CN114875004B - High-stereoselectivity R-transketolase mutant and encoding gene and application thereof - Google Patents

Abstract

The invention discloses a high-stereoselectivity R-ketolase mutant, a coding gene and application thereof, and relates to the technical field of enzyme engineering, wherein the amino acid sequence of the high-stereoselectivity R-ketolase mutant is SEQ ID No.1, and the nucleotide sequence of the coding gene is SEQ ID No.2; a recombinant expression vector containing the coding gene; a host cell containing the recombinant expression vector; a method for preparing the high stereoselective R transketolase mutant; the application of the R transketolase mutant in catalyzing aromatic aldehyde to form high-stereoselectivity R-aromatic dihydroxyketone; the coding gene is applied to catalyzing aromatic aldehyde to form R-aromatic dihydroxyketone with high stereoselectivity. The stereoselectivity of p-methylsulfonyl phenyl dihydroxyketone generated by catalyzing the mutant EcTK1_YYH is 95.2%ee (R), and the mutant has high activity and high stereoselectivity.

Description

High-stereoselectivity R-transketolase mutant and encoding gene and application thereof

Technical Field

The invention relates to the technical field of enzyme engineering, in particular to a high-stereoselectivity R-ketolase mutant and a coding gene and application thereof.

Background

Transketolase is a widely occurring thiamine pyrophosphate dependent enzyme. It links the non-oxidized pentose phosphate pathway with the tricarboxylic acid cycle. Transketolase catalyzes reversible transketosis, and transfers the two carbon units of the ketol donor into an aldehyde acceptor to form chiral dihydroxyketone compounds. In the transketosis reaction, beta-hydroxypyruvate is often used as a ketol donor because beta-hydroxypyruvate is transketoized to produce volatile carbon dioxide, thus rendering the reaction irreversible. Recently, the literature reports detection methods of dihydroxyketone compounds, including colorimetry and HPLC methods, and chiral GC and HPLC methods have been established to detect enantioselectivity of dihydroxyketone compounds. The establishment of these detection methods is advantageous for the modification of the catalytic activity and stereoselectivity of the transketolase mutants.

The stereoselectivity is reflected in the enantiomeric excess ee, which represents the optical purity of a chiral compound, and the higher the ee value, the higher the optical purity. The 2008 Helen C.Hailes subject group reports that wild-type E.coli transketolase catalyzes the production of L-erythrulose with high stereoselectivity and an enantiomeric excess of 95% ee (S) is used to represent the optical purity of a chiral compound. The higher the ee value, the higher the optical purity, but catalyzing valeraldehyde produces a moderately stereoselective hydroxyketone product. There have been studies to increase and reverse the stereoselectivity of the transketolase to catalyze valeraldehyde by site-directed mutagenesis of the transketolase D469 and H26 sites. Wherein the S selectivity of D469E is improved (ee (S) =90%) and the H26Y mutant catalyzes the production of (R) -hydroxyketone (ee=88%) [ Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single-Point Mutations, adv. Synth. Catalyst. 2008,350,2631-2638]. Paul A. Dalby group report in 2019 that the resulting mutants were screened for the ability to catalyze pyruvate donors by subjecting E.coli-derived transketolase to saturation and combination mutations. When pentanal and hexanal are used as receptors, highly stereoselective (S) -hydroxyketones (ee > 98%) are produced [ Engineering transketolase to accept both unnatural donor and acceptor substrates and produce. Alpha. -hydroxyketones, the FEBS Journal 287 (2020) 1758-1776]. In summary, the transketolase has higher catalytic activity on fatty aldehydes (such as propionaldehyde, valeraldehyde and hexanal), and the transketolase mutant obtained by enzyme evolution has higher S selectivity (ee=90% -98%) and R selectivity (ee=88%).

