# US 2013/0157342 A1 · Novel aldolase and production process of substituted alpha-keto acids

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- Number: US 2013/0157342 A1
- Document ID: US20130157342A1
- Status: Published application
- Kind: A1
- Application number: 13/533,317
- Filed: 2012-06-26
- Published: 2013-06-20
- Earliest claimed: 2002-08-26
- Assignees: Ajinomoto Co., Inc.
- Inventors: Masakazu SUGIYAMA; Kunihiko Watanabe; Shigeru Kawahara
- Web page: https://patentgrep.com/patent/US20130157342A1

## CPC

- C12N 9/88: Lyases (4.)
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 7/50: having keto groups, e.g. 2-ketoglutaric acid
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 17/10: Nitrogen as only ring hetero atom
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## 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.

## Claims

### 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.

### Claim 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.

### Claim 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.

### Claim 4 (depends on 3)

An isolated cell transformed by the recombinant DNA according to claim 3.

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 1)

The isolated polynucleotide of claim 1, wherein said polynucleotide has the nucleotide sequence of SEQ ID NO: 15.

### Claim 7 (depends on 1)

The isolated polynucleotide of claim 1, wherein said polynucleotide has the nucleotide sequence of nucleotides 398 to 1141 of SEQ ID NO: 15.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 2)

The isolated polynucleotide of claim 2, wherein said polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 16.

### Claim 11 (depends on 2)

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.