Rational modification of L-threonine transaldolase and its integrated system for efficient synthesis of β-hydroxy-α-amino acids

Abstract

The present invention discloses the rational transformation of L-threonine transaldolase and its integrated system for the efficient synthesis of β-hydroxy-α-amino acids, belonging to the field of biocatalytic engineering. The present invention clones the gene of L-threonine transaldolase from Burkholderia diffusa and heterologously expresses it in E.coli BL21 (DE3) to study the enzymatic properties of its recombinant enzyme and the function of catalytic conversion of L-threo-methylsulfonylphenylserine. At the same time, the key amino acid sites of L-threonine transaldolase are mutated to obtain mutants with significantly improved catalytic efficiency and stereoselectivity. The present invention also introduces an L-threonine transaldolase-mediated catalytic system into an efficient by-product acetaldehyde elimination system to construct an integrated system; not only the inhibitory effect of by-products on the catalytic reaction is eliminated, but also the solubility of the substrate is improved by adding the auxiliary substrate isopropanol, while achieving the in situ regeneration of the cofactor NADH required for the reaction.

Description

Technical Field

[0001] The present invention relates to the rational transformation of L-threonine transaldolase and its integrated system for efficiently synthesizing beta-hydroxy-alpha-amino acids, and belongs to the field of biocatalytic engineering. Background Art

[0002] Due to their diverse structures and functions, β-hydroxy-α-amino acids (βHAAs) have played a crucial role in the development of natural product synthesis, peptide research, drug development, and agricultural prevention. For example, L-threo-dihydroxyphenylserine is a chiral auxiliary in the chemical synthesis of Parkinson's disease treatments and other compounds, such as β-lactams. L-threo-p-methylsulfonylphenylserine is an important intermediate in the synthesis of thiamphenicol, a broad-spectrum antibiotic in the chloramphenicol class. It exhibits strong antibacterial activity against most Gram-negative bacteria and relatively strong antibacterial activity against Gram-positive bacteria such as hemolytic Streptococci, gonococci, meningococci, Pneumocystis carinii, Vibrio cholerae, Shigella dysenteriae, and Haemophilus influenzae. It also has limited antibacterial activity against anaerobic bacilli, Rickettsiae, and amoebas. Clinically, it is primarily used to treat respiratory, urinary, and intestinal infections caused by susceptible bacteria such as Haemophilus influenzae, Escherichia coli, and Salmonella. L-threo-p-nitrophenylserine is an important intermediate in the synthesis of odorless chloramphenicol. Odorless chloramphenicol, also known as palmitoyl chloramphenicol, belongs to the chloramphenicol class of antibiotics and possesses strong antibacterial properties. Suitable for oral administration in children, it gradually releases chloramphenicol in the intestine, resulting in a more sustained effect than standard chloramphenicol. In addition, other βHAAs, such as p-fluorophenylserine and phenylserine, play important roles in pharmaceuticals and peptide development.

[0003] With the rapid development of society, my country's demand for APIs has grown significantly, both for domestic synthesis and for export. It is understood that my country's imports and exports of chloramphenicol drugs are 50 / 50. Currently, these key intermediates are primarily synthesized domestically using chemical methods. For example, chloramphenicol is primarily synthesized using the p-nitroacetophenone method, which involves ethylbenzene undergoing nitration-oxidation-bromination-salification-hydrolysis-acetylation-addition-reduction-decomposition-splitting-and-dichloroacetylation. Chemical synthesis methods are complex, produce numerous byproducts, have poor stereoselectivity, and are environmentally unfriendly. Biocatalysis, due to its mild reaction conditions, environmental friendliness, near-zero byproduct generation, and high stereoselectivity, has attracted considerable attention.

[0004] L-Threonine aldolase uses L-threonine as the donor substrate and various aromatic aldehydes as the acceptor substrates. It exhibits high stereoselectivity, resulting in efficient synthesis of target βHAAs and high optical purity. However, only a few threonine aldolases have been reported and characterized. Furthermore, low catalytic efficiency, poor substrate tolerance, and byproduct inhibition by natural enzymes significantly limit their industrial application. Therefore, it is necessary to identify and characterize novel threonine aldolases and, through rational enzyme design, improve their catalytic efficiency and industrial applicability. Ultimately, this could lead to the efficient preparation of βHAAs, breaking the monopoly of foreign technology and establishing a biocatalytic preparation technology with independent intellectual property rights. Summary of the Invention

[0005] The present invention discovered a novel L-threonine transaldolase from Burkholderia diffusa, cloned it into Escherichia coli BL21 (DE3) for efficient expression, and verified the enzymatic properties and functions of the recombinant enzyme. To further improve the industrial properties of the enzyme, specific amino acid sites of the L-threonine transaldolase were mutated through homology modeling, molecular docking, and multiple sequence alignment methods to obtain mutants with significantly improved catalytic efficiency and stereoselectivity. On this basis, a highly efficient byproduct acetaldehyde elimination system was introduced into the L-threonine transaldolase-mediated catalytic system to construct an integrated system. This eliminates the inhibitory effects of byproducts such as acetaldehyde on the reaction, improving the solubility of the substrate. At the same time, the byproduct elimination mediator can catalyze the auxiliary substrate and achieve in situ regeneration of the cofactor NADH. The integrated system significantly improves the catalytic efficiency and stereoselectivity of the L-threonine transaldolase, achieving efficient biosynthesis of βHAAs and laying a solid foundation for its industrial production.

