A ketoreductase mutant for preparing (S)-3,4-dihydroxy-2'-chlorophenethyl alcohol

Abstract

The invention discloses a ketoreductase mutant, which can convert 3,4-dihydroxy-2'-chloroacetophenone into (S)-3,4-dihydroxy-2'-chlorophenylethanol, and then prepare S-norepinephrine and S-epinephrine. The ketoreductase mutant has a conversion substrate concentration of up to 200g / L, a conversion rate of more than 95%, an ee value of>99%, a simple reaction process, low cost, low pollution, and can selectively obtain S-epinephrine and S-norepinephrine.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a ketoreductase mutant, which can be used for preparing (S)-3,4-dihydroxy-2'-chlorophenylethanol, an intermediate of no S-norepinephrine and S-epinephrine. Background Art

[0002] Norepinephrine, chemical name (R)-4-(2-amino-1-hydroxyethyl)-1,2-benzenediol, is a substance formed by removing the N-methyl group from adrenaline. It also belongs to catecholamines in chemical structure. Norepinephrine is an anti-shock vasoactive drug, mainly used to rescue shock caused by acute hypotension and peripheral vasodilation.

[0003] Norepinephrine is optically active, and the efficacy of the levorotatory isomer is 27 times greater than that of the dextrorotatory isomer. Therefore, R-norepinephrine is widely used in clinical practice to rescue acute hypotension and shock caused by peripheral vasodilation. In the production and preparation of R-norepinephrine, quality analysis and related biological experiments, S-norepinephrine molecules play an important role as standard samples or control experiments. However, there is currently no dedicated synthesis process for S-norepinephrine. Instead, the optical isomers of S-norepinephrine are prepared by splitting the racemic norepinephrine molecules with the help of the original R-norepinephrine synthesis route. And with the continuous advancement of S-norepinephrine in clinical research, the market demand for S-norepinephrine has also been further expanded. However, with the improvement of norepinephrine synthesis technology, more and more manufacturers use asymmetric synthesis methods to directly prepare R-type norepinephrine, and are unable to obtain S-type norepinephrine by splitting methods, resulting in a decrease in the output of S-type norepinephrine and an increase in the supply price.

[0004] According to the existing reported process for preparing norepinephrine, it can be seen that the method for preparing S-type norepinephrine using the synthetic route shown in Scheme 1 is the most concise, but the premise is that the preparation process problem of (S)-3,4-dihydroxy-2'-chlorophenylethanol needs to be solved.

[0005]

[0006] In addition, (S)-3,4-dihydroxy-2'-chlorophenethyl alcohol is also an important intermediate for the preparation of (S)-epinephrine.

[0007] In summary, there is an urgent need for a process for efficiently preparing the chiral compound (S)-3,4-dihydroxy-2'-chlorophenethanol to meet the growing market demand, and the key lies in the selection of ketoreductase. Summary of the invention

[0008] The purpose of the present invention is to provide a new ketoreductase mutant in view of the deficiencies of the prior art.

[0009] On the one hand, the present invention utilizes protein engineering technology to perform semi-rational and rational design on the ketoreductase from Lactobacillus kefiri, and obtains two ketoreductase mutants Mut1 and Mut2 with high catalytic efficiency under high-throughput screening.

[0010] Furthermore, the amino acid sequences of the ketoreductase mutants Mut1 and Mut2 are shown in SEQ ID NOs. 3 and 5, respectively.

[0011] Furthermore, the ketoreductase mutant Mut1 is a ketoreductase mutant having some sites mutated based on the wild-type sequence, specifically, the 14th site is Ala, the 72nd site is Phe, the 94th site is Thr, the 116th site is Ile, the 143rd site is Ser, the 147th site is Leu, the 199th site is His, the 202nd site is Leu, and the 237th site is Pro.

[0012] Furthermore, the ketoreductase mutant Mut2 is obtained by substitution and deletion based on the sequence of the ketoreductase mutant Mut1.

[0013] Furthermore, the ketoreductase mutant is derived from the wild-type Lactobacillus kefiri, and the accession number of the wild-type template NCBI is 4RF2_A.

[0014] Furthermore, the ketoreductase mutant is constructed in vitro onto a recombinant expression vector, preferably a PET series vector.

[0015] Furthermore, the host cell of the ketoreductase mutant is Escherichia coli, and the cell is BL21 (DE3), PLysS or Origami B (DE3).

[0016] Furthermore, after expression in the host cells under IPTG induction, proteins of two mutants were obtained. In the presence of high concentration of isopropanol, catalysis of high concentration substrates was also achieved using whole cells.

