Luliconazole intermediate precursor reductase mutant, gene, engineering bacterium and application

Abstract

The invention relates to the technical field of biology, in particular to a luliconazole intermediate precursor reductase mutant, a gene, an engineering bacterium and application. According to the luliconazole intermediate precursor reductase mutant, the gene, the engineering bacterium and application provided by the embodiment of the invention, the amino acid sequences of the luliconazole intermediate precursor reductase mutant are shown as SEQ ID NO: 5-SEQ ID NO: 9, the stereoselectivity of the luliconazole intermediate precursor reductase mutant on asymmetric reduction catalysis of the luliconazole intermediate precursor 2, 2 ', 4'-trichloroacetophenone is improved, and the yield of the luliconazole intermediate precursor reductase mutant is increased. According to the method disclosed by the invention, the S-type luliconazole intermediate (S)-2-chloro-1-(2, 4-dichlorophenyl) ethanol with high chiral purity is obtained, and the synthesis efficiency of the luliconazole intermediate (S)-2-chloro-1-(2, 4-dichlorophenyl) ethanol is favorably improved.

Description

Technical Field

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

[0002] Luliconazole, whose scientific name is 4-(2,4-dichlorophenyl)-1,3-dithiopentane-2-ide-1-imidazolylacetonitrile, has the following chemical structural formula: .

[0003] Luliconazole is a commonly used imidazole-based topical antifungal drug in clinical practice. Its molecule usually contains one or more key stereocenters, and the absolute configuration of these stereocenters may directly determine the drug's activity, selectivity, and safety.

[0004] Currently, an increasing number of drugs and intermediates can be synthesized via biocatalysis. Luriconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol is an important intermediate in the chemical synthesis of luriconazole. Luriconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol can be obtained by the reduction catalysis of 2,2',4'-trichloroacetophenone.

[0005] 2,2',4'-Trichloroacetophenone is a prochiral ketone compound, and the luriconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol is an S-type chiral hydroxyl compound. In the background art, the luriconazole intermediate precursor reductase exhibits low stereoselectivity and catalytic activity in the asymmetric reductive hydrogenation of the luriconazole intermediate precursor 2,2',4'-trichloroacetophenone, which is detrimental to improving the synthesis efficiency of luriconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol. Summary of the Invention

[0006] In view of the above problems, this application provides a ruriconazole intermediate precursor reductase mutant, gene, engineered bacteria, and application to solve the above-mentioned technical problems that are not conducive to improving the synthesis efficiency of ruriconazole intermediates.

[0007] In a first aspect, embodiments of this application provide a luriconazole intermediate precursor reductase mutant, characterized in that the amino acid sequence of the luriconazole intermediate precursor reductase mutant is shown in SEQ ID NO: 5 to SEQ ID NO: 9.

[0008] Secondly, embodiments of this application provide a ruriconazole intermediate precursor reductase mutant gene, wherein the ruriconazole intermediate precursor reductase mutant gene encodes the aforementioned ruriconazole intermediate precursor reductase mutant.

[0009] Thirdly, embodiments of this application provide a recombinant vector comprising the aforementioned louriconazole intermediate precursor reductase mutant gene.

[0010] Fourthly, embodiments of this application provide a recombinant engineered bacterium, which includes the recombinant vector described above.

[0011] Fifthly, embodiments of this application provide a synthesis system for a luriconazole intermediate, the synthesis system comprising recombinant engineered bacteria and isopropanol, wherein the recombinant engineered bacteria is the aforementioned recombinant engineered bacteria.

[0012] Sixthly, embodiments of this application provide a method for synthesizing a luriconazole intermediate. In a reaction system with a luriconazole intermediate precursor concentration of 50 g / L to 300 g / L, the luriconazole intermediate precursor is used as a substrate, and the aforementioned recombinant engineered bacteria are added. The reaction is carried out at 40°C to 50°C in a reaction system with a pH of 5.0 to 7.0 to obtain the product.

[0013] The embodiments of this application provide a luriconazole intermediate precursor reductase mutant, gene, engineered bacteria, and application. The amino acid sequence of the luriconazole intermediate precursor reductase mutant is shown in SEQ ID NO: 5 to SEQ ID NO: 9. It improves the stereoselectivity of asymmetric reduction catalysis of the luriconazole intermediate precursor 2,2',4'-trichloroacetophenone, and obtains the S-type luriconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol with high chiral purity, which is beneficial to improving the synthesis efficiency of luriconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol.

[0014] These or other aspects of this application will become more apparent in the following description of the embodiments. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the reaction mechanism for the synthesis of louliconazole intermediates.

