Alcohol dehydrogenase and method for preparing (R)-6-hydroxy-8-chloro ethyl caprylate under catalysis of alcohol dehydrogenase

Abstract

The invention discloses alcohol dehydrogenase and a method for preparing (R)-6-hydroxy-8-chloro ethyl caprylate through catalysis of the alcohol dehydrogenase, and the alcohol dehydrogenase is selected from one or more of mutants of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, ADH10 and ADH10; the amino acid sequences of the ADH1-ADH10 are as shown in SEQ ID NO. 1 to SEQ ID NO. 10. According to the invention, ethyl 6-carbonyl-8-chloro caprylate can be catalyzed to generate ethyl (R)-6-hydroxy-8-chloro caprylate or ethyl (S)-6-hydroxy-8-chloro caprylate, the reaction conversion rate of the mutant ADH10-A184L / N87F / A61L of ADH10 reaches 99%, and the e.e. Value of the R type is 99.2%.

Description

Technical Field

[0001] The present invention relates to the field of biosynthesis technology, and particularly relates to an alcohol dehydrogenase and a method for catalytically preparing (R)-ethyl 6-hydroxy-8-chlorooctanoate. Background Art

[0002] Ethyl 6-hydroxy-8-chlorooctanoate is a key organic intermediate, mainly used in the synthesis of chiral drugs, such as anti-inflammatory drugs, anti-tumor drugs, etc., especially in the process of preparing (R)-α-lipoic acid. α-Lipoic acid has significant antioxidant properties and can scavenge free radicals that accelerate aging and cause diseases, similar to the function of vitamins. Due to the special biological activity of (R)-α-lipoic acid, the enantiomeric purity is crucial during its synthesis. Therefore, ethyl 6-hydroxy-8-chlorooctanoate, as a chiral precursor for preparing (R)-α-lipoic acid, has important application value.

[0003] Traditional synthesis methods usually use octanoic acid or its derivatives as starting materials, and prepare ethyl 6-hydroxy-8-chlorooctanoate through steps such as chlorination, hydroxylation, and esterification. However, this method often has the following problems: Many reaction steps and low overall yield.

[0004] Difficult chiral control and hard to obtain products with high optical purity.

[0005] Using toxic or expensive reagents, with relatively high environmental and economic costs.

[0006] In order to obtain (R)-ethyl 6-hydroxy-8-chlorooctanoate with high optical purity, asymmetric synthesis technology has become a research hotspot. The following are some key strategies: Chiral catalytic hydrogenation: Using chiral catalysts (such as BINAP-Ru, Rh complexes, and various metal catalysts) to perform asymmetric hydrogenation on the precursor, directly introducing a chiral center. This method has the characteristics of high efficiency and high selectivity.

[0007] Enzyme-catalyzed reaction: Using biocatalysts such as lipases and oxidoreductases to achieve highly enantioselective hydroxylation or esterification reactions under mild conditions. The existing problems with the enzyme method are: insufficient chirality to meet the drug requirements, or low conversion rate, etc.; Chiral auxiliary method: By introducing a chiral auxiliary (such as a chiral alcohol or amine), controlling the stereochemistry in the reaction, and finally removing the auxiliary to obtain the target product (CN115992107A).

[0008] The main synthetic routes of ethyl 6-hydroxy-8-chlorooctanoate include the MPV reduction method of ethyl 6-hydroxy-8-oxooctanoate (CN114149324A); reduction with sodium borohydride using methanol as a solvent; preparation of (R)-ethyl 6-hydroxy-8-chlorooctanoate by lipase hydrolysis and resolution of the racemic lipoic acid intermediate ethyl 6-hydroxy-8-chlorooctanoate; obtaining the (R)-enantiomer by kinetic resolution of racemic ethyl 6-hydroxy-8-chlorooctanoate through enzymatic acetylation reaction, etc. Traditional methods such as reduction with sodium borohydride yield a racemate, and subsequently, the (R)-enantiomer needs to be obtained through enzymatic resolution. This not only increases the process complexity but also theoretically has a maximum yield of only 50%, which is not conducive to large-scale production. Multi-step chemical conversions are usually accompanied by a relatively high amount of waste generation, which does not conform to the principles of green chemistry.

[0009] In summary, the existing chemical synthesis methods have cumbersome steps, low product yields, and high pollution, are not suitable for large-scale production, and the synthesis methods with high optical purity often have high costs. In the current biological preparation methods, during the catalytic process, the cofactor NADP + has an excessive addition amount, a long catalytic time, a low yield, and may generate various by-products during the synthesis process, affecting the product purity. Moreover, the synthesis methods at the laboratory scale are difficult to be directly applied to industrial production, which are not conducive to the inexpensive preparation of this intermediate. Summary of the Invention

[0010] The object of the present invention is to provide an alcohol dehydrogenase and a method for catalytically preparing (R)-ethyl 6-hydroxy-8-chlorooctanoate.

[0011] The technical solution adopted by the present invention to solve its technical problems is: An alcohol dehydrogenase, selected from one or more of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, ADH10, and mutants of ADH10; The amino acid sequences of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, and ADH10 are shown in SEQ ID NO.1 - SEQ ID NO.10.

