CN116103259A - Rational modification of L-threonine transaldolase and its integrated system for efficient synthesis of β-hydroxy-α-amino acids - Google Patents

Abstract

The invention discloses the rational transformation of L-threonine transaldolase and its integration system to efficiently synthesize β-hydroxy-α-amino acids, belonging to the field of biocatalysis engineering. The present invention clones L-threo-aldolase from Burkholderia diffusa, expresses it heterologously to E.coli BL21 (DE3), studies the enzymatic properties of its recombinant enzyme, and catalyzes the transformation of L-threo-pair Methylsulfonylphenylserine function. At the same time, the key amino acid sites of L-threonine transaldolase were mutated to obtain mutants with significantly improved catalytic efficiency and stereoselectivity. The invention also introduces the efficient by-product acetaldehyde elimination system into the catalytic system mediated by L-threonine transaldolase to construct an integrated system; Alcohols increase the solubility of substrates while achieving in situ regeneration of the cofactor NADH required for the reaction.

Description

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

technical field

The invention relates to the rational transformation of L-threonine transaldolase and its integrated system for efficiently synthesizing β-hydroxyl-α-amino acids, belonging to the field of biocatalysis engineering.

Background technique

β-Hydroxy-α-amino acids (βHAAs) play an important role in the research progress of natural product synthesis, peptide research, drug development, agricultural prevention, etc. due to their structural and functional diversity. For example, L-threo-dihydroxyphenylserine is a chiral auxiliary for the chemical synthesis of drugs for Parkinson's disease and other compounds such as β-lactam. L-threo-p-methylsulfonylphenylserine is an important intermediate in the synthesis of thiamphenicol, a broad-spectrum antibiotic of the chloramphenicol class, which has a strong antibacterial effect on most Gram-negative bacteria , has strong antibacterial effect on Gram-positive bacteria such as hemolytic streptococcus, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides pneumophila, Vibrio cholerae, Shigella bacillus and influenza bacillus, etc. Body, amoeba, etc. also have certain antibacterial effect. Clinically, it mainly treats respiratory tract, urinary tract, and intestinal infections caused by sensitive bacteria such as Haemophilus influenzae, Escherichia coli, and Salmonella. L-threo-p-nitrophenylserine is an important intermediate for the synthesis of odorless chloramphenicol, which is a kind of chloramphenicol, also known as palm chloramphenicol, which belongs to chloramphenicol antibiotics and also has many Strong antibacterial effect, suitable for children to take orally, after oral administration, chloramphenicol is gradually released in the intestinal tract, and the effect is more durable than ordinary chloramphenicol. In addition, other βHAAs such as p-fluphenylserine and phenylserine play important roles in medicine and peptide development.

With the rapid development of society, my country's demand for raw materials has greatly increased. On the one hand, it is used for domestic synthesis, and at the same time, the export volume to foreign countries has also greatly increased. It is understood that my country's import and export of chloramphenicol drugs account for half of each. . At present, these key intermediates are mainly synthesized by chemical methods in China. For example, chloramphenicol is mainly synthesized by p-nitroacetophenone chemical method. Formation-reduction-decomposition-resolution-dichloroacetylation to obtain chloramphenicol. The synthetic steps of the chemical method are complicated, the by-products are many, the stereoselectivity is poor and the environment is not friendly. Biocatalysis has attracted much attention due to its mild reaction conditions, environmental friendliness, almost no by-product formation and high stereoselectivity.

L-threonine transaldolase uses L-threonine as a donor substrate and various aromatic aldehydes as acceptor substrates. It has high stereoselectivity, and can synthesize target βHAAs with high efficiency and high optical purity. However, the number of threonine transaldolases reported and characterized so far is very small. At the same time, due to the low catalytic efficiency of natural enzymes, poor substrate tolerance, and by-product inhibition, their industrial applications are greatly limited. Therefore, it is necessary to excavate and characterize new threonine transaldolase, improve its catalytic efficiency and industrial application properties through enzyme rational design, and finally realize the efficient preparation of βHAAs, break the monopoly of foreign technology, and realize the biocatalytic preparation technology with independent intellectual property rights .

Contents of the invention

In the present invention, a novel L-threonine transaldolase from Burkholderia diffusa was excavated, cloned into Escherichia coli BL21 (DE3) to achieve high-efficiency expression, and the enzymatic properties and functions of the recombinant enzyme were verified. In order to further improve the industrial properties of the enzyme, the specific amino acid sites of L-threonine transaldolase were mutated by methods such as homology modeling, molecular docking and multiple sequence alignment, and the catalytic efficiency and stereoselectivity were significantly improved. mutant strain. On this basis, an efficient by-product acetaldehyde elimination system was introduced into the catalytic system mediated by L-threonine transaldolase to construct an integrated system. Eliminate the inhibitory effect of by-products such as acetaldehyde on the reaction, improve the solubility of the substrate, and at the same time, the by-product elimination mediating enzyme can catalyze the auxiliary substrate to realize the in situ regeneration of the cofactor NADH. The integrated system significantly improves the catalytic efficiency and stereoselectivity of L-threonine transaldolase, realizes the efficient biosynthesis of βHAAs, and lays a solid foundation for its industrial production.

