L-threonine aldolase mutant and application of L-threonine aldolase mutant in catalytic synthesis of p-methylsulfonylphenylserine

Abstract

The invention provides an L-threonine aldolase mutant and application of the L-threonine aldolase mutant in catalytic synthesis of p-methylsulfonylphenylserine, the L-threonine aldolase mutant is derived from pseudomonas, threonine and p-methylsulfonylbenzaldehyde can be used as substrates, and the L-threonine aldolase mutant can be synthesized by using a one-step method. According to the present invention, L-threonine aldolase is adopted as a raw material, pyridoxal phosphate and reduced nicotinamide adenine dinucleotide are adopted as coenzymes, the synthesis of florfenicol intermediate product p-methylsulfonyl phenyl serine is obtained through catalysis, and compared with the wild type L-threonine aldolase, the mutant enzyme has high activity, can effectively improve the conversion efficiency, and can shorten the conversion time; the enzyme and the technical method provided by the invention have the advantages of mild reaction conditions and less pollution, and have good industrial prospects in the production of florfenicol synthesis intermediates.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to an L-threonine aldolase mutant and application thereof in catalyzing the synthesis of p-methylsulfonylphenylserine. Background Art

[0002] Florfenicol is a veterinary chloramphenicol-type broad-spectrum antibacterial drug developed by the American company Senlin-Boya. It has good resistance to Gram-negative and Gram-positive bacteria and is used for broad-spectrum antibacterial treatment of bacterial diseases in cattle, pigs, poultry, etc. Currently, florfenicol has been widely used in veterinary clinical prevention and treatment of bacterial diseases.

[0003] (2S,3R)-4-Methylsulfonylphenylserine [(2S,3R)-MPS] is the key chiral precursor of the antibiotic florfenicol. This amino acid intermediate is then ethyl esterified to obtain the target product (2S,3R)-p-methylsulfonylphenylserine ethyl ester. This precursor can be asymmetric synthesized by L-threonine transaldolase (LTTA) and acetaldehyde elimination system. In recent years, the synthesis of (2S,3R)-MPS includes chemical and enzymatic pathways. The chemical pathway requires chiral resolution, which is inefficient and the resolution steps are very cumbersome. In the process of route synthesis, a large amount of copper salts are used, which harms the environment. The enzymatic method is obviously the most feasible method for synthesizing (2S.3R)-MPS because of its mild reaction conditions and eco-friendly nature. β-Hydroxy-α-amino acids play an important role in chemical synthesis and drug manufacturing. Threonine aldolase (TA) is a pyridoxal-dependent enzyme. According to the stereospecificity of the Cα atom of its product, it can be divided into L-threonine aldolase (LTA) and D-threonine aldolase (DTA). Since almost all active intermediates are L-isomers,

[0004] Therefore, for industrial applications, it is necessary to discover and characterize novel L-threonine aldolases with high stereospecificity to expand the production of β-hydroxy-α-amino acid intermediates. Summary of the invention

[0005] The present invention aims to provide an L-threonine aldolase mutant and its application in catalytic synthesis of p-methylsulfonylphenylserine, and screen out an L-threonine aldolase mutant, which can improve the yield of (2S, 3R)-MPS enzymatic synthesis.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a L-threonine aldolase mutant is provided, wherein the L-threonine aldolase mutant is obtained by replacing the cysteine ​​at position 57 with asparagine, the histidine at position 69 with tyrosine, and the aspartic acid at position 391 with glutamine in the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence of the L-threonine aldolase mutant is shown in SEQ ID NO.2.

[0008] In the second aspect of the present invention, a nucleic acid molecule encoding the L-threonine aldolase mutant is provided.

[0009] In the third aspect of the present invention, an L-threonine aldolase mutant expression vector is provided, wherein the expression vector comprises the nucleic acid molecule.

[0010] Furthermore, the expression vector comprises one of a prokaryotic expression vector and a viral vector.

[0011] In the fourth aspect of the present invention, a recombinant bacterium or an engineered cell line comprising the expression vector is provided.

[0012] In the fifth aspect of the present invention, an engineered strain is provided, wherein the engineered strain is a genetically engineered bacterium expressing a mutant enzyme by co-expressing an L-threonine aldolase mutant having an amino acid sequence as shown in SEQ ID NO.2 and a yeast alcohol dehydrogenase ADH1 having an amino acid sequence as shown in SEQ ID NO.3, using Escherichia coli as a host bacterium.

[0013] As a specific embodiment, the nucleotide sequence of the nucleic acid molecule encoding the wild-type L-threonine aldolase of the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.4. The nucleotide sequence of the nucleic acid molecule encoding the mutant L-threonine aldolase of the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.5.

