WO2022166843A1 - 一种高立体选择性r转酮酶突变体及其编码基因和应用 - Google Patents

一种高立体选择性r转酮酶突变体及其编码基因和应用 Download PDF

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WO2022166843A1
WO2022166843A1 PCT/CN2022/074751 CN2022074751W WO2022166843A1 WO 2022166843 A1 WO2022166843 A1 WO 2022166843A1 CN 2022074751 W CN2022074751 W CN 2022074751W WO 2022166843 A1 WO2022166843 A1 WO 2022166843A1
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transketolase
mutant
highly stereoselective
stereoselective
highly
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PCT/CN2022/074751
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English (en)
French (fr)
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林双君
刘琦
邓子新
黄婷婷
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浙江普洛康裕制药有限公司
普洛药业股份有限公司
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Priority claimed from CN202110162619.2A external-priority patent/CN114875004B/zh
Application filed by 浙江普洛康裕制药有限公司, 普洛药业股份有限公司 filed Critical 浙江普洛康裕制药有限公司
Priority to KR1020237029610A priority Critical patent/KR20230137996A/ko
Priority to EP22749122.2A priority patent/EP4289947A1/en
Priority to MX2023009214A priority patent/MX2023009214A/es
Priority to BR112023015718A priority patent/BR112023015718A2/pt
Priority to CN202280019980.2A priority patent/CN117157395A/zh
Publication of WO2022166843A1 publication Critical patent/WO2022166843A1/zh

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01001—Transketolase (2.2.1.1)

Definitions

  • the invention relates to the technical field of enzyme engineering, relates to a transketolase mutant, an Escherichia coli transketolase mutant and its encoding gene and application, in particular to a highly stereoselective R-transketolase mutant and its encoding gene and application.
  • Transketolase is a ubiquitous thiamine pyrophosphate-dependent enzyme. It links the non-oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Transketolase catalyzes the reversible transketone reaction, which transfers the two-carbon unit of the ketol donor to the aldehyde acceptor to form chiral dihydroxyketones. In the transketone reaction, ⁇ -hydroxypyruvate is often used as the ketol donor because ⁇ -hydroxypyruvate is transketoneized to generate volatile carbon dioxide, making the reaction irreversible.
  • Stereoselectivity is reflected by the enantiomeric excess rate ee, which represents the optical purity of a chiral compound.
  • ee represents the optical purity of a chiral compound.
  • the research group of Helen C. Hailes reported that the wild-type Escherichia coli transketolase can catalyze the production of L-erythrulose, and its stereoselectivity is relatively high.
  • Optical purity of a chiral compound The higher the ee, the higher the optical purity, but the catalysis of valeraldehyde to produce a moderately stereoselective hydroxyketone product.
  • transketolases have been engineered to catalyze aromatic aldehydes.
  • the research group of Helen C. Hailes reported the site-directed mutation of D469 and F434 on transketolase derived from Escherichia coli, and realized the catalysis of p-benzaldehyde and m-hydroxybenzaldehyde, but the catalytic efficiency was low, and the conversion rate was ⁇ 10%.
  • the hydroxyketone products catalyzed by its mutants are mainly R configuration (ee ⁇ 82%) [ ⁇ , ⁇ ′-Dihydroxyketone formation using aromatic and heteroaromatic aldehydes with evolved transketolase enzymes, Chem.
  • transketolase mutant L382N/D470S obtained by directed evolution has good catalytic activity towards benzaldehyde substrates, but its stereoselectivity has not been reported [Second generation engineering of transketolase for polar aromatic aldehyde substrates, Green Chem., 2017, 19, 481–489].
  • the catalytic activity of transketolase towards aromatic aldehydes has been improved by the modification of transketolase, which catalyzes aromatic aldehyde substrates such as phenylacetaldehyde, phenylpropionaldehyde and phenoxyacetaldehyde to produce the S stereostructure.
  • R-aromatic dihydroxyketones are important synthetic building blocks for ketoses, chiral aminodiols and other high value compounds, such as R-p-methylsulfonyl phenyl dihydroxyketones are thiamphenicol and fluorobenzene
  • the chiral intermediate of Nico, R-p-nitrophenyl dihydroxyketone is a chiral intermediate of chloramphenicol
  • the R-phenylhydroxyketone compound is a chiral intermediate of norephedrine and norephedrine . Therefore, the R-transketolase mutant of the present invention can be used for the biosynthesis of chiral drug intermediates, and has certain application value.
  • the technical problem to be solved by the present invention is how to obtain a high activity and high stereoselectivity R-ketolase, which is applied to the synthesis of R-aromatic dihydroxyketones and can improve the substrate conversion rate. And the optical purity of the product R-aromatic dihydroxyketone meets the requirements of industrial production.
  • a transketolase mutant which has a sequence in which the amino acid mutation of the sequence shown in SEQ ID NO: 16 occurs, and the amino acid mutation that occurs is H26Y or any one of the following combined mutations: H26Y+F434G, H26Y+ F434A, H26Y+F434L, H26Y+F434I, H26Y+F434V, H26Y+F434P, H26Y+F434M, H26Y+F434W, H26Y+F434S, H26Y+F434Q, H26Y+F434T, H26Y+F434C, H26Y+F434N, H26Y+F434N H26Y+F434D, H26Y+F434E, H26Y+F434K, H26Y+F434R, H26Y+F434H, H26Y+F434Y+L466F, H26Y+F434Y+L466G, H26
  • transketolase mutant according to [1] wherein the transketolase mutant is a transketolase mutant derived from Escherichia coli.
  • transketolase mutant according to any one of [1] to [3], which is a highly stereoselective R-transketolase mutant, wherein its amino acid sequence is SEQ ID NO: 1 shown.
  • Step 1 using the gene derived from the prokaryotic Escherichia coli transketolase as a template, through PCR site-directed mutagenesis and saturation mutation to obtain a highly stereoselective R transketolase mutant DNA molecule;
  • Step 2 constructing a recombinant expression vector containing the highly stereoselective R-ketolase mutant DNA molecule obtained in step 1;
  • Step 3 mass-producing the host cells containing the recombinant expression vector obtained in step 2 by means of IPTG induction to obtain a highly stereoselective R-ketolase mutant with biological activity;
  • Step 4 Separating and purifying the biologically active and highly stereoselective R-transketolase mutant protein obtained in step 3 by affinity chromatography to obtain a highly active and highly stereoselective R-transketolase mutant protein .
  • the invention provides a kind of Escherichia coli transketolase mutant TK/D469T/R520Q/S385Y reported in the literature as the parent (its sequence is as shown in SEQ ID NO:16) after three rounds Iterative saturation mutation was used to obtain benzaldehyde derivatives with high catalytic efficiency, including p-methylsulfonylbenzaldehyde, p-fluorobenzaldehyde, p-chlorobenzaldehyde, p-bromobenzaldehyde, p-nitrobenzaldehyde, p-methylbenzaldehyde and Benzaldehyde produces R-hydroxyketone transketolase mutants such as EcTK1_YYH.
  • an Escherichia coli transketolase mutant which has a sequence of amino acid mutation of the sequence shown in SEQ ID NO: 16, and the amino acid mutation that occurs is H26Y or any combination of the following Mutation: H26Y+F434G, H26Y+F434A, H26Y+F434L, H26Y+F434I, H26Y+F434V, H26Y+F434P, H26Y+F434M, H26Y+F434W, H26Y+F434S, H26Y+F434Q, H26Y+F434T, H26Y+F434T H26Y+F434N, H26Y+F434Y, H26Y+F434D, H26Y+F434E, H26Y+F434K, H26Y+F434R, H26Y+F434H, H26Y+F434Y+L466F, H26Y+F
  • the present invention provides a highly stereoselective R-transketolase mutant whose amino acid sequence is shown in SEQ ID NO: 1 (based on Escherichia coli transketolase mutant TK/D469T/ R520Q/S385Y parent, amino acid mutation H26Y+F434Y+L466H); the nucleotide sequence of its coding gene is shown in SEQ ID NO:2.
  • SEQ ID NO: 1 sequence is shown below:
  • the SEQ ID NO:2 sequence is shown below:
  • the present invention also provides a recombinant expression vector for the encoding gene of the highly stereoselective R-transketolase mutant.
  • the present invention also provides a host cell containing a recombinant expression vector encoding a gene encoding a highly stereoselective R-transketolase mutant.
  • a highly stereoselective R-transketolase mutant or a DNA molecule encoding the mutant or a recombinant expression plasmid containing a DNA molecule encoding the mutant or a host cell containing the above-mentioned recombinant expression plasmid can be used for R-aromatic two Hydroxyketone synthesis.
  • the present invention also provides a method for preparing a highly stereoselective R-transketolase mutant, comprising the following steps:
  • Step 1 using the gene derived from prokaryotic Escherichia coli transketolase as a template, obtain high stereoselectivity R-ketolase mutant DNA molecule by PCR site-directed mutagenesis and saturation mutation;
  • Step 2 constructing a recombinant expression vector containing the highly stereoselective R-ketolase mutant DNA molecule obtained in step 1;
  • Step 3 mass-producing the host cells containing the recombinant expression vector obtained in step 2 by means of IPTG induction to obtain a highly stereoselective R-ketolase mutant with biological activity;
  • Step 4 Separating and purifying the biologically active and highly stereoselective R-transketolase mutant protein obtained in step 3 by affinity chromatography to obtain a highly active and highly stereoselective R-transketolase mutant protein .
