e.g., U.S. Pat. No. 5,830,716 and Nissen et al., Yeast 18:19-32 (2001)).

In addition, a single exogenous nucleic acid molecule can encode one or more than one polypeptide. For example, a single exogenous nucleic acid molecule can contain sequences that encode three different polypeptides. Further, the cells described herein can contain a single copy, or multiple copies (e.g., about 5, 10, 20, 35, 50, 75, 100 or 150 copies), of a particular exogenous nucleic acid molecule. For example, a particular cell can contain about 50 copies of an exogenous nucleic acid molecule X. Again, the cells described herein can contain more than one particular exogenous nucleic acid molecule. For example, a particular cell can contain about 50 copies of exogenous nucleic acid molecule X as well as about 75 copies of exogenous nucleic acid molecule Y.

In one embodiment, the invention provides a cell containing an exogenous nucleic acid molecule that encodes a polypeptide having enzymatic activity that leads to the formation of ascorbic acid. It is noted that the produced ascorbic acid can be secreted from the cell, eliminating the need to disrupt cell membranes to retrieve the organic compound. Typically, the cell of the invention produces the organic compound with the concentration being at least about 0.1 grams per L (e.g., at least about 1 g/L, 5 g/L, 10 g/L, or 80 g/L). When determining the yield of organic compound production for a particular cell, any method can be used. See, e.g., Kiers et al., Yeast , 14(5):459-469 (1998). Typically, a cell within the scope of the invention such as a microorganism catabolizes a hexose carbon source such as glucose. A cell, however, can catabolize a variety of carbon sources such as pentose sugars (e.g., ribose, arabinose, xylose, and lyxose), glycerols, or myo-inositol. In other words, a cell within the scope of the invention can utilize a variety of carbon sources.

In another embodiment, a cell within the scope of the invention can contain an exogenous nucleic acid molecule that encodes a polypeptide having myo-inositol oxygenase activity. Such cells can have any level of myo-inositol oxygenase activity. For example, a cell containing an exogenous nucleic acid molecule that encodes a polypeptide having myo-inositol oxygenase activity can have myo-inositol oxygenase activity with a specific activity greater than about 5 mg glucuronic acid formed per gram dry cell weight per hour (e.g., greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 500, or more mg glucuronic acid formed per gram dry cell weight per hour). Alternatively, a cell can have myo-inositol oxygenase activity such that a cell extract from 1×10 ⁶ cells has a specific activity greater than about 5 μg glucuronic acid formed per 10 mg total protein per 10 minutes (e.g., greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 500, or more μg glucuronic acid formed per 10 mg total protein per 10 minutes).

A nucleic acid molecule encoding a polypeptide having enzymatic activity can be identified and obtained using any method. For example, standard nucleic acid sequencing techniques and software programs that translate nucleic acid sequences into amino acid sequences based on the genetic code can be used to determine whether or not a particular nucleic acid has any sequence homology with known enzymatic polypeptides. Sequence alignment software such as MEGALIGN® (DNASTAR, Madison, Wis., 1997) can be used to compare various sequences. In addition, nucleic acid molecules encoding known enzymatic polypeptides can be mutated using common molecular cloning techniques (e.g., site-directed mutageneses). Possible mutations include, without limitation, deletions, insertions, and base substitutions, as well as combinations of deletions, insertions, and base substitutions. Further, nucleic acid and amino acid databases (e.g., Genank®) can be used to identify a nucleic acid sequence that encodes a polypeptide having enzymatic activity. Briefly, any amino acid sequence having some homology to a polypeptide having enzymatic activity, or any nucleic acid sequence having some homology to a sequence encoding a polypeptide having enzymatic activity can be used as a query to search GenBank®. The identified polypeptides then can be analyzed to determine whether or not they exhibit enzymatic activity.

Nucleic acid molecules that encode a polypeptide having enzymatic activity can be identified and obtained using common molecular cloning or chemical nucleic acid synthesis procedures and techniques, including PCR. PCR refers to a procedure or technique in which target nucleic acid is amplified in a manner similar to that described in U.S. Pat. No. 4,683,195, and subsequent modifications of the procedure described therein. Generally, sequence information from the ends of the region of interest or beyond are used to design oligonucleotide primers that are identical or similar in sequence to opposite strands of a potential template to be amplified. Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. For example, a nucleic acid sequence can be isolated by PCR amplification from total cellular RNA, total genomic DNA, and cDNA as well as from bacteriophage sequences, plasmid sequences, viral sequences, and the like. When using RNA as a source of template, reverse transcriptase can be used to synthesize complimentary DNA strands.

