Alcohol dehydrogenase mutant and application thereof

Abstract

The invention discloses an alcohol dehydrogenase mutant and application thereof, wherein the alcohol dehydrogenase mutant is obtained by mutating the 258th asparagine of the amino acid sequence shown in SEQ ID NO.1 into cysteine. The invention finds a key amino acid residue that affects the activity of alcohol dehydrogenase, and based on (2S, 3S)-2,3-butanediol dehydrogenase (BDH, NCBI accession number: WP_211439413) derived from Corynebacterium glutamicum, a rationally designed molecular modification method is used to solve the problem that the wild-type enzyme has no enzyme activity or low enzyme activity on natural or non-natural substrates, and the obtained mutant can catalyze the preparation of chiral diols (2S, 5S)-2,5-hexanediol and (2S, 3S)-2,3-butanediol with extremely high optical purity, which provides broad application prospects for the biotransformation production of pharmaceutical precursors (2S, 5S)-2,5-hexanediol and (2S, 3S)-2,3-butanediol.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to an alcohol dehydrogenase mutant and application thereof. Background Art

[0002] Chiral diols are a very important class of compounds, which are very important in drug synthesis, agricultural chemical synthesis, functional material preparation, fragrance and essential oil additives. The chemical method for preparing chiral diols is complicated and cumbersome, and the catalyst is expensive, toxic and environmentally unfriendly. The biological method for catalytic asymmetric reduction of diketones to synthesize chiral diols has attracted the attention of researchers at home and abroad due to its good catalytic activity, stereoselectivity and environmental friendliness. (2S,3S)-2,3-Butanediol is a compound containing two ortho chiral centers, which is the cornerstone of asymmetric synthesis and can also be used to produce printing inks and solvents. (2S,5S)-2,5-Hexanediol, as an optically active alcohol with a specific group, is an important chiral building block for the synthesis of many chiral drugs and other chiral chemicals, and has broad application prospects in the fields of medicine and chemical industry.

[0003] (2S,3S)-2,3-butanediol dehydrogenase (EC 1.1.1.304) is a key enzyme in the synthesis of (2S,3S)-2,3-butanediol. It participates in the synthesis and catabolism of (2S,3S)-2,3-butanediol and is widely present in all living organisms. (2S,3S)-2,3-dehydrogenase has great application value and potential in the field of chiral diol synthesis due to its wide source and strict stereoselectivity. However, the activity of wild (2S,3S)-2,3-butanediol dehydrogenase on the natural substrate (2S,3S)-2,3-butanediol is very low and cannot meet the requirements of industrial applications. It has strong substrate specificity and has almost no activity on (2S,5S)-2,5-hexanediol. Therefore, improving the catalytic ability of wild-type (2S,3S)-2,3-butanediol dehydrogenase to produce (2S,3S)-2,3-butanediol and (2S,5S)-2,5-hexanediol through molecular modification will help it to be better applied in industry.

[0004] In view of this, further in-depth research is needed to solve the problem of no enzyme activity or low enzyme activity of NAD(H)-specific (2S,3S)-2,3-butanediol dehydrogenase in the production of (2S,3S)-2,3-butanediol and (2S,5S)-2,5-hexanediol through structure-based enzyme design. Summary of the invention

[0005] The invention provides an alcohol dehydrogenase mutant and application thereof. The alcohol dehydrogenase mutant solves the problem that the original (2S, 3S)-2,3-butanediol dehydrogenase (amino acid sequence is shown in SEQ ID NO.1) derived from (Corynebacterium glutamicum) has no enzyme activity or has low enzyme activity to prochiral diketones, provides a (2S, 3S)-2,3-butanediol dehydrogenase mutant and application thereof in preparing different (2S, 5S)-2,5-hexanediols and (2S, 3S)-2,3-butanediols.

[0006] The specific technical solutions are as follows:

[0007] The present invention provides an alcohol dehydrogenase mutant, wherein the mutant is obtained by mutating the 258th asparagine in the amino acid sequence shown in SEQ ID NO.1 into cysteine.

