Alcohol dehydrogenase mutant and application thereof in synthesis of (S)-6-hydroxy-8-chloro ethyl caprylate with high optical purity

Abstract

The invention discloses an alcohol dehydrogenase mutant and application of the alcohol dehydrogenase mutant in asymmetric synthesis of (S)-6-hydroxy-8-chloro ethyl caprylate with optical purity. The alcohol dehydrogenase or the mutant thereof provided by the invention has extremely high conversion rate and can effectively improve the optical purity of the product. Moreover, based on the mutant, low-cost, efficient and rapid production of single-configuration ethyl 6-hydroxy-8-chloro caprylate can be realized, a multi-configuration mixture is not generated, and the problems of tedious steps, pollution and the like caused by a conventional chemical synthesis method are also avoided.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis, in particular to an alcohol dehydrogenase mutant and application thereof in synthesizing high optical purity (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester. Background Art

[0002] Alpha-Lipoic Acid (α-LA) belongs to the B vitamins. It is the only "universal antioxidant" that is both fat-soluble and water-soluble. It can efficiently remove free radicals that are prone to cause disease and accelerate aging, inhibit lipid oxidation in nerve tissue, and prevent protein glycosylation. The antioxidant capacity of α-lipoic acid is 5-10 times that of grape seeds and 60 times that of vitamin E, and it can be absorbed by various tissues and organs. Clinically, α-lipoic acid can be used to treat diabetic peripheral neuropathy and diabetic nephropathy. α-lipoic acid has a chiral center, so it has two configurations, R and S. Among them, only the R-configuration of α-lipoic acid can be covalently conjugated to lysine residues through amide bonds to play the function of a coenzyme, so in fact only the R-configuration has effective physiological activity. However, artificially synthesized α-lipoic acid is a mixture (racemic product) composed of equal proportions of R-configuration and S-configuration, and the cost of obtaining R-configuration α-lipoic acid by splitting is too high, making the price of R-configuration α-lipoic acid more than 5 times that of its racemic product, so most of the products on the market are actually limited to racemic products. In order to reduce the burden of S-isomers in human body metabolism racemic products, efficiently preparing optically pure R-configuration α-lipoic acid has important application value and broad market prospects.

[0003] Existing methods for synthesizing (R)-α-lipoic acid can be divided into chemical methods and biological methods. Elliott et al. carried out asymmetric synthesis of (R)-α-lipoic acid by induction of chiral auxiliary agents, and the total yield can reach 37%, but the reaction conditions are too harsh and the cost of the reagents used is too high, which limits the application of this method in industry (Tetrahedron Letters, 1985, 26 (21): 2535-2538). Gopalan et al. tried to generate (R)-α-lipoic acid by microbial enzyme catalysis, but the total yield was only 10%, and the operability was not strong (Journal of the Chemical Society, Perkin Trans., 1990, 7: 1897-1900). One of the most studied synthesis methods is enantiomeric resolution, which uses a chiral resolution agent or esterase / lipase to resolve racemic α-lipoic acid or its precursor, and then converts it into (R)-α-lipoic acid. However, (S)-α-lipoic acid is generated during this process, so its theoretical maximum yield is only 50%, and the resolution yield in actual industrial applications is only about 30%. Constructing the chiral center first during the synthesis process has become an important step to break through the resolution yield constraint.

[0004] The preparation of optically active 6-hydroxy-8-chlorooctanoate intermediates by enzymatic reduction has gradually attracted attention from the industry. In theory, high optical purity (R)-α-lipoic acid can be obtained by constructing R- or S-configuration 6-hydroxy-8-chlorooctanoate ethyl ester and further reacting it. The current mainstream reported methods focus on the R configuration. For example, Olbrich et al. used Geotrichum candidum whole cells to catalyze the asymmetric reduction of 6-carbonyl-8-chlorooctanoate ethyl ester to generate (R)-6-hydroxy-8-chlorooctanoate ethyl ester; Müller et al. used alcohol dehydrogenase TbADH from Thermoanaerobium brokii to catalyze the production of R-type products; Werner et al. used NgADH from Nocardia globulera to prepare (R)-6-hydroxy-8-chlorooctanoate ethyl ester, and the construction of S-configuration 6-hydroxy-8-chlorooctanoate has not been reported. In particular, the (S)-6-hydroxy-8-chlorooctanoate intermediate with high optical purity and cost advantage has important scientific and industrial value. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide an alcohol dehydrogenase mutant and its use in asymmetric synthesis of optically pure (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester. In particular, the alcohol dehydrogenase mutant of the present invention can effectively overcome the need for a large amount of cofactor NADP when using carbonyl reductase to prepare (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester. +The problems of too long catalytic time and too low yield were solved, and the production of (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester with low cost, high efficiency, rapidity, high yield and high purity was achieved. It also avoided the problems of cumbersome steps and pollution caused by conventional chemical synthesis methods.

[0006] The first aspect of the present invention provides an alcohol dehydrogenase or a mutant thereof, wherein the alcohol dehydrogenase or a mutant thereof comprises:

[0007] (1) using the alcohol dehydrogenase shown in any one of SEQ ID NOs: 1-6 as a template, replacing 0 to 10 amino acid residues therein to obtain an alcohol dehydrogenase or a mutant thereof; or

[0008] (2) The alcohol dehydrogenase or its mutant in (1) wherein amino acids are added or deleted and the alcohol dehydrogenase or its mutant still retains the dehydrogenation effect.

