# US 12,760,342 B2 · Bacillus subtilis genetically engineered bacterium for producing tagatose and method for preparing tagatose

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- Number: US 12,760,342 B2
- Document ID: US12760342B2
- Status: Granted
- Kind: B2
- Application number: 18/260,333
- Filed: 2021-07-28
- Published: 2024-08-01
- Granted: 2026-10-13
- Earliest claimed: 2021-01-05
- Assignees: Tianjin Yeahe Biotechnology Co., Ltd.
- Applicants: Tianjin Yeahe Biotechnology Co., Ltd.
- Inventors: Yanhe Ma; Ting Shi; Yunjie Li; Pingping Han; Yuan Li
- Web page: https://patentgrep.com/patent/US12760342B2

## CPC

- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/90: Isomerases (5.)
- C12N 11/00: Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N 15/75: for Bacillus
- C12Y 204/01001: Phosphorylase (2.4.1.1)
- C12Y 301/03: Phosphoric monoester hydrolases (3.1.3)
- C12Y 501/03: acting on carbohydrates and derivatives (5.1.3)
- C12Y 503/01009: Glucose-6-phosphate isomerase (5.3.1.9)
- C12Y 504/02002: Phosphoglucomutase (5.4.2.2)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Provided are a Bacillus subtilis genetically engineered bacterium for producing tagatose and a method for preparing tagatose. The genetically engineered bacterium comprises constructing thermostable α-glucan phosphorylases, thermostable glucose phosphomutases, thermostable glucose phosphate isomerases, thermostable 6-tagatose phosphate epimerases, and thermostable 6-tagatose phosphate phosphatases which are independently expressed or co-expressed. The usage of the genetically engineered bacterium can effectively convert starch into tagatose. Compared with existing methods for producing tagatose, the method has advantages such as suitability for whole-cell recycling, high safety, high yield, simple production process, low cost, and easiness in large-scale preparation.

## Claims

### Claim 1

A Bacillus subtilis genetically engineered bacterium for producing tagatose, characterized in that said genetically engineered bacterium is a Bacillus subtilis genetically engineered bacterium co-expressing an α-glucan phosphorylase gene from Thermococcus kodakarensis, a glucose phosphomutase gene from Thermococcus kodakarensis, a glucose phosphate isomerase gene from Thermus thermophilus, a 6-tagatose phosphate epimerase gene from Thermoanaerobacter indiensis, and a 6-tagatose phosphate phosphatase gene from Archaeoglobus fulgidus, or a mixture of Bacillus subtilis genetically engineered bacteria respectively expressing an α-glucan phosphorylase gene from Thermococcus kodakarensis, a glucose phosphomutase gene from Thermococcus kodakarensis, a glucose phosphate isomerase gene from Thermus thermophilus, a 6-tagatose phosphate epimerase gene from Thermoanaerobacter indiensis, and a 6-tagatose phosphate phosphatase gene from Archaeoglobus fulgidus.

### Claim 2 (depends on 1)

The genetically engineered bacterium according to claim 1, characterized in that a starting strain of said Bacillus subtilis is a protease-knockout strain of Bacillus subtilis strain.

### Claim 3 (depends on 1)

The genetically engineered bacterium according to claim 1, characterized in that said α-glucan phosphorylase, glucose phosphomutase, glucose phosphate isomerase, 6-tagatose phosphate epimerase, and 6-tagatose phosphate phosphatase are respectively thermostable α-glucan phosphorylase, thermostable glucose phosphomutase, thermostable glucose phosphate isomerase, thermostable 6-tagatose phosphate epimerase, and thermostable 6-tagatose phosphate phosphatase, and wherein being thermostable refers to having enzyme activity above 40 °C.

### Claim 4 (depends on 1)

The genetically engineered bacterium according to claim 1, characterized in that endogenous uracil phosphoribosyltransferase gene, α-amylase gene, sporulating RNA polymerase σF factor gene, and surface-active peptide synthase subunit 3 gene are all inactivated or knocked out in said genetically engineered bacterium.

### Claim 5

An expression vector, characterized in that it comprises an α-glucan phosphorylase gene from Thermococcus kodakarensis, a glucose phosphomutase gene from Thermococcus kodakarensis, a glucose phosphate isomerase gene from Thermus thermophilus, a 6-tagatose phosphate epimerase gene from Thermoanaerobacter indiensis, and a 6-tagatose phosphate phosphatase gene from Archaeoglobus fulgidus and is capable of co-expressing these genes.

