Alcohol dehydrogenase mutants and their application in the synthesis of high optical purity (S)-6-hydroxy-8-chlorooctanoate ethyl ester

Abstract

This invention discloses an alcohol dehydrogenase mutant and its application in the asymmetric synthesis of optically pure (S)-6-hydroxy-8-chlorooctanoate ethyl ester. The alcohol dehydrogenase or its mutant in this invention exhibits extremely high conversion rates and effectively improves the optical purity of the product. Furthermore, based on this mutant, low-cost, efficient, and rapid production of a single configuration of 6-hydroxy-8-chlorooctanoate ethyl ester is achieved, avoiding the generation of multi-configuration mixtures and the problems of cumbersome steps and contamination associated with conventional chemical synthesis methods.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, and in particular to alcohol dehydrogenase mutants and their application in the synthesis of high optical purity (S)-6-hydroxy-8-chlorooctanoate ethyl ester. Background Technology

[0002] Alpha-lipoic acid (α-LA) belongs to the B vitamins and is a unique "universal antioxidant" that is both fat-soluble and water-soluble. It effectively scavenge free radicals that cause disease and accelerate aging, inhibits lipid oxidation in nerve tissue, and prevents protein glycosylation. The antioxidant capacity of α-lipoic acid is 5-10 times that of grape seed extract and 60 times that of vitamin E, and it can be absorbed by various tissues and organs. Clinically, α-lipoic acid is used to treat diabetic peripheral neuropathy and diabetic nephropathy. α-Lipoic acid has a chiral center, thus having two configurations: R and S. Only the R configuration of α-lipoic acid can covalently attach to a lysine residue via an amide bond to function as a coenzyme; therefore, only the R configuration actually possesses effective physiological activity. However, synthetically produced α-lipoic acid is a mixture (racemic) composed of equal proportions of the R and S configurations. Furthermore, the cost of obtaining the R-configuration α-lipoic acid through separation is prohibitively high, making its price more than five times that of its racemic counterpart. Therefore, most commercially available products are effectively limited to racemic formulations. To alleviate the burden caused by the S-isomer in racemic formulations metabolized by the human body, the efficient preparation of optically pure R-configuration α-lipoic acid has significant application value and broad market prospects.

[0003] Existing methods for synthesizing (R)-α-lipoic acid can be divided into chemical and biological methods. Elliott et al. performed an asymmetric synthesis of (R)-α-lipoic acid by induction with chiral auxiliaries, achieving an overall yield of 37%. However, the reaction conditions were too harsh and the cost of the reagents was too high, limiting the application of this method in industry (Tetrahedron Letters, 1985, 26(21): 2535-2538). Gopalan et al. attempted to catalyze the production of (R)-α-lipoic acid using microbial enzymes, but the overall yield was only 10%, and the method was not very practical (Journal of the Chemical Society, Perkin Trans., 1990, 7: 1897-1900). One of the more widely studied synthetic methods is enantiomeric resolution, which involves using chiral resolving agents or esterases / lipases to resolve racemic α-lipoic acid or its precursors, thereby converting it into (R)-α-lipoic acid. However, this process is accompanied by the formation of (S)-α-lipoic acid, resulting in a theoretical maximum yield of only 50%, while in practical industrial applications, the resolution yield is only about 30%. Constructing a chiral center during the synthesis process is a crucial step in overcoming the limitations of resolution yield.

[0004] The enzymatic reduction preparation of optically active 6-hydroxy-8-chlorooctanoate intermediates has gradually attracted industry attention. Theoretically, high-purity (R)-α-lipoic acid can be obtained by further reacting ethyl 6-hydroxy-8-chlorooctanoate with either the R or S configuration. Currently, the mainstream reported methods focus on the R configuration. For example, Olbrich et al. used whole-cell catalysis from Geotrichum candidum to asymmetricly reduce ethyl 6-carbonyl-8-chlorooctanoate to (R)-6-hydroxy-8-chlorooctanoate; Müller et al. used the alcohol dehydrogenase TbADH from Thermoanaerobium brokii to catalyze the production of the R-type product; Werner et al. used NgADH from Nocardia globulera to prepare (R)-6-hydroxy-8-chlorooctanoate. The construction of the S configuration of 6-hydroxy-8-chlorooctanoate has not been reported. The development of a high-optical-purity and cost-effective (S)-6-hydroxy-8-chlorooctanoate intermediate has significant scientific and industrial value. Summary of the Invention

