2,5-diketo-D-gluconic acid (2,5-DKG) permeases
View Patent ↗The invention provides isolated nucleic acid molecules encoding polypeptides having 2,5-DKG permease activity, and oligonucleotides therefrom. The isolated nucleic acid molecules can be expressed in appropriate bacterial cells to enhance the production of 2-KLG, which can subsequently be converted to ascorbic acid. Further provided are isolated polypeptides having 2,5-DKG permease acitivity, immunogenic peptides therefrom, and antibodies specific therefor. The invention also provides methods of identifying novel 2,5-DKG permeases.
1. An isolated nucleic acid molecule comprising a nucleotide sequence which encodes a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:4, wherein said polypeptide has 2,5-diketo-gluconate (2,5-DKG) permease activity.
2. The isolated nucleic acid molecule of claim 1 which encodes a polypeptide having the amino acid sequence of SEQ ID NO:4.
3. The isolated nucleic acid molecule of claim 1 operatively linked to a promoter of gene expression.
4. The isolated nucleic acid molecule of claim 3 , wherein said promoter is a lac promoter.
5. A vector comprising the isolated nucleic acid molecule of claim 3 .
6. A bacterial cell comprising the vector of claim 5 .
7. The bacterial cell of claim 6 , wherein said isolated nucleic acid molecule comprises a nucleotide sequence which encodes a polypeptide having an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:4.
8. The bacterial cell of claim 6 , which is of the genus Kiebsiella.
9. The bacterial cell of claim 6 , which is of the genus Pantoea.
10. The bacterial cell of claim 6 , which expresses an enzyme that catalyzes the conversion of 2.5-DKG to 2-keto-L-gulonic acid (2-KLG).
11. The bacterial cell of claim 10 , which expresses enzymes that catalyze the conversion of glucose to 2,5-DKG.
12. A method of enhancing 2-keto-L-gulonic acid (2-KLG) production, comprising a) introducing the isolated nucleic acid molecule of claim 1 into a bacterial cell which expresses an enzyme that catalyzes the conversion of 2,5-DKG to 2-KLG, b) allowing expression of the polypeptide encoded by said nucleic acid molecule end c) culturing the bacterial cell under suitable conditions to produce 2-KLG.
13. The method of claim 12 , wherein said bacterial cell further expresses enzymes that catalyze the conversion of glucose to 2,5-DKG.
14. The method of claim 12 , wherein said bacterial cell is of the genus Pantoea.
15. The method of claim 12 , further comprising converting said 2-KLG to ascorbic acid.
16. The bacterial cell of claim 6 , which is an E. coil cell.
17. The method of claim 12 , wherein the nucleic add molecule encodes a polypeptide having the sequence of SEQ ID NO: 4.
18. The method of claim 12 , wherein the nucleic acid molecule has the sequence of SEQ ID NO: 3 or a sequence having at least 95% sequence identity thereto.
19. The isolated nucleic acid of claim 1 which encodes a polypeptide having at least 98% sequence identity to SEQ ID NO: 4.
20. The method according to claim 12 , wherein the nucleic acid molecule encodes a polypeptide having at least 98% sequence identity to SEQ ID NO: 4.
21. The vector of claim 5 , comprising a spectinomycin resistance gene.