  • step 1 the DNA molecule is obtained by PCR site-directed mutagenesis and saturation mutagenesis using the gene derived from the prokaryotic Escherichia coli transketolase as a template.
  • step 1 the recombinant plasmid pET28a-TK is used as a template.
  • double primer pairs shown in D469T-F, D469T-R, R520Q-F, R520Q-R, S385Y-F and S385Y-R for PCR site-directed mutagenesis D469T-F, D469T-R, R520Q-F , R520Q-R, S385Y-F and S385Y-R nucleotide sequences are respectively as SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:8, SEQ ID NO:5 ID NO: 9.
  • PCR saturation mutations were performed using the double primer pairs indicated by H26-F, H26-R, F434-F, F434-R, L466-F and L466-R, H26-F, H26-R, F434-F, F434-
  • the nucleotide sequences of R, L466-F and L466-R are shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:11, respectively: 15 shown.
  • the transketolase mutant is produced by site-directed mutagenesis and iterative saturation mutation of the amino acid sequence of prokaryotic Escherichia coli transketolase; the amino acid sequence of prokaryotic Escherichia coli transketolase is mutated to tyrosine at position 385.
  • amino acid, aspartic acid at position 469 is mutated to threonine
  • arginine at position 520 is mutated to glutamine (that is, to obtain the sequence shown in SEQ ID NO: 16)
  • histidine at position 26 is mutated To tyrosine
  • phenylalanine at position 434 was mutated to tyrosine
  • leucine at position 466 was mutated to histidine.
  • step 4 obtained the R-transketolase mutant EcTK1_YYH with high activity and high stereoselectivity.
  • the present invention also provides the application of the highly stereoselective R-ketolase mutant in catalyzing aromatic aldehydes to form highly stereoselective R-aromatic dihydroxyketones.
  • p-methylsulfonylbenzaldehyde was used to synthesize R-p-methylsulfonylphenyl dihydroxyketone.
  • the present invention also provides the use of the encoding gene of the high stereoselective R-ketolase mutant in catalyzing the formation of high stereoselective R-aromatic dihydroxyketones from aromatic aldehydes.
  • a recombinant expression vector containing the encoding gene of the highly stereoselective R-transketolase mutant was constructed; the host cells containing the recombinant expression vector were produced in large quantities by means of IPTG induction to obtain a high-stereoselective R-ketolase with biological activity
  • the mutant; the high stereoselectivity R transketolase mutant protein with biological activity was separated and purified by the method of affinity chromatography, and the high activity and high stereoselectivity R transketolase mutant protein was obtained; Sulfonylbenzaldehyde is used as a substrate, and the R-p-methylsulfonyl phenyl dihydroxyketone is synthesized from p-methylsulfonylbenzaldehyde by using a highly active and high stereoselective R-transketolase mutant protein as a catalytic enzyme.
  • Example 1 of the preferred embodiment of the present invention the site-directed mutagenesis and saturation mutagenesis of the natural transketolase derived from prokaryotic Escherichia coli and the screening of high activity and high stereoselectivity R transketolase are described in detail.
  • Example 2 of another preferred embodiment of the present invention the expression and purification process of the highly stereoselective R-transketolase mutant is described in detail.
  • Example 3 of another preferred embodiment of the present invention the detection process of the highly stereoselective R-transketolase mutant EcTK1 catalyzed by p-methylsulfonylbenzaldehyde to obtain a highly stereoselective product is described in detail.
  • the amino acid sequence of the natural transketolase derived from prokaryotic Escherichia coli is transformed through the techniques of site-directed mutagenesis and iterative saturation mutagenesis to obtain an R transketolase mutant EcTK1_YYH with high activity and high stereoselectivity, which catalyzes The resulting stereoselectivity to p-methylsulfonyl phenyl dihydroxyketone was 95.2% ee(R).
  • Its beneficial technical effects are as follows: 1. High activity of R-ketolase mutant; 2. Can catalyze aromatic aldehyde substrates to produce R-aromatic dihydroxyketone; 3. High stereoselectivity of R-aromatic dihydroxyketone 4.
  • R-aromatic dihydroxy ketones are important chiral drug intermediates, and R-transketolase mutants can be used in the biosynthesis of chiral drug intermediates.
  • the mutant obtained in the present invention lays a foundation for its application in the synthesis of chiral R-aromatic dihydroxyketones. At present, this is the first report on the efficient catalysis of aromatic aldehydes to form R-aromatic dihydroxyketones with high stereoselectivity by transketolase.
  • Fig. 1 is in a preferred embodiment of the present invention 3, the schematic diagram of the transketone reaction of high stereoselectivity R transketolase catalyzed p-methylsulfonylbenzaldehyde;
  • Fig. 2 is a preferred embodiment 3 of the present invention, the reaction schematic diagram that high stereoselectivity R transketolase catalyzes p-methylsulfonylbenzaldehyde to obtain high stereoselectivity product and converts it into amino alcohol diastereomer;
  • Fig. 3 is the liquid phase diagram of the reaction product under C18 column condition with p-methanesulfonyl benzaldehyde as substrate;
  • Fig. 4 is the liquid phase diagram of the threo-type reaction product under chiral liquid phase condition with p-methanesulfonyl benzaldehyde as substrate;
  • Fig. 5 is the hydrogen spectrum of reaction product (1R, 2R)-AMPP
  • Fig. 6 is the carbon spectrum of reaction product (1R, 2R)-AMPP
  • Fig. 7 is the liquid phase diagram of the reaction product with benzaldehyde as a substrate under C18 column conditions, namely the high performance liquid chromatography identification of the enzyme cascade catalyzed benzaldehyde reaction product;
  • A is the HPLC of the product of the enzyme cascade reaction Chromatography;
  • B is the standard substance of (1R,2R)-phenylserinol;
  • C is the mixture standard substance of Su Shi and erythro products;
  • A is an enzyme grade HPLC chromatogram of the combined reaction product
  • B is the standard of (1R,2R)-p-methylphenylserinol
  • C is the standard of the mixture of Thresh and erythro products.
  • n any base of a, t, c and g
  • k represents t or g base
  • the nnk degenerate codon can encode 20 random amino acids
  • m represents a or c base
  • mnn is complementary to nnk.
  • extract pET28a-TK/D469T/R520Q plasmid take this plasmid as template, use S385Y-F and S385Y-R as primers to amplify the whole plasmid, repeat the above method to construct pET28a-TK/D469T/R520Q/S385Y (pET28a-EcTK1 ) plasmid.
  • pET28a-EcTK1 pET28a-EcTK1
  • the reaction product of (4) is detected by liquid chromatography, and the ratio of (1S,2R)-aminodiol and (1R,2R)-aminodiol is investigated.
  • EcTK1_Y had the highest R stereoselectivity among the saturated mutants, and its activity was also high. Therefore, EcTK1_Y was selected as the template for the next round of saturation mutations, and F434-F/R was used as the primer for whole plasmid PCR, and the above plasmids were repeated. Construction, expression and detection methods. The R-stereoselectivity of EcTK1_YY was the highest in the second round of saturation mutants, so it was used as the template for the third round of saturation mutants, and the above steps were repeated with L466-F/R as primers. The EcTK1_YYH mutant was finally obtained.
  • the expression and purification method of the mutant EcTK1_YYH pick a single colony of E. coli BL21 (DE3) containing the recombinant plasmid on LB solid medium and inoculate it into 40ml LB liquid medium (containing 50 ⁇ g/ml kanamycin antibiotic) , 37 °C, 220rpm culture overnight. Transfer 7.5ml of bacterial culture solution to a 2L shake flask containing 500ml of liquid LB medium, inoculate two flasks, cultivate at 37°C, 220rpm until OD600 reaches 0.6-0.8, add 0.4mM IPTG for induction, and induce culture at 30°C and 200rpm. 5h.
  • the filtered sample was loaded with 2ml of nickel packing pre-equilibrated with nickel column binding buffer, and the impurity protein was washed with 10 times the column volume of 50mM imidazole elution buffer, and then the target protein was eluted with 5ml of 250mM imidazole elution buffer. , connect the samples in separate tubes, each tube is 500 ⁇ l.
  • EcTK1 catalyzes the detection process of p-methylsulfonylbenzaldehyde to obtain highly stereoselective products, and the involved chemical reactions include transketone reaction and transamination reaction.
  • the principle of the transketone reaction is to use p-methylsulfonylbenzaldehyde as the substrate and the mutant of the transketolase as the catalyst, which is represented by "TK".
  • TK mutant of the transketolase
  • S- dihydroxyketone and R-dihydroxyketone products S-dihydroxyketone is represented by "2a:S” and R-dihydroxyketone product is represented by "2b:R”.
  • the principle of the transamination reaction is to instantaneously convert the transketone reaction product dihydroxyketone into a stable chiral aminodiol compound, and the stereoselectivity of dihydroxyketone is investigated by detecting the stereoselectivity of aminodiol.
