# US 11,441,163 B2 · Enzyme-catalyzed synthesis of (1S,5R)-bicyclolactone

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- Number: US 11,441,163 B2
- Document ID: US11441163B2
- Status: Granted
- Kind: B2
- Application number: 17/038,081
- Filed: 2020-09-30
- Published: 2021-11-18
- Granted: 2022-09-13
- Earliest claimed: 2020-05-14
- Assignees: Fudan University
- Applicants: Fudan University
- Inventors: Fener Chen; Kejie Zhu; Zedu Huang; Dang Cheng; Jiaqi Wang; Yuan Tao
- Web page: https://patentgrep.com/patent/US11441163B2

## CPC

- C12P 17/04: containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C}
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/0073: {with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- 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)

## Abstract

An enzyme-catalyzed synthesis of (1S,5R)-bicyclolactone. A first genetically-engineered bacterium containing Baeyer-Villiger monooxygenase gene and a second genetically-engineered bacterium containing glucose dehydrogenase gene are constructed and then suspended with culture medium to prepare a first suspension and a second suspension, respectively. The first and second suspensions are centrifuged to respectively produce a first supernatant containing Baeyer-Villiger monooxygenase and a second supernatant containing glucose dehydrogenase, which are mixed. The mixed supernatant is then mixed with a raceme of a substituted bicyclo[3.2.0]-hept-2-en-6-one, a solvent, a hydrogen donor and a cofactor to perform an asymmetric Baeyer-Villiger oxidation to produce the (1S,5R)-bicyclolactone, where an amino acid sequence of the Baeyer-Villiger monooxygenase is shown in SEQ ID NO:1.

## Claims

### Claim 1

A method of synthesizing (1S,5R)-bicyclolactone, comprising:
(1) constructing a first genetically-engineered bacterium containing a Baeyer-Villiger monooxygenase gene and a second genetically-engineered bacterium containing a glucose dehydrogenase gene;
(2) preparing a first suspension of resting cells of the first genetically-engineered bacterium and a second suspension of resting cells of the second genetically-engineered bacterium;
(3) preparing a first supernatant containing Baeyer-Villiger monooxygenase and a second supernatant containing glucose dehydrogenase; and
(4) mixing the first supernatant and the second supernatant to obtain a mixed supernatant; mixing the mixed supernatant with a raceme of a substituted bicyclo[3.2.0]-hept-2-en-6-one, a solvent, a hydrogen donor and a cofactor for an asymmetric Baeyer-Villiger oxidation to produce the (1S,5R)-bicyclolactone;
wherein the Baeyer-Villiger monooxygenase has the amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having an identity of more than 90% to SEQ ID NO:1.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleotide sequence of the Baeyer-Villiger monooxygenase gene is shown in SEQ ID NO:2, and the nucleotide sequence of the glucose dehydrogenase gene is shown in SEQ ID NO:3.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first genetically-engineered bacterium and the second genetically-engineered bacterium are constructed from Escherichia coli BL21 (DE3) containing an expression vector pET-28a.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first suspension is prepared through steps of:
inoculating the first genetically-engineered bacterium to a first kanamycin-containing medium followed by activation on a shaker;
culturing the first genetically-engineered bacterium to an optical density OD600 of 0.8-1.2, and adding a first inducer followed by continuous culture;
centrifuging a culture system of the first genetically-engineered bacterium, and collecting cells of the first genetically-engineered bacterium; and
suspending the cells of the first genetically-engineered bacterium with a first buffered solution to prepare the first suspension; and
the second suspension is prepared through steps of:
inoculating the second genetically-engineered bacterium to a second kanamycin-containing medium followed by activation on the shaker;
culturing the second genetically-engineered bacterium to an optical density OD600 of 0.8-1.2, and adding a second inducer followed by continuous culture;
centrifuging a culture system of the second genetically-engineered bacterium, and collecting cells of the second genetically-engineered bacterium; and
suspending the cells of the second genetically-engineered bacterium with a second buffered solution to prepare the second suspension.