[0012] The mutants of ADH10 are mutants with 1 - 3 amino acid mutations, specifically including: ADH10 - A184L, ADH10 - A184F, ADH10 - A184W, ADH10 - A184C, ADH10 - V224A, ADH10 - V224C, ADH10 - A194I, ADH10 - A194M, ADH10 - A194T, ADH10 - A194Q, ADH10 - V142A, ADH10 - V142Q, ADH10 - V142I, ADH10 - V142K, ADH10 - A190V, ADH10 - A190I, ADH10 - P183I, ADH10 - P183A, ADH10 - P183Q, ADH10 - A185V, ADH10 - A185C, ADH10 - A185I, ADH10 - A185R, ADH10 - L201I, ADH10 - L201K, ADH10 - A61L, ADH10 - A61Q, ADH10 - N87L, ADH10 - N87F, ADH10 - N87P, ADH10 - A184L / N87F, ADH10 - A184L / V224A, ADH10 - N87F / A61L, ADH10 - A184L / N87F / A61L.

[0013] A method for synthesizing ethyl (R)-6-hydroxy-8-chlorooctanoate by biocatalysis, comprising the following steps: Add alcohol dehydrogenase, formate dehydrogenase, buffer, ethyl 6-oxo-8-chlorooctanoate, co-solvent, ammonium formate and coenzyme into a reaction vessel to form an enzyme-catalyzed reaction system. Stir and react at 25 - 35 °C for 2 - 24 hours, then perform extraction and rotary evaporation to obtain ethyl (R)-6-hydroxy-8-chlorooctanoate; The alcohol dehydrogenase is selected from one or more of ADH2, ADH4, ADH5, ADH7, ADH9, ADH10, and mutants of ADH10; The amino acid sequences of ADH2, ADH4, ADH5, ADH7, ADH9, and ADH10 are shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, and SEQ ID NO.10.

[0014] In the above method, the alcohol dehydrogenase and formate dehydrogenase can be free enzymes (for example, used in the form of enzyme powder), or can be used in the form of cells expressing the aldehyde-ketone reductase / alcohol dehydrogenase catalyst (for example, wet cells expressing the alcohol dehydrogenase catalyst). Or other forms, such as the cell lysate supernatant expressing the alcohol dehydrogenase catalyst. The engineering strain expressing the alcohol dehydrogenase catalyst can be Escherichia coli, or other engineering strains such as Saccharomyces cerevisiae or Bacillus subtilis.

[0015] The mutants of ADH10 are mutants with 1 - 3 amino acid mutations, specifically including: ADH10 - A184L, ADH10 - A184F, ADH10 - A184W, ADH10 - A184C, ADH10 - V224A, ADH10 - V224C, ADH10 - A194I, ADH10 - A194M, ADH10 - A194T, ADH10 - A194Q, ADH10 - V142A, ADH10 - V142Q, ADH10 - V142I, ADH10 - V142K, ADH10 - A190V, ADH10 - A190I, ADH10 - P183I, ADH10 - P183A, ADH10 - P183Q, ADH10 - A185V, ADH10 - A185C, ADH10 - A185I, ADH10 - A185R, ADH10 - L201I, ADH10 - L201K, ADH10 - A61L, ADH10 - A61Q, ADH10 - N87L, ADH10 - N87F, ADH10 - N87P, ADH10 - A184L / N87F, ADH10 - A184L / V224A, ADH10 - N87F / A61L, ADH10 - A184L / N87F / A61L.

[0016] Taking ADH10 - A184L as an example, this mutant is a mutant with 1 amino acid mutation, that is, the 184th amino acid A of ADH10 (SEQ ID NO.10) is mutated to L; the others are expressed with reference to this meaning; Taking ADH10 - A184L / N87F as an example, this mutant is a mutant with 2 amino acid mutations, that is, the 184th amino acid A of ADH10 (SEQ ID NO.10) is mutated to L, and the 87th amino acid N is mutated to F; the others are expressed with reference to this meaning; Taking DH10 - A184L / N87F / A61L as an example, this mutant is a mutant with 3 amino acid mutations, that is, the 184th amino acid A of ADH10 (SEQ ID NO.10) is mutated to L, the 87th amino acid N is mutated to F, and the 61st amino acid A is mutated to L.

[0017] The alcohol dehydrogenase is added in the form of wet cells of recombinant Escherichia coli expressing alcohol dehydrogenase, and the concentration of wet cells of recombinant Escherichia coli expressing alcohol dehydrogenase in the enzyme-catalyzed reaction system is 10-200 g / L; The formate dehydrogenase is added in the form of wet cells of recombinant Escherichia coli expressing formate dehydrogenase, and the concentration of wet cells of recombinant Escherichia coli expressing formate dehydrogenase in the enzyme-catalyzed reaction system is 10-200 g / L.

[0018] For other forms of addition of alcohol dehydrogenase and formate dehydrogenase, the amounts are calculated based on the amount corresponding to the wet cells. Further preferably, the concentration of wet cells of recombinant Escherichia coli expressing alcohol dehydrogenase in the enzyme-catalyzed reaction system is 25-100 g / L; the concentration of wet cells of recombinant Escherichia coli expressing formate dehydrogenase in the enzyme-catalyzed reaction system is 25-100 g / L.

[0019] The addition amount of ethyl 6-oxo-8-chlorooctanoate in the enzyme-catalyzed reaction system is 20 g / L - 200 g / L.

[0020] The concentration of ammonium formate in the enzyme-catalyzed reaction system is 0.2 M - 1.5 M. The concentration of ammonium formate is further preferably 1.2 M.

[0021] The buffer solution is selected from one of TEA buffer solution, PB buffer solution, and Tris-HCl buffer solution. The concentration of the PB buffer solution is 0.05-0.20 M, and the pH value of the PB buffer solution is 6-8. Further preferably on this basis, the concentration of the PB buffer solution is 0.05-0.10 M, and the pH value of the PB buffer solution is 6.5-7.5. More preferably, the concentration of the PB buffer solution is 0.1 M and the pH value is 7; the concentration of the TEA buffer solution is 0.1 M and the pH value is 7, and the concentration of the Tris-HCl buffer solution is 0.1 M and the pH value is 7.

