Remegipant intermediate precursor ketoreductase mutant, engineered bacteria and application

Abstract

The application relates to the field of biotechnology, in particular to a remegpa intermediate precursor ketoreductase mutant, an engineering bacterium and application. The remegpa intermediate precursor ketoreductase mutant, the engineering bacterium and the application of the embodiment of the application, the amino acid sequence of the remegpa intermediate precursor ketoreductase mutant is shown in SEQ ID NO:1-SEQ ID NO:5, the stereoselectivity of the asymmetric reduction hydrogenation catalyzed by the remegpa intermediate precursor ketoreductase mutant is improved, the R-type remegpa intermediate with high chiral purity is obtained, and the synthesis efficiency of the remegpa intermediate is improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a mutant ketone reductase precursor of retinoic acid intermediate, engineered bacteria and its application. Background Technology

[0002] Rimegepant (also known as rimegepan or rimegepan) is a small molecule calcitonin gene-related peptide (CGRP) receptor antagonist, generally used for the acute treatment of migraines in adults. Its chemical structure is shown below:

[0003] .

[0004] Currently, more and more drugs or intermediates can be synthesized through biocatalysis. (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptan[B]pyridine-5-one contains the structural unit of retemapam and is a key intermediate in the synthesis of retemapam. (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptan[B]pyridine-5-one can be obtained by reducing catalysis of cycloheptan[B]pyridine-5,9-dione.

[0005] Cycloheptano[B]pyridin-5,9-dione is a prochiral ketone compound, and (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridin-5-one is an R-type chiral hydroxyl compound. In the background art, the reductase for the intermediate precursor ketone of rememegapan exhibits low stereoselectivity and catalytic activity in the asymmetric reductive hydrogenation of the intermediate precursor ketone, which is detrimental to improving the synthesis efficiency of rememegapan intermediates. Summary of the Invention

[0006] Based on this, this application provides a mutant ketone reductase precursor of retrimazole intermediate, an engineered strain, and its application, to solve the technical problem in the prior art that is not conducive to improving the synthesis efficiency of retrimazole intermediate.

[0007] In one aspect, an embodiment of this application provides a mutant of retinoic acid intermediate precursor ketone reductase, the amino acid sequence of which is shown in SEQ ID NO: 1 to SEQ ID NO: 5.

[0008] Secondly, one embodiment of this application provides a ketone reductase mutant gene, which encodes the aforementioned ketone reductase mutant of the intermediate precursor of Remepiride.

[0009] Thirdly, one embodiment of this application provides a recombinant vector comprising the aforementioned ketone reductase mutant gene.

[0010] Fourthly, one embodiment of this application provides an engineered bacterium, which includes the recombinant vector described above.

[0011] Fifthly, one embodiment of this application provides a synthesis system for a retinoic acid intermediate, comprising a recombinant engineered bacterium and isopropanol, wherein the recombinant engineered bacterium is a recombinant engineered bacterium containing a gene encoding a retinoic acid intermediate precursor ketone reductase mutant, and the amino acid sequence of the retinoic acid intermediate precursor ketone reductase mutant is shown in SEQ ID NO: 1 to SEQ ID NO: 5.

[0012] Sixthly, one embodiment of this application provides a method for synthesizing a rememegapan intermediate, wherein the aforementioned rememegapan intermediate precursor ketone reductase mutant or a recombinant engineered bacterium containing the encoding gene of the aforementioned rememegapan intermediate precursor ketone reductase mutant is contacted with a rememegapan intermediate precursor ketone to convert the rememegapan intermediate precursor ketone into a rememegapan intermediate.

[0013] The embodiments of this application describe the rememegapan intermediate precursor ketone reductase mutant, engineered bacteria, and their applications. The amino acid sequence of the rememegapan intermediate precursor ketone reductase mutant is shown in SEQ ID NO: 1 to SEQ ID NO: 5. It exhibits improved stereoselectivity for asymmetric reduction catalysis of rememegapan intermediate precursor ketone, yielding R-type rememegapan intermediates with high chiral purity, which is beneficial for improving the synthesis efficiency of rememegapan intermediates. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the reaction principle of the synthesis system for the intermediate of retinoic acid.

[0015] Figure 2 This is a reaction principle diagram of another embodiment of the synthesis system for the intermediate of retinoic acid.

[0016] Figure 3 This is a chiral HPLC analysis spectrum of the racemic product standard in the synthesis method of the intermediate of retinoic acid in this application embodiment.

[0017] Figure 4 The image shows the chiral HPLC analysis spectrum of the standard S configuration in the synthesis method of the retinoic acid intermediate in this application embodiment.

[0018] Figure 5 The chiral HPLC analysis spectrum of the standard R configuration in the synthesis method of the retinoic acid intermediate in this application embodiment is shown.

[0019] Figure 6 The image shows the chiral HPLC analysis spectrum of the reaction product in one embodiment.

[0020] Figure 7Figure 1 shows the results of the experiment optimizing the reaction temperature of ketone reductase, the intermediate precursor of wild-type Remegpam.

[0021] Figure 8 The figure shows the results of the pH optimization experiment for the intermediate precursor ketone reductase of wild-type Remegpam.

[0022] Figure 9 The figure shows the experimental results of the optimal reaction of wild-type Remegran intermediate precursor ketone reductase with the amount of isopropanol added.

[0023] Figure 10 This is a comparison chart of the product concentration changes in Examples 69, 70, and 71 of the synthesis method of Remepiride intermediate.

[0024] Figure 11 The figure shows the experimental results of optimizing the reaction temperature in the synthesis method of the rimex intermediate in the application embodiment.

[0025] Figure 12 The figure shows the experimental results of optimizing the reaction pH in the synthesis method of the rimexam intermediate in the application embodiment.

[0026] Figure 13 The figure shows the experimental results of the optimal amount of isopropanol added in the synthesis method of the rimexam intermediate in the application embodiment.

[0027] Figure 14 This is a comparison graph showing the changes in substrate and product at the first substrate concentration.

[0028] Figure 15 This is a comparison graph showing the changes in substrate and product at the second substrate concentration.

