# US 10,308,945 B2 · Bacterium producing monophosphoryl lipid A and method of producing monophosphoryl lipid A by using bacterium

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- Number: US 10,308,945 B2
- Document ID: US10308945B2
- Status: Granted
- Kind: B2
- Application number: 15/378,681
- Filed: 2016-12-14
- Published: 2017-07-06
- Granted: 2019-06-04
- Earliest claimed: 2016-01-06
- Assignees: Korea Institute of Science and Technology
- Applicants: Korea Institute of Science and Technology
- Inventors: Hak Suk Chung; Eun Gyeong Yang; Dohyeon Hwang
- Web page: https://patentgrep.com/patent/US10308945B2

## CPC

- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12P 19/12: Disaccharides
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)}
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

A bacterium producing monophosphoryl lipid A (MLA) comprising a genetic modification that increases expression of a gene encoding LpxE polypeptide and a method of producing MLA are provided. According to the present invention, MLA may be produced in a simple manner without acid hydrolysis and/or base hydrolysis.

## Claims

### Claim 1

A recombinant bacterium that produces monophosphoryl lipid A (MLA) not conjugated to a 2-keto-3-deoxy-D-manno-octulosonate (Kdo) moiety,
wherein the recombinant bacterium is transformed with at least one exogenous polynucleotide encoding a Lipid A-1 phosphatase (LpxE) polypeptide,
wherein the recombinant bacterium is modified by 1) transformation with at least one of an exogenous polynucleotide encoding a lipid A biosynthesis lauroyltransferase (LpxL) polypeptide, an exogenous polynucleotide encoding a lipid A biosynthesis myristoyltransferase (LpxM) polypeptide, or a combination thereof, or 2) a genetic modification in a 5′-non-coding sequence or a 3′-non-coding sequence for an endogenous polynucleotide encoding a LpxL polypeptide, an endogenous polynucleotide encoding a LpxM polypeptide, or a combination thereof, wherein the recombinant bacterium has increased expression of the polynucleotide encoding the LpxL polypeptide, the polynucleotide encoding the LpxM polypeptide, or a combination thereof as compared to a corresponding recombinant bacterium without the modification of 1) and 2), and
wherein the recombinant bacterium has a disruption in a gene encoding a polypeptide involved in Kdo biosynthesis selected from a gene encoding a KdtA polypeptide, a gene encoding a KdsB polypeptide, a gene encoding a KdsC polypeptide, a gene encoding a KdsA polypeptide, a gene encoding a GutQ polypeptide, a gene encoding a KpsF polypeptide, a gene encoding a KpsU polypeptide, a gene encoding a KdsD polypeptide, or a combination thereof.

### Claim 2 (depends on 1)

The recombinant bacterium of claim 1, wherein the MLA comprises from 2 acyl chains to 7 acyl chains.

### Claim 3 (depends on 1)

The recombinant bacterium of claim 1, wherein the MLA comprises 1-dephospho-lipid A, 1-dephospho-tetra-acylated lipid A, 1-dephospho-penta-acylated lipid A, or a combination thereof.

### Claim 4 (depends on 1)

The recombinant bacterium of claim 1, wherein the LpxE polypeptide belongs to EC 3.1.3.-.

### Claim 5 (depends on 1)

The recombinant bacterium of claim 1, wherein the LpxE polypeptide is a polypeptide having Lipid A-1 phosphatase activity and comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 17.

### Claim 6 (depends on 3)

The recombinant bacterium of claim 3, wherein the LpxL polypeptide belongs to EC 2.3.1.241 and the LpxM polypeptide belongs to EC 2.3.1.243.

### Claim 7 (depends on 3)

The recombinant bacterium of claim 3, wherein the LpxL polypeptide is a polypeptide having lipid A biosynthesis lauroyltransferase activity and comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 and the LpxM polypeptide is a polypeptide having lipid A biosynthesis myristoyltransferase activity and comprising 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 5.

### Claim 8 (depends on 1)

The recombinant bacterium of claim 1, wherein the bacterium is a Gram-negative bacterium.

### Claim 9 (depends on 1)

The recombinant bacterium of claim 1, wherein the bacterium is selected from the group consisting of Escherichia genus bacterium, Shigella genus bacterium, Salmonella genus bacterium, Campylobacter genus bacterium, Neisseria genus bacterium, Haemophilus genus bacterium, Aeromonas genus bacterium, Francisella genus bacterium, Yersinia genus bacterium, Klebsiella genus bacterium, Bordetella genus bacterium, Legionella genus bacterium, Corynebacterium genus bacterium, Citrobacter genus bacterium, Chlamydia genus bacterium, Brucella genus bacterium, Pseudomonas genus bacterium, Helicobacter genus bacterium, Burkholderia genus bacterium, Agrobacterium genus bacterium, Chlorobium genus bacterium, Rhodospirillum genus bacterium, Magnetospirillum genus bacterium, Chlorobaculum genus bacterium, Pelodictyon genus bacterium, Pseudovibro genus bacterium, Phaeospirillum genus bacterium, Syntrophobacter genus bacterium, Bradyrhizobium genus bacterium, Porphyromonas genus bacterium, Rhizobium genus bacterium, Mesorhizobium genus bacterium, Vibrio genus bacterium, Ralstonia genus bacterium, Limnohabitans genus bacterium, and Thermodesulfobacterium genus bacterium.

### Claim 10 (depends on 1)

The recombinant bacterium of claim 1, wherein the recombinant bacterium has a disruption in a gene selected from a gene encoding a LpxT polypeptide, a gene encoding a PagP polypeptide, a gene encoding a KdtA polypeptide, or a combination thereof.

### Claim 11 (depends on 1)

A method of producing MLA, the method comprising:
culturing the recombinant bacterium of claim 1 to obtain a culture comprising MLA; and
isolating MLA from the culture.

### Claim 12 (depends on 11)

The method of claim 11, wherein isolating the MLA from the culture comprises isolating MLA from the recombinant bacterium.

### Claim 13 (depends on 11)

The method of claim 11, wherein the culturing comprises culturing in batch, fed-batch, or continuous mode.

### Claim 14 (depends on 11)

The method of claim 11, wherein the MLA comprises 1-dephospho-lipid A, 1-dephospho-penta-acylated lipid A, 1-dephospho-tetra-acylated lipid A, or a combination thereof.