[0006] In one embodiment of the present invention, the mutant comprises one or more amino acid substitutions at positions 231, 262, 268, 35, and 57, wherein the amino acid at position 231 is substituted with S, T, more preferably with S, the amino acid at position 262 is substituted with N, S, more preferably with S, and the amino acid at position 268 is preferably substituted with S. The amino acid at position 35 is substituted with S, T, A, more preferably with S, and the amino acid at position 57 is substituted with S, N, more preferably with N. The mutant comprises one or more combinations of the preferred substitutions at the above positions, with the most preferred substitution being N35S-V57N-C262S-N268S.

[0007] In one embodiment of the present invention, the catalytic efficiency of the L-threonine transaldolase mutant is measured by performing a whole-cell reaction with L-threonine and p-methylsulfonylbenzaldehyde as substrates and detecting the product peak area by HPLC.

[0008] The present invention provides an L-threonine transaldolase mutant, wherein the mutant is obtained by mutating the valine at position 57 of the L-threonine transaldolase with the amino acid sequence shown in SEQ ID NO. 2 to asparagine, and is named V57N.

[0009] Alternatively, the mutant is obtained by mutating the 57th position of the L-threonine transaldolase in the amino acid sequence shown in SEQ ID NO. 2 to serine, and is named V57S;

[0010] Alternatively, the mutant is obtained by mutating the valine at position 57 of the L-threonine transaldolase shown in the amino acid sequence of SEQ ID NO. 2 to asparagine, the asparagine at position 35 to serine, and the asparagine at position 268 to serine, and is named N35S-V57N-N268S;

[0011] Alternatively, the mutant is obtained by mutating the valine at position 57 of the L-threonine transaldolase shown in the amino acid sequence of SEQ ID NO. 2 to asparagine, the asparagine at position 35 to serine, and the cysteine at position 262 to serine, and is named N35S-V57N-C262S;

[0012] Alternatively, the mutant is obtained by mutating the valine at position 57 of the L-threonine transaldolase as shown in SEQ ID NO. 2 to asparagine, the asparagine at position 231 to serine, and the cysteine at position 262 to serine, and is designated as V57N-N231S-C262S;

[0013] Alternatively, the mutant is obtained by mutating the valine at position 57 of the L-threonine transaldolase shown in the amino acid sequence of SEQ ID NO. 2 to asparagine, mutating the asparagine at position 35 to serine, mutating the cysteine at position 262 to serine, and mutating the asparagine at position 268 to serine, and is named N35S-V57N-C262S-N268S.

[0014] The parent enzyme L-threonine transaldolase is derived from Burkholderia diffusa.

[0015] In one embodiment of the present invention, the nucleotide sequence encoding the parent enzyme L-threonine transaldolase is shown as SEQ ID NO.1.

[0016] The present invention also provides a gene encoding the mutant.

[0017] The present invention also provides a recombinant vector carrying the mutant or the gene.

[0018] In one embodiment of the present invention, the recombinant vector uses pET28a, pRSF-Duet1, pET21a or pGEX-6P-1 as an expression vector.

[0019] The present invention also provides a recombinant cell expressing the mutant, carrying the gene, or carrying the recombinant vector.

[0020] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0021] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as an expression host.

[0022] The present invention provides an L-threonine transaldolase. The nucleotide sequence encoding the L-threonine transaldolase is shown in SEQ ID NO.1.

[0023] The present invention provides an L-threonine aldolase, wherein the amino acid sequence of the L-threonine aldolase includes but is not limited to an amino acid sequence having a homology of ≥90% with the sequence shown in SEQ ID NO.1.

[0024] The present invention provides a study and demonstration of the enzymatic properties of the pure L-threonine transaldolase enzyme, and studying the enzymatic properties of the pure enzyme plays an important role in its subsequent whole-cell biocatalysis.

[0025] In one embodiment of the present invention, the pure L-threonine transaldolase has high catalytic activity at pH 7.0-7.5 and temperature 30-50°C, wherein the metal ion Mg 2+ , Ca 2+ It promotes the activity of L-threonine transaldolase.

[0026] In one embodiment of the present invention, the L-threonine transaldolase activity is determined by using L-threonine as a donor substrate, p-methylsulfonylbenzaldehyde as an acceptor substrate, and pyridoxal phosphate as a coenzyme, and monitoring the decrease of NADH at 340 nm by coupling acetaldehyde dehydrogenase.

[0027] The present invention also provides a recombinant vector carrying L-threonine transaldolase.

[0028] In one embodiment of the present invention, the recombinant vector uses pET28a, pRSF-Duet1, pET21a or pGEX-6P-1 as an expression vector.

[0029] The present invention also provides a recombinant cell expressing the L-threonine transaldolase or containing the recombinant vector.

[0030] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0031] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as an expression host.

[0032] The present invention provides a recombinant Escherichia coli, which expresses the mutant or the L-threonine transaldolase and alcohol dehydrogenase.

[0033] In one embodiment of the present invention, the alcohol dehydrogenase is derived from Komagataella kurtzmanii.

[0034] In one embodiment of the present invention, the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO.3, and the nucleotide sequence encoding the alcohol dehydrogenase is shown as SEQ ID NO.4.