In one embodiment of the present invention, the mutant contains one or more amino acid substitutions at positions 231, 262, 268, 35, and 57, wherein the amino acid substitution at position 231 is S, T, a more preferred substitution It is S, the amino acid at position 262 is replaced by N, S, the more preferred substitution is S, and the preferred substitution at position 268 is S. The 35th amino acid is replaced by S, T, A, the more preferred replacement is S, the 57th amino acid is replaced by S, N, and the more preferred replacement is N. The mutant is one or more combinations of more preferred substitutions of the above positions, and the most preferred one is N35S-V57N-C262S-N268S.

In one embodiment of the present invention, the catalytic efficiency of the L-threonine transaldolase mutant is to carry out a whole-cell reaction with L-threonine and p-methylsulfonylbenzaldehyde as substrates, and it is detected by HPLC Its product peak area is measured.

The present invention provides a mutant of L-threonine transaldolase, wherein the mutant is the valine at the 57th position of L-threonine transaldolase shown in SEQID NO.2. Obtained by mutation to asparagine, named V57N;

Or the mutant is obtained by mutating the valine at the 57th position of L-threonine aldolase shown in SEQ ID NO.2 to serine, named V57S;

Or the mutant is that the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 is mutated into asparagine, and the asparagine at position 35 is mutated into Serine, obtained by mutating asparagine at position 268 to serine, named N35S-V57N-N268S;

Or the mutant is that the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 is mutated into asparagine, and the asparagine at position 35 is mutated into Serine, obtained by mutating cysteine at position 262 to serine at the same time, named N35S-V57N-C262S;

Or the mutant is that the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 is mutated into asparagine, and the asparagine at position 231 is mutated into Serine, obtained by mutating cysteine at position 262 to serine at the same time, named V57N-N231S-C262S;

Or the mutant is that the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 is mutated into asparagine, and the asparagine at position 35 is mutated into Serine, obtained by mutating cysteine at position 262 to serine, and asparagine at position 268 to serine, named N35S-V57N-C262S-N268S.

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

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

The present invention also provides genes encoding the above mutants.

The present invention also provides a recombinant vector carrying the mutant or the above-mentioned gene.

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

The present invention also provides a recombinant cell expressing the above-mentioned mutant, carrying the above-mentioned gene, or carrying the above-mentioned recombinant vector.

In one embodiment of the present invention, the recombinant cells use bacteria or fungi as expression hosts.

In one embodiment of the present invention, the recombinant cells use Escherichia coli as the expression host.

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

The present invention provides an L-threonine transaldolase, the amino acid sequence of the L-threonine transaldolase includes, but not limited to, an amino acid sequence with ≥90% homology to the sequence shown in SEQ ID NO.1.

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

In one embodiment of the present invention, the pure L-threonine transaldolase enzyme has relatively high catalytic activity at pH 7.0-7.5 and temperature 30-50°C, wherein metal ions Mg ²⁺ , Ca ^{2 +} Promote the activity of L-threonine transaldolase.

In one embodiment of the present invention, the assay of the L-threonine transaldolase activity uses L-threonine as a donor substrate, p-methylsulfonylbenzaldehyde as an acceptor substrate, and phosphoric acid Pyridoxal is a coenzyme, and the decrease of NADH at 340nm is monitored by coupling acetaldehyde dehydrogenase.

The invention also provides a recombinant vector carrying L-threonine transaldolase.

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

The present invention also provides recombinant cells expressing the above-mentioned L-threonine transaldolase, or containing the above-mentioned recombinant vector.

In one embodiment of the present invention, the recombinant cells use bacteria or fungi as expression hosts.

In one embodiment of the present invention, the recombinant cells use Escherichia coli as the expression host.

The invention provides a recombinant Escherichia coli, which expresses the above-mentioned mutant or the above-mentioned L-threonine transaldolase, and simultaneously expresses alcohol dehydrogenase.

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

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

The present invention also provides a method for preparing β-hydroxyl-α-amino acids in whole cells. The method is as follows: L-threonine is used as a donor substrate, benzaldehyde and its derivatives are used as an acceptor substrate, The β-hydroxy-α-amino acid is prepared by transforming the above-mentioned recombinant cells or the whole cells of the above-mentioned recombinant Escherichia coli, and the β-hydroxy-α-amino acid is phenylserine and its derivatives.