### Claim 6 (depends on 1)

A method for producing tagatose by utilizing a whole cell of the genetically engineered bacterium according to claim 1 to catalyze starch, comprising the steps of
(1) fermenting said Bacillus subtilis genetically engineered bacterium to obtain whole cells;
(2) subjecting the whole cells of Bacillus subtilis obtained in step (1) to a cell membrane permeability treatment to obtain permeable whole cells;
(3) catalyzing starch by utilizing the permeable whole cells obtained in step (2) to produce tagatose, wherein for co-expressing whole cells of Bacillus subtilis engineered bacterium, the whole cells are used directly for catalysis, and for whole cells of Bacillus subtilis engineered bacterium expressing various enzymes separately, the whole cells are mixed for catalysis.

### Claim 7 (depends on 6)

The method according to claim 6, characterized in further comprising immobilizing the Bacillus subtilis permeable whole cells obtained in step (2) to obtain immobilized whole cells, or a mixture of immobilized whole cells, which can then be used for catalysis.

### Claim 8 (depends on 6)

The method according to claim 6, characterized in that the preparation of whole cells of said step (1) is obtained by means of a fermentation method suitable for expression of exogenous protein.

### Claim 9 (depends on 6)

The method according to claim 6, characterized in that the cell membrane permeability treatment of said step (2) is obtained by heat treatment, addition of organic solvents or addition of surfactant treatment.

### Claim 10 (depends on 9)

The method according to claim 9, characterized in that said organic solvent is selected from acetone, acetonitrile; said surfactant is selected from cetyl trimethyl ammonium bromide, Tween-80.

### Claim 11 (depends on 9)

The method according to claim 9, characterized in that said heat treatment is carried out at a temperature of 45 to 100 °C and a heat treatment time of 10 to 100 min; and cell concentration at the time of treatment is OD600=10 to 300.

### Claim 12 (depends on 11)

The method according to claim 11, characterized in that said heat treatment is carried out at a temperature of 70 to 80 °C and a heat treatment time of 50 to 70 min; and cell concentration at the time of treatment is OD600=30 to 150.

### Claim 13 (depends on 9)

The method according to claim 9, characterized in that said heat treatment is carried out in a buffer selected from HEPES buffer, phosphate buffer, Tris buffer, and acetate buffer.

### Claim 14 (depends on 6)

The method according to claim 6, characterized in that concentration of the substrate starch in the reaction system catalyzed in step (3) is 50 to 300 g/L; reaction conditions are: 0.5 to 96 h at pH 5.0 to 8.0 and 40 to 80 °C.

### Claim 15 (depends on 14)

The method according to claim 14, characterized in that concentration of the substrate starch in the reaction system catalyzed in step (3) is 100 to 200 g/L; reaction conditions are: 12 to 60 h at pH 6.5 to 7.5 and 45 to 75 °C.

### Claim 16 (depends on 6)

The method according to claim 6, characterized in that the reaction catalyzed in said step (3) is carried out in a buffer selected from HEPES buffer, phosphate buffer, Tris buffer, and acetate buffer.

### Claim 17 (depends on 6)

The method according to claim 6, characterized in that for Bacillus subtilis engineered bacterium permeable whole cells expressing various enzymes separately, the mixture is made by mixing permeable whole cells expressing α-glucan phosphorylase, permeable whole cells expressing glucose phosphomutase, permeable whole cells expressing glucose phosphate isomerase, permeable whole cells expressing 6-tagatose phosphate epimerase, and permeable whole cells expressing 6-tagatose phosphate phosphatase in a ratio of (0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-10).

### Claim 18 (depends on 6)

The method according to claim 6, characterized in that said permeable whole cells are immobilized by resuspending said permeable whole cells with sodium phosphate or potassium phosphate buffer, adding inorganic clay and stirring well; subsequently adding polyethyleneimine aqueous solution for flocculation, then adding cross-linking agent for cross-linking; then filtering to obtain a filter cake layer which is washed with deionized water and squeezed to prepare into pellets, dried to obtain the immobilized whole cells.

### Claim 19 (depends on 18)

The method according to claim 18, characterized in that said inorganic clay is selected from montmorillonite, diatomaceous earth, kaolin and bentonite; said crosslinker is selected from glutaraldehyde, trihydroxymethylphosphine, N,N-methylenebisacrylamide, epichlorohydrin and genipin.