  • Transketone reaction products S-dihydroxy ketone and R-dihydroxy ketone are converted into (1S,2R)-amino alcohol and (1R,2R)- Amino alcohol, wherein (1S,2R)-aminoalcohol is represented by "3a:(1S,2R)", and (1R,2R)-aminoalcohol is represented by "3b:(1R,2R)".
  • the concentrations of (1S,2R)-aminoalcohol and (1R,2R)-aminoalcohol were detected in liquid phase to characterize the concentrations of transketone products 2a and 2b, respectively.
  • reaction system 50 mM Tris-Cl buffer, pH 7
  • 5 mM p-methylsulfonylbenzaldehyde 25 mM LiHPA
  • 9 mM MgCl 2 9 mM MgCl 2
  • 4.8 mM ThDP 100 ⁇ M transketolase mutant pure protein
  • (1R,2R)-AMPP ((1R,2R)-2-amino-1-(4-(methylsulfonyl)phenyl)propane-1,3-diol, also known as (1R,2R)-1 , 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane) and a method for synthesizing its derivatives, using benzaldehyde derivatives (formula 1) as substrates, the effect of Escherichia coli transketolase mutants Carry out the transketone reaction to obtain the reaction product shown in formula 2, and then use the compound shown in formula 2 as the substrate to carry out the transamination reaction under the action of the transaminase ATA117 mutant, and the amino donor is D-alanine, D-Glycine, D-Valine, D-Leucine, D-Isoleucine, D-Methionine, D-Proline, D-Tryptophan, D-Serine
  • the nucleotide sequence of the above-mentioned amino acid mutation in the sequence shown in SEQ ID NO: 17 is introduced into the vector pET28a, and into the host E.coli BL21; then on the LB solid medium, pick the Escherichia coli E.coli BL21 containing the recombinant plasmid on the LB solid medium
  • a single colony was inoculated into 40ml LB liquid medium (containing 50 ⁇ g/ml kanamycin antibiotic), and cultured overnight at 37°C and 220rpm; 10ml of bacterial culture was transferred to a 2L shake flask containing 500ml liquid LB medium, and two cells were inoculated. bottle, continue to culture at 37°C, 220rpm until OD600 reaches 0.6-0.8, add 0.2mM IPTG for induction, and induce culture at 30°C, 200rpm for 5h;
  • the crushed mixture was centrifuged at 12,000 rpm for 30 min, and the supernatant was filtered through a 0.22 ⁇ m membrane filter; then, the filtered sample was loaded onto 2 ml of nickel filler that had been pre-equilibrated with a nickel column binding buffer, using 20 times A column volume of 50 mM imidazole elution buffer was used to wash the impurity protein, and then 5 ml of 250 mM imidazole elution buffer was used to elute the target protein. Measure the protein concentration of each tube, combine several tubes of protein solution with higher concentration, dilute or concentrate to 2.5ml, and load the sample into the desalting column equilibrated with glycerol buffer. After the protein solution is drained, add 3.5ml of glycerol buffer to elute protein to obtain the pure protein of Escherichia coli transketolase mutant.
  • the molar extinction coefficient of the enzyme was predicted by the software Vector NTI.
  • the nucleotide sequence of the above-mentioned amino acid mutation in the sequence shown in SEQ ID NO: 19 is introduced into the vector pRSFDuet, and into the host E.coli BL21; then on the LB solid medium, pick the Escherichia coli E.coli containing the recombinant plasmid on the LB solid medium
  • a single colony of BL21 was inoculated into 40ml LB liquid medium (containing 50 ⁇ g/ml kanamycin antibiotic), and cultured overnight at 37°C and 220rpm; 10ml of bacterial culture was transferred to a 2L shake flask containing 500ml liquid LB medium, inoculated Two bottles, continue to culture at 37°C, 220rpm until OD600 reaches 0.6-0.8, add 0.2mM IPTG for induction, and induce culture at 20°C, 200rpm for 15h.
  • the crushed mixture was centrifuged at 12,000 rpm for 30 min, and the supernatant was filtered through a 0.22 ⁇ m membrane filter; then, the filtered sample was loaded on a nickel column combined with 2 ml of nickel filler that had been pre-equilibrated with the buffer, using a 10-fold column.
  • a volume of 50 mM imidazole elution buffer was used to wash the impurity protein, and then 5 ml of 250 mM imidazole elution buffer was used to elute the target protein.
  • the yellow color is due to the binding of the transaminase to the cofactor PLP.
  • the conversion rate is 0-40%*, the conversion rate is 40-60%**, the conversion rate is 60-80%**, and the conversion rate is >80%****.