In one embodiment of the present invention, the general structural formula of described benzaldehyde and derivatives thereof is:

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

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

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

In one embodiment of the present invention, when the whole cell is a recombinant Escherichia coli expressing alcohol dehydrogenase at the same time, the substrate will additionally add isopropanol; Dehydrogenase consumes, provides NADH required for the reaction, and reduces the amount of NADH in the entire acetaldehyde elimination system; special attention is that the introduction of the co-substrate can not only provide NADH regeneration cycle, itself as an organic solvent, for L- Threonine transaldolase whole-cell catalyzed synthesis of β-hydroxy-α-amino acid efficiency has been improved to a certain extent.

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

The general reaction formula is as shown in formula 1:

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 group, mercapto group, nitro group and halogen; R group position is ortho, meta or para.

In one embodiment of the present invention, the whole-cell catalytic system of the L-threonine transaldolase is 120 mM of L-threonine, 30 mM of benzaldehyde and its derivatives, 1 mM of CaCl ₂ , pyridoxal phosphate 0.1mM, the reaction time is 8h, and the reaction buffer is 50mM ammonium formate solution (pH 7.0).

In one embodiment of the present invention, the whole-cell catalytic system of L-threonine transaldolase is obtained by optimizing external conditions with L-threonine and p-methylsulfonylbenzaldehyde as substrates. The external conditions include but are not limited to temperature, the amount of L-threonine transaldolase used in whole cells, and the ratio of substrates (L-threonine: p-methylsulfonylbenzaldehyde).

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

The present invention constructs an efficient by-product acetaldehyde elimination system by introducing alcohol dehydrogenase (sequence shown in SEQ ID NO.3) from Komagataellakurtzmanii. At the same time, the co-catalytic substrate isopropanol is introduced, which can be consumed by alcohol dehydrogenase to provide NADH required for the reaction and reduce the amount of NADH in the entire acetaldehyde elimination system. It should be noted that the introduction of the co-substrate can not only provide the NADH regeneration cycle, but also act as an organic solvent to improve the efficiency of L-threonine transaldolase whole-cell catalyzed synthesis of β-hydroxy-α-amino acids.

In one embodiment of the present invention, the high-efficiency by-product acetaldehyde elimination system uses L-threonine as a donor substrate, benzaldehyde and its derivatives as an acceptor substrate, and isopropyl Alcohol is used as a co-substrate, and β-hydroxyl-α-amino acids are prepared by catalyzing whole cells of recombinant Escherichia coli, and the β-hydroxyl-α-amino acids are phenylserine and its derivatives;

The recombinant Escherichia coli expresses the above-mentioned mutant or the above-mentioned L-threonine transaldolase, and simultaneously expresses alcohol dehydrogenase.

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

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

Beneficial effect

(1) Through amino acid sequence and structure comparison, the present invention excavated a novel L-threonine transaldolase derived from Burkholderiadiffusa, cloned it into Escherichia coli, realized efficient heterologous expression and purification, and studied its enzymatic properties, Optimizing conditions for whole-cell catalytic synthesis of βHAAs. At the same time, through homology modeling, molecular docking and multiple sequence alignment, the key amino acid sites of L-threonine transaldolase were mutated to obtain mutants with significantly improved catalytic efficiency. On this basis, the present invention also introduces an efficient by-product acetaldehyde elimination system and in-situ circulation of cofactors to construct an integrated system of whole-cell catalysis, realizes the efficient preparation of βHAAs, and lays a solid research foundation for its industrial production .

(2) The present invention uses cheap L-threonine and benzaldehyde derivatives as substrates and undergoes one-step catalysis by L-threonine transaldolase to obtain βHAAs with high conversion efficiency and stereoselectivity. The whole-cell biocatalytic reaction does not require cumbersome steps, no redundant by-products are generated, the reaction conditions are mild, and it is environmentally friendly. The invention obtains the target product through a one-step method of biocatalysis, and is a green and efficient method for biosynthesizing βHAAs.

Description of drawings

Figure 1: SDS-PAGE image of purified L-threonine transaldolase; among them, Line 1 is the pure enzyme band of purified L-threonine transaldolase.

Fig. 2: The influence of pH and temperature on the pure enzymatic activity of L-threonine transaldolase; Wherein, Fig. 2A represents the enzyme activity stability of L-threonine transaldolase at different pH; Fig. 2B represents L - Enzyme activity of threonine transaldolase at different pH; FIG. 2C shows temperature stability of L-threonine transaldolase; FIG. 2D shows enzymatic activity of L-threonine transaldolase at different temperatures.

Figure 3: Effect of metal ion addition on the enzymatic activity of L-threonine transaldolase pure enzyme.

Figure 4: The liquid phase detection diagram of the synthesis of L-threo-p-methylsulfonylphenylserine catalyzed by L-threonine transaldolase in whole cells.