[0016] Figure 2 This is a reaction schematic diagram of another embodiment of the synthesis system for the luliconazole intermediate.

[0017] Figure 3 The image shows the chiral GC analysis spectrum of the racemic product standard in the synthesis method of the luliconazole intermediate in this application.

[0018] Figure 4 The chiral GC analysis spectrum of the standard S configuration in the synthesis method of the luliconazole intermediate in this application embodiment is shown.

[0019] Figure 5The chiral GC analysis spectrum of the standard R configuration in the synthesis method of the luliconazole intermediate in this application embodiment is shown.

[0020] Figure 6 This is a chiral GC analysis spectrum of the reaction product of an embodiment of this application.

[0021] Figure 7 The figure shows the results of the experiment on the optimal reaction temperature optimization of the wild-type luliconazole intermediate precursor reductase.

[0022] Figure 8 The figure shows the results of the pH optimization experiment for the reductase of wild-type luriconazole intermediate precursor.

[0023] Figure 9 Figure shows the results of the experiment optimizing the volume concentration of isopropanol, the intermediate precursor reductase of wild-type louliconazole.

[0024] Figure 10 This graph shows the changes in the concentration of the reductase product of the wild-type luriconazole intermediate precursor.

[0025] Figure 11 The figure shows the results of the optimal reaction temperature optimization experiment for the reductase mutant of the luteconazole intermediate precursor.

[0026] Figure 12 The figure shows the results of the pH optimization experiment for the reductase mutant of the intermediate precursor of luteconazole.

[0027] Figure 13 This graph shows the concentration changes of the product from the reductase mutant of the ruliconazole intermediate precursor.

[0028] Figure 14 The figure shows the results of the experiment on the optimization of isopropanol volume concentration for the reductase mutant of the luteconazole intermediate precursor.

[0029] Figure 15 The figure shows the results of an experiment optimizing the volume concentration of isopropanol for another ruriconazole intermediate precursor reductase mutant. 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 "luriconazole intermediate precursor reductase" and "luriconazole intermediate precursor reductase mutant" refer to enzymes exhibiting asymmetric reduction activity of the prochiral ketone compound 2,2',4'-trichloroacetophenone. These enzymes can asymmetricly reduce the prochiral ketone compound 2,2',4'-trichloroacetophenone to the chiral hydroxyl compound (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol. 2,2',4'-trichloroacetophenone is a luriconazole intermediate precursor, and (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol is a luriconazole intermediate.

[0035] 2,2',4'-Trichloroacetophenone (CAS: 4252-78-2).

[0036] (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol (CAS: 126534-31-4).

[0037] The reaction principle for converting 2,2',4'-trichloroacetophenone to (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol using the aforementioned luliconazole intermediate precursor reductase mutant is as follows: .

[0038] The description herein refers to "a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO: n". Obviously, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO: n, even with some sequence deletions, modifications, substitutions, conservative 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 conservative 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 subjected to the addition of the aforementioned sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conservative 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, where n is a natural number.

[0039] This application provides a ruriconazole intermediate precursor reductase mutant, the amino acid sequence of which is shown in SEQ ID NO: 5 to SEQ ID NO: 9.

[0040] The amino acid sequence of the wild-type luriconazole intermediate precursor reductase is shown in SEQ ID NO: 1. The wild-type luriconazole intermediate precursor reductase exhibits poor stereoselectivity in asymmetric reduction catalysis of luriconazole intermediate precursor, and the chiral purity of the S-type luriconazole intermediate is approximately 69.73%.

[0041] Among them, the amino acid sequence shown in SEQ ID NO: 1 (WP_035452557) is derived from Agrilactobacillus composti The amino acid sequence shown in SEQ ID NO: 1 was used to predict the three-dimensional structure of the protein. Analysis of the predicted three-dimensional structure of the protein revealed that positions 94, 96, 145, 147, 165, 196, 199, 202 and 226 in the amino acid sequence shown in SEQ ID NO: 1 are key catalytic sites.

[0042] During the mutation screening process, it was found that the mutation of lysine K at position 96 to valine V, phenylalanine F at position 165 to leucine L, and glutamic acid E at position 202 to leucine L in the amino acid sequence shown in SEQ ID NO: 1 was beneficial to improving stereoselectivity, and the chiral purity of the S-type luliconazole intermediate was improved compared with the wild type.