In addition, nucleic acid hybridization techniques can be used to identify and obtain a nucleic acid molecule that encodes a polypeptide having enzymatic activity. Briefly, any nucleic acid molecule that encodes a known enzymatic polypeptide, or fragment thereof, can be used as a probe to identify a similar nucleic acid molecules by hybridization under conditions of moderate to high stringency. Such similar nucleic acid molecules then can be isolated, sequenced, and analyzed to determine whether the encoded polypeptide has enzymatic activity.

Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, that hybridizes to a probe. The probe can be labeled with a radioisotope such as ³² P, an enzyme, digoxygenin, or by biotinylation. The DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory, Plainview, N.Y. Typically, a probe is at least about 20 nucleotides in length. For example, a probe corresponding to a 20 nucleotide sequence that encodes a mammalian myo-inositol oxygenase can be used to identify a nucleic acid molecule that encodes a fungal polypeptide having myo-inositol oxygenase activity. In addition, probes longer or shorter than 20 nucleotides can be used.

Expression cloning techniques also can be used to identify and obtain a nucleic acid molecule that encodes a polypeptide having enzymatic activity. For example, a substrate known to interact with a particular enzymatic polypeptide can be used to screen a phage display library containing that enzymatic polypeptide. Phage display libraries can be generated as described elsewhere (Burritt et al., Anal. Biochem . 238:1-13 (1990)), or can be obtained from commercial suppliers such as Novagen (Madison, Wis.).

Further, polypeptide sequencing techniques can be used to identify and obtain a nucleic acid molecule that encodes a polypeptide having enzymatic activity. For example, a purified polypeptide can be separated by gel electrophoresis, and its amino acid sequence determined by, for example, amino acid microsequencing techniques. Once determined, the amino acid sequence can be used to design degenerate oligonucleotide primers. Degenerate oligonucleotide primers can be used to obtain the nucleic acid encoding the polypeptide by PCR Once obtained, the nucleic acid can be sequenced, cloned into an appropriate expression vector, and introduced into a microorganism.

Any method can be used to introduce an exogenous nucleic acid molecule into a cell. In fact, many methods for introducing nucleic acid into microorganisms such as bacteria and yeast are well known to those skilled in the art. For example, heat shock, lipofection, electroporation, conjugation, fusion of protoplasts, and biolistic delivery are common methods for introducing nucleic acid into bacteria and yeast cells. See, e.g., Ito et al., J. Bacterol . 153:163-168 (1983); Durrens et al., Curr. Genet . 18:7-12 (1990); and Becker and Guarente, Methods in Enzymology 194:182-187 (1991).

An exogenous nucleic acid molecule contained within a particular cell of the invention can be maintained within that cell in any form. For example, exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episomal state. In other words, a cell of the invention can be a stable or transient transformant. In addition, a microorganism described herein can contain a single copy, or multiple copies (e.g., about 5, 10, 20, 35, 50, 75, 100 or 150 copies), of a particular exogenous nucleic acid molecule as described above.

Methods for expressing an amino acid sequence from an exogenous nucleic acid molecule are well known to those skilled in the art. Such methods include, without limitation, constructing a nucleic acid such that a regulatory element promotes the expression of a nucleic acid sequence that encodes a polypeptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. Thus, regulatory elements include, without limitation, promoters, enhancers, and the like. Any type of promoter can be used to express an amino acid sequence from an exogenous nucleic acid molecule. Examples of promoters include, without limitation, constitutive promoters, tissue-specific promoters, and promoters responsive or unresponsive to a particular stimulus (e.g., light, oxygen, chemical concentration, and the like). For example, a promoter that is unresponsive to lactose can be used to express a polypeptide having myo-inositol oxygenase activity. Moreover, methods for expressing a polypeptide from an exogenous nucleic acid molecule in cells such as bacterial cells and yeast cells are well known to those skilled in the art. For example, nucleic acid constructs that are capable of expressing exogenous polypeptides within E. coli are well known. See, e.g., Sambrook et al., Molecular cloning: a laboratory manual, Cold Spring Harbour Laboratory Press, New York, USA, second edition (1989).

