D-threonine aldolase mutant and application thereof in preparation of D-configuration beta-hydroxyl-alpha-amino acid
By mutating the gene of D-threonine aldolase, a highly selective D-threonine aldolase mutant was constructed, which solved the problem of low catalytic selectivity of D-configuration β-hydroxy-α-amino acids in the existing technology, and realized a production process with high optical purity and environmental protection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-01-23
- Publication Date
- 2026-05-01
AI Technical Summary
Existing technologies are difficult to efficiently catalyze the generation of single-configuration D-type β-hydroxy-α-amino acids, and natural D-threonine aldolases have low selectivity for β-carbons, resulting in complex product separation and low optical purity.
By mutating the gene of D-threonine aldolase and introducing specific amino acid substitutions, a D-threonine aldolase mutant was constructed. In the presence of pyridoxal phosphate and manganese ions, a condensation reaction was carried out using glycine and benzaldehyde derivatives to generate a D-configuration β-hydroxy-α-amino acid.
It achieves highly selective catalytic generation of D-configuration β-hydroxy-α-amino acids, simplifies the production process, improves the optical purity and atom utilization of the product, conforms to the concept of green chemistry, and reduces the difficulty of separation and purification.
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Abstract
Description
D-threonine aldolase mutant and its application in the preparation of D-configuration β-hydroxy-α-amino acids Technical Field
[0001] This invention relates to the field of enzyme engineering technology, specifically to D-threonine aldolase mutants and their application in the preparation of D-configuration β-hydroxy-α-amino acids. Background Technology
[0002] β-hydroxy-α-amino acids are important chiral intermediates widely used in the synthesis of pharmaceuticals, pesticides, and fine chemicals. β-hydroxy-α-amino acids have two chiral centers and four isomers: L-syn-β-hydroxy-α-amino acids, L-anti-β-hydroxy-α-amino acids, D-syn-β-hydroxy-α-amino acids, and D-anti-β-hydroxy-α-amino acids. Currently, there are several reports on the synthesis of high-optically pure L-syn-β-hydroxy-α-amino acids and L-anti-β-hydroxy-α-amino acids catalyzed by L-threonine aldolase, but research on the synthesis of D-configuration β-hydroxy-α-amino acids is relatively limited.
[0003] There are few methods for producing D-configuration β-hydroxy-α-amino acids. Generally, a chemical synthesis method is used, employing copper sulfate as a catalyst. Utilizing the complexation effect of metal ions, aldehydes and glycine selectively react to produce two cis-products in a 1:1 ratio: L-syn-β-hydroxy-α-amino acid copper and D-syn-β-hydroxy-α-amino acid copper. These are then resolved and purified. This method cannot produce L-anti-β-hydroxy-α-amino acids and D-anti-β-hydroxy-α-amino acids.
[0004] In recent years, a new biosynthetic method has emerged using D-threonine aldolase to catalyze the synthesis of D-configuration β-hydroxy-α-amino acids. However, currently reported D-threonine aldolases exhibit high selectivity for the α-carbon (ee (%) > 99%), but low selectivity for the β-carbon. Studies have shown that most natural D-threonine aldolase reactions simultaneously produce D-syn-β-hydroxy-α-amino acids and D-anti-β-hydroxy-α-amino acids, with a preference for D-syn-β-hydroxy-α-amino acids. syn The concentration values are between 20% and 80%, and no D-threonine aldolases that preferentially generate D-anti-β-hydroxy-α-amino acids have been reported. Furthermore, there are currently no research reports on D-threonine aldolases capable of naturally catalyzing the generation of single-configuration D-syn-β-hydroxy-α-amino acids and D-anti-β-hydroxy-α-amino acids. Therefore, obtaining de-oxidized D-threonine aldolases through industrial modification is a promising prospect. syn Value close to 100% and de antiThe D-threonine aldolase mutant with a value close to 100% is of great significance. Summary of the Invention
[0005] This invention addresses the shortcomings of existing D-configuration β-hydroxy-α-amino acid synthesis processes by providing a D-threonine aldolase mutant and its application in the preparation of D-configuration β-hydroxy-α-amino acids.