[0008] The present invention performs AlphaFold 2 tetramer modeling on a mutant (N258C) of (2S, 3S)-2,3-butanediol dehydrogenase (BDH, NCBI accession number: WP_211439413, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) derived from Corynebacterium glutamicum, C258 between chain A and chain D and between chain B and chain C forms two pairs of interchain disulfide bonds, increases the stability of polymer binding, improves the activity of the enzyme to prochiral diketones; and performs activity assays on other non-natural substrates to expand the application of the mutant. Wherein, N258C means: the 258th position, i.e., the last amino acid of the enzyme, is mutated from asparagine to cysteine.

[0009] The present invention also provides a gene encoding the alcohol dehydrogenase mutant.

[0010] The present invention also provides a recombinant vector comprising the coding gene. Preferably, the original expression vector of the recombinant vector is pET28a-SUMO.

[0011] The present invention also provides a genetically engineered bacterium comprising the coding gene. Preferably, the host cell of the genetically engineered bacterium is E. coli BL21 (DE3).

[0012] The present invention also provides the use of the alcohol dehydrogenase mutant or the genetically engineered bacteria in catalyzing the synthesis of (2S, 5S)-2,5-hexanediol from 2,5-hexanedione.

[0013] The present invention also provides the use of the alcohol dehydrogenase mutant or the genetically engineered bacteria in catalyzing the synthesis of (2S, 3S)-2,3-butanediol from 2,3-butanedione.

[0014] The present invention also provides the use of the alcohol dehydrogenase mutant or the genetically engineered bacteria in catalyzing the synthesis of (2S, 5S)-2,5-hexanedione from 2,5-hexanedione.

[0015] The present invention also provides the use of the alcohol dehydrogenase mutant or the genetically engineered bacteria in catalyzing the synthesis of (2S, 3S)-2,3-butanediol from acetoin.

[0016] The present invention also provides the use of the alcohol dehydrogenase mutant or the genetically engineered bacteria in catalyzing the synthesis of (3S)-acetoin from (2S, 3S)-2,3-butanediol.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention is based on (2S,3S)-2,3-butanediol dehydrogenase (BDH, NCBI accession number: WP_211439413) from Corynebacterium glutamicum. Through a rationally designed molecular modification method, the problem that the wild-type enzyme has no enzyme activity or low enzyme activity on natural or non-natural substrates is solved. The obtained mutant can catalyze the preparation of chiral diols (2S,5S)-2,5-hexanediol and (2S,3S)-2,3-butanediol, the enzyme activity is increased by 10-100 times, and it shows very high stereoselectivity.

[0019] (2) The rational design method used in the present invention quickly obtains a (2S,3S)-2,3-butanediol dehydrogenase mutant with high catalytic activity against the chiral diols (2S,5S)-2,5-hexanediol and (2S,3S)-2,3-butanediol.

[0020] (3) The method of the present invention uses NAD(H) as a coenzyme, which is cheaper than NADP(H), thus greatly reducing the production cost of chiral diols.

[0021] (4) The present invention discovered a key amino acid residue that affects the activity of alcohol dehydrogenase, which has important guiding significance for improving the activity of alcohol dehydrogenase. It can not only catalyze multiple substrates, but also the products (2S,5S)-2,5-hexanediol and (2S,3S)-2,3-butanediol obtained by the reaction have extremely high optical purity, which provides broad application prospects for the biotransformation production of pharmaceutical precursors (2S,5S)-2,5-hexanediol and (2S,3S)-2,3-butanediol. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the non-denaturing protein gel electrophoresis image of wild-type alcohol dehydrogenase CgBDH.

[0023] Figure 2 The catalytic mechanism of alcohol dehydrogenase CgBDH and the defined pre-reaction state conformation, where the distance

[0024] Figure 3 The electrophoresis analysis of purified alcohol dehydrogenase CgBDH on SDS-PAGE;

[0025] Among them, 1 is the alcohol dehydrogenase CgBDH without SUMO tag removal; 2 is the alcohol dehydrogenase CgBDH with SUMO tag removal.

[0026] Figure 4 It is the optimum temperature of wild-type alcohol dehydrogenase CgBDH and its mutants.

[0027] Figure 5 It is the optimal pH of wild-type alcohol dehydrogenase CgBDH and its mutants; among them, A, C, E, G, I: reduction reaction; B, D, F, H, J: oxidation reaction. AB: PB buffer; CD: Tris-HCl; EF: Glycine-NaOH; GH: CAPSO; IJ: CAPS.