[0005] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide an alcohol dehydrogenase mutant and its application in the asymmetric synthesis of optically pure (S)-6-hydroxy-8-chlorooctanoate ethyl ester. In particular, the alcohol dehydrogenase mutant of this invention can effectively overcome the need for a large amount of the cofactor NADP when using carbonyl reductase to prepare (S)-6-hydroxy-8-chlorooctanoate ethyl ester. +This method overcomes the problems of excessively long catalytic time and low yield, achieving low-cost, efficient, rapid, high-yield, and high-purity production of (S)-6-hydroxy-8-chlorooctanoic acid ethyl ester. It also avoids the cumbersome steps and pollution associated with conventional chemical synthesis methods.

[0006] A first aspect of the invention provides an alcohol dehydrogenase or a mutant thereof, said alcohol dehydrogenase or a mutant thereof comprising:

[0007] (1) Using any of the alcohol dehydrogenases shown in SEQ ID NO: 1-6 as a template, replacing 0-10 amino acid residues therein to obtain an alcohol dehydrogenase or its mutant; or

[0008] (2) The alcohol dehydrogenase or its mutant in (1) with added or deleted amino acids, and still retaining 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 present invention, 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.

[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 substitution 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 substitutions are selected from S58C, S58R, S91T, S91L, G105A, G105F, T106I, T106F, T106A, S117L, S117I, S117A, S131A, S131L, S131I, L147A, L147C, L147F, and 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 described in (2) has at least 90% sequence identity with the alcohol dehydrogenase or its mutant described in (1).

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

[0015] A second aspect of the invention provides a nucleic acid molecule encoding the alcohol dehydrogenase or a mutant thereof described in the first aspect of the invention.

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

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

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

[0019] In some embodiments of the present invention, the nucleic acid molecule includes SEQ ID NO: 7-12.

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

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

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

[0023] (3) Transformers containing the expression described in (1).

[0024] In some embodiments of the present invention, the expression unit includes 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 present invention, the plant cells and animal cells do not include reproductive 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, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, crospovidone, 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 may be in the form of lyophilized powder, bacterial solution, or granular inoculum. It should be understood that those skilled in the art can also rationally select appropriate forms for use based on actual application needs and existing processing techniques, including but not limited to the aforementioned lyophilized powder, bacterial solution, and granular inoculum.

[0030] A fourth aspect of the invention provides the use of the alcohol dehydrogenase described in the first aspect of the invention or a mutant thereof in the synthesis of ethyl 6-hydroxy-8-chlorooctanoate.

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

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

[0033] The alcohol dehydrogenase described in the first aspect of this invention or its mutant, or a product expressing the alcohol dehydrogenase described in the first aspect of this invention or its mutant, or a combination thereof, is mixed with ethyl 6-carbonyl-8-chlorooctanoate, formate dehydrogenase or a product expressing formate dehydrogenase, or a combination thereof, ethyl 6-carbonyl-8-chlorooctanoate, 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 includes ammonium formate, nicotinamide adenine dinucleotide phosphate (NADP), etc. + ) 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, may be a free enzyme (e.g., used in the form of enzyme powder), or in the form of cells expressing the alcohol dehydrogenase catalyst (e.g., wet bacterial cells expressing the alcohol dehydrogenase catalyst). Alternatively, it may be in other forms, such as cell lysate supernatant expressing the alcohol dehydrogenase catalyst or immobilized enzyme.

[0036] In some embodiments of the present invention, the preparation method of the S-configuration ethyl 6-hydroxy-8-chlorooctanoate specifically includes:

[0037] The engineered bacteria expressing the alcohol dehydrogenase or its mutant as described in the first aspect of the present invention were mixed with ethyl 6-carbonyl-8-chlorooctanoate, engineered bacteria expressing formate dehydrogenase, ethyl 6-carbonyl-8-chlorooctanoate, ammonium formate, solubilizer, buffer, and NADP. + Mix and react at 25-35℃ for 1-24 hours to obtain the final product.

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

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

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

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

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

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

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

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

[0046] In some embodiments of the present invention, the engineered bacteria expressing the alcohol dehydrogenase or its mutant as described in the first aspect of the present invention are recombinant Escherichia coli with alcohol dehydrogenase.