In addition to catalyzing fatty aldehydes, transketolase enzymes may also catalyze aromatic aldehydes after being engineered. The Helen C.Hailes subject group in 2010 reports that site-directed mutagenesis of D469 and F434 is performed on ketolase derived from escherichia coli, so that catalysis of benzaldehyde and m-hydroxybenzaldehyde is realized, but the catalysis efficiency is low, and the conversion rate is less than or equal to 10%. The mutant is catalyzed to produce hydroxy ketone product with R configuration (ee less than or equal to 82%) [ alpha, alpha' -Dihydroxyketone formation using aromatic and heteroaromatic aldehydes with evolved transketolase enzymes, chem.Commun.,2010,46,7608-7610]. Paul A. Dalby subject group 2015 reports that iterative saturation mutation of R520 and S385 is performed on the basis of escherichia coli TK/D469T, and the obtained mutant EcTK/D469T/R520Q/S385Y p-benzaldehyde derivative comprises m-carboxybenzaldehyde, the m-carboxybenzaldehyde and the p-carboxybenzaldehyde have higher catalytic activity, the substrate spectrum of the transketolase is widened, but the stereoselectivity is unknown [ Second generation engineering of transketolase for polar aromatic aldehyde substrates, enzyme and Microbial TecH morphology 71 (2015) 45-52]. The Wolf-Dieter Fessner topic group 2017 reports that the catalytic efficiency of the transketolase on phenylacetaldehyde, phenylpropionaldehyde, phenoxyacetaldehyde and benzyloxy acetaldehyde is improved by directionally evolving the transketolase from bacillus stearothermophilus, the yield is 60-72%, and the hydroxyketone product has absolute S stereoselectivity (ee > 99%). In addition, the ketolase mutant L382N/D470S obtained by directed evolution has good catalytic activity on benzaldehyde substrates, but the stereoselectivity thereof is not reported [ Second generation engineering of transketolase for polar aromatic aldehyde substrates, green chem.,2017,19,481-489]. In summary, the literature reports that the catalytic activity of transketolase on aromatic aldehydes is enhanced by modification of transketolase, which catalyzes aromatic aldehyde substrates such as phenylacetaldehyde, phenylpropionaldehyde and phenoxyacetaldehyde to produce hydroxyketone products of S-stereoconfiguration, while the configuration of the products catalyzing benzaldehyde is unknown. There is thus no document reporting of a transketolase that catalyzes the production of an R-configuration hydroxyketone product from an aromatic aldehyde, in particular a benzaldehyde derivative.

The R-aromatic dihydroxyketone with high stereoselectivity is an important synthetic block of ketose, chiral amino diol and other high-value compounds, such as R-p-methylsulfonyl phenyl dihydroxyketone is a chiral intermediate of thiamphenicol and florfenicol, R-p-nitrophenyl dihydroxyketone is a chiral intermediate of chloramphenicol, and R-phenyl hydroxyketone compound is a chiral intermediate of norephedrine and norpseudoephedrine. Therefore, the R transketolase mutant can be used for biosynthesis of chiral drug intermediates and has a certain application value.

Therefore, the person skilled in the art is dedicated to develop an R-transketolase with high activity and high stereoselectivity, so that the R-transketolase is applied to the synthesis of R-aromatic dihydroxyketone, the substrate conversion rate and the optical purity of the product R-aromatic dihydroxyketone are improved, and the requirement of industrial production is met.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention is to solve the technical problem of how to obtain a high-activity and high-stereoselectivity R-transketolase, which is applied to synthesis of R-aromatic dihydroxyketone, and can improve substrate conversion rate and optical purity of the product R-aromatic dihydroxyketone, thereby meeting the requirements of industrial production.

In order to achieve the aim, the invention provides a ketolase mutant EcTK 1_YH which takes an escherichia coli ketolase mutant TK/D469T/R520Q/S385Y reported in a literature as a female parent and can catalyze benzaldehyde derivatives with high efficiency through three rounds of iterative saturation mutation, wherein the ketolase mutant EcTK 1_YH can be used for producing R-hydroxyketone from p-methylsulfonylbenzaldehyde, p-fluorobenzaldehyde, p-chlorobenzaldehyde, p-bromobenzaldehyde, p-nitrobenzaldehyde, p-methylbenzaldehyde and benzaldehyde.

A high stereoselectivity R transketolase mutant has an amino acid sequence shown as SEQ ID No. 1; the nucleotide sequence of the coding gene is shown as SEQ ID No. 2.

The invention also provides a recombinant expression vector of the coding gene of the high stereoselectivity R transketolase mutant.

The invention also provides a host cell containing the recombinant expression vector of the coding gene of the high stereoselective R-transketolase mutant.

Furthermore, the high stereoselectivity R transketolase mutant, the DNA molecule encoding the mutant, the recombinant expression plasmid containing the DNA molecule encoding the mutant or the host cell containing the recombinant expression plasmid can be used for synthesizing the R-aromatic dihydroxyketone.

The invention also provides a method for preparing the high stereoselectivity R transketolase mutant, which comprises the following steps:

step 1, taking a gene derived from prokaryotic escherichia coli ketolase as a template, and obtaining a high stereoselectivity R ketolase mutant DNA molecule through PCR site-directed mutagenesis and saturation mutagenesis;

step 2, constructing a recombinant expression vector containing the high stereoselectivity R transketolase mutant DNA molecule obtained in the step 1;

step 3, generating a large amount of host cells containing the recombinant expression vector obtained in the step 2 in an IPTG induction mode to obtain a high stereoselectivity R transketolase mutant with bioactivity;

and 4, separating and purifying the high-stereoselectivity R-transketolase mutant protein with biological activity obtained in the step 3 by an affinity chromatography method to obtain the high-stereoselectivity R-transketolase mutant protein with high activity.