[0006] The specific technical solution is as follows: In a first aspect, the present invention provides a D-threonine aldolase mutant, wherein the mutant has 7 or fewer substitutions relative to SEQ ID NO.2 and has less than 100% sequence identity with SEQ ID NO.2, wherein the substitutions are selected from: Q171A, Q171C, Q171D, Q171E, Q171F, Q171G, Q171H, Q171I, Q171K, Q171L, Q171M, Q171P, Q171R, Q171S, Q171T, Q171V, Q171Y, A266C, A266D, A266E, A266G, A266H, A266I, A266K, A266L, A266M, A266N, A266P, A266R, A266S, A266T, A266V, A266Y, S251A, S251C, S251D, S251G, S251H, S251K, S251L, S251M, S251N, S251P, S251Q, S251T, S251V, S251W, S251Y, S206P, N336S, N336A, H209K, C319Y and C319K, or combinations thereof.
[0007] Further, the substituted material is selected from: Q171A, Q171F, Q171H, Q171T, Q171Y, A266C, A266K, A266P, A266V, S251A, S251G, S251T, S206P, N336S, N336A, H209K, C319Y, and C319K, or combinations thereof.
[0008] In a second aspect, the present invention provides a gene encoding the D-threonine aldolase mutant.
[0009] In a third aspect, the present invention provides a recombinant vector comprising a gene encoding a D-threonine aldolase mutant.
[0010] Furthermore, the recombinant vector is a pET28a plasmid containing a gene encoding a D-threonine aldolase mutant.
[0011] In a fourth aspect, the present invention provides a recombinant microorganism comprising the aforementioned gene or recombinant vector.
[0012] In a fifth aspect, the present invention provides a catalyst comprising the aforementioned D-threonine aldolase mutant or recombinant microorganism; the catalyst being an enzyme preparation of the mutant, an immobilized enzyme, or a whole cell or cell lysate of the recombinant microorganism.
[0013] In a sixth aspect, the present invention provides the use of the D-threonine aldolase mutant, the recombinant microorganism, or the catalyst in the preparation of D-β-hydroxy-α-amino acids; wherein the D-β-hydroxy-α-amino acid is a D-syn-β-hydroxy-α-amino acid or a D-anti-β-hydroxy-α-amino acid.
[0014] In a seventh aspect, the present invention provides a method for preparing D-configuration β-hydroxy-α-amino acids, using glycine and a benzaldehyde derivative as substrates, and in the presence of pyridoxal phosphate and manganese ions, using the catalyst, to carry out a condensation reaction to generate D-configuration β-hydroxy-α-amino acids; further, the benzaldehyde derivative is one of p-methylsulfonylbenzaldehyde, p-nitrobenzaldehyde, and 2,4-dihydroxybenzaldehyde.
[0015] Furthermore, the D-β-hydroxy-α-amino acid is a D-syn-β-hydroxy-α-amino acid or a D-anti-β-hydroxy-α-amino acid.
[0016] Furthermore, the reaction is carried out in an organic solvent-water mixture or an aqueous solution.
[0017] More preferably, the organic solvent is DMSO.
[0018] More preferably, the amount of DMSO added does not exceed 20% of the total volume.
[0019] Compared with the prior art, the present invention has the following advantages: (1) The production process is simple and the reaction conditions are mild; (2) The D-threonine aldolase mutant has high selectivity and high optical purity of the product, and no separation is required; (3) The atom utilization rate is high, the production process is environmentally friendly, and it conforms to the concept of green chemistry; (4) The product separation and purification are simple and the subsequent processing is easy. Attached Figure Description
[0020] Figure 1 shows schematic diagrams of the molecular structures of two configurations of the D-configuration β-hydroxy-α-amino acid.
[0021] Figure 2 shows the equation for the biosynthesis of D-configuration β-hydroxy-α-amino acids.
[0022] Figure 3 shows the high-performance liquid chromatography (HPLC) spectra of D-anti-β-hydroxy-α-amino acid and D-syn-β-hydroxy-α-amino acid standards.
[0023] Figure 4 shows the high-performance liquid chromatography (HPLC) spectra of substrates and products in the reaction solution after the preparation of D-syn-β-hydroxy-α-amino acids by D-threonine aldolase.
[0024] Figure 5 shows the high-performance liquid chromatography (HPLC) spectra of substrates and products in the reaction solution after the preparation of D-anti-β-hydroxy-α-amino acids by D-threonine aldolase. Detailed Implementation
[0025] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.
[0026] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.
[0027] Unless otherwise specified, 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 application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.
[0028] The reagents used in the catalytic process, including glycine, p-methylsulfonylbenzaldehyde, p-nitrobenzaldehyde, 2,4-dihydroxybenzaldehyde, pyridoxal phosphate, and manganese chloride, were all commercially available analytical grade.