Figure 5: Optimization of biocatalytic conditions for whole-cell biocatalysis of methylsulfonylphenylserine for synthesis of L-threonine; among them, Figure 5A shows the effect of temperature on whole-cell catalysis of L-threonine transaldolase; Figure 5B shows the concentration of whole cells Effects on whole-cell catalysis of L-threonine transaldolase; Figure 5C shows the effect of substrate ratio on whole-cell catalysis of L-threonine transaldolase.

Figure 6: The homology modeling structure and molecular docking structure display of L-threonine transaldolase.

Figure 7: Multiple sequence alignment of L-threonine transaldolases.

Figure 8: Display of the structural formula of the acceptor substrate aromatic aldehyde for the synthesis of βHAAs.

Figure 9: Comparison of the effect of L-threonine transaldolase coupled with the acetaldehyde elimination system in whole-cell catalysis.

Figure 10: Schematic diagram of the reaction of the acetaldehyde elimination system.

Detailed ways

The detection methods involved in the following examples are as follows:

The liquid phase detection method of the product L-threo-p-methylsulfonylphenylserine:

Using o-phthalaldehyde (OPA)/N-acetyl-L-cysteine (NAC) derivatization method, weigh 20mg of OPA and NAC respectively, dissolve in 4mL boric acid buffer (0.2M, pH 9.8) and 1mL pure acetonitrile The derivatizer is formulated in the medium, the whole cell catalytic sample is processed and mixed with the derivatizer in a 1:4 manner, and then diluted appropriately for liquid phase detection, mobile phase: 50mM potassium dihydrogen phosphate buffer (pH 8.0): pure acetonitrile =83:17. The detection conditions are: flow rate 1mL min ^-1 , column temperature 40°C, detection wavelength 338nm. As shown in Figure 4, the peak eluting time of L-threo-p-methylsulfonylphenylserine is 4.5min, and the peak eluting time of L-erythro-p-methylsulfonylphenylserine is 5.2min. Wherein L-threo-p-methylsulfonylphenylserine diastereomer selectivity (de value) calculation formula is: de=(L-threo-p-methylsulfonylphenylserine peak area+L-erythro-p-methylsulfonylphenyl Serine)/(L-threo-p-methylsulfonylphenylserine peak area-L-erythro-p-methylsulfonylphenylserine)×100%.

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

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

Example 1: Obtaining and expression purification of L-threonine transaldolase gene

Specific steps are as follows:

(1) Obtain the amino acid sequence of the L-threonine transaldolase BuLTTA from Burkholderia diffusa from NCBI (the NCBI database gene accession number is WP_059467200.1, and the amino acid sequence is shown in SEQ ID NO.2), according to Escherichia coli Codon preference, optimize the codon of the gene (the nucleotide sequence is shown in SEQ ID NO.1), entrust the biological company to chemically synthesize, construct the optimized sequence on the vector pGEX-6p-1, and further transform it into The recombinant strain E.coli/pGEX-BuLTTA was obtained from E.coli BL21(DE3) competent cells.

(2) The protein expression induction condition is: 0.2mM IPTG, cultured at 25°C for 14-16 hours, and the obtained whole cells of L-threonine transaldolase were purified by GSH Purose 4 Fast Flow column (purchased from Qianchun Biological Company) according to the instructions and GST tag enzyme digestion to obtain a single-band protein. The construction, induced expression, and purification of the strain are all routine operations; the purified protein is shown in FIG. 1 .

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

Example 2: Study on enzymatic properties of pure L-threonine transaldolase enzyme.

Specific steps are as follows:

(1) Determination of the enzymatic activity of L-threonine transaldolase: the enzymatic activity of LTTAs was determined by NADH and ADH coupling assay. At 25°C, the rate of decrease in absorbance at 340 nm (ε=6220M ⁻¹ cm ⁻¹ ) was monitored using a multi-functional microplate reader (BioTek, Vermont, USA). The reaction system was a reaction mixture (190 μL) consisting of 10 mM p-methylsulfonylbenzaldehyde, 100 mM L-threonine, 50 μM PLP, 0.2 mM NADH and 10 U ADH, and Tris-HCl buffer (20 mM Tris-HCl, pH 7.0 , 10% dimethyl sulfoxide (DMSO), incubated at 25°C for 1 minute. Introduce 15 μg LTTAs enzyme (in 10 μL Tris-HCl buffer), and monitor at 340 nm for 1.5 min. One unit of transaldolase activity ( U) is defined as the amount of enzyme that catalyzes the conversion of 1 μmol of L-threo-p-methylsulfonylserine per minute, and all experiments were repeated three times; on this basis, L-threoline and p-methylsulfonylserine Formaldehyde was used as a substrate to measure the enzymatic activity of LTTAs.

(2) Use phosphate (pH 6.0-8.0), Tris-HCl (pH 8.0-9.0) and carbonate-bicarbonate (pH 9.0-10.0) as buffer reagents. The pH and temperature dependence of LTTAs at different pH values (pH 6.0-10.0) and temperatures (10-80 °C) were determined.

