ω-transaminase TA13 mutant and its application

Abstract

This invention provides an ω-transaminase TA13 mutant and its applications. Specifically, this invention provides an ω-transaminase TA13 mutant, which, relative to the wild-type ω-transaminase TA13 shown in SEQ ID NO:1, has any amino acid substitution among G228A, Q232M, V237A, I260V, T288A, and M380L, or any combination thereof.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an ω-transaminase mutant with enhanced catalytic activity and its applications. Background Technology

[0002] 3-Aminopropanol is an important fine chemical intermediate widely used in pharmaceuticals, pesticides, and dyes, playing a crucial role in the synthesis of drugs such as cyclophosphamide and cyclophosphamide, as well as key raw materials such as vitamin B5 (panthenol). With the improvement of people's living standards, the application of panthenol in daily chemical products is increasing, particularly in hair care products and specialized cosmetics, thus significantly boosting the market demand for 3-aminopropanol.

[0003] In the production of 3-aminopropanol, ω-transaminase (ω-TA), as a biocatalyst, can specifically catalyze the reversible transfer of amino groups between amino donors (such as amino acids, alkylamines, aromatic amines, etc.) and carbonyl compounds (such as aldehydes, ketones, keto acids, etc.). Compared with traditional chemical synthesis methods, the biosynthetic route using ω-TA has advantages such as being environmentally friendly, efficient, and sustainable. In previous work, the inventors screened and obtained several ω-transaminases from specific sources, finding that they possessed catalytic activity for the conversion of 3-hydroxypropanal to 3-aminopropanol, but they suffered from insufficient activity and low catalytic efficiency. In the industrial production of 3-aminopropanol, the catalytic activity of ω-transaminase is a crucial factor; therefore, developing ω-TAs with higher catalytic activity or efficiency is essential for the industrial preparation of 3-aminopropanol. Summary of the Invention

[0004] In their previous work, the inventors discovered that wild-type ω-TA13 (NCBI: WP_127113105.1, amino acid sequence as shown in SEQ ID NO: 1) can catalyze the conversion of 3-hydroxypropanal to 3-aminopropanol, but the catalytic efficiency is not high. Therefore, this invention aims to solve the problem of insufficient activity of wild-type ω-TA13 under natural conditions. This problem is particularly significant in the synthesis of 3-aminopropanol, an important chemical product, by ω-TA, which limits its efficiency and yield in industrial production.

[0005] Existing ω-TA modification methods, such as random mutations or empirical adjustments, often lack specific targets and efficiency, failing to effectively improve enzyme activity and potentially affecting other important enzyme properties, such as substrate specificity and catalytic efficiency. This invention employs a rational design strategy to modify ω-TA13 based on its sequence and structure to enhance its activity. This invention utilizes an efficient and precise modification strategy to obtain a series of ω-TA13 mutants with increased catalytic efficiency, thereby enhancing the application potential of ω-TA in the industrial production of compounds such as 3-aminopropanol.

[0006] Specifically, this invention first constructs a three-dimensional structural model of ω-TA13 using AlphaFold2, performs molecular docking using the AutoDock vina tool, and conducts sequence conservation analysis to predict amino acid residues that significantly affect activity. Based on the prediction results, a series of ω-TA13 mutants are constructed and expressed under experimental conditions. Finally, activity tests are performed on these mutants to screen for those exhibiting optimal performance under specific conditions.

[0007] In this invention, the mutant involved is any one of the following (a) to (l):

[0008] (a) Mutate glycine at position 228 of ω-transaminase ω-TA13 with accession number WP_127113105.1 in the NCBI database to alanine;

[0009] (b) Mutate the proline at position 230 of WP_127113105.1 in the amino acid sequence to alanine;

[0010] (c) Mutate glutamine at position 232 of WP_127113105.1 in the amino acid sequence to methionine;

[0011] (d) Mutate the glycine at position 236 of WP_127113105.1 in the amino acid sequence to alanine;

[0012] (e) Mutate the valine at position 237 of the amino acid sequence WP_127113105.1 to alanine;

[0013] (f) Mutate the isoleucine at position 260 of the amino acid sequence WP_127113105.1 to valine or alanine;

[0014] (g) Mutate the cysteine ​​at position 266 of the amino acid sequence WP_127113105.1 to threonine;

[0015] (h) Mutate the glycine at position 276 of WP_127113105.1 in the amino acid sequence to alanine;

[0016] (i) Mutate the methionine at position 287 of the amino acid sequence WP_127113105.1 to isoleucine;

[0017] (j) Mutate the threonine at position 288 of the amino acid sequence WP_127113105.1 to either cysteine ​​or alanine;

[0018] (k) Mutate the isoleucine at position 289 of WP_127113105.1 in the amino acid sequence to leucine or valine;

[0019] (l) Mutate the methionine at position 380 of the amino acid sequence WP_127113105.1 to leucine;

[0020] Subsequently, using wild-type ω-TA13 as a control and the above-mentioned ω-TA13 mutants as the experimental group, their activity in the transamination-catalyzed synthesis of 3-aminopropanol was verified. It was found that the catalytic activities of mutants G228A, Q232M, V237A, I260V, T288A and M380L were all higher than those of wild-type.