[0029] In this invention, the reaction in which glycine and the above-mentioned aldehyde are converted into D-configuration β-hydroxy-α-amino acids under the catalysis of D-threonine aldolase is shown in the following formula:
[0030] This invention uses high-performance liquid chromatography (HPLC) to analyze the concentrations of substrates and products in the reaction solution and monitor the progress of the reaction.
[0031] The HPLC analysis method was as follows: The chromatographic column was CHIRALPAK ZWIX (-) (DAICELCO., LTD, Japan); the mobile phase was 1 L methanol, 1.9 mL formic acid, and 2.6 mL ethylenediamine; the flow rate was 0.5 mL / min; the column temperature was 40℃; and the detection wavelength was 225 nm. The peak characteristics are shown in Figure 3-5: aldehyde approximately 2.9 min, D-syn-β-hydroxy-α-amino acid approximately 6.2 min, and D-anti-β-hydroxy-α-amino acid approximately 7.4 min.
[0032] This invention characterizes the selectivity of D-threonine aldolase and its mutants using the de value, as shown in the following formula:
[0033] Example 1: Construction of Wild-Type D-Threonine Aldolase Engineered Bacteria. The D-threonine aldolase gene was retrieved from the NCBI database, and the D-threonine aldolase (XD74) with accession number WP_048940648.1 was selected. Its base sequence is shown in SEQ ID NO.1. The five amino acid sequences were converted into nucleotide sequences through codon optimization, as shown in SEQ ID NO.2. The XD74 nucleotide sequence was chemically synthesized (Qingke Biotechnology) and integrated between the BamHI and HindIII restriction sites of the expression vector pET-28a(+). Finally, the constructed plasmid was introduced into *E. coli* BL21(DE3) host cells to construct an engineered bacterium with high amino substrate recognition of D-threonine aldolase.
[0034]
[0035] SEQ ID NO.2: EFMDSGRCAERTLVTTRLSHLSNVLTLPDPALPGQPLAQVDTPSLVLDLDVFEANLGAMQQWADRHGVALRPHAKAHKCPEIARRQLALGARGICCQKVSEALPFLEAGIRDIHISNEVVGPAKLALLAQIAVRADVSVCVDNMTNLHALAAAMAQAGARITVLVEVDVGQGRCGVTDDHDVLALARAAEAMQGLR FGGLQAYHGSVQHVRGHAERAAICAQAAARAAGYASLLRAHGITCERITGGGTGSAEFDAASGVYTELQAGSYAFMDGDYGANEWSGPLSFGTSLFLLSTVMSTPAPGRVILDAGLKSVTIECGLPQVHDRPGLVYAAANDEHGVVRVQEGAVAPALGEVLRLVVPHVDPGFNLHDTLVAYRDETVVGLWPISARGLSRLE.
[0036] Example 2 Construction of D-Threonine Aldolase Mutant 1. Activation and Plasmid Extraction of Engineered Bacteria The engineered bacteria (E. coli BL21(DE3), obtained in Example 1) were activated and cultured using LB medium with the following formula: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, dissolved in deionized water and brought to a final volume. The mixture was then sterilized at 121°C for 20 min and set aside for use. The solid culture medium was LB medium with 1-2% agar added.
[0037] The preserved engineered bacterial glycerol tubes were inoculated into test tubes containing 10 mL of LB medium and cultured overnight at 37°C and 220 rpm. After obtaining the cultured bacterial cells, plasmids were extracted according to the instructions of the Axygen plasmid extraction kit. The obtained plasmids can be used directly for point mutagenesis or stored long-term at -20°C.
[0038] 2. Site-directed gene mutation: Gene mutation was obtained by whole plasmid PCR.
[0039] Table 1 PCR amplification system (50 μL)
[0040] PCR amplification program: (1) Pre-denaturation: 98℃ for 5 min; (2) Denaturation: 98℃ for 30 s; Annealing: 60℃ for 30 s; Extension: 72℃ for 90 s; 30 cycles in total; (3) Post-extension: 72℃ for 10 min; (4) Store at 4℃.
[0041] After PCR amplification, the amplification product was detected by 0.9% agarose gel electrophoresis. The results showed that the amplification product was a single band, approximately 6500 bp in size. The amplification product was purified and recovered using a DNA purification kit. The specific steps were followed according to the kit's instructions. The primers are shown in Table 2.
