A TA2 transaminase mutant modified through rational design and its application

Abstract

This invention provides a rationally designed and modified ω-transaminase TA2 mutant and its applications. Specifically, this invention provides an ω-transaminase TA2 mutant, which, relative to the wild-type ω-transaminase TA2 shown in SEQ ID NO:1, has an amino acid substitution at any position selected from 56, 272, and / or 295, or any combination thereof. The mutant successfully enhances the catalytic activity of wild-type ω-TA2, thereby increasing its catalytic efficiency in the biosynthesis of 3-aminopropanol, and has significant industrial application potential and considerable commercial value.

Description

Technical Field

[0001] This invention relates to a rationally modified ω-transaminase TA2 mutant, belonging to the field of enzyme engineering. 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 like vitamin B5 (panthenol). With rising living standards, the application of panthenol in daily chemical products is increasing, particularly in hair care products and specialized cosmetics, significantly boosting market demand for 3-aminopropanol. Currently, the mainstream process for preparing 3-aminopropanol uses 3-hydroxypropionitrile as a raw material, employing a catalyst for hydrogenation reduction to obtain 3-aminopropanol. However, this process generally suffers from problems such as poor catalyst activity, low 3-aminopropanol yield, high byproduct selectivity, difficult product separation, harsh reaction conditions, low safety, and demanding equipment requirements.

[0003] ω-transaminases (hereinafter referred to as ω-TAs) are biocatalysts that 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 pathway utilizing ω-TAs has advantages such as being environmentally friendly, efficient, and sustainable. In previous work, the applicant screened and obtained several ω-transaminases from specific sources, discovering that they possess catalytic activity for the conversion of 3-hydroxypropanal to 3-aminopropanol.

[0004] However, the catalytic activity of ω-transaminase is an important factor in the industrial production of 3-aminopropanol. Developing ω-TA with higher catalytic activity or efficiency is crucial for the industrial preparation of 3-aminopropanol. Summary of the Invention

[0005] In previous studies, the applicant identified ω-transaminase ω-TA2 (with amino acid sequence shown in SEQ ID NO: 1) in the PDB database as having catalytic activity for the conversion of 3-hydroxypropanal to 3-aminopropanol, but its activity was insufficient. This invention aims to address the problem of insufficient activity of ω-TA2, which is particularly significant in the ω-TA2-catalyzed synthesis of important chemical products such as 3-aminopropanol, limiting its efficiency and yield in industrial production.

[0006] This invention uses ω-TA2 as a wild-type ω-transaminase. Molecular simulation techniques were used to predict the binding mode between the substrate and ω-TA2, identifying key amino acid sites for catalytic activity. Further amino acid conservation analysis was used to determine potential mutation sites, and by combining homologous protein sequence comparison, an ω-TA2 mutant with enhanced activity was successfully designed. This mutation improves the catalytic activity and stability of ω-TA2, optimizes the biosynthesis of 3-aminopropanol, and achieves a significant increase in yield and catalytic efficiency.

[0007] This invention provides an efficient and precise modification strategy to obtain ω-TA2 mutants with enhanced activity, thereby enhancing the application of ω-TA2 in the industrial production of compounds such as 3-aminopropanol.

[0008] In this invention, the mutant involved is any one of the following (a) to (c):

[0009] (a) Mutate the phenylalanine at position 56 of the amino acid sequence SEQ ID NO: 1 to alanine;

[0010] (b) Mutate leucine at position 272 of the amino acid sequence SEQ ID NO: 1 to methionine;

[0011] (c) The threonine at position 295 in the amino acid sequence SEQ ID NO:1 is mutated to alanine.

[0012] Specifically, the present invention provides the following technical solutions:

[0013] 1. A mutant of ω-transaminase TA2, having an amino acid substitution at any position selected from 56, 272 and / or 295 or any combination thereof, relative to the wild-type ω-transaminase TA2 shown in SEQ ID NO: 1.

[0014] 2. The ω-transaminase TA2 mutant according to Project 1, which, relative to the wild-type ω-transaminase TA2 shown in SEQ ID NO: 1, has any one of the amino acid substitutions F56A, L272M and T295A or any combination thereof.