  • the present invention further detects the reaction product on an Agilent 1200 liquid chromatograph, and the specific detection method includes: using a C18 column (4.6 ⁇ 150 mm, particle size 3 ⁇ m), the column temperature is 30° C., 0.5 ml/min, phase A: H 2 O (10 mM KH 2 PO 4 , pH 8.5), Phase B: Acetonitrile.
  • Fig. 3 is the liquid phase diagram of the reaction product of experimental group 5 using p-methanesulfonyl benzaldehyde as a substrate under the condition of C18 column, in Fig. 3, the upper part is (erythro)-p-methanesulfonyl phenylserine
  • Fig. 4 is the liquid phase diagram of (threo)-p-methylsulfosulfonyl phenylserinol product under chiral liquid column conditions, in Fig.
  • the top is the erythro+threo standard control
  • the second row is the threo standard
  • the third row is the (1R, 2R)-p-methylsulfonyl phenylserinol standard control
  • the fourth row is the last row (threo)-p-methylsulfonyl phenylserinol product
  • the calculated ee value is greater than 99%, wherein: RR and SS represent (1R, 2R)- and (1S, 2S)-p-methanesulfonyl phenylserinol.
  • Fig. 5 is the hydrogen spectrum of the reaction product (1R, 2R)-AMPP
  • Fig. 6 is the carbon spectrum of the reaction product (1R, 2R)-AMPP.
  • the present invention synthesizes (1R, 2R)-AMPP, and combined with de value and ee value, the (1R, 2R)-AMPP prepared by the method provided by the present invention has high stereoselectivity.
  • Fig. 7 is the liquid phase diagram of the reaction product of experimental group 9 using benzaldehyde as a substrate under the condition of C18 column, in Fig. 7, the upper part is the reaction product obtained by experimental group 9, and the middle is (1R, 2R)-p-methanesulfonic acid
  • E. coli transketolase mutant TK/D469T/R520Q/S385Y; SEQ ID NO: 16 Amino acid sequence of E. coli transketolase (i.e. E. coli transketolase mutant TK/D469T/R520Q/S385Y; SEQ ID NO: 16):
  • E. coli transketolase mutant TK/D469T/R520Q/S385Y Nucleotide sequence of E. coli transketolase (i.e. E. coli transketolase mutant TK/D469T/R520Q/S385Y) (SEQ ID NO: 17):

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Abstract

提供一种转酮酶突变体及其编码基因和应用，涉及酶工程技术领域。提供的转酮酶突变体具有SEQ ID NO:16所示序列发生氨基酸突变的序列。提供了一种高立体选择性R转酮酶突变体，其氨基酸序列为SEQ ID NO:1；含有该编码基因的重组表达载体；含有该重组表达载体的宿主细胞；制备该高立体选择性R转酮酶突变体的方法；该R转酮酶突变体在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用；其编码基因在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。突变体EcTK1_YYH催化产生的对甲砜基苯基二羟酮的立体选择性为95.2％ee(R)，具有高活性和高立体选择性。

Description

一种高立体选择性R转酮酶突变体及其编码基因和应用
本发明要求于2021年2月5日提交中国专利局、申请号为202110162619.2、发明名称为“一种高立体选择性R转酮酶突变体及其编码基因和应用”和2021年5月24日提交中国专利局、申请号为202110567238.2、发明名称为“一种利用酶级联反应合成(1R,2R)-AMPP的方法”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明涉及酶工程技术领域，涉及一种转酮酶突变体、大肠杆菌转酮酶突变体及其编码基因和应用，尤其涉及一种高立体选择性R转酮酶突变体及其编码基因和应用。
背景技术
转酮酶是一种广泛存在的焦磷酸硫胺素依赖的酶。它连接了非氧化的磷酸戊糖途径和三羧酸循环。转酮酶催化可逆的转酮反应，将酮醇供体的二碳单位转移到醛受体中，形成手性的二羟酮类化合物。在转酮反应中，β-羟基丙酮酸通常用作酮醇供体，因为β-羟基丙酮酸被转酮后生成挥发性的二氧化碳，从而使反应不可逆。近来，文献报道了二羟酮化合物的检测方法，包括比色法和HPLC法，并建立了手性GC和HPLC法来检测二羟酮化合物的对映选择性。这些检测方法的建立有利于改造转酮酶突变体的催化活性和立体选择性。
立体选择性以对映体过剩率ee来反映，ee表示一种手性化合物的光学纯度，ee值越高，光学纯度也越高。2008年Helen C.Hailes课题组报道野生型的大肠杆菌转酮酶能催化产生L-赤藓酮糖，其立体选择性较高，为95％ee(S)对映体过剩率用来表示一种手性化合物的光学纯度。ee值越高，光学纯度也越高，但催化戊醛产生中等立体选择性的羟酮产物。有研究通过对转酮酶D469和H26位点的定点突变，提高并反转了转酮酶催化戊醛的立体选择性。其中，D469E的S选择性得到了提高(ee(S)＝90％)，而H26Y突变体催化产生(R)-羟酮(ee＝88％)[Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single-Point Mutations,Adv.Synth.Catal.2008,350,2631–2638]。Paul A.Dalby课题组在2019年报道通过对大肠杆菌来源的转酮酶进行饱和突变和组合突变，筛选所得突变体能催化丙酮酸供体。当戊醛和己醛作为受体时，产生高立体选择性的(S)-羟酮(ee＞98％)[Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α-hydroxyketones,The FEBS Journal 287(2020)1758–1776]。综上，转酮酶对脂肪醛(比如丙醛，戊醛和己醛)具有较高的催化活性，并通过酶进化获取的转酮酶突变体具有较高的S选择性(ee＝90％-98％)和R选择性(ee＝88％)。
除了催化脂肪醛，转酮酶被改造后还可以催化芳香醛。2010年Helen C.Hailes课题组报道对大肠杆菌来源的转酮酶进行了D469和F434的定点突变，实现了对苯甲醛和间羟基苯甲醛的催化，但催化效率较低，转化率≤10％。其突变体催化产生的羟酮产物主要为R构型(ee≤82％)[α,α′-Dihydroxyketone formation using aromatic and heteroaromatic aldehydes with evolved transketolase enzymes,Chem.Commun.,2010,46,7608–7610]。Paul A.Dalby课题组2015年报道在大肠杆菌TK/D469T的基础上作R520和S385的迭代 饱和突变，筛选所得突变体EcTK/D469T/R520Q/S385Y对苯甲醛衍生物包括间羧基苯甲醛，间羟基苯甲醛和对羧基苯甲醛均具有较高的催化活性，拓宽了转酮酶的底物谱，但其立体选择性未知[Second generation engineering of transketolase for polar aromatic aldehyde substrates,Enzyme and Microbial TecH26ology 71(2015)45–52]。Wolf-Dieter Fessner课题组2017年报道通过对嗜热脂肪芽孢杆菌来源的转酮酶定向进化，提高了转酮酶对苯基乙醛，苯基丙醛，苯氧基乙醛，苄氧基乙醛的催化效率，产率为60-72％，其羟酮产物具有绝对的S立体选择性(ee＞99％)。另外，定向进化所得的转酮酶突变体L382N/D470S对苯甲醛底物具有良好的催化活性，但未报道其立体选择性[Second generation engineering of transketolase for polar aromatic aldehyde substrates,Green Chem.,2017,19,481–489]。综上，文献报道通过对转酮酶的改造提高了转酮酶对芳香醛的催化活性，其催化芳香醛底物如苯基乙醛，苯基丙醛和苯氧基乙醛产生S立体构型的羟酮产物，而催化苯甲醛的产物构型未知。因此未有文献报道能催化芳香醛尤其是苯甲醛衍生物产生R构型羟酮产物的转酮酶。
