An alcohol dehydrogenase mutant with improved catalytic activity, a genetically engineered bacterium and its application

Abstract

The present invention discloses an alcohol dehydrogenase mutant, a genetically engineered bacterium and their applications. The alcohol dehydrogenase mutant includes: using the wild-type alcohol dehydrogenase y29 shown in SEQ ID NO.1 as a template, a mutant L196M in which the leucine L at position 196 is mutated to methionine M, and the amino acid sequence is as shown in SEQ ID NO.2; an alcohol dehydrogenase mutant L196M / Y153F in which the leucine L at position 196 is mutated to methionine M and the tyrosine Y at position 153 is mutated to phenylalanine F, and the amino acid sequence is as shown in SEQ ID NO.3. Compared with the wild-type alcohol dehydrogenase, the alcohol dehydrogenase mutant provided by the present invention can catalyze the substrate ethyl 4-chloroacetoacetate (COBE) to prepare (S)-CHBE with high activity and high stereoselectivity, and uses cheap isopropanol as a cosubstrate to achieve efficient coenzyme recycling, having good industrial application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and relates to an alcohol dehydrogenase mutant with improved catalytic activity, an alcohol dehydrogenase mutant gene, a recombinant expression plasmid and a recombinant expression transformant containing the gene, a preparation method of the alcohol dehydrogenase mutant, and an application of the alcohol dehydrogenase mutant in the synthesis of (S)-ethyl 4-chloro-3-hydroxybutyrate. Technical Background

[0002] As an important chiral building block, chiral alcohols play an important role not only as pharmaceutical intermediates but also in the fields of agrochemicals, fragrances, food additives, etc. At present, the synthesis methods of chiral alcohols are mainly divided into two types: chemical methods and biocatalytic methods. The biocatalytic method using alcohol dehydrogenase has been widely used in the synthesis of chiral alcohols due to its advantages such as mild reaction conditions, environmental friendliness, and excellent stereoselectivity.

[0003] (S)-Ethyl 4-chloro-3-hydroxybutyrate is a key chiral intermediate for the preparation of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors such as statins. Statins are the best-selling cholesterol-lowering and lipid-lowering drugs in the world at present. Therefore, (S)-ethyl 4-chloro-3-hydroxybutyrate, as a key chiral intermediate of statins, has a high demand and a wide range of applications.

[0004] Using ethyl 4-chloroacetoacetate (COBE) as the prochiral substrate for the reduction reaction is easy to synthesize and has a low price. It is a very economical and effective preparation route to obtain (S)-CHBE through an asymmetric reduction reaction with it as the substrate.

[0005] So far, there have been many research reports on the asymmetric reduction of COBE to prepare chiral CHBE. Generally speaking, there are mainly chemical synthesis methods and biocatalytic methods. The chemical synthesis method has a low yield and serious pollution. In contrast, the biocatalytic method has better development prospects. Among the reductases selected for the biocatalytic method, those that are NAD-dependent and can use isopropanol as a hydrogen donor are the best. Because NAD is cheaper than NADP, and compared with glucose, isopropanol will not produce acidic substances after oxidation and is not easy to cause emulsion of the reaction solution, which is beneficial to the next extraction process. In the selection of the reaction system for this process, a single aqueous phase reaction system is better. The large-scale use of organic solvents in production may increase environmental and safety risks. Obtaining a high substrate concentration in a single aqueous phase reaction system is a hot topic in current research.

[0006] Chinese invention patent applications, such as application numbers 200810124754.2, 201010213724.6, 201110225388.1, etc., have disclosed several methods for producing (S)-ethyl 4-chloro-3-hydroxybutyrate by ketoreductase. However, the cofactors used in these methods are all expensive NADP, and glucose needs to be added as a hydrogen donor. Although the substrate concentration can reach 30%, a large amount of organic solvent must be added to form a two-phase system, and the substrate needs to be added in batches, making the operation cumbersome.

[0007] Generally speaking, most enzymes tend to catalyze their natural substrates and will show the disadvantage of low activity when catalyzing non-natural substrates. Therefore, it is necessary to perform molecular modification on them to enable them to reach the level of industrial preparation. With the development of structural biology and bioinformatics, more and more protein engineering technologies, such as semi-rational design, etc., are used to modify proteins, and the catalytic performance of enzymes is improved by changing certain specific amino acids. Summary of the Invention

[0008] The object of the present invention is to provide an alcohol dehydrogenase mutant with improved activity, a genetically engineered bacterium thereof, and their applications, so as to solve the problem that the activity of alcohol dehydrogenase for catalytic reduction of COBE to prepare (S)-CHBE in the prior art is not high, and thus the application of alcohol dehydrogenase in industry is limited.