[0043] During the mutation screening process, it was found that, based on the mutation of lysine K at position 96 to valine V, phenylalanine F at position 165 to leucine L, and glutamic acid E at position 202 to leucine L in the amino acid sequence shown in SEQ ID NO: 1, the mutation of glycine G at position 145 to alanine A was further observed, which is beneficial to improving stereoselectivity. The chiral purity of the S-type luliconazole intermediate was increased to over 96%.

[0044] During the mutation screening process, it was found that, based on the mutation of lysine K at position 96 to valine V, phenylalanine F at position 165 to leucine L, glutamic acid E at position 202 to leucine L, and glycine G at position 145 to alanine A in the amino acid sequence shown in SEQ ID NO: 1, the mutation of serine S at position 199 to histidine H was further observed. This is beneficial to improving stereoselectivity, and the chiral purity of the S-type luliconazole intermediate was increased to over 99%.

[0045] In this embodiment, a luriconazole intermediate precursor reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: lysine K at position 96 is mutated to valine V, phenylalanine F at position 165 is mutated to leucine L, glutamic acid E at position 202 is mutated to leucine L, and glycine G at position 145 is mutated to alanine A (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: 1 are replaced by other amino acids. The aforementioned luriconazole intermediate precursor reductase mutant has the amino acid sequence shown in SEQ ID NO: 5.

[0046] In this embodiment, a luriconazole intermediate precursor reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: lysine K at position 96 is mutated to valine V, phenylalanine F at position 165 is mutated to leucine L, glutamic acid E at position 202 is mutated to leucine L, glycine G at position 145 is mutated to alanine A, and isoleucine I at position 147 is mutated to leucine L (SEQ ID NO: 6). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 1 are replaced by other amino acids. The aforementioned luriconazole intermediate precursor reductase mutant has the amino acid sequence shown in SEQ ID NO: 6.

[0047] In this embodiment, a luriconazole intermediate precursor reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: lysine K at position 96 is mutated to valine V, phenylalanine F at position 165 is mutated to leucine L, glutamate E at position 202 is mutated to leucine L, glycine G at position 145 is mutated to alanine A, and glutamate E at position 196 is mutated to leucine L (SEQ ID NO: 7). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 1 are replaced by other amino acids. The aforementioned luriconazole intermediate precursor reductase mutant has the amino acid sequence shown in SEQ ID NO: 7.

[0048] In this embodiment, a luriconazole intermediate precursor reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: lysine K at position 96 is mutated to valine V, phenylalanine F at position 165 is mutated to leucine L, glutamic acid E at position 202 is mutated to leucine L, glycine G at position 145 is mutated to alanine A, and serine S at position 199 is mutated to histidine H (SEQ ID NO: 8). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 1 are replaced by other amino acids. The aforementioned luriconazole intermediate precursor reductase mutant has the amino acid sequence shown in SEQ ID NO: 8.

[0049] In this embodiment, a luliconazole intermediate precursor reductase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: lysine K at position 96 is mutated to valine V, phenylalanine F at position 165 is mutated to leucine L, glutamic acid E at position 202 is mutated to leucine L, glycine G at position 145 is mutated to alanine A, and cysteine ​​C at position 226 is mutated to valine V (SEQ ID NO: 9). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 1 are replaced by other amino acids. The aforementioned luliconazole intermediate precursor reductase mutant has the amino acid sequence shown in SEQ ID NO: 9.

[0050] In this embodiment, the amino acid sequence of the luriconazole intermediate precursor reductase mutant is shown in SEQ ID NO: 5 to SEQ ID NO: 9. It improves the stereoselectivity of the asymmetric reductive hydrogenation catalysis of the luriconazole intermediate precursor, resulting in a high-chiral-purity S-type luriconazole intermediate (the chiral purity of the S-type luriconazole intermediate is above 94.00%), which is beneficial to improving the synthesis efficiency of the luriconazole intermediate.

[0051] This application provides a ruriconazole intermediate precursor reductase mutant gene, which encodes the aforementioned ruriconazole intermediate precursor reductase mutant.

[0052] The luriconazole intermediate precursor reductase mutant gene can be a polynucleotide. This polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides. This polynucleotide has the nucleotide sequences corresponding to SEQ ID NO: 5 to SEQ ID NO: 9.

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

[0054] This application provides a recombinant vector comprising the aforementioned louriconazole intermediate precursor reductase mutant gene.

[0055] For example, the recombinant vector comprises the polynucleotide encoding the ruliconazole intermediate precursor reductase mutant described above.

[0056] The recombinant vector is any naturally or artificially constructed expression vector encoding the nucleus molecule of the luriconazole intermediate precursor reductase mutant, which can be catalyzed by cellular transcriptases and / or translatases.

