Alcohol dehydrogenase mutant with improved ethyl (R)-4-chloro-3-hydroxybutyrate production capacity and application thereof

Abstract

The invention belongs to the field of enzyme engineering, and discloses an alcohol dehydrogenase mutant with improved production capacity of (R)-4-chloro-3-hydroxybutyric acid ethyl ester, which is characterized in that after an active center, a substrate channel and a far-end hot spot of alcohol dehydrogenase are subjected to semi-rational design transformation, the 151st, 215th and 167th sites of an amino acid sequence shown as SEQ ID NO.1 are subjected to combined mutation to obtain a mutant W151F / F215Y / S167A, and the mutant W151F / F215Y / S167A is used for producing the (R)-4-chloro-3-hydroxybutyric acid ethyl ester. The mutant enzyme with further enhanced (R)-4-chloro-3-hydroxybutyric acid ethyl ester synthesis capability is obtained. The specific enzyme activity of the optimal mutant W151F / F215Y / S167A is 4.55 times that of a wild type. The application of the alcohol dehydrogenase mutant disclosed by the invention has important industrial application significance on green and efficient preparation of (R)-4-chloro-3-ethyl hydroxybutyrate.

Description

Technical Field

[0001] The invention relates to an alcohol dehydrogenase mutant with improved catalytic activity and application thereof, and belongs to the field of enzyme engineering. Background Art

[0002] 4-Chloro-3-hydroxybutyric acid ethyl ester (CHBE) is a chiral organic alcohol and an important intermediate for the synthesis of a series of pharmaceuticals, pesticides and fine chemicals by reduction, substitution or introduction of additional functional groups. 4-Chloro-3-hydroxybutyric acid ethyl ester (CHBE) contains two enantiomers: (S)-4-chloro-3-hydroxybutyric acid ethyl ester and (R)-4-chloro-3-hydroxybutyric acid ethyl ester. (S)-4-chloro-3-hydroxybutyric acid ethyl ester is often used in the synthesis of chiral side chains of statins, while (R)-4-chloro-3-hydroxybutyric acid ethyl ester is a valuable intermediate for the production of afatinib, (R)-carnitine and macrolide A as well as [+]-negatinib.

[0003] At present, the demand for 4-chloro-3-hydroxybutyric acid ethyl ester of R configuration is very large but it is difficult to obtain. Therefore, it is of great significance to study the selective production of optically active drug intermediate (R)-4-chloro-3-hydroxybutyric acid ethyl ester. (R)-4-chloro-3-hydroxybutyric acid ethyl ester is currently mainly prepared by chemical method and biological method. Chemical method usually uses chemical catalyst containing rare metals, reducing metal catalyst containing ammonia or flammable and explosive chemical catalyst (such as sodium borohydride) to catalyze 4-chloroacetoacetic acid ethyl ester to synthesize (R)-4-chloro-3-hydroxybutyric acid ethyl ester. These methods need to use expensive rare metals as chemical catalysts, and the reaction conditions are harsh, and the optical purity of the product is not high. Compared with traditional chemical methods, biosynthesis has the advantages of good enantioselectivity, fast reaction rate, mild reaction conditions, and low cost.