[0029] Figure 16 This is a comparison graph showing the changes in substrate and product at the third substrate concentration. Detailed Implementation

[0030] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0031] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0032] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] In this article, the terms "remegapan intermediate precursor ketone reductase" and "remegapan intermediate precursor ketone reductase mutant" refer to enzymes exhibiting asymmetric reduction activity of prochiral ketone compounds. These enzymes can asymmetricly reduce prochiral ketone compounds, converting them into chiral hydroxyl compounds. Specifically, the remegapan intermediate precursor ketone, which is a prochiral ketone compound, is asymmetrically reduced to generate the remegapan intermediate, which is a chiral hydroxyl compound. The remegapan intermediate precursor ketone is cycloheptano[B]pyridin-5,9-dione, and the remegapan intermediate is (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridin-5-one.

[0035] Cyclohepta[B]pyridine-5,9-dione (CAS: 39713-40-1).

[0036] (R)-9-hydroxy-6,7,8,9-tetrahydrocyclohepta[b]pyridin-5-one (CAS: 1190363-44-0).

[0037] The reaction principle for converting cycloheptano[B]pyridine-5,9-dione to (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridine-5-one using the aforementioned ketoreductase mutant intermediate precursor of retinoic acid is as follows:

[0038] .

[0039] This document describes a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO: n, where n is a natural number. Clearly, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO: n, even with some sequence deletions, modifications, substitutions, conserved substitutions, or additions, can also be used in this application, as long as they exhibit the same or corresponding activity as the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO: n. For example, it is not excluded to add sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions before or after the "polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO: n"; and polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO: n, when they are supplemented with the aforementioned sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions, also fall within the scope of this application, as long as they exhibit the same or corresponding activity as the amino acid sequence shown in SEQ ID NO: n after the addition of the aforementioned sequences.

[0040] One embodiment of this application provides a mutant of retinoic acid intermediate precursor ketone reductase, the amino acid sequence of which is shown in SEQ ID NO: 1 to SEQ ID NO: 5.

[0041] The ketoreductase mutant of the intermediate precursor of Remegpam contains at least the following mutations as shown in SEQ ID NO: 69: cysteine ​​C at position 190 is mutated to valine V, isoleucine I at position 199 is mutated to phenylalanine F, and leucine L at position 195 is mutated to alanine A.

[0042] Among them, the amino acid sequence shown in SEQ ID NO: 69 is derived from Paucilactobacillus wasatchensis The amino acid sequence shown in SEQ ID NO: 69 was used to predict the three-dimensional structure of the protein. Analysis of the predicted three-dimensional structure revealed that positions 190, 199, 195, 153, 173, 226, 144, 115, 165, 202, 99, 150, 196, 96, 157, 131, 94, 80, 147, 87, 71, 64, 40, 29, 25, and 17 in the amino acid sequence shown in SEQ ID NO: 69 are key catalytic sites.

[0043] In this embodiment, a mutant of the retemapam intermediate precursor ketoreductase contains the following mutations in the amino acid sequence corresponding to SEQ ID NO: 69: cysteine ​​C at position 190 is mutated to valine V, isoleucine I at position 199 is mutated to phenylalanine F, and leucine L at position 195 is mutated to alanine A (SEQ ID NO: 1). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 69 are replaced by other amino acids. The aforementioned mutant of the retemapam intermediate precursor ketoreductase has the amino acid sequence shown in SEQ ID NO: 1.

[0044] In this embodiment, a mutant of the retemapam intermediate precursor ketoreductase contains the following mutations in the amino acid sequence corresponding to SEQ ID NO: 69: cysteine ​​C at position 190 is mutated to valine V, isoleucine I at position 199 is mutated to phenylalanine F, leucine L at position 195 is mutated to alanine A, leucine L at position 153 is mutated to glycine G, and aspartic acid D at position 173 is mutated to histidine H (SEQ ID NO: 2). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 69 are replaced by other amino acids. The aforementioned mutant of the retemapam intermediate precursor ketoreductase has the amino acid sequence shown in SEQ ID NO: 2.

[0045] In this embodiment, a mutant of the retemapam intermediate precursor ketoreductase contains the following mutations in the amino acid sequence corresponding to SEQ ID NO: 69: cysteine ​​C at position 190 is mutated to valine V, isoleucine I at position 199 is mutated to phenylalanine F, leucine L at position 195 is mutated to alanine A, leucine L at position 153 is mutated to cysteine ​​C, and aspartic acid D at position 173 is mutated to histidine H (SEQ ID NO: 3). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 69 are replaced by other amino acids. The aforementioned mutant of the retemapam intermediate precursor ketoreductase has the amino acid sequence shown in SEQ ID NO: 3.

[0046] In this embodiment, a mutant of the retemapam intermediate precursor ketoreductase contains the following mutations in the amino acid sequence corresponding to SEQ ID NO: 69: cysteine ​​C at position 190 is mutated to valine V, isoleucine I at position 199 is mutated to phenylalanine F, leucine L at position 195 is mutated to alanine A, leucine L at position 153 is mutated to cysteine ​​C, and aspartic acid D at position 173 is mutated to asparagine N (SEQ ID NO: 4). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 69 are replaced by other amino acids. The aforementioned mutant of the retemapam intermediate precursor ketoreductase has the amino acid sequence shown in SEQ ID NO: 4.

[0047] In this embodiment, a mutant of the retemapam intermediate precursor ketoreductase contains the following mutations in the amino acid sequence corresponding to SEQ ID NO: 69: cysteine ​​C at position 190 is mutated to valine V, isoleucine I at position 199 is mutated to phenylalanine F, leucine L at position 195 is mutated to alanine A, leucine L at position 153 is mutated to threonine T, and aspartic acid D at position 173 is mutated to histidine H (SEQ ID NO: 5). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 69 are replaced by other amino acids. The aforementioned mutant of the retemapam intermediate precursor ketoreductase has the amino acid sequence shown in SEQ ID NO: 5.