[0017] On the other hand, the ketoreductase mutant provided by the present invention can convert 3,4-dihydroxy-2'-chloroacetophenone into (S)-3,4-dihydroxy-2'-chlorophenethanol, and the technical scheme adopted is as follows:

[0018]

[0019] Furthermore, the concentration of the 3,4-dihydroxy-2'-chloroacetophenone can reach 200 g / L.

[0020] Furthermore, the ketoreductase mutant is ketoreductase enzyme powder or whole cells or cell disrupted liquid containing the ketoreductase, preferably whole cells of ketoreductase.

[0021] Furthermore, a coenzyme system needs to be added to the reaction, preferably NADPH, with a concentration of less than 0.1 mM.

[0022] Furthermore, the reaction utilizes the reverse reaction of the ketoreductase mutant to achieve the regeneration of the coenzyme NADPH using isopropanol as a substrate.

[0023] Furthermore, the concentration of isopropanol in the reaction is controlled between 20% and 80%, preferably 80%.

[0024] Furthermore, the reaction temperature of the reaction is controlled at 25-45°C, preferably 37°C.

[0025] Furthermore, the pH of the reaction is controlled between 5.0 and 7.5, preferably 6.0.

[0026] The beneficial effects of the present invention are that the present invention provides a new ketoreductase mutant, the conversion substrate concentration is as high as 200g / L, the conversion rate is more than 95.5%, the chiral ee value is greater than 99%, the reaction process is simple, the cost is low, the pollution is small, and S-epinephrine and S-norepinephrine can be selectively obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] none DETAILED DESCRIPTION

[0028] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0029] Example 1 Construction of ketoreductase KRED mutation library

[0030] Taking the structure of the wild-type ketoreductase of SEQ ID No.1 (the corresponding nucleotide sequence is SEQ ID No.2) as a reference, through computer simulation, it was docked with the substrate and the S product respectively to obtain the optimal structure and energy. In order to allow the S product to exist better in the active pocket of the ketoreductase KRED, we performed saturation mutation screening on the amino acids that produce steric hindrance and constructed a library capacity of 11,000 single clones. By designing saturation primers for these sites, the full plasmid amplification reaction of the recombinant plasmid pET-21a-kred was achieved with the participation of a high-fidelity polymerase, and then the PCR product was digested with the FD DpnI restriction endonuclease as a template. After purification, it was transferred into the competent state of Escherichia coli BL21 (DE3), and then 1 mL of LB medium was added for incubation at 37°C, and then it was coated on a plate containing 50ug / L Amp + The plates were inverted and cultured in a 37°C incubator for 18 h to grow full plates of single clones. These plates were the constructed mutation libraries for later screening.

[0031] Example 2 Screening of ketoreductase KRED mutants

[0032] 188 single clones were randomly selected from each plate for 96-well plate shaking culture. Two wells of each 96-well plate were inoculated with wild-type strains for later analysis as a control group, totaling 20 96-well plates. The specific operation was as follows: 400uL of LB medium was added to a sterile 96-well plate, cultured at 37°C for about 12h, and then transferred to a second 96-well plate for culture at a 10% inoculation volume. The culture was continued for about 3h, and IPTG with a final concentration of 0.1mM was added to induce expression for 16h, and the induction temperature was 25°C. After the culture was completed, the supernatant was discarded by centrifugation and stored in a -20°C refrigerator for later use.

[0033] Prepare the transformation system according to Table 1, use a pistol to transfer 300uL into the above-mentioned 96-well plate after centrifugation, react at 300rpm in a constant temperature oscillator at 37°C for 4h (this is the reaction time for the control group to transform about half of the substrate), and inactivate at 80°C. Finally, all transformation reaction solutions were subjected to absorbance detection at 340nm and chiral HPLC analysis, and the catalytic efficiency and chirality values ​​were increased by more than 10% as positive, and the mutant information was sequenced and analyzed, and the next iterative mutation was performed on the basis of this mutation, and so on, gradually completing the iterative saturation mutation of all sites, and the final mutant with high catalytic activity and chiral selectivity was taken as the best result of this evolution.

