Transketolase, transaminase and method for preparing (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane by using transketolase and transaminase

Abstract

The invention discloses transketolase, transaminase and a method for preparing (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane.The amino acid sequence of the transketolase is shown as SEQ ID NO: 6, the amino acid sequence of the transaminase is shown as SEQ ID NO: 10, the conversion rate of the transketolase used for preparing the (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane is 99.9%, and the conversion rate of the transaminase used for preparing the (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane is 99.9%. The method has the advantages that the de value is 97.0%, the ee value is 99.5%, the reaction cost is low, the product has high optical purity, the mode of sequentially adding enzymes along with the reaction process in a one-pot method system is adopted, the reaction system does not need to be prepared repeatedly, the operation steps are simplified, and therefore the method is beneficial to reduction of by-products and suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological catalysis, and particularly relates to a transketolase, a transaminase and a method for preparing (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane by using the transketolase and the transaminase. BACKGROUND

[0002] Florfenicol is also known as flumethoxynil, chlorosulonil and the like, and is a new generation of animal-specific broad-spectrum antibacterial drug. Its structure is similar to that of thiamphenicol, but its antibacterial capacity can reach 10 times that of thiamphenicol. Moreover, it has a broad antibacterial spectrum, strong bactericidal effect, high safety and efficiency, and does not cause aplastic anemia, teratogenicity, carcinogenicity and mutagenicity, and is therefore widely used. The chiral amino alcohol intermediate (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane ((1R, 2R)-AMPP) is a key intermediate in the synthesis of florfenicol, and its chemical structural formula is shown as formula 1.

[0003]

[0004] Since the chemical synthesis process is often complicated and the conditions are complex, and serious environmental pollution may be caused, therefore, it is of broad application prospect to establish an efficient and mild enzymatic synthesis route of (1R, 2R)-AMPP.

[0005] CN114875084B provides a method for synthesizing (1R, 2R)-AMPP by using enzyme cascade reaction. With p-methylsulfonyl benzaldehyde and lithium hydroxypropionylate as substrates, ketone transfer reaction and amino transfer reaction occur successively under the action of transketolase EcTK1_YYH and transaminase ATA117_ACHH, and finally (1R, 2R)-AMPP is synthesized, and the ee value is greater than 99%. However, lithium hydroxypropionylate is expensive, and the reaction cost is high, which is not conducive to industrial production.

[0006] CN117646044A discloses a method for efficiently synthesizing (1R, 2R)-AMPP by one-pot method of multi-enzyme cascade reaction. With D-serine, p-methylsulfonyl benzaldehyde and D-alanine as substrates, wet mycelium catalyzing reaction by respectively expressing or co-expressing amino acid oxidase RtDAAO, pyruvate decarboxylase SwPDC and transaminase CeTA is used to synthesize the end product (1R, 2R)-AMPP, the conversion rate is 99.9%, and the ee value reaches more than 99%. However, the price of D-serine is still high, which is not conducive to reducing the reaction cost. SUMMARY

[0007] The technical problem to be solved by the present application is that the existing enzymatic synthesis method of the chiral aminohydrol intermediate (1R, 2R)-AMPP of florfenicol has high cost and is not conducive to industrial production.

[0008] The present application solves the above technical problem by the following technical scheme:

[0009] The present application provides a transketolase, wherein the amino acid sequence of the transketolase is shown as SEQ ID NO: 6.

[0010] The present application provides a transaminase, wherein the amino acid sequence of the transaminase is shown as SEQ ID NO: 10.

[0011] The present application provides a multi-enzyme system, wherein the multi-enzyme system comprises at least two enzymes, and comprises the transketolase according to the first aspect of the present application and / or the transaminase according to the second aspect of the present application.

[0012] In some preferred embodiments of the present application, the multi-enzyme system further comprises one or more of D-threonine aldolase, catalase and D-amino acid oxidase.

[0013] In some more preferred embodiments of the present application, the multi-enzyme system is selected from any one of the following groups:

[0014] (1) the D-amino acid oxidase and the transketolase;

[0015] (2) the D-amino acid oxidase, the transketolase and the catalase;

[0016] (3) the D-threonine aldolase, the D-amino acid oxidase and the transketolase;

[0017] (4) the D-threonine aldolase, the D-amino acid oxidase, the transketolase and the catalase;

[0018] (5) the transketolase and the transaminase;

[0019] (6) the D-amino acid oxidase, the transketolase and the transaminase;

[0020] (7) the D-amino acid oxidase, the transketolase, the transaminase and the catalase;

[0021] (8) the D-threonine aldolase, the D-amino acid oxidase, the transketolase and the transaminase;

[0022] (9) the D-threonine aldolase, the D-amino acid oxidase, the transketolase, the transaminase, and the catalase.

[0023] In some further more preferred embodiments of the present application, the amino acid sequence of the D-threonine aldolase is set forth in SEQ ID NO: 1; and the amino acid sequence of the D-amino acid oxidase is set forth in SEQ ID NO: 2.

[0024] The fourth aspect of the present application provides an isolated nucleic acid or a nucleic acid composition, the nucleic acid encoding the transketolase of the first aspect of the present application or the transaminase of the second aspect of the present application; the nucleic acid composition consisting of nucleic acids encoding the enzymes in the multi-enzyme system of the third aspect of the present application.

[0025] The fifth aspect of the present application provides a recombinant expression vector comprising the nucleic acid or the nucleic acid composition of the fourth aspect of the present application.

[0026] In some embodiments of the present application, the recombinant expression vector comprises a pET28a plasmid backbone.

[0027] The sixth aspect of the present application provides a transformant comprising: a host cell; and, the isolated nucleic acid or the nucleic acid composition of the fourth aspect of the present application, or the recombinant expression vector of the fifth aspect of the present application, introduced into the host cell.

[0028] In some preferred embodiments of the present application, the host cell comprises eukaryotic cells and prokaryotic cells.

[0029] In some more preferred embodiments of the present application, the host cell is a bacterium.

[0030] In some further more preferred embodiments of the present application, the bacterium is Escherichia coli, for example, Escherichia coli BL21 (DE3).

[0031] The seventh aspect of the present application provides a reaction system comprising the transketolase of the first aspect of the present application and / or the transaminase of the second aspect of the present application.

[0032] In some preferred embodiments of the present application, the reaction system further comprises any one or more of the following: glycine, formaldehyde, a divalent metal ion compound, a D-threonine aldolase, pyridoxal 5'-phosphate, a D-amino acid oxidase, p-tosylbenzaldehyde, thiamine pyrophosphate, an amino donor, and a catalase.

[0033] In some more preferred embodiments of the present application, the reaction system is selected from any one of the following groups:

[0034] (1) the reaction system comprises hydroxy pyruvic acid, p-methylsulfonyl benzaldehyde, thiamine pyrophosphate and the transketolase; preferably, the reaction system further comprises a divalent metal ion compound;

[0035] (2) the reaction system comprises R-p-methylsulfonyl phenyl diketone, an amino donor, 5'-phosphopyridoxal and the transaminase;

[0036] (3) the reaction system comprises glycine, formaldehyde, D-threonine aldolase, D-amino acid oxidase, p-methylsulfonyl benzaldehyde, thiamine pyrophosphate, 5'-phosphopyridoxal and the transketolase; preferably, the reaction system further comprises a divalent metal ion compound and / or catalase;

[0037] (4) the reaction system comprises D-serine, D-amino acid oxidase, p-methylsulfonyl benzaldehyde, thiamine pyrophosphate and the transketolase; preferably, the reaction system further comprises a divalent metal ion compound and / or catalase;

[0038] (5) the reaction system comprises glycine, formaldehyde, D-threonine aldolase, 5'-phosphopyridoxal, D-amino acid oxidase, p-methylsulfonyl benzaldehyde, thiamine pyrophosphate, an amino donor, the transketolase and the transaminase; preferably, the reaction system further comprises a divalent metal ion compound and / or catalase;

[0039] (6) the reaction system comprises hydroxy pyruvic acid, p-methylsulfonyl benzaldehyde, thiamine pyrophosphate, an amino donor, 5'-phosphopyridoxal, the transketolase and the transaminase; preferably, the reaction system further comprises a divalent metal ion compound;

[0040] (7) the reaction system comprises D-serine, D-amino acid oxidase, p-methylsulfonyl benzaldehyde, thiamine pyrophosphate, an amino donor, 5'-phosphopyridoxal, the transketolase and the transaminase; preferably, the reaction system further comprises a divalent metal ion compound and / or catalase.

[0041] In some further more preferred embodiments of the present application, the amino donor is D-alanine, isopropylamine or phenylethylamine; the divalent metal ion compound is magnesium chloride or magnesium sulfate; the amino acid sequence of the D-threonine aldolase is shown in SEQ ID NO: 1; the amino acid sequence of the D-amino acid oxidase is shown in SEQ ID NO: 2.