Further, the DNA molecule in the step 1 is obtained by PCR site-directed mutagenesis and saturation mutagenesis by taking a gene derived from a prokaryote escherichia coli transketolase as a template.

Further, in the step 1, the recombinant plasmid pET28a-TK is used as a template.

Further, the PCR site-directed mutagenesis uses a double primer pair as shown in D469T-F, D469T-R, R Q-F, R520Q-R, S385Y-F and S385Y-R, and the nucleotide sequences of D469T-F, D469T-R, R Q-F, R520Q-R, S385Y-F and S385Y-R are shown as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, respectively. The PCR saturation mutation adopts a double primer pair shown as H26-F, H26-R, F434-F, F434-R, L466-F and L466-R, and the nucleotide sequences of H26-F, H26-R, F434-F, F434-R, L466-F and L466-R are respectively shown as ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14 and SEQ ID No. 15.

Further, the transketolase mutant is generated by site-directed mutagenesis and iterative saturation mutagenesis of the amino acid sequence of the prokaryote E.coli transketolase; serine at 385 of the amino acid sequence of the prokaryote E.coli transketolase is mutated to tyrosine, aspartic acid at 469 is mutated to threonine, arginine at position 520 is mutated to glutamine, histidine at position 26 is mutated to tyrosine, phenylalanine at position 434 is mutated to tyrosine, and leucine at position 466 is mutated to histidine.

Further, step 4 yielded a highly active and highly stereoselective R transketolase mutant ectk1_yyh.

Further, the result shows that the enzyme of the EcTK1_YYH mutant catalyzes the synthesis of p-methylsulfonyl benzaldehyde into p-methylsulfonyl phenyl dihydroxyketone with the stereoselectivity of 95.2%ee (R).

The invention also provides application of the high-stereoselectivity R-transketolase mutant in catalyzing aromatic aldehyde to form high-stereoselectivity R-aromatic dihydroxyketone.

Further, p-methylsulfonyl benzaldehyde is used as a substrate, and a high-stereoselectivity R transketolase mutant is used as a catalytic enzyme, so that R-p-methylsulfonyl phenyl dihydroxyketone is synthesized from the p-methylsulfonyl benzaldehyde.

The invention also provides application of the coding gene of the high-stereoselectivity R-transketolase mutant in catalyzing aromatic aldehyde to form high-stereoselectivity R-aromatic dihydroxyketone.

Further, constructing a recombinant expression vector containing a coding gene of the high stereoselectivity R transketolase mutant; host cells containing the recombinant expression vector are used for generating a large amount of R transketolase mutants with high stereoselectivity and bioactivity in an IPTG induction mode; separating and purifying the high-stereoselectivity R-transketolase mutant protein with biological activity by an affinity chromatography method to obtain the high-stereoselectivity R-transketolase mutant protein with high activity; synthesizing the R-p-methylsulfonyl phenyl dihydroxyketone by using p-methylsulfonyl benzaldehyde as a substrate and using high-activity high-stereoselectivity R-transketolase mutant protein as a catalytic enzyme.

In example 1 of the preferred embodiment of the present invention, site-directed mutagenesis and saturation mutagenesis of a native transketolase derived from a prokaryote E.coli and screening for highly active and highly stereoselective R transketolase are described in detail.

In example 2, which is another preferred embodiment of the present invention, the expression purification process of the highly stereoselective R transketolase mutant is described in detail.

In another preferred embodiment example 3 of the present invention, the detection process of the high stereoselectivity R transketolase mutant EcTK1 for catalyzing p-methylsulfonyl benzaldehyde to obtain high stereoselectivity product is described in detail.

The invention modifies the amino acid sequence of natural transketolase from prokaryotic escherichia coli by site-directed mutagenesis and iterative saturation mutagenesis technology to obtain a high-activity and high-stereoselectivity R transketolase mutant EcTK1_YYH, and the stereoselectivity of p-methylsulfonyl phenyl dihydroxyketone produced by catalysis is 95.2%ee (R). The beneficial technical effects are as follows: high activity of r transketolase mutants; 2. can catalyze aromatic aldehyde substrates to produce R-aromatic dihydroxyketone; 3.R-high stereoselectivity of aromatic dihydroxyketones; 4.R-aromatic dihydroxyketone is an important chiral drug intermediate, and the R transketolase mutant can be used for biosynthesis of the chiral drug intermediate. The mutant obtained by the invention lays a foundation for the application of the mutant in the synthesis of chiral R-aromatic dihydroxyketone. At present, the first research report of high-efficiency catalytic aromatic aldehyde formation of R-aromatic dihydroxyketone with high stereoselectivity by using transketolase is presented.