高立体选择性的R-芳香二羟酮是酮糖，手性氨基二醇和其他高价值化合物的重要合成砌块，比如R-对甲砜基苯基二羟酮是甲砜霉素和氟苯尼考的手性中间体，R-对硝基苯基二羟酮是氯霉素的手性中间体，而R-苯基羟酮化合物是去甲麻黄碱和去甲伪麻黄碱的手性中间体。因此本发明的R转酮酶突变体能用于手性药物中间体的生物合成，具有一定的应用价值。
因此，本领域的技术人员致力于开发一种高活性和高立体选择性的R转酮酶，使其应用于合成R-芳香二羟酮，提高底物转化率和产物R-芳香二羟酮的光学纯度，满足工业化生产的要求。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何获得一种高活性和高立体选择性的R转酮酶，应用于合成R-芳香二羟酮，能提高底物转化率和产物R-芳香二羟酮的光学纯度，满足工业化生产的要求。
本发明提供了以下技术方案：
[1].一种转酮酶突变体，其具有SEQ ID NO:16所示序列发生氨基酸突变的序列，所述发生的氨基酸突变为H26Y或如下任一种组合突变：H26Y+F434G、H26Y+F434A、H26Y+F434L、H26Y+F434I、H26Y+F434V、H26Y+F434P、H26Y+F434M、H26Y+F434W、H26Y+F434S、H26Y+F434Q、H26Y+F434T、H26Y+F434C、H26Y+F434N、H26Y+F434Y、H26Y+F434D、H26Y+F434E、H26Y+F434K、H26Y+F434R、H26Y+F434H、H26Y+F434Y+L466F、H26Y+F434Y+L466G、H26Y+F434Y+L466A、H26Y+F434Y+L466I、H26Y+F434Y+L466V、H26Y+F434Y+L466P、H26Y+F434Y+L466M、H26Y+F434Y+L466W、H26Y+F434Y+L466S、H26Y+F434Y+L466Q、H26Y+F434Y+L466T、H26Y+F434Y+L466C、H26Y+F434Y+L466N、H26Y+F434Y+L466Y、H26Y+F434Y+L466D、H26Y+F434Y+L466E、H26Y+F434Y+L466K、H26Y+F434Y+L466R、H26Y+F434Y+L466H、H26Y+F434Y+L466H+H261F、H26Y+F434Y+L466H+H261G、H26Y+F434Y+L466H+H261A、H26Y+F434Y+L466H+H261L、H26Y+F434Y+L466H+H261I、H26Y+F434Y+L466H+H261V、H26Y+F434Y+L466H+H261P、H26Y+F434Y+L466H+H261M、H26Y+F434Y+L466H+H261W、H26Y+F434Y+L466H+H261S、H26Y+F434Y+L466H+H261Q、H26Y+F434Y+L466H+H261T、 H26Y+F434Y+L466H+H261C、H26Y+F434Y+L466H+H261N、H26Y+F434Y+L466H+H261Y、H26Y+F434Y+L466H+H261D、H26Y+F434Y+L466H+H261E、H26Y+F434Y+L466H+H261K、H26Y+F434Y+L466H+H261R、H26Y+F434Y+L466H+H461F、H26Y+F434Y+L466H+H461G、H26Y+F434Y+L466H+H461A、H26Y+F434Y+L466H+H461L、H26Y+F434Y+L466H+H461I、H26Y+F434Y+L466H+H461V、H26Y+F434Y+L466H+H461P、H26Y+F434Y+L466H+H461M、H26Y+F434Y+L466H+H461W、H26Y+F434Y+L466H+H461S、H26Y+F434Y+L466H+H461Q、H26Y+F434Y+L466H+H461T、H26Y+F434Y+L466H+H461C、H26Y+F434Y+L466H+H461N、H26Y+F434Y+L466H+H461Y、H26Y+F434Y+L466H+H461D、H26Y+F434Y+L466H+H461E、H26Y+F434Y+L466H+H461K、H26Y+F434Y+L466H+H461R。
[2].根据[1]所述转酮酶突变体，其中，所述的转酮酶突变体为源自于大肠杆菌的转酮酶突变体。
[3].根据[1]或[2]所述转酮酶突变体，其中，所述转酮酶突变体为R转酮酶突变体。
[4].根据[1]～[3]中任一项所述的转酮酶突变体，其为一种高立体选择性R转酮酶突变体，其中，其氨基酸序列为SEQ ID NO:1所示。
[5].如[4]所述的转酮酶突变体，其中，其编码基因的核苷酸序列为SEQ ID NO:2所示。
[6].含有[5]所述的转酮酶突变体的编码基因的重组表达载体。
[7].含有[6]所述的转酮酶突变体的编码基因的重组表达载体的宿主细胞。
[8].一种制备[1]～[5]中任一项所述转酮酶突变体的方法，其中，所述方法包括以下步骤：
步骤1、以来源于原核生物大肠杆菌转酮酶的基因作为模板，通过PCR定点突变和饱和突变得到高立体选择性R转酮酶突变体DNA分子；
步骤2、构建含有步骤1得到的高立体选择性R转酮酶突变体DNA分子的重组表达载体；
步骤3、将含有步骤2得到的重组表达载体的宿主细胞通过IPTG诱导的方式大量产生获得具有生物活性的高立体选择性R转酮酶突变体；
步骤4、通过亲和层析方法，对步骤3获得的具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化，得到了高活性的高立体选择性R转酮酶突变体蛋白。
[9].如[8]所述的方法，其中，所述转酮酶突变体是由所述原核生物大肠杆菌转酮酶的氨基酸序列通过所述定点突变和迭代的所述饱和突变而产生的；所述原核生物大肠杆菌转酮酶的氨基酸序列第385位的丝氨酸突变为酪氨酸，第469位的天冬氨酸突变为苏氨酸，第520位的精氨酸突变为谷氨酰胺，第26位的组氨酸突变为酪氨酸，第434位的苯丙氨酸突变为酪氨酸，第466位的亮氨酸突变为组氨酸。
[10].[1]～[4]中任一项所述的转酮酶突变体在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
[11].[5]所述的转酮酶突变体的编码基因在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
[12].根据[10]所述的应用，其中，以对甲砜基苯甲醛为底物，以所述转酮酶突变体为催化酶，将所述对甲砜基苯甲醛合成R-对甲砜基苯基二羟酮。
[13].根据[11]所述的应用，其中，构建含有所述高立体选择性R转酮酶突变体的编码基因的重组表达载体；含有所述重组表达载体的宿主细胞通过所述IPTG诱导的方式大量产生获得所述具有生物活性的高立体选择性R转酮酶突变体；通过所述亲和层析方法，对所述具有生物活性的高立体选择性R转酮酶突变 体蛋白进行分离纯化，得到所述高活性的高立体选择性R转酮酶突变体蛋白；以所述对甲砜基苯甲醛为底物，以所述高活性的高立体选择性R转酮酶突变体蛋白为催化酶，将所述对甲砜基苯甲醛合成所述R-对甲砜基苯基二羟酮。
进一步地，为实现上述目的，本发明提供了一种以文献报道的大肠杆菌转酮酶突变体TK/D469T/R520Q/S385Y为母本(其序列如SEQ ID NO:16所示)经过三轮迭代的饱和突变，获取了能高效催化苯甲醛衍生物，包括对甲砜基苯甲醛，对氟苯甲醛，对氯苯甲醛，对溴苯甲醛，对硝基苯甲醛，对甲基苯甲醛和苯甲醛产生R羟酮的转酮酶突变体，例如EcTK1_YYH。
在本发明的一些具体实施方案中，提供了大肠杆菌转酮酶突变体，其具有SEQ ID NO:16所示序列发生氨基酸突变的序列，所述发生的氨基酸突变为H26Y或如下任一种组合突变：H26Y+F434G、H26Y+F434A、H26Y+F434L、H26Y+F434I、H26Y+F434V、H26Y+F434P、H26Y+F434M、H26Y+F434W、H26Y+F434S、H26Y+F434Q、H26Y+F434T、H26Y+F434C、H26Y+F434N、H26Y+F434Y、H26Y+F434D、H26Y+F434E、H26Y+F434K、H26Y+F434R、H26Y+F434H、H26Y+F434Y+L466F、H26Y+F434Y+L466G、H26Y+F434Y+L466A、H26Y+F434Y+L466I、H26Y+F434Y+L466V、H26Y+F434Y+L466P、H26Y+F434Y+L466M、H26Y+F434Y+L466W、H26Y+F434Y+L466S、H26Y+F434Y+L466Q、H26Y+F434Y+L466T、H26Y+F434Y+L466C、H26Y+F434Y+L466N、H26Y+F434Y+L466Y、H26Y+F434Y+L466D、H26Y+F434Y+L466E、H26Y+F434Y+L466K、H26Y+F434Y+L466R、H26Y+F434Y+L466H、H26Y+F434Y+L466H+H261F、H26Y+F434Y+L466H+H261G、H26Y+F434Y+L466H+H261A、H26Y+F434Y+L466H+H261L、H26Y+F434Y+L466H+H261I、H26Y+F434Y+L466H+H261V、H26Y+F434Y+L466H+H261P、H26Y+F434Y+L466H+H261M、H26Y+F434Y+L466H+H261W、H26Y+F434Y+L466H+H261S、H26Y+F434Y+L466H+H261Q、H26Y+F434Y+L466H+H261T、H26Y+F434Y+L466H+H261C、H26Y+F434Y+L466H+H261N、H26Y+F434Y+L466H+H261Y、H26Y+F434Y+L466H+H261D、H26Y+F434Y+L466H+H261E、H26Y+F434Y+L466H+H261K、H26Y+F434Y+L466H+H261R、H26Y+F434Y+L466H+H461F、H26Y+F434Y+L466H+H461G、H26Y+F434Y+L466H+H461A、H26Y+F434Y+L466H+H461L、H26Y+F434Y+L466H+H461I、H26Y+F434Y+L466H+H461V、H26Y+F434Y+L466H+H461P、H26Y+F434Y+L466H+H461M、H26Y+F434Y+L466H+H461W、H26Y+F434Y+L466H+H461S、H26Y+F434Y+L466H+H461Q、H26Y+F434Y+L466H+H461T、H26Y+F434Y+L466H+H461C、H26Y+F434Y+L466H+H461N、H26Y+F434Y+L466H+H461Y、H26Y+F434Y+L466H+H461D、H26Y+F434Y+L466H+H461E、H26Y+F434Y+L466H+H461K、H26Y+F434Y+L466H+H461R。