[0042] In some embodiments of the present application, in the (1), the molar concentration of hydroxy pyruvic acid is 50-500 mM, for example 206 mM; the molar ratio of p- methylsulfonylbenzaldehyde to hydroxy pyruvic acid is (0.05-1): 1, for example 0.05: 1; the molar ratio of thiamine pyrophosphate to hydroxy pyruvic acid is (0.01-0.05): 1, for example 0.024: 1; the mass ratio of the transketolase to hydroxy pyruvic acid is (0.05-1): 1, for example 0.12: 1; the molar ratio of the divalent metal ion compound to hydroxy pyruvic acid is (0.01-0.5): 1, for example 0.024: 1;

[0043] In the (2), the molar concentration of R-p-methylsulfonylphenyldihydroxy ketone is 5-100 mM, for example 9.7 mM; the molar ratio of the amino donor to R-p- methylsulfonylphenyldihydroxy ketone is (1-20): 1, for example 10.3: 1; the molar ratio of 5'-phosphopyridoxal to R-p-methylsulfonylphenyldihydroxy ketone is (0.1-0.5): 1, for example 0.26: 1; the mass ratio of the transaminase to R-p-methylsulfonylphenyldihydroxy ketone is (0.1-2): 1, for example 1: 1;

[0044] In the (3), the molar concentration of glycine is 50-500 mM, for example 200 mM; the molar ratio of formaldehyde to glycine is (0.5-5): 1, for example 1: 1; the mass ratio of the D-threonine aldolase to glycine is (0.1-2): 1, for example 1.3: 1; the molar ratio of 5'-phosphopyridoxal to glycine is (0.002-0.02): 1, for example 0.004: 1; the mass ratio of the D-amino acid oxidase to glycine is (0.1-2): 1, for example 1.3: 1; the molar ratio of p-methylsulfonylbenzaldehyde to glycine is (0.05-1): 1, for example 0.1: 1; the molar ratio of thiamine pyrophosphate to glycine is (0.01-0.05): 1, for example 0.025: 1; the mass ratio of the transketolase to glycine is (0.05-1): 1, for example 0.7: 1; the molar ratio of the divalent metal ion compound to glycine is (0.01-0.5): 1, for example 0.16: 1; the enzyme activity unit of the catalase is 50-500 U / mL, for example 150 U / mL;

[0045] In the (4), the molar concentration of D-serine is 50 to 500 mM, for example, 197 mM; the mass ratio of the D-amino acid oxidase to D-serine is (0.05 to 2): 1, for example, 1:1; the molar ratio of p-methylthioformaldehyde to D-serine is (0.05 to 1): 1, for example, 0.1:1; the molar ratio of thiamine pyrophosphate to D-serine is (0.01 to 0.05): 1, for example, 0.025:1; the mass ratio of the transketolase to D-serine is (0.05 to 1): 1, for example, 0.5:1; the molar ratio of the divalent metal ion compound to D-serine is (0.01 to 0.5): 1, for example, 0.16:1; and the enzyme activity unit of the catalase is 50 to 500 U / mL, for example, 150 U / mL;

[0046] In the (5), the molar concentration of glycine is 50 to 500 mM, for example, 200 mM; the molar ratio of formaldehyde to glycine is (0.5 to 5): 1, for example, 1:1; the mass ratio of the D-threonine aldolase to glycine is (0.1 to 2): 1, for example, 1.3:1; the molar ratio of 5'-phosphopyridoxal to glycine is (0.002 to 0.02): 1, for example, 0.017:1; the mass ratio of the D-amino acid oxidase to glycine is (0.1 to 2): 1, for example, 1.3:1; the molar ratio of p-methylthioformaldehyde to glycine is (0.05 to 1): 1, for example, 0.1:1; the molar ratio of thiamine pyrophosphate to glycine is (0.01 to 0.05): 1, for example, 0.025:1; the molar ratio of the amino donor to glycine is (0.1 to 1): 1, for example, 0.5:1; the mass ratio of the transketolase to glycine is (0.05 to 1): 1, for example, 0.7:1; the mass ratio of the transaminase to glycine is (0.1 to 2): 1, for example, 1.3:1; the molar ratio of the divalent metal ion compound to glycine is (0.01 to 0.5): 1, for example, 0.16:1; and the enzyme activity unit of the catalase is 50 to 500 U / mL, for example, 150 U / mL;

[0047] In the (6), the molar concentration of hydroxy pyruvic acid is 50-500 mM, for example, 193 mM; the molar ratio of p-methylsulfonyl benzaldehyde to hydroxy pyruvic acid is (0.05-1): 1, for example, 0.1:1; the molar ratio of thiamine pyrophosphate to hydroxy pyruvic acid is (0.01-0.05): 1, for example, 0.026:1; the molar ratio of the amino donor to hydroxy pyruvic acid is (0.1-1): 1, for example, 0.5:1; the molar ratio of 5'-phosphopyridoxal to hydroxy pyruvic acid is (0.002-0.02): 1, for example, 0.013:1; the mass ratio of the transketolase to hydroxy pyruvic acid is (0.05-1): 1, for example, 0.5:1; the mass ratio of the transaminase to hydroxy pyruvic acid is (0.1-2): 1, for example, 1:1; the molar ratio of the divalent metal ion compound to hydroxy pyruvic acid is (0.01-0.5): 1, for example, 0.16:1;

[0048] In the (7), the molar concentration of D-serine is 50-500 mM, for example, 197 mM; the molar ratio of 5'-phosphopyridoxal to D-serine is (0.002-0.02): 1, for example, 0.013:1; the mass ratio of the D-amino acid oxidase to D-serine is (0.05-2): 1, for example, 1:1; the molar ratio of p-methylsulfonyl benzaldehyde to D-serine is (0.05-1): 1, for example, 0.1:1; the molar ratio of the amino donor to D-serine is (0.1-1): 1, for example, 0.5:1; the molar ratio of thiamine pyrophosphate to D-serine is (0.01-0.05): 1, for example, 0.025:1; the mass ratio of the transketolase to D-serine is (0.05-1): 1, for example, 0.5:1; the mass ratio of the transaminase to D-serine is (0.1-2): 1, for example, 1:1; the molar ratio of the divalent metal ion compound to D-serine is (0.01-0.5): 1, for example, 0.16:1; the enzyme activity unit of the catalase is 50-500 U / mL, for example, 150 U / mL.

[0049] In the present application, the transaminase, the transketolase, the D-amino acid oxidase, the catalase and / or the D-threonine aldolase are used in the form of liquid enzyme, solid enzyme, immobilized enzyme or cells expressing the enzyme; the liquid enzyme is preferably crude enzyme solution or purified enzyme solution; the crude enzyme solution is obtained by resuspending and homogenizing the wet cells with buffer.

[0050] In the present application, when the enzyme is used in the form of purified enzyme solution or solid purified enzyme, the added mass of the enzyme is calculated based on the mass of the enzyme protein; when other forms of enzyme are used, the added mass of the enzyme is calculated based on the mass of the wet cells expressing the enzyme.

[0051] The eighth aspect of the present application provides a method for preparing R-p-methylsulfonyl phenyl diketone, comprising step 1: reacting p-methylsulfonyl benzaldehyde and hydroxy pyruvic acid catalyzed by the trans-ketolase as described in the first aspect of the present application in the presence of thiamine pyrophosphate to obtain R-p-methylsulfonyl phenyl diketone.

[0052] In some preferred embodiments of the present application, the hydroxy pyruvic acid is obtained from step 2: reacting D-serine catalyzed by D-amino acid oxidase to obtain hydroxy pyruvic acid; preferably, the step 2 further comprises: converting the generated hydrogen peroxide into water and oxygen catalyzed by catalase.

[0053] In some more preferred embodiments of the present application, the D-serine is obtained from step 3: reacting formaldehyde and glycine catalyzed by D-threonine aldolase in the presence of 5'-phosphopyridoxyl to obtain D-serine.

