CN110540977A

CN110540977A - Application of L-threonine transaldolase in synthesis of florfenicol chiral intermediate

Info

Publication number: CN110540977A
Application number: CN201910839203.2A
Authority: CN
Inventors: 林娟; 许炼; 王力超; 陈承滔; 赖凌燕
Original assignee: Fujian Changsheng Biotechnology Development Co Ltd
Current assignee: Fujian Changsheng Biotechnology Development Co Ltd
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2019-12-06
Anticipated expiration: 2039-09-05
Also published as: CN110540977B

Abstract

The invention provides an application of L-threonine transaldolase in florfenicol chiral intermediate synthesis, wherein the L-threonine transaldolase is selected from any one of the following groups: (1) has the sequence shown in SEQ ID NO: 1; (2) has the sequence shown in SEQ ID NO: 1, and the polypeptide has catalytic activity, wherein the amino acid sequence shown in the 1 is more than or equal to 90% of homologous polypeptide; (3) converting SEQ ID NO: 1 by substitution, deletion or addition of 1-5 amino acid residues, and retains catalytic activity. The invention screens new L-threonine transaldolase genes by gene mining, expresses L-threonine transaldolase from recombinant escherichia coli by adopting a genetic engineering means, synthesizes (2S,3R) -p-methylsulfonyl phenyl serine by taking p-methylsulfonylbenzaldehyde and L-threonine as raw materials through a whole-cell catalytic reaction, can obtain the florfenicol key chiral synthesis building block by one-step reaction at normal temperature and normal pressure, is environment-friendly and is a green biosynthesis way.

Description

application of L-threonine transaldolase in synthesis of florfenicol chiral intermediate

Technical Field

The invention belongs to the fields of biotechnology and chemical engineering, and particularly relates to application of L-threonine transaldolase in synthesis of a florfenicol chiral intermediate.

background

The (2S,3R) -p-methylsulfonylphenylserine is a key chiral building block for synthesizing the florfenicol. Florfenicol is a beta-aminoalcohol antibiotic, has the advantages of rapid absorption, wide distribution in vivo, long half-life, no aplastic side effect, difficult generation of drug resistance, no residue, no cross drug resistance and the like, and is a new generation of chloramphenicol antibiotic for replacing chloramphenicol and thiamphenicol. Is mainly used for treating bacterial diseases and mycoplasma infection of pigs, chickens and fish caused by sensitive bacteria, and has unique curative effects on diseases caused by livestock and poultry escherichia coli, infectious pleuropneumonia of pigs, asthma and the like. In recent years, the production and export of florfenicol in China are continuously increased, the export amount is about 1744.4 tons in 2015, the comparably increase is 21.18 percent, the export amount is increased by 39.51 percent, and the export amount of florfenicol in China reaches about 2000 tons in 2017 years, which is a list of varieties with export amount of more than 1 hundred million dollars in Chinese medicinal raw material medicine year.

At present, the main method for industrially producing (2S,3R) -p-methylsulfonylphenylserine ethyl ester at home and abroad takes p-methylsulfonylbenzaldehyde and glycine as raw materials, generates p-methylsulfonylphenylserine through aldol condensation reaction in the presence of copper sulfate and ammonia water, esterifies the p-methylsulfonylphenylserine ethyl ester to obtain (2S,3R) -p-methylsulfonylphenylserine ethyl ester through D-tartaric acid resolution. A large amount of copper sulfate wastewater is generated in the production process of obtaining p-methylsulfonylphenylserine ethyl ester, so that the wastewater treatment cost is high.

At present, only one patent application for synthesizing p-methylsulfonylphenylserine by using transaldolase (application publication No. CN 109836362A) is a method for preparing chiral (2S,3R) -p-methylsulfonylphenylserine ethyl ester (application publication No. CN 109836362A), but the scheme of the invention application is to use pure enzyme to carry out reaction, the production cost of the pure enzyme is high, the reaction time is long, the transaldolase used for the reaction described in the specification of the application is self-made by an applicant, relevant literature reports, patent protection and market sales information about the enzyme cannot be found, and the enzyme cannot be purchased on an official website of the applicant, namely, the technical scheme described in the invention application cannot be realized and verified by a person skilled in the art.