[0035] The present invention also provides a method for preparing β-hydroxy-α-amino acids using whole cells. The method comprises: using L-threonine as a donor substrate and benzaldehyde and its derivatives as an acceptor substrate, and adopting the above-mentioned recombinant cells or the above-mentioned recombinant Escherichia coli whole cells for transformation to prepare the β-hydroxy-α-amino acids, wherein the β-hydroxy-α-amino acids are phenylserine and its derivatives.

[0036] In one embodiment of the present invention, the general structural formula of the benzaldehyde and its derivatives is:

[0037]

[0038] The R groups include, but are not limited to, hydrogen, alkyl, alkoxy, alkylsulfonyl, alkylsulfinyl, alkylthio, sulfonic acid, sulfinic acid, mercapto, nitro, and halogen; and the R group position is ortho, meta, or para.

[0039] In one embodiment of the present invention, the general structural formula of phenylserine and its derivatives is:

[0040]

[0041] The R groups include, but are not limited to, hydrogen, alkyl, alkoxy, alkylsulfonyl, alkylsulfinyl, alkylthio, sulfonic acid, sulfinic acid, mercapto, nitro, and halogen; and the R group position is ortho, meta, or para.

[0042] In one embodiment of the present invention, when the whole cell is a recombinant Escherichia coli that also expresses alcohol dehydrogenase, isopropanol is additionally added to the substrate; the introduction of the co-catalytic substrate isopropanol can be consumed by the alcohol dehydrogenase to provide the NADH required for the reaction, thereby reducing the amount of NADH used in the entire acetaldehyde elimination system; it is particularly noteworthy that the introduction of the co-substrate not only provides an NADH regeneration cycle, but also, as an organic solvent, has a certain improvement in the efficiency of the L-threonine transaldolase whole-cell catalytic synthesis of β-hydroxy-α-amino acids.

[0043] In one embodiment of the present invention, the phenylserine derivatives are obtained by whole-cell biocatalysis of L-threonine transaldolase using L-threonine as a donor substrate and benzaldehyde and its derivatives as an acceptor substrate.

[0044] The general reaction formula is shown in Formula 1:

[0045]

[0046] In one embodiment of the present invention, the R group includes but is not limited to: hydrogen, alkyl, alkoxy, alkylsulfonyl, alkylsulfinyl, alkylthio, sulfonic acid, sulfinic acid, mercapto, nitro and halogen; the R group position is ortho, meta or para.

[0047] In one embodiment of the present invention, the whole-cell catalytic system of the L-threonine transaldolase is 120 mM L-threonine, 30 mM benzaldehyde and its derivatives, 1 mM CaCl2, 0.1 mM pyridoxal phosphate, the reaction time is 8 h, and the reaction buffer is 50 mM ammonium formate solution (pH 7.0).

[0048] In one embodiment of the present invention, the L-threonine transaldolase whole-cell catalytic system is obtained by optimizing external conditions using L-threonine and p-methylsulfonylbenzaldehyde as substrates. The external conditions include, but are not limited to, temperature, L-threonine transaldolase whole-cell dosage, and substrate ratio (L-threonine:p-methylsulfonylbenzaldehyde).

[0049] In one embodiment of the present invention, the optimal conditions obtained by whole cell optimization are a temperature of 35°C and a whole cell dosage of 40 mg mL -1 , substrate ratio (L-threonine: p-methylsulfonylbenzaldehyde = 4:1).

[0050] The present invention constructs a highly efficient byproduct acetaldehyde elimination system by introducing alcohol dehydrogenase from Komagataella kurtzmanii (sequence shown in SEQ ID NO. 3). Simultaneously, a co-catalytic substrate, isopropanol, is introduced, which can be consumed by the alcohol dehydrogenase, providing the NADH required for the reaction and reducing the NADH usage in the entire acetaldehyde elimination system. Of particular note, the introduction of the co-substrate not only provides NADH regeneration but also acts as an organic solvent, significantly improving the efficiency of the L-threonine transaldolase-catalyzed whole-cell synthesis of β-hydroxy-α-amino acids.

[0051] In one embodiment of the present invention, the efficient byproduct acetaldehyde elimination system is a system that uses L-threonine as a donor substrate, benzaldehyde and its derivatives as an acceptor substrate, and isopropanol as a co-substrate, and uses recombinant Escherichia coli whole cells to catalyze the preparation of β-hydroxy-α-amino acids, wherein the β-hydroxy-α-amino acids are phenylserine and its derivatives;

[0052] The recombinant Escherichia coli expresses the mutant or the L-threonine transaldolase and alcohol dehydrogenase at the same time.

[0053] In one embodiment of the present invention, the alcohol dehydrogenase is derived from Komagataella kurtzmanii.

[0054] In one embodiment of the present invention, the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO.3, and the nucleotide sequence encoding the alcohol dehydrogenase is shown as SEQ ID NO.4.

[0055] Beneficial effects

[0056] (1) The present invention discovered a new L-threonine transaldolase from Burkholderia diffusa through amino acid sequence and structure comparison, cloned it into Escherichia coli, achieved efficient heterologous expression and purification, studied its enzymatic properties, and optimized the conditions for the whole-cell catalytic synthesis of βHAAs. At the same time, the key amino acid sites of L-threonine transaldolase were mutated by means of homology modeling, molecular docking, and multiple sequence alignment to obtain mutants with significantly improved catalytic efficiency. On this basis, the present invention also introduced an efficient by-product acetaldehyde elimination system and in situ recycling of cofactors to construct an integrated system for whole-cell catalysis, achieving efficient preparation of βHAAs and laying a solid research foundation for its industrial production.