[0048] In this embodiment, the amino acid sequence of the rememegapan intermediate precursor ketone reductase mutant is shown in SEQ ID NO: 1 to SEQ ID NO: 5. It improves the stereoselectivity of the rememegapan intermediate precursor ketone in asymmetric reductive hydrogenation catalysis, and obtains the R-type rememegapan intermediate with high chiral purity (the chiral purity of the R-type rememegapan intermediate is 99.9%), which is beneficial to improving the synthesis efficiency of rememegapan intermediate.

[0049] One embodiment of this application provides a ketone reductase mutant gene that encodes the aforementioned ketone reductase mutant of the intermediate precursor of Remepiride.

[0050] Among them, the ketone reductase mutant gene can be a polynucleotide.

[0051] The polynucleotide has the nucleotide sequences corresponding to SEQ ID NO: 1 to SEQ ID NO: 5.

[0052] The polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides.

[0053] The polynucleotide only needs to encode the aforementioned ketoreductase mutant of the intermediate precursor of retinoic acid, and any nucleotide in the polynucleotide can be chemically modified.

[0054] This application also provides a recombinant vector comprising the aforementioned ketoreductase mutant gene. Specifically, the recombinant vector comprises the aforementioned polynucleotide encoding the ketoreductase mutant intermediate precursor of retinoic acid.

[0055] The recombinant vector is any natural or artificially constructed expression vector that encodes the nucleic acid molecule of the retemapa intermediate precursor ketone reductase mutant, which can be catalyzed by cellular transcriptases and / or translatases.

[0056] Specifically, a recombinant vector is a DNA preparation containing a polynucleotide sequence encoding a retinoic acid precursor ketone reductase mutant of retinoic acid intermediates. It may also contain a control sequence. In the recombinant vector, the polynucleotide sequence encoding the retinoic acid precursor ketone reductase mutant is operatively linked to a suitable control sequence, allowing the retinoic acid precursor ketone reductase mutant to be expressed in a suitable host. Specifically, the control sequence may include, but is not limited to, promoters capable of initiating transcription, arbitrary operon sequences for regulating transcription, suitable mRNA ribosome binding sites, and sequences for controlling transcription and translation termination. After transformation into a suitable host cell, the recombinant vector can replicate or function independently of the host genome, or it can integrate into the genome itself to replicate or function.

[0057] There are no particular restrictions on the type of recombinant vector; any vector known in the art can be used as long as it can replicate in the host cell. Exemplarily, commonly used vectors in the art can include plasmids, granules, viruses, bacteriophages, or transposons in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, and the pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, and pET system can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0058] This application also provides an engineered bacterium, including the recombinant vector described above.

[0059] In this embodiment, the engineered bacteria serve as the host cell. The recombinant vector described above is transformed into the host cell, enabling the synthesis of the retemapa intermediate precursor ketone reductase mutant within the host cell. The recombinant vector, introduced into the host cell, allows the polynucleotide encoding the retemapa intermediate precursor ketone reductase mutant to be expressed in the host cell, thus enabling the host cell to synthesize the aforementioned retemapa intermediate precursor ketone reductase mutant. This polynucleotide can be inserted into the host cell's chromosome, located outside the host cell's chromosome, or simultaneously inserted into the host cell's chromosome and located outside the chromosome. The polynucleotide can be DNA or RNA, as long as it can be expressed in the host cell. Exemplarily, the recombinant vector can be an expression cassette, including a promoter, transcription termination element, ribosomal domain, and translation termination element operatively linked to the polynucleotide.

[0060] The host cell can be a eukaryotic cell or a prokaryotic cell, and further, the prokaryotic cell can be a bacterial cell.

[0061] As one implementation method, the host cell can be Escherichia coli (Escherichia coli). Escherichia ) genus, Erwinia ( Erwinia ) genus, Serratia ( Serratia ) genus, Providencia ( Providencia ) genus, Corynebacterium ( Corynebacterium ) genus or short bacilli ( Brevibacterium ) genus; for example, the host cell can be *Escherichia coli* (E. coli). Escherichia coli Bacillus subtilis ( Bacillus subtilis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ) or Aspergillus oryzae ( Aspergillus oryzae ).

[0062] This application provides a synthesis system for a retinoic acid intermediate, comprising a recombinant engineered bacterium and a co-substrate. The recombinant engineered bacterium is a recombinant engineered bacterium containing a gene encoding a retinoic acid intermediate precursor ketone reductase mutant. The amino acid sequence of the retinoic acid intermediate precursor ketone reductase mutant is shown in SEQ ID NO: 1 to SEQ ID NO: 5.

[0063] Cycloheptano[B]pyridine-5,9-dione (Remegapan intermediate precursor ketone) is a prochiral ketone compound. Cycloheptano[B]pyridine-5,9-dione (Remegapan intermediate precursor ketone) is used as the substrate, and a Remegapan intermediate precursor ketone reductase mutant is used as the catalyst. Utilizing the asymmetric reduction catalytic activity of the Remegapan intermediate precursor ketone reductase mutant, the substrate cycloheptano[B]pyridine-5,9-dione is converted to (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridine-5-one (Remegapan intermediate), and (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridine-5-one (Remegapan intermediate) is used as the product.

[0064] Among them, the asymmetric reduction catalytic reaction of the retinoic acid intermediate precursor ketone reductase mutant requires the presence of a coenzyme.

[0065] Coenzymes can be NADP + / NADPH, NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate, which structurally contains an additional hydride (i.e., a negatively charged hydrogen atom); NADP + It is the oxidized state of nicotinamide adenine dinucleotide phosphate, which does not have an additional hydride (i.e., a negatively charged hydrogen atom), and therefore exhibits a positive charge. NADP + It mainly acts as an electron acceptor in cells, participating in a variety of redox reactions.

[0066] like Figure 1 As shown, in the asymmetric reductive hydrogenation reaction of the substrate cycloheptane[B]pyridine-5,9-dione catalyzed by the ketor reductase mutant of the rememegapan intermediate precursor to generate (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptane[B]pyridine-5-one (rememegapan intermediate), NADPH is converted to NADP. + .