[0022] The coenzyme is NADP + or NAD + and the concentration of the coenzyme in the enzyme-catalyzed reaction system is 0.1 mM - 2.5 mM. The concentration of the coenzyme is further preferably 0.25 mM.

[0023] The cosolvent is one or a combination of ethyl acetate, butyl acetate, ethyl laurate, DMSO, methyl tert-butyl ether, dimethyl phthalate, methanol, isopropanol, etc., and the amount of the cosolvent accounts for 5-50% of the total volume of the enzyme-catalyzed reaction system. Further preferably, the amount of the cosolvent accounts for 20% of the total volume of the enzyme-catalyzed reaction system.

[0024] The beneficial effects of the present invention are: Biocatalysis not only features mild reaction conditions, environmental friendliness, high regioselectivity and stereoselectivity, but also avoids heavy metal residues in the products, exactly making up for the deficiencies of chemical methods. Therefore, in recent years, the application of biocatalytic asymmetric reduction of ketones in the asymmetric synthesis of chiral alcohols has received increasing attention. The present invention provides 10 alcohol dehydrogenases, namely ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, and ADH10, as well as mutants of the preferred enzymes, which can catalyze ethyl 6-oxo-8-chlorooctanoate to produce (R)-ethyl 6-hydroxy-8-chlorooctanoate or (S)-ethyl 6-hydroxy-8-chlorooctanoate. Among them, the mutant ADH10-A184L / N87F / A61L of ADH10 has a reaction conversion rate of 99% and an R-type e.e. value of 99.2%. This method has high stereoselectivity, can accurately recognize the substrate and catalyze specific reactions to generate a single enantiomer; the reaction conditions are mild, avoiding the high temperature, high pressure or strong acid and strong base conditions required by chemical methods, reducing equipment requirements and safety risks; it is environmentally friendly and conforms to the concept of green chemistry; it has high catalytic efficiency, can proceed efficiently at low concentrations, reduce raw material consumption and production costs; it is easy to optimize, and through protein engineering and directed evolution, the activity, stability and selectivity of the enzyme can be optimized to meet different needs; it simplifies the process, with fewer steps, reduces the complexity of intermediate separation and purification, simplifies the process flow, and has broad application prospects and considerable market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the technical roadmap of the present invention.

[0026] Figure 2 is the HPLC detection chart of (S)-ethyl 6-hydroxy-8-chlorooctanoate of the present invention.

[0027] Figure 3 is the HPLC detection chart of (R)-ethyl 6-hydroxy-8-chlorooctanoate of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] The following are specific examples to further illustrate the technical solutions of the present invention.

[0029] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0030] The technical roadmap of the present invention is as Figure 1 shown.

[0031] Example 1: Screening of Alcohol Dehydrogenase The steps are as follows: S1. Using the alcohol dehydrogenases reported in the prior art (literature names: 1. Efficient reductive desymmetrization of bulky 1,3-cyclodiketones enabled by structure-guided directed evolution of a carbonyl reductase; 2. Enantioselective Cascade Biocatalysis via Epoxide Hydrolysis and Alcohol Oxidation: One-Pot Synthesis of (R)-α-Hydroxy Ketones from Meso- or Racemic Epoxides; 3. Induced Axial Chirality in Biocatalytic Asymmetric Ketone Reduction; 4. Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases; 5. Reengineered carbonyl reductase for reducing methyl-substituted cyclohexanones; 6. Efficient preparation of the chiral intermediate of luliconazole with Lactobacillus kefir alcohol dehydrogenase through rational rearrangement of the substrate binding pocket) as gene probes, search for homologous sequences through the GenBank database, mine alcohol dehydrogenases, select according to 60%-90% homology, find putative sequences, and make them in FASTA format.

[0032] S2. Use Clustal Omega to perform multiple sequence alignments on the mined gene sequences, export the data, compare the consistency of different sequences, and exclude gene sequences with a similarity > 95%. According to other reported alcohol dehydrogenases, these sequences all contain conserved sequences and catalytic triads, and sequences without conserved sequences are removed. Use Espript for alignment. Select according to the phylogenetic tree made by MEGA in connection with the homology of the reported alcohol dehydrogenases and the Espript sequence alignment. Only one of the genes with more than 80% sequence identity is retained, and appropriate sequences are screened out.

[0033] S3. According to the codon preference of Escherichia coli, the genes of ten screened alcohol dehydrogenases (ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, ADH10) were codon-optimized (SEQ ID No.11 - SEQ ID No.20). The full genes were synthesized by Tsingke Biological and integrated into the pET28a vector. The vector containing the full genes was transferred into E. coli BL21(DE3) cells to prepare recombinant E. coli engineering bacteria for alcohol dehydrogenase, and stored at -80 °C for the next experiment.

[0034] Preparation of recombinant E. coli engineering bacteria for formate dehydrogenase: (1) The formate dehydrogenase gene (GenBank accession number: AXT18256.1) was synthesized by Tsingke Biological and integrated into the pET28a vector to complete the construction of the expression vector.

