An ene reductase mutant and its use in the production of a boviguat intermediate

Abstract

The application discloses an ene reductase mutant and application thereof in production of a boviguatin intermediate, and belongs to the technical field of biological catalysis. The ene reductase mutant is obtained by Y76I, Y76M or N290P mutation of wild-type ene reductase with an amino acid sequence as shown in SEQ ID NO. 1, and the amino acid sequence of the ene reductase mutant is as shown in any one of SEQ ID NO. 3-5. The ene reductase mutant provided by the application can be used for catalyzing 4-propyl furan-2(5 H ) ketone to generate a boviguatin intermediate ( R ) 4-propyl dihydrofuran-2( 3H ) ketone, the ee value of the product reaches more than 99%, and the application provides a potential biological catalyst for enzymatic synthesis of a boviguatin pharmaceutical intermediate.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, and in particular to an olefin reductase mutant and its application in the production of buvastan intermediates. Background Technology

[0002] Brivaracetam, chemical name (2 S )-2-[(4 R [2-oxo-4-propylpyrrolidone-1-yl]butyramide is a new-generation antiepileptic drug that was approved by the US FDA on February 19, 2016, for adjunctive treatment of partial seizures (with or without secondary generalized seizures) in patients aged 16 years and older with epilepsy. Brivaracetam is chemically similar to levetiracetam but possesses significantly optimized pharmacological properties: it exerts its effect through highly selective binding to synaptic vesicle protein 2A (SV2A), with an affinity 15-30 times that of levetiracetam, allowing for a clinical dose reduction of approximately 10-fold. In addition to its therapeutic uses, brivaracetam is also widely used in neuroscience research and drug development.

[0003] Boisstein's structure contains two chiral centers (2 S， 4 R ). Among them 2 S Chiral centers can be obtained through chiral raw materials (2 S The introduction of 2-aminobutyramide, and 4 R The construction of chiral centers is achieved through ( R )-4-propyldihydrofuran-2(3 H )-ketone is introduced as an intermediate.

[0004] Currently, various chemical synthesis methods have been reported for the preparation of buvasidan intermediates. R )-4-propyldihydrofuran-2(3 H )-ketone. Chromatographic separation begins with the condensation of pentanal and glyoxylic acid, followed by... S After reaction and hydrogen reduction of )-diaminobutyramide, it was separated by chromatographic column chromatography to obtain ( R )-4-propyldihydrofuran-2(3 H )-ketones, although this route uses inexpensive and readily available raw materials and has simple steps, the yield is extremely low, only 4.6%; the chiral source synthesis method uses ( R Using 2-aminovaleric acid as a raw material, it is synthesized through steps such as substitution, reduction, protection, and cyclization. R )-4-propyldihydrofuran-2(3 H )-ketones, with relatively high yields, reaching 43.8%; the asymmetric synthesis method uses p-toluenesulfinic acid as a raw material, and synthesizes intermediates through multiple steps such as esterification, Grignard reaction, and Pummer rearrangement, and then combines with ( SThe product was obtained by the reaction of 2-diaminobutyramide, but this route involved numerous steps, a long reaction time, and a yield of only 11.4%. The enzymatic resolution method used dimethyl 2-propylmalonate as a raw material. After substitution and decarboxylation reactions, it was catalytically resolved using Bacillus subtilis protease, followed by reduction and cyclization to obtain (…). R )-4-propyldihydrofuran-2(3 H )-ketone, this route is relatively short, but the yield is only 42%, and the product ee The value is 97%.