[0021] Specifically, the present invention provides the following technical solutions:

[0022] 1. A mutant of ω-transaminase TA13, which, relative to the wild-type ω-transaminase TA13 shown in SEQ ID NO: 1, has any amino acid substitution of G228A, Q232M, V237A, I260V, T288A and M380L or any combination thereof.

[0023] 2. Nucleic acid molecules encoding the ω-transaminase TA13 mutant as described in Project 1.

[0024] 3. An expression vector comprising the nucleic acid molecule described in item 2; optionally, the expression vector is a vector suitable for expression in bacteria or yeast; preferably, the expression vector is a pET, pGEX, or pYES expression vector.

[0025] 4. A host cell comprising the nucleic acid molecule described in item 2 or the expression vector described in item 3; optionally, the host cell is a bacterium or fungus; preferably, the host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

[0026] 5. The use of the ω-transaminase TA13 mutant as described in Project 1, the nucleic acid molecule as described in Project 2, the expression vector as described in Project 3, or the host cell as described in Project 4 in the preparation of 3-aminopropanol.

[0027] 6. A method for preparing 3-aminopropanol by a biological enzymatic method, the method comprising adding the ω-transaminase TA13 mutant described in Project 1 or the host cell described in Project 4 to the reaction system.

[0028] 7. The method according to Project 6, wherein the reaction system further includes 3-hydroxypropanal as a substrate;

[0029] Preferably, the reaction system further includes a coenzyme; more preferably, the coenzyme is pyridoxal 5′-phosphate.

[0030] 8. The method according to Project 7, wherein the reaction system further includes an amino donor, wherein the amino donor includes L-alanine or isopropylamine;

[0031] Preferably, the molar ratio of the amino donor to the substrate is 2-6:1, the reaction temperature is 35-40℃, and the pH is 7-9.

[0032] Beneficial effects

[0033] This invention enhances the catalytic activity of wild-type ω-TA13 through rational design, thereby increasing its catalytic efficiency in the industrial biosynthesis of 3-aminopropanol, which has great application and commercial value. Attached Figure Description

[0034] Figure 1 The three-dimensional structure of wild-type ω-TA13 and a schematic diagram of the catalytic pocket are shown, with darker colors indicating amino acids in the catalytic pocket.

[0035] Figure 2 Candidate sites in the wild-type ω-TA13 catalytic pocket that may affect activity were shown.

[0036] Figure 3 The relative yield of 3-aminopropanol produced by a single point mutant is shown relative to the yield of 3-aminopropanol produced by wild-type ω-TA13.

[0037] Figure 4 The relative yield of 3-aminopropanol produced by the two-site mutant is shown relative to the yield of 3-aminopropanol produced by the wild-type ω-TA13. Detailed Implementation

[0038] The present invention is further illustrated by the following embodiments, but no embodiment or combination thereof should be construed as limiting the scope or embodiments of the invention. The scope of the invention is limited by the appended claims. Based on this specification and general knowledge in the art, those skilled in the art can clearly understand the scope of the claims. Without departing from the spirit and scope of the invention, those skilled in the art can make any modifications and alterations to the technical solutions of the invention, and such modifications and alterations are also included within the scope of the invention.

[0039] Unless otherwise stated, the experimental methods used in the following examples are conventional methods, such as those described in Molecular Cloning: A Laboratory Manual by J. Sambrook et al.; unless otherwise stated, the reagents and materials used are commercially available.

[0040] In some embodiments, the host cell may be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae, etc.

[0041] The culture media and solutions involved in the following examples are shown below:

[0042] LB solid medium: 10 g·L -1 Tryptone, 5 g / L -1 Yeast extract, 10 g·L -1 Sodium chloride and 2 g·L -1 Agar powder.

[0043] LB liquid medium: 10 g·L -1 Tryptone, 5 g / L -1 Yeast extract and 10 g·L -1 Sodium chloride.