The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

FIG. 1 is a schematic diagram showing a high stereoselective R-transketolase catalyzed transketotion of p-methylsulfonyl benzaldehyde in accordance with a preferred embodiment 3 of the present invention;

FIG. 2 is a schematic representation of the conversion of a highly stereoselective R-transketolase catalyzed p-methylsulfonyl benzaldehyde to an amino alcohol diastereomer in accordance with a preferred embodiment 3 of the present invention.

Detailed Description

The following description of the preferred embodiments of the present invention refers to the accompanying drawings, which make the technical contents thereof more clear and easy to understand. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.

Example 1: site-directed mutagenesis and saturation mutagenesis and screening

Site-directed mutagenesis and saturation mutagenesis of native transketolase from E.coli, a prokaryote, and screening for highly active and highly stereoselective R transketolase. The nucleotide sequences of the primers used are shown in the following table 1, wherein N represents A, T, C and G arbitrary bases, K represents T or G bases, and NNK degenerate codons can encode 20 random amino acids in table 1. M represents an A or C base, and MNN is complementarily paired with NNK.

TABLE 1 primer nucleotide sequences

(1) PCR amplification (95 ℃ C. 5min;95 ℃ C. 30s,65 ℃ C. 30s,72 ℃ C. 7.5min,34 cycles; 72 ℃ C. 10 min) was performed using recombinant plasmid pET28a-TK as a template, the nucleotide sequences of which are shown in SEQ ID No.3, and D469T-F and D469T-R as primers, using Phanta Max polymerase (purchased from Norwezan); the PCR product is directly transformed into E.coli BL21 (DE 3) competent cells after being digested by FD-Dpn I (30 ℃ incubator for 6 hours), the resuscitated solution is fully sucked and mixed evenly, and then about 1/8 resuscitated solution is coated on a kanamycin-resistant LB plate and cultured for 12-16 hours at 37 ℃.

(2) 3 single colonies on the dilution plate are selected and cultured in a kanamycin-resistant LB culture medium for 7-8 hours at 37 ℃, one part of culture solution is used for sequencing, and the other part of culture solution is temporarily stored in a refrigerator at 4 ℃ for a short period of time. The sequencing positive transformant culture medium was transferred to fresh 5mL LB medium containing kanamycin resistance at an inoculum size of 1%. Culturing overnight, extracting plasmid, amplifying whole plasmid with the plasmid (pET 28 a-TK/D469T) as template and R520Q-F and R520Q-R as primers, and repeating the above plasmid construction method. Then extracting pET28a-TK/D469T/R520Q plasmid, using the plasmid as template, using S385Y-F and S385Y-R as primer to amplify whole plasmid, repeating the above-mentioned method to construct pET28a-TK/D469T/R520Q/S385Y (pET 28a-EcTK 1) plasmid. The recombinant library is obtained by amplifying the whole plasmid by taking the pET28a-EcTK1 plasmid as a template and taking H26-F and H26-R as primers and repeating the method.

(3) All single colonies (about 40) on the above dilution plates were picked and cultured in kanamycin-resistant LB medium at 37℃for 7-8 hours, one portion of the culture broth was used for sequencing, and the other portion of the culture broth was temporarily stored in a refrigerator at 4℃for a short period of time. The transformant culture solution positive to sequencing is transferred to a 24-hole deep-hole plate filled with fresh 5mL LB culture medium containing kanamycin resistance in an inoculum size of 1%, and is cultured for 3 hours at 37 ℃, then 0.4mM IPTG inducer is added for 30 ℃ for 5 hours, high-efficiency expression of recombinant genes is induced, and recombinant cells are collected by centrifugation at 4000rpm and preserved at-80 ℃. pET28a-EcTK1 recombinant cells were obtained by the same induction method as a control.

(4) The recombinant cells obtained were suspended in 300ul of 50mM Tris-Cl (pH=7.5), sonicated, and centrifuged at low temperature to obtain the supernatant. To the reaction solution containing 5mM p-methylsulfonyl benzaldehyde and 30uM of R-aminotransferase pure protein, 20% v/v of the supernatant was added, and the mixture was reacted at 30℃for 6 hours. Since the whole bacterium contains ketol isomerase, the racemization of dihydroxyketone is accelerated, in order to accurately examine the stereoselectivity of the ketosis product dihydroxyketone, a stereospecific R-aminotransferase is coupled, dihydroxyketone is instantaneously converted into a stable chiral amino diol compound, and the chirality of dihydroxyketone is examined by detecting the chirality of amino diol.