[0005] To avoid the problems of harsh reaction conditions, cumbersome synthetic routes, and difficulties in separating chiral intermediates in chemical synthesis, stereoselective synthesis has become a feasible approach. In recent years, the technique of biocatalytic asymmetric reduction and activation of pre-chiral double bonds has shown great potential due to its advantages such as high stereoselectivity, mild reaction conditions, and few byproducts. CN119432782 discloses a method derived from... Bacillus halotolerans A complex enzyme composed of olefin reductase, carbonyl reductase, and glucose dehydrogenase, using compound 5-hydroxy-4-n-propyl-2-furanone as a substrate, undergoes double bond reduction and hydroxyl group removal reactions to yield compound ( R )-4-propyldihydrofuran-2(3 H )-ketone, with a product selectivity of 93.0%, can be further obtained by diastereomeric crystallization. ee A product with a purity greater than 99.8%. CN119876055A discloses a product derived from... Novosphingobium nitrogenifigens The olefin reductase mutant W67H catalyzes 4-propylfuran-2(5-) H )-Ketone synthesis R )-4-propyldihydrofuran-2(3 H )-ketone, product ee The value is 96.9%. CN116948997A and CN113444702A respectively disclose a method derived from... Vanderwaltozyma polyspora olefin reductase and a type derived from Saccharomyces pastorianus The olefin reductase OYE1, and its mutants can selectively catalyze 4-propylfuran-2 ( 5H )-Keto formation ( R )-4-propyldihydrofuran-2(3 H )-ketone, ee The value is as high as 99%. This demonstrates the effectiveness of utilizing bioasymmetric synthesis (…). R )-4-propyldihydrofuran-2(3 H The )-ketone method can achieve excellent stereoselectivity, but its catalytic efficiency is generally low, which limits its application.

[0006] In summary, the use of olefin reductase to catalyze substrate synthesis ( R)-4-propyldihydrofuran-2(3 H )-ketone resolved the 4 in the buvasitial structure R The traditional synthesis of chiral centers faces the challenge of poor selectivity, but the low catalytic efficiency of enzymes remains a problem, making low-cost, large-scale industrial production currently difficult. Therefore, the development of 4-propylfuran-2 ( 5H )-ketones possess highly efficient olefin reductases, which are of great significance for achieving the green and efficient synthesis of buvasidan intermediates. Summary of the Invention

[0007] The purpose of this invention is to provide an olefin reductase mutant and its application in the production of brivacertan intermediates. Three olefin reductase mutants are obtained by mutating wild-type olefin reductase with Y76I, Y76M, or N290P, and these mutants are used to synthesize brivacertan intermediates. R )-4-propyldihydrofuran-2(3 H The conversion rate of (R)-4-propyldihydrofuran-2-(-ketone) can reach up to 68.5%, and the ee value is greater than 99%. This invention solves the current problem of preparing (R)-4-propyldihydrofuran-2-(-ketone) with olefin reductase. 3H The problems of low efficiency, high cost, and limited industrial application in the )-ketone process provide a foundation for subsequent modification and application.

[0008] To achieve the above objectives, the present invention provides the following solution: The present invention provides an olefin reductase mutant, which is obtained by mutating the wild-type olefin reductase with amino acid sequence as shown in SEQ ID NO.1 by Y76I, Y76M or N290P, and the amino acid sequence of the olefin reductase mutant is shown in any one of SEQ ID NO.3-5.

[0009] The present invention also provides a gene encoding the olefin reductase mutant described above.

[0010] The present invention also provides recombinant plasmids containing the aforementioned gene.

[0011] The present invention also provides recombinant engineered bacteria comprising the recombinant plasmid described above.

[0012] Preferably, the recombinant engineered bacteria further includes a gene encoding glucose dehydrogenase; the accession number of this glucose dehydrogenase in NCBI is WP_412052519.1. In this embodiment of the invention, the pACYC184-gdh recombinant plasmid pACYC184-gdh and the mutant recombinant vector are jointly introduced into host Escherichia coli BL21(DE3) competent cells to obtain recombinant engineered bacteria containing olefin reductase mutant and glucose dehydrogenase.

[0013] This invention also provides the described olefin reductase mutant, the described gene, the described recombinant plasmid, or the described recombinant engineered bacteria in the production of buvasidan intermediates ( R )-4-propyldihydrofuran-2( 3H Applications of )-ketones.

[0014] This invention also provides the described olefin reductase mutant, the described gene, the described recombinant plasmid, or the described recombinant engineered bacteria in the preparation of buvasidan intermediates ( R )-4-propyldihydrofuran-2( 3H Application of )-ketones in catalysts.