[0057] Specifically, a recombinant vector is a DNA preparation containing a polynucleotide sequence encoding a luriconazole intermediate precursor reductase mutant. It may also contain a control sequence. In the recombinant vector, the polynucleotide sequence encoding the luriconazole intermediate precursor reductase mutant is operatively linked to a suitable control sequence, allowing the luriconazole intermediate precursor 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 for replication or function.

[0058] 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.

[0059] This application provides a recombinant engineered bacterium, which includes the recombinant vector described above.

[0060] In this embodiment, the recombinant engineered bacteria serves as the host cell. The recombinant vector described above is transformed into the host cell, enabling the synthesis of the luriconazole intermediate precursor reductase mutant within the host cell. The recombinant vector is introduced into the host cell, and the polynucleotide encoding the luriconazole intermediate precursor reductase mutant in the recombinant vector is expressed in the host cell, allowing the host cell to synthesize the aforementioned luriconazole intermediate precursor 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. For example, 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.

[0061] The host cell can be a eukaryotic cell or a prokaryotic cell, and further, the prokaryotic cell can be a bacterial cell.

[0062] 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 ).

[0063] For example, the recombinant engineered bacteria is *Escherichia coli*, which is... E. coli BL21(DE3).

[0064] This application provides a synthesis system for a luliconazole intermediate, comprising a recombinant engineered bacterium and a co-substrate. The recombinant engineered bacterium is a recombinant engineered bacterium containing a gene encoding a luliconazole intermediate precursor reductase mutant. The amino acid sequence of the luliconazole intermediate precursor reductase mutant is shown in SEQ ID NO: 5 to SEQ ID NO: 9.

[0065] Among them, 2,2',4'-trichloroacetophenone, a prochiral ketone, is a precursor of the luliconazole intermediate. Using 2,2',4'-trichloroacetophenone as a substrate and a luliconazole intermediate reductase mutant as a catalyst, the asymmetric reduction catalytic activity of the luliconazole intermediate reductase mutant is utilized to convert 2,2',4'-trichloroacetophenone into the luliconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol, with the luliconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol as the product.

[0066] The asymmetric reduction catalytic reaction of the luriconex intermediate precursor reductase mutant requires the presence of a coenzyme.

[0067] 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.

[0068] like Figure 1 As shown, in the asymmetric reductive hydrogenation reaction of the substrate 2,2',4'-trichloroacetophenone catalyzed by the luliconazole intermediate precursor reductase mutant to generate the luliconazole intermediate (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol, NADPH is converted to NADP. + .

[0069] In the embodiments of this application, both the luriconazole intermediate precursor reductase mutant and the wild-type luriconazole intermediate precursor 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.

[0070] 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.

[0071] The recombinant engineered bacteria contain coenzymes within their cells, so the synthesis system for this luriconazole intermediate does not require the addition of additional coenzymes.

[0072] In this embodiment, the luriconazole intermediate precursor 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 S-type chiral hydroxyl compounds with high optical purity (ee of SEQ ID NO: 8 is greater than 99.0%), which has good prospects for industrial application. In this embodiment, the luriconazole intermediate precursor reductase mutant exhibits high conversion rate and high chiral selectivity for the luriconazole intermediate precursor 2,2',4'-trichloroacetophenone.

[0073] As one implementation method, please refer to Figure 2 As shown, the co-substrate can be isopropanol, which is oxidized by the luliconazole intermediate precursor reductase mutant to form acetone.

[0074] 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 recombinant engineered bacteria embodiments, which will not be repeated here.

[0075] In some embodiments, the recombinant engineered bacteria used in the synthesis system can be wet cells obtained by inducing and culturing recombinant engineered bacteria.

[0076] In some embodiments, the recombinant engineered bacteria is Escherichia coli. E. coli BL21(DE3).

[0077] 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.

[0078] In some embodiments, the reaction system further includes a buffer solution, and the volume percentage of isopropanol in the reaction system is 30% to 60%, for example, 40%. Specifically, the reaction system includes isopropanol, a buffer solution, a substrate, 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.

[0079] In some implementations, the buffer is a PBS buffer, and the pH of the reaction system is 5.0 to 7.0.

[0080] This application provides a method for synthesizing a luriconazole intermediate. In a reaction system with a luriconazole intermediate precursor concentration of 50 g / L to 300 g / L, the luriconazole intermediate precursor is used as a substrate, and the above-mentioned recombinant engineered bacteria are added. The reaction is carried out at 40°C to 50°C in a reaction system with a pH of 5.0 to 7.0 to obtain the product.