[0004] So far, methods such as whole-cell catalysis, enzyme immobilization, and directed evolution have been reported to improve the production efficiency of (R)-4-chloro-3-hydroxybutyrate ethyl ester. For example, Lodderomyces elongisporus was used as a catalyst and yeast cells were permeabilized with hexadecyltrimethylammonium bromide to increase the catalytic activity of alcohol dehydrogenase (ADH) in the cells, and finally 447mM 4-chloro-ethyl acetoacetate was completely converted into (R)-4-chloro-3-hydroxybutyrate ethyl ester (yield 99.5%; ee>99%). In addition, in order to improve the stability of aldehyde-keto reductase, the aldehyde-keto reductase LEK and GDH were co-immobilized on MCF cross-linked with 1.0mM p-benzoquinone by covalent co-immobilization combined with microwave radiation assistance. This method established an efficient and stable dual-enzyme cascade biological redox system, and the relative activity of the immobilized enzyme (CoLG-MIA) was 140% of that of free LEK. In order to further improve the catalytic efficiency of aldehyde-keto reductase, two mutants with higher molar conversion rates were obtained by site-directed mutagenesis of aldehyde-keto reductase (LEK) (LEK W28A and LEK S209G ), the coenzyme regeneration system constructed with glucose as the cosubstrate can completely convert 51.8mM ethyl 4-chloro-acetoacetate into (R)-4-chloro-3-hydroxybutyrate ethyl ester (ee>99%). However, they still have obstacles such as low substrate concentration, poor enzyme stability, and high catalyst / substrate (C / S) ratio, which hinder their application in the industrial synthesis of (R)-4-chloro-3-hydroxybutyrate ethyl ester. Therefore, the development of potent biocatalysts with high substrate concentration and specific activity is crucial to improve the manufacturing process and reduce the cost of industrial production. Summary of the invention

[0005] The present invention aims to provide an alcohol dehydrogenase mutant with improved (R)-4-chloro-3-hydroxybutyric acid ethyl ester production ability, a coding gene, a recombinant plasmid, a recombinant genetic engineering bacterium and an application thereof in catalyzing the production of (R)-4-chloro-3-hydroxybutyric acid ethyl ester from a substrate 4-chloro-ethyl acetoacetate.

[0006] The amino acid sequence of alcohol dehydrogenase before mutation is shown in SEQ ID NO.1:

[0007] MAKVAMITGGAQGIGAEIARHLSKDGFDIAIADLPGQKDTAASVIKEVEGAGQKATFVALDVTDQAAFTNAVDQVSNELGGFDVMVNNAGIAKVDKVEEIKQADLELSFKINVFGVLYGIQAAAKKFKQ LGVAGKIINASSIAGMRAFPVWATYSATKAAVISLTQSSANELAHDHITVNAYAPGVVGTTGMWDEIDAKMADINGKPLGQNKADFVDTIPLGRTVEPNDIANMVSFLASEKADFITGQAYAVDGGGLL

[0008] The nucleotide sequence of the gene encoding the alcohol dehydrogenase is shown in SEQ ID NO.2:

[0009] ATGGCTAAAGTTGCGATGATCACCGGTGGTGCGCAGGGTATAGGTGCGGAAATCGCGCGTCACCTGTCTAAAGATGGTTTCGATATCGCGATCGCGGATCTGCCGGGCCAGAAAGATACCGCTGCGTCTGTTATCAAAGAAGTGGAAGGTGCGGGTCAGAAAGCGACCTTCGTTGCGCTGGATGTTACCGATC AGGCGCTTTTACCAACGCGGTTGATCAGGTTTCTAACGAACTGGGCGGTTTTGATGTTATGGTTAACAACGCTGGTATCGCAAAAGTTGATAAAGTTGAAGAAATCAAACAGGCGGATCTGGAACTGAGCTTCAAAATTAACGTGTTCGGTGTTCTGTACGGTATCCAGGCTGCTGCTAAAAAAATTTAAACAG CTGGGTGTTGCGGGCAAAATCATTAACGCAAGCTCTATCGCTGGCATGCGTGCTTTTCCGGTGTGGGCGACCTATTCCGCTACCAAAGCTGCTGTTATTTCTCTGACCCAGAGCTCTGCAAATGAACTGGCACACGATCACATCACCGTTAACGCTTATGCGCCGGGTGTTGTTGGTACCACCGGCATGTGGG ATGAAATCGATGCGAAAATGGCGGATATCAACGGTAAACCGCTGGGTCAAAACAAAGCGGATTTCGTTGATACCATCCCGCTGGGTCGTACCGTTGAACCGAACGATATCGCGAACATGGTTAGCTTCCTGGCGAGCGAAAAAGCGGATTTCATCACCGGTCAGGCGTACGCGGTTGATGGCGGCGGTCTGCTG

[0010] An alcohol dehydrogenase mutant with improved ability to produce ethyl (R)-4-chloro-3-hydroxybutyrate, the alcohol dehydrogenase mutant comprising at least one of mutation point a, mutation point b and mutation point c.