[0015] This invention also provides a method for producing an intermediate for buvacertane ( R )-4-propyldihydrofuran-2( 3H The method for ketones includes the following steps: After fermenting the recombinant engineered bacteria, the bacterial cells were collected by centrifugation and freeze-dried to obtain crude enzyme powder; Using the crude enzyme powder as a catalyst, 4-propylfuran-2(5 H The reaction was carried out using )-ketone as a substrate. After the reaction was completed, the mixture was separated and purified to obtain the ()-ketone. R )-4-propyldihydrofuran-2( 3H )-ketone.

[0016] Preferably, the reaction system also includes NADP. + The reaction mixture was prepared with glucose, and the reaction medium was PBS buffer. The reaction conditions were shaking at 200 rpm and 37°C for 12 hours.

[0017] The present invention also provides an intermediate for the production of buvacertan ( R )-4-propyldihydrofuran-2( 3H The catalyst for )-ketone, wherein the catalyst is the recombinant engineered bacteria.

[0018] The present invention discloses the following technical effects: This invention obtained three mutants, Y76I, Y76M, and N290P, by site-directed mutagenesis of wild-type olefin reductase. Experiments showed that these mutants catalyze 4-propylfuran-2-( 5H )-keto synthesis of key intermediates for buvasidan ( R )-4-propyldihydrofuran-2( 3HWhen synthesizing )-ketones, the product ee values ​​are all greater than 99%, exhibiting excellent stereoselectivity. Compared with the wild-type enzyme (conversion rate 45.7%), the mutants Y76I, Y76M, and N290P show increased conversion rates of 58.3%, 61.9%, and 68.5%, respectively, demonstrating significantly improved catalytic efficiency. The mutants provided by this invention offer excellent candidate enzymes for the green, efficient, and low-cost biocatalytic synthesis of buvascarb intermediates, with promising prospects for industrial application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The substrate is 4-propylfuran-2 ( 5H )-ketone and product ( R )-4-propyldihydrofuran-2( 3H Gas chromatogram of )-ketone; Figure 2 For products ( R )-4-propyldihydrofuran-2( 3H Stereoselective gas chromatogram of )-ketone. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1: Construction of recombinant engineered bacteria for olefin reductase OpER The target fragment was synthesized from the olefin reductase OpER gene (gene sequence shown in SEQ ID NO.2, amino acid sequence shown in SEQ ID NO.1). The target fragment was inserted into the expression plasmid pET28a(+) to obtain pET28a(+)-OpER. The primer sequences used are shown in Table 1.

[0027] Table 1 Primer information for constructing OpER gene expression strains The PCR amplification system was as follows (50 μL): template DNA 1-5 ng, PrimeSTAR Max Premix (2×) 25 μL, upstream and downstream primers 1.5 μL each, and sterile distilled water to make up to 50 μL.

[0028] PCR reaction parameters: (1) 98℃ pre-denaturation for 2 minutes; (2) 95℃ denaturation for 10 seconds; (3) 55℃ annealing for 5 seconds; (4) 72℃ extension for 2 minutes, repeat steps (2)-(4) 30 times; (5) 72℃ complete extension for 5 minutes, store at 16℃.

[0029] After observing the target band by 1.0% agarose gel electrophoresis, the remaining PCR product was purified to obtain a purified linearized plasmid. 50 ng of the purified linearized plasmid was then used for seamless cloning.

[0030] The seamless cloning system consisted of (20 μL): 50 ng of linearized plasmid, 5 μL of Seamless Cloning Mix (2×), and 10 μL made up with sterile distilled water. The seamless cloning reaction was carried out in a 50°C water bath for 15 minutes.

[0031] 5 μL of the seamless cloning product was heat-transformed into E. coli BL21(DE3) competent cells. After recovery, the cells were plated on LB agar plates containing kanapenem and cultured overnight. Subsequently, 5-10 clones were picked and cultured in LB medium at 37°C for 8 h. The bacterial culture was then sequenced to verify the results, thus obtaining the recombinant engineered olefin reductase strain pET28a-OpER.