### Claim 5 (depends on 4)

The method of claim 4, wherein the first inducer and the second inducer are both 0.05-0.8 mM isopropylthiogalactoside (IPTG); after the first inducer is added, the culture is performed at 15-25° C. for 8-24 h; and after the second inducer is added, the culture is performed at 15-25° C. for 8-24 h.

### Claim 6 (depends on 4)

The method of claim 4, wherein the first buffered solution and the second buffered solution are both a 30-300 mM sodium phosphate buffered solution.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first supernatant is prepared by sequentially ultrasonicating and centrifuging the first suspension; and the second supernatant is prepared by sequentially ultrasonicating and centrifuging the second suspension.

### Claim 8 (depends on 1)

The method of claim 1, wherein in step (4), a volume ratio of the first supernatant to the second supernatant is 3.5-29:1.

### Claim 9 (depends on 8)

The method of claim 8, wherein the volume ratio of the first supernatant to the second supernatant is 20-29:1.

### Claim 10 (depends on 1)

The method of claim 1, wherein the asymmetric Baeyer-Villiger oxidation reaction mixture comprises 1.0-100 g/L of the raceme of the substituted bicyclo[3.2.0]-hept-2-en-6-one; 5-750 g/L of the hydrogen donor and 0-0.5 mM of the cofactor; a cell concentration of the first suspension is 0.1-25 g (wet weight)/L; and a cell concentration of the second suspension is 0.1-25 g (wet weight)/L.

### Claim 11 (depends on 1)

The method of claim 1, wherein the hydrogen donor is glucose, and the cofactor is a combination of NADP+/NADPH and FADH2/FAD.

### Claim 12 (depends on 10)

The method of claim 10, wherein the hydrogen donor is glucose, and the cofactor is a combination of NADP+/NADPH and FADH2/FAD.

### Claim 13 (depends on 1)

The method of claim 1, wherein the asymmetric Baeyer-Villiger oxidation is performed at 20-40° C. and pH 6.0-8.0.

### Claim 14 (depends on 13)

The method of claim 13, wherein the asymmetric Baeyer-Villiger oxidation is performed at 20-25° C. and pH 6.5-7.5.

### Claim 15 (depends on 1)

The method of claim 1, wherein in the asymmetric Baeyer-Villiger oxidation, the solvent is selected from the group consisting of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethyl acetamide, benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene, n-hexane, cyclohexane, acetonitrile, ethyl acetate, dichloromethane, 1,2-dichloroethane, methanol, ethanol, isopropanol and a combination thereof.

### Claim 16 (depends on 1)

The method of claim 1, wherein the solvent is a mixture of toluene, 1,2-dichloroethane and a phosphate buffered solution.

### Claim 17 (depends on 16)

The method of claim 16, wherein in the solvent, a volume ratio of the toluene to the 1,2-dichloroethane to the phosphate buffered solution is 3:2:95.

### Claim 18 (depends on 16)

The method of claim 16, wherein the phosphate buffered solution is a 100-250 mM phosphate buffered solution at pH 6.5-7.5.

### Claim 19 (depends on 1)

The method of claim 1, further comprising:
after the asymmetric Baeyer-Villiger oxidation is completed, subjecting the reaction mixture to extraction with ethyl acetate, and collecting organic phases;
combining the organic phases;
washing the combined organic phase with a saturated sodium bicarbonate solution, water and a saturated sodium chloride solution in sequence; and
drying the washed organic phase with anhydrous sodium sulfate followed by vacuum distillation to produce the (1S,5R)-bicyclolactone.

### Claim 20 (depends on 19)

The method of claim 19, wherein the extraction is performed 3-4 times with an equal volume of ethyl acetate; and the washing is performed twice with the saturated sodium bicarbonate solution, once with water and once with the saturated sodium chloride solution.