[0015] 3. A nucleic acid molecule encoding an ω-transaminase TA2 mutant according to any one of items 1-2.

[0016] 4. An expression vector comprising the nucleic acid molecule described in item 3; 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.

[0017] 5. A host cell comprising the nucleic acid molecule described in item 3 or the expression vector described in item 4; 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.

[0018] 6. The use of the ω-transaminase TA2 mutant as described in any one of Items 1-2, the nucleic acid molecule as described in Item 3, the expression vector as described in Item 4, or the host cell as described in Item 5 in the preparation of 3-aminopropanol.

[0019] 7. A method for preparing 3-aminopropanol by a biological enzymatic process, the method comprising adding the ω-transaminase TA2 mutant described in item 1 or 2, or the host cell described in item 5, to the reaction system.

[0020] 8. The method according to Project 7, wherein the reaction system further includes 3-hydroxypropanal as a substrate;

[0021] Preferably, the reaction system further includes a coenzyme; more preferably, the coenzyme is pyridoxal 5′-phosphate.

[0022] 9. The method according to Project 8, wherein the reaction system further includes an amino donor, wherein the amino donor includes L-alanine, ammonia, or isopropylamine.

[0023] 10. The method according to Project 9, wherein the molar ratio of the amino donor to the substrate is 2-5:1, the reaction temperature is 37-45℃, and the pH is 7-8.

[0024] Beneficial effects

[0025] This invention develops a novel biosynthetic pathway for 3-aminopropanol using ω-TA2, which is simple and environmentally friendly. Through rational design, the catalytic activity of wild-type ω-TA2 was successfully enhanced, thereby increasing its catalytic efficiency in the biosynthesis of 3-aminopropanol, demonstrating significant industrial application potential and substantial commercial value. Attached Figure Description

[0026] Figure 1 The three-dimensional structure of wild-type ω-TA2 is shown.

[0027] Figure 2 The results show the confirmation of the catalytic active sites of wild-type ω-TA2.

[0028] Figure 3 The results of multiple sequence alignment of wild-type ω-TA2 and mutant are shown.

[0029] Figure 4 The results of the ω-TA2 amino acid frequency analysis are shown. Detailed Implementation

[0030] 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.

[0031] 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.; and unless otherwise stated, the reagents and materials used are commercially available. In some embodiments, the host cell may be *Escherichia coli*, *Corynebacterium glutamicum*, *Bacillus subtilis*, *Pichia pastoris*, or *Saccharomyces cerevisiae*, etc.

[0032] The culture media and solutions involved in the following examples are shown below:

[0033] 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.

[0034] LB liquid medium: 10 g·L -1 Tryptone, 5 g / L -1 Yeast extract and 10 g·L -1 Sodium chloride.

[0035] 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.

[0036] Example 1: Design of ω-TA2 mutant

[0037] 1. Construct a 3D structural model of ω-TA2 using AlphaFold:

[0038] 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 3D structure of ω-TA2 by referring to the user manual or standard procedures in existing technologies.

[0039] Specifically, the amino acid sequence of wild-type ω-TA2 (SEQ ID NO: 1) is formatted into the input file format required by AlphaFold. AlphaFold is 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-TA2 based on the input amino acid sequence, as shown in the following figure. Figure 1 As shown.

[0040] 2. Molecular docking simulation

[0041] Based on the obtained ω-TA2 three-dimensional structure, molecular docking was performed using AutoDock Vina (www.autodock.scripps.edu) software to simulate the interaction between the substrate 3-hydroxypropionaldehyde and ω-TA2.

[0042] 3. Active pocket, site confirmation

[0043] The docking results were imported into PyMOL software (written by Warren Lyford DeLan and commercially distributed by DeLanoScientific LLC), and the software was operated according to its standard operating procedures to confirm the relevant active sites, which specifically involved the following steps:

[0044] Loading protein structures: Use PyMOL to import the protein's PDB file or download the relevant protein structure from the RCSB PDB database (www.rcsb.org).