[0035] (2) Escherichia coli BL21(DE3) was used for the expression of the enzyme protein. The externally constructed plasmid was sequentially transformed into the same strain of E. coli cells, and then spread on LB plate medium (added with 50 mg / L kanamycin and 20 mg / L chloramphenicol). The specific implementation is as follows: First, the bacteria transformed with plasmid pET28 were spread on LB plate medium containing kanamycin resistance (50 mg / L kanamycin) and cultured at 37 °C for 12 hours. Then, single colonies were picked and transferred into LB liquid medium (extra added kanamycin to a final concentration of 50 mg / L), and cultured at 37 °C for 12 hours to prepare competent cells. Then, the plasmid pET28 containing the target gene was transformed into the competent cells and spread on LB plate medium containing chloramphenicol (20 mg / L) resistance, and cultured at 37 °C for 12 hours to obtain recombinant bacteria, which were stored at -80 °C for the next experiment.

[0036] Example 2: Screening of alcohol dehydrogenase for 6 - oxo - 8 - chloroethyl octanoate The steps are as follows: S1. Pick single colonies on the plates of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, ADH10 and formate dehydrogenase, and inoculate them into 10 mL LB liquid medium (containing 50 μg / mL antibiotic Kan), and culture them in a temperature-controlled shaker at 37 °C and 200 rpm for 8 - 10 h. The culture solution was inoculated into 100 mL LB liquid medium (containing 50 μg / mL antibiotic Kan) at an inoculation amount of 1%, and also cultured in a temperature-controlled shaker at 37 °C and 180 rpm for 2 - 2.5 h until the cell density OD600 The value is 0.6 - 0.8. Add the inducer IPTG with a final concentration of 0.1 mM, and continue culturing in a temperature-controlled shaker at 24°C and 180 rpm for 12 h. Pour the cultured bacterial solution into a 500 ml large centrifuge cup, centrifuge at 8000 rpm and 4°C in a low-temperature high-speed centrifuge for 10 min, discard all the supernatant, collect the bacterial cells and weigh them for use in subsequent experiments.

[0037] S2. Respectively, take 0.15 g of wet bacterial cells of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, and ADH10 and add them to 2 mL EP tubes, and set up a blank control group.

[0038] S3. Correspondingly, add 0.15 g of wet bacterial cells of formate dehydrogenase to each EP tube. Add 600 μL of PB buffer (0.1 M, pH 7), 100 μL of 7 M ammonium formate solution, 100 μL of 25 mM NADP + solution 100 μL, 200 μL of 0.5 M ethyl 6-oxo-8-chlorooctanoate (containing 100 μL of methyl tert-butyl ether) solution to each EP tube. The total system is 1 mL. Place the EP tubes in a thermostatic mixer and shake and react at 30°C and 1200 rpm for 6 h.

[0039] S4. After the reaction is completed, respectively take 200 μL of the reaction solution, add 200 μL of ethyl acetate, shake and mix well for 2 min, centrifuge at 12000 rpm for 1 min, take the supernatant, and send it for GC detection.

[0040] After GC detection, the conversion rates and e.e. values of each alcohol dehydrogenase are shown in Table 1: Table 1 Aldehyde-ketone reductase / Alcohol dehydrogenase Conversion rate e.e. value ADH1 38.46％ S form, 76.24% ADH2 90.45％ R form, 22.24% ADH3 34.32％ S form, 60.64% ADH4 55.79％ R form, 1.24% ADH5 22.46％ R form, 48.58% ADH6 94.21％ S form, 92.22% ADH7 47.79％ R form, 80.44% ADH8 12.68％ S form, 33.78% ADH9 36.40％ R form, 68.44% ADH10 90.68％ R form, 81.80% The results show that the conversion rate of ADH10 is relatively high at 90.68%, and its chirality is R-type with an e.e. value of 81.80%. The chirality of ADH1, ADH3, ADH6, and ADH8 is S-type ( Figure 2 ).

[0041] Example 3: Construction and screening of an alcohol dehydrogenase mutant library 1. Starting strain: Use the alcohol dehydrogenase ADH10 synthesized after screening in Example 1 as the original strain, activate and extract the plasmid pET28b(+)-ADH-10. Among them, the amino acid sequence of the alcohol dehydrogenase ADH-10 is shown in SEQ ID NO.10, and the coding gene sequence is shown in SEQ ID NO.20.

[0042] 2. Single mutation: (1) Construction of the mutant library The preparation of the ADH-10 mutant library was achieved by site-directed mutagenesis. Using the vector pET28b(+)-ADH-10 in the original strain as a template, polymerase chain reaction (PCR) was carried out. The recombinant plasmid digested with DpnⅠ was transferred into Escherichia coli BL21(DE3) competent cells, and the clones were inoculated into 10 mL of LB plate medium and cultured at 37 °C for 12 - 16 h.

[0043] (2) GC screening Positive clones on the plate and the original strain were randomly selected and inoculated into 10 mL of LB liquid medium (with 50 μg / mL of the antibiotic Kan), and cultured in a temperature-controlled shaker at 37 °C and 200 rpm for 8 - 10 h. The culture solution was inoculated into 100 mL of LB liquid medium (with 50 μg / mL of the antibiotic Kan) at an inoculation amount of 1%, and also cultured in a temperature-controlled shaker at 37 °C and 180 rpm for 2 - 2.5 h (equivalent to when the cell density OD 600 value was 0.6 - 0.8), then an inducer IPTG with a final concentration of 0.1 mM was added, and the culture was continued in a temperature-controlled shaker at 24 °C and 180 rpm for 12 hours. The cultured bacterial solution was poured into an 800 ml large centrifuge tube and centrifuged at 8000 rpm and 4 °C for 10 min in a low-temperature high-speed centrifuge. The supernatant was discarded completely, and the cells were collected and weighed for use in subsequent experiments.