[0009] In some embodiments of the present invention, the alcohol dehydrogenase mutant described in (1) is an alcohol dehydrogenase mutant obtained by replacing 0 to 5 amino acid residues in the alcohol dehydrogenase shown in SEQ ID NO: 5 as a template.

[0010] In some embodiments of the invention, amino acid residue replacements include: S58C, S58R, S91T, S91L, G105A, G105F, T106I, T106F, T106A, S117L, S117I, S117A, S131A, S131L, S131I, L147A, L147C, L147F, V156A, V156L, V164A, V164L, G199W, G199L, L205A, L205F, L205V, S207V, S207L, S207N, Y209I, Y209L, Y209V, Y209F, I223T, I223A and Y240F.

[0011] In some embodiments of the present invention, when SEQ ID When the alcohol dehydrogenase shown in NO:5 is used as a template, the amino acid residue replacement is selected from at least one of S58C, S58R, S91T, S91L, G105A, G105F, T106I, T106F, T106A, S117L, S117I, S117A, S131A, S131L, S131I, L147A, L147C, L147F, V156A, V156L, V164A, V164L, G199W, G199L, L205A, L205F, L205V, S207V, S207L, S207N, Y209I, Y209L, Y209V, Y209F, I223T, I223A and Y240F.

[0012] In some embodiments of the present invention, when the alcohol dehydrogenase shown in SEQ ID NO: 5 is used as a template, the amino acid residue replacement is selected from S58C, S58R, S91T, S91L, G105A, G105F, T106I, T106F, T106A, S117L, S117I, S117A, S131A, S131L, S131I, L147A, L147C, L147F, V15 6A, V156L, V164A, V164L, G199W, G199L, L205A, L205F, L205V, S207V, S207L, S207 N, Y209I, Y209L, Y209V, Y209F, I223T, I223A, Y240F, T106F / L205A and T106A / L205A.

[0013] In some embodiments of the present invention, the alcohol dehydrogenase or its mutant in (2) has at least 90% sequence identity with the alcohol dehydrogenase or its mutant in (1).

[0014] In some embodiments of the present invention, the alcohol dehydrogenase or its mutant in (2) has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the alcohol dehydrogenase or its mutant in (1).

[0015] The second aspect of the present invention provides a nucleic acid molecule encoding the alcohol dehydrogenase or a mutant thereof according to the first aspect of the present invention.

[0016] In some embodiments of the present invention, the nucleic acid molecule further contains a non-coding sequence.

[0017] In some embodiments of the present invention, the non-coding sequence includes a promoter, a terminator, an intron, a cis-acting element, an enhancer, a tailing signal and a repetitive sequence. Of course, those skilled in the art can reasonably add other non-coding sequences according to actual use requirements, including but not limited to the above non-coding sequences.

[0018] In some embodiments of the present invention, the non-coding sequence does not affect the expression of alcohol dehydrogenase or its mutant.

[0019] In some embodiments of the invention, the nucleic acid molecule comprises SEQ ID NOs: 7-12.

[0020] The third aspect of the present invention provides a product, the product comprising at least one of the following (1)-(3):

[0021] (1) an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention;

[0022] (2) A transformant containing the nucleic acid molecule according to the second aspect of the present invention;

[0023] (3) A transformant containing the expression vector described in (1).

[0024] In some embodiments of the invention, the expressor comprises a plasmid.

[0025] In some embodiments of the present invention, the transformants include viruses, bacteria, fungi, plant cells and animal cells.

[0026] In some embodiments of the invention, the plant cells and animal cells do not include propagation material.

[0027] In some embodiments of the present invention, the product further contains pharmaceutically acceptable excipients.

[0028] In some embodiments of the present invention, the pharmaceutically acceptable excipients include but are not limited to diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, cross-linked polyvinylpyrrolidone, etc.), lubricants (such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0029] In some embodiments of the present invention, the transformants in the product are in the form of freeze-dried powder, bacterial liquid, and granular inoculation agent. Of course, it should be understood that those skilled in the art can also reasonably select an appropriate form for use according to actual use requirements and existing processing techniques, including but not limited to the freeze-dried powder, bacterial liquid, and granular inoculation agent mentioned above.

[0030] The fourth aspect of the present invention provides the use of the alcohol dehydrogenase or its mutant described in the first aspect of the present invention in the synthesis of 6-hydroxy-8-chlorooctanoic acid ethyl ester.

[0031] In some embodiments of the present invention, the 6-hydroxy-8-chlorooctanoic acid ethyl ester is 6-hydroxy-8-chlorooctanoic acid ethyl ester of S configuration.

[0032] A fifth aspect of the present invention provides a method for preparing S-configuration 6-hydroxy-8-chlorooctanoic acid ethyl ester, comprising the following steps:

[0033] The alcohol dehydrogenase or its mutants described in the first aspect of the present invention or one or a combination of products expressing the alcohol dehydrogenase or its mutants described in the first aspect of the present invention is mixed with 6-carbonyl-8-chlorooctanoic acid ethyl ester, formate dehydrogenase or one or a combination of products expressing formate dehydrogenase, 6-carbonyl-8-chlorooctanoic acid ethyl ester and formate dehydrogenase substrate, and reacted at 25-35° C. for 1-24 hours to obtain the product.