[0034] Table 1 Reaction solution system for screening saturated mutant library

[0035] raw material concentration Substrate 20g / L Isopropyl alcohol 20％ NADH 2g / L pH 7.0 Potassium Phosphate Buffer 100mM

[0036] Example 4 Recombinant expression of ketoreductase

[0037] The positive mutant strains screened in the early stage were expanded for fermentation preparation. First, BL21 (DE3) cells containing the target gene were inoculated into a culture medium containing Amp + In a resistant LB test tube, culture overnight at 37°C to obtain a primary seed culture solution. The seed culture solution is transferred to a 2YT liquid culture medium containing resistance at a 1% inoculation ratio, placed in a shaker, cultured at 37°C, 200rpm for 3-5h, and when the OD reaches 0.6-0.8, the temperature is lowered to 20°C, IPTG is added, and the concentration is controlled at 0.1mM to induce expression overnight. The fermentation broth is centrifuged and the bacteria are collected, and the collected bacteria are dissolved with 20mM Tris-HCL buffer (pH 6.5), and the cells are broken by ultrasound. After centrifugation at 12000rpm for 10min, the supernatant obtained is the transaminase protein, and the expression of the protein can be seen by electrophoresis.

[0038] Example 4 Catalytic reaction of wild-type ketoreductase

[0039] Add 0.2g 3,4-dihydroxy-2'-chloroacetophenone to a 50mL reaction bottle, dissolve it with 4mL isopropanol, add 6mg coenzyme NADPH, add 16mL water, adjust the pH to 7.0 with 0.1M NaOH, and finally add 1g cells (containing wild-type ketoreductase). Stir the reaction in a 37℃ water bath, and use 0.1M NaOH to control the pH between 6.0 and 7.5 during the reaction. After 24h of reaction, dissolve it with 2 times acetonitrile and perform HPLC analysis. The conversion rate is 65%. The target product is obtained after rotary evaporation at 40℃, and the ee value is only 45%.

[0040] Example 5 Catalytic reaction of ketoreductase mutant Mut1

[0041] Add 4g 3,4-dihydroxy-2'-chloroacetophenone to a 50mL reaction bottle, dissolve it with 16mL isopropanol, add 6mg coenzyme NADPH, add 4mL water, adjust the pH to 6.5 with 0.1M sodium hydroxide solution, and finally add 1g ketoreductase enzyme powder (mutant Mut1). Stir the reaction in a 37℃ water bath, and use 0.1M sodium hydroxide solution to control the pH between 6.0 and 7.5 during the reaction. After 24h of reaction, dissolve it with 2 times acetonitrile and perform HPLC analysis. The conversion rate is 95%. The target product is obtained after rotary evaporation at 40℃, with an ee value of 99%.

