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

Abstract

The present invention provides a kind of L-threonine aldolase mutant and its application in catalytic synthesis of p-methylsulfonylphenylserine, the L-threonine aldolase mutant source Pseudomonas of the present invention, can use threonine and p-methylsulfonylbenzaldehyde as substrate, with pyridoxal phosphate and reduced nicotinamide adenine dinucleotide as coenzyme, catalysis obtains the synthesis of florfenicol intermediate p-methylsulfonylphenylserine, compared to wild-type L-threonine aldolase, mutant enzyme has higher activity, can effectively improve conversion efficiency, shorten conversion time. The enzyme and technical method reaction conditions of the present invention are mild, pollution is small, and there is good industrial prospect in the production of florfenicol synthetic intermediate.

Description

Technical Field

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

[0002] Florfenicol, a broad-spectrum chloramphenicol antibiotic developed by the US-based company Senlin-Plough, is used for veterinary use. It has good resistance to both Gram-negative and Gram-positive bacteria and is used for the broad-spectrum treatment of bacterial diseases in cattle, pigs, and poultry. Currently, florfenicol is widely used in veterinary clinical practice to prevent and treat bacterial diseases.

[0003] (2S, 3R)-4-Methylsulfonylphenylserine [(2S, 3R)-MPS] is a key chiral precursor of the antibiotic florfenicol. This amino acid intermediate can be further ethyl esterified to yield the target product, (2S, 3R)-4-methylsulfonylphenylserine ethyl ester. This precursor can be asymmetric synthesized using L-threonine transaldolase (LTTA) and an acetaldehyde elimination system. In recent years, the synthesis of (2S, 3R)-MPS has been pursued through chemical and enzymatic pathways. The chemical route requires chiral resolution, which is inefficient and cumbersome, and also involves the use of large amounts of copper salts, which pose an environmental risk. Enzymatic methods, however, offer mild reaction conditions and are environmentally friendly, making them the most viable method for synthesizing (2S, 3R)-MPS. β-Hydroxy-α-amino acids play an important role in chemical synthesis and drug manufacturing. Threonine aldolase (TA) is a pyridoxal-dependent enzyme. It can be divided into L-threonine aldolase (LTA) and D-threonine aldolase (DTA) according to the stereospecificity of the Cα atom of its product. 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 catalyzing the synthesis of p-methylsulfonylphenylserine, and screen out an L-threonine aldolase mutant that 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 solutions:

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

[0011] In the fourth aspect of the present invention, a recombinant bacterium or 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 the amino acid sequence shown in SEQ ID NO.2 and a yeast alcohol dehydrogenase ADH1 having the amino acid sequence shown in SEQ ID NO.3, using Escherichia coli as the host bacterium.

[0013] As a specific embodiment, the nucleotide sequence of the nucleic acid molecule encoding the wild-type L-threonine aldolase with 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 with 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 catalyzing the 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 engineered cell line or 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 lyase solution.

[0020] As a specific embodiment, the preparation method of the above-mentioned lyase 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 the 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 culture, 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 has significantly improved activity compared to the wild type. The entire reaction is carried out at 30°C, the reaction conditions are milder, the operation is simple, and 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) exhibits excellent stereoselectivity at the C-β position in the synthesis of amino acid intermediates, making it an ideal catalyst for the asymmetric synthesis of (2S,3R)-MPS. However, the byproduct acetaldehyde inhibits LTTA, hindering the accumulation of (2S,3R)-MPS. To address these technical challenges, this application employed alcohol dehydrogenase to convert the byproduct acetaldehyde into ethanol, constructing a fusion expression enzyme that allows for faster conversion of the toxic intermediate into a non-toxic product. By optimizing the enzymatic conversion system, we were able to increase the yield of (2S,3R)-MPS enzymatic synthesis, providing a promising enzymatic method for the large-scale synthesis of (2S,3R)-MPS, a precursor of florfenicol, and facilitating industrial biosynthetic 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 is a brief introduction to 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 paying any creative work.

[0029] Figure 1 Schematic diagram of the reaction catalyzed by L-threonine aldolase of the present invention.

[0030] Figure 2 This is a diagram of protein purification after whole-cell lysis of the recombinant pET28a-PsLTTA (WT) strain of the present invention. Note: M: maker; 1: D391Q precipitate; 2: D391Q supernatant; 3: D391Q flowthrough; 4: Buffer A Wash; 5: 2.5% imidazole; 6: 25% imidazole; 7: 50% imidazole; 8: Buffer C Wash; 9: 2.5% Buffer D; 10: 25% Buffer D; 11: 50% Buffer D; 12: D391Q ultrafiltration.

