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 1×106 cells comprises a specific activity greater than 150 μg glucuronic acid formed per 10 mg total protein per 10 minutes, where each of the 1×106 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.

المصدر

DOI: 10.6084/m9.figshare.5104873.v1براءة الاختراع: US07326549B2uspto-grants-2008_02