[0045] Select target residues: Use commands or tools in PyMOL to select residues containing active sites within a 5A radius around the ligand-protein binding active site.

[0046] Displaying residues and amino acid side chains: Ensure that the selected residues and surrounding amino acid side chains are displayed for better observation of the active site environment.

[0047] Analyze the interactions between residues: Use tools in PyMOL, such as distance measurement or hydrogen bond viewing, to analyze the interactions between residues in the active site, including hydrogen bonds, hydrophobic interactions, etc.

[0048] Marking important residues: Based on the analysis results, mark important residues in PyMOL for further analysis or visualization.

[0049] The results are as follows Figure 2 As shown, it displays amino acid residues that interact with the substrate located around the binding active site of wild-type ω-TA2.

[0050] 4. Homologous multiple sequence alignment

[0051] The amino acid sequence (SEQ ID NO: 1) of wild-type ω-TA2 was uploaded to the NCBI Blast database, where 5,000 homologous sequences were matched. The homologous sequence set was then downloaded and aligned with the sequence in MEGA.

[0052] The comparison results are as follows Figure 3 As shown, the sequence is highly consistent in most places, but there are differences in the distribution of amino acid residues at some sites, suggesting that they may be related to the activity of ω-TA2. The amino acid distribution frequency at these sites will be statistically analyzed later.

[0053] 5. Mutant Confirmation

[0054] After homologous sequence alignment, mutation sites are screened in the viable pocket based on their conservation, thus obtaining the amino acid frequency distribution results, such as... Figure 4 As shown, sites 56, 272, and 295 are located at the active site and are associated with enzyme activity. Furthermore, co-evolutionary analysis was performed on all sites near the active site, i.e., the frequency distribution of amino acid residues at these sites was studied by comparing a large number of homologous sequences. The results showed that most sites are highly conserved, meaning they have not mutated during evolution, which also indicates that these sites are crucial for enzyme function. However, some sites exhibited variability in frequency. Based on this phenomenon, we analyzed the frequency distribution of these sites before considering whether to mutate them. For example, site 56 may have evolved to be phenylalanine (F) or alanine (A), so the inventors attempted to mutate F to A to explore changes in activity. Finally, in this embodiment, a series of mutants that may affect ω-TA2 activity were obtained, namely, mutants F56A, L272M, and T295A.

[0055] Example 2: Construction of host cells expressing ω-TA2 wild-type and mutants, and expression of ω-TA2 wild-type and mutants.

[0056] 1. Construction of recombinant host cells containing the coding genes of wild-type ω-TA2 and its mutants

[0057] Based on the codon preference of E. coli, the codons of the ω-TA2 mutant and wild-type ω-TA2 sequences were optimized. The codon-optimized coding genes 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-TA2 were used as the control group.

[0058] 2. Expression of ω-TA2 wild-type and its mutants

[0059] 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 TA2 mutant and wild-type coding sequences respectively 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 hours to obtain seed cultures. 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 solution until the optical density (OD600) at 600 nm reached 0.70. After culturing at 25 °C for another 20 hours, the whole E. coli cells were harvested by centrifugation (6000g, 20 minutes) at 4 °C. 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-TA2 enzyme or its mutants, and could be used to express wild-type ω-TA2 enzyme or its mutants in a catalytic system for transamination catalysis to prepare 3-aminopropanol.

[0060] Alternatively, wild-type ω-TA2 enzyme or its mutant can be 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 90-100 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-TA2 enzyme or its mutant) is collected and stored at -20℃ for later use.

[0061] Example 3: Construction of a catalytic reaction system for ω-TA2 enzyme or its mutant

[0062] In this embodiment, a reaction system for producing 3-aminopropanol was constructed.

[0063] In this reaction system, L-alanine is used as the amino donor, 3-hydroxypropanal as the substrate, and wild-type ω-TA2 enzyme or its mutant is used as the catalyst. For wild-type ω-TA2 enzyme or its mutant, whole *E. coli* cells collected in Example 2 can be directly added to the reaction system to express wild-type ω-TA2 enzyme or its mutant, or the supernatant obtained by cell disruption and extraction in Example 2 (containing wild-type ω-TA2 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 wild-type ω-TA2 enzyme or its mutant, and the specific conditions for the catalytic reaction are as follows.