[0014] In the sixth aspect of the present invention, provided is the use of the L-threonine aldolase mutant, the nucleic acid molecule, the expression vector, the recombinant bacteria or engineered cell line or the engineered strain in the catalytic synthesis of p-methylsulfonylphenylserine.

[0015] Furthermore, in the application, the reaction substrates are: p-methylsulfonylbenzaldehyde and L-threonine.

[0016] In a seventh aspect of the present invention, a method for catalytically synthesizing p-methylsulfonylphenylserine is provided, the method comprising:

[0017] Using p-methylsulfonylbenzaldehyde and L-threonine as substrates, the L-threonine aldolase mutant is used to catalyze the synthesis of p-methylsulfonylphenylserine;

[0018] Alternatively, the recombinant bacteria or the engineered cell line or the engineered strain is used to synthesize the product p-methylsulfonylphenylserine in vivo.

[0019] Furthermore, in the in vitro catalytic synthesis reaction, the reaction system for catalytically synthesizing p-methylsulfonylphenylserine contains: threonine, p-methylsulfonylbenzaldehyde, pyridoxal phosphate and reduced nicotinamide adenine dinucleotide coenzyme, Tris-HCl buffer and lysing enzyme solution.

[0020] As a specific embodiment, the preparation method of the above-mentioned lysing enzyme solution includes:

[0021] (1) The nucleotide sequences encoding the proteins shown in SEQ ID NO.1 and SEQ ID NO.2 were cloned into the pET28a vector, and the sequence gene shown in SEQ ID NO.3 was cloned into the pET15b vector to construct recombinant expression plasmids pET28a-PsLTTA (WT), pET28a-PsLTTA (MUT) and pET15b-ADE1 respectively.

[0022] (2) The above-mentioned recombinant plasmids pET28a-PsLTTA(WT) / pET15b-ADE1 and pET28a-PsLTTA(MUT) / pET15b-ADE1 were combined in pairs and then transformed into Escherichia coli BL21(DE3) to obtain recombinant genetically engineered bacteria. After fermentation and cultivation, the recombinant bacteria were collected by centrifugation to obtain the whole cells required for the catalytic reaction, and ultrasonically disrupted to obtain the lytic enzyme solution.

[0023] Preferably, the reaction temperature is 30° C. and the reaction time is 24 h.

[0024] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0025] 1. The L-threonine aldolase mutant provided by the present invention can have significantly improved activity compared with the wild type. The entire reaction is carried out at 30°C, the reaction conditions are milder, the operation is simple, and the separation is convenient. The yield is increased to more than 98%, and it has better industrial prospects.

[0026] 2. The present application provides an engineered strain, in which L-threonine aldolase (PsLTTA) and alcohol dehydrogenase (ADH1) from Pseudomonas putida are cloned into two expression vectors, pET28a and pET15b, respectively, and co-transformed into Escherichia coli BL21 (DE3) for co-expression, thereby obtaining a genetically engineered bacterium that co-expresses L-threonine aldolase and alcohol dehydrogenase. The enzyme encoded by the bacterium can catalyze the condensation of p-methylsulfonylbenzaldehyde and threonine to synthesize p-methylsulfonylphenylserine, and the conversion efficiency can reach more than 92%.

[0027] L-threonine aldolase (LTTA) has good stereoselectivity on C-β in the synthesis of amino acid intermediates and is an ideal catalyst for the asymmetric synthesis of (2S, 3R)-MPS. At the same time, the by-product acetaldehyde will inhibit LTTA, which is not conducive to the accumulation of (2S, 3R)-MPS. In order to solve these technical problems, the present application uses alcohol dehydrogenase to convert the by-product acetaldehyde into ethanol, constructs a fusion expression enzyme, and converts the toxic intermediate into a non-toxic product faster. By optimizing the enzymatic conversion system, we can increase the yield of the enzymatic synthesis of (2S, 3R)-MPS, and provide a promising enzymatic method for the large-scale synthesis of the florfenicol precursor product (2S, 3R)-MPS, which is helpful for industrial biosynthesis applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 It is a schematic diagram of the L-threonine aldolase catalytic reaction of the present invention.

[0030] Figure 2 This is a protein purification diagram after the whole cell lysis of the recombinant bacteria pET28a-PsLTTA (WT) of the present invention. Note: M: maker; 1: D392Q precipitation; 2: D392Q supernatant; 3: D392Q flow-through; 4: BufferA Wash 5: 2.5% imidazole; 6: 25% imidazole; 7: 50% imidazole; 8: BufferC Wash; 9: 2.5% BufferD; 10: 25% BufferD; 11: 50% BufferD; 12: D392Q ultrafiltration.