In summary, a suitable and efficient L-threonine transaldolase needs to be found for the catalytic synthesis process of florfenicol chiral intermediate (2S,3R) -p-methylsulfonylphenylserine, so as to reduce the production cost, improve the production efficiency and reduce the environmental pollution.

the invention content is as follows:

The invention aims to provide application of L-threonine transaldolase in synthesis of a florfenicol chiral intermediate, and steps of a method for synthesizing the florfenicol chiral intermediate by catalyzing the L-threonine transaldolase, and aims to solve the technical problems of complex production steps, low efficiency, high production cost, great environmental pollution and the like of (2S,3R) -p-methylsulfonylphenylserine in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

The application of L-threonine transaldolase in synthesizing florfenicol chiral intermediates, wherein the L-threonine transaldolase is selected from any one of the following groups:

(1) Has the sequence shown in SEQ ID NO: 1;

(2) Has the sequence shown in SEQ ID NO: 1, and the polypeptide has catalytic activity, wherein the amino acid sequence shown in the 1 is more than or equal to 90% of homologous polypeptide;

(3) Converting SEQ ID NO: 1 by substitution, deletion or addition of 1-5 amino acid residues, and retains catalytic activity.

Preferably, the L-threonine transaldolase has catalytic activity for asymmetric catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine from p-methylsulfonylbenzaldehyde and L-threonine.

preferably, the L-threonine transaldolase is encoded by an LTTA gene derived from Pseudomonas sp, the sequence of the LTTA gene being shown in SEQ ID NO: 2, respectively.

Preferably, the reaction system for asymmetric catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine by p-methylsulfonylbenzaldehyde and L-threonine comprises: p-methylsulfonylbenzaldehyde, L-threonine, pyridoxal phosphate, magnesium chloride, and L-threonine transaldolase whole cells, wherein the reaction buffer is Tris-HCl buffer (pH7.0) containing 10% ethyl acetate (v/v), and the reaction is carried out at 15-30 ℃ for 12-24 h.

Preferably, the L-threonine transaldolase is a recombinant Escherichia coli whole cell expressing LTTA gene by using a genetic engineering means.

Preferably, the mass molar concentration ratio of each raw material in the reaction system is p-methylsulfonylbenzaldehyde: l-threonine: pyridoxal phosphate: 30 parts of magnesium chloride: 100: 0.2: 10, the whole cell concentration of the recombinant Escherichia coli in the reaction system is 6.25mg/mL, and the Tris-HCl buffer solution (pH7.0) is 100 mM.

preferably, the preparation method of the recombinant escherichia coli whole cell comprises the following steps:

S1, constructing a polypeptide containing a sequence shown as SEQ ID NO: 2, a recombinant expression plasmid vector of the LTTA gene;

S2, transforming the recombinant expression plasmid vector in the step S1 into competent cells of escherichia coli, culturing at 37 ℃ overnight, and then culturing at 0.2mM IPTG and 28 ℃ for 16 h;

S3, collecting the Escherichia coli thallus cultured in the step S2 to obtain the L-threonine transaldolase whole cell.

Preferably, the preparation method of the reaction system for asymmetric catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine by using the L-threonine transaldolase comprises the following steps: adding Tris-HCl buffer solution (pH7.0) into a reaction container, then respectively weighing p-methylsulfonylbenzaldehyde, L-threonine, pyridoxal phosphate and magnesium chloride, sequentially putting into a reaction bottle, fully and uniformly mixing, then adding whole cells of recombinant escherichia coli, and carrying out oscillation reaction.

Preferably, after the reaction system for asymmetrically catalyzing and synthesizing the (2S,3R) -p-methylsulfonylphenylserine by the L-threonine transaldolase reaches the preset reaction time, the reaction solution is centrifuged for 5-10min at 8000-10000rpm, cell precipitates are removed, and the collected supernatant is the catalytic product containing the (2S,3R) -p-methylsulfonylphenylserine.