[0081] In one implementation method, the reaction system also includes isopropanol, and the volume ratio of isopropanol to the reaction system is 0.30 to 0.60:1.

[0082] In some embodiments, the volume ratio of isopropanol to the reaction system is 0.4:1.

[0083] This application provides the use of the above-mentioned luriconazole intermediate precursor reductase mutant in the preparation of luriconazole.

[0084] This application provides the application of the above-mentioned recombinant engineered bacteria in the preparation of ruliconazole.

[0085] Preparation of Luliconazole intermediate precursor reductase mutant The encoding gene for the luliconazole intermediate precursor reductase mutant and the luliconazole intermediate precursor reductase can be called the LKCR gene. The LKCR gene contains the nucleotide sequence corresponding to the amino acid sequence of the aforementioned luliconazole intermediate precursor reductase mutant or luliconazole intermediate precursor reductase. For example, the LKCR gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 5 to SEQ ID NO: 9. The LKCR gene is constructed in the pET-28a plasmid to obtain a recombinant vector.

[0086] The recombinant vector is a pET-28a plasmid containing the LKCR gene, hereinafter referred to as pET-28a-LKCR; 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: 5 to SEQ ID NO: 9 in the various embodiments of this application and Comparative Example 1 are then used as the target expression gene to construct the vector.

[0087] Expression of the gene encoding the luteconazole intermediate precursor reductase mutant a. Transform pET-28a-LKCR into E. coli E. coli In BL21(DE3), select a single clone of pET-28a-LKCR 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex mixer.

[0093] 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 the luliconazole intermediate precursor reductase mutant.

[0094] 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 ℃.

[0095] Purification of the mutant protein of ruliconazole intermediate precursor reductase 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 the luriconazole intermediate precursor reductase mutant.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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 ℃.

[0101] Electrophoretic analysis of the mutant protein of ruliconazole intermediate precursor reductase 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.

[0102] 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.

[0103] 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.

[0104] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0105] Enzyme-catalyzed reactions In vitro enzyme catalytic reaction conditions: Buffer for the reaction (may not include buffer): PBS buffer at concentrations of 100mM, 200mM, and 300mM; Reaction pH: pH5, pH6, pH7; The concentrations of 2,2',4'-trichloroacetophenone were 50 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L, and 300 g / L. The volume concentrations of isopropanol are 4 mL / 10 mL, 5 mL / 10 mL, 6 mL / 10 mL, 7 mL / 10 mL, 8 mL / 10 mL, 9 mL / 10 mL, and 10 mL / 10 mL. The amount of *E. coli* expressing the gene encoding the ruriconazole intermediate precursor reductase mutant (the whole-cell catalyst obtained in the above steps) added was 50 g / L and 100 g / L. Reaction temperatures: 35℃, 40℃, 45℃; 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.

[0106] Detection of (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol Detection method: BGB-174 chiral column; Mobile phase: nitrogen / hydrogen; flow rate: 0.6 ml / min; Temperature program: 100℃ held for 1 min, heating rate 25℃ / min to 160℃, held for 1 min, heating rate 5℃ / min to 210℃, held for 5 min; Detector: FID; in, Figure 3 , Figure 4 and Figure 5 Spectral analysis was performed on the purchased standard samples for result comparison. Figure 3 The chiral GC (Gas Chromatography) spectrum of the racemic mixture of the product standard is used to compare the chiral differences between the reaction product and the racemic mixture. Figure 4 The chiral GC analysis spectrum of the standard S-configuration is used to confirm that the reaction product is S-configuration. Figure 5 The chiral GC spectrum of the standard R configuration is used to identify the reaction byproducts of the R configuration and to compare the chiral characteristics of the reaction products; according to Figure 3 , Figure 4 and Figure 5 The comparison confirms the S-configuration reaction product, which elutes at approximately 16.58 min, while the R-configuration reaction byproduct elutes at approximately 16.49 min. Figure 6 Here is an example of the chiral GC analysis spectrum of the reaction products in one embodiment (Example 7). The S-configuration reaction product elutes at approximately 16.575 min, the R-configuration reaction byproduct elutes at approximately 16.49 min, and the substrate elutes at approximately 14.6 min. Figure 6 As shown, both the reaction byproducts and substrates of the R configuration are below the detection limit. The concentrations of the substrate, the reaction products of the S configuration, and the reaction byproducts of the R configuration can be obtained by chiral GC analysis. The chiral purity of the S configuration can be calculated by the ratio of the concentrations of the reaction products of the S configuration and the reaction byproducts of the R configuration.