[0067] In the embodiments of this application, both the mutant and wild-type retemapam intermediate precursor ketone reductase are short-chain dehydrogenases. While possessing the aforementioned asymmetric reduction catalytic activity, they also exhibit oxidation catalytic activity, capable of oxidizing the co-substrate to form byproducts. During the oxidation of the co-substrate to form byproducts, NADP... + It is converted into NADPH.

[0068] In the asymmetric reduction catalytic reaction, NADPH is converted to NADP. + NADP in oxidative catalysis + It is converted into NADPH, coenzyme NADP.+ / NADPH is recycled.

[0069] The recombinant engineered bacteria contain coenzymes within their cells, so the synthesis system for this Remepiride intermediate does not require the addition of additional coenzymes.

[0070] In this embodiment, the retemapa intermediate precursor ketone reductase mutant synthesized by recombinant engineered bacteria efficiently catalyzes the asymmetric reduction of prochiral ketone compounds in a system without the addition of any coenzymes, generating chiral hydroxyl compounds with high optical purity (ee=99.9%), which has good prospects for industrial application. In this embodiment, the retemapa intermediate precursor ketone reductase mutant has the characteristics of high conversion rate and high chiral selectivity for cycloheptano[B]pyridine-5,9-dione (retemapa intermediate precursor ketone).

[0071] As one implementation method, please refer to Figure 2 As shown, the co-substrate can be isopropanol, which is oxidized by the ketone reductase mutant of the retinoic acid intermediate precursor to form acetone.

[0072] Specifically, the recombinant engineered bacteria can be the engineered bacteria described in the above embodiments. The construction of the recombinant engineered bacteria can be referred to the description of the above engineered bacteria embodiments, which will not be repeated here.

[0073] In some embodiments, the recombinant engineered bacteria used in the synthesis system can be wet cells obtained by inducing and culturing recombinant engineered bacteria.

[0074] In some embodiments, the recombinant engineered bacteria is Escherichia coli. E. coli BL21(DE3).

[0075] In some embodiments, the recombinant engineered bacteria used in the synthesis system may be the crude enzyme solution obtained by breaking down the wet bacterial cells, or the immobilized cells prepared from the wet bacterial cells.

[0076] In some embodiments, the reaction system further includes a buffer solution, wherein the volume percentage of isopropanol in the reaction system is 40%–50%. Specifically, the reaction system includes isopropanol, a buffer solution, and recombinant engineered bacteria, wherein isopropanol and the buffer solution are in liquid form, and the volume of the reaction system is mainly determined by the volume of isopropanol and the volume of the buffer solution. The volume of the reaction system can be understood as the sum of the volumes of isopropanol and the buffer solution.

[0077] In some embodiments, the buffer solution is a Tris-HCl buffer solution, and the pH of the reaction system is 8.0 to 9.0.

[0078] This application provides a method for synthesizing a rememegapan intermediate, which involves contacting the aforementioned rememegapan intermediate precursor ketone reductase mutant or a recombinant engineered bacterium containing the encoding gene of the aforementioned rememegapan intermediate precursor ketone reductase mutant with a rememegapan intermediate precursor ketone to convert the rememegapan intermediate precursor ketone into a rememegapan intermediate.

[0079] Cycloheptano[B]pyridine-5,9-dione (Remegapan intermediate precursor ketone) is a prochiral ketone compound. Cycloheptano[B]pyridine-5,9-dione (Remegapan intermediate precursor ketone) is used as the substrate, and a Remegapan intermediate precursor ketone reductase mutant is used as the catalyst. Utilizing the asymmetric reductive hydrogenation catalytic activity of the Remegapan intermediate precursor ketone reductase mutant, the substrate cycloheptano[B]pyridine-5,9-dione is converted to (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridine-5-one (Remegapan intermediate), and (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridine-5-one (Remegapan intermediate) is used as the product.

[0080] In one implementation method, in a reaction system containing isopropanol, recombinant engineered bacteria containing the encoding gene of the retemapa intermediate precursor ketone reductase mutant are used to convert the retemapa intermediate precursor ketone into a retemapa intermediate, wherein the retemapa intermediate precursor ketone is cycloheptano[B]pyridine-5,9-dione, and the retemapa intermediate is (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridine-5-one.

[0081] In this embodiment, the ketone reductase mutant of the intermediate precursor of remdesivir synthesized by recombinant engineered bacteria efficiently catalyzes the asymmetric reduction of prochiral ketone compounds in a system without the addition of any coenzymes, generating chiral hydroxyl compounds with high optical purity (ee=99.9%).

[0082] In some embodiments, the reaction system further includes a buffer solution, wherein the volume ratio of isopropanol to the reaction system is 0.40–0.50:1; and / or, the buffer solution is a Tris-HCl buffer solution, and the pH of the reaction system is 8.0–9.0; and / or, the reaction temperature is 30°C–40°C; and / or, the ratio of the concentration of the recombinant engineered bacteria to the initial concentration of cycloheptano[B]pyridine-5,9-dione is 50 g / L: 50 g / L–300 g / L.

[0083] For example, the ratio of the concentration of the recombinant engineered bacteria to the initial concentration of cycloheptano[B]pyridine-5,9-dione is 50 g / L: 50 g / L to 200 g / L, or the ratio of the concentration of the host cell to the initial concentration of cycloheptano[B]pyridine-5,9-dione is 50 g / L: 200 g / L to 300 g / L.

[0084] Preparation of Remepiquat intermediate precursor ketone reductase mutant

[0085] The gene encoding the retinoic acid intermediate precursor ketone reductase mutant and the retinoic acid intermediate precursor ketone reductase can be called the KRED gene. The KRED gene contains the nucleotide sequence corresponding to the amino acid sequence of the aforementioned retinoic acid intermediate precursor ketone reductase mutant or retinoic acid intermediate precursor ketone reductase. For example, the KRED gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 69. The KRED gene is constructed in the pET-28a plasmid to obtain a recombinant vector.

[0086] The recombinant vector is a pET-28a plasmid containing the KRED gene, hereinafter referred to as pET-28a-KRED; the host cell used in the various embodiments and comparative examples of this application is Escherichia coli. The structure and sequence of the pET-28a plasmid can be found in CN202410706921.3. The nucleotide sequences corresponding to the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 69 in the various embodiments of this application and Comparative Example 1 are then used as the target expression gene to construct the pET-28a plasmid.