[0009] To solve the above technical problems, the present invention adopts the following technical solutions:

[0010] According to the first aspect of the present invention, there is provided a single-point mutant of alcohol dehydrogenase with improved catalytic activity, including an alcohol dehydrogenase mutant formed by mutating the 196th amino acid residue as follows using the wild-type alcohol dehydrogenase y29 shown in SEQ ID NO.1: the alcohol dehydrogenase mutant L196M in which the leucine L at the 196th position is mutated to methionine M, and the amino acid sequence is as shown in SEQ ID NO.2; using the wild-type alcohol dehydrogenase y29 shown in SEQ ID NO.1 as a template, the leucine L at the 196th position is mutated to methionine M, and the tyrosine Y at the 153rd position is mutated to phenylalanine F, the alcohol dehydrogenase mutant L196M / Y153F, and the amino acid sequence is as shown in SEQ ID NO.3.

[0011] According to the second aspect of the present invention, there is provided a recombinant vector containing the coding gene of the above-mentioned alcohol dehydrogenase mutant with improved catalytic activity, and the recombinant vector is formed by inserting the coding gene of the alcohol dehydrogenase mutant into a vector plasmid.

[0012] According to the third aspect of the present invention, there is provided a genetically engineered bacterium containing the above-mentioned recombinant vector.

[0013] According to the fourth aspect of the present invention, there is provided an application of the above-mentioned alcohol dehydrogenase mutant or the above-mentioned genetically engineered bacterium in catalyzing a ketone substrate to produce a corresponding chiral alcohol.

[0014] Preferably, the application includes the application of catalyzing the substrate COBE to prepare (S)-CHBE.

[0015] According to a preferred embodiment of the present invention, the application includes: S1: culturing the genetically engineered bacterium in LB medium, inducing expression to obtain a crude enzyme solution; S2: using ethyl 4-chloroacetoacetate as a substrate, adding the crude enzyme solution, coenzyme NAD + and isopropanol as cosubstrates, mixing evenly and then placing it in a shaker for reaction to achieve the preparation of ethyl (S)-4-chloro-3-hydroxybutyrate.

[0016] According to the present invention, first, the alcohol dehydrogenase y29 gene cloned from the strain S. maltophilia was selected as a template. The amino acid sequence of the alcohol dehydrogenase y29 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4. The spatial structure of the alcohol dehydrogenase y29 was analyzed by homology modeling, and the substrate COBE was docked into the alcohol dehydrogenase y29. The amino acid residues (sites 196 and 153) that play a key role in catalytic activity were selected by means of the HotSpot Wizard website for site-directed saturation mutagenesis. After obtaining the mutants, the catalytic activities of the mutants were determined according to the bioreduction reaction; the mutant L196M with the highest enzyme activity was used as a template, and the amino acid residues at site 153 were subjected to the next round of iterative combinatorial mutagenesis, and the catalytic activities of the mutants were determined according to the bioreduction reaction. Finally, the best double-point mutant y29-L196M / Y153F was obtained. Through protein engineering strategies, mutants with improved catalytic performance of alcohol dehydrogenase were obtained, and the obtaining of these mutants is very beneficial for industrial production.

[0017] Furthermore, the present invention synthesizes chiral alcohols by means of asymmetric reduction. Preferably, the alcohol dehydrogenase mutant L196M / Y153F of the present invention catalyzes the reduction of 200 g / L of the substrate COBE, and uses isopropanol as a cosubstrate to achieve efficient coenzyme recycling. A conversion rate of 99% can be achieved within 6 hours, and excellent stereoselectivity is maintained, with the ee value of the product being 99.8%.

[0018] The present invention has the following advantages compared with the prior art:

[0019] The method of the present invention effectively improves the disadvantages of low substrate concentration and long enzyme reaction time in the traditional enzyme-catalyzed production process by adding inexpensive and widely sourced auxiliary materials, and realizes the preparation of (S)-CHBE with high efficiency and low cost. Moreover, the added auxiliary materials do not affect the separation and purification of the product. The substrate and other substances can be added at one time, and no other organic solvents are required. The operation is simple and it is particularly suitable for popularization and application in the pharmaceutical industry.