[0011] Mutation point a: The mutant W151F was obtained by mutating the tryptophan at position 151 of the amino acid sequence shown in SEQ ID NO.1 to phenylalanine;

[0012] Mutation point b: the mutant F215Y obtained by mutating the phenylalanine at position 215 of the amino acid sequence shown in SEQ ID NO.1 to tyrosine;

[0013] Mutation point c: The mutant S167A was obtained by mutating the serine at position 167 of the amino acid sequence shown in SEQ ID NO.1 to alanine.

[0014] Preferably, the alcohol dehydrogenase mutant comprises mutation point a, mutation point b and mutation point c, that is, the 151st tryptophan, the 215th phenylalanine and the 167th serine of the amino acid sequence shown in SEQ ID NO.1 are combined and mutated to obtain the mutant W151F / F215Y / S167A.

[0015] Furthermore, the coding gene of the alcohol dehydrogenase mutant, the recombinant plasmid containing the coding gene of the alcohol dehydrogenase mutant, the genetically engineered bacteria prepared by transformation of the recombinant plasmid, and the production of the alcohol dehydrogenase mutant using genetically engineered bacteria are all within the protection scope of this patent.

[0016] The alcohol dehydrogenase LCRIII used in the present invention is derived from Lactobacillus curieae S1L19, the amino acid sequence of the enzyme is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. The inventor uses AlphaFold3 for structural modeling, inputs the LCRIII amino acid sequence into AlphaFold3 to evaluate the rationality of the model, generates an alcohol dehydrogenase LCRIII model, docks the substrate into the alcohol dehydrogenase LCRIII model, analyzes the substrate-enzyme docking complex conformation, and formulates corresponding protein engineering strategies for the active center, substrate channel and distal hotspot of the alcohol dehydrogenase LCRIII to obtain mutants with improved enzyme activity. Through these protein engineering strategies, the present invention obtains multiple single mutants and combined mutants with improved catalytic efficiency of alcohol dehydrogenase to generate (R)-4-chloro-3-hydroxybutyric acid ethyl ester, and the acquisition of these mutants is of great significance to the catalytic synthesis of (R)-4-chloro-3-hydroxybutyric acid ethyl ester.

[0017] The beneficial effects achieved by the present invention are:

[0018] 1) The present invention molecularly modified LCRIII through a semi-rational design strategy, and constructed a variety of single-point and multi-point mutants of alcohol dehydrogenase that can catalyze 4-chloro-ethyl acetoacetate to generate (R)-4-chloro-3-hydroxybutyrate ethyl ester. The catalytic activities of the single-point mutants W151F, F215Y, and S167A were increased from 5.05 U / mg of the wild enzyme to 13.25 U / mg, 17.10 U / mg, and 6.86 U / mg, respectively. The catalytic activities of the multi-point mutants W151F / F215Y, W151F / S167A, F215Y / S167A, and W151F / F215Y / S167A were increased from 5.05 U / mg of the wild enzyme to 20.04 U / mg, 16.25 U / mg, 17.31 U / mg, and 23.00 U / mg, respectively.

[0019] 2) The engineered strain containing the multi-point mutants W151F / F215Y / S167A constructed by the present invention was subjected to fed-batch fermentation in a 1L reactor under optimal transformation conditions (30°C, pH 6, glucose / COBE (mmol / mmol) of 1.5, and bacterial dosage of 15 gDCW / L). It was able to completely catalyze up to 296.26 g / L of 4-chloro-ethyl acetoacetate to produce (R)-4-chloro-3-hydroxybutyric acid ethyl ester in 32 hours, with an ee value of 99.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 Schematic diagram of the construction of the recombinant plasmid pET28a-LCRIII-GDH;

[0022] Figure 2 The SDS-PAGE diagram of alcohol dehydrogenase LCRIII expressed alone and LCRIII co-expressed with GDH;