[0034] In some embodiments of the present invention, the formate dehydrogenase substrate comprises ammonium formate, nicotinamide adenine dinucleoside phosphate (NADP + ) and nicotinamide adenine dinucleotide (NAD + ).

[0035] In some embodiments of the present invention, the alcohol dehydrogenase or its mutant or the product expressing the alcohol dehydrogenase or its mutant can be a free enzyme (e.g., in the form of enzyme powder), or in the form of cells expressing the alcohol dehydrogenase catalyst (e.g., wet bacteria expressing the alcohol dehydrogenase catalyst). Or in other forms, such as supernatant of cell disruption expressing the alcohol dehydrogenase catalyst or immobilized enzyme.

[0036] In some embodiments of the present invention, the preparation method of the S-configuration 6-hydroxy-8-chlorooctanoic acid ethyl ester specifically comprises:

[0037] The engineered bacteria expressing the alcohol dehydrogenase or its mutant according to the first aspect of the present invention are mixed with 6-carbonyl-8-chlorooctanoic acid ethyl ester, the engineered bacteria expressing formate dehydrogenase, 6-carbonyl-8-chlorooctanoic acid ethyl ester, ammonium formate, a cosolvent, a buffer and NADP + Mix and react at 25-35°C for 1-24h to obtain the product.

[0038] In some embodiments of the present invention, the buffer is selected from any one of TEA buffer, PB buffer and Tris-HCl buffer.

[0039] In some embodiments of the present invention, the buffer is PB buffer.

[0040] In some embodiments of the present invention, the final concentration of the buffer is 0.05-0.20M.

[0041] In some embodiments of the present invention, the final concentration of the buffer is 0.05-0.10M.

[0042] In some embodiments of the present invention, the final concentration of the buffer is 0.1M.

[0043] In some embodiments of the present invention, the pH value of the buffer is 6-8.

[0044] In some embodiments of the present invention, the pH value of the buffer is 6-7.

[0045] In some embodiments of the present invention, the pH value of the buffer is 6.5.

[0046] In some embodiments of the present invention, the engineered bacteria expressing the alcohol dehydrogenase or a mutant thereof according to the first aspect of the present invention is alcohol dehydrogenase recombinant Escherichia coli.

[0047] In some embodiments of the present invention, the final concentration of the alcohol dehydrogenase recombinant Escherichia coli wet cells is 10-250 g / L.

[0048] In some embodiments of the present invention, the final concentration of the alcohol dehydrogenase recombinant Escherichia coli wet cells is 25-100 g / L.

[0049] In some embodiments of the present invention, the engineered bacteria expressing formate dehydrogenase is formate dehydrogenase recombinant Escherichia coli.

[0050] In some embodiments of the present invention, the final concentration of the formate dehydrogenase recombinant Escherichia coli is 10-200 g / L.

[0051] In some embodiments of the present invention, the final concentration of the formate dehydrogenase recombinant Escherichia coli is 25-75 g / L.

[0052] In some embodiments of the present invention, the concentration of ammonium formate is 0.1-1M.

[0053] In some embodiments of the present invention, the concentration of ammonium formate is 0.7M.

[0054] In some embodiments of the invention, the NADP + or NAD + The concentration is 0.01-1.0mM.

[0055] In some embodiments of the invention, the NADP + or NAD + The concentration is 0.1 mM.

[0056] In some embodiments of the present invention, the co-solvent is selected from at least one of ethyl acetate, butyl acetate, dibutyl phthalate, DMSO (dimethyl sulfoxide), methyl tert-butyl ether, dimethyl phthalate, and methanol.

[0057] In some embodiments of the present invention, the volume of the co-solvent accounts for 5-50% of the total volume of the reaction system.

[0058] In some embodiments of the present invention, no co-solvent may be added.

[0059] In some embodiments of the present invention, when preparing (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester, the alcohol dehydrogenase or its mutant includes α-LA-ADH2, α-LA-ADH5, α-LA-ADH6, ADH5-S58C / R, ADH5-S91T / L, ADH5-G105A / F, ADH5-T106I / F / A, ADH5-S117L / I / A, ADH5-S131A / L / I, ADH5-S132A / L / I, ADH5-S133A / L / I, ADH5-S134B / L / I, ADH5-S135C / R, ADH5-S136C / R, ADH5-S137C / R, ADH5-S138A / L / I, ADH5-S139A / L / I, ADH5-S140A / L / I, ADH5-S141A / L / I, ADH5-S142A / L / I, ADH5-S143B / L / I, ADH5-S144C / R, ADH5-S145B / L / I, ADH5-S146C / R, ADH5-S147C / R, ADH5-S148A / L / I, ADH5-S149A / L / I, ADH5-S150A / F, ADH5-S151 DH5-L147A / C / F, ADH5-V156A / L, ADH5-V164A / L, ADH5-G199W / L, ADH5-L205A / F / V, ADH5-S207V / L / N, ADH5-Y209I / L / V / F, ADH5-I223T / A, ADH5-Y240F, ADH5-T106F / S131A and ADH5-T106A / S131A.