[0107] It should be noted that in the following examples and comparative examples, the product specifically refers to the S-configuration reaction product ((S)-2-chloro-1-(2,4-dichlorophenyl)ethanol), and the substrate specifically refers to 2,2',4'-trichloroacetophenone. During the measurement of product and substrate concentrations, the dilution of the reaction solution and liquid phase detection will introduce detection errors, leading to differences in the same concentration at different measurement points in the product or substrate concentration change curves.

[0108] 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%.

[0109] Catalytic conditions of wild-type luliconazole intermediate precursor reductase a) Wild-type reaction temperature comparison experiment The reaction temperatures were 30℃, 35℃, 40℃, 45℃, and 50℃, respectively. Other reaction conditions are as follows: The reaction buffer was a 100 mM PBS buffer with a volume concentration of 6 mL / 10 mL. Reaction pH: pH 6; The initial concentration of 2,2',4'-trichloroacetophenone was 50 g / L; The volume concentration of isopropanol is 4 mL / 10 mL; The amount of *E. coli* expressing the gene encoding the luliconazole intermediate precursor reductase (the whole-cell catalyst obtained in the above steps, with the amino acid sequence shown in SEQ ID NO: 1) added was 50 g / L. Reaction time: 30 minutes; The reaction system is 10 mL.

[0110] Please see Figure 7 As shown, the reductase of the intermediate precursor of ruliconazole exhibits relatively good activity at reaction temperatures of 40℃ to 50℃, with the activity being optimal at a reaction temperature of 45℃.

[0111] b) Wild-type pH comparison experiment The reaction buffers and their pH values ​​were: pH 4.0 (acetic acid-sodium acetate buffer), pH 5.0 (acetic acid-sodium acetate buffer), pH 5.0 (PBS buffer), pH 6.0 (PBS buffer), pH 7.0 (PBS buffer), pH 8.0 (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 2,2',4'-trichloroacetophenone was 50 g / L; The volume concentration of isopropanol is 4 mL / 10 mL; The buffer concentration is 6 mL / 10 mL; The amount of *E. coli* expressing the gene encoding the luliconazole intermediate precursor reductase (the whole-cell catalyst obtained in the above steps, with the amino acid sequence shown in SEQ ID NO: 1) added was 50 g / L. Reaction temperature: 45℃; Reaction time: 30 minutes; The reaction system is 10 mL.

[0112] Please see Figure 8 As shown, the reductase of the intermediate precursor of luteconazole exhibits relatively good activity in PBS buffer at a reaction pH of 5.0–7.0; the reductase of the intermediate precursor of luteconazole exhibits optimal activity in PBS buffer at a reaction pH of 6.0.

[0113] c) Comparison experiment of different isopropanol volume concentrations in wild-type plants Isopropanol volume concentrations: 3 mL / 10 mL (30% volume concentration), 4 mL / 10 mL (40% volume concentration), 5 mL / 10 mL (50% volume concentration), 6 mL / 10 mL (60% volume concentration). Other reaction conditions are as follows: The initial concentration of 2,2',4'-trichloroacetophenone was 50 g / L; The buffer solution is PBS buffer; Reaction pH: pH 6; The amount of wild-type luriconazole intermediate precursor reductase (whole-cell catalyst) added was 50 g / L; Reaction temperature: 45℃; Reaction time: 30 minutes; The reaction system is 10 mL.

[0114] Please see Figure 9 As shown, the reductase of the ruliconazole intermediate precursor exhibits the best activity at a volume concentration of 40% isopropanol.

[0115] d) Wild-type product concentration change experiment The initial concentration of 2,2',4'-trichloroacetophenone was 50 g / L; The volume concentration of isopropanol is 4 mL / 10 mL; The PBS buffer concentration is 6 mL / 10 mL. Reaction pH: pH 6; The amount of wild-type luriconazole intermediate precursor reductase (whole-cell catalyst) added was 50 g / L; Reaction temperature: 45℃; Reaction time: 24 hours; The reaction system is 10 mL.

[0116] Samples 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 for chiral GC analysis. The product mass concentration was measured, and the results are as follows: Figure 10 As shown.

[0117] Screening experiments on mutant enzyme activity and R conformation chiral purity The amino acid sequences of the luliconazole intermediate precursor reductase mutants in Examples 1 to 8 are shown in SEQ ID NO: 2 to SEQ ID NO: 9, and the amino acid sequence of the luliconazole intermediate precursor reductase in Comparative Example 1 is shown in SEQ ID NO: 1.