[0044] Preparation of kanamycin resistance solution: Accurately weigh 10 mg of kanamycin and dissolve it in 100 mL of sterile water. Filter the solution through a 0.22 μm microporous membrane in a laminar flow hood to remove bacteria. Aliquot the solution into sterile 2 mL centrifuge tubes and store at -20°C for later use.

[0045] Example 1: Design of various mutants of ω-TA13

[0046] I. Three-dimensional structural modeling and catalytic pocket confirmation of wild-type ω-TA13 (WT)

[0047] 1. Construct a 3D structural model of ω-TA13 using AlphaFold:

[0048] AlphaFold (www.deepmind.com / research / highlightedresearch / alphafold) uses deep learning algorithms to predict the spatial structure of proteins, especially providing accurate 3D protein structure predictions when experimental crystal structure data is lacking. The tool was used to model the structure of ω-TA13, following the user manual or standard procedures of existing technologies.

[0049] Specifically, following standard operating procedures, the amino acid sequence (SEQ ID NO:1) of wild-type ω-TA13 was formatted into the input file format required by AlphaFold. AlphaFold was then run using the prepared input file to perform predictions. AlphaFold utilizes its deep learning model to predict the three-dimensional structure of wild-type ω-TA13 based on the input amino acid sequence. After the run, a series of files (PDB files) were generated. The structural model with the highest confidence level was selected, and the results are as follows: Figure 1 As shown.

[0050] 2. Assessment of protein conformation rationality

[0051] Following conventional methods, the structural rationality of the protein model obtained in Step 1 was evaluated using the SAVESS v6.0 scoring system (accessible at https: / / saves.mbi.ucla.edu), and the results were presented in a Laplace plot. Generally, if the proportion of amino acid residues in the allowed and maximum allowed regions of a protein exceeds 90%, the model structure is considered to obey stereochemical rules. If the proportion of residues in the optimal region exceeds 90%, the model is considered high quality; and a proportion exceeding 80% indicates a reliable model. The evaluation results show that the model predicted using AlphaFold2 in Step 1 has 91.71% of its residues located in this region. Therefore, the AlphaFold2-predicted model can be used for subsequent experiments.

[0052] 3. Confirmation of the active site of wild-type ω-TA13 (WT)

[0053] Following conventional methods, the molecular docking software AutoDock vina was used to load the molecular structure of the substrate 3-hydroxypropionaldehyde and the structural information of the wild-type ω-TA13 (WT) active center into the software platform to simulate the binding mode of 3-hydroxypropionaldehyde and the ω-TA13 active center. After molecular docking, the binding mode and binding capacity of 3-hydroxypropionaldehyde to the ω-TA13 (WT) active center were determined by evaluating the score, binding sites, and interaction information such as hydrogen bonding and hydrophobic interactions. Based on the docking results and analysis, 44 amino acid sites within 8 Å of the ω-TA13 active center were identified, centered on the 3-hydroxypropionaldehyde binding site. The results are shown in Table 1. Among them, the sites interacting with the substrate are listed below. Figure 2 As shown.

[0054] Table 1. Amino acid sites within 8 Å of the active site of wild-type ω-TA13 (WT)

[0055]

[0056] II. Conservatism Analysis of ω-TA13 Based on Sequence

[0057] 1. Obtain homologous sequences of ω-TA13:

[0058] Using conventional methods of existing technology, sequence alignment was performed on the public database of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search tool (BLAST, https: / / blast.ncbi.nlm.nih.gov) to obtain homologous sequences of ω-TA13.

[0059] 2. Amino acid conservation analysis

[0060] Multiple sequence alignment was performed on the homologous sequences obtained above using MUSCLE, and conservation analysis was performed using the consurf tool (https: / / consurf.tau.ac.il / consurf_index.php). The results showed that among the 44 amino acid sites within 8 Å of the ω-TA13 active site, 32 amino acid sites were highly conserved, and 12 amino acid sites had an average level of conservation and were non-highly conserved residues. This indicated that the 12 amino acid sites were suitable for subsequent experiments, and the final mutant library with a total of 15 mutants was obtained (see Table 2).

[0061] Table 2 shows the obtained mutation library.

[0062]

[0063] Example 2: Construction of host cells expressing wild-type ω-TA13 and its mutants, and expression of wild-type ω-TA13 and its mutants

[0064] 1. Construction of recombinant host cells containing the coding genes of wild-type ω-TA13 and its mutants

[0065] Based on the codon preference of E. coli, the coding sequences of wild-type ω-TA13 and the above 15 mutants were optimized. The codon-optimized coding sequences were then synthesized by a commercial company, ligated into the vector pRSFDuet-1 (purchased from Hongxun Biotechnology), and transformed into host cells E. coli BL21(DE3) (purchased from Sangon Biotech) to obtain recombinant host cells. Host cells transformed with wild-type ω-TA13 were used as the control group.

