Novel 2,5-diketo-l-gluconic acid reductases and methods of use

Abstract

Described herein are novel nucleic acids, proteins and methods that can be used to provide new catalysts with desirable traits for industrial processes. In particular, novel reductases isolated from the environment using PCR methods are described.

1 . An isolated nucleic acid molecule comprising a nucleic acid sequence which encodes a peptide having an amino acid sequence which has at least about 60% sequence identity to an amino acid sequence as set forth in  FIG. 2A  or  2 B. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1  wherein said amino acid sequence has at least about 70% sequence identity to said amino acid sequence of  FIG. 2A  or  2 B. 
     
     
         3 - 6 . (canceled) 
     
     
         7 . The nucleic acid of  claim 1  wherein said nucleic acid encodes at least a fragment of a protein, wherein said protein comprises 2,5-diketo-D-gluconic acid reductase activity. 
     
     
         8 . An expression vector comprising the nucleotide sequence of  claim 1 . 
     
     
         9 . A host cell comprising the vector of  claim 8 . 
     
     
         10 . The host cell of  claim 9  wherein said cell is  Pantoea.    
     
     
         11 . An isolated polypeptide comprising an amino acid sequence having at least about 60% identity to an amino acid sequence as set forth in  FIG. 2A  or  2 B, wherein said polypeptide comprises 2,5-diketo-D-gluconic acid reductase activity. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . The polypeptide of  claim 11  wherein said polypeptide has an amino acid sequence as set forth in  FIG. 2A . 
     
     
         15 . The polypeptide of  claim 11  wherein said polypeptide has an amino acid sequence as set forth in  FIG. 2B . 
     
     
         16 . The polypeptide of  claim 11  wherein said polypeptide has a Q at a position corresponding to position 232 of the amino acid sequence shown in  FIG. 2A . 
     
     
         17 . The polypeptide of  claim 11  wherein said polypeptide has a Q at a position corresponding to position 238 of the amino acid sequence shown in  FIG. 2A . 
     
     
         18 . The polypeptide of  claim 11  wherein said polypeptide has improved catalytic efficiency. 
     
     
         19 . The polypeptide of  claim 11  wherein said polypeptide has NADH dependent activity. 
     
     
         20 . The polypeptide of  claim 11  wherein said polypeptide has improved thermal stability. 
     
     
         21 . The polypeptide of  claim 11  wherein said polypeptide has increased solvent tolerance. 
     
     
         22 . The polypeptide of  claim 11  wherein said polypeptide has an altered pH optimum. 
     
     
         23 . A process for converting glucose to ascorbic acid comprising culturing the host cell of  claim 8  under conditions suitable for the expression of 2,5-diketo-D-gluconic acid reductase. 
     
     
         24 . A method for identifying a 2,5-diketo-L-gluconic acid reductase comprising:
 a) isolating nucleic acid molecules having homology to 2,5-diketo-L-gluconic acid reductases from uncultured microorganisms; and   b) screening said molecules for 2,5-diketo-D-gluconic acid reductase activity, wherein said molecules having 2,5-diketo-D-gluconic acid reductase activity are identified as a 2,5-diketo-L-gluconic acid reductase.