[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 a recombinant formate dehydrogenase-producing 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 the ammonium formate is 0.1-1M.

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

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

[0055] In some embodiments of the present invention, the NADP + or NAD + The concentration was 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 reaction system volume.

[0058] In some embodiments of the present invention, a co-solvent may not be added.

[0059] In some embodiments of the present invention, when preparing (S)-6-hydroxy-8-chlorooctanoate 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, A 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 ethyl (S)-6-hydroxy-8-chlorooctanoate, the alcohol dehydrogenase or its mutant is ADH5-T106F / L205A.

[0061] A sixth aspect of the present invention provides the use of the S-configuration ethyl 6-hydroxy-8-chlorooctanoate 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, ethyl (S)-6-hydroxy-8-chlorooctanoate prepared by the preparation method according to 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, the obtained (R)-α-lipoic acid has higher optical purity than (R)-α-lipoic acid obtained by conventional methods.

[0065] The beneficial effects of this invention are:

[0066] 1. This invention provides various alcohol dehydrogenases and their mutants, which can catalyze the formation of (S)-6-hydroxy-8-chlorooctanoate ethyl ester from 6-carbonyl-8-chlorooctanoate. Among them, the ADH5 mutants ADH5-S131A, ADH5-S131L, ADH5-S131I, and ADH5-T106F / S131A achieve a conversion rate of 99% and an optical purity of greater than 99% for the S-type.

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

[0068] 3. The biosynthesis method of this invention requires only one reaction to obtain the target product, uses fewer reagents, has mild reaction conditions, and high catalytic activity, which reduces production costs and enables large-scale production. It has broad application prospects and considerable market value. Attached Figure Description

[0069] Figure 1 This is a technical roadmap for the enzymatic synthesis of (S)-6-hydroxy-8-chlorooctanoate using an alcohol dehydrogenase mutant, as described in this invention.

[0070] Figure 2 This is the GC spectrum of (S)-6-hydroxy-8-chlorooctanoate ethyl ester prepared using an alcohol dehydrogenase mutant in an embodiment of the present invention. Detailed Implementation

[0071] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0072] Definitions and Explanations

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

[0074] In this invention, the term "vector" includes nucleic acid vectors, such as DNA vectors, such as plasmids, viscera, 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 foreign proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbialand Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the products and methods of this 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 transgenes as described in this invention include vectors containing regulatory sequences (such as promoter and enhancer regions) that guide gene transcription. Other available vectors for expressing transgenes contain polynucleotide sequences that improve the translation rate of the transgene or enhance the stability of mRNA transcribed from the gene or nuclear export. These sequence elements include, for example, untranslated regions at 5' and 3' and polyadenylation signal sites to guide efficient transcription of the gene carried on the expression vector. Expression vectors suitable for use with the products and methods described in this 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 norethisterone.

[0075] In this invention, the term "freeze-dried powder" refers to a product obtained by pre-freezing the water in a liquid medicine using a vacuum freeze-drying machine, and then sublimating the frozen water in the liquid medicine under a vacuum sterile environment.

[0076] In this invention, the term "granular inoculated microbial agent" refers to a microbial agent used to prevent powdered microbial agents from coming into direct contact with fungicides or chemical fertilizers during use, which would reduce their effectiveness. It usually refers to a type of microbial agent obtained by mixing microbial liquid with granular carriers (such as biochar, vermiculite, etc.).

[0077] "Amino acids" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (e.g., the α-carbon bound to hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds whose structure differs from that of typical amino acids, but which function similarly to naturally occurring amino acids.

[0078] The natural amino acids and their various expression forms described in this article are well known to those skilled in the art, and their specific correspondences are shown in the table below:

[0079]

[0080]

[0081] Example 1: Screening of alcohol dehydrogenase templates

[0082] In this embodiment, alcohol dehydrogenases were screened from the following six alcohol dehydrogenases to serve as templates for alcohol dehydrogenase mutants.