[0023] Figure 3 It is a schematic diagram of the relative enzyme activity of the wild type and mutant of alcohol dehydrogenase LCRIII;

[0024] Figure 4 This is a diagram showing the reaction process of synthesizing (R)-4-chloro-3-hydroxybutyric acid ethyl ester from 4-chloro-ethyl acetoacetate catalyzed by an engineered strain containing the optimal combination mutants W151F / F215Y / S167A. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0026] The formula of LB liquid culture medium is: peptone 10g / L, sodium chloride 10g / L, yeast extract 5g / L. Add a certain amount of deionized water to the weighed three substances and stir them thoroughly. The pH value is natural. The prepared culture medium is sterilized at 121℃ for 20 minutes using a high-pressure steam sterilizer.

[0027] LB solid medium can be prepared by adding 1.5% agar to LB liquid medium, and the sterilization conditions are the same as above.

[0028] 4-chloro-ethyl acetoacetate and (R)-4-chloro-3-hydroxybutyric acid ethyl ester were determined by high performance liquid chromatography. The conditions for high performance liquid chromatography analysis were: CHIRALCEL OB-OH column, 5 μm, column temperature: 30°C, detection wavelength: 215 nm, injection volume: 10 μL, flow rate: 0.6 mL / min, mobile phase: n-hexane / isopropanol = 75:25 (V / V).

[0029] Example 1

[0030] Alcohol dehydrogenase LCRIII and GDH (GDH is Glutamate Dehydrogenase (glutamate dehydrogenase) gene sequence Get the column:

[0031] The LCRIII gene sequence from Lactobacillus curieae S1L19 (GenBank accession number: WP_035165810.1) was retrieved from the NCBI database. The sequence is shown in SEQ ID No. 2. The sequence was synthesized by Sangon Biotech (Shanghai). The GDH gene sequence is shown in SEQ ID NO. 3: ATGTATCCGGATTTAAAAGGAAAAGTCGTCGCTATT ACAGGAGCTGCTTCAGGGCTCGGAAAGGCGATGGCCATTCGCTTCGGCAAGGAGCAGGCAAAAGTGGTTATCAACT ATTATAGTAATAAACAAGATCCGAACGAGGTAAAAGAAGAGGTCATCAAGGCGGGCGGTGAAGCTGTTGTCGTCCA AGGAGATGTCACGAAAGAGGAAGATGTAAAAAAATATCGTGCAAACGGCAATTAAGGAGTTCGGCACACTCGATATT ATGATTAATAATGCCGGTCTTGAATTCCCTGTGCCATCTCACGAAATGCCGCTCAAGGATTGGGATAAAGTCATCG GCACGAACTTAACGGGTGCCTTTTTAGGAAGCCGTGAAGCGATTAAATATTTCGTAGAAAACGATATCAAGGGAAA TGTCATTAACATGTCCAGTGTGCACGAAGTGATTCCTTGGCCGTTATTTGTCCACTATGCGGCAAGTAAAGGCGGG ATAAAGCTGATGACAGAAACATTAGCGTTGGAATACGCGCCGAAGGGCATTCGCGTCAATAATAATTGGGCCAGGTG CGATCTCGACGGCCAATCAATGCTGGCAAATTCGCTGACCCTAAACAGAAAGCTGATGTAGAAAGCATGATTCCAAT GGGATATATCGGCGAACCGGAGGAGATCGCCGCAGTAGCAGCCTGGCTTGCTTCGAAGGAAGCCAGCTACGTCACA GGCATCACGTTATTCGCGGACGGCGGTATGACACAATATCCTTCATTCCAGGCAGGCCGCGGT.