[0087] Expression of the gene encoding the intermediate precursor ketone reductase mutant of retinoic acid.

[0088] a. Transformation of pET-28a-KRED into E. coli E. coli In BL21(DE3), select a single clone of pET-28a-KRED or a strain preserved at -80℃, streak it onto the surface of LB solid medium containing the corresponding antibiotic (such as kanamycin, final concentration 50 μg / mL) in a clean bench, and then place it in a 37 ℃ constant temperature incubator for 12 h until a clear single colony is formed.

[0089] b. Use a sterile inoculation loop to pick a single colony from a fresh plate and inoculate it into 10 mL of LB liquid medium containing the same antibiotic. Incubate at 37 ℃ and 200 rpm for 12 h with shaking to obtain a seed culture with OD600≈3.0, ensuring that the bacteria are in the logarithmic growth phase.

[0090] c. Transfer the seed culture at an inoculum of 1% (v / v) to an Erlenmeyer flask containing 50 mL of LB liquid medium (kanamycin, final concentration 50-100 μg / mL), and culture with shaking at 37 ℃ and 200 rpm for 2 h. Monitor the cell growth until the OD600 is approximately 0.6-0.8, providing highly active cells for subsequent catalytic reactions.

[0091] d. Lower the temperature of the shaker to 16 ℃-18 ℃. After the temperature of the cultured bacterial solution has decreased, add isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 h.

[0092] e. After expression is complete, collect the above culture solution into a bottle, pre-cool the centrifuge to 4°C, and centrifuge at 5500 rpm for 10 min.

[0093] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex mixer.

[0094] g. Obtaining whole-cell catalyst: Centrifuge the resuspended bacterial cells again at 5500 rpm for 10 min, discard the supernatant, and collect the wet bacterial cells as the whole-cell catalyst. This wet bacterial cell can be used directly as the catalyst for the ketone reductase mutant intermediate precursor of Remepiride.

[0095] h. Preservation of whole-cell catalyst: Add wet bacterial cells to 30 mL of protein purification buffer, vortex the bacterial cells (there should be no solid particles), pour into a 50 mL centrifuge tube, and store at -80 ℃.

[0096] Purification of the mutant protein of retinoic acid intermediate precursor ketone reductase

[0097] a. Preparation of crude enzyme solution: 1.0 g of collected wet bacterial cells (whole-cell catalyst) were added to 20 mL of equilibration buffer for resuspending. The resuspended cells were then disrupted using a cell disruptor set to 300 W to prevent excessive temperature from affecting enzyme activity. The disruption program was set to run for 1 second and pause for 3 seconds. The disruption solution was continuously cooled with an ice-water mixture until the suspension became clear and transparent. The disruption solution was then centrifuged at 4 ℃ and 12000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 µm filter to obtain the crude enzyme solution. This crude enzyme solution can be used directly as a catalyst for the ketone reductase mutant intermediate precursor of Remegapan.

[0098] b. Regeneration and equilibration of ion exchange chromatography column: Protein purification was performed using a DEAE Sepharose Fast Flow anion exchange column. The column was washed with a high-salt buffer (containing 1-2 M NaCl) at a flow rate of 1 mL / min for 3-5 column volumes, followed by washing with 0.1 M NaOH for 3-5 column volumes, then washing with elution buffer for 3-5 column volumes, and finally washing with equilibration buffer until the detector parameters such as OD280, conductivity, and pH value stabilized.

[0099] c. Loading and elution of crude enzyme solution: Load the prepared crude enzyme solution at a loading rate of 0.5 mL / min, with a loading volume of 20 mL. After loading, wash with equilibration buffer for 3-5 column volumes, then elute using an increasing salt concentration gradient with elution buffer. Collect each fraction and confirm by protein electrophoresis. If the purification effect is unsatisfactory, this step can be repeated, or purification can be performed again using agarose gel G75 FF.

[0100] d. Protein concentration: The collected target protein was concentrated using ultrafiltration membrane concentration method. The concentration was carried out using a 10 kDa protein concentration tube and centrifuged at 4 ℃ and 5000 rpm for 30 min.

[0101] e. Protein desalting: Dilute the concentrated protein with an appropriate amount of PBS buffer (20 mM, pH 7.0) and place it in a dialysis bag (molecular weight cutoff 8~14 kDa). Use 20 mM, pH 7.0 PBS dialysate and let it stand overnight at 4 ℃. The dialysate needs to be changed once during the process.

[0102] f. Storage of ion exchange chromatography columns: After use, the ion exchange chromatography column should be rinsed with 1 M NaOH for 3-5 column volumes, then rinsed with 20% ethanol, and stored in a refrigerator at 4 ℃.

[0103] Electrophoretic analysis of the mutant protein of retinoic acid intermediate precursor ketone reductase

[0104] a. Protein sample processing: Add the purified protein solution and 5 × loading buffer at a ratio of 1:4 (v / v), heat in boiling water for 10 min, and set aside for later use.

[0105] b. Sample loading and electrophoresis: Place the precast protein gel (Genscript, SurePAGE, 4%~20%) in the electrophoresis tank, and add the protein sample and marker to the sample wells of the protein gel using a pipette.

[0106] c. Staining and destaining: Remove the outer shell of the pre-cast gel after electrophoresis, and automatically destain and stain using a protein staining and destaining instrument for 15 minutes.

[0107] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0108] Enzyme-catalyzed reactions

[0109] In vitro enzyme catalytic reaction conditions:

[0110] Reaction buffer: 100mM, 200mM, or 300mM acetate buffer, PBS buffer, or Tris-HCl buffer.

[0111] Reaction pH: pH5, pH6, pH7, pH8, pH9;

[0112] The concentrations of cycloheptano[B]pyridine-5,9-dione were 50 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L, and 300 g / L.

[0113] Isopropanol volume concentrations were 4 mL / 10 mL, 4.5 mL / 10 mL, and 5 mL / 10 mL;

[0114] The amount of *E. coli* expressing the gene encoding the mutant ketone reductase intermediate precursor of retinoic acid (the whole-cell catalyst obtained in the above steps) added was 50 g / L and 100 g / L.