[0054] In some embodiments of the present application, the reaction conditions of the step 1 comprise one or more of the following: temperature is 20-35℃, for example 25℃; pH is 7.0-8.0, for example 7.5; reaction time is 0.5-20h, for example 2h;

[0055] The reaction system of the step 1 is the (1) in the reaction system as described in the seventh aspect of the present application;

[0056] The reaction conditions of the step 2 comprise one or more of the following: temperature is 20-35℃, for example 25℃; pH is 7.0-8.0, for example 7.5; reaction time is 0.5-20h, for example 14h;

[0057] The reaction system of the step 2 comprises D-serine and the D-amino acid oxidase; preferably, the reaction system further comprises the catalase; wherein the molar concentration of D-serine is 0.1-1.5M, for example 1.05M; the added mass ratio of the D-amino acid oxidase to D-serine is (0.05-2):1, for example 0.09:1; the enzyme activity unit of the catalase is 50-500U / mL, for example 150U / mL;

[0058] The reaction conditions of the step 3 comprise one or more of the following: temperature is 25-40℃, for example 35℃; pH is 7.0-8.0, for example 7.3; reaction time is 1-30h, for example 24h;

[0059] The reaction system of step 3 comprises the D-threonine aldolase, glycine, formaldehyde and pyridoxal 5'-phosphate; preferably, the reaction system further comprises a divalent metal ion compound; more preferably, the divalent metal ion compound is magnesium chloride or magnesium sulfate; wherein the molar concentration of glycine is 0.1-3M, for example 1.33M; the molar ratio of the divalent metal ion compound to glycine is (0.01-0.5):1, for example 0.012:1; the molar ratio of formaldehyde to glycine is (0.5-5):1, for example 1.2:1; the mass ratio of the D-threonine aldolase to glycine is (0.1-2):1, for example 0.15:1; and the molar ratio of pyridoxal 5'-phosphate to glycine is (0.05-0.5):1000, for example 0.15:1000.

[0060] The ninth aspect of the present application provides a method for preparing R-p-tosylphenyldihydroxyl ketone, which comprises: (i) catalyzing the reaction of glycine, formaldehyde and p-tosylbenzaldehyde with the (3) or (4) in the multi-enzyme system as described in the third aspect of the present application in the presence of pyridoxal 5'-phosphate and thiamine pyrophosphate to obtain R-p-tosylphenyldihydroxyl ketone;

[0061] or, (ii) catalyzing the reaction of D-serine and p-tosylbenzaldehyde with the (1) or (2) in the multi-enzyme system as described in the third aspect of the present application in the presence of thiamine pyrophosphate to obtain R-p-tosylphenyldihydroxyl ketone.

[0062] In some preferred embodiments of the present application, the method is a one-pot method.

[0063] The reaction system of (i) is the (3) in the reaction system as described in the seventh aspect of the present application.

[0064] The reaction system of (ii) is the (4) in the reaction system as described in the seventh aspect of the present application.

[0065] The components of the reaction system are added in one step or in stages.

[0066] In some more preferred embodiments of the present application, the step of adding the components of the reaction system of (i) in stages is: (i') adding glycine, formaldehyde, the D-threonine aldolase and pyridoxal 5'-phosphate, and reacting for 0.5-3h, for example 2h; (ii') adding the D-amino acid oxidase, and reacting for 0.5-3h, for example 1h; (iii') adding p-tosylbenzaldehyde, thiamine pyrophosphate and the transketolase.

[0067] The reaction conditions of the method comprise one or more of the following: a temperature of 25-40°C, for example 30°C or 35°C; a pH of 7.0-8.0, for example 7.5; a total reaction time of 1-30h, for example 19h.

[0068] In some preferred embodiments of the present application, the divalent metal ion compound is further added in the (i’) and / or (iii’); and / or, the catalase is further added in the (ii’);

[0069] The tenth aspect of the present application provides a method for preparing (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane, which comprises: catalyzing the reaction of an amino donor and R-p-methylsulfonylphenyl diketone by the transaminase as described in the second aspect of the present application in the presence of pyridoxal 5’-phosphate to obtain (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane.

[0070] In some preferred embodiments of the present application, the reaction conditions of the method comprise one or more of the following: a temperature of 20-35°C, for example 25°C; a pH of 7.0-8.0, for example 7.5; a reaction time of 0.5-20h, for example 5h;

[0071] The reaction system of the method is the (2) in the reaction system as described in the seventh aspect of the present application; preferably, the amino donor is D-alanine, isopropylamine or phenylethylamine.

[0072] In some more preferred embodiments of the present application, the R-p-methylsulfonylphenyl diketone is prepared by the method as described in the eighth aspect or the ninth aspect of the present application.

[0073] The eleventh aspect of the present application provides a method for preparing (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane, which comprises: (i) catalyzing the reaction of glycine, formaldehyde, p-methylsulfonylbenzaldehyde and an amino donor by the (8) or (9) in the multi-enzyme system as described in the third aspect of the present application in the presence of pyridoxal 5’-phosphate and thiamine pyrophosphate to obtain (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane;

[0074] Or, (ii) catalyzing the reaction of hydroxypropanoic acid, p-methylsulfonylbenzaldehyde and an amino donor by the (5) in the multi-enzyme system as described in the third aspect of the present application in the presence of pyridoxal 5’-phosphate and thiamine pyrophosphate to obtain (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane;

[0075] Alternatively, (iii) in the presence of pyridoxal 5'-phosphate and thiamine pyrophosphate, D-serine, p-methylsulfonylbenzaldehyde and an amino donor are catalyzed by (6) or (7) in the multi-enzyme system as described in the third aspect of the invention to obtain (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane.

[0076] In some preferred embodiments of the present invention, the method is a one-pot method;

[0077] The reaction system of (i) is (5) as described in the reaction system of the seventh aspect of the present invention;

[0078] The reaction system described in (ii) is (6) of the reaction system described in the seventh aspect of the present invention;

[0079] The reaction system described in (iii) is (7) of the reaction system described in the seventh aspect of the present invention;

[0080] The components of the reaction system are added either in one step or in stages.

[0081] In some more preferred embodiments of the present invention, the step of adding each component of the reaction system in stages in step (i) is as follows: (i') adding glycine, formaldehyde, the D-threonine aldolase and pyridoxal 5'-phosphate, and reacting for 0.5-3 h, for example, 2 h; (ii') adding the D-amino acid oxidase, and reacting for 0.5-3 h, for example, 1 h; (iii') adding p-methylsulfonylbenzaldehyde, thiamine pyrophosphate, the amino donor, pyridoxal 5'-phosphate, the transketolase and the transaminase;

[0082] The reaction conditions of the method include one or more of the following: temperature of 25-40℃, for example 30℃ or 35℃; pH of 7.0-8.0, for example 7.5; and total reaction time of 1-30h, for example 19h.

[0083] In some preferred embodiments of the present invention, the divalent metal ion compound is further added to (i') and / or (iii'); and / or, the catalase is further added to (ii').

[0084] The twelfth aspect of the present invention provides a reaction solution comprising R-p-methylsulfonylphenyl dihydroxyone, said reaction solution being prepared by the method described in the eighth or ninth aspect of the present invention.

[0085] The thirteenth aspect of the present invention provides a reaction solution comprising (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane, said reaction solution being prepared by the method as described in the tenth or eleventh aspect of the present invention.

[0086] The fourteenth aspect of the present invention provides the use of the transketolase as described in the first aspect of the present invention, the multi-enzyme system as described in the third aspect of the present invention, the nucleic acid or nucleic acid composition as described in the fourth aspect of the present invention, the recombinant expression vector as described in the fifth aspect of the present invention, the transformant as described in the sixth aspect of the present invention, or the reaction system as described in the seventh aspect of the present invention in the preparation of R-p-methylsulfonylphenyldihydroxyone.

[0087] The fifteenth aspect of the present invention provides the use of the transketolase as described in the first aspect of the present invention, the transaminase as described in the second aspect of the present invention, the multi-enzyme system as described in the third aspect of the present invention, the nucleic acid or nucleic acid composition as described in the fourth aspect of the present invention, the recombinant expression vector as described in the fifth aspect of the present invention, the transformant as described in the sixth aspect of the present invention, the reaction system as described in the seventh aspect of the present invention, or the reaction solution as described in the twelfth aspect of the present invention in the preparation of (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane.

[0088] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0089] The reagents and raw materials used in this invention are all commercially available.

[0090] By adopting the above technical solution, the present invention achieves the following technical effects:

[0091] This invention uses inexpensive and readily available formaldehyde and glycine as substrates, and synthesizes the final product (1R,2R)-AMPP through a four-step enzyme cascade reaction catalyzed by D-threonine aldolase, D-amino acid oxidase, transketolase, and transaminase. The conversion rate is 99.9%, the de value is 97.0%, and the ee value is 99.5%. The reaction route used in this invention has low cost, the product has high optical purity, and the use of a one-pot system with enzymes added sequentially according to the reaction process eliminates the need for repeated preparation of the reaction system, simplifying the operation steps and thus reducing by-products, making it suitable for industrial production. Attached Figure Description

[0092] Figure 1A schematic diagram of the reaction route for preparing the final product (1R,2R)-1,3-dihydroxy-2-amino-1-p-methylsulfonylphenylpropane ((1R,2R)-AMPP, compound 6) using formaldehyde and glycine (compound 1) as substrates. In this diagram, DTA represents D-threonine aldolase; DAAO represents D-amino acid oxidase; PDC / TK represents pyruvate decarboxylase or transketolase; and ATA represents amine transaminase. Detailed Implementation

[0093] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0094] Unless otherwise specified, all materials and reagents used in the embodiments of the present invention are commonly used materials and reagents in the art and can be obtained through conventional commercial channels.