[0032] Embodiment 2:

[0033] Construction of recombinant plasmid:

[0034] The alcohol dehydrogenase LCRIII gene sequence and the GDH gene sequence were connected by RBS, and then inserted into the pET-28a plasmid vector using the BamH I and HindIII restriction endonuclease sites to construct the recombinant plasmid pET-28a-LCRIII-GDH. The schematic diagram of the construction of the recombinant plasmid pET28a-LCRIII-GDH is shown in Figure 1In addition, in order to purify the target protein, a 6×His tag was introduced at the N-terminus of the enzyme. The primers with RBS sequences and homology arms are shown in Table 1. Figure 2 The SDS-PAGE diagram of alcohol dehydrogenase LCRIII expressed alone and LCRIII co-expressed with GDH.

[0035] Table 1 Primer sequences

[0036]

[0037] Construction of recombinant bacteria, heterologous expression of alcohol dehydrogenase and protein purification:

[0038] Example 3: Selection of mutation sites

[0039] In order to improve the catalytic efficiency of LCRIII, we first analyzed the LCRIII sequence data set and amino acid conformation, and then developed a protein modification strategy to comprehensively regulate the active center, substrate channel and distal hotspots of LCRIII. Finally, a small and precise mutation library containing 15 site-directed mutation sites and 2 saturation mutation sites was established, and mutants with improved enzyme activity were efficiently screened. Finally, the reasonable mutation sites A92C, I142V, A143V, W151I, W151M, W151F, and V186I were determined.

[0040] Table 2 Primer sequences

[0041]

[0042]

[0043]

[0044] The specific experimental methods are as follows:

[0045] First, we used the sequence information of the LCRIII family to determine the mutation sites in the active center according to consensus design. The LCRIII amino acid sequence was entered into Consensus Finder, which found 406 similar sequences from the NCBI database and provided a table of amino acid frequencies and counts at each position. In order to reduce the number of candidate amino acids, we applied the following three criteria to screen mutants:

[0046] 1) The non-conserved amino acids around the active site may be related to the catalytic performance of the enzyme, so we selected the active site The residues within were studied;

[0047] 2) To avoid contingency in consensus evolution, mutations with a consensus amino acid count of 5 or more were selected;

[0048] 3) LCRIII is a hydrophobic active pocket. The hydrophobic effect is key to protein assembly and stability, so mutations that may introduce hydrophilic amino acids into the protein core were removed.

[0049] Next, according to the molecular docking results of LCRIII and substrate, the substrate channel entrance was engineered. The active pocket of the enzyme was drawn using Pymol software. The study found that the four amino acid residues I142, F148, V186 and F215 formed a narrow bottleneck at the channel entrance, which was not conducive to substrate entry and product release. Therefore, we optimized these four amino acid residues. Through alanine scanning, these four sites were first mutated to I142A, F148A, V186A, and F215A, and the wild-type and mutant purified enzymes were extracted and the relative enzyme activity was determined. The results showed that a positive mutation was obtained at F215. Then, the NNK SM library of the F215 site was constructed, and finally the best mutant F215Y at the modified site was obtained, with a relative enzyme activity of 17.10U / mg, which is 3.39 times that of the wild type (5.05U / mg).

[0050] Finally, in order to further improve the enzymatic activity of LCRIII, the distal mutation "hotspots" were efficiently identified based on sequence alignment and conservative analysis. The T69, V72, S163, and S167 sites of LCRIII were finally located as potential mutation "hotspots" for improving activity. Therefore, we first performed alanine scanning on these four sites and mutated them to T69A, V72A, S163A, and S167A, respectively. The experimental results showed that a positive mutation was obtained at S167. Subsequently, the NNK SM library of the S167 site was constructed, and finally the best mutant S167A at this site was obtained, with a specific enzyme activity of 6.86U / mg, which is 1.36 times that of the wild type (5.05U / mg).

[0051] In order to verify whether different mutants have synergistic or cooperative effects, we tried to further construct double and triple mutants of LCRIII by combining mutations. The specific enzyme activities of mutants W151F / F215Y, W151F / S167A, F215Y / S167A and W151F / F215Y / S167A were 20.04U / mg, 16.25U / mg, 17.31U / mg and 23.00U / mg, respectively, which were 3.97 times, 3.22 times, 3.43 times and 4.55 times that of the wild type, respectively. This result shows that we designed mutant libraries located in the active center, near the active center and far from the active center through protein engineering strategies such as consensus evolution, structural analysis and sequence alignment. These mutant libraries all contain positive mutants and have synergistic effects with each other, which ultimately significantly improves the catalytic performance of LCRIII. Figure 3 Schematic diagram of the enzyme activity of wild-type and mutant alcohol dehydrogenase LCRIII.