在本发明的一些实施方案中，本发明提供了一种高立体选择性R转酮酶突变体，其氨基酸序列为SEQ ID NO:1所示(基于大肠杆菌转酮酶突变体TK/D469T/R520Q/S385Y母本，发生氨基酸突变H26Y+F434Y+L466H)；其编码基因的核苷酸序列为SEQ ID NO:2所示。
SEQ ID NO:1序列如下所示：
Figure PCTCN2022074751-appb-000001
Figure PCTCN2022074751-appb-000002
SEQ ID NO:2序列如下所示：
Figure PCTCN2022074751-appb-000003
本发明还提供一种高立体选择性R转酮酶突变体的编码基因的重组表达载体。
本发明还提供一种含有高立体选择性R转酮酶突变体的编码基因的重组表达载体的宿主细胞。
进一步地，高立体选择性R转酮酶突变体或编码该突变体的DNA分子或含有编码该突变体的DNA分子的重组表达质粒或含有上述重组表达质粒的宿主细胞均可用于R-芳香二羟酮合成。
本发明还提供一种制备高立体选择性R转酮酶突变体的方法，包括以下步骤：
步骤1、以来源于原核生物大肠杆菌转酮酶的基因作为模板，通过PCR定点突变和饱和突变得到高立 体选择性R转酮酶突变体DNA分子；
步骤2、构建含有步骤1得到的高立体选择性R转酮酶突变体DNA分子的重组表达载体；
步骤3、将含有步骤2得到的重组表达载体的宿主细胞通过IPTG诱导的方式大量产生获得具有生物活性的高立体选择性R转酮酶突变体；
步骤4、通过亲和层析方法，对步骤3获得的具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化，得到了高活性的高立体选择性R转酮酶突变体蛋白。
进一步地，步骤1中DNA分子以来源于原核生物大肠杆菌转酮酶的基因作为模板，通过PCR定点突变和饱和突变得到。
进一步地，步骤1中以重组质粒pET28a-TK为模板。
进一步地，PCR定点突变采用的如D469T-F、D469T-R、R520Q-F、R520Q-R、S385Y-F和S385Y-R所示的双引物对，D469T-F、D469T-R、R520Q-F、R520Q-R、S385Y-F和S385Y-R的核苷酸序列分别如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9所示。PCR饱和突变采用的是H26-F、H26-R、F434-F、F434-R、L466-F和L466-R所示的双引物对，H26-F、H26-R、F434-F、F434-R、L466-F和L466-R的核苷酸序列分别如SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15所示。
进一步地，转酮酶突变体是由原核生物大肠杆菌转酮酶的氨基酸序列通过定点突变和迭代的饱和突变而产生的；原核生物大肠杆菌转酮酶的氨基酸序列第385位的丝氨酸突变为酪氨酸，第469位的天冬氨酸突变为苏氨酸，第520位的精氨酸突变为谷氨酰胺(即获得SEQ ID NO:16所示序列)，第26位的组氨酸突变为酪氨酸，第434位的苯丙氨酸突变为酪氨酸，第466位的亮氨酸突变为组氨酸。
进一步地，步骤4获得了高活性和高立体选择性的R转酮酶突变体EcTK1_YYH。
进一步地，结果表明，EcTK1_YYH突变体酶催化对甲砜基苯甲醛合成对甲砜基苯基二羟酮的立体选择性为95.2％ee(R)。
本发明还提供高立体选择性R转酮酶突变体在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
进一步地，以对甲砜基苯甲醛为底物，以高立体选择性R转酮酶突变体为催化酶，将对甲砜基苯甲醛合成R-对甲砜基苯基二羟酮。
本发明还提供高立体选择性R转酮酶突变体的编码基因在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
进一步地，构建含有高立体选择性R转酮酶突变体的编码基因的重组表达载体；含有重组表达载体的宿主细胞通过IPTG诱导的方式大量产生获得具有生物活性的高立体选择性R转酮酶突变体；通过亲和层析方法，对具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化，得到了高活性的高立体选择性R转酮酶突变体蛋白；以对甲砜基苯甲醛为底物，以高活性的高立体选择性R转酮酶突变体蛋白为催化酶，将对甲砜基苯甲醛合成所述R-对甲砜基苯基二羟酮。
在本发明的较佳实施方式实施例1中，详细说明了对来源于原核生物大肠杆菌的天然转酮酶的定点突变和饱和突变及筛选高活性和高立体选择性的R转酮酶。
在本发明的另一较佳实施方式实施例2中，详细说明了高立体选择性R转酮酶突变体的表达纯化过程。
在本发明的另一较佳实施方式实施例3中，详细说明了高立体选择性R转酮酶突变体EcTK1催化对甲砜基苯甲醛获得高立体选择性产物的检测过程。
本发明通过定点突变和迭代的饱和突变的技术对来源于原核生物大肠杆菌的天然转酮酶的氨基酸序列进行改造，获得了高活性和高立体选择性的R转酮酶突变体EcTK1_YYH，其催化产生的对甲砜基苯基二羟酮的立体选择性为95.2％ee(R)。其有益的技术效果体现为：1.R转酮酶突变体的高活性；2.可以催化芳香醛底物，产生R-芳香二羟酮；3.R-芳香二羟酮的高立体选择性；4.R-芳香二羟酮是重要的手性药物中间体，R转酮酶突变体能用于手性药物中间体的生物合成。本发明获得的突变体为其在手性R-芳香二羟酮合成中的应用奠定了基础。目前，这是转酮酶高效催化芳香醛形成高立体选择性的R-芳香二羟酮的首次研究报道。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例3中，高立体选择性R转酮酶催化对甲砜基苯甲醛的转酮反应示意图；
图2是本发明的一个较佳实施例3，高立体选择性R转酮酶催化对甲砜基苯甲醛获得高立体选择性产物转换成氨醇非对映异构体的反应示意图；
图3为以对甲磺砜基苯甲醛为底物的反应产物在C18柱条件下的液相图；
图4为以对甲磺砜基苯甲醛为底物的苏式反应产物在手性液相条件下的液相图；
图5为反应产物(1R,2R)-AMPP的氢谱；
图6为反应产物(1R,2R)-AMPP的碳谱；
图7为以苯甲醛为底物的反应产物在C18柱条件下的液相图，即酶级联催化苯甲醛反应产物的高效液相色谱鉴定；其中，A为酶级联反应的产物的HPLC色谱；B为(1R,2R)-苯基丝氨醇的标准品；C为苏氏和赤式产物的混合物标品；
图8为以对甲基苯甲醛为底物的反应产物在C18柱条件下的液相图，即酶级联催化对甲基苯甲醛反应产物的高效液相色谱鉴定；其中，A为酶级联反应产物的HPLC色谱；B为(1R,2R)-对甲基苯基丝氨醇的标准品；C为苏氏和赤式产物的混合物标品。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
实施例1：定点突变和饱和突变及筛选
对来源于原核生物大肠杆菌的天然转酮酶的定点突变和饱和突变及筛选高活性和高立体选择性的R转酮酶。所使用到的引物核苷酸序列如下表1所示，表1中n代表a、t、c和g任意碱基，k代表t或g碱基，nnk简并密码子可编码随机20种氨基酸。m代表a或c碱基，mnn与nnk互补配对。
表1引物核苷酸序列
Figure PCTCN2022074751-appb-000004
(1)以重组质粒pET28a-TK为模板，其核苷酸序列如SEQ ID NO:3所示，以D469T-F和D469T-R为引物，利用Phanta Max聚合酶(购自诺唯赞)进行PCR扩增(95℃5min；95℃30s，65℃30s，72℃7.5min，34个循环；72℃10min)；PCR产物经FD-Dpn I消化(30℃培养箱，6h)后直接转化至E.coli BL21(DE3)感受态细胞，复苏液充分吹吸混匀，涂布约1/8复苏液于卡那霉素抗性LB平板，37℃培养12-16h。
SEQ ID NO:3：
Figure PCTCN2022074751-appb-000005
Figure PCTCN2022074751-appb-000006
Figure PCTCN2022074751-appb-000007
Figure PCTCN2022074751-appb-000008
(2)挑取上述稀释平板上的3个单菌落于卡那霉素抗性LB培养基中37℃培养7-8h，一部分培养液用于测序，另一部分培养液暂时置于4℃冰箱短期保存。测序阳性的转化子培养液以1％的接种量转接至新鲜的含卡那霉素抗性的5mL LB培养基中。培养过夜，抽提质粒，以该质粒(pET28a-TK/D469T)为模板，以R520Q-F和R520Q-R为引物扩增全质粒，重复以上质粒构建方法。继而抽提pET28a-TK/D469T/R520Q质粒，以该质粒为模板，以S385Y-F和S385Y-R为引物扩增全质粒，重复上述方法构建pET28a-TK/D469T/R520Q/S385Y(pET28a-EcTK1)质粒。以pET28a-EcTK1质粒为模板，以H26-F和H26-R为引物扩增全质粒，重复上述方法，获取重组子文库。
(3)挑取上述稀释平板上的所有单菌落(约40个)于卡那霉素抗性LB培养基中37℃培养7-8h，一部分培养液用于测序，另一部分培养液暂时置于4℃冰箱短期保存。测序阳性的转化子培养液以1％的接种量转接至装有新鲜的含卡那霉素抗性的5mL LB培养基的24孔深孔板中，37℃条件下培养3h后，加入0.4mM IPTG诱导剂30℃培养5h，诱导重组基因的高效表达，重组细胞经4000rpm离心收集菌体，-80℃保藏。以同样诱导方法获得pET28a-EcTK1重组菌体作为对照。
(4)将获得的重组菌体加入300μl的50mM Tris-Cl(pH＝7.5)悬浮，超声破碎，低温离心，获取上清。在含有5mM对甲砜基苯甲醛和30μM R-转氨酶纯蛋白的反应液中加入20％v/v的上清，30℃反应6h。由于全菌中含有酮醇异构酶，后者会加速二羟酮的消旋，因此为了准确考察转酮产物二羟酮的立体选择性，偶联了一个立体专一性的R-转氨酶，将二羟酮瞬时转化成稳定的手性氨基二醇化合物，通过检测氨基二醇的手性来考察二羟酮的手性。