[0060] In some embodiments of the present invention, when preparing (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester, the alcohol dehydrogenase or its mutant is ADH5-T106F / L205A.

[0061] The sixth aspect of the present invention provides the use of S-configuration 6-hydroxy-8-chlorooctanoic acid ethyl ester prepared by the preparation method described in the fifth aspect of the present invention in the preparation of α-lipoic acid.

[0062] In some embodiments of the present invention, the (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester prepared according to the preparation method of the fifth aspect of the present invention is subjected to operations such as chlorination according to conventional synthesis methods in the art to further obtain α-lipoic acid.

[0063] In some embodiments of the present invention, the α-lipoic acid is (R)-α-lipoic acid.

[0064] In some embodiments of the present invention, in the application, the obtained (R)-α-lipoic acid has a higher optical purity than that of the (R)-α-lipoic acid obtained by conventional methods.

[0065] The beneficial effects of the present invention are:

[0066] 1. The present invention provides a variety of alcohol dehydrogenases and mutants thereof, which can catalyze 6-carbonyl-8-chlorooctanoic acid ethyl ester to produce (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester, wherein the ADH5 mutants ADH5-S131A, ADH5-S131L, ADH5-S131I, and ADH5-T106F / S131A have a reaction conversion rate of 99%, and the S-type optical purity is greater than 99%.

[0067] 2. Based on the alcohol dehydrogenase and its mutants in the present invention, efficient biosynthesis of (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester can be achieved. Moreover, the synthesis method not only has mild reaction conditions, is environmentally friendly, has high regioselectivity and stereoselectivity, but also avoids heavy metal residues in the product, thus overcoming the shortcomings of existing chemical methods.

[0068] 3. The biosynthesis method of the present invention only requires one step of reaction to obtain the target product, and uses fewer reagents, mild reaction conditions, and high catalytic activity, which reduces production costs and can be used for large-scale production. It has broad application prospects and considerable market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The technical roadmap of the enzymatic synthesis method for preparing (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester using alcohol dehydrogenase mutants in the embodiments of the present invention.

[0070] Figure 2 This is the GC spectrum of (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester prepared using the alcohol dehydrogenase mutant in the examples of the present invention. DETAILED DESCRIPTION

[0071] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0072] Definition and Explanation

[0073] The term "pharmaceutically acceptable" in the present invention refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0074] In the present invention, the term "vector" includes nucleic acid vectors, for example, DNA vectors, such as plasmids, cosmids or artificial chromosomes, RNA vectors, viruses or any other suitable replicons (e.g., viral vectors). A variety of vectors have been developed in the prior art for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described in, for example, Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the products and methods of the present invention contain polynucleotide sequences, and, for example, other sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express transgenics as described in the present invention include vectors containing regulatory sequences (such as promoter and enhancer regions) that direct gene transcription. Other available vectors for expressing transgenics contain polynucleotide sequences that increase the translation rate of the transgenic or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the products and methods of the present invention may also contain polynucleotides encoding markers for selecting cells containing the vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseoin.

[0075] In the present invention, the term "lyophilized powder" refers to a freeze-dried product obtained by pre-freezing the water in a drug solution by a vacuum freeze-drying method using a freeze dryer, and then sublimating the frozen water in the drug solution in a vacuum and sterile environment.

[0076] In the present invention, the term "granular inoculation agent" refers to an agent used to prevent powdered agents from directly contacting fungicides or chemical fertilizers during use, resulting in reduced effects. It usually refers to the type of agent obtained by mixing a bacterial solution with a granular carrier (such as biochar, vermiculite, etc.).

[0077] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (e.g., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as naturally occurring amino acids, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds whose structure is different from the general amino acid chemical structure, but function similarly to naturally occurring amino acids.

[0078] The natural amino acids and their various expressions described herein are well known to those skilled in the art, and their specific corresponding situations are shown in the following table:

[0079]

[0080]

[0081] Example 1 Screening of alcohol dehydrogenase template

[0082] In this Example, alcohol dehydrogenases used as templates for alcohol dehydrogenase mutants were screened from the following 6 types of alcohol dehydrogenases.

[0083] Among them, the six alcohol dehydrogenases are:

[0084] α-LA-ADH1, the corresponding amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the nucleic acid molecule encoding the alcohol dehydrogenase is shown in SEQ ID NO: 7.

[0085] α-LA-ADH2, the corresponding amino acid sequence is shown in SEQ ID NO: 2, and the nucleotide sequence of the nucleic acid molecule encoding the alcohol dehydrogenase is shown in SEQ ID NO: 8.

[0086] α-LA-ADH3, the corresponding amino acid sequence is shown in SEQ ID NO: 3, and the nucleotide sequence of the nucleic acid molecule encoding the alcohol dehydrogenase is shown in SEQ ID NO: 9.

[0087] α-LA-ADH4, the corresponding amino acid sequence is shown in SEQ ID NO: 4, and the nucleotide sequence of the nucleic acid molecule encoding the alcohol dehydrogenase is shown in SEQ ID NO: 10.