(3) In order to study the stability of pH, the enzyme solution was incubated in various pH buffers for 12 hours at 4°C, and the residual activity was determined at 30°C.

To assess thermostability, enzyme solutions were incubated in 20 mM Tris buffer (pH 7.0) at different temperatures (10-80°C) for 1 h, and the remaining enzyme activity was determined at 30°C after cooling.

At the same time, in the presence of different metal ions with the final concentration of 1mM and 5mM, the effect of metal ions on the activity of L-threonine transaldolase was studied. The L-threonine transaldolase activity measured in the absence of metal ions was used as a control.

The results are shown in Figure 2, Figures 2A and 2B show that studies have shown that L-threonine transaldolase has stable enzyme activity at a temperature of 10-60°C, and the optimum reaction temperature is 50°C; the pH value is 7.0-60°C. 7.5, it has stable and high activity (Figure 2C, 2D); at the same time, as shown in Figure 3, the addition of Ca ²⁺ and Mg ²⁺ can significantly promote the activity of L-threonine aldolase, especially When 1mM Ca ²⁺ or Mg ²⁺ was added, the enzyme activity was 1.51 times and 1.26 times that of the control, respectively.

Example 3: Optimization of conditions for whole-cell synthesis of L-threo-p-methylsulfonylphenylserine by L-threonine transaldolase

Specific steps are as follows:

(1) The initial reaction conditions for whole-cell synthesis of L-threo-p-methylsulfonylphenylserine by L-threonine transaldolase are: the reaction system is 1mL, containing 30mM p-methylsulfonylbenzaldehyde, 100mM L-threoline acid, 50 μM PLP, 1 mM CaCl ₂ and 30 mg mL ⁻¹ wet cells (E.coli/pGEX-BuLTTA prepared in 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 with a rotation speed of 200 rpm, a temperature of 30° C., and a reaction time of 5 hours. After the reaction was completed, the whole cells were removed by high-speed centrifugation to obtain a supernatant.

Reaction samples were analyzed by a modified OPA/NAC derivatization method. 20 mg NAC and 20 mg OPA were dissolved in 5 mL of derivatization buffer (4 mL, 0.2M boric acid, 1 mL acetonitrile, pH 9.8) to give OPA/NAC reagent. Agilent-1260HPLC system (Agilent Technologies Inc., Palo Alto, USA) was used for chromatographic analysis, and the ultraviolet detection wavelength was 338nm. Samples were pre-column derivatized using an autosampler program. Chromatographic column: Hypersil ODS-2 reverse phase chromatography column (250×4.6mm 2.5μm), mobile phase: 50mm KH ₂ PO ₄ , pH8.0 acetonitrile (83/17), flow rate: 1ml min ^-1 , temperature: 40℃ . The results are shown in Figure 5, wherein the peak eluting time of L-threo-p-methylsulfonylphenylserine is 4.547min, and that of L-erythro-p-methylsulfonylphenylserine is 5.225min.

(2) Optimized the temperature of L-threonine whole-cell catalytic reaction

Reaction system: The reaction system is 1 mL, containing 30 mM p-methylsulfonylbenzaldehyde, 100 mM L-threonine, 50 μMPLP, 1 mM CaCl ₂ and 30 mg mL ^-1 wet cells (E.coli/pGEX- BuLTTA) with 10% DMSO added as a co-solvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out in a shaker with a rotation speed of 200rpm, the temperature was set at 10-40°C, the interval was 5°C, and the reaction time was 5h. After the reaction was completed, the whole cells were removed by high-speed centrifugation, and the supernatant was properly diluted for liquid phase detection. All experiments All were repeated three times to obtain the average value.

The results show that: as shown in Figure 5A, the increase of temperature can promote the conversion rate of methanesulfonylbenzaldehyde catalyzed by L-threonine transaldolase in whole cells, but with the increase of temperature, the de value of the product decreases Trend, in order to balance the problem of product yield and de value, choose 35 ℃ as the optimum reaction temperature, at this time, the substrate conversion rate is 55.1%, and the de value of the target product is 80.2%.

(3) Optimized the whole cell concentration of the reaction

Reaction system: The reaction system is 1 mL, containing 30 mM p-methylsulfonylbenzaldehyde, 100 mM L-threonine, 50 μMPLP, 1 mM CaCl ₂ and 10-60 mg mL ^-1 wet cells (E.coli/ pGEX-BuLTTA), with the addition of 10% DMSO as a co-solvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out in a shaker with a rotation speed of 200 rpm, a temperature of 35°C, and a reaction time of 5 hours. After the reaction was completed, the whole cells were removed by high-speed centrifugation, and the supernatant was properly diluted for liquid phase loading. All experiments were repeated three times to obtain the average value.