The scheme of the invention has at least the following beneficial effects:

The invention screens new L-threonine transaldolase genes by gene mining, and expresses the L-threonine transaldolase derived from recombinant escherichia coli by adopting a genetic engineering means. L-threonine transaldolase (EC 2.1.2.1, LTTA) is pyridoxal phosphate (PLP) dependent enzyme, can directly catalyze different aldehydes to react with L-threonine to form asymmetric C-C bonds, thereby obtaining beta-hydroxy-alpha-amino acid with high added value, and has good application prospect in the aspect of biosynthesizing drugs.

The method takes cheap and easily-obtained p-methylsulfonylbenzaldehyde as a raw material, directly adopts L-threonine transaldolase to synthesize (2S,3R) -p-methylsulfonylphenylserine in a whole-cell catalytic manner, has mild reaction conditions and short reaction time, only needs to react for 12 hours at the temperature of 20 ℃, the enantiomeric excess ee of a target product (2S,3R) -p-methylsulfonylphenylserine is more than 99.9 percent, and the diastereomeric excess de is 94.5 percent; and the whole cells are used as the catalytic carrier, so that pure enzyme does not need to be prepared, preparation links such as enzyme separation and purification, drying and the like are saved, and the production cost is greatly reduced. The traditional organic synthesis method needs expensive metal catalysts and has serious heavy metal pollution, the invention adopts a biological catalysis method to replace a chemical synthesis method, and can obtain the florfenicol key chiral synthesis building block (2S,3R) -p-methylsulfonylphenylserine through one-step reaction at normal temperature and normal pressure, and meanwhile, the product is environment-friendly, thus being a green biological synthesis method.

Drawings

FIG. 1 is the result of comparison of the similarity between the L-threonine transaldolase gene (GenBank No. CVTX01000156) and the reported L-threonine transaldolase gene (Nature communications,2017,8:15935.) in example 1;

FIG. 2 is a graph showing the results of detecting the expression of L-threonine transaldolase protein in recombinant E.coli by SDS-PAGE in example 1;

FIG. 3 is a graph showing the effect of different substrate p-methylsulfonylbenzaldehyde concentrations and cell concentrations on the conversion and de values of the product (2S,3R) -p-methylsulfonylphenylserine in Experimental example 2, wherein: (A) substrate concentration 10mM, (B) substrate concentration 20mM, (C) substrate concentration 30mM, (D) substrate concentration 40 mM;

FIG. 4 is a graph showing the effect of different reaction temperatures on the conversion rate and de value of the product (2S,3R) -p-methylsulfonylphenylserine in Experimental example 4;

FIG. 5 is a HPLC check chart of the product (2S,3R) -p-methylsulfonylphenylserine of the catalytic reaction in example 1; wherein: (A) detecting (2S,3R) -p-methylsulfonylphenylserine standard product at 340nm, wherein T (2S,3R) ═ 6.902 min; (B) detecting at 340nm, wherein T (2S,3R) is 6.909min, and T (2S,3S) is 8.261 min; (C) detecting at 236nm, wherein T (2S,3R) ═ 6.909min, T (2S,3S) ═ 8.260min, and T-methyl sulfone benzaldehyde ═ 11.074 min;

FIG. 6 is a LC-MS characterization of the product (2S,3R) -p-methylsulfonylphenylserine from the catalytic reaction in example 1; wherein: (A) detecting (2S,3R) -p-methylsulfonylphenylserine by liquid chromatography; (B) detecting (2S,3R) -p-methylsulfonylphenylserine by mass spectrum. (ESI +) M/z C10H13NO5S [ M + H ] +, 260.0557;

FIG. 7 is a NMR chart of the product (2S,3R) -p-methylsulfonylphenylserine catalyzed in example 1; 1H NMR (400MHz, D2O) δ 7.74(D, J ═ 7.5Hz,2H),7.46(D, J ═ 7.7Hz,2H),4.62(s,1H),4.60(s,1H),3.10(D, J ═ 6.3Hz, 1H).