[0028] Figure 6 Shown are the thermal denaturation curves of wild-type alcohol dehydrogenase CgBDH and its mutants. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

[0030] The experimental methods in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

[0031] Reagents used in upstream genetic engineering: DpnI used in the examples of the present invention was purchased from TaKaRa, Takara Bioengineering (Dalian) Co., Ltd.; plasmid extraction kits and DNA recovery and purification kits were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21 (DE3), plasmid pET28a-SUMO, etc. were purchased from Novagen; DNA marker, low molecular weight standard protein, agarose electrophoresis reagents were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; primer synthesis and sequence sequencing were completed by Qingke Bioengineering Co., Ltd. The use of the above reagents refers to the product instructions.

[0032] Reagents used in the catalytic reaction: (2S,5S)-2,5-hexanediol and (2S,3S)-2,3-butanediol, NAD +and NADH were purchased from MacLean. Standard assay for (2S,3S)-2,3-butanediol dehydrogenase activity: appropriate amount of crude enzyme solution (1-5μg / mL), 12.5mM substrate (2,5-hexanedione and 2,3-butanedione), 0.56mM NAD+, total system 1000μL, reaction medium glycine-NaOH buffer (20mM, pH 9.2). Reaction at 20℃ for 5min, NADH generated / consumed in the sample was quantitatively analyzed by microplate reader, and a data point was taken every 30s. Definition of enzyme activity unit (U): Under standard reaction conditions, enzyme activity unit (U) is defined as the amount of enzyme required to consume or generate 1μmol NADH per minute.

[0033] Example 1 Design of N258C mutant

[0034] Purify different batches of alcohol dehydrogenase CgBDH, the purification process is:

[0035] (1) After the recombinant cells containing alcohol dehydrogenase CgBDH were disrupted, the supernatant was transferred to a pre-cooled centrifuge tube and placed in an ice bath for later use. The liquid flow rate during the entire purification process was maintained at 1 mL / min. The pre-loaded column was equilibrated with a nickel column using a Tris-HCl buffer (20 mM, pH = 8.0) containing 3 mM imidazole at a volume of ten times the column.

[0036] (2) The supernatant flows through the nickel column. During this process, the target enzyme containing the His tag and some impurity proteins will specifically bind to the nickel.

[0037] (3) The protein in the nickel column was washed with ten column volumes of Tris-HCl buffer (20 mM, pH = 8.0) containing 20 mM imidazole.

[0038] (4) The nickel column was eluted with 500 mM imidazole Tris-HCl buffer (20 mM, pH = 8.0) to collect the protein.

[0039] The purification results are as follows Figure 1 As shown, the molecular weight of the monomer His6-SUMO-CgBDH protein is 40.84 kDa. The molecular weight analysis shows that the enzyme is in a dynamic equilibrium of monomers, dimers, tetramers and octamers. According to some inactive mutants, it was found that alcohol dehydrogenase CgBDH plays a catalytic role when it is a polymer.

[0040] Therefore, it is proposed to improve the enzyme activity by increasing the stability between polymers. Among them, N258 is found to be a key site. It is the last amino acid of the enzyme. When forming a tetramer, the four N258 residues converge in the inner core of the protein; it is proposed to mutate N258 to C, construct interchain disulfide bonds to improve the binding stability between polymers; AlphaFold2 was used to predict the tetrameric structure of the N258C mutant, and it was found that two pairs of disulfide bonds were formed between chains A and D and between chains B and C.

[0041] All-atom molecular dynamics simulations were performed on the wild-type alcohol dehydrogenase CgBDH and its mutants to investigate the stability of the designed interchain disulfide bonds of alcohol dehydrogenase CgBDH and the structural mechanism of improved enzyme activity.