[0044] Consistent with the screening method of alcohol dehydrogenase in Example 2, GC was used to detect the concentration and chirality of ethyl 6-oxo-8-chlorooctanoate and (R) / (S)-6-hydroxy-8-chlorooctanoate, and the conversion rate was calculated. Using the conversion rate and chirality as indicators, superior strains were screened.

[0045] The obtained dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored in an -80°C refrigerator. The finally screened dominant mutants were ADH10-A184L, ADH10-A184F, ADH10-A184W, ADH10-A184C, ADH10-V224A, ADH10-V224C, ADH10-A194I, ADH10-A194M, ADH10-A194T, ADH10-A194Q, ADH10-V142A, ADH10-V142Q, ADH10-V142I, ADH10-V142K, ADH10-A190V, ADH10-A190I, ADH10-P183I, ADH10-P183A, ADH10-P183Q, ADH10-A185V, ADH10-A185C, ADH10-A185I, ADH10-A185R, ADH10-L201I, ADH10-L201K, ADH10-A61L, ADH10-A61Q, ADH10-N87L, ADH10-N87F, ADH10-N87P.

[0046] 3. Iterative mutation Using vectors pET28b(+)-ADH10-A184L, ADH10-A184F, ADH10-A184W, ADH10-A184C, ADH10-V224A, ADH10-V224C, ADH10-A194I, ADH10-A194M, ADH10-A194T, ADH10-A194Q, ADH10-V142A, ADH10-V142Q, ADH10-V142I, ADH10-V142K, ADH10-A190V, ADH10-A190I, ADH10-P183I, ADH10-P183A, ADH10-P183Q, ADH10-A185V, ADH10-A185C, ADH10-A185I, ADH10-A185R, ADH10-L201I, ADH10-L201K, ADH10-A61L, ADH10-A61Q, ADH10-N87L, ADH10-N87F, ADH10-N87P as templates, polymerase chain reaction (PCR) was carried out. Further screening for dominant mutations was performed on the basis of single-point mutants according to the steps of GC screening.

[0047] 4. Catalytic activity detection Using the cell suspensions of single mutant strains, iterative mutant strains, and control strains as catalysts respectively, and ethyl 6-oxo-8-chlorooctanoate as the substrate, the catalytic activities of each mutant were compared. The reaction system was selected to be 1 mL. 0.15 g of wet cells of formate dehydrogenase and wet cells of ADH10 mutant were added to each EP tube respectively. 600 μL of PB buffer (0.1 M, pH 7), 100 μL of 7 M ammonium formate solution, 100 μL of 25 mM NADP + solution 100 μL, 200 μL of 0.5 M ethyl 6-oxo-8-chlorooctanoate (containing 100 μL of methyl tert-butyl ether) solution were added. The reaction was carried out with vortex oscillation at 30 °C and 1200 rpm for 2 h. 200 μL of the reaction solution was taken and 50 μL of 6 M hydrochloric acid was added (to terminate the reaction), and then extracted twice with 200 μL of ethyl acetate. The ethyl acetate phases were combined. For the ethyl acetate sample, the concentrations and chirality of ethyl 6-oxo-8-chlorooctanoate and (R) / (S)-6-hydroxy-8-chlorooctanoate were detected by GC and the conversion rate was calculated.

[0048] The screening results are shown in Table 2: Table 2 Finally, through further screening, the most dominant strain of the ADH10 mutant was obtained as: ADH10-A184L / N87F / A61L.

[0049] Example 4: Screening of cosolvents The steps are as follows: S1. 1 g of wet cells of ADH10-A184L / N87F / A61L and 1 g of wet cells of formate dehydrogenase, 6 mL of PB buffer (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP + solution 1 mL, 2 mL of 500 g / L ethyl 6-oxo-8-chlorooctanoate (containing 1 mL of cosolvent) solution were added to the reaction flask. Using ethyl acetate, butyl acetate, ethyl laurate, DMSO (dimethyl sulfoxide), methyl tert-butyl ether, dimethyl phthalate, methanol, isopropanol, etc. as cosolvents respectively, and without adding cosolvent as the control group, the reaction was carried out at 30 °C and 800 rpm.

[0050] S2. When the reaction reached 2 h, 1 mL of the reaction solution was taken, extracted with an equal volume of ethyl acetate, and the upper organic layer was taken by centrifugation and sent for GC detection. The results are shown in Table 3: Table 3 Cosolvent Conversion rate e.e. value None >99％ R form, 98.88% Ethyl acetate 68.44％ R form, 88.86% Butyl acetate 78.26％ R form, 90.82% Ethyl laurate 90.42％ R form, 95.84% DMSO (Dimethyl sulfoxide) 85.47％ R form, 78.22% Methyl tert-butyl ether >99％ R form, 99.2% Dimethyl phthalate >99％ R form, 98.40% Methanol 78.86％ R form, 90.74% Isopropanol 88.98％ R form, 96.26% The results show that when the cosolvent is methyl tert-butyl ether, the conversion rate is the highest at >99%, and its chirality is R type with an e.e. value of 99.2%.

[0051] Example 5: Buffer and pH Optimization Buffer Optimization: S1. Add 1 g of each of ADH10 - A184L / N87F / A61L wet cells and formate dehydrogenase wet cells, 6 mL of buffer (0.1 M), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP + solution, and 2 mL of 500 g / L ethyl 6 - oxo - 8 - chloro - octanoate (containing 1 mL of methyl tert - butyl ether) solution into the reaction flask. The buffers are set as PB, Tris - HCl, and TEA buffers with pH 7.0, and react at 30 °C and 800 rpm.