As described herein, a cell within the scope of the invention can contain an exogenous nucleic acid molecule that encodes a polypeptide having enzymatic activity that leads to the formation of ascorbic acid. Methods of identifying cells that contain exogenous nucleic acid are well known to those skilled in the art. Such methods include, without limitation, PCR and nucleic acid hybridization techniques such as Northern and Southern analysis. In some cases, immunohistochemistry and biochemical techniques can be used to determine if a cell contains a particular nucleic acid by detecting the expression of the encoded enzymatic polypeptide encoded by that particular nucleic acid molecule. For example, an antibody having specificity for an encoded enzyme can be used to determine whether or not a particular cell contains that encoded enzyme. Further, biochemical techniques can be used to determine if a cell contains a particular nucleic acid molecule encoding an enzymatic polypeptide by detecting an organic product produced as a result of the expression of the enzymatic polypeptide. For example, detection of ascorbic acid after introduction of exogenous nucleic acid that encodes a polypeptide having L-gulono-γ-lactone oxidase activity into a cell that does not normally express such a polypeptide can indicate that that cell not only contains the introduced exogenous nucleic acid molecule but also expresses the encoded enzymatic polypeptide from that introduced exogenous nucleic acid molecule. Methods for detecting specific enzymatic activities or the presence of particular organic products are well known to those skilled in the art. For example, the presence of ascorbic acid can be determined as described elsewhere. See, Sullivan and Clarke, J. Assoc. Offic. Agr. Chemists , 38:514-518 (1955).

The invention also provides genetically modified cells having reduced polypeptide activity. The term “reduced” as used herein with respect to a cell and a particular polypeptide's activity refers to a lower level of activity than that measured in a comparable cell of the same species. For example, a particular microorganism lacking enzymatic activity X is considered to have reduced enzymatic activity X if a comparable microorganism has at least some enzymatic activity X. It is noted that a cell can have the activity of any type of polypeptide reduced including, without limitation, enzymes, transcription factors, transporters, receptors, signal molecules, and the like. For example, a cell can contain an exogenous nucleic acid molecule that disrupts a regulatory and/or coding sequence of a polypeptide having myo-inositol oxygenase activity. Disrupting myo-inositol oxygenase expression can lead to the accumulation of D-myo-inositol or derivatives. It is also noted that reduced polypeptide activities can be the result of lower polypeptide concentration, lower specific activity of a polypeptide, or combinations thereof. Many different methods can be used to make a cell having reduced polypeptide activity. For example, a cell can be engineered to have a disrupted regulatory sequence or polypeptide-encoding sequence using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Sterns, Cold Spring Harbor Press (1998). Alternatively, antisense technology can be used to reduce the activity of a particular polypeptide. For example, a cell can be engineered to contain a cDNA that encodes an antisense molecule that prevents a polypeptide from being translated. The term “antisense molecule” as used herein encompasses any nucleic acid molecule or nucleic acid analog (e.g., peptide nucleic acids) that contains a sequence that corresponds to the coding strand of an endogenous polypeptide. An antisense molecule also can have flanking sequences (e.g., regulatory sequences). Thus, antisense molecules can be ribozymes or antisense oligonucleotides. A ribozyme can have any general structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the molecule cleaves RNA.

A cell having reduced activity of a polypeptide can be identified using any method. For example, biological assays such as the assay described in Example 3 for measuring myo-inositol oxygenase activity can be used to identify cells having reduced myo-inositol oxygenase activity.

In one embodiment, the invention provides microorganisms that contain reduced myo-inositol transporter activity. Microorganisms containing reduced myo-inositol transporter activity can produce inositol and inositol-related products (e.g., myo-inositol, meso-inositol, hexahydroxycyclohexane, and Vitamin B ₈ ) in the presence of inositol. In other words, inositol-1-phosphate synthase activity (EC 5.5.1.4) is not regulated by inositol in microorganisms lacking myo-inositol transporter activity. In general, such microorganisms can be produced by reducing the activity of itr1, itr2, opi1, or similar polypeptides. Again, microorganisms containing reduced myo-inositol transporter activity can be produced by any method including, without limitation, mutagenesis, knock-out, and anti-sense technology. It is noted that nucleic acid that encodes itr1 from S. cerevisiae is set forth in GenBank® Accession Number D90352, and nucleic acid that encodes itr2 from S. cerevisiae is set forth in GenBank® Accession Number D90353.