[0031] Figure 3This is a protein purification diagram after the whole cell lysis of the recombinant bacteria pET28a-PsLTTA (MUT) of the present invention. Note: M: maker; 1: PsLTTA supernatant; 2: PsLTTA flow-through; 3: BufferA Wash; 4: 5% imidazole; 5: 25% imidazole; 6: 50% imidazole; 7: PsLTTA ultrafiltration; 8: D392Q ultrafiltration.

[0032] Figure 4 This is a diagram of protein purification after the whole cell lysis of the recombinant bacteria pET28a-ADE1 of the present invention. Note: 1: ADH1 supernatant; 2: ADH1 precipitate; 3: ADH1 whole cell lysate; M: maker.

[0033] Figure 5 It is a retention time peak diagram of the catalytic reaction substrate p-methylsulfonylbenzaldehyde and the product p-methylsulfonylphenylserine of the present invention.

[0034] Figure 6 The HPLC chromatogram of p-methylsulfonylphenylserine obtained after the wild-type L-threonine aldolase of the present invention catalyzes the reaction for 24 hours.

[0035] Figure 7 The HPLC chromatogram of the mutant L-threonine aldolase of the present invention after 24 hours of catalytic reaction compared with the wild type. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0037] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods. The pET28a and pET15b of the present application are conventional commercially available vectors.

[0039] The following is a detailed description of an L-threonine aldolase mutant of the present application and its application in catalyzing the synthesis of p-methylsulfonylphenylserine in combination with examples and experimental data.

[0040] Example 1 Construction of composite L-threonine aldolase and its mutant engineering bacteria

[0041] 1. Select wild-type L-threonine aldolase from Pseudomonas putida and alcohol dehydrogenase from yeast, obtain the original sequence by prokaryotic Escherichia coli codon optimization gene synthesis, clone and insert the synthesized gene sequence into pET28a and pET15b vectors to obtain a fusion plasmid vector. The amino acid sequence corresponding to the synthesized nucleic acid sequence is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the insertion sites are BamHI and XhoI to obtain a recombinant plasmid. The nucleotide sequence of the nucleic acid molecule encoding the wild-type L-threonine aldolase of the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.4. The nucleotide sequence of the nucleic acid molecule encoding the mutant L-threonine aldolase of the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.5. The sequence is as follows:

[0042] Table 1 Synthetic sequences

[0043]

[0044] Note: SEQ ID NO.1: L-threonine aldolase PsLTTA wild-type amino acid sequence; SEQ ID NO.2: L-threonine aldolase PsLTTA mutant amino acid sequence; SEQ ID NO.3: yeast alcohol dehydrogenase ADH1 amino acid sequence.

[0045] (1) The cysteine ​​at position 57 of the amino acid sequence shown in SEQ ID NO. 1 is replaced with asparagine.

[0046] (2) The histidine at position 69 of the amino acid sequence shown in SEQ ID NO.1 is replaced with tyrosine.

[0047] (3) The aspartic acid at position 391 of the amino acid sequence shown in SEQ ID NO.1 is replaced with glutamine.

[0048] Depend on Figure 7 As a result, it can be seen that the catalytic activity of the mutant of the present application is significantly improved compared with the wild type.

[0049] 2. The recombinant plasmids constructed above were transformed into E. coli DH5α, plated, and single colonies were selected for colony PCR to obtain positive transformants and sequenced to confirm that the recombinant plasmids containing the correct sequence were obtained. Recombinant expression plasmids pET28a-PsLTTA (WT), pET28a-PsLTTA (MUT) and pET15b-ADE1 were constructed respectively.

[0050] 3. The correctly sequenced recombinant expression plasmids pET28a-PsLTTA (MUT) and pET15b-ADE1 were co-transformed into Escherichia coli BL21 (DE3), plated, and single colonies were selected for culture to construct genetically engineered bacteria expressing mutant enzymes. They were stored in a final concentration of 30% glycerol and placed in a -80°C refrigerator for use. At the same time, the correctly sequenced recombinant expression plasmids pET28a-PsLTTA (WT) and pET15b-ADE1 were co-transformed into Escherichia coli BL21 (DE3), plated, and single colonies were selected for culture to construct wild-type genetically engineered bacteria as controls.

[0051] Example 2: Fermentation and expansion of bacterial cells

[0052] 1. The wild-type genetically engineered bacteria and mutant genetically engineered bacteria of L-threonine aldolase prepared in Example 1 were fermented and expanded in a 5L small fermentation tank.

[0053] Table 2 Ratio of fermentation ingredients

[0054]

[0055] 2. Add the single clone to 5 ml of resistant LB medium overnight. Take 3 ml and add it to 300 ml of resistant LB medium and culture for 4 hours until OD 600 =0.8-1.0 Take 300ml of the first-grade seed solution and add it to 2700ml of the resistance fermentation medium. Parameter settings: speed 150, pH 7.0, pressure 0.05MPa, temperature 37℃. Induce culture for about 8h, OD 600 =20, add IPTG for induction, 30℃ for 12-16h, OD 600 =40 Harvest bacteria.