[0057] (2) The present invention uses inexpensive L-threonine and benzaldehyde derivatives as substrates, and through a one-step catalysis by L-threonine transaldolase, βHAAs with high conversion efficiency and stereoselectivity can be obtained. The whole-cell biocatalytic reaction does not require cumbersome steps, no unnecessary by-products are generated, the reaction conditions are mild, and it is environmentally friendly. The present invention obtains the target product through a one-step biocatalytic method, which is a green and efficient method for the biosynthesis of βHAAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 : SDS-PAGE image of purified L-threonine aldolase; wherein, Line 1 is the pure enzyme band of purified L-threonine aldolase.

[0059] Figure 2 : The effect of pH and temperature on the pure enzyme activity of L-threonine transaldolase; wherein, Figure 2 A represents the enzyme activity stability of L-threonine transaldolase at different pH; Figure 2 B represents the enzyme activity of L-threonine transaldolase at different pH; Figure 2 C represents the temperature stability of L-threonine transaldolase; Figure 2 D represents the enzyme activity of L-threonine transaldolase at different temperatures.

[0060] Figure 3 : Effects of metal ion addition on the activity of pure L-threonine transaldolase enzyme.

[0061] Figure 4 :Liquid phase detection diagram of the synthesis of L-threo-p-methylsulfonylphenylserine catalyzed by whole-cell L-threonine transaldolase.

[0062] Figure 5 : Optimization of whole-cell biocatalytic conditions for the synthesis of methylsulfonylphenylserine from L-threonine; Figure 5 A represents the effect of temperature on whole-cell catalysis of L-threonine transaldolase; Figure 5 B represents the effect of whole cell concentration on whole cell catalysis of L-threonine transaldolase; Figure 5 C represents the effect of substrate ratio on the whole-cell catalysis of L-threonine transaldolase.

[0063] Figure 6 : Homology modeling structure and molecular docking structure display of L-threonine transaldolase.

[0064] Figure 7 : Multiple sequence alignment of L-threonine transaldolase.

[0065] Figure 8 :Display of the structural formula of aromatic aldehydes, the acceptor substrates for the synthesis of βHAAs.

[0066] Figure 9: Comparison of the effects of whole-cell catalysis of L-threonine transaldolase coupled with acetaldehyde elimination system.

[0067] Figure 10 : Schematic diagram of acetaldehyde elimination system reaction. DETAILED DESCRIPTION

[0068] The detection methods involved in the following embodiments are as follows:

[0069] Liquid phase detection method of the product L-threo-p-methylsulfonylphenylserine:

[0070] The o-phthalaldehyde (OPA) / N-acetyl-L-cysteine (NAC) derivatization method was used. 20 mg of OPA and NAC were weighed and dissolved in 4 mL of boric acid buffer (0.2 M, pH 9.8) and 1 mL of pure acetonitrile to prepare the derivatization reagent. The resulting whole-cell catalytic sample was treated and mixed with the derivatization reagent in a 1:4 ratio. After appropriate dilution, liquid chromatography detection was performed. The mobile phase was 50 mM potassium dihydrogen phosphate buffer (pH 8.0): pure acetonitrile = 83:17. The detection conditions were: flow rate 1 mL min -1 , column temperature 40℃, detection wavelength 338nm. Figure 4 As shown, the elution time of L-threo-p-methylsulfonylphenylserine is 4.5 min, and the elution time of L-erythro-p-methylsulfonylphenylserine is 5.2 min. The diastereomeric selectivity (de value) of L-threo-p-methylsulfonylphenylserine is calculated as follows: de = (peak area of L-threo-p-methylsulfonylphenylserine + L-erythro-p-methylsulfonylphenylserine) / (peak area of L-threo-p-methylsulfonylphenylserine - L-erythro-p-methylsulfonylphenylserine) × 100%.

[0071] The primer sequences involved in the following examples are as follows:

[0072] Table 1: Primers required for single-point mutation of L-threonine transaldolase

[0073]

[0074] Example 1: Acquisition and expression purification of L-threonine transaldolase gene

[0075] The specific steps are as follows:

[0076] (1) The amino acid sequence of L-threonine transaldolase BuLTTA from Burkholderia diffusa was obtained (the amino acid sequence is shown in SEQ ID NO. 2), and the gene was codon-optimized according to the codon preference of Escherichia coli (the nucleotide sequence is shown in SEQ ID NO. 1). The optimized sequence was chemically synthesized by a biotechnology company, and the optimized sequence was constructed into the vector pGEX-6p-1, which was further transformed into E. coli BL21 (DE3) competent cells to obtain the recombinant bacterium E. coli / pGEX-BuLTTA.

[0077] (2) Protein expression induction conditions were: 0.2 mM IPTG, culture at 25°C for 14-16 h, and the obtained L-threonine transaldolase whole cells were purified by GSH Purose 4 Fast Flow column (purchased from Qianchun Biotechnology Co., Ltd.) according to the instructions and the GST tag was cleaved to obtain a single band of protein. The construction of the strain, induction expression and purification were all routine operations; the purified protein was as follows Figure 1 shown.