[0042] Specifically:

[0043] All molecular dynamics simulations were performed using Amber18 software; the ff14SB force field was used for proteins, and NAD + and NADH cofactor using the force field constructed by Holmberg et al.; adding explicit TIP3P water molecules, the protein is 2.5 Å away from the edge of the box. Electroneutralization was then performed using an ion concentration of 0.15 M NaCl. The energy minimization was performed in three stages. The first stage minimized only the positions of solvent molecules and ions; the second stage minimized hydrogen atoms; and the third stage minimized all atoms in the simulation system without constraints. Each stage of minimization consisted of 2500 steepest descent steps and 2500 conjugate gradient steps. The system was then gently heated by increasing the temperature from 0 to 300 K with constant volume and periodic boundary conditions. The force constants are applied to proteins and small molecules, and the temperature is controlled by the Langevin thermostat method. The PME method is used to model long-range electrostatic interactions. Lennard-Jones and electrostatic interactions are modeled using The bond lengths involving hydrogen atoms were constrained using the SHAKE algorithm. NPT-MD was then run for 400 ps at a constant pressure of 1 atm and temperature of 300 K with a time step of 2 fs, followed by an additional 100 ns simulation under unconstrained conditions. The results show that the designed interchain disulfide bonds are stable and that the number of pre-reaction conformations of the N258C mutant is about 2.95 times that of the wild type. The pre-reaction conformation is defined as Figure 2 shown.

[0044] Example 2 Construction of single point mutations

[0045] The wild-type alcohol dehydrogenase CgBDH was subjected to a single-point mutation of N258C. The specific method is as follows:

[0046] 1. Whole plasmid PCR

[0047] Using the pET28a-SUMO-CgBDH plasmid as a template, upstream and downstream primers covering the mutation point were designed (Table 1) for full plasmid PCR:

[0048] Table 1 Primers used for construction of single point mutation library

[0049]

[0050] PCR amplification system:

[0051]

[0052] PCR amplification conditions:

[0053] 1) Pre-denaturation: 95℃ for 5min;

[0054] 2) Denaturation: 98°C for 10 s; annealing: 60°C for 15 s; extension: 72°C for 10 s; 30 cycles in total;

[0055] 3) Post-extension: 72°C for 10 min;

[0056] 4) Store at 4℃.

[0057] 2. Template digestion:

[0058] The PCR product was subjected to agarose gel electrophoresis and then digested with DpnI enzyme. The digestion system of the plasmid template was as follows: 1 μL DpnI enzyme, 45 μL PCR product, and 4 μL Buffer. The template digestion was completed at 37°C for 1 hour.

[0059] 3. Conversion and verification:

[0060] The digested product was verified by nucleic acid agarose gel electrophoresis and then transformed into E. coli BL21 (DE3) competent cells using the 42°C heat shock method. The specific process is as follows:

[0061] (1) Thaw the competent cells on ice for 5 minutes;

[0062] (2) Under sterile conditions, add 10 μL of DNA to 100 μL of competent cells and mix gently, then place on ice for 30 min;

[0063] (3) The EP tube was placed in a 42°C metal bath for 90 seconds for heat shock and then cooled on ice for 2 minutes.

[0064] (4) Add 500 μL of LB medium to the EP tube and mix well with a pipette tip, then incubate at 37°C in a shaker at 220 rpm for 60 min;

[0065] (5) After concentration, take an appropriate volume and apply it to the corresponding resistance plate. After culturing in a 37°C incubator for 12-16 hours, colonies will appear; pick out 3-4 single colonies from each plate for culture, and sequence to verify whether the mutation is successful.

[0066] 4. Mutant strain culture and protein expression

[0067] After the mutant strains were successfully sequenced, they were activated by streaking on the plate, and a single colony was inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking for 12 h. A 2% inoculum was transferred to 200 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking until OD 600 When it reached about 0.6-0.8, IPTG was added to a final concentration of 0.5 mM and induced at 37°C for 6 h.

[0068] After the culture is completed, the culture solution is centrifuged at 4000g for 15min at 4℃, the supernatant is discarded, and the cells are collected. The collected cells are washed twice with 20mM Tris-HCl buffer at pH 8.0, resuspended in Tris-HCl buffer, and ultrasonically disrupted 30 times at 400W power, each ultrasonication lasting 3s, with a rest of 7s. The cell disruption solution is centrifuged at 12000g for 30min at 4℃ to remove the precipitate, and the supernatant obtained is the crude enzyme solution.