In another embodiment, the invention provides cells that can have the ability, or the enhanced ability, to transport or produce substrates. These cells can have nucleic acid sequences that encode polypetides with transporter activity (e.g., itr1 from S. cerevisiae ) and/or inositol-1-phophate synthase activity as described herein.

In another embodiment, the invention provides cells having reduced L-gulonate 3-dehydrogenase activity (E.C. 1.1.1.45). Such cells also can contain a polypeptide having phytase activity, a polypeptide having phosphatase activity (or a mixture of polypeptides having different phosphatase activities), and/or a mixture of polypeptides having phytase activity and polypeptides having phosphatase activity (or polypeptides having different phosphatase activities). In addition, such cells can contain exogenous nucleic acid molecules that encode a polypeptide having phytase activity, a polypeptide having phosphatase activity (or a mixture of polypeptides having different phosphatase activities), and/or a mixture of polypeptides having phytase activity and polypeptides having phosphatase activity (or polypeptides having different phosphatase activities). For example, a cell can contain polypeptides having multiple inositol polyphosphate phosphatase activities or exogenous nucleic acid molecules that encode polypeptides having multiple inositol polyphosphate phosphatase activities. Cells having phytase activity, phosphatase activity, and/or mixtures thereof as well as reduced L-gulonate 3-dehydrogenase activity can be used to produce increased levels of myo-inositol from phytic acid.

8. Organic Compound Production and Culturing Methods

The invention provides methods for producing an organic compound. For example, the methods and materials described herein can be used to produce D-glucose, D-glucose-1-phosphate, D-glucose-6-phosphate, UDP-D-glucose, D-myo-inositol, D-myo-inositol-1-phosphate, D-glucuronate, D-glucuronate-1-phosphate, UDP-D-glucuronate, D-glucurono-3,6-lactone, L-gulonate, L-gulono-γ-lactone, glucaric acid, and L-ascorbate. Other examples of compounds that can be produced include, without limitation, L-dehydroascorbate, L-threonate, and 3-dehydro-L-threonate. It is noted that a produced compound can be in the D or L configuration. In addition, a polypeptide having a particular enzymatic activity can be used such that the desired organic compound is optically pure (e.g., about 75, 80, 85, 90, 95, 99, or 99.9 percent pure).

A cell described herein can be used to produce a particular organic compound such as myo-inositol, ascorbic acid, or glucaric acid. For example, a microorganism containing all the polypeptides necessary to produce ascorbic acid from glucose as depicted in FIG. 1 can be used to produce ascorbic acid. Alternatively, different microorganisms can be used to produce a particular, organic compound. For example, three different microorganisms each containing a different set of polypeptides necessary to produce ascorbic acid from glucose can be used to produce ascorbic acid. In other words, one or more than one group of cells can be used to produce a particular organic compound.

In addition, purified polypeptides having enzymatic activity can be used alone or in combination with cells to produce organic compounds. For example, with reference to FIG. 1, a microorganism containing polypeptides necessary to catalyze steps one through six can be used to produce L-gulono-γ-lactone from glucose, while a purified polypeptide having gulono-γ-lactone oxidase activity (EC 1.1.3.8) can be used to convert L-gulono-γ-lactone into L-ascorbate. Any method can be used to purify a particular polypeptide. For example, size fractionation, ion exchange, HPLC, and affinity chromatography can be used to purify a polypeptide having enzymatic activity. In addition, purified polypeptides can be used on a solid support (e.g., glass beads, polymer structures, and other plastics), or in solution.

Further, cell free extracts containing a polypeptide having enzymatic activity can be used alone or in combination with purified polypeptides and/or cells to produce organic compounds. For example, with reference to FIG. 1, a microorganism containing polypeptides necessary to catalyze steps one through five can be used to produce L-gulonate from glucose, while a cell-free extract containing a polypeptide having 1,4-lactone hydroxyacylhydrolase activity (EC 3.1.1.25) is used to convert L-gulonate into L-gulono-γ-lactone, and a purified polypeptide having gulono-γ-lactone oxidase activity (EC 1.1.3.8) is used to convert L-gulono-γ-lactone into L-ascorbate. Any method can be used to produce a cell-free