Enzyme-catalyzed synthesis of (1S,5R)-bicyclolactone

Patent metadata

Field	Value
Publication number	US-11441163-B2
Application number	US-202017038081-A
Country	US
Kind code	B2
Filing date	Sep 30, 2020
Priority date	May 14, 2020
Publication date	Sep 13, 2022
Grant date	Sep 13, 2022

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.

First claim

What is claimed is:

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.

2. 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.

3. 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.

4. 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 OD 600 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 OD 600 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.

5. 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.

6. 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.

7. 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.

8. 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.

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

10. 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.

11. The method of claim 1 , wherein the hydrogen donor is glucose, and the cofactor is a combination of NADP + /NADPH and FADH 2 /FAD.

12. The method of claim 10 , wherein the hydrogen donor is glucose, and the cofactor is a combination of NADP + /NADPH and FADH 2 /FAD.

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

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

15. 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.

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

17. 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.

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

19. 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.

20. 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.

Assignees

• Univ Fudan

Inventors

• Chen Fener
• Zhu Kejie
• Huang Zedu
• Cheng Dang
• Wang Jiaqi
• Tao Yuan

Classifications

• C12P17/04 Primary

  containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C} · CPC title

• C12N9/0073 Primary

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title

• C12Y101/9901

  Glucose dehydrogenase (acceptor) (1.1.99.10) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12Y114/13022

  Cyclohexanone monooxygenase (1.14.13.22) · CPC title