[0052] Example 4 Construction of mutants

[0053] Site-directed mutagenesis was performed using the recombinant plasmid pET-28a-LCRIII-GDH as a template using Phanta DNA polymerase (Vazyme, Nanjing, China). The primers for site-directed mutagenesis are listed in Table 2 below. The PCR program included an initial denaturation step (98°C for 30 seconds); followed by 30 cycles (98°C for 10 seconds denaturation, 60°C for 5 seconds annealing, and 72°C for 35 seconds extension); and finally an extension at 72°C for 1 minute. The PCR product was digested with endonuclease Dpn I (TaKaRa) at 37°C for 1 hour, Dpn I was inactivated at 70°C for 15 minutes, and the mixture was transformed into Escherichia coli JM109, and positive mutants were confirmed by sequencing (Sangon Biotech). The positive mutant plasmid was transformed into Escherichia coli BL21 (DE3) for expression.

[0054] The wild type, recombinant plasmids containing single and multiple mutations were transformed into E. coli BL21 (DE3) competent cells, and cultured at 37°C for 12 hours in 5 mL of Luria-Bertani (LB) liquid medium containing 50 μg / mL kanamycin. The culture was then transferred to a 250 mL conical flask containing 50 mL of LB medium (50 μg / mL kanamycin was added). When the culture reached OD 600 = 0.6, isopropyl-β-d-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM, and the cells were shaken at 20°C for 20 hours to induce protein expression.

[0055] E. coli cells were harvested by centrifugation at 6000 × g for 10 minutes at 4°C. The cell pellet was resuspended in PBS buffer (100 mM, pH 7.2) and sonicated for 20 minutes (2 second on / off cycle) at 4°C to promote cell lysis. The cell lysate was then centrifuged at 6000 × g for 30 minutes at 4°C to separate cell debris. The supernatant was applied to a Ni-NTA agarose column equilibrated with dialysis buffer. After thoroughly washing the column with dialysis buffer, the target protein was eluted using elution buffer (100 mM Tris-HCl, 200 mM imidazole, pH 7.0).

[0056] After protein purification, the purity of the protein was assessed by Coomassie Brilliant Blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined using a BCA protein assay kit (Beyotime).

[0057] Enzyme activity measurement method:

[0058] Enzyme activity was determined at 30°C by measuring the decrease in NAD(P)H absorbance at 340 nm. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the reduction of 1.0 μmol NAD+ or the oxidation of 1.0 μmol NADH per minute at the optimal temperature. The reaction system consisted of 100 mM phosphate buffer (pH 6.0), 0.2 mM NADH, 5 mM substrate, and purified enzyme or dry cells in a total volume of 200 μL. Reactions without enzyme addition served as controls.

[0059] The results of enzyme activity assay showed that the specific enzyme activity of W151F was 13.25 U / mg, which was 2.62 times that of the wild type (5.05 U / mg).

[0060] Example 5 Catalytic conversion

[0061] In order to maximize the synthesis rate of (R)-4-chloro-3-hydroxybutyric acid ethyl ester, the reaction conditions such as reaction temperature, initial pH, glucose ratio, and cell dose were first optimized in a 50 mL system. Several reaction parameters (glucose concentration, reaction pH, reaction temperature, and cell dose) that affect biotransformation were evaluated by adding different concentrations of glucose (0.5-2.5mmol glucose / (mmol COBE)) and cell doses (5-30g DCW / L) in 50 mL phosphate buffer. Unless otherwise specified, each assay was stirred at 30°C for 6 hours. The effects of reaction temperature and pH on biosynthesis were studied at different temperatures (25-60°C) and different pH (4.0-9.0). Based on the experimental results, the optimal transformation conditions were determined: 30°C, pH 6, glucose / COBE (mmol / mmol) of 1.5, and bacterial dosage of 15g DCW / L.

