Ketoreductase mutant for synthesizing remegapam intermediate and application of ketoreductase mutant

Abstract

The invention relates to the technical field of biology, in particular to a ketoreductase mutant used for synthesizing a remegapam intermediate and application of the ketoreductase mutant, and discloses the ketoreductase mutant used for synthesizing the remegapam intermediate and application of the ketoreductase mutant used for synthesizing the remegapam intermediate. The stereoselectivity of asymmetric reduction catalysis of the ketoreductase mutant on the remegapam intermediate precursor ketone is improved, the R-type remegapam intermediate with high chiral purity is obtained, and the synthesis efficiency of the remegapam intermediate is improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a ketone reductase mutant for the synthesis of retinoic acid intermediates and its applications. 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] 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.

[0004] 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

[0005] Based on this, this application provides a ketone reductase mutant for synthesizing rememegapan intermediates and its application, in order to solve the technical problem in the prior art that is not conducive to improving the synthesis efficiency of rememegapan intermediates.

[0006] In a first aspect, embodiments of this application provide a ketone reductase mutant for synthesizing the intermediate of retinoic acid, characterized in that it generates four mutations in the amino acid sequence shown in SEQ ID NO: 14, namely, 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, and leucine L at position 153 is mutated to threonine T, valine V, phenylalanine F, cysteine ​​C, or glycine G.

[0007] Secondly, embodiments of this application provide a ketone reductase mutant gene, which encodes the aforementioned ketone reductase mutant used for the synthesis of the intermediate of retinoic acid.

[0008] Thirdly, embodiments of this application provide a recombinant vector comprising the aforementioned ketone reductase mutant gene.

[0009] Fourthly, embodiments of this application provide a recombinant engineered bacterium, the engineered bacterium including the recombinant vector described above.

[0010] Fifthly, embodiments of this application provide a composition for preparing a remedypa intermediate, comprising: The ketone reductase mutant used to synthesize the intermediate of retinoic acid, or the recombinant engineered bacteria mentioned above.

[0011] Sixthly, embodiments of this application provide a method for the biological preparation of a remedypa intermediate, comprising: Using a certain concentration of rimex intermediate precursor ketone as a substrate, recombinant engineered bacteria were added, and the reaction was carried out at 30℃~40℃ in a reaction system with pH 8.0~9.0. After the reaction was completed, the product was obtained. The recombinant engineered bacteria are engineered bacteria containing the coding gene of the ketone reductase mutant used to synthesize the intermediate of retinoic acid.

[0012] The ketone reductase mutant used in the synthesis of rememegapan intermediates and its application in this application improve the stereoselectivity of asymmetric reduction catalysis of rememegapan intermediate precursor ketones, resulting in R-type rememegapan intermediates with high chiral purity, which is beneficial to improving the synthesis efficiency of rememegapan intermediates. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the reaction mechanism of the intermediate of Remepiride.

[0014] Figure 2 This is a reaction principle diagram of another embodiment of the retinoic acid intermediate.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Figure 6 The image shows the chiral HPLC analysis spectrum of the reaction product in one embodiment.

[0019] Figure 7 The figure shows the experimental results of optimizing the reaction temperature of ketone reductase, the intermediate precursor of wild-type Remegpam.

[0020] Figure 8 The figure shows the results of the pH optimization experiment for the intermediate precursor ketone reductase of wild-type Remegpam.

[0021] 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.

[0022] Figure 10 This is a graph showing the product concentration variation in Example 14 of the synthesis method of the intermediate of retinoic acid. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] In this article, the terms "ketone reductase" and "ketone reductase mutant" refer to enzymes exhibiting asymmetric reduction activity of prochiral ketones, which can asymmetricly reduce prochiral ketones to chiral hydroxyl compounds. Specifically, the precursor ketone of the rememegapan intermediate, which is a prochiral ketone, is asymmetrically reduced to generate the rememegapan intermediate, which is a chiral hydroxyl compound. The precursor ketone of the rememegapan intermediate is cycloheptano[B]pyridin-5,9-dione, and the rememegapan intermediate is (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridin-5-one.

[0028] 7,8-dihydro-5H-cyclohepta[b]pyridine-5,9(6H)-dione (CAS: 39713-40-1).

[0029] (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[B]pyridin-5-one (CAS: 1190363-44-0).

[0030] The reaction principle for converting cycloheptano[B]pyridin-5,9-dione to (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridin-5-one using the aforementioned ketone reductase mutant is as follows: .

[0031] The description herein refers to "a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO:". Obviously, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO:, 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:. 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:". Furthermore, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO:, when subjected to the addition of 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: after the addition of the aforementioned sequences.