[0115] Reaction temperatures: 40℃, 45℃, 50℃;

[0116] Reaction time: 2h, 3.5h, 4h, 6h, 8h, 10h, 12h, 15h, 16h, 18h, 20h, 22h, or 24h;

[0117] The total volume of the conversion reaction is 10 mL.

[0118] Detection of (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[B]pyridin-5-one

[0119] Detection method: OD-H chiral column;

[0120] Mobile phase: n-hexane:isopropanol 70:30;

[0121] Detector: 2998 PDA;

[0122] in, Figure 3 , Figure 4 and Figure 5 Spectral analysis was performed on the purchased standard samples for result comparison. Figure 3This is the chiral HPLC chromatogram of the racemic mixture of the product standard, used to compare the chiral differences between the reaction product and the racemic mixture; Figure 4 The chiral HPLC chromatogram of the S-configuration standard is used to identify the reaction byproducts of the S-configuration and to compare the chiral characteristics of the reaction products. Figure 5 This is the chiral HPLC chromatogram of the standard R configuration, used to confirm that the reaction product is of the R configuration; according to Figure 3 , Figure 4 and Figure 5 The comparison confirms the reaction product of the R configuration, which elutes at approximately 11 min, while the reaction byproduct of the S configuration elutes at approximately 12 min. Figure 6 Here is an example of a chiral HPLC analysis spectrum of the reaction products in one embodiment. The R-configuration reaction product elutes at approximately 11 min, the S-configuration reaction byproduct elutes at approximately 12 min, and the substrate elutes at approximately 22 min. The concentrations of the substrate, the R-configuration reaction product, and the S-configuration reaction byproduct can be obtained from the chiral HPLC analysis spectrum. Based on the concentrations of the R-configuration reaction product and the S-configuration reaction byproduct, the chiral purity of the R-configuration can be calculated by the proportion.

[0123] It should be noted that in the following examples and comparative examples, the product specifically refers to the reaction product of the R configuration ((R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptan[B]pyridine-5-one), and the substrate specifically refers to cycloheptan[B]pyridine-5,9-dione. As the reaction proceeds, isopropanol in the reaction system is converted into acetone. Since acetone is highly volatile at room temperature, and isopropanol is somewhat volatile at room temperature, the volatilization and reflux of isopropanol and acetone will continue throughout the entire reaction process. Therefore, during the measurement of product and substrate concentrations, especially in the middle and later stages of the reaction (after 8 hours of reaction), the amount of acetone in the reaction system increases. The volatilization and reflux of isopropanol and acetone will cause volume fluctuations in the reaction system, which in turn will lead to fluctuations in the same concentration at different measurement points in the product concentration change curve or substrate concentration change curve. In the early stage of the reaction (within 2 hours of reaction), since the amount of acetone is relatively small, the volume fluctuations in the reaction system caused by the volatilization and reflux of isopropanol and acetone are small, and their impact on the detection of product and substrate concentrations can be basically ignored. Furthermore, during the measurement of product and substrate concentrations, the dilution of the test reaction solution and liquid phase detection will introduce detection errors, which will lead to differences in the same concentration at different measurement points in the product concentration change curve or substrate concentration change curve.

[0124] Calculation of relative enzyme activity

[0125] Relative enzyme activity = (activity of the tested enzyme / activity of the standard enzyme) × 100% = (product production of the tested enzyme / product production of the standard enzyme) × 100%.

[0126] Catalytic conditions of wild-type Remegapan intermediate precursor ketone reductase

[0127] Reaction temperature comparison experiment

[0128] The reaction temperatures were 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃, respectively.

[0129] Other reaction conditions are as follows:

[0130] The reaction buffer was a 100 mM Tris-HCl buffer.

[0131] Reaction pH: pH 8;

[0132] The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L;

[0133] The volume concentration of isopropanol is 5 mL / 10 mL;

[0134] The amount of *E. coli* expressing the gene encoding the intermediate precursor ketone reductase of retinoic acid (the whole-cell catalyst obtained in the above steps, with the amino acid sequence shown in SEQ ID NO: 69) added was 50 g / L;

[0135] Reaction time: 4 hours;

[0136] The reaction system is 10 mL.

[0137] Please see Figure 7 As shown, the ketone reductase intermediate precursor of Remegpam exhibits relatively good activity at reaction temperatures of 40℃ to 50℃, with the activity being optimal at 40℃.

[0138] Comparison experiment at different pH levels

[0139] The reaction buffers and their pH values ​​are as follows: pH 4.0 (acetic acid buffer), pH 5.0 (acetic acid buffer), pH 5.0 (PBS buffer), pH 6.0 (PBS buffer), pH 7.0 (PBS buffer), pH 8.0 (PBS buffer), pH 9.0 (PBS buffer), pH 8.0 (Tris-HCl buffer), and pH 9.0 (Tris-HCl buffer).

[0140] Other reaction conditions are as follows:

[0141] The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L;

[0142] The volume concentration of isopropanol is 5 mL / 10 mL;

[0143] The amount of *E. coli* expressing the gene encoding the intermediate precursor ketone reductase of retinoic acid (the whole-cell catalyst obtained in the above steps, with the amino acid sequence shown in SEQ ID NO: 69) added was 50 g / L;

[0144] Reaction temperature: 35℃;

[0145] Reaction time: 4 hours;

[0146] The reaction system is 10 mL.

[0147] Please see Figure 8 As shown, the ketone reductase, an intermediate precursor of Remepiride, exhibits relatively good activity in Tris-HCl buffer at a pH of 8.0–9.0.

[0148] Isopropanol addition experiment

[0149] The amounts of isopropanol added were 2 mL (20% by volume), 3 mL (30% by volume), 4 mL (40% by volume), 5 mL (50% by volume), and 6 mL (60% by volume).

[0150] Other reaction conditions are as follows:

[0151] The reaction buffer was a 100 mM Tris-HCl buffer.