[0066] 2. Expression of ω-TA13 wild-type and its mutants

[0067] Add kanamycin resistance solution to LB liquid medium to a final concentration of 100.0 μg / mL. After activating the recombinant host cells containing the ω-TA13 mutant and wild-type ω-TA13 coding sequences by streaking on LB agar plates, they were seeded into 5 ml of LB liquid medium and cultured at 37°C and 220 rpm / min for 10–12 h. Then, 2 ml of the seed culture was transferred to 200 ml of LB liquid medium in a 1000 ml flask. The culture was induced with 0.3 mM IPTG until the optical density (OD600) at 600 nm reached 0.70. After culturing at 25 °C for 20 h, the whole E. coli cells were harvested by centrifugation at 4 °C (6000 g, 20 min). The cells were washed twice with phosphate buffer (100 mM, pH 7.5). The obtained whole E. coli cells were strains capable of expressing wild-type ω-TA13 enzyme or its mutant, which can be used to express wild-type ω-TA13 enzyme or its mutant in a catalytic system for transamination catalysis to prepare 3-aminopropanol.

[0068] Alternatively, wild-type ω-TA13 enzyme or its mutant can be directly extracted from the *E. coli* and added to the catalytic system for transamination catalysis. In this case, the *E. coli* cells need to be lysed. The cells can be resuspended in the same buffer and the suspension placed on ice. The cells are sonicated at 400 W for 90 times with a 4-second interval to obtain a cell-free extract. After centrifugation (12000×g, 4℃), the supernatant of the cell lysate (i.e., wild-type ω-TA13 enzyme or its mutant) is stored at -20℃ for later use.

[0069] Example 3: Construction of a catalytic reaction system for ω-TA13 enzyme or its mutant

[0070] In this embodiment, a reaction system for producing 3-aminopropanol was constructed.

[0071] In this reaction system, L-alanine is used as the amino donor, 3-hydroxypropanal as the substrate, and ω-TA13 enzyme or its mutant is used as the catalyst. For ω-TA13 enzyme or its mutant, whole *E. coli* cells collected in Example 2 can be directly added to the reaction system to express ω-TA13 enzyme or its mutant, or the supernatant obtained by cell disruption and extraction in Example 2 (containing ω-TA13 enzyme or its mutant) can be added to the reaction system. In this example, whole *E. coli* cells collected in Example 2 are directly added to the reaction system to express ω-TA13 enzyme or its mutant, and the specific conditions for the catalytic reaction are as follows.

[0072] During the aforementioned research, the inventors discovered that ω-TA13 enzyme can catalyze the reaction of 3-hydroxypropanal and the amino donor L-alanine to 3-aminopropanol. Furthermore, when the molar ratio of amino donor to substrate is 5:1 (3-hydroxypropanal is 13.5 mmol / L), and the reaction is carried out in a 2 ml reaction system at 37°C for 24 h, 0.96 g / L of 3-aminopropanol can be obtained.

[0073] To evaluate the catalytic activity of the TA13 mutant, this example assesses the improvement in enzyme activity by measuring the concentration of the target product under high substrate concentration and short catalytic time. The specific procedures are as follows:

[0074] Maintaining a molar ratio of amino donor (i.e., L-alanine) to reaction substrate (i.e., 3-hydroxypropanal) of 2.4:1, the whole E. coli cells collected in Example 2 above were placed in a 2 mL reactor for culture. After preparing the transformation system according to the table below, the centrifuge tubes were placed in a shaker at 220 rpm and 37 °C. The pH was adjusted to 7.5 with PBS, and the transamination reaction of the substrate was carried out for 3 h.

[0075] Table 3 Enzyme Conversion Reaction System

[0076]

[0077] Example 4: Yield analysis of 3-aminopropanol produced by wild-type ω-TA13 and its mutants

[0078] In this embodiment, the yields of ω-TA13 wild-type and its mutants under the same conditions were determined. Using the catalytic system containing ω-TA13 wild-type and its mutants constructed in Example 3, the content of 3-aminopropanol generated by wild-type ω-TA13 and its mutants under the same conditions was determined by HPLC.

[0079] Three parallel experiments were designed for each mutant, and the average value of the final results from each group was calculated. Figure 3 As shown, it displays the relative yield of 3-aminopropanol produced by each mutant compared to the yield of 3-aminopropanol produced by the wild-type ω-TA13. The wild-type ω-TA13 produced 0.56 g / L of 3-aminopropanol. The mutants G228A, Q232M, V237A, I260V, T288A, and M380L produced higher yields of 3-aminopropanol than the wild-type, at 113%, 109%, 107%, 115%, 119%, and 103% of the wild-type yield, respectively. Figure 3 Furthermore, the data from each group showed significant differences, with P values ​​all < 0.05. This indicates that the above mutants exhibited improved catalytic efficiency compared to the wild type.