[0095] LB liquid medium: peptone 10 g / L, yeast extract 18 g / L, glycerol 0.4% (v / v), KH2PO4 (anhydrous) 2.31 g / L, K2HPO4 (trihydrate) 16.43 g / L. Here, g / L refers to the ratio of the mass of a component added (g) to the total volume (L) of TB liquid medium; % (v / v) refers to the ratio of the volume of a component added (L) to the total volume (L) of TB liquid medium, and so on.

[0096] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0097] LB agar plate: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 18 g / L, pH 7.2 (adjusted with 1 mol / L NaOH).

[0098] The reagent information used in the examples is shown in Table 1 below.

[0099] Table 1 Reagent Information

[0100]

[0101] The detection method used in this embodiment is as follows:

[0102] 1. HPLC detection method for conversion rate in Examples 2.1 and 2.2

[0103] Fmoc-Cl derivatization procedure: Transfer 1 mL of the reaction solution dilution solution (a certain amount of reaction solution diluted with purified water), pH 9.0 sodium borate buffer, and Fmoc-Cl derivatization solution (2.8 mg / mL acetonitrile solution) to a 5 mL centrifuge tube, shake for 15 min, then transfer 1 mL of adamantane solution (5.2 mg / mL acetonitrile aqueous solution), shake for another 15 min, and finally add 70 μL of 1 mol / L hydrochloric acid solution to the derivatization solution, shake well, and filter through an organic filter membrane for sample injection.

[0104] Column: Diamonsil C18(2) (5μm, 250×4.6mm);

[0105] Mobile phase: Mobile phase A: 10 mM ammonium acetate aqueous solution (pH adjusted to 3.3 with formic acid), Mobile phase B: acetonitrile, gradient elution was performed according to Table 2.

[0106] Table 2. HPLC gradient elution procedure for conversion detection in steps one and two.

[0107]

[0108]

[0109] Detection wavelength: 265 nm; flow rate: 1 mL / min; injection volume: 5 μL; column temperature: 30 ℃.

[0110] The retention time for glycine was 13.3 min; the retention time for serine was 8.9 min. The conversion rates in Example 2.1 were calculated based on the glycine content, and the conversion rates in Example 2.2 were calculated based on the serine content. The content was calculated using the area normalization method based on the peak area ratio of the compound in the HPLC chromatogram (the same applies below).

[0111] 2. HPLC method for determining the ee value of D-serine (compound 2)

[0112] Marfey derivatization procedure: Transfer 1 mL of product solution (0.5 mg / mL aqueous solution) to a glass vial, add 1 mL of Marfey derivatization solution (3 mg / mL acetonitrile solution) and 100 μL of sodium bicarbonate solution (84 mg / mL aqueous solution), shake well, wrap with aluminum foil to protect from light, and then place in a 60°C oven for 1 hour. After derivatization, remove the glass vial and add 100 μL of hydrochloric acid solution, shake well, and take a portion of the derivatization solution to dilute five times with 50% acetonitrile-water diluent. Filter and prepare for sample injection.

[0113] Column: Diamonsil C18(2) (5μm, 250×4.6mm);

[0114] Mobile phase: Mobile phase A: 10 mM ammonium acetate aqueous solution (pH adjusted to 3.3 with formic acid), Mobile phase B: acetonitrile, gradient elution was performed according to Table 3.

[0115] Table 3. HPLC gradient elution procedure for D-serine eee value determination

[0116]

[0117]

[0118] Detection wavelength: 340 nm; flow rate: 1 mL / min; injection volume: 5 μL; column temperature: 35 ℃.

[0119] The retention time for D-serine was 10.2 min; the retention time for L-serine was 8.7 min.

[0120] 3. HPLC detection methods for conversion rates in Examples 2.3, 2.4 and Example 3.

[0121] Column: Agilent Eclipse plus C18 (3.5μm, 150×4.6mm);

[0122] Mobile phase: Mobile phase A: 0.1% TFA aqueous solution, Mobile phase B: 0.1% TFA acetonitrile solution, gradient elution according to Table 4:

[0123] Table 4. HPLC gradient elution procedures for reaction conversion detection in steps 3 and 4.

[0124] Time (min) Mobile phase A % Mobile phase B % 0.00 90 10 10.00 60 40 11.00 60 40 13.00 90 10 18.00 90 10

[0125] Detection wavelength: 210 nm; flow rate: 1 mL / min; injection volume: 10 μL; column temperature: 35 ℃.

[0126] The retention time of p-methylsulfonylbenzaldehyde (compound 4) was 6.7 min; the retention time of R-p-methylsulfonylphenyldihydroxyone (compound 5) was 3.4 min. The conversion rates in Examples 2.3 and 3 were calculated based on the content of p-methylsulfonylbenzaldehyde, and the conversion rate in Example 2.4 was calculated based on the content of R-p-methylsulfonylphenyldihydroxyone.

[0127] 4. HPLC method for determining the de value of Soviet-AMPP

[0128] Column: ACE SuperC18 (3μm, 150×4.6mm);

[0129] Mobile phase: Mobile phase A: 10 mM KH2PO4 aqueous solution, pH 8.5; Mobile phase B: acetonitrile; gradient elution was performed according to Table 5.

[0130] Table 5. HPLC gradient elution procedure for de value detection.

[0131] Time (min) Mobile phase A % Mobile phase B % 0.00 98 2 15.00 90 10 18.00 70 30 24.00 70 30 25.00 98 2 31.00 98 2

[0132] Detection wavelength: 224 nm; flow rate: 0.5 mL / min; injection volume: 5 μL; column temperature: 30 °C.

[0133] The retention time of the erythro-AMPP was 13.4 min, and the retention time of the sulphro-AMPP was 14.2 min.

[0134] Formula for calculating the de value:

[0135] Among them, A 苏式 A represents the peak area of ​​the Soviet-AMPP (i.e., (1R,2R)-AMPP and (1S,2S)-AMPP). 赤式 This represents the peak area of ​​erythro-AMPP (i.e., (1S,2R)-AMPP and (1R,2S)-AMPP).

[0136] 5. HPLC method for determining the ee value of (1R,2R)-AMPP (compound 6)

[0137] Collect Soviet-AMPP samples, concentrate them, and separate them using a chiral liquid chromatography column.

[0138] Chromatographic column: Chiralpak chiral IG column;

[0139] Chromatographic conditions: pure methanol (containing 0.1% diethylamine), 15 min;

[0140] Detection wavelength: 224 nm; flow rate: 0.5 mL / min; column temperature: 25 °C.

[0141] The retention time of (1R,2R)-AMPP was 5.6 min; the retention time of (1R,2R)-AMPP was 9.4 min.

[0142] Formula for calculating the ee value:

[0143] Among them: A 1R,2R A represents the peak area of ​​(1R,2R)-AMPP. 1S,2S This represents the peak area of ​​(1S,2S)-AMPP.

[0144] Example 1: Preparation of enzymes required for the reaction

[0145] 1.1 Acquisition and synthesis of enzyme genes

[0146] For the enzymatic preparation of (1R,2R)-AMPP, the amino acid sequences of D-threonine aldolase, D-amino acid oxidase, pyruvate decarboxylase, transketolase, and transaminase used in this patent are shown in Table 6. The genes encoding each enzyme were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (698 Xiangmin Road, Songjiang District, Shanghai) through nucleotide sequence optimization and cloned into the pET28a vector between the NdeI and HindIII restriction sites, enabling the expression of the N-terminal 6×His tag.

[0147] Table 6. Enzyme sources and sequences

[0148]

[0149] 1.2 Transformation of enzyme genes

[0150] The synthesized recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells, and then the recombinant Escherichia coli bacterial suspensions were plated on LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 16 h until single colonies of recombinant Escherichia coli formed on the LB plates.

[0151] 1.3 Expression of enzyme genes

[0152] Single colonies of recombinant *E. coli* from LB agar plates were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C for 5 h. Then, they were transferred at a 1% (v / v) inoculation rate to TB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 220 rpm until OD500. 600 At a concentration of approximately 0.4-0.6, add IPTG to a final concentration of 0.1 mM to induce enzyme expression. Induce at 25°C and 250 rpm for 16 h, then centrifuge at 4000 rpm for 20 min, discard the supernatant, and collect the wet cells.