[0069] 5. Enzyme activity determination

[0070] The enzyme activity of the above mutants on substrates 2,5-hexanedione and 2,3-butanedione was measured by an ELISA instrument. The results are shown in Table 2:

[0071] Table 2 Determination of enzyme activity of (2S,3S)-2,3-butanediol dehydrogenase and its single-point mutants on different substrates

[0072]

[0073] Example 3

[0074] 1. Purification of wild-type CgBDH and its mutants

[0075] The purification process of alcohol dehydrogenase CgBDH and its mutants is as follows:

[0076] (1) After the recombinant cells containing the mutant (N258C) were disrupted, the supernatant was transferred to a pre-cooled centrifuge tube and placed in an ice bath for later use. The liquid flow rate during the entire purification process was maintained at 1 mL / min. The pre-loaded column was equilibrated with a nickel column using a Tris-HCl buffer (20 mM, pH = 8.0) containing 3 mM imidazole at a volume of ten times the column.

[0077] (2) The supernatant flows through the nickel column. During this process, the target enzyme containing the His tag and some impurity proteins will specifically bind to the nickel.

[0078] (3) The protein in the nickel column was washed with ten column volumes of Tris-HCl buffer (20 mM, pH = 8.0) containing 20 mM imidazole.

[0079] (4) The nickel column was eluted with 500 mM imidazole Tris-HCl buffer (20 mM, pH = 8.0) to collect the protein.

[0080] After the enzyme is purified, 3 volumes of Tris-HCl buffer (20 mM, pH = 8.0) are added to dilute the high concentration of imidazole. The SUMO-Tag is removed by ULP1 protease; ULP1 protease has high specificity and maintains high activity in a wide range of reaction environment systems. To ensure the activity of alcohol dehydrogenase CgBDH, enzyme cleavage is performed at 4°C. Figure 3 As shown. The molecular weight of the monomeric His6-SUMO-CgBDH protein is 40.84 kDa. After the protein is cleaved by ULP1 protease, ULP1 protease, His6-SUMO tag (apparent molecular weight 13 kDa) and uncleaved protein remain in the affinity chromatography column. The molecular weight of the cleaved CgBDH protein is 27.05 kDa. SDS-PAGE analysis showed that the protein was successfully purified to more than 95% homogeneity ( Figure 3 ).

[0081] 2. Establishment of the optimal reaction conditions

[0082] Determine the optimal conditions for enzyme-catalyzed reactions: including temperature and pH, etc.

[0083] The optimum temperature determination method for alcohol dehydrogenase is as follows: the pure enzyme and the components of the enzyme activity detection system are incubated in an ELISA plate for 5 minutes, the incubation temperature range is 20-65°C, and an experimental point is set every 5°C. The detection system is: the purified pure enzyme solution (1-5 μg / mL) of the first part of the embodiment, 0.56 mM NAD + , the total system is 200μL, and the reaction medium is glycine-NaOH buffer (20mM, pH 9.2). After the incubation, 12.5mM substrate 2,3-butanedione was added, and the amount of NADH generated in the sample was measured by an ELISA reader at a wavelength of 340nm, taking a data point every 30s. The temperature corresponding to the experimental group with the greatest activity is the optimal temperature of the enzyme. At the optimal temperature, the enzyme activity here is defined as 100%, and the temperature-activity curve is made by different temperatures and relative enzyme activities. The optimal temperature finally obtained is 50℃.

[0084] The optimum pH determination method of alcohol dehydrogenase is as follows: alcohol dehydrogenase is incubated in phosphate buffer (20mM, pH6.0-8.0), Tris-HCl buffer (20mM, pH 7.0-9.0) and glycine-NaOH buffer (20mM, pH 8.5-10.5) respectively, and the detection temperature is room temperature (25°C). The reaction system is 200μL, which also includes the purified pure enzyme solution (1-5μg / mL) and 0.56mM NAD + . After incubation for 5 minutes, 12.5mM 2,3-butanedione was added, and the amount of NADH generated in the sample was measured by a microplate reader at a wavelength of 340nm, with a data point taken every 30 seconds. The pH corresponding to the buffer with the highest activity was taken as the optimal pH for alcohol dehydrogenase. The enzyme activity-pH curve was made by plotting the relative activity of the enzyme at different pH values. The optimal pH for the oxidation reaction of the enzyme was 10.5, and the optimal pH for the reduction reaction was 7.0. The activity of the N258C mutant was higher than that of the wild type in the pH range of 5.5-11, which was approximately 1.6-7 times higher.