[0080] Example 5: Design and yield analysis of ω-TA13 combined multi-point mutants

[0081] 1. Two-point mutant design

[0082] This invention designs 14 double-point mutants by combining the 6 effective single-point mutants obtained in Example 4, as shown in Table 4.

[0083] Table 4. Mutation libraries obtained from two-site mutations.

[0084]

[0085] 2. Construction and expression of host cells expressing the ω-TA13 dual-site mutant, and construction of the catalytic reaction system.

[0086] Referring to Example 2, host cells expressing the two-site mutants described in Table 4 above were constructed, and the two-site mutants were expressed; and a catalytic reaction system for the two-site mutants was constructed according to the method of Example 3.

[0087] 3. Detection and analysis of ω-TA13 multi-site mutant yield

[0088] In this embodiment, we aim to determine the yield of 3-aminopropanol produced by wild-type ω-TA13 and its mutants under the same conditions. Using the catalytic reaction system constructed in Example 5.2, the relative yield of 3-aminopropanol produced by each dual-site mutant relative to the yield of 3-aminopropanol produced by wild-type ω-TA13 was determined by HPLC, wherein the yield of 3-aminopropanol produced by wild-type ω-TA13 was 0.56 g / L.

[0089] Three parallel experiments were designed for each mutant, and the average value of the final results from each group was calculated. Figure 4 As shown, the two-site mutants tested all increased the yield of 3-aminopropanol, and the data of each group were significantly different, with P values ​​all < 0.05; among them, V237A / T288A produced the highest yield of 3-aminopropanol, which was 121% of that of the wild type, and the catalytic effect was significantly improved.

[0090] sequence

[0091] SEQ ID NO. 1: Wild-type ω-TA13 protein sequence

[0092] MNMITNHMPTAELQALDAAHHIHPFTTQDDLTAKGARIITRATGVTLTDSEGTEILDAMAGLWCVNIGYGRDELAEVAARQMRELPYYNTFFQTTHIPAIALSAKLAELAPGDLN HVFYAGSGSEANDTNMRMVRTYWAQKGKPEKKIIISRKNAYHGSTMAGASLGGMTPMHEQGGLPIPDVHHIDQPHWYSEGGDMSREEFGLQRAQELEKAILELGEDKVAAFIGEPI QGAGGVVIPPATYWPEIQRICDKYEILLIADEVICGFGRTGNWFGSETVGIKPHIMTIAKGLSSGYAPIGGSIVCDEVAEVIGACEFNHGYTYSGHPVAAAVALENLRILEEEGI VDRVREETAPYLAEKWSSLADHPLVGEARSVGLMGTLALTPNKETRASFAGDAGTIGYICREFCFANNLVMRHVGDRMIISPPLVITKAEIDTLVERARLALDLTLEKIKADGLYK

[0093] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A mutant of ω-transaminase TA13, which, relative to the wild-type ω-transaminase TA13 shown in SEQ ID NO: 1, has any amino acid substitution of G228A, Q232M, V237A, I260V, T288A and M380L or any combination thereof.

2. A nucleic acid molecule encoding the ω-transaminase TA13 mutant according to claim 1.

3. An expression vector comprising the nucleic acid molecule of claim 2; optionally, the expression vector is a vector suitable for expression in bacteria or yeast; preferably, the expression vector is a pET, pGEX, or pYES expression vector.

4. A host cell comprising the nucleic acid molecule of claim 2 or the expression vector of claim 3; optionally, the host cell is a bacterium or a fungus; preferably, the host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

5. The use of the ω-transaminase TA13 mutant as described in claim 1, the nucleic acid molecule as described in claim 2, the expression vector as described in claim 3, or the host cell as described in claim 4 in the preparation of 3-aminopropanol.

6. A method for preparing 3-aminopropanol by a bioenzymatic method, the method comprising adding the ω-transaminase TA13 mutant of claim 1 or the host cell of claim 4 to the reaction system.

7. The method according to claim 6, wherein, The reaction system also includes 3-hydroxypropionaldehyde as a substrate; Preferably, the reaction system further includes a coenzyme; more preferably, the coenzyme is pyridoxal 5′-phosphate.

8. The method according to claim 7, wherein, The reaction system also includes an amino donor, wherein the amino donor includes L-alanine or isopropylamine.

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