Detailed Description

the following preferred embodiments of the present invention are provided to aid in a further understanding of the invention. It should be understood by those skilled in the art that the description of the embodiments of the present invention is by way of example only, and not by way of limitation.

Example 1

in the embodiment, the L-threonine transaldolase derived from recombinant escherichia coli is expressed by using a genetic engineering means, so that the L-threonine transaldolase is obtained; meanwhile, a whole cell is used as a carrier, p-methylsulfonylbenzaldehyde and L-threonine are asymmetrically catalyzed to synthesize (2S,3R) -p-methylsulfonylbenzserine, and a set of technological conditions for synthesizing (2S,3R) -p-methylsulfonylbenzserine by using L-threonine transaldolase in whole cell asymmetric catalysis are established by optimizing reaction conditions, wherein ee of enantiomer excess is more than 99.9%, de of diastereomer excess is 94.5%, and conversion rate of substrate p-methylsulfonylbenzaldehyde is 67.1%. The specific operation steps comprise:

(1) Whole cell for obtaining L-threonine transaldolase by genetic engineering method

A new L-threonine transaldolase gene (GenBank No. CVT01000156) was screened by gene mining, SEQ ID NO: 2, the gene sequence and the reported L-threonine transaldolase gene (An L-threonine transaldolase is required for L-threo-b-hydroxy-a-amino acid assembly during synthesis of beta-fetuin biosynthesis, Nature communications,2017,8:15935.) have 95.84% similarity (see the alignment result in the attached figure 1), but the article reports that when the function of the P.fluoroscedases ATCC 39502 biosynthesis gene cluster is studied, the L-threonine transaldolase can catalyze the synthesis of (2S,3R) -p-nitroanilide from p-nitroacetal and L-threonine, but whether other aldehydes can be used as substrates or not is not further studied.

The L-threonine transaldolase in this example was encoded by a Pseudomonas sp-derived LTTA gene (GenBank No. CVTX01000156) having the base sequence of SEQ ID NO: 2, the encoded amino acid sequence is SEQ ID NO: 1. the applicant optimizes the LTTA gene to obtain an optimized LTTA gene sequence, wherein the base sequence is SEQ ID NO: 3. the optimized LTTA gene DNA sequence is artificially synthesized, the LTTA gene is further connected to an expression plasmid pET28a to construct a recombinant expression plasmid pET28a-LTTA, and escherichia coli BL21(DE3) is transformed to heterologously express L-threonine transaldolase.

A single clone containing the recombinant plasmid was picked up and inoculated into 20mL of LB liquid medium (containing 50. mu.g/mL of kanamycin), and cultured overnight at 37 ℃ and 180 rpm. 10mL of the culture medium was pipetted into 1L of LB liquid medium (containing 50. mu.g/mL kanamycin), and cultured at 37 ℃ with shaking at 200 rpm.

When the OD600 of the recombinant Escherichia coli culture solution is 0.5, IPTG (Isopropyl Thiogalactoside) is added to the final concentration of 0.2mM, the culture is stopped after 16h of induction at 28 ℃, and the thalli are harvested by centrifugation at 5000rpm for 10min at 4 ℃. The expression of the L-threonine transaldolase protein was detected by SDS-PAGE, and the results are shown in FIG. 2, in which Control is recombinant E.coli not induced by IPTG; LTTA is recombinant E.coli induced with the addition of 0.2mM IPTG. As can be seen, the LTTA group showed the desired band at around 48kDa, whereas the Control group did not show the band, indicating that the LTTA protein was successfully expressed in recombinant E.coli. The operations of codon optimization, construction of recombinant expression plasmid vector, transformation of escherichia coli competence, culture, induction and the like involved in the embodiment belong to general technical operations in the technical field of genetic engineering, and therefore, the steps and processes are not described in detail herein.