[0032] One embodiment of this application provides a ketone reductase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1 to SEQ ID NO: 13.

[0033] The ketone reductase mutant is generated by at least the following mutations as shown in SEQ ID NO: 14: 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.

[0034] Among them, the amino acid sequence shown in SEQ ID NO: 14 is derived from Paucilactobacillus wasatchensis The amino acid sequence shown in SEQ ID NO: 14 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: 14 are key catalytic sites.

[0035] In this embodiment, a ketone reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 14: 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: 14 are replaced by other amino acids. The aforementioned ketone reductase mutant has the amino acid sequence shown in SEQ ID NO: 1.

[0036] In this embodiment, a ketone reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 14: 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, and leucine L at position 153 is mutated to threonine T. In these mutations, the corresponding amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 14 are replaced by other amino acids. The aforementioned ketone reductase mutant has the amino acid sequence shown in SEQ ID NO: 2.

[0037] In this embodiment, a ketone reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 14: 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, and leucine L at position 153 is mutated to valine V. In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 14 are replaced by other amino acids. The aforementioned ketone reductase mutant has the amino acid sequence shown in SEQ ID NO: 5.

[0038] In this embodiment, a ketone reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 14: 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, and leucine L at position 153 is mutated to phenylalanine F. In these mutations, the corresponding amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 14 are replaced by other amino acids. The aforementioned ketone reductase mutant has the amino acid sequence shown in SEQ ID NO: 6.

[0039] In this embodiment, a ketone reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 14: 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, and leucine L at position 153 is mutated to cysteine ​​C. In these mutations, the corresponding amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 14 are replaced by other amino acids. The aforementioned ketone reductase mutant has the amino acid sequence shown in SEQ ID NO: 8.

[0040] In this embodiment, a ketone reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 14: 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, and leucine L at position 153 is mutated to glycine G. In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 14 are replaced by other amino acids. The aforementioned ketone reductase mutant has the amino acid sequence shown in SEQ ID NO: 12.

[0041] Wild-type ketone reductase exhibits poor stereoselectivity, with the chiral purity of the R-type rememegapan intermediate being only 49%. The ketone reductase mutants shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 12 all demonstrate extremely high stereoselectivity, with the R-type rememegapan intermediate possessing a chiral purity of 99.9%. Furthermore, compared to the wild-type, their catalytic activity is enhanced. Moreover, compared to the mutant shown in SEQ ID NO: 1, the catalytic activity of all the above ketone reductase mutants is improved to varying degrees.

[0042] In this embodiment, the ketone reductase mutant described above improves the stereoselectivity of asymmetric reductive hydrogenation catalysis of the precursor ketone of rememegapan intermediate, resulting in a high-chiral-purity R-type rememegapan intermediate (the chiral purity of the R-type rememegapan intermediate is 99.9%), which is beneficial to improving the synthesis efficiency of rememegapan intermediate.

[0043] One embodiment of this application provides a ketone reductase mutant gene that encodes the aforementioned ketone reductase mutant.

[0044] Among them, the ketone reductase mutant gene can be a polynucleotide.

[0045] The polynucleotide has the nucleotide sequences corresponding to SEQ ID NO: 1 to SEQ ID NO: 13.

[0046] The polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides.

[0047] The polynucleotide only needs to encode the aforementioned ketone reductase mutant, and any nucleotide in the polynucleotide can be chemically modified.

[0048] 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.

[0049] The recombinant vector is any natural or artificially constructed expression vector that encodes the nucleic acid molecule of the ketone reductase mutant, which can be catalyzed by cellular transcriptases and / or translatases.

[0050] Specifically, a recombinant vector is a DNA preparation containing a polynucleotide sequence encoding a ketoreductase mutant. It may also contain control sequences. In the recombinant vector, the polynucleotide sequence encoding the ketoreductase mutant is operatively linked to a suitable control sequence, allowing the ketoreductase 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 for replication or function.

[0051] 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.

[0052] This application also provides an engineered bacterium, including the recombinant vector described above.

[0053] In this embodiment, the engineered bacteria serves as the host cell. The recombinant vector described above is transformed into the host cell, enabling the synthesis of the ketone reductase mutant within the host cell. The recombinant vector is introduced into the host cell, and the polynucleotide encoding the ketone reductase mutant in the recombinant vector can be expressed in the host cell, allowing the host cell to synthesize the aforementioned 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.

[0054] The host cell can be a eukaryotic cell or a prokaryotic cell, and further, the prokaryotic cell can be a bacterial cell.

[0055] 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 ).

