A mutant of ω-transaminase TA12 modified through rational design

Abstract

This invention provides a rationally designed mutant of transaminase TA12. Specifically, this invention provides a TA12 mutant of ω-transaminase that, relative to the wild-type ω-transaminase TA12 shown in SEQ ID NO:1, has an amino acid substitution at any position selected from 26, 74, and / or 136, or any combination thereof. The mutant successfully enhances the catalytic activity of wild-type ω-TA12, 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 mutant of ω-transaminase TA12 with enhanced activity through rational design modification, 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, such as ω-TA12, and found that they have catalytic activity in converting 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 research, the applicant identified ω-transaminase ω-TA12 (amino acid sequence shown in SEQ ID NO: 1) with amino acid sequence accession number WP_076629700.1 in the NCBI database as having catalytic activity for the conversion of 3-hydroxypropanal to 3-aminopropanol. However, it faces issues with activity and stability, which limits its application in the production of 3-aminopropanol. This invention aims to address the problems of insufficient activity and stability of ω-TA12, which is particularly significant in the TA12-catalyzed synthesis of important chemical products such as 3-aminopropanol, limiting its efficiency and yield in industrial production.

[0006] To overcome this challenge, this invention employs a rational design strategy, using molecular simulation techniques to predict the binding mode between the substrate and ω-TA12 and identifying key amino acid sites for catalytic activity. Further, amino acid conservation analysis was used to identify potential mutation sites, and by combining homologous protein sequence comparison, an ω-12 mutant with enhanced activity was successfully designed. This mutation improves the activity and stability of TA12, optimizes the biosynthesis of 3-aminopropanol, increases yield and catalytic efficiency, thereby enhancing the application of TA12 in the industrial production of compounds such as 3-aminopropanol.

[0007] Specifically, this invention first identifies relevant sites near the catalytic active site of the enzyme through molecular docking; secondly, it analyzes the conservation of amino acid sequences based on co-evolution theory, and performs homologous protein multiple sequence alignment on the wild-type sequence. Finally, it identifies and constructs a series of ω-transaminase mutants, and expresses these mutants under experimental conditions. Finally, it conducts activity tests on these mutants under the same conditions to screen for mutants exhibiting optimal performance.

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

[0009] (a) Mutate the threonine at position 26 of SEQ ID NO: 1 in the amino acid sequence to serine;

[0010] (b) Mutate the leucine at position 74 of SEQ ID NO: 1 in the amino acid sequence to methionine;

[0011] (c) Mutate the threonine at position 136 of SEQ ID NO: 1 in the amino acid sequence to histidine;

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

[0013] 1. A mutant of ω-transaminase TA12, having an amino acid substitution at any position selected from 26, 74 and / or 136 or any combination thereof, relative to the wild-type ω-transaminase TA12 shown in SEQ ID NO: 1.

[0014] 2. The ω-transaminase TA12 mutant according to Project 1, which, relative to the wild-type ω-transaminase TA12 shown in SEQ ID NO: 1, has any one of the amino acid substitutions T26S, L74M and T136H or any combination thereof.

[0015] 3. A nucleic acid molecule encoding the ω-transaminase TA12 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 epitope 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 TA12 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 bioenzymatic method, the method comprising adding the ω-transaminase TA12 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 pyridoxal coenzyme 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-TA12, which is simple and environmentally friendly. Through rational design, the catalytic activity of wild-type ω-TA12 is enhanced, increasing its catalytic efficiency in the industrial 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-TA12 and a schematic diagram of the catalytic pocket are shown.

[0027] Figure 2 The results show the confirmation of the catalytic active sites of wild-type ω-TA12.

[0028] Figure 3 The results of multiple sequence alignment of wild-type ω-TA12 and mutant are shown.

[0029] Figure 4 The results of the ω-TA12 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.; unless otherwise stated, the reagents and materials used are commercially available.

[0032] In some embodiments, the host cell may be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae, etc.

[0033] The culture media and solutions involved in the following examples are shown below:

[0034] 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.

[0035] LB liquid medium: 10 g·L -1 Tryptone, 5 g / L -1 Yeast extract and 10 g·L -1 Sodium chloride.

[0036] 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.

[0037] Example 1: Design of various mutants of ω-TA12

[0038] 1. Construct a 3D structural model of ω-TA2 using AlphaFold:

[0039] 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-TA12, following the user manual or standard procedures of existing technologies.