[0088] α-LA-ADH5, the corresponding amino acid sequence is shown in SEQ ID NO: 5, and the nucleotide sequence of the nucleic acid molecule encoding the alcohol dehydrogenase is shown in SEQ ID NO: 11.

[0089] α-LA-ADH6, the corresponding amino acid sequence is shown in SEQ ID NO: 6, and the nucleotide sequence of the nucleic acid molecule encoding the alcohol dehydrogenase is shown in SEQ ID NO: 12.

[0090] The specific screening scheme is:

[0091] According to the above amino acid sequence, 6 kinds of alcohol dehydrogenases were synthesized, and then used to construct expression vectors (such as using pET28b) according to conventional methods in the art, and the corresponding expression bacteria (Escherichia coli) were obtained after transfection. Then 0.15g of expression bacteria were taken in 2mL EP tubes, and a blank control group (empty bacteria) was set up. 700μL of PB buffer (0.1M, pH 7), 100μL of 7M ammonium formate solution, and 25mM NADP were added to each EP tube. + 100 μL of solution and 100 μL of 0.5M ethyl 6-carbonyl-8-chlorooctanoate (in methyl tert-ether) solution. Place the EP tube in a constant temperature mixer and shake at 1200 rpm for 6 hours at 30°C. After the reaction, take 200 μL of the reaction solution, add 200 μL of ethyl acetate, shake and mix for 2 minutes, centrifuge at 12000 rpm for 1 minute, take the supernatant, and perform GC (gas chromatography) detection.

[0092] After GC detection, the conversion rates and ee values ​​of the above six alcohol dehydrogenases are shown in Table 1:

[0093] Table 1 Conversion rates and ee values ​​of six alcohol dehydrogenases

[0094]

[0095]

[0096] The results showed that the conversion rate of α-LA-ADH4 was higher, reaching 97.66%, but its chirality was R-type with an ee value of 82.60%. The conversion rate of α-LA-ADH-5 was higher, reaching 98.66%, and its chirality was S-type with an ee value of 99.20%.

[0097] Therefore, α-LA-ADH-5 was selected as a template for alcohol dehydrogenase mutants.

[0098] Example 2 Construction of alcohol dehydrogenase mutants

[0099] The above-mentioned α-LA-ADH-5 was used as a template for the alcohol dehydrogenase mutant (the nucleotide sequence encoding α-LA-ADH-5 is shown in SEQ ID NO: 11), and the plasmid vector pET28b(+)-ADH-5 was constructed according to the same method.

[0100] In this embodiment, the mutants of α-LA-ADH-5 include single mutation site mutants and multiple mutation site mutants.

[0101] The specific construction method is:

[0102] (1) Single mutation site mutants:

[0103] The synthesis of the whole gene was completed by Beijing Qingke Biotechnology Co., Ltd., and the genes of alcohol dehydrogenase ADH1, ADH2, ADH3, ADH4, ADH5 and ADH6 were codon optimized, and the corresponding restriction sites were added at both ends of the gene, and constructed into the corresponding vector to obtain the recombinant plasmid vector of alcohol dehydrogenase, and the obtained recombinant plasmid vector was transferred into the host such as Escherichia coli BL21 by conventional methods, and the plasmid vector pET28b(+)-ADH-5 constructed by Qingke Biotechnology was used as a template, and primers were used for PCR amplification to obtain the amplified fragment. The recombinant plasmid digested with DpnⅠ was transferred to the competent cells of Escherichia coli BL21 (DE3) to obtain clones, and then the clones were inoculated into 10mL LB plate medium and cultured at 37℃ for 12-16h.

[0104] Randomly select the positive clones (i.e., clones containing the above-mentioned amplified fragments) and the original strain on the plate, inoculate them in 10mL liquid LB medium (add the corresponding antibiotic Kan in the test tube), and place them in a temperature-controlled shaker at 37°C and 200rpm for 8-10h. The culture solution is inoculated into 100mL LB medium (add the corresponding antibiotic in the shake bottle) at an inoculation volume of 1%, and placed in a temperature-controlled shaker at 37°C and 180rpm for 2-2.5h (equivalent to when the strain density OD600 value is 0.6-0.8), add the inducer IPTG with a final concentration of 0.1mM, and then place it in a temperature-controlled shaker at 24°C and 180rpm for 12 hours. The cultured bacterial solution is transferred to an 800mL centrifuge cup, centrifuged at 8000rpm and 4°C for 10min in a low-temperature high-speed centrifuge, discard the supernatant, collect the bacteria and weigh them for subsequent experiments.

[0105] The same method as in Example 1 was used to measure the concentration and chirality of 6-carbonyl-8-chlorooctanoic acid ethyl ester and (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester by GC detection and calculate the conversion rate. The dominant strain was screened out using the conversion rate and chirality as indicators.

[0106] The obtained dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored in a -80 ° C refrigerator. The dominant mutants finally screened were ADH5-S58C / R, ADH5-S91T / L, ADH5-G105A / F, ADH5-T106I / F / A, ADH5-S117L / I / A, ADH5-S131A / L / I, ADH5-L147A / C / F, ADH5-V156A / L, ADH5-V164A / L, ADH5-G199W / L, ADH5-L205A / F / V, ADH5-S207V / L / N, ADH5-Y209I / L / V / F, ADH5-I223T / A and ADH5-Y240F. The naming method of each mutant is: template-mutation site, such as ADH5-T106I, which means that α-LA-ADH5 is used as a template, and the 106th position of its amino acid sequence is mutated from T (threonine) to I (isoleucine).