[0062] Substrate fed-batch strategy and conversion:

[0063] Subsequently, the optimized conversion conditions were used to carry out fed-batch fermentation in a 1L reactor with a volume of 600 mL to evaluate the industrial synthesis potential of (R)-4-chloro-3-hydroxybutyric acid ethyl ester by the engineered strain containing the optimal combination of mutants W151F / F215Y / S167A. Figure 4 The reaction system comprises phosphate buffer (100 mM, pH 6), 4-chloro-ethyl acetoacetate (360 mmol), glucose (540 mmol) and E. coli / pet28a-LCRIII W151F / F215Y / S167A-GDH (9 g DCW) with a final volume of 600 mL was mixed in a bioreactor equipped with two electrodes for monitoring temperature and pH. The resulting mixture was stirred at 200 rpm for 32 hours. During the reaction, the temperature was maintained at 30°C and the pH was adjusted to 6 with 2M NaOH. 4-Chloro-ethyl acetoacetate (180 mmol) and glucose (270 mmol) were added to the reaction mixture at 6 hours, 12 hours, 18 hours, and 24 hours. After the reaction was completed, the two layers were separated and the aqueous layer was extracted twice with ethyl acetate. The extraction layer was combined with the original organic layer, washed with NaHCO3, and dried over anhydrous Na2SO4. The resulting solvent was evaporated in vacuo to obtain the final product as an oily liquid. The liquid phase detection results showed that E.coli / pet28a-LCRIII W151F / F215Y / S167A -GDH can efficiently catalyze 296.26 g / L of ethyl 4-chloro-acetoacetate to produce (R)-4-chloro-3-hydroxybutyrate ethyl ester (ee>99%).

[0064] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An alcohol dehydrogenase mutant having improved production capacity of ethyl (R)-4-chloro-3-hydroxybutyrate, characterized in that: The amino acid sequence of the alcohol dehydrogenase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO.1, and the mutation site includes at least one of mutation point a, mutation point b and mutation point c; Mutation point a is the mutant W151F obtained by mutation of tryptophan at position 151 to phenylalanine; Mutation point b is the mutant F215Y obtained by mutation of phenylalanine at position 215 to tyrosine; Mutation point c is the mutant S167A obtained by mutation of serine at position 167 to alanine.

2. The alcohol dehydrogenase mutant with improved (R)-4-chloro-3-hydroxybutyrate ethyl production capacity according to claim 1, characterized in that It also includes mutation point a, mutation point b and mutation point c.

3. A gene encoding the alcohol dehydrogenase mutant having improved (R)-4-chloro-3-hydroxybutyrate ethyl production ability according to claim 1 or 2.

4. A recombinant plasmid comprising the coding gene according to claim 3.

5. The recombinant plasmid according to claim 4, characterized in that It also contains the GDH gene.

6. A recombinant genetically engineered bacterium comprising the recombinant plasmid according to claim 4 or 5.

7. The method for constructing a recombinant genetically engineered bacterium according to claim 5, characterized in that: The steps include: The sequence of the alcohol dehydrogenase mutant of claim 1 is connected to the GDH gene sequence by RBS, the connected fragments are inserted into a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid is introduced into a host cell for culture to obtain the recombinant genetic engineering bacteria.

8. The method for constructing a recombinant genetically engineered bacterium according to claim 7, characterized in that: The plasmid vector is pET-28a; the host cell is Escherichia coli BL21 (DE3).

9. Use of the recombinant genetically engineered bacteria according to claim 6 in the preparation of alcohol dehydrogenase mutants.

10. Use of the alcohol dehydrogenase mutant according to claim 1 or 2 in catalyzing the production of (R)-4-chloro-3-hydroxybutyric acid ethyl ester from ethyl 4-chloroacetoacetate.

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