[0152] Reaction pH: pH 8;

[0153] The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L;

[0154] The addition amount of the intermediate precursor ketone reductase mutant (whole-cell catalyst) of Remepiride was 50 g / L;

[0155] Reaction temperature: 40℃;

[0156] Reaction time: 4 hours;

[0157] The reaction system is 10 mL.

[0158] Please see Figure 9 As shown, the activity of ketone reductase, the intermediate precursor of Remegapan, is relatively better when the isopropanol volume concentration is 3 mL / 10 mL to 5 mL / 10 mL, and the activity is relatively optimal when the isopropanol volume concentration is 5 mL / 10 mL.

[0159] Enzyme activity and R configuration chiral purity screening experiments

[0160] The amino acid sequences of the ketoreductase mutants of the intermediate precursor of retinoic acid in Examples 1 to 68 are shown in SEQ ID NO: 1 to SEQ ID NO: 68, and the amino acid sequence of the ketoreductase intermediate precursor of retinoic acid in Comparative Example 1 is shown in SEQ ID NO: 69.

[0161] Catalytic reaction conditions for each embodiment and Comparative Example 1

[0162] The reaction buffer was a 100 mM Tris-HCl buffer.

[0163] Reaction pH: pH 8;

[0164] The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L;

[0165] The volume concentration of isopropanol is 5 mL / 10 mL;

[0166] The addition amount of the intermediate precursor ketone reductase mutant (whole-cell catalyst) of Remepiride was 50 g / L;

[0167] Reaction temperature: 40℃;

[0168] Reaction time: 4 hours;

[0169] The reaction system is 10 mL.

[0170] Samples of Example 1 and Comparative Example 1 were taken during a reaction time of 4 hours and subjected to chiral HPLC analysis to measure the substrate residue and R-configuration chiral purity of Example 1 and Comparative Example 1. The results are shown in Table 1.

[0171] Samples from Examples 1 to 68 were taken during a reaction time of 1 hour and subjected to chiral HPLC analysis to measure the relative activity and chiral purity of the R configuration of Examples 1 to 68. The results are shown in Tables 2-1, 2-2 and 2-3.

[0172] The results of Example 1 and Comparative Example 1 are shown in Table 1. It can be seen that the chiral purity of the wild-type ketoreductase for the R-configuration of the ketoreductase intermediate in Comparative Example 1 was only 49%, while the ketoreductase mutant for the ketoreductase intermediate in Example 1 had three mutation sites (cysteine ​​C at position 190 was mutated to valine V, isoleucine I at position 199 was mutated to phenylalanine F, and leucine L at position 195 was mutated to alanine A). The chiral purity of the R-configuration of the ketoreductase intermediate of the mutant in Example 1 was 99.9%. The substrate residue of the mutant in Example 1 after 4 hours of reaction was slightly less than that of the wild type in Comparative Example 1. However, since the chiral purity of the R configuration in Example 1 was 99.9%, which was much higher than that of the R chiral purity in Comparative Example 1, the amount of product ((R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[B]pyridin-5-one) in Example 1 was much greater than that in Comparative Example 1. That is, the amount of product generated by the mutant in Example 1 was greater than that of the wild type in Comparative Example 1.

[0173] Table 1. Parameters of Example 1 and Comparative Example

[0174]

[0175] The results of Examples 1 to 68 are shown in Tables 2-1, 2-2, and 2-3. In Example 1, the relative activity of the ketoreductase mutant of the retinoic acid intermediate precursor was 1. In Examples 2 to 68, the mutants had a mutation at position 190 (cysteine ​​C) to valine V, position 199 (isoleucine I) to phenylalanine F, and position 195 (leucine L) to alanine A, respectively. In addition to the above three mutation sites, the mutants in Examples 2 to 68 also had 1 to 2 other mutation sites. The relative activities of the retinoic acid intermediate precursor ketoreductase mutants in Examples 2 to 68 are shown based on those in Example 1.

[0176] Table 2-1 Parameters of Examples 1 to 23

[0177]

[0178] Table 2-2 Parameters of Examples 24 to 46

[0179]

[0180] Table 2-3 Parameters of Examples 47 to 68

[0181]

[0182] The mutants of Examples 2 to 5, in addition to the above three mutations, also have mutations at position 153 and position 173. The chiral purity of the R configuration of the mutants of Examples 2 to 5 is 99%, and the enzyme activity of the mutants of Examples 2 to 5 is significantly improved compared with Example 1.

[0183] Product concentration change experiment

[0184] Reaction conditions

[0185] The reaction buffer was a 100 mM Tris-HCl buffer.

[0186] Reaction pH: pH 8;

[0187] The initial concentration of cycloheptano[B]pyridine-5,9-dione was 200 g / L;

[0188] The volume concentration of isopropanol is 5 mL / 10 mL;

[0189] The addition amount of the intermediate precursor ketone reductase mutant (whole-cell catalyst) of Remepiride was 50 g / L;

[0190] Reaction temperature: 40℃;

[0191] Reaction time: 24 hours;

[0192] The reaction system is 10 mL.

[0193] Table 3 Sequences of each embodiment

[0194]

[0195] Samples from Examples 69, 70, and 71 were taken at reaction times of 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, and 24 hours, respectively, for chiral HPLC analysis. The product mass concentration was measured, and the results are as follows: Figure 10 As shown.

[0196] like Figure 10 As shown, throughout the reaction process, the product concentrations of Examples 70 and 71 were consistently higher than those of Example 69. Examples 69, 70, and 71 exhibited relatively rapid product formation rates from 1 to 6 hours, relatively slower product formation rates from 6 to 16 hours, and product formation rates tended to stabilize after 16 hours.

[0197] Reaction temperature comparison experiment

[0198] The reaction conditions for Examples 72 to 77 are as follows:

[0199] The reaction buffer was a 100 mM Tris-HCl buffer.

[0200] Reaction pH: pH 8;

[0201] The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L;

[0202] The volume concentration of isopropanol is 5 mL / 10 mL;

[0203] The addition amount of the intermediate precursor ketone reductase mutant (whole-cell catalyst) of Remepiride was 50 g / L;

[0204] Reaction time: 3.5 hours;

[0205] The total reaction volume is 10 mL.