[0083] The six alcohol dehydrogenases are as follows:

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

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

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

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

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

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

[0090] The specific screening scheme is as follows:

[0091] Six alcohol dehydrogenases were synthesized according to the above amino acid sequence, and then used to construct expression vectors (e.g., using pET28b) according to conventional methods in the art. After transfection, the corresponding expression bacteria (E. coli) were obtained. Then, 0.15 g of the expression bacteria were placed in 2 mL EP tubes, and a blank control group (empty vector bacteria) was set up. Each EP tube was supplemented with 700 μL of PB buffer (0.1 M, pH 7), 100 μL of 7 M ammonium formate solution, and 25 mM NADP. + 100 μL of the solution and 100 μL of 0.5 M ethyl 6-carbonyl-8-chlorooctanoate (in tertiary methyl ether) solution were added. The EP tube was placed in a constant temperature mixer and reacted at 30 °C and 1200 rpm for 6 h with shaking. After the reaction was completed, 200 μL of the reaction solution was taken and 200 μL of ethyl acetate was added. The mixture was shaken and mixed for 2 min, centrifuged at 12000 rpm for 1 min, and the supernatant was collected for GC (gas chromatography) detection.

[0092] The conversion rates and ee values ​​of the above six alcohol dehydrogenases were determined by GC analysis, as shown in Table 1.

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

[0094]

[0095]

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

[0097] Therefore, α-LA-ADH-5 was chosen as the template for the alcohol dehydrogenase mutant.

[0098] Example 2 Construction of alcohol dehydrogenase mutant

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

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

[0101] The specific construction method is as follows:

[0102] (1) Single mutation site mutant:

[0103] The synthesis of the complete genome was completed by Beijing Qingke Biotechnology Co., Ltd. Codon optimization was performed on the genes of alcohol dehydrogenases ADH1, ADH2, ADH3, ADH4, ADH5, and ADH6. Corresponding restriction enzyme sites were added to both ends of the genes, and the resulting recombinant plasmid vectors of alcohol dehydrogenases were constructed. These recombinant plasmid vectors were then transformed into hosts such as *E. coli* BL21 using conventional methods. Using the plasmid vector pET28b(+)-ADH-5 constructed by Qingke Biotechnology as a template, PCR amplification was performed using primers to obtain the amplified fragments. The recombinant plasmids, digested with DpnI, were transferred into *E. coli* BL21(DE3) competent cells to obtain clones. The clones were then inoculated into 10 mL LB agar plates and cultured at 37°C for 12–16 h.

[0104] Randomly select positive clones (i.e., clones containing the amplified fragments described above) and the original bacterial strain from the plate, inoculate them into 10 mL of liquid LB medium (with the corresponding antibiotic Kan added to the test tube), and incubate at 37°C and 200 rpm in a temperature-controlled shaker for 8-10 h. Inoculate 1% of the culture solution into 100 mL of LB medium (with the corresponding antibiotic added to the shake flask), and incubate at 37°C and 180 rpm in a temperature-controlled shaker for 2-2.5 h (equivalent to when the bacterial density OD600 value is 0.6-0.8). Add IPTG inducer to a final concentration of 0.1 mM, and then continue incubation at 24°C and 180 rpm in a temperature-controlled shaker for 12 hours. Transfer the cultured bacterial solution to an 800 mL centrifuge cup, centrifuge at 8000 rpm and 4°C for 10 min in a low-temperature high-speed centrifuge, discard the supernatant, collect the bacterial cells and weigh them for subsequent experiments.

[0105] Using the same method as in Example 1, the concentrations and chirality of ethyl 6-carbonyl-8-chlorooctanoate and ethyl (S)-6-hydroxy-8-chlorooctanoate were determined by GC detection, and the conversion rate was calculated. The dominant strains were screened based on the conversion rate and chirality.

[0106] The obtained dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored at -80℃. The final dominant mutants were identified as 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 convention for each mutant is: template-mutation site, such as ADH5-T106I, which means that α-LA-ADH5 is used as a template, and at position 106 of its amino acid sequence, T (threonine) is mutated to I (isoleucine).

[0107] (2) Multiple mutation sites mutants:

[0108] Using the single-mutation site mutants obtained above as templates, 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 were constructed using conventional methods in the art. The following primers were used: 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. Using the constructed recombinant vector as a template, PCR amplification was performed using primers to obtain the target amplified fragment. The primers used were designed using conventional software in the field or employing the following design method: Upstream primer: approximately 15 bp of nucleotide sequence before the mutation site + replacement nucleotide sequence at the mutation site + approximately 10 bp of nucleotide sequence after the mutation site; Downstream primer: approximately 25 bp of reverse nucleotide sequence before the mutation site. After design, the resulting upstream and downstream primers were validated, with the Tm value difference being [not specified]. < 3 is sufficient.