[0032] Example 2: Construction of olefin reductase mutant Point mutations were performed at tyrosine residue at position 76 and asparagine residue at position 290 in the amino acid sequence of the wild-type olefin reductase to obtain the Y76I, Y76M, and N290P mutants. Using the recombinant plasmid pET28a-OpER containing the target fragment constructed above as a template, the mutated plasmids were amplified by PCR. The primer sequences used are shown in Table 2.

[0033] Table 2 Primer information for constructing mutants Following the procedures described in Example 1, recombinant vectors pET28a-OpER / Y76I, pET28a-OpER / Y76M, and pET28a-OpER / N290P containing the olefin reductase mutant gene were obtained. These vectors were then transformed into E. coli BL21(DE3) competent cells, and after revival, they were plated on LB plates containing kanamycin and cultured overnight.

[0034] Subsequently, 5-10 clones were selected and cultured in LB medium at 37°C for 8 hours. The bacterial culture was then sequenced for verification to obtain the olefin reductase mutant recombinant engineered bacteria E. coli BL21(DE3) / pET28a-OpER / Y76I, E. coli BL21(DE3) / pET28a-OpER / Y76M, and E. coli BL21(DE3) / pET28a-OpER / N290P. The amino acid sequences of the olefin reductase mutants are shown in SEQ ID NO. 3-5.

[0035] Example 3: Preparation and Induction Expression of Double-Plasmid Genetically Engineered Bacteria The mutant recombinant vector obtained in Example 2 and the glucose dehydrogenase recombinant vector pACYC184-gdh (which has been disclosed in the literature, see: Baoqi Zhang, Han Du, Yanqiu Zheng, Jiale Sun, Yu Shen, Jinping Lin and Dongzhi Wei. Design and engineering of whole-cell biocatalyst for efficient synthesis of (R)-citronellal[J]. Microbial Biotechnology, 2022, 15(5), 1486–1498) were cultured overnight in LB liquid medium containing the corresponding resistance. After plasmid extraction, the cells were introduced into host Escherichia coli BL21(DE3) competent cells and cultured on LB plates containing kanamycin and chloramphenicol. Single colonies were picked and cultured in LB medium containing kanamycin and chloramphenicol to finally obtain recombinant engineered bacteria containing olefin reductase mutant and glucose dehydrogenase.

[0036] The recombinant engineered bacteria containing the olefin reductase mutant and glucose dehydrogenase were inoculated into LB liquid medium containing the screening antibiotic and cultured at 37°C for 12 h. The seed culture was then inoculated into 200 mL of LB liquid medium containing the screening antibiotic and cultured at 37°C until the bacterial OD... 600 Once the pH reaches 0.6, add IPTG to a final concentration of 0.2 mM and incubate at 20°C for 12 hours. Centrifuge the culture medium, collect the cells, and obtain resting cells. Freeze-dry the collected resting cells to obtain freeze-dried crude enzyme powder.

[0037] Example 4: Activity and stereoselectivity of wild-type OpER and its mutants Using the freeze-dried crude enzyme powder from Example 3 as a catalyst, it was used to catalyze 4-propylfuran-2(5 H The reduction reaction of )-ketones was used to detect the catalytic activity and stereoselectivity of wild-type OpER and its mutants.

[0038] The catalytic reaction system consisted of: 100 mM phosphate buffer (pH 7.5), 20 g / L crude enzyme powder, and 8 mM 4-propylfuran-2 (5... H )-ketone, 0.2mM NADP + Add 16mM glucose. The prepared reaction solution was shaken at 200rpm and 37℃ for 12h.

[0039] After the reaction was completed, the mixture was extracted with an equal volume of ethyl acetate containing n-octanol. After centrifugation at 14,000 rpm for 5 min, the upper organic phase was collected, dried with anhydrous sodium sulfate, and then centrifuged at 14,000 rpm for 5 min. An appropriate amount of supernatant was collected, and the activity and selectivity were detected by gas chromatography (GC).