[0078] The results showed that SDS-PAGE showed a single band with a molecular weight of approximately 48 kDa, which was consistent with the theoretical molecular weight of the recombinant protein of 48.6 kDa.

[0079] Example 2: Study on the enzymatic properties of pure L-threonine transaldolase.

[0080] The specific steps are as follows:

[0081] (1) Determination of L-threonine transaldolase activity: The enzyme activity of LTTAs was determined by NADH and ADH coupling assay. The activity was monitored at 340 nm (ε = 6220 nm) using a multifunctional microplate reader (BioTek, Vermont, USA) at 25°C. -1 cm -1 ) is the rate of decrease in absorbance at 340 nm. The reaction system consisted of a 190 μL reaction mixture consisting of 10 mM p-methylsulfonylbenzaldehyde, 100 mM L-threonine, 50 μM PLP, 0.2 mM NADH, and 10 U ADH. Tris-HCl buffer (20 mM Tris-HCl, pH 7.0, 10% dimethyl sulfoxide (DMSO)) was added and incubated at 25°C for 1 minute. 15 μg of LTTAs enzyme (in 10 μL Tris-HCl buffer) was introduced and monitored at 340 nm for 1.5 minutes. One unit (U) of transaldolase activity was defined as the amount of enzyme that catalyzes the conversion of 1 μmol of L-threo-p-methylsulfonylphenylserine per minute. All experiments were repeated three times. On this basis, the enzyme activity of LTTAs was determined using L-threonine and p-methylsulfonylbenzaldehyde as substrates.

[0082] (2) Using phosphate (pH 6.0-8.0), Tris-HCl (pH 8.0-9.0), and carbonate-bicarbonate (pH 9.0-10.0) as buffers, the pH and temperature dependence of LTTAs at different pH values (pH 6.0-10.0) and temperatures (10-80°C) were determined.

[0083] (3) To study the pH stability, the enzyme solution was incubated in various pH buffers at 4 °C for 12 h, and the residual activity was measured at 30 °C.

[0084] To evaluate thermal stability, enzyme solutions were incubated in 20 mM Tris buffer (pH 7.0) at different temperatures (10-80°C) for 1 h, cooled, and the remaining enzyme activity was measured at 30°C.

[0085] The effect of metal ions on L-threonine transaldolase activity was also investigated in the presence of different metal ions at final concentrations of 1 mM and 5 mM. The L-threonine transaldolase activity measured in the absence of metal ions served as a control.

[0086] The results are as follows Figure 2 As shown, Figure 2 A, 2B show that studies have shown that L-threonine transaldolase has stable enzyme activity at temperatures between 10 and 60°C, with the optimum reaction temperature being 50°C; and has stable and high activity at pH values between 7.0 and 7.5 ( Figure 2 C, 2D); at the same time Figure 3 As shown, Ca 2+ With Mg 2+ The addition of Ca2+ significantly promoted the activity of L-threonine transaldolase, especially when 1 mM Ca2+ was added. 2+ or Mg 2+ , and their enzyme activities were 1.51 times and 1.26 times that of the control, respectively.

[0087] Example 3: Optimization of conditions for the synthesis of L-threo-methylsulfonylphenylserine by whole-cell L-threonine transaldolase

[0088] The specific steps are as follows:

[0089] (1) The initial reaction conditions for the synthesis of L-threo-p-methylsulfonylphenylserine by L-threonine transaldolase whole cells were as follows: the reaction system contained 30 mM p-methylsulfonylbenzaldehyde, 100 mM L-threonine, 50 μM PLP, 1 mM CaCl2, and 30 mg mL -1Wet cells (E. coli / pGEX-BuLTTA prepared in Example 1) were supplemented with 10% DMSO as a cosolvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out on a shaker at 200 rpm, 30°C, and for 5 hours. After completion of the reaction, the whole cells were removed by high-speed centrifugation to obtain the supernatant.

[0090] The reaction samples were analyzed using the improved OPA / NAC derivatization method. 20 mg of NAC and 20 mg of OPA were dissolved in 5 mL of derivatization buffer (4 mL, 0.2 M boric acid, 1 mL of acetonitrile, pH 9.8) to obtain the OPA / NAC reagent. Chromatographic analysis was performed using an Agilent-1260 HPLC system (Agilent Technologies Inc., Palo Alto, USA) with UV detection at a wavelength of 338 nm. The samples were pre-column derivatized using an automatic injection program. Chromatographic column: Hypersil ODS-2 reversed-phase column (250×4.6 mm 2.5 μm), mobile phase: 50 mm KH2PO4, pH 8.0 acetonitrile (83 / 17), flow rate: 1 ml min -1 , temperature: 40℃. The results are as follows Figure 5 As shown, the peak time of L-threo-p-methylsulfonylphenylserine is 4.547 min, and the peak time of L-erythro-p-methylsulfonylphenylserine is 5.225 min.

[0091] (2) Optimization of the temperature of the L-threonine whole-cell catalytic reaction

[0092] Reaction system: The reaction system for 1 mL contains 30 mM p-methylsulfonylbenzaldehyde, 100 mM L-threonine, 50 μM PLP, 1 mM CaCl2 and 30 mg mL -1 Wet cells (E. coli / pGEX-BuLTTA prepared in Example 1) were supplemented with 10% DMSO as a cosolvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). Reactions were performed on a shaker at 200 rpm, set at 10-40°C with a 5°C interval, and lasted for 5 h. After completion of the reaction, whole cells were removed by high-speed centrifugation. The supernatant was appropriately diluted and loaded onto a liquid sample for analysis. All experiments were repeated three times and the average value was calculated.

