Myo-inositol oxygenases

Abstract

The invention provides methods and materials related to the production of organic products such as glucuronic acid, ascorbic acid, and glucaric acid. Specifically, the invention provides cells, methods for culturing cells, isolated nucleic acid molecules, and methods and materials for producing various organic products such as glucuronic acid, ascorbic acid, and glucaric acid.

1 . An isolated cell comprising myo-inositol and one or more exogenous nucleic acid molecules, wherein said one or more exognous nucleic acid molecules encodes at least one polypeptide having myo-inositol oxygenase activity, and wherein said cell expresses said at least one polypeptide. 
     
     
         2 . The cell of  claim 1 , wherein said cell is a prokaryotic cell. 
     
     
         3 . The cell of  claim 2 , wherein said prokaryotic cell is selected from the group consisting of  Pseudomonas, Escherichia, Bacillus, Lactobacillus, Lactococcus , and  Corynebacterium  cells. 
     
     
         4 . The cell of  claim 1 , wherein said cell is a eukaryotic cell. 
     
     
         5 . The cell of  claim 4 , wherein said eukaryotic cell is selected from the group consisting of yeast, fungi, insect, and mammalian cells. 
     
     
         6 . The cell of  claim 1 , wherein said cell is selected from the group consisting of  Saccharomyces, Pichia, Aspergillus, Cryptococcus, Schwanniomyces, Schizosaccharomyces, Spodoptera, Cricetulus , and  Homo sapiens  cells. 
     
     
         7 . The cell of  claim 1 , wherein said cell is a plant cell. 
     
     
         8 . The cell of  claim 1 , wherein said at least one polypeptide comprises 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. 
     
     
         9 . The cell of  claim 1 , said cell further comprising a second polypeptide encoded by said one or more exogenous nucleic acid molecules. 
     
     
         10 . The cell of  claim 9 , wherein said second polypeptide comprises glucuronate reductase activity. 
     
     
         11 . The cell of  claim 10 , wherein said second polypeptide comprises an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:36. 
     
     
         12 . The cell of  claim 9 , wherein said second polypeptide comprises 1,4-lactone hydroxyacylhydrolase activity, D-glucono-1,5-lactone lactonohydrolase activity, hexose oxidase activity, or uronolactonase activity. 
     
     
         13 . The cell of  claim 12 , wherein said second polypeptide comprises an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:37 or 38. 
     
     
         14 . The cell of  claim 9 , wherein said second polypeptide comprises gulono-γ-lactone oxidase activity, galactono-γ-lactone oxidase activity, or gulono-γ-lactone dehydrogenase activity. 
     
     
         15 . The cell of  claim 14 , wherein said second polypeptide comprises an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:39 or 40. 
     
     
         16 . The cell of  claim 9 , wherein said second polypeptide comprises phosphatase activity. 
     
     
         17 . The cell of  claim 16 , wherein said second polypeptide comprises an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:41 or 44. 
     
     
         18 . The cell of  claim 9 , wherein said second polypeptide comprises phytase activity. 
     
     
         19 . The cell of  claim 18 , wherein said second polypeptide comprises an amino acid sequence at least about 50 percent identical to the amino acid sequence set forth in SEQ ID NO:42 or 43. 
     
     
         20 . The cell of  claim 1 , said cell further comprising a second polypeptide and a third polypeptide encoded by said one or more exogenous nucleic acid molecules. 
     
     
         21 . The cell of  claim 20 , wherein said second polypeptide comprises an activity selected from the group consisting of glucuronate reductase, 1,4-lactone hydroxyacylhydrolase, D-glucono-1,5-lactone lactonohydrolase, gulono-γ-lactone oxidase, gulono-γ-lactone dehydrogenase, uronolactonase, galactono-γ-lactone oxidase, pyridine nucleotide transhydrogenase, phytase, and phosphatase activities. 
     
     
         22 . The cell of  claim 20 , wherein said third polypeptide comprises an activity selected from the group consisting of glucuronate reductase, 1,4-lactone hydroxyacylhydrolase, D-glucono-1,5-lactone lactonohydrolase, gulono-γ-lactone oxidase, gulono-γ-lactone dehydrogenase, uronolactonase, galactono-γ-lactone oxidase, pyridine nucleotide transhydrogenase, phytase, and phosphatase activities. 
     
     
         23 . The cell of  claim 1 , wherein said cell lacks L-gulonate 3-dehydrogenase activity. 
     
     
         24 . The cell of  claim 1 , wherein said cell comprises a genetic modification that reduces L-gulonate 3-dehydrogenase activity. 
     
     
         25 . The cell of  claim 24 , wherein said genetic modification comprises a nucleic acid deletion in the genome of said cell. 
     
     
         26 . The cell of  claim 1 , wherein said cell produces ascorbic acid, glucaronic acid, or glucaric acid. 
     
     
         27 . The cell of  claim 26 , wherein said cell comprises pyridine nucleotide transhydrogenase activity. 
     
     
         28 . The cell of  claim 1 , wherein said cell comprises myo-inositol oxygenase activity with a specific activity greater than 40 mg glucuronic acid per gram dry cell weight per hour. 
     
     
         29 . The cell of  claim 1 , wherein said cell comprises myo-inositol oxygenase activity such that an extract from 1×06 cells comprises a specific activity greater than 150 μg glucuronic acid formed per 10 mg total protein per 10 minutes, wherein each of said 1×10 6  cells is said cell or a progeny of said cell. 
     
     
         30 . The cell of  claim 1 , wherein said exogenous nucleic acid molecule comprises a promoter that is lactose unresponsive. 
     
     
         31 . The cell of  claim 1 , wherein said polypeptide lacks an N-terminal polyhistidine tag. 
     
     
         32 . The cell of  claim 1 , wherein said polypeptide lacks glutathione-5-transferase sequence. 
     
     
         33 . The cell of  claim 1 , wherein said exogenous nucleic acid molecule is integrated into the genome of said cell. 
     
     
         34 . A cell comprising a genetic modification that reduces myo-inositol oxygenase activity. 
     
     
         35 . The cell of  claim 34 , wherein said cell is a eukaryotic cell. 
     
     
         36 . The cell of  claim 34 , wherein said cell is a plant cell. 
     
     
         37 . The cell of  claim 34 , wherein said genetic modification comprises a nucleic acid deletion in the genome of said cell. 
     
     
         38 . The cell of  claim 34 , wherein said cell lacks said myo-inositol oxygenase activity. 
     
     
         39 . A cell comprising a genetic modification that reduces L-gulonate 3-dehydrogenase activity. 
     
     
         40 . The cell of  claim 39 , wherein said cell is a eukaryotic cell. 
     
     
         41 . The cell of  claim 39 , wherein said genetic modification comprises a nucleic acid deletion in the genome of said cell. 
     
     
         42 . The cell of  claim 39 , wherein said cell lacks said L-gulonate 3-dehydrogenase activity. 
     
     
         43 . An isolated nucleic acid molecule comprising a nucleic acid sequence at least about 50 percent identical to the sequence set forth in SEQ ID NO: 1 and wherein the polypeptide comprises myo-inositol oxygenase activity. 
     
     
         44 . The isolated nucleic acid molecule of  claim 43 , wherein said nucleic acid sequence is as set forth in SEQ ID NO: 1. 
     
     
         45 . 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 and wherein the polypeptide comprises myo-inositol oxygenase activity. 
     
     
         46 . The isolated nucleic acid molecule of  claim 45 , wherein said amino acid sequence comprises SEQ ID NO: 19.