(5) The reaction product of (4) was detected by liquid chromatography, and the ratio of (1S, 2R) -amino diol to (1R, 2R) -amino diol was examined. The liquid phase analysis conditions were: column super-C18 (250 mm. Times.4.6 mm, particle size 5 μm), phase A: h ₂ O (containing 10mM KH) ₂ PO ₄ Ph=8.5), phase B: acetonitrile, chromatographic conditions: the retention times of (1S, 2R) -amino diol and (1R, 2R) -amino diol were 9.7 and 10.9min, respectively, with 0min 98:2 to 20min 90:10 linear change, 1ml/min,224 nm. The result shows that the EcTK1_Y has the highest R stereoselectivity in the saturated mutant and higher activity, so that the EcTK1_Y is selected as a template for the next round of saturated mutation, F434-F/R is used as a primer for carrying out whole plasmid PCR, and the plasmid construction, expression and detection methods are repeated. The R stereoselectivity of EcTK1_YY was highest in the second round of saturation mutant, and therefore it was regarded as the third round of saturation mutationThe steps are repeated by using L466-F/R as a primer of the template. Finally, the EcTK1_YYH mutant is obtained.

Example 2: expression purification of transketolase mutants

Expression purification method of mutant EcTK1_YYH: e.coli BL21 (DE 3) single colonies containing the recombinant plasmid were picked up on LB solid medium and inoculated into 40ml LB liquid medium (containing 50. Mu.g/ml kanamycin antibiotic) and cultured overnight at 37℃at 220 rpm. 7.5ml of the bacterial culture was transferred to a 2L shaking flask containing 500ml of liquid LB medium, two flasks were inoculated, incubated at 37℃and 220rpm until OD600 reached 0.6-0.8, induced by the addition of 0.4mM IPTG, and induced at 30℃and 200rpm for 5h. 1L of fermentation broth was collected, cells were collected by centrifugation at 5000rpm for 20min, and the collected cells were resuspended in 30mL of nickel column binding buffer and placed in an ice-water mixture. Ultrasonic crushing conditions: the operation is carried out for 5s, the pause is carried out for 10s, and the total time is 30min. The crushed mixture was centrifuged at 12,000rpm for 1 hour, and the supernatant was filtered through a 0.22 μm filter. The filtered sample was loaded with 2ml of nickel packing pre-equilibrated with nickel column binding buffer, the heteroproteins were washed with 10 column volumes of 50mM imidazole elution buffer, then the target proteins were eluted with 5ml 250mM imidazole elution buffer, and the samples were pipetted 500ul each. And (3) measuring the protein concentration of each tube by a micro ultraviolet-visible light spectrophotometer (NanoDrop), combining a plurality of tubes of protein liquid with higher concentration, diluting or concentrating to 2.5ml, loading the protein liquid into a desalting column balanced by glycerol buffer solution, drying the protein liquid, and adding 3.5ml of glycerol buffer solution to elute the protein.

Example 3: investigation of stereoselectivity of the EcTK1 mutant to catalyze p-methylsulfonyl benzaldehyde to obtain a product

The high stereoselectivity R transketolase mutant EcTK1 catalyzes a detection process of obtaining a high stereoselectivity product of the p-methylsulfonyl benzaldehyde, and the related chemical reactions comprise a transketolase reaction and an ammonia-converting reaction.

As shown in FIG. 1, the principle of the transketolase reaction is to take p-methylsulfonyl benzaldehyde as a substrate, wherein the substrate is expressed as '1', the transketolase mutant is taken as a catalyst, the catalyst is expressed as 'TK', and LiHPA and MgCl ₂ And in the presence of ThDP, S-dihydroxyketone and R-dihydroxyketone are formedThe product, S-dihydroxyketone, was prepared as described in "2a: s "represents the R-dihydroxyketone product as" 2b: R ".

As shown in fig. 2, the principle of the transamination reaction is to transiently convert the dihydroxyketone, which is a product of the transketotion reaction, into a stable chiral amino diol compound, and examine the stereoselectivity of dihydroxyketone by detecting the stereoselectivity of amino diol. The transketolase reaction products S-dihydroxyketone and R-dihydroxyketone are converted into (1S, 2R) -amino alcohol and (1R, 2R) -amino alcohol under the action of D-Ala, PLP, NADH, LDH and the aminotransferase ATA117_AC, respectively, wherein (1S, 2R) -amino alcohol is represented by the formula "3a: (1S, 2R) "means (1R, 2R) -amino alcohol as" 3b: (1R, 2R) "means. The liquid phase detects the concentration of (1 s,2 r) -amino alcohol and (1 r,2 r) -amino alcohol to characterize the concentration of the transketolase products 2a and 2b, respectively.

