Metabolically engineered bacterial strains having enhanced 2-keto-D-gluconate accumulation
OwnerTimothy Dodge, M. Rashid +1inventors
InventorsTimothy Dodge, M. Rashid, Fernando Valle
Life of this patent
Estimating the term…
Abstract
The present invention relates to a method of altering bacterial host cells to accumulate 2-keto-D-gluconic acid (2-KDG) by inactivating an endogenous membrane bound 2-keto-D-gluconate dehydrogenase (2-KDGDH), which prior to inactivation catalyzed the conversion of 2-KDG to 2,5-diketogluconate (2,5-DKG).
Claims26
A method for increasing the accumulation of 2-keto-D-gluconic acid (2-KDG) in a bacterial host cell comprising,
- a) inactivating in a bacterial host cell, which is capable of producing 2,5 diketogluconate (2,5-DKG) from the enzymatic conversion of 2-KDG in the presence of a carbon source, at least one endogenous, gene necessary for 2-keto-D-gluconate dehydrogenase (2-KDGDH) activity to obtain an altered bacterial cell;
- and
- b) culturing the altered bacterial cell under suitable culture conditions to produce 2-KDG.
A method for accumulating 2-keto-D-gluconic acid (2-KDG) in a bacterial host cell comprising,
- a) inactivating in a bacterial host cell, which is capable of producing 2,5-diketogluconate (2,5-DKG) from the enzymatic conversion of 2-KDG in the presence of glucose, an operon which encodes a 2-keto-D-gluconate dehydrogenase (2-KDGDH) enzyme to obtain an altered bacterial cell, wherein the operon includes a dehydrogenase gene and a cytochrome c gene;
- b) culturing the altered bacterial cell under suitable culture conditions to produce 2-KDG;
- and
- c) allowing the accumulation of 2-KDG in the altered bacterial cell.
An altered bacterial cell which is capable of producing 2-keto-D-gluconate from a carbon source genetically engineered to comprise a nonfunctional 2-keto-D-gluconate dehydrogenase (2-KDGDH) enzyme.
A method of increasing the availability of 2-keto-D-gluconic acid (2-KbG) in a bacterial culture comprising,
- a) inactivating in a bacterial host cell, which is capable of producing 2,5-diketogluconate (2,5-DKG) from the enzymatic conversion of 2-KDG in the presence of glucose, an operon which encodes a 2-keto-D-gluconate dehydrogenase (2-KDGDH) enzyme to obtain an altered bacterial cell, wherein the operon includes a dehydrogenase gene and a cytochrome c gene;
- b) culturing the altered bacterial cell under suitable culture conditions to produce 2-KDG.
An isolated polynucleotide encoding an enzyme having 2-keto-D-gluconate dehydrogenase (2-KDGDH) activity, wherein said enzyme is comprised of three subunits, a first subunit, subunit A having the amino acid sequence of at least 96% identity to SEQ ID NO:
- 2;
- a second subunit, subunit B having the amino acid sequence of SEQ ID NO:
- 4;
- and a third subunit, subunit C having at least 95% sequence identity to SEQ ID NO:
- 6.
Dependencies are inferred from the claim text.