[0052] S2. Take 1 mL of the reaction solution at 1 h of reaction, add an equal volume of ethyl acetate for extraction, centrifuge, and take the upper organic layer for GC detection. The results are shown in Table 4: Table 4 Buffer solution Conversion rate e.e. value PB buffer solution at pH 7.0 87.43％ R form, 99.16% Tris-HCl buffer solution at pH 7.0 67.66％ R form, 99.14% TEA buffer solution at pH 7.0 70.24％ R form, 99.16% The results show that the conversion rate is the highest when the buffer is PB buffer with pH 7.0.

[0053] pH Optimization: S1. Add 1 g of each of ADH10 - A184L / N87F / A61L wet cells and formate dehydrogenase wet cells, 6 mL of buffer (0.1 M), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP + solution, and 2 mL of 500 g / L ethyl 6 - oxo - 8 - chloro - octanoate (containing 1 mL of methyl tert - butyl ether) solution into the reaction flask. The buffers are set as PB buffers with pH 6.0, 6.5, 7.0, 7.5, and 8.0, and react at 30 °C and 800 rpm.

[0054] S2. Take 1 mL of the reaction solution at 2 h of reaction, add an equal volume of ethyl acetate for extraction, centrifuge, and take the upper organic layer for GC detection. The results are shown in Table 5: Table 5 Buffer solution Conversion rate PB buffer solution at pH 6.0 89.46％ PB buffer solution at pH 6.5 95.44％ PB buffer solution at pH 7.0 99％ PB buffer solution at pH 7.5 80.32％ PB buffer solution at pH 8.0 62.22％ The results show that the highest conversion rate is >99% when the buffer is PB buffer with pH 7.0, which is the most preferred.

[0055] Example 6: Reaction Temperature Optimization The steps are as follows: S1. Add 1 g of each of ADH10 - A184L / N87F / A61L wet cells and formate dehydrogenase wet cells, 6 mL of PB buffer (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP +1 mL of solution, 2 mL of 500 g / L 6-carbonyl-8-chlorooctanoic acid ethyl ester (containing 1 mL of methyl tert-butyl ether) solution. The reaction temperatures were set at 20, 25, 30, 35, and 40° C., respectively, and the reaction was carried out at 800 rpm.

[0056] S2. After 6 hours of reaction, 1 mL of the reaction solution was taken and extracted with an equal volume of ethyl acetate. The upper organic phase was centrifuged and sent to GC for detection. The results are shown in Table 6: Table 6 Temperature (°C) Conversion rate e.e. value 20 81.66％ R form, >99% 25 90.88％ R form, >99% 30 >99％ R form, >99% 35 94.35％ R form, 96.88% 40 82.33％ R form, 90.42% The results show that the highest conversion rate is >99% when the temperature is 30°C, and its chirality is R type with an ee value of >99%, which is the most preferred.

[0057] Example 7: Scale-up experiment and post-processing S1. Take a 2000mL three-necked flask, add 50g each of ADH10-A184L / N87F / A61L wet bacteria and formate dehydrogenase wet bacteria, add 600mL of PB buffer (0.1M, pH7.0), 100mL of 11.3M ammonium formate solution, and 3mM NADP + The solution is 100 mL, containing 100 g of ethyl 6-carbonyl-8-chlorooctanoate (containing 100 mL of methyl tert-butyl ether), about 200 mL, and the total reaction system is 1 L. Place in a 30°C water bath and stir at 300 rpm for reaction.

[0058] S2. Take 200 μL of the reaction solution at 2h, 4h and 6h of reaction, add an equal volume of ethyl acetate for extraction and then perform TLC detection.

[0059] S3, react for 6 hours. After detection of complete conversion, the reaction solution is extracted with ethyl acetate. The same volume of ethyl acetate is added for the first extraction, and 1 / 2 volume of ethyl acetate is added for the second and third extractions (extraction: after shaking and mixing, centrifuge at 8000rpm for 5min, aspirate the supernatant, and use anhydrous Na2SO4 to absorb water). After the extraction, the extract is rotary evaporated.

[0060] S4. The extract is rotary evaporated by first vacuum pump and then oil pump. The product is obtained after rotary evaporation and used for the next step of chlorination.

[0061] The GC test results of the product are as attached. Figure 3 As shown, the product is (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester.

[0062] ADH10-A184L / N87F / A61L can catalyze 6-carbonyl-8-chlorooctanoic acid ethyl ester to produce (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester, with a reaction conversion rate of >99% and an ee value of greater than 99%.

[0063] Embodiment 8: The difference between this example and Example 7 is as follows: S1. Take a 2000 mL three-necked flask, add 10 g each of ADH10 - A184L / N87F / A61L wet cells and formate dehydrogenase wet cells, add 730 mL of PB buffer (0.1 M, pH 7.0), 100 mL of 2 M ammonium formate solution, 100 mL of 1 mM NADP + solution, about 70 mL of a system containing 20 g of ethyl 6-oxo-8-chlorooctanoate (containing 50 mL of methyl tert-butyl ether), and the total reaction system is 1 L. Place it in a 30 °C water bath and stir the reaction at 300 rpm.