The results show: As shown in Figure 5B, the wet cell concentration is positively correlated with the conversion rate of the substrate and the de value of the product within a certain range. When the wet cell concentration is greater than 40 mg mL ^-1 , the conversion rate and de value begin to rise slowly, indicating that this At this time, the amount of enzyme contained in the whole cell has reached the maximum amount of enzyme, so 40mg mL ^-1 is selected as the most suitable amount for the whole cell reaction of L-threonine transaldolase. At this time, the conversion rate of the substrate is 63.5%, and the de value of the target product is was 83.2%.

(4) Optimized the substrate ratio of the reaction

Reaction system: The reaction system is 1 mL, containing 30 mM p-methylsulfonylbenzaldehyde, 30-210 mM L-threonine, 50 μM PLP, 1 mM CaCl ₂ and 40 mg mL ^-1 wet cells (E.coli prepared in Example 1 /pGEX-BuLTTA), adding 10% DMSO as a co-solvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out in a shaker with a rotation speed of 200rpm, a temperature of 35°C, and a reaction time of 5 hours. After the reaction was completed, the whole cells were removed by high-speed centrifugation. After the supernatant was mixed with the derivative agent, it was properly diluted for loading and testing. All experiments were repeated three times to obtain the average value. The results show that:

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

Embodiment 4: Rational modification of L-threonine transaldolase

Specific steps are as follows:

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

Design single-point mutation primers (Table 1), use the wild-type transaldolase sequence (amino acid sequence as shown in SEQ ID NO.2) as a template for full-plasmid PCR, and perform template digestion and product purification on the obtained PCR products, followed by transformation In the competent cloning host E.coli JM109, clones were picked for sequencing verification, and p-methylsulfonylbenzaldehyde and L-threonine were used as substrates to carry out further whole-cell transformation of the correctly sequenced mutant strains.

(2) Reaction conditions: the reaction system was 1 mL, containing 30 mM p-methylsulfonylbenzaldehyde, 120 mM L-threonine, 50 μM PLP, 1 mM CaCl ₂ and 40 mg ^mL wet cells (the mutant was prepared according to Example 1 The whole cell of E.coli/pGEX-BuLTTA-M6, add 10% DMSO as cosolvent.Reaction buffer is 50mM ammonium formate (pH 7.0).Reaction is carried out in shaker, rotating speed 200rpm, temperature is set to 35 ℃, The reaction time is 5h. The results are shown in Table 2.

Table 2: Comparison of conversion efficiency of wild-type enzyme and mutant enzyme to L-threo-p-methylsulfonylphenylserine

The results showed that by detecting the content and de value of the target product by liquid phase, 5 mutants with significantly improved catalytic conversion product efficiency were finally obtained: amino acid-directed mutations at positions 231, 262, 268, 35, and 57; Body N231S, C262S, N268S, N35S, and C57N have the best effect. The conversion rate of whole-cell catalyzed L-threo-p-methylsulfonylphenylserine reaches 75.6-80.2%, and the de value of the target product reaches 88.2-91.1%.

(3) Perform combined mutations on the obtained single point mutations, and test their whole-cell transformation effects; the primer sequences are shown in Table 1, and the construction method is: use the amino acid sequence of a single point mutation as a template, and design another mutation site The PCR primers of the whole plasmid were selected, and the double mutant plasmid with the correct mutation was obtained through template digestion, product purification, transformation and sequencing of selected clones. Then use the plasmid as a template to construct the next mutation site, so as to obtain three mutations and four mutation genes. The catalytic reaction was carried out according to the method of step (2), and the results are shown in Table 3.

Table 3: Conversion efficiency of L-threo-p-methylsulfonylphenylserine catalyzed by beneficial mutation site combination mutations

The results show that Table 3 shows the obtained forward combination mutants. The superposition of these mutation sites has obtained multiple mutants with doubled effects, wherein the mutant N35S-V57N-C262S-N268S (named M6) whole-cell catalyzes the synthesis of L- The conversion rate of threo-p-methylsulfonylphenylserine reached 97.6%, and the de value reached 96.8%.

Example 5: Whole-cell catalyzed synthesis of βHAAs by L-threonine transaldolase mutant M6

Using whole cells of L-threonine transaldolase mutant M6 as a catalyst, different βHAAs were synthesized. As shown in Figure 8, they are different acceptor substrates, aromatic aldehydes, mainly groups with different positions on the benzene ring ( They are named 1a-18a). The various βHAAs detection methods adopt the liquid phase detection method described in Example 3.