[0042] Example 6 Catalytic reaction of ketoreductase mutant Mut2

[0043] Add 4g 3,4-dihydroxy-2'-chloroacetophenone to a 50mL reaction bottle, dissolve it with 16mL isopropanol, add 6mg coenzyme NADPH, add 4mL water, adjust the pH to 6.5 with 0.1M sodium hydroxide solution, and finally add 1g cells (containing ketoreductase mutant Mut2). Stir the reaction in a 37℃ water bath, and use 0.1M sodium hydroxide solution to control the pH between 6.0 and 7.5 during the reaction. After 24h of reaction, dissolve it with 2 times acetonitrile and perform HPLC analysis. The conversion rate is 99%. The target product is obtained after rotary evaporation at 40℃, with an ee value of 99%. Sequence Listing <110> Shangke Biopharmaceuticals (Shanghai) Co., Ltd. <120> A ketoreductase mutant for preparing (S)-3,4-dihydroxy-2'-chlorophenethyl alcohol <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 252 <212> PRT <213> Lactobacillus kefiri <400> 1 Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr 1 5 10 15 Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala 20 25 30 Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala 35 40 45 Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala 50 55 60 Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala 65 70 75 80 Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser 85 90 95 Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser 100 105 110 Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg 115 120 125 Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile 130 135 140 Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys 145 150 155 160 Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu 165 170 175 Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala 210 215 220 Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly 225 230 235 240 Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln 245 250 <210> 2 <211> 759 <212> DNA / RNA <213> Lactobacillus kefir <400> 2 atgactgatc gtttaaaagg caaagtagca attgtaactg gcggtacctt gggaattggc 60 ttggcaatcg ctgataagtt tgttgaagaa ggcgcaaagg ttgttattac cggccgtcac 120 gctgatgtag gtgaaaaagc tgccaaatca atcggcggca cagacgttat ccgttttgtc 180 caacacgatg cttctgatga agccggctgg actaagttgt ttgatacgac tgaagaagca 240 tttggcccag ttaccacggt tgtcaacaat gccggaattg cggtcagcaa gagtgttgaa 300 gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttggatgg tgctttcttc 360 ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420 atgtcatcta tcgaaggttt tgttggtgat ccaactctgg gtgcatacaa cgcttcaaaa 480 ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540 gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcttgaa 600 ggggcagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660 aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaatt tgccactggt 720 gcagaattcg ttgtcgatgg tggatacact gctcaataa 759 <210> 3 <211> 252 <212> PRT <213> Artificial Sequence <400> 3 Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Ala Gly Thr 1 5 10 15 Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala 20 25 30 Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala 35 40 45 Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala 50 55 60 Ser Asp Glu Ala Gly Trp Thr Phe Leu Phe Asp Thr Thr Glu Glu Ala 65 70 75 80 Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser 85 90 95 Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser 100 105 110 Val Asn Leu Ile Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg 115 120 125 Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile 130 135 140 Glu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys 145 150 155 160 Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu 165 170 175 Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp His Glu Gly Leu Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala 210 215 220 Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Pro Ala Thr Gly 225 230 235 240 Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln 245 250 <210> 4 <211> 759 <212> DNA / RNA <213> Artificial Sequence <400> 4 atgactgatc gtttaaaagg caaagtagca attgtaactg ccggtacctt gggaattggc 60 ttggcaatcg ctgataagtt tgttgaagaa ggcgcaaagg ttgttattac cggccgtcac 120 gctgatgtag gtgaaaaagc tgccaaatca atcggcggca cagacgttat ccgttttgtc 180 caacacgatg cttctgatga agccggctgg actttcttgt ttgatacgac tgaagaagca 240 tttggcccag ttaccacggt tgtcaacaat gccggaatta cggtcagcaa gagtgttgaa 300 gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttgattgg tgtcttcttc 360 ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420 atgtcatcta tcgaaggtct tgttggtgat ccaactctgg gtgcatacaa cgcttcaaaa 480 ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540 gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcatgaa 600 gggctagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660 aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaacc tgccactggt 720 gcagaattcg ttgtcgatgg tggatacact gctcaataa 759 <210> 5 <211> 247 <212> PRT <213> Artificial Sequence <400> 5 Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Ala Gly Thr 1 5 10 15 Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala 20 25 30 Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala 35 40 45 Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala 50 55 60 Ser Asp Glu Ala Gly Trp Thr Phe Leu Phe Asp Thr Thr Glu Glu Ala 65 70 75 80 Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser 85 90 95 Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser 100 105 110 Val Asn Leu Ile Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg 115 120 125 Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile 130 135 140 Glu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys 145 150 155 160 Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu 165 170 175 Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp His Glu Gly Leu Glu Glu Met Met Ser Gln 195 200 205 Arg Thr His Ile Gly Glu Pro Asn Asp Ile Ala Trp Ile Cys Val Tyr 210 215 220 Leu Ala Ser Asp Glu Ser Lys Pro Ala Thr Gly Ala Glu Phe Val Val 225 230 235 240 Asp Gly Gly Tyr Thr Ala Gln 245 <210> 6 <211> 744 <212> DNA / RNA <213> Artificial Sequence <400> 6 atgactgatc gtttaaaagg caaagtagca attgtaactg ccggtacctt gggaattggc 60 ttggcaatcg ctgataagtt tgttgaagaa ggcgcaaagg ttgttattac cggccgtcac 120 gctgatgtag gtgaaaaagc tgccaaatca atcggcggca cagacgttat ccgttttgtc 180 caacacgatg cttctgatga agccggctgg actttcttgt ttgatacgac tgaagaagca 240 tttggcccag ttaccacggt tgtcaacaat gccggaatta cggtcagcaa gagtgttgaa 300 gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttgattgg tgtcttcttc 360 ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420 atgtcatcta tcgaaggtct tgttggtgat ccaactctgg gtgcatacaa cgcttcaaaa 480 ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540 gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcatgaa 600 gggctagaag aaatgatgtc acagcggacc catatcggtg aacctaacga tatcgcttgg 660 atctgtgttt acctggcatc tgacgaatct aaacctgcca ctggtgcaga attcgttgtc 720 gatggtggat acactgctca 744

Claims

1. A ketoreductase mutant for preparing (S)-3,4-dihydroxy-2'-chlorophenethyl alcohol, characterized in that: The amino acid sequence of the ketoreductase mutant is shown in SEQ ID NO. 3 or 5.

2. The ketoreductase mutant according to claim 1, characterized in that The gene nucleotide sequence of the ketoreductase mutant is shown in SEQ ID NO. 4 or 6.

3. The ketoreductase mutant according to claim 1, characterized in that The ketoreductase mutants were expressed in BL21(DE3), BL21(DE3)PLysS or Origami B(DE3).

4. The ketoreductase mutant according to claim 1, characterized in that The ketoreductase mutant can convert 3,4-dihydroxy-2'-chloroacetophenone into (S)-3,4-dihydroxy-2'-chlorophenethanol.

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