# US 11,078,473 B2 · Lysine decarboxylases having modifications at titratable amino acids

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- Number: US 11,078,473 B2
- Document ID: US11078473B2
- Status: Granted
- Kind: B2
- Application number: 16/473,215
- Filed: 2016-12-30
- Published: 2019-10-31
- Granted: 2021-08-03
- Earliest claimed: 2016-12-30
- Assignees: Cathay Biotech Inc.; Cibt America Inc.
- Applicants: Cathay Biotech Inc.; Cibt America Inc.
- Inventors: Howard Chou; Ling Chen; Wenqiang Lu; Xiucai Liu
- Web page: https://patentgrep.com/patent/US11078473B2

## CPC

- C12N 9/88: Lyases (4.)
- C12P 13/001: {Amines; Imines}
- C12Y 401/01018: Lysine decarboxylase (4.1.1.18)
- 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)}
- C12Y: ENZYMES

## Abstract

The invention provides CadA polypeptides with mutations that increase activity in alkaline pH compared to the wild-type lysine decarboxylase. The invention also provides methods of generating such mutant polypeptides, microorganisms genetically modified to overexpress the mutant polypeptides, and methods of generating such microorganisms.

## Claims

### Claim 1

A product, which is one of the following products I) through VI):
I) a CadA variant polypeptide comprising at least one amino acid substitution in a region corresponding to amino acids 455 to 483 as determined with reference to SEQ ID NO: 2, wherein the at least one amino acid substitution is at a histidine residue at position 458 and/or at an aspartic acid residue at position 457 and/or at an aspartate residue at position 460; and wherein the CadA variant polypeptide has at least 95% identity to any one of SEQ ID NOS: 2 to 5, wherein the at least one amino acid substitution at the histidine residue at position 458 is H458A/C/E/F/G/M/P/T/V/W/Y, the at least one amino acid substitution at the aspartic acid residue at position 457 is D457A/C/F/H/I/K/L/M/N/S/W/Y, and the at least one amino acid substitution at the aspartate residue at position 460 is D460F/ V/W/Y;
II) a polynucleotide comprising a nucleic acid sequence encoding a CadA variant polypeptide of I);
III) an expression vector comprising a polynucleotide of II);
IV) a genetically modified host cell comprising a CadA variant polypeptide of I);
V) a genetically modified host cell comprising a polynucleotide of II), wherein the nucleic acid sequence encoding the CadA variant polypeptide is integrated into the host cell chromosome; or
VI) a genetically modified host cell comprising an expression vector of III).

### Claim 2 (depends on 1)

A product of claim 1, which is IV) the genetically modified host cell, wherein the host cell is genetically modified to over express one or more lysine biosynthesis polypeptides.

### Claim 3 (depends on 1)

A product of claim 1, which is IV) the genetically modified host cell, wherein the host cell is a bacterium.

### Claim 4 (depends on 3)

The product of claim 3, wherein the host cell is from the genus Escherichia, Hafnia or Corynebacterium.

### Claim 5 (depends on 3)

The product of claim 3, wherein the host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Hafnia alvei.

### Claim 6 (depends on 1)

A product of claim 1, which is V) the genetically modified host cell, wherein the host cell is a bacterium.

### Claim 7 (depends on 6)

The product of claim 6, wherein the host cell is from the genus Escherichia, Hafnia or Corynebacterium.

### Claim 8 (depends on 6)

The product of claim 6, wherein the host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Hafnia alvei.

### Claim 9 (depends on 1)

A product of claim 1, which is VI) the genetically modified host cell, wherein the host cell is from the genus Escherichia, Hafnia or Corynebacterium.

### Claim 10 (depends on 9)

The product of claim 9, wherein the host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Hafnia alvei.

### Claim 11

A method, which is one of the following methods I) through III):
I) a method of producing cadaverine, the method comprising culturing a genetically modified host cell under conditions in which the CadA variant polypeptide is expressed, wherein the genetically modified host cell comprises a CadA variant polypeptide comprising at least one amino acid substitution in a region corresponding to amino acids 455 to 483 as determined with reference to SEQ ID NO: 2, wherein the at least one amino acid substitution is at a histidine residue at position 458 and/or at an aspartic acid residue at position 457 and/or at an aspartate residue at position 460; and the CadA variant polypeptide has at least 95% identity to any one of SEQ ID NOS: 2 to 5, wherein the at least one amino acid substitution at the histidine residue at position 458 is H458A/C/E/F/G/M/P/TN/W/Y, the at least one amino acid substitution at the aspartic acid residue at position 457 is D457A/C/F/H/I/K/L/M/N/S/W/Y, and the at least one amino acid substitution at the aspartate residue at position 460 is D460F/ V/W/Y;
II) a method of producing cadaverine, the method comprising culturing a genetically modified host cell under conditions in which the CadA variant polypeptide is expressed, wherein the genetically modified host cell comprises a polynucleotide comprising a nucleic acid sequence encoding a CadA variant polypeptide comprising at least one amino acid substitution in a region corresponding to amino acids 455 to 483 as determined with reference to SEQ ID NO: 2, wherein the at least one amino acid substitution is at a histidine residue at position 458 and/or at an aspartic acid residue at position 457 and/or at an aspartate residue at position 460; and the CadA variant polypeptide has at least 95% identity to any one of SEQ ID NOS: 2 to 5, wherein the nucleic acid sequence encoding the CadA variant polypeptide is integrated into the host cell chromosome, wherein the at least one amino acid substitution at the histidine residue at position 458 is H458A/C/E/F/G/M/P/T/V/W/Y, the at least one amino acid substitution at the aspartic acid residue at position 457 is D457A/C/F/H/I/K/L/M/N/S/W/Y, and the at least one amino acid substitution at the aspartate residue at position 460 is D460F/V/W/Y; or
III) a method of producing cadaverine, the method comprising culturing a genetically modified host cell under conditions in which the CadA variant polypeptide is expressed, wherein the genetically modified host cell comprises an expression vector comprising a polynucleotide comprising a nucleic acid sequence encoding a CadA variant polypeptide comprising at least one amino acid substitution in a region corresponding to amino acids 455 to 483 as determined with reference to SEQ ID NO: 2, wherein the at least one amino acid substitution is at a histidine residue at position 458 and/or at an aspartic acid residue at position 457 and/or at an aspartate residue at position 460; and the CadA variant polypeptide has at least 95% identity to any one of SEQ ID NOS: 2 to 5, wherein the at least one amino acid substitution at the histidine residue at position 458 is H458A/C/E/F/G/M/P/TN/W/Y, the at least one amino acid substitution at the aspartic acid residue at position 457 is D457A/C/F/H/I/K/L/M/N/S/W/Y, and the at least one amino acid substitution at the aspartate residue at position 460 is D460F/V/W/Y.

### Claim 12 (depends on 11)

The method of claim 11, wherein, in I), II) or III), the host cell is a bacterium.

### Claim 13 (depends on 11)

The method of claim 11, wherein, in I), II) or III), the host cell is from the genus Escherichia, Hafnia or Corynebacterium.

### Claim 14 (depends on 11)

The method of claim 11, wherein, in I), II) or III), the host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Hafnia alvei.