[0040] Specifically, the amino acid sequence of wild-type ω-TA12 (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-TA based on the input amino acid sequence, as shown in the figure. Figure 1 As shown.

[0041] 2. Molecular docking simulation

[0042] Based on the three-dimensional structure obtained above, molecular docking was performed using AutoDock Vina (www.autodock.scripps.edu) software to simulate the interaction between the substrate 3-hydroxypropionaldehyde and ω-TA12.

[0043] 3. Active pocket, site confirmation

[0044] 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 standard procedures to confirm the relevant active sites. The results are as follows: Figure 2 As shown, it displays amino acid residues that interact with the substrate surrounding the binding active site of wild-type ω-TA12.

[0045] 4. Homologous multiple sequence alignment

[0046] The amino acid sequence of wild-type ω-TA12 (SEQ ID NO: 1) was uploaded to the NCBI Blast database, where 5000 homologous sequences were matched. The homologous sequence set was then downloaded and aligned using MEGA. The results are as follows: Figure 3As 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-TA12. The amino acid distribution frequency at these sites will be statistically analyzed later.

[0047] 5. Mutant Confirmation

[0048] 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, positions 74, 26, and 136 are located at the active site, indicating their association with enzyme activity. Furthermore, co-evolutionary analysis was performed on all sites near the active site, comparing the amino acid residue distribution frequencies of these sites with a large number of homologous sequences. The results show that most sites are highly conserved, meaning they have not undergone mutations during evolution, which also indicates that these sites are crucial for enzyme function. However, some sites exhibit varying frequencies of variation. Based on this phenomenon, we analyze the distribution frequency of these sites before considering mutation. For example, the leucine L at position 74 may have evolved to M or S, so we could try mutating L to M or S.

[0049] Finally, in this embodiment, a series of mutants that may affect ω-TA12 activity were obtained, namely, mutants T26S, L74M and T136H (their amino acid sequences are shown in SEQ ID NO:2-4, respectively).

[0050] Example 2: Construction of host cells expressing wild-type ω-TA12 and its mutants, and expression of wild-type and mutant ω-TA2.

[0051] 1. Construction of recombinant host cells containing the coding genes of wild-type ω-TA12 and its mutants

[0052] Based on the codon preference of E. coli, the codons of the ω-TA12 mutant and wild-type ω-TA12 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 E. coli BL21(DE3) host cells (purchased from Sangon Biotech) to obtain recombinant host cells. Host cells transformed with wild-type ω-TA12 were used as the control group.

[0053] 2. Expression of ω-TA12 in wild-type and mutant strains

[0054] 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 TA12 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 h 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 h, the whole E. coli cells were harvested by centrifugation (6000 g, 20 min) 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-TA12 enzyme or its mutant, which can be used to express wild-type ω-TA12 enzyme or its mutant in a catalytic system for transamination catalysis to prepare 3-aminopropanol.

[0055] Alternatively, wild-type ω-TA12 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-TA12 enzyme or its mutant) is stored at -20℃ for later use.

[0056] Example 3: Construction of a catalytic reaction system for ω-TA12 enzyme or its mutant

[0057] In this embodiment, a reaction system for producing 3-aminopropanol was constructed.

[0058] In this reaction system, L-alanine is used as the amino donor, 3-hydroxypropanal as the substrate, and wild-type ω-TA12 enzyme or its mutant is used as the catalyst. For wild-type ω-TA12 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-TA12 enzyme or its mutant, or the supernatant obtained by cell disruption and extraction in Example 2 (containing wild-type ω-TA12 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-TA12 enzyme or its mutant, and the specific conditions for the catalytic reaction are as follows.

[0059] During the aforementioned research, the inventors discovered that the wild-type ω-TA12 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 reaction 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, 0.70 g / L of 3-aminopropanol can be obtained.

[0060] To evaluate the catalytic activity of the ω-TA12 mutant, in this example, the improvement in enzyme activity was assessed by measuring the concentration of the target product under high substrate concentration and short catalytic time. The specific procedures are as follows:

[0061] Maintaining a molar ratio of amino donor to reaction substrate of 2.4:1, the whole Escherichia coli cells collected in Example 2 were cultured in a 2 mL reactor under the conditions described in Table 1, at 200 rpm, 37-45°C, and pH 7.0-8.0, for substrate transammoniation for 3 h.