[0206] The reaction temperatures in each embodiment are shown in Table 4:

[0207] Table 4 Reaction temperatures of each example

[0208]

[0209] Please see Figure 11 As shown, the relative activity of the ketoreductase mutant of the retinoic acid intermediate precursor in Example 75 was 1. The relative activities of the ketoreductase mutants of the retinoic acid intermediate precursor in Examples 72-74 and Examples 76-77 are shown based on that of Example 75. A reaction temperature of 40°C to 50°C is relatively preferred, and a reaction temperature of 40°C is relatively optimal.

[0210] Comparison experiment at different pH levels

[0211] The reaction conditions for Examples 78 to 84 are as follows:

[0212] The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L;

[0213] The volume concentration of isopropanol is 5 mL / 10 mL;

[0214] The addition amount of the intermediate precursor ketone reductase mutant (whole-cell catalyst) of Remepiride was 50 g / L;

[0215] Reaction temperature: 35℃;

[0216] Reaction time: 16 hours;

[0217] The total reaction volume is 10 mL.

[0218] The reaction pH values ​​in each embodiment are shown in Table 5:

[0219] Table 5. pH values ​​of the reactions in each example

[0220]

[0221] Please see Figure 12 As shown, the relative activity of the ketoreductase mutant of the retinoic acid intermediate precursor in Example 83 was 1. The relative activities of the ketoreductase mutants of the retinoic acid intermediate precursor in Examples 78-82 and Example 84 were shown based on that of Example 83. The buffer was Tris-HCl buffer, and the reaction pH was preferably 8.0-9.0.

[0222] Isopropanol addition experiment

[0223] The reaction conditions for Examples 85 to 89 are as follows:

[0224] The reaction buffer was a 100 mM Tris-HCl buffer.

[0225] Reaction pH: pH 8;

[0226] The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L;

[0227] The addition amount of the intermediate precursor ketone reductase mutant (whole-cell catalyst) of Remepiride was 50 g / L;

[0228] Reaction temperature: 40℃;

[0229] Reaction time: 3.5 hours;

[0230] The total reaction volume is 10 mL.

[0231] The volumes of isopropanol in each embodiment are shown in Table 6:

[0232] Table 6 Isopropanol Volumes in Each Example

[0233]

[0234] Please see Figure 13 As shown, the relative activity of the retemapam intermediate precursor ketone reductase mutant in Example 88 was 1. The relative activities of the retemapam intermediate precursor ketone reductase mutants in Examples 85-87 and Example 89 were shown based on Example 88. An isopropanol volume concentration of 3 mL / 10 mL to 5 mL / 10 mL was relatively preferred, with an isopropanol volume concentration of 5 mL / 10 mL being the most optimal.

[0235] Experiment on the addition of cycloheptano[B]pyridine-5,9-dione

[0236] The reaction conditions for Examples 90 to 92 are as follows:

[0237] The reaction buffer was a 100 mM Tris-HCl buffer.

[0238] Reaction pH: pH 8;

[0239] The volume concentration of isopropanol is 5 mL / 10 mL;

[0240] The addition amount of the intermediate precursor ketone reductase mutant (whole-cell catalyst) of Remepiride was 50 g / L;

[0241] Reaction temperature: 35℃;

[0242] Reaction time: 24 hours;

[0243] The total reaction volume is 10 mL.

[0244] The concentrations of cycloheptano[B]pyridine-5,9-dione in each embodiment are shown in Table 7:

[0245] Table 7. Amount of cycloheptano[B]pyridine-5,9-dione added in each example

[0246]

[0247] Please see Figure 14 , Figure 15 and Figure 16 As shown, the substrate in Example 90 reacted completely in about 16 hours, the substrate in Example 91 reacted completely in about 16 hours, and the substrate in Example 92 reacted completely in about 18 hours.

[0248] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0249] The above embodiments merely illustrate preferred implementations of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Therefore, the scope of protection of this patent application should be determined by the appended claims.

Claims

1. A remegpaπ intermediate pre-ketoreductase mutant characterized by, The amino acid sequence of the remegpa intermediate precursor ketoreductase mutant is shown in SEQ ID NO: 1-5.

2. A ketoreductase mutant gene, wherein the mutant gene comprises a mutation at a position corresponding to position 107 of SEQ ID NO:

1. The ketoreductase mutant gene encodes the remegpa intermediate precursor ketoreductase mutant of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector comprises the ketoreductase mutant gene of claim 2.

4. An engineered bacterium, characterized in that, The engineered bacteria comprise the recombinant vector of claim 3.

5. A system for the synthesis of a remegapant intermediate, characterized by, The recombinant engineered bacteria are Escherichia coli BL21 (DE3) containing a gene encoding a remegpidem intermediate precursor ketoreductase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1-5. E. coli BL21 (DE3).

6. The synthesis system of a remegapant intermediate according to claim 5, wherein, The reaction system further comprises a buffer, and the volume ratio of the isopropyl alcohol to the reaction system is 0.40-0.50:

1.

7. A method of synthesizing a remegapant intermediate, characterized by, In the reaction system containing isopropyl alcohol, the remegpa intermediate precursor ketoreductase mutant containing the coding gene of the remegpa intermediate precursor ketoreductase mutant is used to convert the remegpa intermediate precursor ketone into the remegpa intermediate, the remegpa intermediate precursor ketone is cycloheptano[B]pyridine-5,9-dione, the remegpa intermediate is (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[B]pyridine-5-one, the amino acid sequence of the remegpa intermediate precursor ketoreductase mutant is shown in SEQ ID NO: 1-5, the reaction system further comprises a buffer, and the volume ratio of the isopropyl alcohol to the reaction system is 0.40-0.50:

1.

8. The method of synthesis of a remegapant intermediate according to claim 7, wherein, The pH value of the reaction system is 8.0-9.0; and / or, the reaction temperature is 30-40℃; and / or, the ratio of the concentration of the recombinant engineered bacteria to the initial concentration of the cycloheptano[B]pyridine-5,9-dione is 50 g / L:50 g / L-300 g / L.

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