[0040] The concentrations of substrate and product were determined using an HP-5 column (30m × 0.32 mm × 0.25 µm; Agilent). Figure 1 Injector temperature: 220℃; detector temperature: 250℃; carrier gas (N2): 1 mL / min; split ratio: 1:10. The initial temperature was 80℃, increased to 230℃ at a rate of 15℃ / min. Under these analytical conditions, the substrate retention time was 5.6 min, and the product (… R )-4-propyldihydrofuran-2(3 H The retention time of the )-ketone was 4.8 min.

[0041] The stereoselectivity of the product was determined using a Beta Dex-120 column (30m × 0.25mm × 0.25μm). Figure 2 Injector temperature: 280℃; detector temperature: 280℃; carrier gas (N2): 0.5mL / min; split ratio: 1:50. Initial column oven temperature: 90℃, held at 90℃ for 2 min, then increased to 180℃ at 10℃ / min, and held for 20 min.

[0042] product( R )-4-propyldihydrofuran-2(3 H The retention time of the ketone was 15.6 min. S )-4-propyldihydrofuran-2(3 H The retention time of the )-ketone was 15.7 min.

[0043] The detection results are shown in Table 3. Wild-type OpER and its mutant 4-propylfuran-2(5H)-one exhibit strict stereoselectivity, and the catalytic product ( R )-4-propyldihydrofuran-2(3 H )-ketone ee The values ​​were all greater than 99%. The catalytic activities of mutants Y76I, Y76M and N290P were improved to varying degrees compared with the wild-type enzyme OpER, among which mutant N290P had the highest conversion rate, reaching 68.5%.

[0044] Table 3. Catalytic reaction results of olefin reductase OpER and its mutants The sequence involved in this invention: SEQ ID NO.1(SEQ ID NO.1): MTPSTSLASSNLFKPIKVGKVELKNRLVFAPTTRYRASKDFVPTDSMLKYYEQRAENNGGLLTAATYVDFNFGLYPFTPMIKTPAQVAAWAKIIEAVHKQGSYFSIQLWHLGRAADPKFNKEKGVPFVAPSAIYLDQDSEKAAREAGNELRELTIPEEIPIVKEFAAAAKRAIHEAKADFIELHSAHGYLLDQFIQPNINKRTDKYGGSIENRARLVLEVVDACIEAVGAEHVGIRLSPYAKFQGSEGVDSEINPIASFGYILSELEKARDGNRLAYVSVVEPRVSGNVDSNDQRKFDTSWIREIWKGILFRAGGYLKENEQSLEHDVNQDDRTLIGVSRYYTSNPDLVERLKKGLSLTPYDRSRFYNHSSNDGYLTWPKYGEDEEKYKAVLDVEPKALA 。

[0045] SEQ ID NO.2(SEQ ID NO.2):

[0046] SEQ ID NO.3(SEQ ID NO.3): MTPSTSLASSNLFKPIKVGKVELKNRLVFAPTTRYRASKDFVPTDSMLKYYEQRAENNGGLLTAATYVDFNFGLIPFTPMIKTPPAQVAAWAKIIEAVHKQGSYFSIQLWHLGRAADPKFNKEKGVPFVAPSAIYLDQDSEKAAREAGNELRELTIPEEIPIVKEFAAAAKRAIHEAKADFIELHSAHGYLLDQFIQPNINKRTDKYGGSIENRARLVLEVVDACIEAVGAEHVGIRLSPYAKFQGSEGVDSEINPIASFGYILSELEKARDGNRLAYVSVVEPRVSGNVDSNDQRKFDTSWIREIWKGILFRAGGYLKENEQSLEHDVNQDDRTLIGVSRYYTSNPDLVERLKKGLSLTPYDRSRFYNHSSNDGYLTWPKYGEDEEKYKAVLDVEPKALA 。