[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 (10mM each) 0.5μL plasmid template 1μL Phanta DNA polymerase (purchased from Novizan) 0.5μL Ultrapure water 8.5μL

[0112] The PCR program set according to the Phanta Super-Fidelity DNA Polymerase manual is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 4 min, 30 cycles; 72℃ final extension for 10 min; 16℃ incubation.

[0113] Then, using the single-mutation site mutant construction method, multiple mutant sites were introduced using the above PCR system.

[0114] Following the above embodiments, GC testing was performed again to screen for dominant mutations. The specific steps were as follows: Using conventional methods in the art, the recombinant vectors containing nucleic acid molecules encoding each mutant were transfected into microbial expression vectors to obtain single-mutant and multi-mutant strains. Simultaneously, using the corresponding wild-type (WT, i.e., using the above template) as a catalyst and ethyl 6-carbonyl-8-chlorooctanoate as a substrate, the catalytic activity of each mutant was compared. The catalytic reaction system consisted of 1 mL of the sample. In the system, the strain acting as the catalyst was 100 g / wet weight L, the final substrate concentration was 50 g / L, and 100 mM PB buffer was used as the reaction medium (containing 10% methyl ether as a co-solvent, pH 7.0). The system was vortexed at 30°C and 1200 rpm for 2 h. Then, 200 μL of the reaction solution was taken and 50 μL of 6M hydrochloric acid was added (to terminate the reaction). 200 μL of ethyl acetate was added for extraction twice, and the ethyl acetate phases were combined. The concentrations and chirality of ethyl 6-carbonyl-8-chlorooctanoate and ethyl (S)-6-hydroxy-8-chlorooctanoate in the ethyl acetate sample were determined using the GC detection method described in Example 1, and the conversion rate was calculated.

[0115] The results are shown in Table 3.

[0116] Table 3. Transformation rate and ee value of mutants

[0117]

[0118]

[0119] Based on the above results, the optimal dominant ADH5 mutant strain obtained through screening was ADH5-L205A / Y209L. The actual transformation rates of all mutants were significantly higher than those of the wild type.

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

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

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

[0123] Add 1 g (wet weight) each of the ADH5-L205A / Y209L mutant strain and formate dehydrogenase engineered bacteria (Escherichia coli) obtained in the above examples 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. + 1 mL of solution and 2 mL of 500 g / L ethyl 6-carbonyl-8-chlorooctanoate solution (with or without different co-solvents) were used. In this example, the co-solvents selected were ethyl acetate, butyl acetate, dibutyl phthalate, DMSO (dimethyl sulfoxide), methyl tert-butyl ether, dimethyl phthalate, and methanol. A control group without co-solvents was used. The reaction was carried out at 30 °C and 800 rpm for 2 h. 1 mL of the reaction solution from each group was taken, extracted with an equal volume of ethyl acetate, and the upper organic phase was collected by centrifugation for GC analysis. The results are shown in Table 4.

[0124] Table 4. Effects of cosolvents on the transformation of alcohol dehydrogenase mutants

[0125] Cosolvent Conversion rate ee value none >99％ S-shaped, 99.48% Ethyl acetate 25.78％ S-shaped, 86.82% Butyl acetate 68.34％ S-shaped, 92.26% Dibutyl phthalate 80.24％ S-shaped, 96.88% DMSO (dimethyl sulfoxide) >99％ S-shaped, 69.16% Methyl tert-butyl ether >99％ S-shaped, 99.56% Dimethyl phthalate >99％ S-shaped, 98.22% methanol 80％ S-shaped, 98.14%

[0126] The above results indicate that the highest conversion rate (>99%) was achieved when methyl tert-butyl ether was used as the co-solvent, with a chirality of S-type and an ee value of 99.56%. However, the conversion rate of the control group without co-solvent was also >99%. Therefore, it can be concluded that the above-mentioned alcohol dehydrogenase mutant can be used without adding a co-solvent, and under these conditions, the conversion rate is not significantly reduced, thus improving the green and environmentally friendly nature of the reaction.