[0020] In summary, the present invention provides a mutant alcohol dehydrogenase with improved activity, a genetically engineered bacterium thereof, and their applications. The alcohol dehydrogenase involved in the present invention is based on the wild-type alcohol dehydrogenase y29 shown in SEQ ID NO.1, and on this basis, the amino acid sites crucial for activity are determined, and single-site saturation mutagenesis and iterative combinatorial mutagenesis are carried out to form it. The mutant obtained according to the present invention has a great improvement compared with the wild-type alcohol dehydrogenase y29. This mutant alcohol dehydrogenase has high activity, high stereoselectivity and high tolerance to the substrate, and can efficiently catalyze the substrate ethyl 4-chloroacetoacetate to prepare (S)-ethyl 4-chloro-3-hydroxybutyrate, which is of great significance for the production of intermediates of statin drugs such as atorvastatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shows the detection results of the conversion rate of the substrate ethyl 4-chloroacetoacetate;

[0022] Figure 2 Shows the standard curve of the concentration of the product (S)-ethyl 4-chloro-3-hydroxybutyrate;

[0023] Figure 3 Shows the stereoselectivity detection of the product (S)-ethyl 4-chloro-3-hydroxybutyrate;

[0024] Figure 4 Shows the reaction time curves of the wild type, single-site mutant L196M and double-site mutant L196M / Y153F using isopropanol as the hydrogen donor. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following further describes the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional operations in the art.

[0026] The present invention selects the alcohol dehydrogenase y29 gene cloned from the strain S. maltophilia; the amino acid sequence of the alcohol dehydrogenase y29 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4. The present invention constructs a mutant library by performing site-directed saturation mutagenesis on the wild-type alcohol dehydrogenase y29 obtained by screening. The obtained mutants are screened through biocatalytic reduction reactions, and the mutant L196M with the highest improved conversion rate is obtained. Using this as a template, a second round of iterative combinatorial mutagenesis is carried out, and finally the double-point mutant L196M / Y153F with further improved enzyme activity is obtained.

[0027] Example 1 Selection of Mutation Sites

[0028] In order to obtain the spatial structure of alcohol dehydrogenase y29, its protein sequence and the SWISS-MODEL (SWISS-MODEL (expasy.org)) website are used for homology modeling. Finally, the crystal structure of alcohol dehydrogenase from Aromatoleum aromaticum EbN1 (PDB ID: 4URE) with a similarity of 49% is used as a template to construct the structure of y29. Then, ERRAT and Ramachandran plot are used to evaluate the results obtained from the modeling. After obtaining a reliable model, the coenzyme NADH is superimposed into the y29 model through the Discovery Studio software. In order to make the superposition more accurate, the coenzyme NADH is first subjected to energy minimization and structural modification, and finally the y29 / NADH complex is obtained. Then, the energy-minimized substrate COBE is docked into the y29 / NADH complex.

[0029] The structural sequence information of the y29 / NADH complex is analyzed through the website HotSpot Wizard to identify its flexible residues; information such as solvent-accessible surface area, binding pocket size, substrate channel, sequence identity, etc. are calculated to identify the hot-spot amino acid residues that may have non-covalent interactions with the substrate COBE or affect the protein conformational dynamics and thus change the enzyme activity. Then, the next step of site-directed saturation mutagenesis and iterative combinatorial mutagenesis is carried out.

[0030] Example 2 Construction and Screening of Mutants

[0031] First, cultivate the wild-type y29 and extract the plasmid of this bacterium as the template for PCR. Use Quickchange (https: / / www.agilent.com / store / primerDesign Program.jsp) to design primers online. The specific method is as follows: First, paste the text or the base sequence of alcohol dehydrogenase y29 in FASTA format into the text box of the browser; Second, click "Upload Translated", and the system will automatically translate the base sequence into an amino acid sequence; Third, there is a ticked rectangular box in front of each amino acid after translation, select the site to be mutated; Fourth, select the target amino acid to be mutated into at the position of "change amino acid(s)to"; Finally, click "Design Primers", and the system will automatically generate primers and include information such as primer length and annealing temperature.

[0032] The DNA polymerase used is a Taq enzyme with high fidelity. This high-fidelity enzyme can achieve perfect amplification when the target fragment is relatively long (about 10 kb). The full-length recombinant plasmid obtained after PCR with the primers designed by the Quickchange method is about 6 kb in length including the target fragment. Therefore, a high-fidelity enzyme is selected for plasmid amplification. The primers used for mutation are shown in Table 1 below:

[0033] Table 1

[0034]

[0035] The PCR reaction system is shown in Table 2 below:

[0036] Table 2

[0037]

[0038] Total 25

[0039] The PCR program is shown in Table 3 below:

[0040] Table 3

[0041]

[0042] After PCR, add 0.5 μL of DpnI enzyme to the obtained PCR product, gently pipette and mix it evenly with a pipette gun, and then place it in a 37°C water bath for 3 hours for enzymatic digestion to specifically excise the methylated template DNA strand. Transform the enzymatically digested PCR product into competent cells of Escherichia coli E.coli BL21(DE3) to achieve heterologous expression of the enzyme. After transformation, pick a single colony and inoculate it into 5 mL of LB medium, culture it overnight at 37°C, and then sequence the strain.