The reaction system was 1 mL, containing 30 mM 1a-18a, 120 mM L-threonine, 50 μM PLP, 1 mM CaCl ₂ and 40 mg mL ^-1 wet cells (E.coli/pGEX-BuLTTA-M6 prepared according to the method in Example 1 ), adding 10% DMSO as co-solvent. The reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out in a shaker with a rotation speed of 200 rpm, a temperature of 35°C, and a reaction time of 5 hours. After the reaction was completed, the whole cells were removed by high-speed centrifugation. After the supernatant was properly diluted, the sample was loaded and tested according to the method of Example 3. The results are shown in Table 4. .

Table 4: Whole-cell transformation effect of L-threonine transaldolase mutant M6 on different receptor substrates

The results show that, as shown in Table 4, for most of the aldehydes in the selected 18 aromatic aldehydes, the L-threonine aldolase mutant M6 can achieve a higher conversion rate (90.1-98.2%) with stereo The target product was synthesized selectively (90.1～96.7%).

It was found through observation that L-threonine transaldolase can only catalyze aromatic aldehydes with electron-withdrawing groups on the benzene ring, such as nitro, sulfonyl, chlorine, fluorine, etc. Groups of aromatic aldehydes cannot be utilized. In particular, the aromatic aldehyde substrates measured in the present invention include 1a-18a but are not limited thereto. All aromatic ring substrates that can be catalyzed by L-threonine transaldolase and its mutants in the present invention should be included in Inside.

Example 6: Construction of by-product acetaldehyde elimination system and verification of whole cell transformation

L-threonine transaldolase uses L-threonine and various aromatic aldehydes as substrates to efficiently synthesize βHAAs with excellent stereoselectivity. In the reaction product, in addition to containing the isomer of the target product, there is also an equal amount of a molecule of acetaldehyde generated. The accumulation of acetaldehyde may affect the whole-cell conversion of L-threonine aldolase, for example, the occurrence of product inhibition will cause the substrate utilization rate to decrease. Therefore, further elimination of by-products is necessary.

The present invention creates a novel acetaldehyde elimination system. The acetaldehyde elimination system is shown in FIG. 10 . Using the redox property of acetaldehyde dehydrogenase, not only acetaldehyde can be utilized, but also some alcohol compounds can be consumed. Based on this, the present invention screened the alcohol dehydrogenase (NCBI sequence number is KAI0462988.1) from Komagataella kurtzmanii. At the same time, the co-catalytic substrate isopropanol is introduced, which can be consumed by alcohol dehydrogenase, and at the same time convert NAD ⁺ to NADH, providing regeneration of NADH and reducing the amount of NADH in the entire acetaldehyde elimination system. It is particularly emphasized that the by-product elimination system not only has the function of eliminating the by-product acetaldehyde, but also realizes the in-situ circulation of cofactors.

Specific steps are as follows:

(1) Construction of recombinant Escherichia coli: the amino acid sequence of the alcohol dehydrogenase was chemically synthesized by a biological company after codon optimization and constructed on the vector pET28a to obtain the recombinant vector pET-KkADH, and the recombinant vector pET-KkADH was combined with transaldehyde The enzyme pGEX-BuLTTA was transformed into E. coli BL21 competent at the same time, and the transformed solution after incubation was coated with ampicillin and kana antibiotic plate to obtain a co-transformed recombinant E. coli with double enzymes: E.coli/pET-KkADH-pGEX- The induction condition of BuLTTA is 25°C, with the addition of 0.4mM IPTG. Under this condition, a well-expressed soluble recombinant protein can be obtained.

(2) In this implementation example, wild-type BuLTTA was first used to test the effect of coupling with the acetaldehyde elimination system. Reaction system: The reaction system was 1 mL, 30 mM p-methylsulfonylbenzaldehyde, 120 mM L-threonine, 50 μM PLP, 1 mM CaCl ₂ and 40 mg mL ^-1 wet cells (recombinant pET-KkADH co-transformed with pGEX-BuLTTA recombinant large intestine Bacteria: E.coli/pET-KkADH-pGEX-BuLTTA), 10% DMSO, 10% isopropanol and 1 mM NADH; the reaction buffer was 50 mM ammonium formate (pH 7.0). The reaction was carried out in a shaker with a rotation speed of 200 rpm, a temperature set at 35° C., and a reaction time of 5 h. After the reaction, centrifuge to remove whole cells, dilute the obtained supernatant appropriately, and use the sample detection method in Example 3 for detection; at the same time, use no alcohol dehydrogenase as a control: E.coli/pGEX-BuLTTA, use Example 3 Sample testing method for testing;

The results are shown in Figure 9. Compared with the uncoupled acetaldehyde elimination system, the addition of the acetaldehyde elimination system greatly improves the catalytic efficiency of L-threonine transaldolase in whole cells, and the almost maximum can be achieved by adding only 1mM NADH The substrate conversion rate (≥99%), the de value of the target product also has a certain promotion, and finally reaches 88.9%.

(3) The present invention expands the concentration of the substrate aromatic aldehyde to 200mM, and performs 1L-scale bioreactor transformation to test the transformation of L-threonine transaldolase and its mutant M6 when coupled with the acetaldehyde elimination system efficiency.