[0118] Catalytic reaction conditions of Examples 1 to 8 and Comparative Example 1 The initial concentration of 2,2',4'-trichloroacetophenone was 50 g / L; The volume concentration of isopropanol is 4 mL / 10 mL; The PBS buffer concentration is 6 mL / 10 mL. Reaction pH: pH 6; The addition amount of the luliconazole intermediate precursor reductase mutant (whole-cell catalyst) was 50 g / L; Reaction temperature: 45℃; Reaction time: 30 minutes; The reaction system is 10 mL.

[0119] Samples were taken from Examples 1 to 8 and Comparative Example 1 during a reaction time of 30 minutes for chiral GC analysis. The relative enzyme activity and S-configuration chiral purity of Examples 1 to 8 and Comparative Example 1 were measured, and the results are shown in Table 1.

[0120] Table 1 Parameters of each embodiment The results of Examples 1 to 8 and Comparative Example 1 are shown in Table 1. In Comparative Example 1, the relative activity of the luriconazole intermediate precursor reductase is 1. The relative activities of the luriconazole intermediate precursor reductase mutants in Examples 1 to 8 are shown based on Comparative Example 1.

[0121] Table 1 Parameters of Examples 1 to 8 and Comparative Example 1 As shown in Table 1, the S-configuration chiral purity of the mutants in Examples 4 to 8 is above 94%, and the S-configuration chiral purity of the mutant in Example 7 is above 99%. Furthermore, the enzyme activity of the mutants in Examples 4 to 8 is significantly increased compared to Comparative Example 1.

[0122] Catalytic conditions experiment of the luteconazole intermediate precursor reductase mutant a) Mutant reaction temperature comparison experiment The reaction temperatures were 30℃, 35℃, 40℃, 45℃, and 50℃, respectively. Other reaction conditions are as follows: The reaction buffer was a 100 mM PBS buffer with a volume concentration of 6 mL / 10 mL. Reaction pH: pH 6; The initial concentration of 2,2',4'-trichloroacetophenone was 50 g / L; The volume concentration of isopropanol is 4 mL / 10 mL; The amount of *E. coli* expressing the gene encoding the ruriconazole intermediate precursor reductase mutant (the whole-cell catalyst obtained in the above steps, with the amino acid sequence shown in SEQ ID NO: 8) added was 50 g / L. Reaction time: 30 minutes; The reaction system is 10 mL.

[0123] Please see Figure 11 As shown, the ruliconazole intermediate precursor reductase mutant exhibits relatively good activity at reaction temperatures of 40℃ to 50℃, with the optimal activity at a reaction temperature of 45℃.

[0124] b) Comparison experiment of mutants at different pH values The reaction buffers and their pH values ​​were: pH 4.0 (acetic acid-sodium acetate buffer), pH 5.0 (acetic acid-sodium acetate buffer), pH 5.0 (PBS buffer), pH 6.0 (PBS buffer), pH 7.0 (PBS buffer), pH 8.0 (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 2,2',4'-trichloroacetophenone was 50 g / L; The volume concentration of isopropanol is 4 mL / 10 mL; The buffer concentration is 6 mL / 10 mL; The amount of *E. coli* expressing the gene encoding the ruriconazole intermediate precursor reductase mutant (the whole-cell catalyst obtained in the above steps, with the amino acid sequence shown in SEQ ID NO: 8) added was 50 g / L. Reaction temperature: 45℃; Reaction time: 30 minutes; The reaction system is 10 mL.

[0125] Please see Figure 12 As shown, the reductase of the intermediate precursor of luteconazole exhibits relatively good activity in PBS buffer at a reaction pH of 5.0–7.0; the reductase of the intermediate precursor of luteconazole exhibits optimal activity in PBS buffer at a reaction pH of 6.0.

[0126] C) Experiment on the concentration change of mutant products The initial concentration of 2,2',4'-trichloroacetophenone was 400 g / L; The volume concentration of isopropanol is 4 mL / 10 mL; The PBS buffer concentration is 6 mL / 10 mL. Reaction pH: pH 6; The addition amount of the luliconazole intermediate precursor reductase mutant (whole-cell catalyst) was 50 g / L; The amino acid sequence of the luliconazole intermediate precursor reductase mutant is SEQ ID NO: 8; Reaction temperature: 45℃; Reaction time: 24 hours; The reaction system is 10 mL.

[0127] Samples were taken at reaction times of 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, and 24 hours for chiral GC analysis. The product mass concentration was measured, and the results are as follows: Figure 13 As shown.