[0056] This application provides a composition for preparing a retinoic acid intermediate, comprising the above-mentioned ketone reductase mutant or the above-mentioned recombinant engineered bacteria.

[0057] Furthermore, the composition also includes an auxiliary substrate.

[0058] In one embodiment, the composition includes a recombinant engineered bacterium and a co-substrate, wherein the recombinant engineered bacterium is a recombinant engineered bacterium containing a gene encoding a ketone reductase mutant, and the amino acid sequence of the ketone reductase mutant is shown in SEQ ID NO: 1 to SEQ ID NO: 13.

[0059] 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 ketone reductase mutant is used as the catalyst. Utilizing the asymmetric reduction catalytic activity of the ketone reductase mutant, the substrate cycloheptano[B]pyridine-5,9-dione is converted into (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.

[0060] Among them, the catalytic reaction of asymmetric reduction by ketone reductase mutants requires the presence of a coenzyme.

[0061] 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.

[0062] like Figure 1 As shown, in the asymmetric reductive hydrogenation reaction of the substrate cycloheptane and [B]pyridine-5,9-dione catalyzed by the ketoreductase mutant to generate (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptane[B]pyridine-5-one (the intermediate of retemaciopran), NADPH is converted to NADP. + .

[0063] In the embodiments of this application, both the ketone reductase mutant and the wild-type ketone reductase belong to short-chain dehydrogenases. In addition to the aforementioned asymmetric reduction catalytic activity, they also possess 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.

[0064] 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.

[0065] The recombinant engineered bacteria contain coenzymes within their cells, so the reaction system for this Remepiride intermediate does not require the addition of additional coenzymes.

[0066] In this embodiment, the 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 ketone reductase mutant has the characteristics of high conversion rate and high chiral selectivity for cycloheptano[B]pyridine-5,9-dione (remegapan intermediate precursor ketone).

[0067] 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 to form acetone.

[0068] 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.

[0069] In some embodiments, the recombinant engineered bacteria used in the reaction system can be wet cells obtained by inducing and culturing recombinant engineered bacteria.

[0070] In some embodiments, the recombinant engineered bacteria is Escherichia coli. E. coli BL21(DE3).

[0071] In some embodiments, the recombinant engineered bacteria used in the reaction 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.

[0072] 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.

[0073] 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.

[0074] This application provides a biological preparation method for rememegapan intermediate, which involves contacting the aforementioned ketone reductase mutant or a recombinant engineered bacterium containing the encoding gene of the aforementioned ketone reductase mutant with rememegapan intermediate precursor ketone to convert rememegapan intermediate precursor ketone into rememegapan intermediate.

[0075] 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 ketone reductase mutant is used as the catalyst. Utilizing the asymmetric reductive hydrogenation catalytic activity of the ketone reductase mutant, the substrate cycloheptano[B]pyridine-5,9-dione is converted into (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.

[0076] In one implementation method, in a reaction system containing isopropanol, a recombinant engineered bacterium containing the coding gene of the ketone reductase mutant is used to convert the precursor ketone of rememegapan intermediate into rememegapan intermediate, wherein the precursor ketone of rememegapan intermediate is cycloheptano[B]pyridine-5,9-dione, and the rememegapan intermediate is (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cycloheptano[B]pyridine-5-one.

[0077] In this embodiment, the 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%).

[0078] 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.

[0079] 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.

[0080] Preparation of ketone reductase mutants The ketone reductase mutant and the gene encoding ketone reductase can be called the KRED gene. The KRED gene contains the nucleotide sequence corresponding to the amino acid sequence of the aforementioned ketone reductase mutant or 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: 14. The KRED gene is constructed in the pET-28a plasmid to obtain a recombinant vector.

[0081] 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 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: 14 in the embodiments of this application and Comparative Example 1 are then used as the target expression gene to construct the pET-28a plasmid.

[0082] Expression of the gene encoding ketone reductase mutant 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.

[0083] 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.

[0084] c. Transfer the seed culture at a 1% (v / v) inoculation rate to an Erlenmeyer flask containing 50 mL of LB liquid medium (kanamycin, final concentration 50–100 μg / mL), and culture with shaking at 37 °C 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.

[0085] 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.

[0086] 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.

[0087] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex mixer.

[0088] 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 a catalyst for ketone reductase mutants.

[0089] 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 ℃.

[0090] Purification of ketone reductase mutant protein 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 membrane to obtain the crude enzyme solution. This crude enzyme solution can be used directly as a catalyst for ketone reductase mutants.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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 ℃.

[0096] Electrophoretic analysis of ketone reductase mutant protein 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.

[0097] 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.