(1) The reaction system was 100. Mu.l (50 mM Tris-Cl buffer, pH 7), 5mM p-methylsulfonyl benzaldehyde, 25mM LiHPA,9mM MgCl ₂ 4.8mM ThDP, 100. Mu.M transketolase mutant pure protein, reaction at 25℃for 20min, chelating Mg with 18mM EDTA ²⁺ The transketotion reaction is terminated. Mu.l of the transketolase reaction solution was pipetted into a new 100. Mu.l system, 200mM D-Ala,2mM PLP,20mM NADH,90U/ml LDH and 200. Mu.M aminotransferase ATA117_AC were added and reacted at 25℃for 30min. The S-and R-hydroxyketone products are thus completely converted into the (1S, 2R) -and (1R, 2R) -aminoalcohol diastereomers. The concentration of the hydroxyketone enantiomer was investigated by detecting the concentration of the amino alcohol diastereomer.

(2) Analysis of the amino alcohol product used Agilent liquid phase, column super-C18 column (4.6X1250 mm, particle size 5 μm), phase A: h ₂ O (containing 10mM KH) ₂ PO ₄ Ph=8.5), phase B: the retention times of acetonitrile, 1ml/min,224nm, (1S, 2R) -and (1R, 2R) -amino alcohol were 9.7 and 10.9min, respectively. The chromatographic conditions are shown in table 2:

TABLE 2 chromatographic conditions

(3) The key mutants of the saturated mutation in three rounds of iteration by taking the escherichia coli EcTK1 as a female parent respectively catalyze 5mM of p-methylsulfonyl benzaldehyde substrate at 25 ℃ for 20min, and the conversion rate C (%) and the stereoselectivity ee (%) of the produced R-hydroxyketone are shown in Table 3.

Table 3 shows the conversion C (%) and stereoselectivity ee (%)

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Sequence listing

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gccaacgcag tcggtatggc gattgcagaa aaaacgctgg cggcgcagtt taaccgtccg 420