[0093] The results show that: Figure 5As shown in Figure A, increasing temperature promotes the conversion of p-methylsulfonylbenzaldehyde catalyzed by L-threonine transaldolase whole cells. However, the de value of the product shows a downward trend with increasing temperature. In order to balance the problem of product yield and de value, 35°C was selected as the optimal reaction temperature. At this time, the substrate conversion rate is 55.1% and the de value of the target product is 80.2%.

[0094] (3) Optimized the whole cell concentration of the reaction

[0095] Reaction system: The reaction system for 1 mL contains 30 mM p-methylsulfonylbenzaldehyde, 100 mM L-threonine, 50 μM PLP, 1 mM CaCl2 and 10-60 mg mL -1 Wet cells (E. coli / pGEX-BuLTTA prepared in Example 1) were supplemented with 10% DMSO as a cosolvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). Reactions were performed on a shaker at 200 rpm and 35°C for 5 h. After completion of the reaction, whole cells were removed by high-speed centrifugation. The supernatant was appropriately diluted and loaded onto a liquid chromatography plate for analysis. All experiments were repeated three times and the average value was calculated.

[0096] The results show that: Figure 5 As shown in B, the wet cell concentration is positively correlated with the substrate conversion rate and product de value within a certain range. When the wet cell concentration is greater than 40 mg mL -1 The conversion rate and de value began to rise slowly, indicating that the enzyme content of the whole cell had reached the maximum enzyme content. Therefore, 40 mg mL -1 As the most suitable amount of L-threonine transaldolase for the whole-cell reaction, the substrate conversion rate was 63.5% and the de value of the target product was 83.2%.

[0097] (4) Optimized the substrate ratio of the reaction

[0098] Reaction system: The reaction system for 1 mL contains 30 mM p-methylsulfonylbenzaldehyde, 30-210 mM L-threonine, 50 μM PLP, 1 mM CaCl2 and 40 mg mL -1 Wet cells (E. coli / pGEX-BuLTTA prepared in Example 1) were supplemented with 10% DMSO as a cosolvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out on a shaker at 200 rpm and 35°C for 5 hours. After completion of the reaction, the whole cells were removed by high-speed centrifugation. The supernatant was mixed with the derivatization reagent and diluted appropriately for sample loading and testing. All experiments were repeated three times and the average value was calculated. The results show:

[0099] like Figure 5As shown in Figure C, when the ratio of L-threonine to p-methylsulfonylbenzaldehyde is greater than 4:1, the conversion rate and de value remain stable. Therefore, the most suitable substrate ratio is determined to be L-threonine: p-methylsulfonylbenzaldehyde 4:1. At this time, the substrate conversion rate reaches 70.1% and the de value of the target product reaches 86.3%.

[0100] Example 4: Rational modification of L-threonine transaldolase

[0101] The specific steps are as follows:

[0102] (1) The present invention uses L-threonine transaldolase from Burkholderia diffusa as the wild-type enzyme ( Figures 6-7 ), and identified 14 mutation sites. The specific steps are:

[0103] Single-point mutation primers were designed (Table 1). Whole-plasmid PCR was performed using the wild-type transaldolase sequence (amino acid sequence shown in SEQ ID NO. 2) as a template. The resulting PCR product was digested and purified, then transformed into the competent cloning host E. coli JM109. Clones were selected for sequencing verification. Correctly sequenced mutants were further transformed with whole cells using p-methylsulfonylbenzaldehyde and L-threonine as substrates.

[0104] (2) Reaction conditions: The reaction system was 1 mL, containing 30 mM p-methylsulfonylbenzaldehyde, 120 mM L-threonine, 50 μM PLP, 1 mM CaCl2, and 40 mg mL -1 Wet cells (whole cells of mutant E. coli / pGEX-BuLTTA-M6 were prepared according to Example 1, and 10% DMSO was added as a cosolvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out in a shaker at 200 rpm and 35°C for 5 h. The results are shown in Table 2.

[0105] Table 2: Comparison of the efficiency of wild-type enzyme and mutant enzyme in catalyzing the conversion of L-threo-methylsulfonylphenylserine

[0106]

[0107] The results showed that by liquid phase detection of the content and de value of the target product, five site mutations were finally obtained with significantly improved catalytic conversion efficiency: amino acid-directed mutations at sites 231, 262, 268, 35, and 57; among them, the mutants N231S, C262S, N268S, N35S, and C57N had the best effects, with their whole-cell catalytic conversion rates of L-threo-methylsulfonylphenylserine reaching 75.6-80.2%, and the de values of the target product reaching 88.2-91.1%.

[0108] (3) Combinatorial mutations were performed on the obtained single-point mutations, and their whole-cell transformation effects were tested; the primer sequences are shown in Table 1. The construction method is as follows: using the amino acid sequence of a single-point mutation as a template, whole-plasmid PCR primers for other mutation sites were designed, and double-mutation plasmids with correct mutations were obtained by template digestion, product purification, transformation, and clone selection and sequencing verification. This plasmid was then used as a template to construct the next mutation site, thereby obtaining triple-mutation and quadruple-mutation genes. The catalytic reaction was carried out according to the method of step (2), and the results are shown in Table 3.