[0107] (2) Multiple mutation site mutants:

[0108] The single mutation site mutants obtained above were used as templates to construct the corresponding recombinant vectors pET28b(+)-ADH5-S58C / R, pET28b(+)-ADH5-S91T / L, pET28b(+)-ADH5-G105A / F, pET28b(+)-ADH5-T106I / F / A, pET28b(+)-ADH5-S117L / I / A, pET28b(+)-ADH5-S131A / L / I, and pET28b(+)-ADH5-L14 7A / C / F, pET28b(+)-ADH5-V156A / L, pET28b(+)-ADH5-V164A / L, pET28b(+)-ADH5-G199W / L, pET28b(+)-ADH5-L205A / F / V, pET28b(+)-ADH5-S207V / L / N, pET28b(+)-ADH5-Y209I / L / V / F, pET28b(+)-ADH5-I223T / A and pET28b(+)-ADH5-Y240F. The constructed recombinant vector was used as a template and primers were used for PCR amplification to obtain the target amplified fragment. The primers used were designed using conventional software in the art or in the following design method: upstream primer: select the nucleotide sequence of about 15 bp before the mutation site + the replacement nucleotide sequence of the mutation site + the nucleotide sequence of about 10 bp after the mutation site; downstream primer: select the reverse nucleotide sequence of about 25 bp before the mutation site. After the design is completed, the obtained upstream primer and downstream primer are verified, and the Tm value difference is < 3 will do.

[0109] The PCR amplification system is shown in Table 2.

[0110] Table 2 PCR amplification system (25 μL)

[0111] Components content Forward primer (100 μM) 1μL Reverse primer (100 μM) 1μL 2×Phanta buffer 12.5μL dNTP mixture (10 mM each) 0.5μL Plasmid template 1μL DNA polymerase Phanta (purchased from Novazon) 0.5μL Ultrapure water 8.5μL

[0112] The PCR program was set according to the manual of Phanta Super-Fidelity DNA polymerase: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 4 min, 30 cycles; final extension at 72°C for 10 min; and insulation at 16°C.

[0113] Then, a single mutation site mutant construction method was used to introduce multiple mutant sites using the above PCR system.

[0114] According to the above embodiment, GC test screening for dominant mutations was performed again, and the specific steps were as follows: according to conventional methods in the art, the obtained recombinant vector containing nucleic acid molecules encoding each mutant was transfected into a microbial expression vector to obtain a single mutant strain and a multiple mutant strain, and at the same time, the corresponding wild type (WT, i.e., using the above template) was used as a catalyst, and 6-carbonyl-8-chlorooctanoic acid ethyl ester was used as a substrate to compare the catalytic activity of each mutant. Wherein, the catalytic reaction system is 1mL. In the system, the strain as a catalyst is 100g / wet weight L, the final concentration of the substrate is 50g / L, and 100mM PB buffer is the reaction medium (containing 10% methyl tert-ether as a cosolvent in a volume ratio of 10%, pH 7.0). The system was vortexed and oscillated at 30°C and 1200rpm for 2h. Then 200μL of the reaction solution was taken and 50μL of 6M hydrochloric acid (for termination of the reaction) was added. 200μL of ethyl acetate was added to extract twice, and the ethyl acetate phase was combined. The GC detection method in Example 1 was used to detect the concentration and chirality of 6-carbonyl-8-chlorooctanoic acid ethyl ester and (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester in the ethyl acetate sample and calculate the conversion rate.

[0115] The results are shown in Table 3.

[0116] Table 3 Conversion rate and ee value of mutants

[0117]

[0118]

[0119] Based on the above results, the best ADH5 mutant strain obtained by screening is ADH5-L205A / Y209L. The actual transformation rates of all mutants are significantly greater than that of the wild type.

[0120] Example 3 Effects of different factors on the conversion of alcohol dehydrogenase mutants

[0121] In this embodiment, the effects of different influencing factors on the conversion of alcohol dehydrogenase mutants were further tested.

[0122] (1) Effect of cosolvent on the transformation of alcohol dehydrogenase mutants:

[0123] Add 1 g (wet weight) of the ADH5-L205A / Y209L mutant strain and formate dehydrogenase engineered bacteria (Escherichia coli) constructed in the above example to the reaction flask. Then add 6 mL of PB buffer (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, and 10 mM NADP + Solution 1mL, 500g / L 6-carbonyl-8-chlorooctanoic acid ethyl ester (containing different cosolvents or without cosolvents) solution 2mL. In the present embodiment, cosolvents are selected from ethyl acetate, butyl acetate, dibutyl phthalate, DMSO (dimethyl sulfoxide), methyl tert-butyl ether, dimethyl phthalate and methanol, and no cosolvent is added as a control group, and the reaction is carried out at 30°C and 800rpm for 2h. Take 1mL of reaction solution from each group, add an equal volume of ethyl acetate to extract, and centrifuge to take the upper organic phase for GC detection. The results are shown in Table 4.