[0098] 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.

[0099] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0100] Enzyme-catalyzed reactions In vitro enzyme catalytic reaction conditions: Reaction buffer: 100mM, 200mM, or 300mM acetate buffer, PBS buffer, or Tris-HCl buffer. Reaction pH: pH5, pH6, pH7, pH8, pH9; 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. Isopropanol volume concentrations were 4 mL / 10 mL, 4.5 mL / 10 mL, and 5 mL / 10 mL; The amount of *E. coli* expressing the gene encoding the ketone reductase mutant (the whole-cell catalyst obtained in the above steps) added was 50 g / L and 100 g / L. Reaction temperatures: 40℃, 45℃, 50℃; Reaction time: 2h, 3.5h, 4h, 6h, 8h, 10h, 12h, 15h, 16h, 18h, 20h, 22h, or 24h; The total volume of the conversion reaction is 10 mL.

[0101] Detection of (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[B]pyridin-5-one Detection method: OD-H chiral column; Mobile phase: n-hexane:isopropanol 70:30; Detector: 2998 PDA; in, Figure 3 , Figure 4 and Figure 5 Spectral analysis was performed on the purchased standard samples for result comparison. Figure 3 This 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.

[0102] 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.

[0103] Calculation of relative enzyme activity 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%.

[0104] Catalytic conditions of wild-type ketone reductase Reaction temperature comparison experiment The reaction temperatures were 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃, respectively. Other reaction conditions are as follows: The reaction buffer was a 100 mM Tris-HCl buffer. Reaction pH: pH 8; The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L; The volume concentration of isopropanol is 5 mL / 10 mL; The amount of *E. coli* expressing the ketone reductase encoding gene (the whole-cell catalyst obtained in the above steps, with the expressed amino acid sequence shown in SEQ ID NO: 14) added was 50 g / L; Reaction time: 4 hours; The reaction system is 10 mL.

[0105] Please see Figure 7 As shown, ketone reductase exhibits relatively better activity at reaction temperatures between 40℃ and 50℃, with 40℃ being the optimal temperature for its activity.

[0106] Comparison experiment at different pH levels 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). Other reaction conditions are as follows: The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L; The volume concentration of isopropanol is 5 mL / 10 mL; The amount of *E. coli* expressing the ketone reductase encoding gene (the whole-cell catalyst obtained in the above steps, with the expressed amino acid sequence shown in SEQ ID NO: 14) added was 50 g / L; Reaction temperature: 35℃; Reaction time: 4 hours; The reaction system is 10 mL.

[0107] Please see Figure 8 As shown, ketone reductase exhibits relatively better activity in Tris-HCl buffer and at a pH of 8.0–9.0.

[0108] Isopropanol addition experiment 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). Other reaction conditions are as follows: The reaction buffer was a 100 mM Tris-HCl buffer. Reaction pH: pH 8; The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L; The addition amount of ketone reductase mutant (whole-cell catalyst) was 50 g / L; Reaction temperature: 40℃; Reaction time: 4 hours; The reaction system is 10 mL.

[0109] Please see Figure 9 As shown, wild-type ketone reductase exhibits relatively good activity at isopropanol concentrations of 3 mL / 10 mL (30% isopropanol volume ratio) to 5 mL / 10 mL (50% isopropanol volume ratio), with the activity being relatively optimal at an isopropanol volume concentration of 5 mL / 10 mL. Figure 9 The relative activity at 50% isopropanol volume ratio is shown as 100%, and the relative activities at other points are calculated based on the relative activity at 50% isopropanol volume ratio.

[0110] Enzyme activity and R configuration chiral purity screening experiments The amino acid sequences of the ketone reductase mutants in Examples 1 to 13 are shown in SEQ ID NO: 1 to SEQ ID NO: 13, and the amino acid sequence of the ketone reductase in Comparative Example 1 is shown in SEQ ID NO: 14.

[0111] Catalytic reaction conditions for each embodiment and Comparative Example 1 The reaction buffer was a 100 mM Tris-HCl buffer. Reaction pH: pH 8; The initial concentration of cycloheptano[B]pyridine-5,9-dione was 50 g / L; The volume concentration of isopropanol is 5 mL / 10 mL; The addition amount of ketone reductase mutant (whole-cell catalyst) was 50 g / L; Reaction temperature: 40℃; Reaction time: 4 hours; The reaction system is 10 mL.

[0112] Samples of Example 1 and Comparative Example 1 were taken during the 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.

[0113] Samples from Examples 1 to 13 were taken during a reaction time of 1 hour and subjected to chiral HPLC analysis. The relative activity and chiral purity of the R configuration of Examples 1 to 13 were measured, and the results are shown in Table 2.