[0109] Table 3: Conversion efficiency of beneficial mutation site combinations to L-threo-methylsulfonylphenylserine

[0110]

[0111] The results show that Table 3 shows the obtained forward combination mutants. The superposition of these mutation sites obtained multiple mutants with doubled effects. Among them, the mutant N35S-V57N-C262S-N268S (named M6) catalyzed the synthesis of L-threo-methylsulfonylphenylserine in whole cells with a conversion rate of 97.6% and a de value of 96.8%.

[0112] Example 5: Synthesis of βHAAs by L-threonine transaldolase mutant M6 whole cells

[0113] Using the L-threonine transaldolase mutant M6 whole cells as catalyst, different βHAAs were synthesized, such as Figure 8 The following are different acceptor substrate aromatic aldehydes, mainly different groups at different positions on the benzene ring (named 1a to 18a). The liquid phase detection method described in Example 3 was used for the detection of various βHAAs.

[0114] The reaction system was 1 mL, containing 30 mM 1a-18a, 120 mM L-threonine, 50 μM PLP, 1 mM CaCl2, and 40 mg mL -1 Wet cells (E. coli / pGEX-BuLTTA-M6 prepared according to the method of Example 1) were supplemented with 10% DMSO as a cosolvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out on a shaker at 200 rpm and 35°C for 5 hours. After completion of the reaction, the whole cells were removed by high-speed centrifugation. The supernatant was appropriately diluted and tested according to the method of Example 3. The results are shown in Table 4.

[0115] Table 4: Whole-cell conversion effects of L-threonine transaldolase mutant M6 on different receptor substrates

[0116]

[0117] The results showed that, as shown in Table 4, for most of the 18 selected aromatic aldehydes, the L-threonine transaldolase mutant M6 could synthesize the target products with high conversion rates (90.1-98.2%) and stereoselectivity (90.1-96.7%).

[0118] Observations have shown that L-threonine transaldolase can only catalyze the reaction of aromatic aldehydes with electron-withdrawing groups on the benzene ring, such as nitro, sulfonyl, chloro, and fluoro groups. It is ineffective against aromatic aldehydes with electron-donating groups on the benzene ring. It is important to note that the aromatic aldehyde substrates assayed in the present invention include, but are not limited to, 1a-18a. This assay encompasses all aromatic aldehyde substrates catalyzed by the L-threonine transaldolase and its mutants described herein.

[0119] Example 6: Construction of byproduct acetaldehyde elimination system and whole-cell transformation verification

[0120] L-Threonine transaldolase uses L-threonine and various aromatic aldehydes as substrates, efficiently synthesizing βHAAs with excellent stereoselectivity. The reaction products contain not only isomers of the target product but also an equivalent amount of acetaldehyde. Acetaldehyde accumulation can affect the whole-cell conversion of L-Threonine transaldolase, such as product inhibition, which can lead to decreased substrate utilization. Therefore, further efforts to eliminate these byproducts are necessary.

[0121] The present invention creates a novel acetaldehyde elimination system, the acetaldehyde elimination system is as follows Figure 10 As shown, the redox property of acetaldehyde dehydrogenase can not only utilize acetaldehyde, but also consume some alcohol compounds. Based on this, the present invention screened the alcohol dehydrogenase from Komagataella kurtzmanii (NCBI sequence number KAI0462988.1). At the same time, the co-catalytic substrate isopropanol was introduced, which can be consumed by alcohol dehydrogenase, and NAD + It is converted into NADH, providing NADH regeneration and reducing the NADH usage in the entire acetaldehyde elimination system. It is particularly emphasized that this byproduct elimination system not only has the function of eliminating the byproduct acetaldehyde, but also realizes the in-situ circulation of the cofactor.

[0122] The specific steps are as follows:

[0123] (1) Construction of recombinant Escherichia coli: The alcohol dehydrogenase amino acid sequence was codon-optimized and chemically synthesized by a biological company and constructed into the vector pET28a to obtain the recombinant vector pET-KkADH. The recombinant vector pET-KkADH and the transaldolase pGEX-BuLTTA were simultaneously transformed into the competent Escherichia coli BL21. The incubated transformation liquid was coated on ampicillin and kanamycin plates to obtain co-transformed recombinant Escherichia coli with two enzymes: E. coli / pET-KkADH-pGEX-BuLTTA. The induction conditions were 25°C and the addition of 0.4 mM IPTG. Under these conditions, a well-soluble recombinant protein could be obtained.

[0124] (2) This embodiment first tests the effect of coupling with the acetaldehyde elimination system using wild-type BuLTTA. Reaction system: The reaction system consists of 1 mL, 30 mM p-methylsulfonylbenzaldehyde, 120 mM L-threonine, 50 μM PLP, 1 mM CaCl2, and 40 mg mL -1 Wet cells (recombinant pET-KkADH and pGEX-BuLTTA co-transformed recombinant Escherichia coli: E. coli / pET-KkADH-pGEX-BuLTTA), 10% DMSO, 10% isopropanol, and 1 mM NADH; the reaction buffer is 50 mM ammonium formate (pH 7.0). The reaction is carried out in a shaker at 200 rpm and 35°C for 5 hours. After the reaction is completed, the whole cells are centrifuged and removed. The obtained supernatant is appropriately diluted and tested using the sample detection method of Example 3. At the same time, a control without the addition of alcohol dehydrogenase: E. coli / pGEX-BuLTTA, is tested using the sample detection method of Example 3.