(5)将(4)所述反应产物采用液相色谱进行检测，考察(1S,2R)-氨基二醇和(1R,2R)-氨基二醇的比例。液相分析条件为：色谱柱super-C18(250mm×4.6mm,粒径5μm)，A相：H 2O(含10mM KH 2PO 4，pH＝8.5)，B相：乙腈，色谱条件：0min 98:2到20min 90:10线性变化，1ml/min,224nm，(1S,2R)-氨基二醇和(1R,2R)-氨基二醇的保留时间分别为9.7和10.9min。结果表明EcTK1_Y在饱和突变体中的R立体选择性最高，并且其活性也较高，因此选用EcTK1_Y为下一轮饱和突变的模板，以F434-F/R为引物进行全质粒PCR，重复上述质粒构建，表达和检测方法。在第二轮饱和突变体中EcTK1_YY的R立体选择性最高，因此将其作为第三轮饱和突变的模板，以L466-F/R为引物，重复上述步骤。最终获得EcTK1_YYH突变体。
实施例2：转酮酶突变体的表达纯化
突变体EcTK1_YYH的表达纯化方法：在LB固体培养基上挑取含重组质粒的大肠杆菌E.coli BL21(DE3)单菌落，接种至40ml LB液体培养基(含50μg/ml卡那霉素抗生素)，37℃，220rpm培养过夜。将7.5ml细菌培养液转移至含500ml液体LB培养基的2L摇瓶中，接种两瓶，37℃，220rpm培养至OD600达到0.6-0.8，加入0.4mM IPTG诱导，于30℃，200rpm下诱导培养5h。收集1L发酵液，于5000rpm，离 心20min收集细胞，将收集得到的细胞重悬于30mL镍柱结合缓冲液中，并置于冰水混合物中。超声破碎条件：工作5s，停顿10s，总计30min。将经过破碎处理后的混合物，于12,000rpm离心1h后，上清液过0.22μm滤膜过滤。将过滤后的样品上样经镍柱结合缓冲液预先平衡过的2ml镍填料，用10倍柱体积的50mM咪唑洗脱缓冲液冲洗杂蛋白，然后用5ml 250mM咪唑洗脱缓冲液洗脱目的蛋白，分管接样，每管500μl。微量紫外-可见光分光光度计(NanoDrop)测定每管的蛋白浓度，合并浓度较高的几管蛋白液，稀释或者浓缩至2.5ml，上样经甘油缓冲液平衡过的脱盐柱，蛋白液流干后，加入3.5ml甘油缓冲液洗脱蛋白。
实施例3：考察EcTK1突变体催化对甲砜基苯甲醛获得产物的立体选择性
高立体选择性R转酮酶突变体EcTK1催化对甲砜基苯甲醛获得高立体选择性产物的检测过程，所涉及到的化学反应包括转酮反应和转氨反应。
如图1所示，转酮反应的原理是以对甲砜基苯甲醛为底物，转酮酶突变体为催化剂，该催化剂以“TK”表示，LiHPA、MgCl 2和ThDP存在下，生成S-二羟酮和R-二羟酮产物，S-二羟酮以“2a：S”表示，R-二羟酮产物以“2b:R”表示。
如图2所示，转氨反应的原理是将转酮反应产物二羟酮瞬时转化成稳定的手性氨基二醇化合物，通过检测氨基二醇的立体选择性来考察二羟酮的立体选择性。转酮反应产物S-二羟酮和R-二羟酮，在D-Ala、PLP、NADH、LDH和转氨酶ATA117_AC的作用下，分别转化成(1S,2R)-氨醇和(1R,2R)-氨醇，其中(1S,2R)-氨醇以“3a：(1S,2R)”表示，(1R,2R)-氨醇以“3b：(1R,2R)”表示。液相检测(1S,2R)-氨醇和(1R,2R)-氨醇的浓度，用以分别表征转酮产物2a和2b的浓度。
(1)反应体系共100μl(50mM Tris-Cl buffer，pH 7)，5mM对甲砜基苯甲醛，25mM LiHPA，9mM MgCl 2，4.8mM ThDP，100μM转酮酶突变体纯蛋白，25℃反应20min，加入18mM EDTA螯合Mg 2+终止转酮反应。吸取20μl转酮反应液于新的100μl体系，加入200mM D-Ala,2mM PLP,20mM NADH,90U/ml LDH和200μM转氨酶ATA117_AC于25℃反应30min。S-和R-羟酮产物因此完全转化成(1S,2R)-和(1R,2R)-氨醇非对映异构体。通过检测氨醇非对映异构体的浓度来考察羟酮对映异构体的浓度。
(2)氨醇产物的分析采用安捷伦液相，色谱柱super-C18柱(4.6×250mm，粒径5μm)，A相：H 2O(含10mM KH 2PO 4,pH＝8.5)，B相：乙腈，1ml/min，224nm，(1S,2R)-和(1R,2R)-氨醇的保留时间分别为9.7和10.9min。色谱条件如表2所示：
表2色谱条件
Figure PCTCN2022074751-appb-000009
(3)以大肠杆菌EcTK1为母本，三轮迭代的饱和突变中，其关键突变体分别催化5mM对甲砜基苯甲醛底物，25℃20min，所产生R-羟酮的转化率C(％)和立体选择率ee(％)如表3所示。
表3 R-羟酮的转化率C(％)和立体选择率ee(％)
Figure PCTCN2022074751-appb-000010
实施例4：EcTK1突变体应用于(1R,2R)-AMPP及其衍生物的合成
(1R,2R)-AMPP((1R,2R)-2-氨基-1-(4-(甲磺酰)苯基)丙烷-1,3-二醇，也称为(1R,2R)-1,3-二羟基-2-氨基-1-对甲砜基苯丙烷)及其衍生物的合成方法，以苯甲醛衍生物(式1)为底物，在大肠杆菌转酮酶突变体的作用下进行转酮反应，得到式2所示的反应产物，接着以式2所示的化合物为底物，在转氨酶ATA117突变体的作用下进行转氨反应，氨基供体为D-丙氨酸、D-甘氨酸、D-缬氨酸、D-亮氨酸、D-异亮氨酸、D-甲硫氨酸、D-脯氨酸、D-色氨酸、D-丝氨酸、D-酪氨酸、D-半胱氨酸、D-苯丙氨酸、D-天冬酰胺、D-谷氨酰胺、D-苏氨酸、D-天冬氨酸、D-谷氨酸、D-赖氨酸、D-精氨酸、D-组氨酸或异丙胺，合成式3所示的(1R,2R)-AMPP及其衍生物。
所涉及的反应式如下：
Figure PCTCN2022074751-appb-000011
以下就该方法进行详细阐述：
步骤1、大肠杆菌转酮酶突变体的表达纯化
将SEQ ID NO:17所示序列发生上述氨基酸突变的核苷酸序列导入载体pET28a中，并导入宿主E.coli BL21；随后在LB固体培养基上挑取含重组质粒的大肠杆菌E.coli BL21单菌落，接种至40ml LB液体培养基(含50μg/ml卡那霉素抗生素)，37℃、220rpm培养过夜；将10ml细菌培养液转移至含500ml液体LB培养基的2L摇瓶中，接种两瓶，继续在37℃、220rpm培养至OD600达到0.6-0.8，加入0.2mM IPTG诱导，于30℃、200rpm下诱导培养5h；
培养结束后，收集1L发酵液，于5000rpm，离心20min收集细胞，将收集得到的细胞重悬于30mL镍柱结合缓冲液中，置于冰水混合物中进行超声破碎，超声破碎条件为工作5s，停顿10s，总计30min。
将经过破碎处理后的混合物，于12000rpm离心30min后，上清液过0.22μm滤膜过滤；随后，将过滤后的样品上样经镍柱结合缓冲液预先平衡过的2ml镍填料，用20倍柱体积的50mM咪唑洗脱缓冲液冲洗杂蛋白，然后用5ml 250mM咪唑洗脱缓冲液洗脱目的蛋白，分管接样，每管500μl。测定每管的蛋白浓度，合并浓度较高的几管蛋白液，稀释或者浓缩至2.5ml，上样经甘油缓冲液平衡过的脱盐柱，蛋白液流干后，加入3.5ml甘油缓冲液洗脱蛋白，得到大肠杆菌转酮酶突变体纯蛋白。
步骤1中，蛋白浓度的测定使用Thermo Scientific Nanodrop 8000型检测器检测280nm处的吸光值E， 目标蛋白浓度根据摩尔消光系数换算得到，即蛋白浓度(mg/mL)＝E/摩尔消光系数，重组酶的摩尔消光系数通过软件Vector NTI预测获得。
步骤2、转氨酶ATA117突变体的表达纯化
将如SEQ ID NO:19所示序列发生上述氨基酸突变的核苷酸序列导入载体pRSFDuet中，并导入宿主E.coli BL21；随后在LB固体培养基上挑取含重组质粒的大肠杆菌E.coli BL21单菌落，接种至40ml LB液体培养基(含50μg/ml卡那霉素抗生素)，37℃、220rpm培养过夜；将10ml细菌培养液转移至含500ml液体LB培养基的2L摇瓶中，接种两瓶，继续在37℃、220rpm培养至OD600达到0.6-0.8，加入0.2mM IPTG诱导，于20℃，200rpm下诱导培养15h。
培养结束后，收集1L发酵液，于5000rpm，离心20min收集细胞，将收集得到的细胞重悬于30mL镍柱结合缓冲液中，置于冰水混合物中进行超声破碎，超声破碎条件为工作5s，停顿10s，总计30min。
将经过破碎处理后的混合物，于12000rpm离心30min,上清液过0.22μm滤膜过滤；随后，将过滤后的样品上样经镍柱结合缓冲液预先平衡过的2ml镍填料，用10倍柱体积的50mM咪唑洗脱缓冲液冲洗杂蛋白，然后用5ml 250mM咪唑洗脱缓冲液洗脱目的蛋白，分管接样，每管500μl。由于转氨酶结合了辅因子PLP，因此呈现出黄色。合并黄色较深的几管蛋白液，稀释或者浓缩蛋白液至2.5ml，上样经甘油缓冲液平衡过的层析柱，蛋白液流干后，加入3.5ml甘油缓冲液洗脱蛋白，得到转氨酶突变体纯蛋白。
步骤3、合成(1R,2R)-AMPP及其衍生物
在50μl，含100mM的Tris-HCl buffer(pH 7.5)中加入10mM苯甲醛衍生物、30mM羟基丙酮酸锂(LiHPA，作为转酮供体)、4.8mM硫胺素焦磷酸(TPP，作为转酮反应的辅因子)、18mM MgCl 2(作为转酮反应的金属离子)、100μM大肠杆菌转酮酶突变体，在25℃下反应1-3h；
加入100mM Tris-HCl buffer(pH 7.5)、200mM D-Ala、或D-甘氨酸、或D-缬氨酸、或D-亮氨酸、或D-异亮氨酸、或D-甲硫氨酸、或D-脯氨酸、或D-色氨酸、或D-丝氨酸、或D-酪氨酸、或D-半胱氨酸、或D-苯丙氨酸、或D-天冬酰胺、或D-谷氨酰胺、或D-苏氨酸、或D-天冬氨酸、或D-谷氨酸、或D-赖氨酸、或D-精氨酸、或D-组氨酸、或异丙胺(作为转氨供体)、2mM磷酸吡哆醛(PLP，作为转氨反应的辅因子)、50μM转氨酶ATA117突变体，扩充反应体系至100μl，在25℃下反应3-6h，得到(1R,2R)-AMPP及其衍生物。
表4级联反应产物的转化率和立体选择性
Figure PCTCN2022074751-appb-000012
转化率在0-40％的*，转化率在40-60％的**，转化率在60-80％的***，转化率在>80％的****。
de在0-40％的*，de在40-70％的**，de在70-90％的***，de在>90％的****。