[0064] Example 9: The difference between this example and Example 7 is as follows: S1. Take a 2000 mL three-necked flask, add 200 g each of ADH10 - A184L / N87F / A61L wet cells and formate dehydrogenase wet cells, add 100 mL of PB buffer (0.1 M, pH 7.0), 100 mL of 15 M ammonium formate solution, 100 mL of 25 mM NADP + solution, about 700 mL of a system containing 200 g of ethyl 6-oxo-8-chlorooctanoate (containing 500 mL of methyl tert-butyl ether), and the total reaction system is 1 L. Place it in a 30 °C water bath and stir the reaction at 300 rpm.

[0065] Amino acid sequence: SEQ ID NO.1 MSTAGKVIKCKAAVLWEEKKPFSIEEVEVAPPKAHEVRIKMVATGICRSDDHVVSGTLVTPL PVIAGHEAAGIVESIGEGVTTVRPGDKVIPLFTPQCGKCRVCKHPEGNFCLKNDLSMPRGT MQDGTSRFTCRGKPIHHFLGTSTFSQYTVVDEISVAKIDAASPLEKVCLIGCGFSTGYGSAV KVAKVTQGSTCAVFGLGGVGLSVIMGCKAAGAARIIGVDINKDKFAKAKEVGATECVNPQ DYKKPIQEVLTEMSNGGVDFSFEVIGRLDTMVTALSCCQEAYGVSVIVGVPPDSQNLSMNP MLLLSGRTWKGAIFGGFKSKDSVPKLVADFMAKKFALDPLITHVLPFEKINEGFDLLRSGESI RTILTFHHHHHH SEQ ID NO.2 MRAVVFENKERVAVKEVNAPRLQHPLDALVRVHLAGICGSDLHLYHGKIPVLPGSVLGHEF VGQVEAVGEGIQDLQPGDWVVGPFHIACGTCPYCRRHQYNLCERGGVYGYGPMFGNLQG AQAEILRVPFSNVNLRKLPPNLSPERAIFAGDILSTAYGGLIQGQLRPGDSVAVIGAGPVGLM AIEVAQVLGASKILAIDRIPERLERAASLGAIPINAEQENPVRRVRSETNDEGPDLVLEAVGG AATLSLALEMVRPGGRVSAVGVDNAPSFPFPLASGLVKDLTFRIGLANVHLYIDAVLALLAS GRLQPERIVSHYLPLEEAPRGYELFDRKEALKVLLVVRGHHHHHHSEQ ID NO.3 MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTSDIHTVFEGAIGERHNMILGHEAV GEVVEVGSEVKDFKPGDRVVVPAITPDWRTSEVQRGYHQHSGGMLAGWKFSNVKDGVFG EFFHVNDADMNLAHLPKEIPLEAAVMIPDMMTTGFHGAELADIELGATVAVLGIGPVGLMA VAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDIVNYKDGPIESQIMNLTEGKGVDAAIIAG GNADIMATAVKIVKPGGTIANVNYFGEGEVLPVPRLEWGCGMAHKTIKGGLCPGGRLRME RLIDLVFYKRVDPSKLVTHVFRGFDNIEKAFMLMKDKPKDLIKPVVILAHHHHHHSEQ ID NO.4 MADTMLAAVVREFGKPLSIERLPIPDIKPHQILVKVDTCGVCHTDLHAARGDWPSKPNPPFI PGHEGVGHIVAVGSQVGDFVKTGDVVGVPWLYSACGHCEHCLGGWETLCEKQDDTGYTV NGCFAEYVVADPNYVAHLPSTIDPLQASPVLCAGLTVYKGLKMTEARPGQWVAVSGVGGL GQMAVQYAVAMGMNVVAVDIDDEKLATAKKLGASLTVNAKDTDPARFIQQQIGGAHGALV TAVGRTAFSQAMGYARRGGTIVLNGLPPGDFPVSIFDMVMNGTTIRGSIVGTRLDMIEAMD FFARGKVKSVVTPGKLENINTIFDDLQNGRLEGRTVLDFRSHHHHHHSEQ ID NO.5 MSIPSSQYGFVFNKQSGLNLRNDLPVHKPKAGQLLLKVDAVGLCHSDLHVIYEGLDCGDN YVMGHEIAGTVAAVGDDVINYKVGDRVACVGPNGCGGCKYCRGAIDNVCKNAFGDWFGL GYDGGYQQYLLVTRPRNLSRIPDNVSADVAAASTDAVLTPYHAIKMAQVSPTSNILLIGAG GLGGNAIQVAKAFGAKVTVLDKKKEARDQAKKLGADAVYETLPESISPGSFSACFDFVSVQ ATFDVCQKYVEPKGVIMPVGLGAPNLSFNLGDLALREIRILGSFWGTTNDLDDVLKLVSEG KVKPVVRSAKLKELPEYIEKLRNNAYEGRVVFNPHHHHHH SEQ ID NO.6 MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQ HDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFG TRLGIQRMKNKGLGASIINMSSIEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVR VNTVHPGYIKTPLVDDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEF VVDGGYTAQHHHHHH SEQ ID NO.7 MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQH DSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTR LGIQRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVN TVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVD GGYTAQHHHHHH SEQ ID NO.8 MINMRNRVAIVTGGAMGMGNGCARKLAEAGAKVYLIDRSNLVAAAAQDMREAGLNANH VQVDITDQESLTSAYDGIAAESGRLDVLVNAAGVGDSRMFLDVDDAHYKKVIDVNVRGT WNSCRAAVPHMLSNKHGRIINFGSISGPIVADPGWTVYALSKGAIFGFTKALASEFAGQNILV NAILPGSMDTPMMRAAAADTNPADPQSVIDEIAAAVPLKRLGTIDDAGNLALFLASDLASY LTGQAIVLDGAFTLAEYQSGGLEAPAETPALVNHHHHHH SEQ ID NO.9 MYRLLNKTAVITGGNSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADV TKLEDLDRLYAIVREQRGSIDVLFANSGAVEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALP LLRDGGSVILTSSVAGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTP SLENNVSTQEEADELRAKAAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLT QVHHHHHH SEQ ID NO.10 MTNLPTALITGASSGIGATYAERFARRGHNLVMVARDKARMDLLASRLREETKVTIDVIQA DLTQQEDLAEIESRLREDTSIGILINNAGMGQSGAFIQQNAQSIDRLVMLNTTAPTRLAAAVA ARFAQEGKGSIVNIGSVVGFAPELGMTIYGATKAFVLFLSQGLNLELGSKGIYVQAVLPAAT RTEIWSRAGMDINSLPEVMDVNELVDAALVGFDRKELVTIPPLHVAERWDELEQARQGLMSEIRQAHAAERYLPQAHHHHHH。