[0153] Add 50 mM pH 7.0 phosphate buffer at a ratio of 1:10 (1 g of bacterial cells to 10 mL of buffer) to suspend the bacterial cells separately. Hybridize using a high-pressure homogenizer (4℃, 600 bar), centrifuge at 12000 rpm for 5 min, discard the precipitate, and obtain the supernatant, which is the crude enzyme solution for each enzyme. Store at -20℃ for later use. For each type of crude enzyme solution, each 1 mL of crude enzyme solution (mother liquor) contains the equivalent of 100 mg of wet bacterial cells.

[0154] The crude enzyme solutions were centrifuged at 12,000 rpm for 20 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtered samples were then purified using a nickel column. First, contaminating proteins were washed with 10 column volumes of 25 mM imidazole elution buffer, followed by elution of the target protein with 250 mM imidazole elution buffer. The target protein was then concentrated by dialyzing to prepare pure enzyme solutions. Protein concentration was determined using the Bradford method, and the solutions were stored at -80°C with added glycerol for later use.

[0155] Example 2: Stepwise preparation of (1R,2R)-AMPP

[0156] The reaction route for preparing (1R,2R)-AMPP is as follows: Figure 1 As shown.

[0157] 2.1 First step reaction: Preparation of D-serine (compound 2)

[0158] In a 2L three-necked flask, add 800mL of water, 100g of glycine (1.33M), and 1.5g of anhydrous magnesium chloride (15.8mM). Heat to 35℃ and stir until fully dissolved. Then add 15.7mL of formaldehyde solution (37% aqueous solution, density 1.09g / mL). Adjust the pH to 7.4-7.5 with sodium hydroxide. Add 15g of wet bacterial cells expressing D-threonine aldolase E1 and 50mg of pyridoxal 5'-phosphate (PLP, 0.2mM). Control the temperature at 35℃. Add approximately 100mL of formaldehyde solution (37% aqueous solution), bringing the total reaction volume to approximately 1L. Maintain the pH at 7.3 and react for 24 hours. Filter the reaction solution to remove cells and other impurities. Perform HPLC analysis on the resulting supernatant. The conversion rate of glycine was calculated to be 98.5%, and the ee value of D-serine was 99.55%.

[0159] 2.2 Second step reaction: Preparation of hydroxypyruvic acid (compound 3)

[0160] Take 8 mL of the supernatant from the first step of reaction E1, add 50 mM Tris-HCl (pH 7.5) buffer to adjust the pH to 7.5, then add 1 mL of crude D-amino acid oxidase E2 solution and 150 U / mL catalase to prepare a 10 mL reaction system. React at 25 °C for 14 h. Centrifuge the reaction solution to remove precipitates and other impurities. Perform HPLC analysis on a sample of the supernatant, calculating the conversion rate of D-serine to be 98.0%.

[0161] 2.3 Third step reaction: Preparation of R-p-methylsulfonylphenyl dihydroxyone (compound 5)

[0162] Take 2 mL of the supernatant from the second step of the E2 reaction, add 50 mM Tris-HCl (pH 7.5) buffer to adjust the pH to 7.5, then add 10 mM p-methylsulfonylbenzaldehyde, 5 mM thiamine pyrophosphate (TPP), 5 mM magnesium chloride, and a pure enzyme solution containing 25 mg of pyruvate decarboxylase or transketolase to prepare a 10 mL reaction system. React at 25 °C for 2 h, and perform HPLC analysis on a sample of the reaction solution to calculate the conversion rate of p-methylsulfonylbenzaldehyde, as shown in Table 7.

[0163] Table 7 Catalytic effects of pyruvate decarboxylase and transketolase

[0164] Enzyme number Conversion rate E3 89.5％ E4 93.1％ E5 94.8％ E6 97.2％

[0165] 2.4 Fourth step reaction: Preparation of (1R,2R)-AMPP (compound 6)

[0166] Continuing with the third step of reaction in E6, add 100 mM D-alanine, 2.5 mM PLP, and a pure enzyme solution containing 25 mg of transaminase to the reaction solution. The total volume of the reaction system is approximately 10 mL. Continue the reaction at 25 °C for 5 h. Take a sample of the reaction solution for HPLC analysis and calculate the conversion rate of R-p-methylsulfonylphenyl dihydroxyone, the de value of threo-AMPP, and the ee value of (1R,2R)-AMPP, as shown in Table 8.

[0167] Table 8 Catalytic effects of transaminases

[0168] Enzyme number Conversion rate de value ee value E7 87.4％ 95.8％ 98.9％ E8 96.0％ 95.6％ 99.0％ E9 95.1％ 95.0％ 98.6％ E10 99.9％ 96.5％ 99.4％

[0169] Example 3: One-pot preparation of (1R,2R)-AMPP

[0170] The total volume of the one-pot reaction system was 50 mL. Weigh 0.75 g glycine (200 mM), 0.81 g formaldehyde solution (37% aqueous solution), and 0.15 g anhydrous magnesium chloride (31.5 mM). Add 100 mM Tris-HCl (pH 7.5) buffer to adjust the pH to 7.5. Add 10 mL of crude D-threonine aldolase E1 enzyme solution and 10 mg PLP (0.8 mM), and react at 35 °C for 2 h. Add 10 mL of crude D-amino acid oxidase E2 enzyme solution and 150 U / mL catalase, and continue the reaction at 30 °C for 1 h. Then add 20 mM p-methylsulfonylbenzaldehyde, 5 mM TPP, 100 mM D-alanine, and 2.5 mM... PLP, 5 mL of crude transketolase E6 enzyme solution and 10 mL of crude transaminase E10 enzyme solution, and water were added to a total volume of 50 mL. The reaction was continued at 30 °C for 16 h. The reaction solution was sampled and analyzed by HPLC. The conversion rate of p-methylsulfonylbenzaldehyde was calculated to be 99.9%, the de value of threo-AMPP was 97.0%, and the ee value of (1R,2R)-AMPP was 99.5%.

[0171] Sequence:

[0172] SEQ ID NO:1 (D-threonine aldolase E1)

[0173] MSQEVIRGIVLPPPAQAGDPLAGVDTPSLVLDLAAFEANLRAMQAWADRHEVALRPHAKAHKCPEVARRQLALGARGICCQKVSEAVPFVAAGITDIHISNEVVGPAKLRLLAQLARAAKLSVCVDNAANLARISQAMAAAGAEIDVLVEVDVGQGRCGVSDDATVLALAQQARDLPGVTFVGLQAYHGSVQHLRTREERAAVCRQAARIAASYQLLLRESGIACDIITGGGTGSAEFDAASGVYTELQAGSYAFMDGDYGANEWDGALAFQNSLFVLSTVMSTPAPDRVILDAGLKSTTAECGPPAIHGAQGLQYAAINDEHGVVRVAPDAQPPALGDTLLLVPSHVDPTFNLHDGLVVYRDGIVQDIWEISARGFSR

[0174] SEQ ID NO:2 (D-amino acid oxidase E2)

[0175] MAKIVVIGAGVAGLTTALQLLRKGHEVTIVSEFTPGDLSIGYTSPWAGANWLTFYDGGKLADYDAVSYPILRELARSSPEAGIRLINQRSHVLKRDLPKLEGAMSAICQRNPWFKNTVDSFEIIEDRSRIVHDDVAYLVEFASVCIHTGVYLNWLMSQCLSLGATVVKRRVNHIKDANLLHSSGSRPDVIVNCSGLFARFLGGVEDKKMYPIRGQVVLVRNSLPFMASFSSTPEKENEDEALYIMTRFDGTSIIGGCFQPNNWSSEPDPSLTHRILSRALDRFPELTKDGPLDIVRECVGHRPGREGGPRVELEKIPGVGFVVHNYGAAGAGYQSSYGMADEAISYVERALTRPNL

[0176] SEQ ID NO:3 (pyruvate decarboxylase E3)

[0177] MKQRVGQYLMDAVNAAGVDKIFGVPGDFNLAFLDDIISHDQVEWIGNTNELNASYAADGYARINGLGALVTTFGVGELSAVNGIAGSYAERVPVIAITGAPTRAVESAGKYVHHSLGEGTFDDYRKMFEPITTAQGYITPENATTEIPRLIQAAINERRPVHLHLPIDVAMTEIDVPKSFQPEARDDQDVSHYIQMIEDKLNSAKQPVIITGHEINSFGLHSELEQFVNQTHIPVAQLSLGKGAFNEENEHYIGIFDGSIAEENVKNYVNQSDAILNIGAKLTDSATAGFSFEFDIDDVVMINHNYFKMNETISEQVALPHLIKGLMSISYKNKSEFPMYQRPKEHDYQVDHEPLTQATYFKMMQDFLQLDDILIAEQGSSFFGAYDLALYKDNTFIGQPLWGSIGYTLPATLGTQIAAPHRRNVLLIGDGSLQLTVQSLSTMIRQQLKPIIFVVNNDGYTVERLIHGMKEPYNDIHMWDYKTLPAVFGGDNVVVHDVNTSHELKETFEKINAHSDCMHFVEVKMAIEDAPAKLSDIAKAFASQNK