ggccacgaca ttgtcgacca ctacacctac gccttcatgg gcgacggctg catgatggaa 480

ggcatctccc acgaagtttg ctctctggcg ggtacgctga agttgggtaa actgattgca 540

ttctacgatg acaacggtat ttctatcgat ggtcacgttg aaggctggtt caccgacgac 600

accgcaatgc gtttcgaagc ttacggctgg cacgttattc gcgacatcga cggtcatgac 660

gcggcatcta tcaaacgcgc agtagaagaa gcgcgcgcag tgactgacaa accttccctg 720

ctgatgtgca aaaccatcat cggtttcggt tccccgaaca aagccggtac ccacgactcc 780

cacggtgcgc cgctgggcga cgctgaaatt gccctgaccc gcgaacaact gggctggaaa 840

tatgcgccgt tcgaaatccc gtctgaaatc tatgctcagt gggatgcgaa agaagcaggc 900

caggcgaaag aatccgcatg gaacgagaaa ttcgctgctt acgcgaaagc ttatccgcag 960

gaagccgctg aatttacccg ccgtatgaaa ggcgaaatgc cgtctgactt cgacgctaaa 1020

gcgaaagagt tcatcgctaa actgcaggct aatccggcga aaatcgccag ccgtaaagcg 1080

tctcagaatg ctatcgaagc gttcggtccg ctgttgccgg aattcctcgg cggttctgct 1140

gacctggcgc cgtataacct gaccctgtgg tctggttcta aagcaatcaa cgaagatgct 1200

gcgggtaact acatccacta cggtgttcgc gagttcggta tgaccgcgat tgctaacggt 1260

atctccctgc acggtggctt cctgccgtac acctccacct atctgatgtt cgtggaatac 1320

gcacgtaacg ccgtacgtat ggctgcgctg atgaaacagc gtcaggtgat ggtttacacc 1380

cacgactcca tcggtcatgg cgaaaccggc ccgactcacc agccggttga gcaggtcgct 1440

tctctgcgcg taaccccgaa catgtctaca tggcgtccgt gtgaccaggt tgaatccgcg 1500

gtcgcgtgga aatacggtgt tgagcgtcag gacggcccga ccgcactgat cctctcccaa 1560

cagaacctgg cgcagcagga acgaactgaa gagcaactgg caaacatcgc gcgcggtggt 1620

tatgtgctga aagactgcgc cggtcagccg gaactgattt tcatcgctac cggttcagaa 1680

gttgaactgg ctgttgctgc ctacgaaaaa ctgactgccg aaggcgtgaa agcgcgcgtg 1740

gtgtccatgc cgtctaccga cgcatttgac aagcaggatg ctgcttaccg tgaatccgta 1800

ctgccgaaag cggttactgc acgcgttgct gtagaagcgg gtattgctga ctactggtac 1860

aagtatgttg gcctgaacgg tgctatcgtc ggtatgacca ccttcggtga atctgctccg 1920

gcagagctgc tgtttgaaga gttcggcttc actgttgata acgttgttgc gaaagcaaaa 1980

gaactgctgt aa 1992

<210> 3

<211> 7288

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100

agcagcggcc tggtgccgcg cggcagccat atgatgtcct cacgtaaaga gcttgccaat 5160

gctattcgtg cgctgagcat ggacgcagta cagaaagcca aatccggtca cccgggtgcc 5220

cctatgggta tggctgacat tgccgaagtc ctgtggcgtg atttcctgaa acacaacccg 5280

cagaatccgt cctgggctga ccgtgaccgc ttcgtgctgt ccaacggcca cggctccatg 5340

ctgatctaca gcctgctgca cctcaccggt tacgatctgc cgatggaaga actgaaaaac 5400

ttccgtcagc tgcactctaa aactccgggt cacccggaag tgggttacac cgctggtgtg 5460

gaaaccacca ccggtccgct gggtcagggt attgccaacg cagtcggtat ggcgattgca 5520

gaaaaaacgc tggcggcgca gtttaaccgt ccgggccacg acattgtcga ccactacacc 5580

tacgccttca tgggcgacgg ctgcatgatg gaaggcatct cccacgaagt ttgctctctg 5640

gcgggtacgc tgaagctggg taaactgatt gcattctacg atgacaacgg tatttctatc 5700

gatggtcacg ttgaaggctg gttcaccgac gacaccgcaa tgcgtttcga agcttacggc 5760

tggcacgtta ttcgcgacat cgacggtcat gacgcggcat ctatcaaacg cgcagtagaa 5820

gaagcgcgcg cagtgactga caaaccttcc ctgctgatgt gcaaaaccat catcggtttc 5880

ggttccccga acaaagccgg tacccacgac tcccacggtg cgccgctggg cgacgctgaa 5940

attgccctga cccgcgaaca actgggctgg aaatatgcgc cgttcgaaat cccgtctgaa 6000

atctatgctc agtgggatgc gaaagaagca ggccaggcga aagaatccgc atggaacgag 6060

aaattcgctg cttacgcgaa agcttatccg caggaagccg ctgaatttac ccgccgtatg 6120

aaaggcgaaa tgccgtctga cttcgacgct aaagcgaaag agttcatcgc taaactgcag 6180

gctaatccgg cgaaaatcgc cagccgtaaa gcgtctcaga atgctatcga agcgttcggt 6240

ccgctgttgc cggaattcct cggcggttct gctgacctgg cgccgtctaa cctgaccctg 6300

tggtctggtt ctaaagcaat caacgaagat gctgcgggta actacatcca ctacggtgtt 6360

cgcgagttcg gtatgaccgc gattgctaac ggtatctccc tgcacggtgg cttcctgccg 6420

tacacctcca ccttcctgat gttcgtggaa tacgcacgta acgccgtacg tatggctgcg 6480

ctgatgaaac agcgtcaggt gatggtttac acccacgact ccatcggtct gggcgaagac 6540

ggcccgactc accagccggt tgagcaggtc gcttctctgc gcgtaacccc gaacatgtct 6600

acatggcgtc cgtgtgacca ggttgaatcc gcggtcgcgt ggaaatacgg tgttgagcgt 6660

caggacggcc cgaccgcact gatcctctcc cgtcagaacc tggcgcagca ggaacgaact 6720

gaagagcaac tggcaaacat cgcgcgcggt ggttatgtgc tgaaagactg cgccggtcag 6780

ccggaactga ttttcatcgc taccggttca gaagttgaac tggctgttgc tgcctacgaa 6840

aaactgactg ccgaaggcgt gaaagcgcgc gtggtgtcca tgccgtctac cgacgcattt 6900

gacaagcagg atgctgctta ccgtgaatcc gtactgccga aagcggttac tgcacgcgtt 6960

gctgtagaag cgggtattgc tgactactgg tacaagtatg ttggcctgaa cggtgctatc 7020

gtcggtatga ccaccttcgg tgaatctgct ccggcagagc tgctgtttga agagttcggc 7080

ttcactgttg ataacgttgt tgcgaaagca aaagaactgc tgtaactcga gcaccaccac 7140

caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc 7200

accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 7260

ttgctgaaag gaggaactat atccggat 7288

<210> 4

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tccatcggtc tgggcgaaac cggcccgact caccagccgg ttga 44

<210> 5

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ggctggtgag tcgggccggt ttcgcccaga ccgatggagt cgtg 44