[0043] After ensuring that the sequence is correct, appropriate amounts of bacterial liquid were drawn from the seed tubes for the wild-type enzyme and the correctly sequenced mutant enzyme, respectively, and inoculated into 2 mL EP tubes (sterilized) containing 500 μL LB liquid culture medium (with kanamycin resistance added), and then the EP tubes were placed in a shaker at 37°C and 200 rpm for overnight culture; the next day, 10 μL of bacterial liquid was drawn from the EP tube on the clean bench and transferred to a 2 mL EP tube (sterilized) containing 500 μL LB liquid culture medium (with kanamycin resistance added), and cultured in a shaker at 37°C and 200 rpm for about 2 hours; then 5 μL of 0.01 M IPTG inducer was added, and the tubes were placed in a shaker at 20°C and 200 rpm for induction culture for 20 hours.

[0044] Centrifuge the induced expression bacterial solution at 14,000 rpm for 2 minutes, and discard the supernatant; add 150 μL of Tris-HCl buffer (pH 8.0, 100 mM) to the EP tube, and gently pipette to resuspend the cells (try not to stain the tube wall with liquid). Then add 180 μL of Tris-HCl buffer and 20 μL of the resuspended bacterial solution to the ELISA plate, gently pipette to mix it evenly, and then put it into the ELISA reader to measure the OD 600 The measured OD 600 The amount of bacteria with the largest value was set at 100 μL, and the amounts of other bacteria were calculated to ensure that the bacterial concentrations in each reaction system were the same (the amount of bacteria less than 100 μL was supplemented with buffer).

[0045] According to the screening reaction system in Table 4, each substance was added to a 2 mL EP tube in sequence, mixed evenly, and then placed in a shaker at 30°C and 200 rpm for reaction for 24 hours; after the reaction, 800 μL of ethyl acetate was added to the EP tube, mixed thoroughly, and centrifuged at 14,000 rpm for 10 min, and the supernatant ethyl acetate layer was aspirated into a new EP tube; an appropriate amount of anhydrous Na2SO4 was added for drying, and the mixture was centrifuged at 14,000 rpm for 5 min, and the supernatant ethyl acetate layer was aspirated and divided into two equal parts for gas phase detection, one for detecting its conversion rate, and the other for detecting its ee value after derivatization. The specific derivatization steps are as follows: put the above sample dried with anhydrous Na2SO4 into a fume hood, and after it is completely evaporated, add appropriate amounts of acetic anhydride and pyridine, and heat it in a boiling water bath for 1 hour; after it is cooled to room temperature, add a certain amount of ethyl acetate, blow and suck with a pipette to mix it evenly, and then centrifuge at 14,000 rpm for 10 minutes, and draw 200 μL of supernatant into a liquid phase vial for ee value detection.

[0046] The detection method is as follows. The conversion rate of the substrate COBE is detected by gas chromatography using the internal standard method. 2-Hydroxyacetophenone is selected as the internal standard. The gas chromatography column used is an Agilent HP-5 capillary column (30 m × 0.25 mm, 0.25 μm). The analysis method is as follows: The inlet and detector temperatures are both set at 250 °C, the column oven temperature is 80 °C, the carrier gas is N2, the flow rate is 1 mL / min, the split ratio is 30:1, and the injection volume is 3 μL. The temperature programming is to hold at 80 °C for 1 min, then increase the temperature to 140 °C at a rate of 10 °C / min and hold for 2 min. As Figure 1 shown, under these analysis conditions, the retention time of the substrate COBE is 6.552 min, the retention time of the product CHBE is 6.983 min, and the retention time of the internal standard 2-hydroxyacetophenone is 8.523 min. The conversion rate is calculated according to the following formula: Conversion rate (%) = (Concentration of CHBE in the reaction system / Initial concentration of the substrate COBE) × 100%.

[0047] First, prepare CHBE with different concentrations (0 mM, 25 mM, 50 mM, 75 mM, 100 mM) and 10 mM 2-hydroxyacetophenone (using ethyl acetate as the solvent). Then, add 100 μL of CHBE with different concentrations and 900 μL of Tris-HCl buffer into 2 mL EP tubes respectively. After thorough mixing, perform the treatment (extract with ethyl acetate containing 10 mM 2-hydroxyacetophenone). After gas phase detection, plot a standard curve with the concentration of the product CHBE as the abscissa and the peak area ratio of CHBE to the internal standard as the ordinate, as Figure 2 shown. The regression equation is: y = 0.0864x + 0.1961, R 2 = 0.9977.