[0085] 3. Activity determination of wild-type CgBDH and its mutants

[0086] At 340 nm, the oxidation of NADH or NAD + The catalytic activity of CgBDH and its mutants was determined by the reduction of NADH. The protein concentration was determined using the BCA method with bovine serum albumin as the standard protein. Diacetyl, acetoin, meso-2,3-butanediol and (2S,3S)-2,3-butanediol were used as substrates to determine the reaction kinetic parameters. The reduction reaction was measured in Tris-HCl buffer (20 mM, pH = 7.0) with a NADH concentration of 0.50 mM, and the kinetics were measured using a series of substrate concentrations (0.025, 0.05, 0.125, 0.25, 0.5, 1.25, 2.5, 5, 12.5 mM). The oxidation reaction was measured in Gly-NaOH (20 mM, pH = 10.5) with a NAD+ concentration of 0.56 mM. The parameters of (2S,3S)-2,3-butanediol were analyzed using a series of substrate concentrations (0.025, 0.05, 0.125, 0.25, 0.5, 1.25, 2.5, 5, 12.5 mM). Both reactions were performed at 25 °C. K m and V max The values ​​were fitted using nonlinear regression models using R language.

[0087] The N258C mutant had improved activity on all six substrates tested (Table 3). m The value is doubled, but k catThe value is about 4 times that of the wild type, and the final catalytic efficiency is about 2 times that of the wild type. The k cat The catalytic efficiency was increased by about 8 times (Table 4).

[0088] 4. Stability determination of wild-type CgBDH and its mutants

[0089] Thermodynamic stability determination: nanoDSF differential scanning fluorescence uses the fluorescent amino acids (tryptophan and tyrosine) in the protein to analyze the changes in fluorescence intensity and peak value during the unfolding process of the protein, thereby analyzing the thermal stability of the protein. The melting temperature (T) was determined by recording the fluorescence intensity at 330nm and 350nm using a Prometheus NT.Plex instrument. m The concentration of wild-type CgBDH and its mutants was 500 μg / mL, and the experimental temperature gradient was 2°C / min, increasing from 25°C to 95°C.

[0090] Kinetic stability determination: 200 μL of enzyme was placed in a 50°C metal bath. Samples were taken out at different times (0, 2, 5, 10 min) and then cooled to 25°C to determine the half-life of the enzyme activity.

[0091] The thermal denaturation curve of N258C showed two T m The values ​​of CgBDH at 50°C and 50°C were 2°C and 19.7°C higher than those of wild type, respectively. In addition, the thermal denaturation curve is different from that of wild type, which also shows the influence of disulfide bonds on protein conformation. The kinetic stability of N258C has also been significantly improved. After heating at 50°C for 5 minutes, the wild type completely lost its activity, while N258C still maintained 70% of its activity after heating for 20 minutes. The half-life of CgBDH at 50°C is t 1 / 2,50℃ is 0.91min; the half-life of CgBDH-N258C at 50℃ is t 1 / 2,50℃ It is 13.45min.

[0092] Table 3 Enzyme activities of wild-type CgBDH and its mutants

[0093]

[0094] Table 4 Kinetic constants of wild-type CgBDH and its mutants

[0095]

Claims

1. An alcohol dehydrogenase mutant, characterized in that The amino acid sequence is obtained by mutating the 258th asparagine in the amino acid sequence shown in SEQ ID NO.1 into cysteine.

2. A gene encoding the alcohol dehydrogenase mutant according to claim 1.

3. A recombinant vector comprising the coding gene according to claim 2.

4. A genetically engineered bacterium comprising the coding gene according to claim 2.

5. Use of the alcohol dehydrogenase mutant according to claim 1 or the genetically engineered bacteria according to claim 4 in catalyzing 2,5-hexanedione.

6. Use of the alcohol dehydrogenase mutant according to claim 1 or the genetically engineered bacteria according to claim 4 in catalyzing 2,3-butanedione.

7. Use of the alcohol dehydrogenase mutant according to claim 1 or the genetically engineered bacteria according to claim 4 in catalyzing acetoin.

8. Use of the alcohol dehydrogenase mutant according to claim 1 or the genetically engineered bacteria according to claim 4 in catalyzing (2S, 3S)-2,3-butanediol.

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