[0056] The total amount of bacterial sludge collected from the 3.3L fermentation tank was about 150g, and the content of the collected bacterial sludge was about 50g / L. The fermentation inoculation amount was 10%, the induction expression temperature was 30℃, and the fermentation time was 22h. The large amount of bacterial sludge obtained from the fermentation provided a basis for the optimization of the subsequent transformation system.

[0057] Example 3: Synthesis of p-methylsulfonylphenylserine catalyzed by L-threonine aldolase

[0058] 1. p-Methylsulfonylphenylserine (2S, 3R)-MPS is an intermediate product synthesized from p-methylsulfonylbenzaldehyde and threonine by L-threonine aldolase, and its concentration is detected by HPLC. The conversion system includes 100mL reaction buffer containing 100mM Tris-HCl (pH 7.0), a total volume of no more than 5% isopropanol, a final concentration of 0.25mM PLP, 0.25mM NADH, 4g p-methylsulfonylbenzaldehyde, 3.88g L-threonine, and a cell lysate of 4g bacterial mud. The above reactants are added to a 250ml reaction bottle, heated to 30°C and magnetically stirred to mix, and a sample is taken after 24h to detect the conversion rate by HPLC.

[0059] The results are as follows Figure 6 and Figure 7 As shown, the conversion rate of the wild-type enzyme reached about 92%, and the conversion rate of the mutant enzyme reached more than about 98%.

[0060] 2. The mobile phase for HPLC detection is: NaH2PO4 / acetonitrile (4 / 1). The filter is filtered through a 0.22um pore size membrane, and the mobile phase components are ultrasonically degassed for 15 minutes at a flow rate of 1 ml / min. After the reaction of the conversion system, the product (2S, 3R) MPS characteristic peak appears at 2.55 minutes, and the substrate 4-methylsulfonylbenzaldehyde characteristic peak appears at 11.52 minutes.

[0061] 3. Further system suitability solution: Take appropriate amount of p-methylsulfonylbenzaldehyde and p-methylsulfonylphenylserine standard samples, place them in a sample bottle, add appropriate amount of mobile phase solution used for detection to dilute different gradients, and determine the retention time of characteristic peaks of each component of the substrate and product obtained after the conversion system reaction.

[0062] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0063] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0064] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0065] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A L-threonine aldolase mutant, characterized in that: The L-threonine aldolase mutant is obtained by replacing the 57th cysteine ​​with asparagine, the 69th histidine with tyrosine, and the 391st aspartic acid with glutamine in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of the L-threonine aldolase mutant is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the L-threonine aldolase mutant according to claim 1.

3. An expression vector for an L-threonine aldolase mutant, characterized in that: The expression vector comprises the nucleic acid molecule of claim 2.

4. A recombinant bacterium or engineered cell line comprising the expression vector according to claim 3.

5. An engineered strain, characterized in that: The engineered strain is a genetically engineered bacterium expressing a mutant enzyme by co-expressing an L-threonine aldolase mutant having an amino acid sequence shown in SEQ ID NO.2 and a yeast alcohol dehydrogenase ADH1 having an amino acid sequence shown in SEQ ID NO.3, using Escherichia coli as a host bacterium.

6. Use of the L-threonine aldolase mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3, the recombinant bacterium or engineered cell line according to claim 4, or the engineered strain according to claim 5 in catalyzing the synthesis of p-methylsulfonylphenylserine.

7. The application according to claim 6, characterized in that: The reaction substrates of the catalytic synthesis are p-methylsulfonylbenzaldehyde and L-threonine.

8. A catalytic product, characterized in that The invention comprises at least one of the L-threonine aldolase mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3, the recombinant bacteria or engineered cell line according to claim 4, and the engineered Bacillus strain according to claim 5.

9. A method for catalytic synthesis of p-methylsulfonylphenylserine, characterized in that: The method comprises: Using p-methylsulfonylbenzaldehyde and L-threonine as substrates, the L-threonine aldolase mutant according to claim 1 is used to catalyze the synthesis of p-methylsulfonylphenylserine; Alternatively, the recombinant bacteria or engineered cell line according to claim 4 or the engineered strain according to claim 5 is used to synthesize the product p-methylsulfonylphenylserine in vivo.

10. The method for catalytic synthesis of p-methylsulfonylphenylserine according to claim 9, characterized in that: The reaction system for catalyzing the synthesis of p-methylsulfonylphenylserine contains: threonine, p-methylsulfonylbenzaldehyde, pyridoxal phosphate and reduced nicotinamide adenine dinucleotide coenzyme, Tris-HCl buffer and lysing enzyme solution.

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