[0178] SEQ ID NO:4(Transketolase E4)

[0179] MSSRKELANAIRALSMDAVQKAKSGYPGAPMGMADIAEVLWRDFLKHNPQNPSWADRDRFVLSNGHGSMLIYSLLHLTGYDLPMEELKNFRQLHSKTPGHPEVGYTAGVETTTGPLGQGIANAVGMAIAEKTLAAQFNRPGHDIVDHYTYAFMGDGCMMEGISHEVCSLAGTLKLGKLIAFYDDNGISIDGHVEGWFTDDTAMRFEAYGWHVIRDIDGHDAASIKRAVEEARAVTDKPSLLMCKTIIGFGSPNKAGTHDSHGAPLGDAEIALTREQLGWKYAPFEIPSEIYAQWDAKEAGQAKESAWNEKFAAYAKAYPQEAAEFTRRMKGEMPSDFDAKAKEFIAKLQANPAKIASRKASQNAIEAFGPLLPEFLGGSADLAPYNLTLWSGSKAINEDAAGNYIHYGVREFGMTAIANGISLHGGFLPYTSTYLMFVEYARNAVRMAALMKQRQVMVYTHDSIGHGETGPTHQPVEQVASLRVTPNMSTWRPCDQVESAVAWKYGVERQDGPTALILSQQNLAQQERTEEQLANIARGGYVLKDCAGQPELIFIATGSEVELAVAAYEKLTAEGVKARVVSMPSTDAFDKQDAAYRESVLPKAVTARVAVEAGIADYWYKYVGLNGAIVGMTTFGESAPAELLFEEFGFTVDNVVAKAKELL

[0180] SEQ ID NO:5 (Transketolase E5)

[0181] MAHSIEELAITTIRTLSIDAIEKAKSGHPGMPMGAAPMAYTLWTKFMNHNPANPNWFNRDRFVLSAGHGSMLLYSLLHLSGYDVSMDDLKQFRQWGSKTPGHPEYGHTPGVEATTGPLGQGIAMAVGMAMAERHLAATYNRDGFEIINHYTYAICGDGDLMEGVASEAASLAGHLKLGRLIVLYDSNDISLDGELNLSFSENVAQRFQAYGWQYLRVEDGNNIEEIAKALEEARADLSRPTLIEVKTTIGYGAPNKAGTSGVHGAPLGAQEAKLTKEAYRWTFAEDFYVPEEVYAHFRATVQEPGAKKEAKWNEQLAAYEQAHPELAAQLKRAIEGKLPDGWEASLPVYEAGKSLATRSSSGEVINAIAKAVPQLFGGSADLASSNKTLIKGGGNFFPGSYEGRNVWFGVREFAMGAALNGMALHGGLKVFGGTFFVFSDYLRPAIRLAALMGLPVIYVLTHDSIAVGEDGPTHEPIEQLASLRAMPNLSVIRPADANETAAAWRLALESTDKPTALVLTRQDVPTLAATAELAYEGVKKGAYVVSPAKNGAPEALLLATGSEVGLAVKAQEALAAEGIHVSVISMPSWDRFEAQPKSYRDEVLPPAVTKRLAIEMGASLGWERYVGAEGDILAIDRFGASAPGEKIMAEYGFTVDNVVRRTKALLGK

[0182] SEQ ID NO:6 (Transketolase E6)

[0183] MAHSIEELAITTIRTLSIDAIEKAKSGYPGMPMGAAPMAYTLWTKFMNHNPANPNWFNRDRFVLSAGHGSMLLYSLLHLSGYDVSMDDLKQFRQWGSKTPGHPEYGHTPGVEATTGPLGQGIAMAVGMAMAERHLAATYNRDGFEIINHYTYAICGDGDLMEGVASEAASLAGHLKLGRLIVLYDSNDISLDGELNLSFSENVAQRFQAYGWQYLRVEDGNNIEEIAKALEEARADLSRPTLIEVKTTIGYGAPNKAGTSGVHGAPLGAQEAKLTKEAYRWTFAEDFYVPEEVYAHFRATVQEPGAKKEAKWNEQLAAYEQAHPELAAQLKRAIEGKLPDGWEASLPVYEAGKSLATRSSSGEVINAIAKAVPQLFGGSADLASSNKTLIKGGGNFFPGSYEGRNVWFGVREFAMGAALNGMALHGGLKVFGGTYFVFSDYLRPAIRLAALMGLPVIYVLTHDSIAHGEDGPTHEPIEQLASLRAMPNLSVIRPADANETAAAWRLALESTDKPTALVLTRQDVPTLAATAELAYEGVKKGAYVVSPAKNGAPEALLLATGSEVGLAVKAQEALAAEGIHVSVISMPSWDRFEAQPKSYRDEVLPPAVTKRLAIEMGASLGWERYVGAEGDILAIDRFGASAPGEKIMAEYGFTVDNVVRRTKALLGK

[0184] SEQ ID NO:7 (Transaminase E7)

[0185] MAFSADTSEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARISIFDQGYLHSDVTYTAFHVWNGNAFRLDDHIERLFSNAESMRIIPPLTQDEVKEIALELVAKTELREACVSVSITRGYSSTPGERDITKHRPQVYMYAVPYQWHVPFDRIRDGVHAMVAQSVRRTPRSSIDPQVKNFQWGDLIRAVQETHDRGFEAPLLLDGDGLLAEGSGHNVVVIKDGVVRSPGRAALPGITRKTVLEIAESLGHEAILADITLAELLDADEVLGCTTAGGVWPFVSVDGNPISDGVPGPVTQSIIRRYWELNVESSSLLTPVQY

[0186] SEQ ID NO:8 (Transaminase E8)

[0187] MASMDKVFAGYQSRLRVLEASTNPLAQGVAWIEGELVPLSQARIPLMDQGFLHSDLTYDVPAVWDGRFFRLDDHISRLEKSCSKLRLKLPLPRDEVKRVLVDMVARSGIRDAFVELIVTRGLTGVRGAGRPEDLVNNLYMFLQPYLWVMPPETQLVGGSAVITRTVRRTPPGSMDPTVKNLQWGDLTRALLEASDRGASYPFLTDGDANITEGSGYNIVLIKDGAIHTPDRGVLEGVTRKTVFDIAKANGFEVRLEVVPVELAYRADEIFMCTTAGGIMPITSLDGQPVNGGQIGPITKKIWDDYWALHYDPAFSFEIKYDEAGASTNGVNGVHK

[0188] SEQ ID NO:9 (Transaminase E9)

[0189] MATIDKVFAGYYARQKLLERSDNPFSKGIAYVEGKFVLPYEARIPLLDEGFMNSDLTYDTINVWDGRFFRLDDHLQRILESCDKMRLKFPLALSSVKKILVEMVAKSGIRDAMVKIIVTRGLTGVRGRKPEDLYNNNIYLLVLPYIWLMAPEDQSHGGSAI ITRTVRRTPPGAFDPTIKNLQWGDFIKGMFEAKDRGATYPFLTDGDTHLTEGPGFNIVLVKNGILYTPDRGVLEGITRKSVIEVARANSIDVRLEVVPVETAYHADEIFMCSTGGGIMPITLLDGKPVNDGQVGPITKKIWDGYWEMHYNAAYSFPVDYGSD

[0190] SEQ ID NO:10 (Transaminase E10)

[0191] MATIDKVFAGYYARQKLLERSDNPFSKGIAYVEGKFVLPYEARIPLLDEGFMNSDLTYDAINVWDGRFFRLDDHLQRILESCDKMRLKFPLALSSVKKILVEMVAKSGIRDACVKIIVTRGLTGVRGRKPEDLYNNNIYLLVLPYIWFMAPEDQSHGGSAI ITRTVRRTPPGAFDPTIKNLQWGDFIKGMFEAKDRGATYPFLTDGDTHLTEGPGHNIVLVKNGILYTPDRGVLEGITRKSVIEVARANSIDVRLEVVPVETAYHADEIFMCSTGGGIMPITLLDGKPVNDGQVGPITKKIWDGYWEMHYNAAYSFPVDYGSD

[0192] In the above embodiments, the concentration of each component in the reaction system refers to the concentration of that component before the reaction begins, after all of it has been added to the reaction system, i.e., the added concentration. The added concentration is calculated based on the total volume of the reaction system when it is fully prepared.