[0064] During the aforementioned research, the inventors discovered that wild-type ω-TA2 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, 0.72 g / L of 3-aminopropanol can be obtained.

[0065] To evaluate the catalytic activity of the ω-TA2 mutant, the improvement in enzyme activity was assessed by measuring the concentration of the target product, 3-aminopropanol, under conditions of high substrate concentration and short catalytic time. The specific procedures are as follows:

[0066] The enzyme conversion system was prepared under the conditions described in Table 1, maintaining a molar ratio of amino donor to reaction substrate of 2.4:1. The whole cells of Escherichia coli collected in Example 2 were placed in a 2 mL reactor and cultured at 220 rpm, 37 °C, and pH 7.0-8.0 for 3 hours for the transamination of the substrate.

[0067] Table 1 Enzyme Conversion Reaction System

[0068]

[0069] Example 4: Yield detection of ω-TA2 wild type and various mutants

[0070] In this embodiment, the activities of ω-TA2 wild-type and its mutants under the same conditions were determined, and mutants that could enhance activity were identified. The catalytic system containing ω-TA2 wild-type and mutants constructed in Example 3 was used, and the yield of 3-aminopropanol synthesized by wild-type and its mutants was detected by HPLC. In addition to single mutations, this invention also constructed and detected combined mutations, namely, F56A+L272M, F56A+T295A, and L272M+T295A dual-site mutants, and their results were also detected. Three parallel experiments were designed for each mutant, and the final 3-aminopropanol yield (average of the three parallel experiments) results are shown in Table 2. The catalytic efficiency of the single-site mutants F56A, L272M, and T295A was significantly improved compared to the wild-type ω-TA2 enzyme (WT), with mutant F56A showing the best effect. All dual-site mutants exhibited improved catalytic efficiency. Furthermore, significance analysis was performed on the following data, and all P values ​​were <0.01, indicating significant differences.

[0071] Table 2. Yields of 3-aminopropanol obtained from ω-TA2 enzyme and its mutants

[0072]

[0073] sequence list

[0074] SEQ ID NO. 1: Wild-type ω-TA2 protein sequence

[0075] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0076] SEQ ID NO. 2: Mutant ω-TA2-F56A protein sequence

[0077] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQVPPIESTEGDYLIMPDGTRLLDAFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0078] SEQ ID NO. 3: Mutant bodyω-TA2-L272M protein sequence

[0079] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVMTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0080] SEQ ID NO. 4: Mutant ω - TA2 - T295A Protein Sequence

[0081] MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLIMPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFV STGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITE VSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIIAMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVE QAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQ

[0082] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A mutant of ω-transaminase TA2, having an amino acid substitution at any position selected from 56, 272 and / or 295 or any combination thereof, relative to the wild-type ω-transaminase TA2 shown in SEQ ID NO:

1.

2. The ω-transaminase TA2 mutant according to claim 1, which, relative to the wild-type ω-transaminase TA2 shown in SEQ ID NO: 1, has any one of the amino acid substitutions F56A, L272M and T295A or any combination thereof.

3. A nucleic acid molecule encoding an ω-transaminase TA2 mutant according to any one of claims 1-2.

4. An expression vector comprising the nucleic acid molecule of claim 3; 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.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4; 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.

6. The use of the ω-transaminase TA2 mutant as described in any one of claims 1-2, the nucleic acid molecule as described in claim 3, the expression vector as described in claim 4, or the host cell as described in claim 5 in the preparation of 3-aminopropanol.

7. A method for preparing 3-aminopropanol by a bioenzymatic method, the method comprising adding the ω-transaminase TA2 mutant of claim 1 or 2, or the host cell of claim 5, to the reaction system.

8. The method according to claim 7, 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.

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

10. The method according to claim 9, wherein, The molar ratio of the amino donor to the substrate is 2-5:1, the reaction temperature is 37-45℃, and the pH is 7-8.

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