(2) whole-cell asymmetric catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine by L-threonine transaldolase

100mL of reaction system: 0.55g of p-methylsulfonylbenzaldehyde, 1.2g L-threonine, 5mg of pyridoxal phosphate and 10mg of magnesium chloride were weighed out and put into a 250mL reaction flask, and 100mL of 10% ethyl acetate (v/v) in Tris-HCl buffer (100mM, pH7) was added in advance. After mixing well, 0.625g of recombinant E.coli whole cells (wet weight) were added and reacted at 20 ℃ for 12 hours with shaking. After the reaction, the cell precipitate was removed by centrifugation at 10000rpm for 5min, and the supernatant containing the product was collected.

The L-threonine transaldolase expressed by the recombinant escherichia coli is intracellular enzyme, and the recombinant escherichia coli whole cell is adopted in the reaction system, so that the L-threonine transaldolase is obtained and purified without breaking the cell. In the whole cell reaction process, the micromolecular substrate penetrates through the cell membrane to enter the cell to react with the enzyme, the generated product penetrates through the cell membrane to be discharged outside the cell, and the whole catalytic reaction is carried out in the cell, so that the whole catalytic reaction is more efficient, stable and reliable.

(3) OPA/NAC derivatization detection product (2S,3R) -p-methylsulfonylphenylserine

OPA/NAC derivatization reagent preparation: solution A: weighing 10mg of o-phthalaldehyde (OPA) and adding the o-phthalaldehyde (OPA) into 5mL of methanol, and oscillating the mixture at 30 ℃ to fully dissolve the o-phthalaldehyde; and B, liquid B: 10mg of N-acetylcysteine (NAC) was weighed out and dissolved in 20mL of buffer (0.2M boric acid, 0.2M potassium chloride); mixing solution A and solution B, and storing at 4 deg.C in dark.

100 mul of reaction supernatant and 400 mul of OPA/NAC derivatization reagent are mixed evenly, and then the mixture is placed for 10min in a dark place at room temperature for HPLC detection. The HPLC detection conditions are as follows: detection wavelength: 236 and 340 nm; a chromatographic column: agilent C18column (250X 4.6mm,5 μm); mobile phase: 50mM KH2PO4, pH 7.0/acetonitrile (81/19, v/v); flow rate: 1mL/min, temperature: 30 ℃; loading: 20 μ L.

the results are shown in FIG. 5. The peak emergence time of the products (2S,3R) -p-methylsulfonylphenylserine and (2S,3S) -p-methylsulfonylphenylserine is 6.909min and 8.261min respectively, and the enantiomeric excess ee is more than 99.9%; the diastereomer excess de ═ 94.5%, and the substrate conversion (based on the amount of substrate reacted) was 67.1%. The calculation formulas of enantiomeric excess and diastereomer excess are respectively as follows:

Wherein the content of (2R, 3S) and (2R, 3R) is less than 0.01%.

(4) Purification of (2S,3R) -p-methylsulfonylphenylserine

The product (2S,3R) -p-methylsulfonylphenylserine is purified by using a C18 solid-phase extraction column, the eluent is 10% acetonitrile, the effluent is collected according to a 1 mL/tube, the effluent is subjected to HPLC detection after being derivatized by OPA/NAC, the effluent containing (2S,3R) -p-methylsulfonylphenylserine is collected, rotary evaporation and drying are carried out, a colorless viscous liquid is obtained, and the yield of the target product (2S,3R) -p-methylsulfonylphenylserine is 34.8%.

The product yield calculation formula is: the yield of the product is M/M multiplied by 100 percent,

In the formula: m is the actual product mass; m is the theoretical product mass.

(5) (2S,3R) -p-methylsulfonylphenylserine structure characterization

The product obtained in this example was subjected to liquid chromatography-mass spectrometry (LCMS). Wherein (2S,3R) -p-methylsulfonylphenylserine ethyl ester was purchased from a source leafy organism with a purity > 99%, CAS number: 120-47-8, hydrolyzing under alkaline condition to obtain (2S,3R) -p-methylsulfonyl phenyl serine.

the detection conditions of LCMS are as follows: the chromatographic column is Agilent C18column (150X 4.6mm,5 μm); a positive ion mode; the mobile phase is 10% acetonitrile, and the sample loading amount is 20 mu L; the flow rate is 0.3 mL/min; the temperature was 30 ℃. LC detection result shows that the purified substance is a single peak (figure 6A), and MS detection result shows that the purified product is (2S,3R) -p-methylsulfonylphenylserine (figure 6B).