Claims

1. A ketol-acid reductase, characterized in that, The amino acid sequence of the transketolase is shown as SEQ ID NO:

6.

2. A transaminase, characterized in that, The amino acid sequence of the transaminase is shown as SEQ ID NO:

10.

3. A multi-enzyme system, characterized in that, The multi-enzyme system comprises at least two enzymes, and comprises the transketolase according to claim 1 and / or the transaminase according to claim 2; Preferably, the multi-enzyme system further comprises one or more of D-threonine aldolase, catalase and D-amino acid oxidase; More preferably, the multi-enzyme system is selected from any one of the following groups: (1) the D-amino acid oxidase and the transketolase; (2) the D-amino acid oxidase, the transketolase and the catalase; (3) the D-threonine aldolase, the D-amino acid oxidase and the transketolase; (4) the D-threonine aldolase, the D-amino acid oxidase, the transketolase and the catalase; (5) the transketolase and the transaminase; (6) the D-amino acid oxidase, the transketolase and the transaminase; (7) the D-amino acid oxidase, the transketolase, the transaminase and the catalase; (8) the D-threonine aldolase, the D-amino acid oxidase, the transketolase and the transaminase; (9) the D-threonine aldolase, the D-amino acid oxidase, the transketolase, the transaminase and the catalase; Further more preferably, the amino acid sequence of the D-threonine aldolase is shown as SEQ ID NO: 1; and the amino acid sequence of the D-amino acid oxidase is shown as SEQ ID NO:

2.

4. An isolated nucleic acid or nucleic acid composition, comprising, The nucleic acid encodes the transketolase according to claim 1 or the transaminase according to claim 2; and the nucleic acid composition consists of nucleic acids encoding enzymes in the multi-enzyme system according to claim 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid or the nucleic acid composition according to claim 4.

6. A transformant characterized in that, The transformant comprises: a host cell; and, the isolated nucleic acid or the nucleic acid composition according to claim 4, or the recombinant expression vector according to claim 5, introduced into the host cell.

7. A reaction system characterized in that, The reaction system comprises the transketolase according to claim 1 and / or the transaminase according to claim 2; Preferably, the reaction system further comprises any one or more of the following: glycine, formaldehyde, a divalent metal ion compound, D-threonine aldolase, pyridoxal 5'-phosphate, D-amino acid oxidase, p-tosylbenzaldehyde, thiamine pyrophosphate, an amino donor and catalase; More preferably, the reaction system is selected from any one of the following groups: (1) the reaction system comprises hydroxyphosphooxyacetic acid, p-tosylbenzaldehyde, thiamine pyrophosphate and the transketolase; preferably, the reaction system further comprises a divalent metal ion compound; (2) the reaction system comprises R-p-tosylphenyldihydroxyketone, an amino donor, pyridoxal 5'-phosphate and the transaminase; (3) the reaction system comprises glycine, formaldehyde, D-threonine aldolase, D-amino acid oxidase, p-tosylbenzaldehyde, thiamine pyrophosphate, 5'-phosphopyridoxal, and the transketolase; preferably, the reaction system further comprises a divalent metal ion compound and / or catalase; (4) the reaction system comprises D-serine, D-amino acid oxidase, p-tosylbenzaldehyde, thiamine pyrophosphate, and the transketolase; preferably, the reaction system further comprises a divalent metal ion compound and / or catalase; (5) the reaction system comprises glycine, formaldehyde, D-threonine aldolase, 5'-phosphopyridoxal, D-amino acid oxidase, p-tosylbenzaldehyde, thiamine pyrophosphate, an amino donor, the transketolase, and the transaminase; preferably, the reaction system further comprises a divalent metal ion compound and / or catalase; (6) the reaction system comprises hydroxyphosphopyruvic acid, p-tosylbenzaldehyde, thiamine pyrophosphate, an amino donor, 5'-phosphopyridoxal, the transketolase, and the transaminase; preferably, the reaction system further comprises a divalent metal ion compound; (7) the reaction system comprises D-serine, D-amino acid oxidase, p-tosylbenzaldehyde, thiamine pyrophosphate, an amino donor, 5'-phosphopyridoxal, the transketolase, and the transaminase; preferably, the reaction system further comprises a divalent metal ion compound and / or catalase; Further more preferably, the amino donor is D-alanine, isopropylamine, or phenylethylamine; the divalent metal ion compound is magnesium chloride or magnesium sulfate; the amino acid sequence of the D-threonine aldolase is shown in SEQ ID NO: 1; and the amino acid sequence of the D-amino acid oxidase is shown in SEQ ID NO:

2.

8. The reaction system of claim 7, wherein, In the (1), the molar concentration of hydroxyphosphopyruvic acid is 50-500 mM; the molar ratio of p-tosylbenzaldehyde to hydroxyphosphopyruvic acid is (0.05-1):1; the molar ratio of thiamine pyrophosphate to hydroxyphosphopyruvic acid is (0.01-0.05):1; the mass ratio of the transketolase to hydroxyphosphopyruvic acid is (0.05-1):1; and the molar ratio of the divalent metal ion compound to hydroxyphosphopyruvic acid is (0.01-0.5):1; In the (2), the molar concentration of R-p-tosylphenyldihydroxyketone is 5-100 mM; the molar ratio of the amino donor to R-p-tosylphenyldihydroxyketone is (1-20):1; the molar ratio of 5'-phosphopyridoxal to R-p-tosylphenyldihydroxyketone is (0.1-0.5):1; and the mass ratio of the transaminase to R-p-tosylphenyldihydroxyketone is (0.1-2):1; In the (3), the molar concentration of glycine is 50-500 mM; the molar ratio of formaldehyde to glycine is (0.5-5): 1; the mass ratio of the D-threonine aldolase to glycine is (0.1-2): 1; the molar ratio of pyridoxal 5'-phosphate to glycine is (0.002-0.02): 1; the mass ratio of the D-amino acid oxidase to glycine is (0.1-2): 1; the molar ratio of p-methylthio-benzaldehyde to glycine is (0.05-1): 1; the molar ratio of thiamine pyrophosphate to glycine is (0.01-0.05): 1; the mass ratio of the transketolase to glycine is (0.05-1): 1; the molar ratio of the divalent metal ion compound to glycine is (0.01-0.5): 1; and the enzyme activity unit of the catalase is 50-500 U / mL; In the (4), the molar concentration of D-serine is 50-500 mM; the mass ratio of the D-amino acid oxidase to D-serine is (0.05-2): 1; the molar ratio of p-methylthio-benzaldehyde to D-serine is (0.05-1): 1; the molar ratio of thiamine pyrophosphate to D-serine is (0.01-0.05): 1; the mass ratio of the transketolase to D-serine is (0.05-1): 1; the molar ratio of the divalent metal ion compound to D-serine is (0.01-0.5): 1; and the enzyme activity unit of the catalase is 50-500 U / mL; In the (5), the molar concentration of glycine is 50-500 mM; the molar ratio of formaldehyde to glycine is (0.5-5): 1; the mass ratio of the D-threonine aldolase to glycine is (0.1-2): 1; the molar ratio of pyridoxal 5'-phosphate to glycine is (0.002-0.02): 1; the mass ratio of the D-amino acid oxidase to glycine is (0.1-2): 1; the molar ratio of p-methylthio-benzaldehyde to glycine is (0.05-1): 1; the molar ratio of thiamine pyrophosphate to glycine is (0.01-0.05): 1; the molar ratio of the amino donor to glycine is (0.1-1): 1; the mass ratio of the transketolase to glycine is (0.05-1): 1; the mass ratio of the transaminase to glycine is (0.1-2): 1; the molar ratio of the divalent metal ion compound to glycine is (0.01-0.5): 1; and the enzyme activity unit of the catalase is 50-500 U / mL; In the (6), the molar concentration of hydroxy pyruvic acid is 50-500 mM; the molar ratio of p-methylsulfonyl benzaldehyde to hydroxy pyruvic acid is (0.05-1):1; the molar ratio of thiamine pyrophosphate to hydroxy pyruvic acid is (0.01-0.05):1; the molar ratio of the amino donor to hydroxy pyruvic acid is (0.1-1):1; the molar ratio of 5'-phosphopyridoxal to hydroxy pyruvic acid is (0.002-0.02):1; the mass ratio of the transketolase to hydroxy pyruvic acid is (0.05-1):1; the mass ratio of the transaminase to hydroxy pyruvic acid is (0.1-2):1; the molar ratio of the divalent metal ion compound to hydroxy pyruvic acid is (0.01-0.5):1; In the (7), the molar concentration of D-serine is 50-500 mM; the molar ratio of 5'-phosphopyridoxal to D-serine is (0.002-0.02):1; the mass ratio of the D-amino acid oxidase to D-serine is (0.05-2):1; the molar ratio of p-methylsulfonyl benzaldehyde to D-serine is (0.05-1):1; the molar ratio of the amino donor to D-serine is (0.1-1):1; the molar ratio of thiamine pyrophosphate to D-serine is (0.01-0.05):1; the mass ratio of the transketolase to D-serine is (0.05-1):1; the mass ratio of the transaminase to D-serine is (0.1-2):1; the molar ratio of the divalent metal ion compound to D-serine is (0.01-0.5):1; the enzyme activity unit of the catalase is 50-500 U / mL.