[0042] Table 2. Mutation primers for different mutants
[0043] 3. Construction of the mutant engineered bacteria: The purified gene fragment was digested with DpnI to remove the template, and then recombined with recombinase. The recombinant product was transformed into E. coli BL21(DE3) competent cells, plated, and single colonies were picked and cultured in LB liquid. Positive transformants were identified by PCR, and the correctness of the mutation site was verified by sequencing. After verification, sterile glycerol was added to a final concentration of 15%, labeled, and stored at -80℃ for later use.
[0044] Example 3. Cultivation of bacterial cells and preparation of crude enzyme solution. 1. Composition of bacterial cell culture medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, dissolved in deionized water and brought to a final volume, then sterilized at 121°C for 20 min.
[0045] After activating the engineered bacteria from Examples 1 and 2 by streak plating, single colonies were inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking. Then, 2% of the inoculum was transferred to 50 mL of fresh LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD (outlet capacity) was reached. 600 When the concentration reaches approximately 0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and induce culture at 18°C for 18-24 h. After culture, centrifuge the culture at 4000 rpm for 10 min, discard the supernatant, collect the bacterial cells, and wash the cells twice with 10 mM pH 7.4 phosphate buffer. Store at -80°C for later use.
[0046] 2. Preparation of crude enzyme solution: After the culture was completed, the bacterial cells collected were suspended four times in 1M pH 8.0 glycine-NaOH buffer and sonicated at 400W for 30 times, with each sonication lasting 3 seconds and a 7-second interval. The lysate was centrifuged at 12000 rpm at 4℃ for 10 minutes to remove the precipitate. The supernatant obtained was the crude enzyme solution containing D-threonine aldolase.
[0047] Example 4: Preparation of D-syn-p-methylsulfonylbenzylserine from D-threonine aldolase mutant in aqueous solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutant, which synthesize D-syn-β-hydroxy-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1 L) contained 1 M glycine, 50 mM p-methylsulfonylbenzaldehyde, 50 μM pyridoxal phosphate, and 0.1 mM manganese chloride, with 250 mL of crude enzyme solution. The reaction was carried out at 30 °C for 8 h, with samples taken at intervals to determine the concentrations and de values of the substrate and product. The reaction data are shown in Table 3.
[0048] Example 5: Preparation of D-syn-p-methylsulfonylbenzylserine from D-threonine aldolase mutant in water-DMSO solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutant, which synthesize D-syn-β-hydroxy-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M glycine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, along with 250mL of crude enzyme solution and 5-20% DMSO (v / v). The reaction was carried out at 30℃ for 1h, after which the concentrations and de values of the substrate and product were measured. The data at the end of the reaction are shown in Table 3.
[0049] Table 3. De values and final conversion rates of D-syn-p-methylsulfonylbenzylserine prepared by different mutants in different systems.
[0050] Note: "-" in the table indicates mutant inactivation and is used only for data display.
[0051] Example 6: Preparation of D-anti-p-methylsulfonylbenzylserine from D-threonine aldolase mutant in aqueous solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutant, which synthesize D-anti-β-hydroxy-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M glycine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 1h, and the concentrations and de values of the substrate and product were then measured. The data at the end of the reaction are shown in Table 4.
[0052] Example 7: Preparation of D-anti-p-methylsulfonylbenzaldehyde and its mutant D-anti-β-hydroxy-α-amino acid threonine aldolase mutant in water-DMSO solution. Following the method in Example 3, engineered bacteria expressing threonine aldolase and its mutant were cultured, and crude enzyme solution was obtained. The reaction system (1L) contained 1M glycine, 50mM p-methylsulfonylbenzaldehyde, 50μM pyridoxal phosphate, 0.1mM manganese chloride, 250mL crude enzyme solution, and 5-20% DMSO (v / v). The reaction was carried out at 30℃ for 1h, and the concentrations and de values of the substrate and product were then measured. The data at the end of the reaction are shown in Table 4.
[0053] Table 4. de values and final conversion rates of D-anti-p-methylsulfonylbenzylserine prepared by different mutants in different systems.
[0054] Example 8: Preparation of D-syn-p-nitrophenylserine from D-threonine aldolase mutant in aqueous solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutant, which synthesize D-syn-β-hydroxy-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M glycine, 50mM p-nitrobenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 1h, after which the concentrations of substrate and product, and the de value, were measured. The data at the end of the reaction are shown in Table 5.
[0055] Example 9: Preparation of D-syn-2,4-dihydroxybenzylserine from D-threonine aldolase mutant in aqueous solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutant, which synthesize D-syn-β-hydroxy-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M glycine, 50mM 2,4-dihydroxybenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 1h, after which the concentrations of substrate and product, and the de value, were measured. Data at the end of the reaction are shown in Table 5.
