# US 10,501,503 B2 · Modified membrane permeability

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- Number: US 10,501,503 B2
- Document ID: US10501503B2
- Status: Granted
- Kind: B2
- Application number: 15/774,820
- Filed: 2015-11-09
- Published: 2019-01-31
- Granted: 2019-12-10
- Earliest claimed: 2015-11-09
- Assignees: Cathay Biotech Inc.; Cibt America Inc.
- Applicants: Cathay Biotech Inc.; CIBT America Inc.
- Inventors: Howard Chou; Lijun Yu; Xiucai Liu
- Web page: https://patentgrep.com/patent/US10501503B2

## CPC

- C07K 14/245: Escherichia (G)
- C12N 9/88: Lyases (4.)
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12P 13/001: {Amines; Imines}
- C12P 13/08: Lysine; Diaminopimelic acid; Threonine; Valine
- C12Y 401/01018: Lysine decarboxylase (4.1.1.18)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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

Provided are microorganisms genetically modified to overexpress porin polypeptides to enhance the production of lysine and lysine derivatives by the microorganism. Also provided are methods of generating such microorganism, and methods of producing lysine and lysine derivatives using the genetically modified microorganisms.

## Claims

### Claim 1

A genetically modified host cell comprising a heterologous nucleic acid encoding an OMP porin polypeptide, wherein the host cell overexpresses the OMP porin polypeptide and has increased production of an amino acid lysine and cadaverine, and wherein the OMP porin polypeptide is an OmpA, OmpC, OmpF, OmpX, OmpE, OmpG, or OmpW porin polypeptide set forth as SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16 respectively; or wherein the OMP porin polypeptide is a conservatively modified porin polypeptide variant of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16 that encodes the mature OMP porin polypeptide.

### Claim 2 (depends on 1)

The genetically modified host cell of claim 1, wherein the heterologous nucleic acid encoding the OMP porin polypeptide is encoded by an expression vector introduced into the cell, wherein the expression vector comprises the heterologous nucleic acid operably linked to a promoter; and/or
wherein the OMP porin polypeptide is endogenous to the host cell; and/or
wherein the heterologous nucleic acid is integrated into the host chromosome; and/or
wherein the host cell overexpresses a lysine decarboxylase; and/or
wherein the host cell overexpresses one or more lysine biosynthesis polypeptides; and/or
wherein the host cell overexpresses a TetA polypeptide.

### Claim 3 (depends on 1)

The genetically modified host cell of claim 1, wherein the host cell is of the genus Escherichia, Hafnia, or Corynebacterium; and/or wherein the host cell is Escherichia coli, Hafnia alvei, or Corynebacterium glutamicum; and/or wherein the host cell is Escherichia coli or Hafnia alvei.

### Claim 4 (depends on 3)

The genetically modified host cell of claim 3, wherein the Omp porin polypeptide is an OmpA, OmpC, OmpF, or OmpW polypeptide.

### Claim 5 (depends on 4)

The genetically modified host cell of claim 4, wherein the host cell overexpresses a lysine decarboxylase polypeptide; and/or wherein the host cell overexpresses a LysC, DapA, LysA, Asd, DapB, AspC, or TetA polypeptide.

### Claim 6 (depends on 1)

A method of producing an amino acid, the method comprising culturing a host cell of claim 1 under conditions in which the OMP porin polypeptide is overexpressed.