9. A process for the preparation of R-p-tosylphenyldihydro- pyrone, characterized in that, The method comprises step 1: catalyzing the reaction of p-methylsulfonyl benzaldehyde and hydroxy pyruvic acid by the transketolase as claimed in claim 1 in the presence of thiamine pyrophosphate to obtain R-p-methylsulfonyl phenyl dihydroxy ketone; Preferably, the hydroxy pyruvic acid is obtained by step 2: catalyzing the reaction of D-serine by the D-amino acid oxidase to obtain hydroxy pyruvic acid; preferably, the step 2 further comprises: converting the hydrogen peroxide generated in the reaction into water and oxygen by the catalase; More preferably, the D-serine is obtained by step 3: catalyzing the reaction of formaldehyde and glycine by the D-threonine aldolase in the presence of 5'-phosphopyridoxal to obtain D-serine.

10. The method of claim 9, wherein, The reaction conditions of the step 1 comprise one or more of the following: the temperature is 20-35°C; the pH is 7.0-8.0; the reaction time is 0.5-20 h; The reaction system of the step 1 is the (1) in the reaction system as claimed in claim 7 or 8; The reaction conditions of the step 2 comprise one or more of the following: the temperature is 20-35°C; the pH is 7.0-8.0; the reaction time is 0.5-20 h; The reaction conditions of the step 2 comprise one or more of the following: the temperature is 20-35°C; the pH is 7.0-8.0; the reaction time is 0.5-20 h; The reaction system of step 2 comprises D-serine and the D-amino acid oxidase; preferably, the reaction system further comprises the catalase; wherein the molar concentration of D-serine is 0.1-1.5 M; the mass ratio of the D-amino acid oxidase to D-serine is (0.05-2):1; and the catalase has an enzyme activity of 50-500 U / mL; The reaction conditions of step 3 comprise one or more of the following: a temperature of 25-40°C; a pH of 7.0-8.0; and a reaction time of 1-30 h; The reaction system of step 3 comprises the D-threonine aldolase, glycine, formaldehyde, and 5'-phosphopyridoxyl; preferably, the reaction system further comprises a divalent metal ion compound; more preferably, the divalent metal ion compound is magnesium chloride or magnesium sulfate; wherein the molar concentration of glycine is 0.1-3 M; the molar ratio of the divalent metal ion compound to glycine is (0.01-0.5):1; the molar ratio of formaldehyde to glycine is (0.5-5):1; the mass ratio of the D-threonine aldolase to glycine is (0.1-2):1; and the molar ratio of 5'-phosphopyridoxyl to glycine is (0.05-0.5):1000.

11. A process for the preparation of R-p-tosylphenyldihydro- pyrone, characterized in that, The method comprises: (i) catalyzing the reaction of glycine, formaldehyde, and p-methylsulfonylbenzaldehyde by the (3) or (4) in the multi-enzyme system of claim 3 in the presence of 5'-phosphopyridoxyl and thiamine pyrophosphate to obtain R-p-methylsulfonylphenyldihydroxyketone; Or, (ii) catalyzing the reaction of D-serine and p-methylsulfonylbenzaldehyde by the (1) or (2) in the multi-enzyme system of claim 3 in the presence of thiamine pyrophosphate to obtain R-p-methylsulfonylphenyldihydroxyketone; Preferably, the method is a one-pot method; The reaction system of (i) is the (3) in the reaction system of claim 7 or 8; The reaction system of (ii) is the (4) in the reaction system of claim 7 or 8; The substances in the reaction system are added in one step or in stages; More preferably, the step of adding the components of the reaction system of (i) in stages is: (i') adding glycine, formaldehyde, the D-threonine aldolase, and 5'-phosphopyridoxyl, and reacting for 0.5-3 h; (ii') adding the D-amino acid oxidase, and reacting for 0.5-3 h; (iii') adding p-methylsulfonylbenzaldehyde, thiamine pyrophosphate, and the transketolase; preferably, the divalent metal ion compound is further added in (i') and / or (iii'); and / or, the catalase is further added in (ii'); The reaction conditions of the method comprise one or more of the following: a temperature of 25-40°C; a pH of 7.0-8.0; and a total reaction time of 1-30 h.

12. A process for the preparation of (1R,2R)-1,3-dihydroxy-2-amino-1 -p- methylsulfonylphenylpropane, characterized in that, The method comprises: reacting an amino donor and R-p-methylsulfonyl phenyl diketone in the presence of pyridoxal 5'-phosphate to obtain (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane catalyzed by the transaminase as claimed in claim 2; Preferably, the reaction conditions of the method comprise one or more of the following: temperature is 20-35℃; pH is 7.0-8.0; reaction time is 0.5-20h; The reaction system of the method is the (2) in the reaction system as claimed in claim 7 or 8; preferably, the amino donor is D-alanine, isopropylamine or phenylethylamine; More preferably, the R-p-methylsulfonyl phenyl diketone is prepared by the method as claimed in any one of claims 9-11.

13. A process for the preparation of (1R,2R)-1,3-dihydroxy-2-amino-1 -p- methylsulfonylphenylpropane, characterized in that, The method comprises: (i) reacting glycine, formaldehyde, p-methylsulfonyl benzaldehyde and an amino donor in the presence of pyridoxal 5'-phosphate and thiamine pyrophosphate to obtain (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane catalyzed by the (8) or (9) in the multi-enzyme system as claimed in claim 3; Or, (ii) reacting hydroxypyruvic acid, p-methylsulfonyl benzaldehyde and an amino donor in the presence of pyridoxal 5'-phosphate and thiamine pyrophosphate to obtain (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane catalyzed by the (5) in the multi-enzyme system as claimed in claim 3; Or, (iii) reacting D-serine, p-methylsulfonyl benzaldehyde and an amino donor in the presence of pyridoxal 5'-phosphate and thiamine pyrophosphate to obtain (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-methylsulfonyl phenylpropane catalyzed by the (6) or (7) in the multi-enzyme system as claimed in claim 3; Preferably, the method is a one-pot method; The reaction system of the (i) is the (5) in the reaction system as claimed in claim 7 or 8; The reaction system of the (ii) is the (6) in the reaction system as claimed in claim 7 or 8; The reaction system of the (iii) is the (7) in the reaction system as claimed in claim 7 or 8; The components of the reaction system are added in one step or in stages; More preferably, the components of the reaction system of the (i) are added in stages in the following steps: (i') adding glycine, formaldehyde, the D-threonine aldolase and pyridoxal 5'-phosphate, and reacting for 0.5-3h; (ii') adding the D-amino acid oxidase, and reacting for 0.5-3h; (iii') adding p-methylsulfonyl benzaldehyde, thiamine pyrophosphate, the amino donor, pyridoxal 5'-phosphate, the ketol-acid reductoisomerase and the transaminase; preferably, the divalent metal ion compound is also added in the (i') and / or (iii'); and / or, the catalase is also added in the (ii'); The reaction conditions of the method comprise one or more of the following: temperature is 25-40℃; pH is 7.0-8.0; total reaction time is 1-30h.

14. A reaction solution comprising R-p-tosylphenyldihydro- pyrone, characterized in that, The reaction solution is prepared by the method of any one of claims 9-11.

15. A reaction solution comprising (1R, 2R)-1, 3-dihydroxy-2-amino-1-p-tosylphenylpropane, characterized in that, The reaction solution is prepared by the method of claim 12 or 13.

16. Use of a trans-ketolase enzyme according to claim 1, a multi-enzyme system according to claim 3, a nucleic acid or nucleic acid composition according to claim 4, a recombinant expression vector according to claim 5, a transformant according to claim 6, or a reaction system according to claim 7 or 8, for the preparation of R-p-tosylphenyl dihydroxyketone.

17. Use of a trans-ketolase enzyme according to claim 1, a transaminase according to claim 2, a multi-enzyme system according to claim 3, a nucleic acid or nucleic acid composition according to claim 4, a recombinant expression vector according to claim 5, a transformant according to claim 6, a reaction system according to claim 7 or 8, or a reaction solution according to claim 14, for the preparation of (1R,2R)-1,3-dihydroxy-2-amino-1-p-tosylphenylpropane.

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