[0031] Figure 3This is a diagram of protein purification after whole-cell lysis of the recombinant strain pET28a-PsLTTA (MUT). Note: M: maker; 1: PsLTTA supernatant; 2: PsLTTA flow-through; 3: Buffer A Wash; 4: 5% imidazole; 5: 25% imidazole; 6: 50% imidazole; 7: PsLTTA ultrafiltration; 8: D391Q ultrafiltration.

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

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

[0034] Figure 6 The figure is an 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 compared with the wild type after 24 hours of catalytic reaction. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below in conjunction with specific embodiments 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 embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0037] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or can be obtained by existing methods. The pET28a and pET15b in this 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 the catalytic synthesis of p-methylsulfonylphenylserine in combination with examples and experimental data.

[0040] Example 1 Construction of composite L-threonine aldolase and its mutant engineered 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 the synthesized gene sequence and insert it 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. 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 with 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 with 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: amino acid sequence of wild-type L-threonine aldolase PsLTTA; SEQ ID NO.2: amino acid sequence of mutant L-threonine aldolase PsLTTA; SEQ ID NO.3: amino acid sequence of yeast alcohol dehydrogenase ADH1.

[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 The results show 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. Positive transformants were sequenced to confirm the correct sequence of the recombinant plasmid. The recombinant expression plasmids pET28a-PsLTTA (WT), pET28a-PsLTTA (MUT), and pET15b-ADE1 were constructed.

[0050] 3. Transform the correctly sequenced recombinant expression plasmids pET28a-PsLTTA (MUT) and pET15b-ADE1 into E. coli BL21(DE3), plate them, and select a single colony for culture to construct a genetically engineered bacterium expressing the mutant enzyme. Store the bacterium in a final concentration of 30% glycerol and store it in a -80°C freezer until ready for use. Simultaneously, transform the correctly sequenced recombinant expression plasmids pET28a-PsLTTA (WT) and pET15b-ADE1 into E. coli BL21(DE3), plate them, and select a single colony for culture to construct a wild-type genetically engineered bacterium as a control.

[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 5 L small fermentation tank.

[0053] Table 2 Ratio of fermentation ingredients

[0054]

[0055] 2. Add the single clone to 5 ml of resistant LB medium and incubate overnight. Take 3 ml and add it to 300 ml of resistant LB medium and incubate for 4 hours until the OD 600 =0.8-1.0 Take 300ml of the first-grade seed solution and add it to 2700ml of 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, induce at 30℃ for 12-16h, OD 600 =40 harvest bacteria.

[0056] 3. The 3L fermenter culture yielded approximately 150g of sludge, with a sludge content of approximately 50g / L. The inoculum size was 10%, the expression induction temperature was 30°C, and the fermentation time was 22h. This large amount of sludge provided a foundation for optimizing 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 in the synthesis of florfenicol from p-Methylsulfonylbenzaldehyde and threonine, catalyzed by L-threonine aldolase. Its concentration was determined by HPLC. The conversion system consisted of 100 mL of reaction buffer containing 100 mM Tris-HCl (pH 7.0) with no more than 5% isopropanol, a final concentration of 0.25 mM PLP, 0.25 mM NADH, 4 g p-Methylsulfonylbenzaldehyde, 3.88 g L-threonine, and 4 g of bacterial slurry containing cell lysate. These reactants were added to a 250 mL reaction flask, heated to 30°C, and magnetically stirred to mix thoroughly. After 24 hours, samples were collected and the conversion rate was determined 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. HPLC analysis was performed using a mobile phase consisting of NaH₂PO₄ / acetonitrile (4 / 1). Filter the mixture through a 0.22 μm pore size membrane and ultrasonically degas the mobile phase for 15 minutes at a flow rate of 1 ml / min. After the reaction, the resulting components exhibited a characteristic peak for the product (2S,3R) MPS at 2.55 minutes, and a characteristic peak for the substrate, 4-methylsulfonylbenzaldehyde, at 11.52 minutes.

[0061] 3. Further system suitability solution: Take an appropriate amount of p-methylsulfonylbenzaldehyde and p-methylsulfonylphenylserine standard samples, place them in a sample bottle, add an appropriate amount of the mobile phase solution used for detection to dilute different gradients, and determine the retention time of the 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 considered as equivalent replacement methods and are included in the scope of protection 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 list of elements includes not only those elements, but also other elements not expressly listed, or elements 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,

[0066] It is intended that the present invention also includes these modifications and variations.

Claims

1. An L-threonine aldolase mutant, characterized in that 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. 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 catalytically synthesizing 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 catalytically synthesizing p-methylsulfonylphenylserine according to claim 9, characterized in that: 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.

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