[0047] SEQ ID NO.4(SEQ ID NO.4): MTPSTSLASSNLFKPIKVGKVELKNRLVFAPTTRYRASKDFVPTDSMLKYYEQRAENNGGLLTAATYVDFNFGLMPFTPMIKTPAQVAAWAKIIEAVHKQGSYFSIQLWHLGRAADPKFNKEKGVPFVAPSAIYLDQDSEKAAREAGNELRELTIPEEIPIVKEFAAAAKRAIHEAKADFIELHSAHGYLLDQFIQPNINKRTDKYGGSIENRARLVLEVVDACIEAVGAEHVGIRLSPYAKFQGSEGVDSEINPIASFGYILSELEKARDGNRLAYVSVVEPRVSGNVDSNDQRKFDTSWIREIWKGILFRAGGYLKENEQSLEHDVNQDDRTLIGVSRYYTSNPDLVERLKKGLSLTPYDRSRFYNHSSNDGYLTWPKYGEDEEKYKAVLDVEPKALA 。

[0048] SEQ ID NO.5(SEQ ID NO.5): MTPSTSLASSNLFKPIKVGKVELKNRLVFAPTTRYRASKDFVPTDSMLKYYEQRAENNGGLLTAEATYVDFNFGLYPFTPMIKTPAQVAAWAKIIIEAVHK QGSYFSIQLWHLGRAADPKFNKEKGVPFVAPSAIYLDQDSEKAAREAGNELRELTIPEIEPIVKEFAAAAKRAIHEAKADFIELHSAHGYLLDQFIQPNIN KRTDKYGGSIENRARLVLEVVDACIEAVGAEHVGIRLSPYAKFQGSEGVDSEINPIASFGYILSELEKRARDGNRLAYVSVVEPRVSGPVDSNDQRKFDT SWIREIWKGILFRAGGYLKENEQSLEHDVNQDDRTLIGVSRYYTSNPDLVERLKKGLSLTPYDRSRFYNHSSNDGYLTWPKYGEDEEKYKAVLDVEPKALA .

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A olefin reductase mutant, characterized in that, It is obtained by N290P mutation of wild-type olefin reductase with amino acid sequence as shown in SEQ ID NO.1, and the amino acid sequence of the olefin reductase mutant is shown in SEQ ID NO.

5.

2. The gene encoding the olefin reductase mutant of claim 1.

3. A recombinant plasmid comprising the gene of claim 2.

4. Recombinant engineered bacteria comprising the recombinant plasmid as described in claim 3.

5. The recombinant engineered bacteria as described in claim 4, characterized in that, The recombinant engineered bacteria also contain a gene encoding glucose dehydrogenase.

6. The olefin reductase mutant as described in claim 1, the gene as described in claim 2, the recombinant plasmid as described in claim 3, or the recombinant engineered bacteria as described in claim 4 or 5 in the production of buvacertan intermediate ( R )-4-propyldihydrofuran-2( 3H Applications of )-ketones.

7. The olefin reductase mutant as described in claim 1, the gene as described in claim 2, the recombinant plasmid as described in claim 3, or the recombinant engineered bacteria as described in claim 4 or 5, in the preparation of buvacertan intermediates ( R )-4-propyldihydrofuran-2( 3H Application of )-ketones in catalysts.

8. An intermediate for producing Brucine buvacertane ( R )-4-propyldihydrofuran-2( 3H The method for ketones is characterized by, Includes the following steps: After fermenting the recombinant engineered bacteria as described in claim 4 or 5, the bacterial cells are collected by centrifugation and freeze-dried to obtain crude enzyme powder. Using the crude enzyme powder as a catalyst, 4-propylfuran-2(5 H The reaction was carried out using )-ketone as a substrate. After the reaction was completed, the mixture was separated and purified to obtain the ()-ketone. R )-4-propyldihydrofuran-2( 3H )-ketone.

9. The method as described in claim 8, characterized in that, The reaction system also includes NADP. + The reaction mixture was prepared with glucose, and the reaction medium was PBS buffer. The reaction conditions were shaking at 200 rpm and 37°C for 12 hours.

10. An intermediate for the production of Brucine ( R )-4-propyldihydrofuran-2( 3H Catalysts for )-ketones, characterized in that, The catalyst comprises the recombinant engineered bacteria as described in claim 4 or 5.

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