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

[0128] First, the inventors tested the effects of different buffer solutions on the transformation of alcohol dehydrogenase mutants.

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

[0130] Table 5. Effects of buffer solution on the transformation of alcohol dehydrogenase mutants.

[0131]

[0132]

[0133] The above results indicate that PB buffer at pH 7.0 has the highest conversion rate when used as a buffer solution.

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

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

[0136] Table 6. Effect of pH on the transformation of alcohol dehydrogenase mutants

[0137] buffer solution 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 indicate that the highest conversion rate, 99%, was achieved when the buffer solution was PB buffer at pH 6.5.

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

[0140] Add 1 g (wet weight) each of the ADH5-T106F / S131A mutant strain and formate dehydrogenase engineered bacteria obtained in the above examples 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. +1 mL of solution and 2 mL of 500 g / L ethyl 6-carbonyl-8-chlorooctanoate (containing methyl tert-butyl ether) solution were used. The reaction temperatures were set at 20, 25, 30, 35, and 40 °C, respectively. The reaction was carried out at 800 rpm for 6 h. 1 mL of the reaction solution from each group was taken, extracted with an equal volume of ethyl acetate, and the upper organic phase was collected by centrifugation for GC analysis. The results are shown in Table 7.

[0141] Table 7. Effect of temperature on the transformation of alcohol dehydrogenase mutants

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

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

[0144] Example 4: Practical effect of alcohol dehydrogenase mutant in (S)-6-hydroxy-8-chlorooctanoate ethyl ester scale-up experiment

[0145] In this embodiment, the inventors used the ADH5-L205A / Y209L mutant as an example to test the actual effect of the alcohol dehydrogenase mutant in the scale-up experiment of (S)-6-hydroxy-8-chlorooctanoate. The specific test method is as follows:

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

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

[0148] After 6 hours of reaction, TLC analysis showed complete conversion. The reaction solution was extracted with ethyl acetate. An equal volume of ethyl acetate was added for the first extraction, followed by a second and third extraction with half a volume of ethyl acetate. The extraction process included: shaking to mix, centrifuging at 8000 rpm for 5 minutes, collecting the supernatant, and then removing water using anhydrous Na₂SO₄. After extraction, the extract was rotary evaporated (using a vacuum pump followed by an oil pump). The final product obtained after rotary evaporation can be used for further reactions to prepare (R)-α-lipoic acid.

[0149] The final product was subjected to GC analysis, and the results are as follows: Figure 2 As shown, the final product is (S)-6-hydroxy-8-chlorooctanoate ethyl ester. Repeated experiments revealed that other ADH5 mutants exhibited the same effect as ADH5-T106F / S131A. This demonstrates that the mutants in the above examples can catalyze the formation of (S)-6-hydroxy-8-chlorooctanoate ethyl ester from 6-carbonyl-8-chlorooctanoate ethyl ester, with ADH5-L205A / Y209L showing the best performance, achieving a conversion rate >99% and an S-type optical purity >99%.

[0150] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The alcohol dehydrogenase mutant is an alcohol dehydrogenase mutant obtained by L205A and Y209L mutations based on the alcohol dehydrogenase shown in SEQ ID NO:

5.

2. A nucleic acid molecule encoding the alcohol dehydrogenase mutant of claim 1.

3. A product characterized in that, The product includes at least one of the following (1)-(3): (1) Contains an expression unit of the nucleic acid molecule described in claim 2; (2) A transformant containing the nucleic acid molecule described in claim 2; (3) Transformers containing the expression units described in (1); The expression units include plasmids; The transformant was a bacterium.

4. The use of the alcohol dehydrogenase mutant of claim 1 in the synthesis of S-configuration ethyl 6-hydroxy-8-chlorooctanoate.

5. A method for preparing S-configuration ethyl 6-hydroxy-8-chlorooctanoate, comprising the following steps: The alcohol dehydrogenase mutant of claim 1 or the transformant of the product of claim 3 is combined with ethyl 6-carbonyl-8-chlorooctanoate, formate dehydrogenase, ammonium formate and nicotinamide adenine dinucleotide phosphate (NADP). + Mix the ingredients and react them at 15-40℃ for 1-24 hours to obtain the final product.

Images