[0128] like Figure 13 As shown, during the reaction time of 0 to 1 hour, the substrate was consumed at an extremely rapid rate, and the product increased at an extremely rapid rate; during the reaction time of 1 to 2 hours, the substrate was consumed at a relatively rapid rate, and the product increased at a relatively rapid rate; during the reaction time of 2 to 8 hours, the substrate consumption rate slowed down slightly but remained relatively fast, and the product increase rate slowed down slightly but remained relatively fast; during the reaction time of 8 to 18 hours, the substrate consumption rate was relatively slow, and the product increase rate was relatively slow.

[0129] d) Comparison experiment of different isopropanol volume concentrations in mutants Isopropanol volume concentrations: 3 mL / 10 mL (30% volume concentration), 4 mL / 10 mL (40% volume concentration), 5 mL / 10 mL (50% volume concentration), 6 mL / 10 mL (60% volume concentration). Other reaction conditions are as follows: The initial concentration of 2,2',4'-trichloroacetophenone was 50 g / L; The buffer solution is PBS buffer; Reaction pH: pH 6; The addition amount of the luliconazole intermediate precursor reductase mutant (whole-cell catalyst) was 50 g / L; The amino acid sequence of the luliconazole intermediate precursor reductase mutant is SEQ ID NO: 8; Reaction temperature: 45℃; Reaction time: 30 minutes; The reaction system is 10 mL.

[0130] Please see Figure 14 As shown, the ruliconazole intermediate precursor reductase mutant exhibits the best relative activity at an isopropanol volume concentration of 40%.

[0131] e) Comparison experiment of different isopropanol volume concentrations in mutants Isopropanol volume concentrations: 4 mL / 10 mL (40% volume concentration), 5 mL / 10 mL (50% volume concentration), 6 mL / 10 mL (60% volume concentration), 7 mL / 10 mL (70% volume concentration), 8 mL / 10 mL (80% volume concentration), 9 mL / 10 mL (90% volume concentration). Other reaction conditions are as follows: The initial concentration of 2,2',4'-trichloroacetophenone was 300 g / L; The buffer solution is PBS buffer; Reaction pH: pH 6; The addition amount of the luliconazole intermediate precursor reductase mutant (whole-cell catalyst) was 50 g / L; The amino acid sequence of the luliconazole intermediate precursor reductase mutant is SEQ ID NO: 8; Reaction temperature: 45℃; Reaction time: 24 hours; The reaction system is 10 mL.

[0132] Samples were taken at reaction times of 2 hours, 6 hours, and 24 hours for chiral GC analysis to measure the product mass concentration. The results are as follows: Figure 15 As shown.

[0133] Please see Figure 15 As shown, the ruliconazole intermediate precursor reductase mutant can almost completely consume the substrate at isopropanol concentrations ranging from 40% to 90%.

[0134] The above description is merely an embodiment of this application. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of this application, but these improvements all fall within the protection scope of this application.

Claims

1. A luliconazole intermediate precursor reductase mutant characterized by, The amino acid sequences of the luliconazole intermediate precursor reductase mutants are shown in SEQ ID NO: 5 to SEQ ID NO:

9.

2. A mutant gene of a precursor reductase of a luliconazole intermediate, characterized in that, The luliconazole intermediate precursor reductase mutant gene encodes the luliconazole intermediate precursor reductase mutant as described in claim 1.

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

4. A recombinant engineered bacterium, characterized in that, The recombinant 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 system for the synthesis of an intermediate of luliconazole, characterized by, The synthesis system for the luliconazole intermediate includes a recombinant engineered bacterium and isopropanol, wherein the recombinant engineered bacterium is the recombinant engineered bacterium as described in claim 4.

7. The synthesis system of a luliconazole intermediate according to claim 6, characterized by, The volume ratio of isopropanol to the reaction system is 30% to 60%.

8. The synthetic system for the luriconezol intermediate according to claim 7, characterized in that, The reaction system also includes PBS buffer, the pH of which is 5.0 to 7.

0.

9. A method for synthesizing a louriconazole intermediate, characterized in that, In a reaction system with a concentration of 50 g / L to 300 g / L of the luriconazole intermediate precursor, the recombinant engineered bacteria as described in claim 4 were added as a substrate, and the reaction was carried out at 40°C to 50°C in a reaction system with a pH of 5.0 to 7.0 to obtain the product.

10. The method for synthesizing the ruliconazole intermediate according to claim 9, characterized in that, The reaction system includes isopropanol, and the volume ratio of isopropanol to the reaction system is 0.30 to 0.60:1.

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