<210> 6

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

accgcactga tcctctccca acagaacctg gcgcagcagg aacgaa 46

<210> 7

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ctgctgcgcc aggttctgtt gggagaggat cagtgcggtc gggccg 46

<210> 8

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tctgctgacc tggcgccgta taacctgacc ctgtggtctg gttcta 46

<210> 9

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

agaccacagg gtcaggttat acggcgccag gtcagcagaa ccgccg 46

<210> 10

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (15)..(16)

<223> n is a, c, g, or t

<400> 10

aagccaaatc cggtnnkccg ggtgccccta tgggtat 37

<210> 11

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (16)..(17)

<223> n is a, c, g, or t

<400> 11

ataggggcac ccggmnnacc ggatttggct ttctgta 37

<210> 12

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (15)..(16)

<223> n is a, c, g, or t

<400> 12

cgtacacctc caccnnkctg atgttcgtgg aatacgc 37

<210> 13

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (16)..(17)

<223> n is a, c, g, or t

<400> 13

tccacgaaca tcagmnnggt ggaggtgtac ggcagga 37

<210> 14

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (15)..(16)

<223> n is a, c, g, or t

<400> 14

acgactccat cggtnnkggc gaaaccggcc cgactca 37

<210> 15

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (16)..(17)

<223> n is a, c, g, or t

<400> 15

gggccggttt cgccmnnacc gatggagtcg tgggtgt 37

Claims (9)

1. A high-stereoselectivity R transketolase mutant is characterized in that the amino acid sequence is shown as SEQ ID No. 1.

2. The high stereoselective R transketolase mutant according to claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

3. A recombinant expression vector comprising a gene encoding the high stereoselective R transketolase mutant of claim 2.

4. A host cell comprising a recombinant expression vector for a gene encoding the high stereoselective R transketolase mutant of claim 3.

5. A method for preparing the high stereoselective R transketolase mutant according to claim 1, comprising the steps of:

step 1, taking a gene derived from prokaryotic escherichia coli ketolase as a template, and obtaining a high stereoselectivity R ketolase mutant DNA molecule through PCR site-directed mutagenesis;

step 2, constructing a recombinant expression vector containing the high stereoselectivity R transketolase mutant DNA molecule obtained in the step 1;

step 3, generating a large amount of host cells containing the recombinant expression vector obtained in the step 2 in an IPTG induction mode to obtain a high stereoselectivity R transketolase mutant with bioactivity;

step 4, separating and purifying the high-stereoselectivity R-transketolase mutant protein with biological activity obtained in the step 3 by an affinity chromatography method to obtain the high-stereoselectivity R-transketolase mutant protein with high activity; the transketolase mutant is generated by the amino acid sequence of the prokaryote escherichia coli transketolase through the site-directed mutagenesis; serine at 385 th site, aspartic acid at 469 th site, arginine at 520 th site, histidine at 26 th site, phenylalanine at 434 th site, leucine at 466 rd site and histidine of the amino acid sequence of the prokaryotic escherichia coli transketolase are mutated into tyrosine, and the serine at 385 th site, the aspartic acid at 469 th site, the arginine at 520 th site, the histidine at 26 th site, the phenylalanine at 434 th site and the leucine at 466 rd site are mutated into glutamine.

6. Use of a high stereoselective R transketolase mutant according to claim 1 for catalyzing the formation of a high stereoselective R-aromatic dihydroxyketone from an aromatic aldehyde.

7. Use of a gene encoding a high stereoselective R transketolase mutant according to claim 2 for catalyzing the formation of a high stereoselective R-aromatic dihydroxyketone from an aromatic aldehyde.

8. The use according to claim 6, wherein p-methylsulfonyl benzaldehyde is used as a substrate, and the high stereoselectivity R transketolase mutant is used as a catalytic enzyme, and the p-methylsulfonyl benzaldehyde is synthesized into R-p-methylsulfonyl phenyl dihydroxyketone.

9. The use according to claim 7, further comprising constructing a recombinant expression vector comprising a gene encoding said high stereoselective R transketolase mutant; the host cell containing the recombinant expression vector is used for generating a large amount of R transketolase mutants with high stereoselectivity in an IPTG induction mode; separating and purifying the high-stereoselectivity R-transketolase mutant protein by an affinity chromatography method to obtain high-activity high-stereoselectivity R-transketolase mutant protein; the p-methylsulfonyl benzaldehyde is used as a substrate, and the high-activity high-stereoselectivity R transketolase mutant protein is used as a catalytic enzyme, so that R-p-methylsulfonyl phenyl dihydroxyketone is synthesized by the p-methylsulfonyl benzaldehyde.