Novel aldolase and production process of substituted alpha-keto acids

Abstract

4-(Indol-3-ylmethyl)-4-hydroxy-2-oxoglutarate, which is useful as an intermediate in the synthesis of monatin, may be synthesized from indole pyruvic acid and pyruvic acid (and/or oxaloacetic acid) by using a novel aldolase derived from the genus Pseudomonas, Erwinia, Flavobacterium , or Xanthomonas.

We claim: 
     
         1 . An isolated polynucleotide of following (a) or (b):
 (e) a polynucleotide having the nucleotide sequence of SEQ ID NO: 15 or the nucleotide sequence of nucleotides 398 to 1141 of SEQ ID NO: 15; or   (f) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence complementary to the full-length nucleotide sequence of SEQ ID NO: 15 or the nucleotide sequence of nucleotides 398 to 1141 of SEQ ID NO: 15, and encodes a protein having aldolase activity, wherein said stringent conditions comprise a wash step in 0.1×SSC and 0.1% SDS at 65° C.   
     
     
         2 . An isolated polynucleotide of following (c) or (d):
 (g) a polynucleotide that encodes a protein comprising the amino acid sequence of SEQ ID NO: 16; or   (h) a polynucleotide that encodes a protein having an amino acid sequence that contains a substitution, deletion, insertion, or addition of one to ten amino acid residues in the amino acid sequence of SEQ ID NO: 16, and has aldolase activity.   
     
     
         3 . A recombinant DNA produced by ligating into a vector DNA a polynucleotide selected from the group consisting of:
 (e) a polynucleotide having the nucleotide sequence of SEQ ID NO: 15 or the nucleotide sequence of nucleotides 398 to 1141 of SEQ ID NO: 15;   (f) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence complementary to the full-length nucleotide sequence of SEQ ID NO: 15 or the nucleotide sequence of nucleotides 398 to 1141 of SEQ ID NO: 15, and encodes a protein having aldolase activity, wherein said stringent conditions comprise a wash step in 0.1×SSC and 0.1% SDS at 65° C.;   (g) a polynucleotide that encodes a protein comprising the amino acid sequence of SEQ ID NO: 16; and   (h) a polynucleotide that encodes a protein having an amino acid sequence that contains a substitution, deletion, insertion, or addition of one to ten amino acid residues in the amino acid sequence of SEQ ID NO: 16, and has aldolase activity.   
     
     
         4 . An isolated cell transformed by the recombinant DNA according to  claim 3 . 
     
     
         5 . A process for producing a protein having aldolase activity comprising:
 cultivating a culture of cells according to  claim 4  in a medium; and   accumulating a protein having aldolase activity in the any one of medium and cells or both.   
     
     
         6 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide has the nucleotide sequence of SEQ ID NO: 15. 
     
     
         7 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide has the nucleotide sequence of nucleotides 398 to 1141 of SEQ ID NO: 15. 
     
     
         8 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 15, and encodes a protein having aldolase activity, wherein said stringent conditions comprise a wash step in 0.1×SSC and 0.1% SDS at 65° C. 
     
     
         9 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of nucleotides 398 to 1141 of SEQ ID NO: 15, and encodes a protein having aldolase activity, wherein said stringent conditions comprise a wash step in 0.1×SSC and 0.1% SDS at 65° C. 
     
     
         10 . The isolated polynucleotide of  claim 2 , wherein said polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 16. 
     
     
         11 . The isolated polynucleotide of  claim 2 , wherein said polynucleotide encodes a protein having an amino acid sequence that contains a substitution, deletion, insertion, or addition of one to ten amino acid residues in the amino acid sequence of SEQ ID NO: 16.