[0056] Table 5. de values and conversion rates of D-syn threonine aldolase mutants (p-nitrobenzaldehyde, 2,4-dihydroxybenzaldehyde)
[0057] Example 10: Preparation of D-anti-p-nitrophenylserine from D-threonine aldolase mutant in aqueous solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutant, which synthesize D-anti-β-hydroxy-α-amino acids, were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M glycine, 50mM p-nitrobenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 1h, after which the concentrations of substrate and product, and the de value, were measured. The data at the end of the reaction are shown in Table 6.
[0058] Example 11: Preparation of D-anti-2,4-dihydroxybenzylserine mutants in aqueous solution. Following the method described in Example 3, engineered bacteria expressing threonine aldolase and its mutants were cultured to obtain crude enzyme solution. The reaction system (1L) contained 1M glycine, 50mM 2,4-dihydroxybenzaldehyde, 50μM pyridoxal phosphate, and 0.1mM manganese chloride, with 250mL of crude enzyme solution. The reaction was carried out at 30℃ for 1h, and the concentrations and de values of the substrate and product were then measured. The data at the end of the reaction are shown in Table 6.
[0059] Table 6. de values and conversion rates of D-antithreonine aldolase mutants (p-nitrobenzaldehyde, 2,4-dihydroxybenzaldehyde)
Claims
1. A D-threonine aldolase mutant, characterized in that, The mutant has seven or fewer substitutions relative to SEQ ID NO.2 and has less than 100% sequence identity with SEQ ID NO.2, wherein the substitutions are selected from: Q171A, Q171C, Q171D, Q171E, Q171F, Q171G, Q171H, Q171I, Q171K, Q171L, Q171M, Q171P, Q171R, Q171S, Q171T, Q171V, Q171Y, A266C, A266D, A266E, A266G, A266H, A266I, A266K, A266L, A266M, A266N, A266P, A266R, A266S, A266T, A266V, A266Y, S251A, S251C, S251D, S251G, S251H, S251K, S251L, S251M, S251N, S251P, S251Q, S251T, S251V, S251W, S251Y, S206P, N336S, N336A, H209K, C319Y and C319K, or combinations thereof.
2. The D-threonine aldolase mutant as described in claim 1, characterized in that, The substituted material is selected from: Q171A, Q171F, Q171H, Q171T, Q171Y, A266C, A266K, A266P, A266V, S251A, S251G, S251T, S206P, N336S, N336A, H209K, C319Y, and C319K, or combinations thereof.
3. A gene encoding a D-threonine aldolase mutant as described in claim 1 or 2.
4. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 3.
5. A recombinant microorganism, characterized in that, The recombinant microorganism comprises the gene of claim 3 or the recombinant vector of claim 4.
6. A catalyst, characterized in that, The catalyst comprises the D-threonine aldolase mutant of claim 1 or 2 or the recombinant microorganism of claim 5; the catalyst is an enzyme preparation of the mutant, an immobilized enzyme, or a whole cell or cell lysate of the recombinant microorganism.
7. The use of a D-threonine aldolase mutant as described in claim 1 or 2, or a recombinant microorganism as described in claim 5, or a catalyst as described in claim 6, in the preparation of D-β-hydroxy-α-amino acids; wherein, The D-β-hydroxy-α-amino acid is either D-syn-β-hydroxy-α-amino acid or D-anti-β-hydroxy-α-amino acid.
8. A method for preparing D-configuration β-hydroxy-α-amino acids, characterized in that, Using glycine and benzaldehyde derivatives as substrates, pyridoxal phosphate and manganese ions as cofactors, and employing the catalyst described in claim 6, a condensation reaction is carried out to generate D-configuration β-hydroxy-α-amino acids; wherein, the benzaldehyde derivative is one of p-methylsulfonylbenzaldehyde, p-nitrobenzaldehyde, and 2,4-dihydroxybenzaldehyde; and the D-β-hydroxy-α-amino acid is either D-syn-β-hydroxy-α-amino acid or D-anti-β-hydroxy-α-amino acid.
9. The method as described in claim 8, characterized in that, The reaction is carried out in an organic solvent-water mixture or an aqueous solution.
10. The method as described in claim 8, characterized in that, The organic solvent is DMSO, and the amount of organic solvent added does not exceed 20% of the total volume.