Its whole-cell catalytic system is: 800mM L-threonine, 200mM p-methylsulfonylbenzaldehyde, 0.2mM pyridoxal phosphate, 2mM NADH, 40mg mL ^-1 threonine transaldolase (or mutant M6) and ethanol Dehydrogenase co-transformed E. coli whole cells (E.coli/pET-KkADH-pGEX-BuLTTA, E.coli/pET-KkADH-pGEX-M6), the reaction buffer was 50mM ammonium formate (pH 7.0), while adding 10% DMSO and 10% isopropanol. The reaction temperature is controlled by an automatic temperature control device at 35°C, 25% ammonia water is automatically added to control the pH to about 7.0, and the rotation speed is 400 rpm. The reaction is terminated when the conversion rate no longer increases. The conversion effect is shown in Table 5.

Table 5: Expanded reaction results

The result shows that wild-type L-threoline aldolase can only obtain 30.2% conversion rate under the situation that 200mM substrate aldehyde is added, and the titer of product L-threo-p-methylsulfonylphenylserine reaches 60.4mM, Under the coupling of the mutant M6 and the acetaldehyde elimination system, the substrate conversion rate reached 95%, the target product titer was as high as 190mM, and the conversion time was only 4h.

Comparative example 1:

The specific embodiment is the same as the steps (1) to (2) of Example 6, the difference is that the alcohol dehydrogenase is adjusted to be derived from the alcohol dehydrogenase (ApADH) of Acetobacter pasteurianus and the alcohol dehydrogenase (ScADH) of Saccharomyces cerevisiae respectively , Alcohol dehydrogenase (ZpADH) of Zygosaccharomyces parabailii (its sequence numbers on NCBI are respectively: WP_012812267, AJT77739.1, AQZ12029.1). The amount of NADH added in the reaction system was set to 1 mM, and other conditions remained unchanged. The results are shown in Table 6.

Table 6: Reaction results of alcohol dehydrogenase from different sources

The results show that the introduction of different alcohol dehydrogenases has a greater effect on the conversion efficiency of the target product. Among them, the introduction of KkADH got the maximum substrate conversion rate and the maximum product de value.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person 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 defined by the claims.

Claims (10)

1. A L-threonine transaldolase mutant, characterized in that, the mutant is, amino acid sequence such as the 57th valine of the L-threonine transaldolase shown in SEQ ID NO.2 Amino acid is mutated to asparagine; Or the mutant is obtained by mutating the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 to serine; Or the mutant is that the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 is mutated into asparagine, and the asparagine at position 35 is mutated into Serine, obtained by mutating the asparagine at position 268 to serine; Or the mutant is that the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 is mutated into asparagine, and the asparagine at position 35 is mutated into Serine, obtained by mutating the cysteine at position 262 to serine; Or the mutant is that the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 is mutated into asparagine, and the asparagine at position 231 is mutated into Serine, obtained by mutating the cysteine at position 262 to serine; Or the mutant is that the valine at position 57 of the L-threonine transaldolase shown in SEQ ID NO.2 is mutated into asparagine, and the asparagine at position 35 is mutated into Serine is obtained by mutating cysteine at position 262 to serine and asparagine at position 268 to serine. 2. A gene encoding the mutant of claim 1. 3. The mutant carrying the mutant according to claim 1, or the recombinant vector carrying the gene according to claim 2. 4. A recombinant cell expressing the mutant according to claim 1, carrying the gene according to claim 2, or carrying the recombinant vector according to claim 3. 5. A L-threonine transaldolase, characterized in that the nucleotide sequence encoding the L-threonine transaldolase is as shown in SEQ ID NO.1. 6. A recombinant cell, characterized in that it expresses the L-threonine transaldolase according to claim 1. 7. A recombinant escherichia coli, characterized in that, the recombinant escherichia coli has expressed the mutant according to claim 1 or the L-threonine transaldolase according to claim 5, and simultaneously expressed alcohol dehydrogenase . 8. The recombinant escherichia coli as claimed in claim 7, wherein said alcohol dehydrogenase is derived from Komagataella kurtzmanii. 9. A method for preparing β-hydroxyl-α-amino acids from whole cells, characterized in that, the method is as follows: L-threonine is used as a donor substrate, and benzaldehyde and its derivatives are used as acceptor substrates , using the recombinant cells of claim 4 or claim 6 or the recombinant Escherichia coli whole cell transformation described in claim 7 or 8 to obtain β-hydroxyl-α-amino acids, and the β-hydroxyl-α-amino acids are phenylserine and its derivatives. 10. the method as claimed in claim 9, is characterized in that, the general structural formula of described benzaldehyde and derivative thereof is:

The general structure formula of described phenylserine and its derivatives is:

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

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