[0114] 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 in Comparative Example 1 for the R-configuration Remegapan intermediate is only 49%, while the ketoreductase mutant in Example 1 has three mutation sites (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). The chiral purity of the R-configuration Remegapan intermediate of the mutant in Example 1 is 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.

[0115] Table 1. Parameters of Example 1 and Comparative Example Table 2 Parameters of each embodiment The results of Examples 1 to 13 are shown in Table 2. In Example 1, the relative activity of the ketone reductase mutant is 1. In Examples 2 to 13, the mutants have cysteine ​​C at position 190 mutated to valine V, isoleucine I at position 199 mutated to phenylalanine F, and leucine L at position 195 mutated to alanine A, respectively. In addition to the above three mutation sites, the mutants in Examples 2 to 13 also have 1 to 2 mutation sites. The relative activities of the ketone reductase mutants in Examples 2 to 13 are shown based on those in Example 1.

[0116] Table 2 Parameters of each embodiment The mutants of Examples 2, 5, 6, 8 and 12, in addition to the above three mutations, also have a mutation at position 153. The chiral purity of the R configuration of the mutants of Examples 2, 5, 6, 8 and 12 is greater than 99%, and the enzyme activity of the mutants of Examples 2, 5, 6, 8 and 12 is significantly improved compared with Example 1.

[0117] Product concentration change experiment Reaction conditions The reaction buffer was a 100 mM Tris-HCl buffer. Reaction pH: pH 8; The initial concentration of cycloheptano[B]pyridine-5,9-dione was 200 g / L; The volume concentration of isopropanol is 5 mL / 10 mL; The addition amount of ketone reductase mutant (whole-cell catalyst) was 50 g / L; Reaction temperature: 40℃; Reaction time: 24 hours; The reaction system is 10 mL.

[0118] Table 3 Sequences of each embodiment Samples from Example 14 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, and chiral HPLC analysis was performed to measure the product mass concentration. The results are as follows: Figure 10 As shown.

[0119] like Figure 10 As shown, in the entire reaction process, the product generation rate in Example 14 was relatively fast from 1 to 6 hours, the product generation rate slowed down from 6 to 16 hours, and the product generation rate tended to stabilize after 16 hours.

[0120] 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.

[0121] 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 ketoreductase mutant for use in the synthesis of a remegedipam intermediate, characterized in that, It is generated by four mutations in the amino acid sequence shown in SEQ ID NO: 14, namely, 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, and leucine L at position 153 is mutated to threonine T, valine V, phenylalanine F, cysteine ​​C, or glycine G.

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 ketone reductase mutant gene encodes the ketone reductase mutant as described in claim 1 for the synthesis of the intermediate of retinoic acid.

3. A recombinant vector, characterized in that, The recombinant vector includes the ketone reductase mutant gene as described in claim 2.

4. A recombinant engineered bacterium, characterized in that, The engineered bacteria include the recombinant vector as described in claim 3.

5. The recombinant engineered bacteria according to claim 4, characterized in that, The recombinant engineering bacteria is Escherichia coli, and the Escherichia coli is E. coli BL21(DE3).

6. A composition for the preparation of a remegerpam intermediate, characterized in that, include: The ketone reductase mutant for synthesizing the intermediate of retinoic acid as described in claim 1, or the recombinant engineered bacteria as described in claim 1.

7. The composition for preparing a remegedip intermediate according to claim 6, wherein, It also includes isopropanol.

8. A method of bioproduction of a remegapant intermediate, characterized by, include: Using a certain concentration of rimex intermediate precursor ketone as a substrate, recombinant engineered bacteria were added, and the reaction was carried out at 30℃~40℃ in a reaction system with pH 8.0~9.

0. After the reaction was completed, the product was obtained. The recombinant engineered bacteria is an engineered bacterium containing the coding gene of the ketone reductase mutant for synthesizing the intermediate of retinoic acid as described in claim 1.

9. The method for biopreparing the intermediate of retinoic acid according to claim 8, characterized in that, The recombinant engineering bacteria is Escherichia coli, and the Escherichia coli is E. coli BL21(DE3).

10. The method for biopreparing the intermediate of retinoic acid according to claim 9, characterized in that, The mass of the recombinant engineered bacteria is based on the wet weight of the bacterial cells. The ratio of the concentration of the recombinant engineered bacteria to the initial concentration of the intermediate precursor ketone of the recombinant engineered bacteria is 50 g / L: 50 g / L to 300 g / L. The volume ratio of the isopropanol to the reaction system is 0.30 to 0.50:1.

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