ee在0-40％的*，ee在40-70％的**，ee在70-90％的***，ee在90-99％的****,ee在>99％的*****。
本发明进一步对反应产物在Agilent 1200型液相色谱仪上进行检测，具体检测方法包括：使用C18柱(4.6×150mm，粒径3μm)，柱温30℃，0.5ml/min，A相：H 2O(10mM KH 2PO 4，pH8.5)，B相：乙腈。
表5液相色谱的色谱条件
Figure PCTCN2022074751-appb-000013
图3为实验组5以对甲磺砜基苯甲醛为底物的反应产物在C18柱条件下的液相图，图3中，上方为(赤式)-对甲磺砜基苯基丝氨醇和(苏式)-对甲磺砜基苯基丝氨醇标品的液相图，中间为(1R,2R)-对甲磺砜基苯基丝氨醇的标品，最下方为实验组5得到的反应产物，如图3所示，实验组5制备得到的产物中以苏式为主，并根据公式：de＝[(苏式-赤式)/(苏式+赤式)]×100％计算得到de>90％，其中：赤式和苏式代表(赤式)-和(苏式)-对甲磺砜基苯基丝氨醇。
根据液相出峰，收集(苏式)-对甲磺砜基苯基丝氨醇产物，浓缩，进一步用手性液相柱分离，考察产物的对映选择性。对映异构体分析条件为：手性柱IG柱，柱温25℃，0.5ml/min，224nm，色谱条件：纯甲醇(含0.1％二乙胺)，15min。图4为(苏式)-对甲磺砜基苯基丝氨醇产物在手性液相柱条件下的液相图，图4中，最上方为赤式+苏式标品对照，第二行为苏式标品对照，第三行为(1R,2R)-对甲磺砜基苯基丝氨醇标品对照，第四行即最后一行为(苏式)-对甲磺砜基苯基丝氨醇产物，如图4所示，(1R,2R)-和(1S,2S)-对甲磺砜基苯基丝氨醇的保留时间分别为5.6和9.4min，根据公式ee＝[(RR-SS)/(RR+SS)]×100％计算ee值大于99％，其中：RR和SS表示(1R,2R)-和(1S,2S)-对甲磺砜基苯基丝氨醇。
图5为反应产物(1R,2R)-AMPP的氢谱，图6为反应产物(1R,2R)-AMPP的碳谱，根据图5-图6和图3-图4的标品对照可知，本发明合成了(1R,2R)-AMPP，结合de值和ee值，本发明提供的方法制备得到的(1R,2R)-AMPP的立体选择性性较高。
图7为实验组9以苯甲醛为底物的反应产物在C18柱条件下的液相图，图7中，上方为实验组9得到的反应产物，中间为(1R,2R)-对甲磺砜基苯基丝氨醇的标品，下方为(赤式)-苯基丝氨醇和(苏式)-苯基丝氨醇标品，如图7所示，实验组9制备得到的产物以苏式为主，并根据公式：de＝[(苏式-赤式)/(苏式+赤式)]×100％计算得到de>90％，其中：赤式和苏式代表(赤式)-和(苏式)-苯基丝氨醇。
图8为实验组13以对甲基苯甲醛为底物的反应产物在C18柱条件下的液相图，图8中，上方为实验组13得到的反应产物，中间为(赤式)-对甲基苯基丝氨醇，下方为(苏式)-对甲基苯基丝氨醇标品，如图8所示，实验组13制备得到的产物以苏式为主，并根据公式：de＝[(苏式-赤式)/(苏式+赤式)]×100％计算得到de>90％，其中：赤式和苏式代表(赤式)-和(苏式)-对甲基苯基丝氨醇。
大肠杆菌转酮酶(即大肠杆菌转酮酶突变体TK/D469T/R520Q/S385Y)的氨基酸序列；SEQ ID NO:16)：
Figure PCTCN2022074751-appb-000014
大肠杆菌转酮酶的核苷酸序列(即大肠杆菌转酮酶突变体TK/D469T/R520Q/S385Y)(SEQ ID NO:17)：
Figure PCTCN2022074751-appb-000015
转氨酶ATA117的氨基酸序列(SEQ ID NO:18)：
Figure PCTCN2022074751-appb-000016
转氨酶ATA117的核苷酸序列(SEQ ID NO:19)：
Figure PCTCN2022074751-appb-000017
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (11)

  1. 一种高立体选择性R转酮酶突变体，其特征在于，其氨基酸序列为SEQ ID NO:1所示。
  2. 如权利要求1所述的高立体选择性R转酮酶突变体，其特征在于，其编码基因的核苷酸序列为SEQ ID NO:2所示。
  3. 含有权利要求2所述的高立体选择性R转酮酶突变体的编码基因的重组表达载体。
  4. 含有权利要求3所述的高立体选择性R转酮酶突变体的编码基因的重组表达载体的宿主细胞。
  5. 一种制备权利要求1所述高立体选择性R转酮酶突变体的方法，其特征在于，所述方法包括以下步骤：
    步骤1、以来源于原核生物大肠杆菌转酮酶的基因作为模板，通过PCR定点突变和饱和突变得到高立体选择性R转酮酶突变体DNA分子；
    步骤2、构建含有步骤1得到的高立体选择性R转酮酶突变体DNA分子的重组表达载体；
    步骤3、将含有步骤2得到的重组表达载体的宿主细胞通过IPTG诱导的方式大量产生获得具有生物活性的高立体选择性R转酮酶突变体；
    步骤4、通过亲和层析方法，对步骤3获得的具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化，得到了高活性的高立体选择性R转酮酶突变体蛋白。
  6. 如权利要求5所述的一种制备高立体选择性R转酮酶突变体的方法，其特征在于，所述转酮酶突变体是由所述原核生物大肠杆菌转酮酶的氨基酸序列通过所述定点突变和迭代的所述饱和突变而产生的；所述原核生物大肠杆菌转酮酶的氨基酸序列第385位的丝氨酸突变为酪氨酸，第469位的天冬氨酸突变为苏氨酸，第520位的精氨酸突变为谷氨酰胺，第26位的组氨酸突变为酪氨酸，第434位的苯丙氨酸突变为酪氨酸，第466位的亮氨酸突变为组氨酸。
  7. 权利要求1所述的高立体选择性R转酮酶突变体在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
  8. 权利要求2所述的高立体选择性R转酮酶突变体的编码基因在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
  9. 按照权利要求7所述的应用，其特征在于，以对甲砜基苯甲醛为底物，以所述高立体选择性R转酮酶突变体为催化酶，将所述对甲砜基苯甲醛合成R-对甲砜基苯基二羟酮。
  10. 按照权利要求8所述的应用，其特征在于，构建含有所述高立体选择性R转酮酶突变体的编码基因的重组表达载体；含有所述重组表达载体的宿主细胞通过所述IPTG诱导的方式大量产生获得所述具有生物活性的高立体选择性R转酮酶突变体；通过所述亲和层析方法，对所述具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化，得到所述高活性的高立体选择性R转酮酶突变体蛋白；以所述对甲砜基苯甲醛为底物，以所述高活性的高立体选择性R转酮酶突变体蛋白为催化酶，将所述对甲砜基苯甲醛合成所述R-对甲砜基苯基二羟酮。
  11. 一种大肠杆菌转酮酶突变体，其特征在于，所述大肠杆菌转酮酶突变体具有SEQ ID NO:16所示序列发生氨基酸突变的序列，所述发生氨基酸突变的位点为H26Y位点或如下任一种组合突变位点：H26Y+F434G、H26Y+F434A、H26Y+F434L、H26Y+F434I、H26Y+F434V、H26Y+F434P、H26Y+F434M、H26Y+F434W、H26Y+F434S、H26Y+F434Q、H26Y+F434T、H26Y+F434C、 H26Y+F434N、H26Y+F434Y、H26Y+F434D、H26Y+F434E、H26Y+F434K、H26Y+F434R、H26Y+F434H、H26Y+F434Y+L466F、H26Y+F434Y+L466G、H26Y+F434Y+L466A、H26Y+F434Y+L466I、H26Y+F434Y+L466V、H26Y+F434Y+L466P、H26Y+F434Y+L466M、H26Y+F434Y+L466W、H26Y+F434Y+L466S、H26Y+F434Y+L466Q、H26Y+F434Y+L466T、H26Y+F434Y+L466C、H26Y+F434Y+L466N、H26Y+F434Y+L466Y、H26Y+F434Y+L466D、H26Y+F434Y+L466E、H26Y+F434Y+L466K、H26Y+F434Y+L466R、H26Y+F434Y+L466H、H26Y+F434Y+L466H+H261F、H26Y+F434Y+L466H+H261G、H26Y+F434Y+L466H+H261A、H26Y+F434Y+L466H+H261L、H26Y+F434Y+L466H+H261I、H26Y+F434Y+L466H+H261V、H26Y+F434Y+L466H+H261P、H26Y+F434Y+L466H+H261M、H26Y+F434Y+L466H+H261W、H26Y+F434Y+L466H+H261S、H26Y+F434Y+L466H+H261Q、H26Y+F434Y+L466H+H261T、H26Y+F434Y+L466H+H261C、H26Y+F434Y+L466H+H261N、H26Y+F434Y+L466H+H261Y、H26Y+F434Y+L466H+H261D、H26Y+F434Y+L466H+H261E、H26Y+F434Y+L466H+H261K、H26Y+F434Y+L466H+H261R、H26Y+F434Y+L466H+H461F、H26Y+F434Y+L466H+H461G、H26Y+F434Y+L466H+H461A、H26Y+F434Y+L466H+H461L、H26Y+F434Y+L466H+H461I、H26Y+F434Y+L466H+H461V、H26Y+F434Y+L466H+H461P、H26Y+F434Y+L466H+H461M、H26Y+F434Y+L466H+H461W、H26Y+F434Y+L466H+H461S、H26Y+F434Y+L466H+H461Q、H26Y+F434Y+L466H+H461T、H26Y+F434Y+L466H+H461C、H26Y+F434Y+L466H+H461N、H26Y+F434Y+L466H+H461Y、H26Y+F434Y+L466H+H461D、H26Y+F434Y+L466H+H461E、H26Y+F434Y+L466H+H461K、H26Y+F434Y+L466H+H461R。
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