The product obtained by the invention is further characterized by Nuclear Magnetic Resonance (NMR), and the obtained characteristic diagram is shown in figure 7, which shows that the product is (2S,3R) -p-methylsulfonylphenylserine.

Experiments were designed to explore the optimal conditions for the whole-cell catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine by L-threonine transaldolase in experimental examples 1 to 4.

experimental example 1 Effect of different Metal ions (10mM) on LTTA catalytic Activity

In order to explore the influence of different metal ions on the catalytic activity of LTTA, the following experiment was designed. The reaction system and conditions were the same as in example 1 except that the kind of metal ions added was different and the concentration of each metal ion was 10mM, and the experimental results are shown in Table 1 below. It is known that Mg, Cs, Li, Ca and Ba have a promoting effect on LTTA catalytic activity, the best effect is Mg, and the LTTA enzymatic activity is improved by 24%; fe. Ni, Zn and Cu have an inhibiting effect on LTTA, and the de value of LTTA is 17-81% under different metal ion conditions.

TABLE 1 Effect of different Metal ions on LTTA Activity

Experimental example 2 Effect of different substrate and cell concentrations on LTTA conversion and de values

The effects of various substrates on the methylsulfonylbenzaldehyde concentration (10-40mM) and the cell concentration (3.0-50mg/mL) on the LTTA conversion and de value were examined using methylsulfonylbenzaldehyde and L-threonine as substrates, and the reaction system and conditions were the same as in example 1 except that the reaction temperature in this example was 30 ℃ and the organic solvent was added in the form of 10% acetonitrile. The results of the experiment are shown in FIG. 3. It can be seen that the de value of the product decreases with the increase of the cell concentration, and when the cell concentration is 3.0 and 6.25mg/mL, the de value of the product does not change significantly (about 85%), and when the cell concentration is more than 6.25mg/mL, the de value of the product starts to decrease (from 85% to 73%); the conversion of the substrate was significantly higher at a cell concentration of 6.25mg/mL than at a cell concentration of 3.0mg/mL, so the optimal cell concentration was determined to be 6.25 mg/mL. Under this condition, the substrate concentration was 30mM, the product accumulation was the largest, and therefore, the optimum substrate concentration and cell concentration were determined to be 30mM and 6.25mg/mL, respectively, under which the conversion rate reached about 69.5% and the de value was 85%.

Experimental example 3 Effect of different organic solvents on LTTA conversion and de-value

The influence of different organic solvents on the LTTA conversion rate and the de value was examined, the reaction system and conditions were the same as in example 1 except that the reaction temperature was 30 ℃ and the types of the organic solvents added were different, and the experimental results are shown in Table 2 below. It is understood that the conversion and de values were at lower levels (41.6% and 71.3%) without the addition of organic solvent, indicating that organic solvent is necessary for LTTA catalysis; low doses of organic solvent (10% and 20%) favoured the conversion of the product, whereas when the organic solvent was added in an amount greater than 30%, the conversion of the product was inhibited. By taking 10% acetonitrile as a control, the 10% ethyl acetate is found to improve the conversion rate (80%) and the de value (90%); methanol and DMSO can improve the conversion rate of the product, but can not improve the de value; ethanol and acetone are detrimental to the conversion of the product and the de value, so 10% ethyl acetate was chosen as the final organic solvent addition.

TABLE 2 influence of organic solvent on the conversion and de value of the product (2S,3R) -p-methylsulfonylphenylserine

Experimental example 4 Effect of different temperatures on LTTA conversion and de value

The influence of different temperatures on the LTTA conversion rate and the de value is detected, the reaction system and conditions are basically the same as those in example 1, the difference is that the reaction temperature is different, and the experimental result is shown in figure 4. The de value of the product began to decrease (95% to 87%) with increasing temperature. When the reaction temperature is 10, 15 and 20 ℃, the de value of the product is not obviously changed, but the conversion rate of the product is obviously improved. Therefore, 20 ℃ was selected as the optimum reaction temperature.