[0125] The results are as follows Figure 9 As shown in the figure, compared with the uncoupled acetaldehyde elimination system, the addition of the acetaldehyde elimination system greatly improved the efficiency of L-threonine transaldolase whole-cell catalysis, wherein only adding 1 mM NADH can achieve almost the maximum substrate conversion rate (≥99%), and the de value of the target product also increased to a certain extent, ultimately reaching 88.9%.

[0126] (3) The present invention increases the concentration of the substrate aromatic aldehyde to 200 mM and performs a 1 L scale bioreactor conversion to test the conversion efficiency of L-threonine transaldolase and its mutant M6 when coupled to the acetaldehyde elimination system.

[0127] The whole-cell catalytic system is: 800mM L-threonine, 200mM p-methylsulfonylbenzaldehyde, 0.2mM pyridoxal phosphate, 2mM NADH, 40mg mL -1Threonine transaldolase (or mutant M6) and alcohol dehydrogenase were co-transformed into whole Escherichia coli cells (E. coli / pET-KkADH-pGEX-BuLTTA, E. coli / pET-KkADH-pGEX-M6) in a reaction buffer of 50 mM ammonium formate (pH 7.0) supplemented with 10% DMSO and 10% isopropanol. The reaction temperature was maintained at 35°C using an automatic temperature controller. 25% aqueous ammonia was automatically added to maintain the pH at approximately 7.0. The reaction was maintained at 400 rpm and terminated when the conversion rate stopped increasing. The conversion results are shown in Table 5.

[0128] Table 5: Scale-up reaction results

[0129]

[0130] The results showed that the wild-type L-threonine transaldolase could only achieve a conversion rate of 30.2% when 200mM of substrate aldehyde was added, and the titer of the product L-threo-p-methylsulfonylphenylserine reached 60.4mM. When the mutant M6 was coupled with the acetaldehyde elimination system, the substrate conversion rate reached 95%, the titer of the target product was as high as 190mM, and the conversion time was only 4h.

[0131] Comparative Example 1:

[0132] The specific embodiment is the same as steps (1) to (2) of Example 6, except that the alcohol dehydrogenase is adjusted to alcohol dehydrogenase (ApADH) from Acetobacter pasteurianus, alcohol dehydrogenase (ScADH) from Saccharomyces cerevisiae, and alcohol dehydrogenase (ZpADH) from Zygosaccharomyces parabailii (their sequences are numbered as WP_012812267, AJT77739.1, and AQZ12029.1 on NCBI, respectively). The amount of NADH added to the reaction system was set to 1 mM, and other conditions remained unchanged. The results are shown in Table 6.

[0133] Table 6: Reaction results of alcohol dehydrogenase from different sources

[0134]

[0135] The results showed that the introduction of different alcohol dehydrogenases significantly improved the conversion efficiency of the target product. Among them, the introduction of KkADH achieved the highest substrate conversion rate and the highest product de value.

[0136] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An L-threonine transaldolase mutant, characterized in that The mutant is obtained by mutating the valine at position 57 of the L-threonine transaldolase with the amino acid sequence shown in SEQ ID NO. 2 to asparagine, the asparagine at position 35 to serine, and the asparagine at position 268 to serine; or The mutant is obtained by mutating the 57th valine of the L-threonine transaldolase with the amino acid sequence shown in SEQ ID NO. 2 to asparagine, the 35th asparagine to serine, and the 262nd cysteine to serine.

2. An L-threonine transaldolase mutant, characterized in that The mutant is obtained by mutating the valine at position 57 of the L-threonine transaldolase shown in the amino acid sequence of SEQ ID NO. 2 to asparagine, the asparagine at position 35 to serine, the cysteine at position 262 to serine, and the asparagine at position 268 to serine.

3. A gene encoding the mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. A recombinant cell expressing the mutant according to claim 1 or 2, carrying the gene according to claim 3, or carrying the recombinant vector according to claim 4, characterized in that: The recombinant cell uses bacteria or fungi as expression hosts.

6. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli expresses the mutant according to claim 1 and also expresses alcohol dehydrogenase.

7. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli expresses the mutant according to claim 2 and also expresses alcohol dehydrogenase.

8. The recombinant Escherichia coli according to claim 6 or 7, wherein The alcohol dehydrogenase is derived from Komagataella kurtzmanii.

9. A method for preparing L-threo-methylsulfonylphenylserine from whole cells, characterized in that: The method comprises the following steps: using L-threonine as a donor substrate and p-methylsulfonylbenzaldehyde as an acceptor substrate, and adopting the recombinant Escherichia coli whole cell of claim 6 or 8 for transformation.

10. A method for preparing β-hydroxy-α-amino acid using whole cells, characterized in that: The method comprises the following steps: using L-threonine as a donor substrate and a benzaldehyde derivative as an acceptor substrate, and transforming the whole recombinant Escherichia coli cells according to claim 7 to prepare a β-hydroxy-α-amino acid, wherein the β-hydroxy-α-amino acid is a phenylserine derivative; and the structure of the benzaldehyde derivative is any one of 1a, 4a, 5a, 6a, 7a, 10a, 11a, 12a, 16a, and 17a:

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