[0124] Table 4 Effect of cosolvent on conversion of alcohol dehydrogenase mutants

[0125] Solvent Conversion rate ee value none >99％ Type S, 99.48% Ethyl acetate 25.78％ S-type, 86.82% Butyl acetate 68.34％ S-type, 92.26% Dibutyl phthalate 80.24％ S-type, 96.88% DMSO (Dimethyl Sulfoxide) >99％ S-type, 69.16% Methyl tert-butyl ether >99％ Type S, 99.56% Dimethyl phthalate >99％ S-type, 98.22% Methanol 80％ S-type, 98.14%

[0126] The above results show that the highest conversion rate is >99% when the cosolvent is methyl tert-butyl ether, and its chirality is S-type ee value 99.56%. However, the conversion rate of the control group without cosolvent can also be >99%, so it can be explained that the alcohol dehydrogenase mutant can be used without adding cosolvent, and in this case, its conversion rate is not significantly reduced, thereby improving the greenness of the reaction.

[0127] (2) Effects of buffer and pH on conversion of alcohol dehydrogenase mutants:

[0128] First, the inventors tested the effects of different buffers on the conversion of alcohol dehydrogenase mutants.

[0129] Add 1g (wet weight) of the ADH5-L205A / Y209L mutant strain and formate dehydrogenase engineered bacteria constructed in the above example to the reaction flask. Then add 6mL of different buffers (0.1M), 1mL of 7M ammonium formate solution, 10mM NADP +1mL of solution, 2mL of 500g / L 6-carbonyl-8-chlorooctanoic acid ethyl ester (containing methyl tert-butyl ether) solution. Among them, the buffer solution selected was pH 7.0 PB buffer, Tris-HCl buffer and TEA buffer, and the reaction was carried out at 30°C and 800rpm for 1h. 1mL of reaction solution was taken from each group, extracted with an equal volume of ethyl acetate, and the upper organic phase was centrifuged for GC detection. The results are shown in Table 5.

[0130] Table 5 Effect of buffer on conversion of alcohol dehydrogenase mutants

[0131]

[0132]

[0133] The above results show that the conversion rate is highest when PB buffer at pH 7.0 is used as the buffer.

[0134] The inventors further tested the effect of different pH on the conversion of alcohol dehydrogenase mutants.

[0135] Add 1 g (wet weight) of the ADH5-L205A / Y209L mutant strain and formate dehydrogenase engineered bacteria constructed in the above example to the reaction flask. Then add 6 mL of PB buffer (0.1 M) of different pH values, 1 mL of 7 M ammonium formate solution, 10 mM NADP + 1mL of solution, 2mL of 500g / L 6-carbonyl-8-chlorooctanoic acid ethyl ester (containing methyl tert-butyl ether) solution. The pH of the buffer solution was set to 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The reaction was carried out at 30°C and 800rpm for 2h. 1mL of the reaction solution was taken from each group, extracted with an equal volume of ethyl acetate, and the upper organic phase was centrifuged for GC detection. The results are shown in Table 6.

[0136] Table 6 Effect of pH on conversion of alcohol dehydrogenase mutants

[0137] Buffer Conversion rate pH 6.0 PB buffer 90.46％ pH 6.5 PB buffer 99％ pH 7.0 PB buffer 92.56％ pH 7.5 PB buffer 83.48％ pH 8.0 PB buffer 65.44％

[0138] The above results show that the conversion rate is the highest when the buffer is PB buffer at pH 6.5, which is 99%.

[0139] (3) Effect of reaction temperature on conversion of alcohol dehydrogenase mutants:

[0140] Add 1 g (wet weight) of the ADH5-T106F / S131A mutant strain and formate dehydrogenase engineered bacteria constructed in the above example to the reaction flask. Then add 6 mL of PB buffer (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, 10 mM NADP +1mL of solution, 2mL of 500g / L 6-carbonyl-8-chlorooctanoic acid ethyl ester (containing methyl tert-butyl ether) solution. The reaction temperatures were set at 20, 25, 30, 35, and 40°C, respectively. The reaction was carried out at 800rpm for 6h. 1mL of reaction solution was taken from each group, extracted with an equal volume of ethyl acetate, and the upper organic phase was centrifuged for GC detection. The results are shown in Table 7.

[0141] Table 7 Effect of temperature on conversion of alcohol dehydrogenase mutants

[0142] Temperature(℃) Conversion rate ee value 20 72.42％ S type, >99% 25 86.73％ Type S, >99% 30 >99％ S type, >99% 35 78.35％ S-type, 94.44% 40 54.88％ S-type, 82.72%

[0143] The above results show that the conversion rate is the highest at 30°C, which is >99%, and its chirality is S-type, with an ee value of >99%.

[0144] Example 4 Actual effect of alcohol dehydrogenase mutants in (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester amplification experiment

[0145] In this example, the inventors took the ADH5-L205A / Y209L mutant as an example to test the actual effect of the alcohol dehydrogenase mutant in the (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester amplification experiment. The specific test method is as follows:

[0146] Take a 15L liquid tank, add 500g (wet weight) of the ADH5-T106F / S131A mutant strain and formate dehydrogenase engineered bacteria constructed in the above example. Then add 10L of PB buffer (0.1M, pH 6.5) and mix well, then continue to add 2L of 7M ammonium formate solution, 2.5mM NADP + 2L of solution and 1kg of ethyl 6-carbonyl-8-chlorooctanoate were mixed and transferred to a 50L reactor. Water was added to make up the system to 20L, and then the reaction was carried out at 30°C and 300rpm with mechanical stirring. 200μL of the reaction solution was taken at 2h, 4h, and 6h of reaction, and extracted with an equal volume of ethyl acetate and then detected by TLC (thin layer chromatography).