[0048] The stereoselectivity of the product CHBE is also detected by gas chromatography. The gas chromatography column used is an Agilent CP-Chirasil-Dex CB chiral column (25 m × 0.32 mm, 0.25 μm). The analysis method is as follows: The inlet temperature is set at 250 °C, the detector temperature is set at 280 °C, the column oven temperature is 90 °C, the carrier gas is N2, the flow rate is 0.8 mL / min, the split ratio is 30:1, and the injection volume is 1 μL. The temperature programming is to hold at 90 °C for 5 min, then increase the temperature to 135 °C at a rate of 1.5 °C / min, and then increase the temperature to 190 °C at a rate of 40 °C / min and hold for 2 min. As Figure 3As shown, under these analysis conditions, the retention time of the product (R)-CHBE was 20.107 min, and the retention time of (S)-CHBE was 20.645 min. The ee value was calculated according to the following formula: ee value (%) = [(R - S) / (R + S)] × 100%, where R and S represent the peak areas of the R-form and S-form of the product CHBE in the gas chromatogram, respectively.

[0049] The conversion rate and stereoselectivity of the bioreduction reaction of each mutant were detected. Among them, after saturation mutagenesis at position 196, the best mutant y29-L196M was screened and used as a template to construct mutants with iterative combinatorial mutagenesis at position 153. Further, the best double-point mutant L196M / Y153F was obtained, and its k cat / K m was 11.09 times higher than that of the wild type.

[0050] Table 4

[0051]

[0052] Example 3 Mutant Bioreduction of Ethyl 4-Chloroacetoacetate

[0053] To further verify the catalytic performance of the double-point mutant L196M / Y153F, the catalytic reaction was scaled up to 30 mL. Under the condition of a substrate dosage of 200 g / L, the reaction process of the double-point mutant L196M / Y153F was monitored, and the results are as Figure 4 shown. The initial reaction rate of the double-point mutant L196M / Y153F was significantly higher than that of the single-point mutant L196M, and the conversion rate reached 99% after 6 hours of reaction; while the single-point mutant reached equilibrium after 12 hours of reaction, and the conversion rate was only 95%. Therefore, the catalytic performance of the mutants obtained in the present invention has been greatly improved compared with the wild-type alcohol dehydrogenase y29.

[0054] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. An alcohol dehydrogenase mutant with improved catalytic activity, characterized in that, They are respectively: Using the wild-type alcohol dehydrogenase y29 shown in SEQ ID NO.1 as a template, the alcohol dehydrogenase mutant L196M in which the leucine L at the 196th position is mutated to methionine M, and the amino acid sequence is as shown in SEQ ID NO.2; Using the wild-type alcohol dehydrogenase y29 shown in SEQ ID NO.1 as a template, the alcohol dehydrogenase mutant L196M / Y153F in which the leucine L at the 196th position is mutated to methionine M and the tyrosine Y at the 153rd position is mutated to phenylalanine F, and the amino acid sequence is as shown in SEQ ID NO.

3.

2. The coding gene of an alcohol dehydrogenase mutant, characterized in that, The encoding gene encodes the following protein: the amino acid sequence of the alcohol dehydrogenase mutant as described in claim 1.

3. A recombinant vector comprising the coding gene of the alcohol dehydrogenase mutant according to claim 2, characterized in that, The recombinant vector is constructed by inserting the encoding gene of the alcohol dehydrogenase mutant into a vector plasmid.

4. A genetically engineered bacterium comprising the recombinant vector according to claim 3.

5. The genetically engineered bacterium according to claim 4, wherein The host bacterium is Escherichia coli E.coli BL21(DE3).

6. Use of the alcohol dehydrogenase mutant according to claim 1 or the genetically engineered bacterium according to claim 4 in the preparation of (S)-4-chloro-3-hydroxybutyric acid ethyl ester by catalyzing ethyl 4-chloroacetoacetate.

7. The application according to claim 6, characterized in that The use includes: S1: Culturing the genetically engineered bacterium according to claim 4 in an LB medium, inducing expression to obtain a crude enzyme solution; S2: Using ethyl 4-chloroacetoacetate as a substrate, adding the crude enzyme solution, coenzyme NAD+, and isopropanol as a cosubstrate, mixing evenly and then reacting it in a shaker to achieve the preparation of (S)-4-chloro-3-hydroxybutyric acid ethyl ester.

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