[0066] The above-described embodiments are only a preferred solution of the present invention and do not impose any formal restrictions on the present invention. There are other variations and modifications without exceeding the technical solutions described in the claims.

Claims

1. An alcohol dehydrogenase, characterized in that One or more selected from ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, ADH10, and mutants of ADH10; The amino acid sequences of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9 and ADH10 are shown in SEQ ID NO.1 to SEQ ID NO.

10.

2. An alcohol dehydrogenase according to claim 1, characterized in that The mutants of ADH10 are mutants of 1-3 amino acids, specifically including: ADH10-A184L, ADH10-A184F, ADH10-A184W, ADH10-A184C, ADH10-V224A, ADH10-V224C, ADH10-A194I, ADH10-A194M, ADH10-A194T, ADH10-A194Q, ADH10-V142A, ADH10-V142Q, ADH10-V142I, ADH10-V142K, ADH10-A190V, ADH10-A190I, ADH10-P1 83I, ADH10-P183A, ADH10-P183Q, ADH10-A185V, ADH10-A185C, ADH10-A185I, ADH10-A185R, ADH10-L201I, ADH10-L201K, ADH10-A61L, ADH 10-A61Q, ADH10-N87L, ADH10-N87F, ADH10-N87P, ADH10-A184L / N87F, ADH10-A184L / V224A, ADH10-N87F / A61L, ADH10-A184L / N87F / A61L.

3. A method for synthesizing (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester by bioenzymatic method, characterized in that: The following steps are involved: Adding alcohol dehydrogenase, formate dehydrogenase, buffer, 6-carbonyl-8-chlorooctanoic acid ethyl ester, cosolvent, ammonium formate and coenzyme into a reaction container to form an enzyme catalytic reaction system, stirring and reacting at 25-35° C. for 2-24 hours, extracting, and rotary evaporating to obtain (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester; The alcohol dehydrogenase is selected from one or more of ADH2, ADH4, ADH5, ADH7, ADH9, ADH10, and mutants of ADH10; The amino acid sequences of ADH2, ADH4, ADH5, ADH7, ADH9 and ADH10 are shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.

10.

4. The method according to claim 3, characterized in that The mutants of ADH10 are mutants of 1-3 amino acids, specifically including: ADH10-A184L, ADH10-A184F, ADH10-A184W, ADH10-A184C, ADH10-V224A, ADH10-V224C, ADH10-A194I, ADH10-A194M, ADH10-A194T, ADH10-A194Q, ADH10-V142A, ADH10-V142Q, ADH10-V142I, ADH10-V142K, ADH10-A190V, ADH10-A190I, ADH10-P1 83I, ADH10-P183A, ADH10-P183Q, ADH10-A185V, ADH10-A185C, ADH10-A185I, ADH10-A185R, ADH10-L201I, ADH10-L201K, ADH10-A61L, ADH 10-A61Q, ADH10-N87L, ADH10-N87F, ADH10-N87P, ADH10-A184L / N87F, ADH10-A184L / V224A, ADH10-N87F / A61L, ADH10-A184L / N87F / A61L.

5. The method according to claim 3, characterized in that: The alcohol dehydrogenase is added in the form of alcohol dehydrogenase recombinant Escherichia coli wet cells, and the concentration of alcohol dehydrogenase recombinant Escherichia coli wet cells in the enzyme catalytic reaction system is 10-200 g / L; The formate dehydrogenase is added in the form of formate dehydrogenase recombinant Escherichia coli wet cells, and the concentration of formate dehydrogenase recombinant Escherichia coli wet cells in the enzyme catalytic reaction system is 10-200 g / L.

6. The method according to claim 3, characterized in that The addition amount of 6-carbonyl-8-chlorooctanoic acid ethyl ester in the enzyme catalytic reaction system is 20g / L-200g / L.

7. The method according to claim 3, characterized in that The concentration of ammonium formate in the enzyme-catalyzed reaction system is 0.2M-1.5M.

8. The method according to claim 3, characterized in that The buffer is selected from one of TEA buffer, PB buffer and Tris-HCl buffer.

9. The method according to claim 3, characterized in that: The coenzyme is NADP + or NAD + , the concentration of coenzyme in the enzyme-catalyzed reaction system is 0.1mM~2.5mM.

10. The method according to claim 3, characterized in that: The cosolvent is a combination of one or more of ethyl acetate, butyl acetate, ethyl dodecanoate, DMSO, methyl tert-butyl ether, dimethyl phthalate, methanol and isopropanol, and the amount of the cosolvent accounts for 5-50% of the total volume of the enzyme catalytic reaction system.

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