From the above results, the optimum conditions for catalytic reaction were determined to be a cell concentration of 6.25mg/mL, p-methylsulfonylbenzaldehyde 30mM, magnesium chloride 10mM, 10% ethyl acetate, and 20 ℃. The optimized whole-cell catalytic reaction conditions are as follows: 30mM of p-methylsulfonylbenzaldehyde, 100mM of L-threonine, 0.2mM of pyridoxal phosphate, 10mM of magnesium chloride, 6.25mg/mL of Escherichia coli cell concentration, 10% of ethyl acetate (v/v) in Tris-HCl buffer (100mM, pH7.0), 12 hours and 20 ℃ for reaction, respectively.

finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present application and not for limiting the protection scope thereof, and although the present application is described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: numerous variations, modifications, and equivalents will occur to those skilled in the art upon reading the present application and are within the scope of the claims as issued or as granted.

Sequence listing

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gtctgcccgc atttgcagag caactgccac gccgaacaac tgccgccgat gtgggtggca 840

ttcaaggaga tggaactctt cggtcgggac tatgccgcgc agatcgtgag caatgccaag 900

actctggccc gtcacctgca tgagttgggg ctggatgtga ccggagagtc gttcggtttc 960

acccagaccc accaagtgca ctttgccgtg ggtgatctgc aaaaagcact cgatctgtgc 1020

gtcaacagcc tgcatgccgg cgggattcgt tccaccaata tcgaaattcc cggaaaaccg 1080

ggcgtgcatg gcatacgcct gggtgttcag gcgatgacac gccgtggcat gaaagagaaa 1140

gattttgaag tggtcgcacg cttcatcgcc gacctgtact tcaagaaaac cgagccggcc 1200

aaggtcgcgc aacagatcaa ggagttcctt caggctttcc ccctcgcgcc attggcttac 1260

tccttcgata actacctgga tgatgagtta ctggcggcgg tctatcaggg tgctcaacga 1320

tga 1323

<210> 3

<211> 1323

<212> DNA

<213> Pseudomonas sp

<400> 3

atgagcaatg ttaaacagca gaccgcacag attgttgatt ggctgagcag caccctgggt 60

aaagatcatc agtatcgtga agatagcctg agcctgaccg caaatgaaaa ttatccgagc 120

gcactggttc gtctgaccag cggtagcacc gcaggcgcat tttatcattg tagctttccg 180

tttgaagttc cggcaggcga atggcatttt ccggaaccgg gtcatatgaa tgcaattgcc 240

gatcaggttc gtgatctggg taaaaccctg attggtgcac aggcatttga ttggcgtccg 300

aatggtggta gtaccgcaga acaggcactg atgctggcag catgtaaacc tggtgaaggt 360

tttgttcatt ttgcacatcg tgatggtggt cattttgccc tggaaagcct ggcacagaaa 420

atgggtattg aaatttttca tctgccggtt aatccgatca gcctgctgat tgatgttgca 480

aaactggatg aaatggttcg tcgtaatccg catattcgta ttgttattct ggaccagtca 540

tttaaactgc gttggcagcc gctggcagaa attcgtagcg ttctgccgga tagctgtacc 600

ctgacctatg atatgagcca tgatggtggc ctgattatgg gtggtgtttt tgatagtccg 660

ctgagctgtg gtgcagatat tgttcatggt aatacccata aaaccattcc gggtccgcag 720

aaaggttata ttggttttaa aagcgcacag catccgctgc tggttgatac cagcctgtgg 780

gtttgtccgc atctgcagag caattgtcat gccgaacagc tgcctccgat gtgggttgca 840

tttaaagaaa tggaactgtt cggtcgtgat tatgcagccc agattgttag caatgcaaaa 900

accctggcac gtcatctgca tgaactgggt ttagatgtta ccggtgaaag ctttggtttt 960

acacagaccc atcaggtgca ttttgcagtt ggtgatctgc agaaagcact ggatctgtgt 1020

gttaatagcc tgcatgccgg tggtattcgt agcaccaaca ttgaaattcc gggtaaaccg 1080

ggtgttcatg gcattcgtct gggtgtgcag gcaatgaccc gtcgtggtat gaaagaaaaa 1140

gattttgaag tggtggcacg ctttattgcg gatctgtatt tcaaaaaaac cgaaccggca 1200

aaagttgccc agcagattaa agaattcctg caggcatttc cgctggcacc tctggcatat 1260

agctttgata actatctgga tgatgaactg ttagcagcag tttatcaggg tgcacagcgt 1320

taa 1323

Claims

1. The application of L-threonine transaldolase in the synthesis of florfenicol chiral intermediates is characterized in that the L-threonine transaldolase is selected from any one of the following groups:

(1) Has the sequence shown in SEQ ID NO: 1;

2. the use of an L-threonine transaldolase in the synthesis of a chiral intermediate of florfenicol as claimed in claim 1, characterized in that the L-threonine transaldolase has catalytic activity for the asymmetric catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine from p-methylsulfonylbenzaldehyde and L-threonine.

3. The use of an L-threonine transaldolase in the synthesis of a florfenicol chiral intermediate according to claim 2, wherein the L-threonine transaldolase encodes an LTTA gene derived from Pseudomonas (Pseudomonas sp.) having the sequence set forth in SEQ ID NO: 2, respectively.

4. The application of the L-threonine transaldolase in the synthesis of chiral intermediate of florfenicol as claimed in claim 3, wherein the reaction system for asymmetric catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine from p-methylsulfonylbenzaldehyde and L-threonine comprises: p-methylsulfonylbenzaldehyde, L-threonine, pyridoxal phosphate, magnesium chloride, and L-threonine transaldolase whole cells, wherein the reaction buffer is a Tris-HCl buffer containing 10% ethyl acetate (v/v), and the reaction is carried out at 15-30 ℃ for 12-24 h.

5. The application of the L-threonine transaldolase in the synthesis of chiral intermediates of florfenicol as claimed in claim 4, wherein the L-threonine transaldolase is a recombinant Escherichia coli whole cell expressing LTTA gene by using genetic engineering means.

6. The application of the L-threonine transaldolase in the synthesis of florfenicol chiral intermediates according to claim 5, wherein the ratio of the molar mass concentrations of the raw materials in the reaction system is p-methylsulfonylbenzaldehyde: l-threonine: pyridoxal phosphate: 30 parts of magnesium chloride: 100: 0.2: 10, the whole cell concentration of the recombinant escherichia coli in the reaction system is 6.25mg/mL, and the Tris-HCl buffer solution is 100 mM.

7. The use of the L-threonine transaldolase in the synthesis of chiral intermediates of florfenicol as claimed in claim 6, wherein the method for preparing the recombinant E.coli whole cells comprises:

8. The application of the L-threonine transaldolase in the synthesis of the chiral intermediate of florfenicol as claimed in claim 7, wherein the preparation method of the reaction system for the asymmetric catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine by the L-threonine transaldolase is as follows: adding Tris-HCl buffer solution into a reaction container, then respectively weighing p-methylsulfonylbenzaldehyde, L-threonine, pyridoxal phosphate and magnesium chloride, sequentially putting into a reaction bottle, fully and uniformly mixing, then adding whole cells of recombinant escherichia coli, and carrying out oscillation reaction.

9. The application of the L-threonine transaldolase in the synthesis of the chiral intermediate of florfenicol as claimed in claim 8, which is characterized in that after the reaction system for the asymmetric catalytic synthesis of (2S,3R) -p-methylsulfonylphenylserine by the L-threonine transaldolase reaches a predetermined reaction time, the reaction solution is centrifuged at 8000-10000rpm for 5-10min, cell precipitates are removed, and the collected supernatant is the catalytic product containing (2S,3R) -p-methylsulfonylphenylserine.