[0147] The reaction process is as follows Figure 1 shown.

[0148] After 6 hours of reaction, TLC test results showed that the conversion was complete. The reaction solution was extracted with ethyl acetate. The same volume of ethyl acetate was added for the first extraction, and then 1 / 2 volume of ethyl acetate was used for the second and third extractions. The extraction operation included: shaking and mixing, centrifuging at 8000 rpm for 5 minutes, absorbing the supernatant, and then using anhydrous Na 2 SO 4After the extraction is completed, the extract is subjected to rotary evaporation (using a vacuum pump first and then an oil pump for rotary evaporation). The final product is obtained after the rotary evaporation, and can be used for further reaction to prepare (R)-α-lipoic acid.

[0149] The final product was tested by GC, and the results were as follows Figure 2 As shown, it is shown that the final product is (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester. Repeating the experiment, it can be found that the effects of other ADH5 mutants are the same as ADH5-T106F / S131A. It can be shown that the mutants in the above examples can catalyze 6-carbonyl-8-chlorooctanoic acid ethyl ester to produce (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester, among which ADH5-L205A / Y209L has the best effect, with a reaction conversion rate of>99% and an S-type optical purity of greater than 99%.

[0150] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Alcohol dehydrogenase or a mutant thereof, characterized in that The alcohol dehydrogenase or its mutant comprises: (1) using the alcohol dehydrogenase shown in any one of SEQ ID NOs: 1-6 as a template, replacing 0 to 10 amino acid residues therein to obtain an alcohol dehydrogenase or a mutant thereof; or (2) The alcohol dehydrogenase or its mutant in (1) wherein amino acids are added or deleted and the alcohol dehydrogenase or its mutant still retains the dehydrogenation effect.

2. The alcohol dehydrogenase or a mutant thereof according to claim 1, characterized in that The alcohol dehydrogenase mutant described in (1) is an alcohol dehydrogenase mutant obtained by replacing 1 to 5 amino acid residues in the alcohol dehydrogenase shown in SEQ ID NO: 5 as a template.

3. The alcohol dehydrogenase or a mutant thereof according to claim 1 or 2, characterized in that Amino acid residue substitutions include: , S58C, S58R, S91T, S91L, G105A, G105F, T106I, T106F, T106A, S117L, S117I, S117A, S131A, S131L, S131I, L147A, L147C, L147F, V156A, V156L, V164A, V164L, G199W, G199L, L205A, L205F, L205V, S207V, S207L, S207N, Y209I, Y209L, Y209V, Y209F, I223T, I223A, and Y240F.

4. The alcohol dehydrogenase or a mutant thereof according to claim 1 or 2, characterized in that The alcohol dehydrogenase or its mutant described in (2) has at least 90% sequence identity with the alcohol dehydrogenase or its mutant described in (1).

5. A nucleic acid molecule encoding the alcohol dehydrogenase or a mutant thereof according to any one of claims 1 to 4.

6. A product, characterized in that The product comprises at least one of the following (1)-(3): (1) An expression vector containing the nucleic acid molecule according to claim 5; (2) A transformant containing the nucleic acid molecule according to claim 5; (3) A transformant containing the expression vector described in (1); Wherein, the expression element comprises a plasmid; The transformants include viruses, bacteria, fungi, plant cells and animal cells.

7. Use of the alcohol dehydrogenase or a mutant thereof according to any one of claims 1 to 4 in the synthesis of 6-hydroxy-8-chlorooctanoic acid ethyl ester, preferably, the 6-hydroxy-8-chlorooctanoic acid ethyl ester is 6-hydroxy-8-chlorooctanoic acid ethyl ester of S configuration.

8. A method for preparing S-configuration 6-hydroxy-8-chlorooctanoic acid ethyl ester, comprising the following steps: The alcohol dehydrogenase or its mutant according to any one of claims 1 to 4 or one or a combination of products expressing the alcohol dehydrogenase or its mutant according to any one of claims 1 to 4 is mixed with 6-carbonyl-8-chlorooctanoic acid ethyl ester, formate dehydrogenase or one or a combination of products expressing formate dehydrogenase, 6-carbonyl-8-chlorooctanoic acid ethyl ester and formate dehydrogenase substrate, and reacted at 15-40° C. for 1-24 hours to obtain the product.

9. The preparation method according to claim 8, characterized in that: The formate dehydrogenase substrates include ammonium formate, nicotinamide adenine dinucleoside phosphate (NADP + ) and nicotinamide adenine dinucleotide (NAD + ) 10. Use of S-configuration 6-hydroxy-8-chlorooctanoic acid ethyl ester prepared by the preparation method according to claim 8 or 9 in the preparation of α-lipoic acid; preferably, the α-lipoic acid is (R)-α-lipoic acid.

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