[0062] Table 1 Enzyme Conversion Reaction System

[0063]

[0064] Example 4: Yield detection of wild-type ω-TA12 and its mutants

[0065] In this embodiment, the activities of ω-TA12 wild-type and its mutants under the same conditions were determined, and mutants that could enhance the activity were identified. The yield of 3-aminopropanol generated by the wild-type and mutant catalytic systems containing ω-TA12 constructed in Example 3 was detected by HPLC. In addition to single mutations, this invention also constructed and detected combined mutations, specifically combined mutants with two sites: T26S+L74M, T26S+T136H, and L74M+T136H, and their detection was performed.

[0066] Three parallel experiments were designed for each mutant. The final 3-aminopropanol yield results are shown in Table 2, which represents the average of the three parallel experiments. The catalytic efficiency of the single-site mutants T26S, L74M, and T136H was improved compared with the wild-type TA12 enzyme (WT). Among them, the catalytic efficiency of the T136H mutant was increased by 28.9% compared with the wild type. The catalytic efficiency of the two-site mutants T26S + L74M, T26S + T136H, and L74M + T136H were also significantly improved. In addition, significance analysis was performed on the following groups of data, and the P values ​​were all <0.01, indicating significant differences.

[0067] Table 2. Yields of 3-aminopropanol obtained from TA12 enzyme and its mutants

[0068]

[0069] SEQ ID NO. 1: Wild-type ω-TA12 protein sequence

[0070] 1 MPAITNHLPT SELQALDAAH HMHPFTAGGE LAAKGARVIT RANGVFLHDS

[0071] 51 EGNEILDGMA GLWCVNIGYG RGELADVAAR QMRELPYYNT FFQTTHVPAI

[0072] 101ALTQKIAELA PGDLNHVFFA GSGSEANDTN LRMVRTYWAI KGKPDKHIVI

[0073] 151 SRKNAYHGSS VGSGSLGGMT AMHAQGGLPI PGIVHIDQPN WWAEGGSMSR

[0074] 201 EEFGVSRAKQ LEVAILEHGE DKVAAFIAEP IQGAGGVIIP PETYWPEIQR

[0075] 251 ICDKYDILLI ADEVICGFGR TGNWFGSQTL GIKPHIMTIA KGLSSGYQPI

[0076] 301 GGSIVCDEVA EVIGSGEFNH GYTYSGHPVA AAVALENLRI LEEENVLDHV

[0077] 351 RDVAMPALHE MWHGLADHPL VGETTITGMM GSLALTPHKD SRAKFAMDAG

[0078] 401 TAGFMCRERC FANNLVMRHV YDRMVISPPL IITPDEIAEI GRRARTALDE

[0079] 451 CYVQLKDGDM LKPAA

[0080] SEQ ID NO. 2: Mutant ω-TA12-T26S protein sequence

[0081] 1 MPAITNHLPT SELQALDAAH HMHPFSAGGE LAAKGARVIT RANGVFLHDS

[0082] 51 EGNEILDGMA GLWCVNIGYG RGELADVAAR QMRELPYYNT FFQTTHVPAI

[0083] 101ALTQKIAELA PGDLNHVFFA GSGSEANDTN LRMVRTYWAI KGKPDKHIVI

[0084] 151 SRKNAYHGSS VGSGSLGGMT AMHAQGGLPI PGIVHIDQPN WWAEGGSMSR

[0085] 201 EEFGVSRAKQ LEVAILEHGE DKVAAFIAEP IQGAGGVIIP PETYWPEIQR

[0086] 251 ICDKYDILLI ADEVICGFGR TGNWFGSQTL GIKPHIMTIA KGLSSGYQPI

[0087] 301 GGSIVCDEVA EVIGSGEFNH GYTYSGHPVA AAVALENLRI LEEENVLDHV

[0088] 351 RDVAMPALHE MWHGLADHPL VGETTITGMM GSLALTPHKD SRAKFAMDAG

[0089] 401 TAGFMCRERC FANNLVMRHV YDRMVISPPL IITPDEIAEI GRRARTALDE

[0090] 451 CYVQLKDGDM LKPAA

[0091] SEQ ID NO. 3: Mutant ω-TA12-L74M protein sequence

[0092] 1 MPAITNHLPT SELQALDAAH HMHPFTAGGE LAAKGARVIT RANGVFLHDS

[0093] 51 EGNEILDGMA GLWCVNIGYG RGEMADVAAR QMRELPYYNT FFQTTHVPAI

[0094] 101ALTQKIAELA PGDLNHVFFA GSGSEANDTN LRMVRTYWAI KGKPDKHIVI

[0095] 151 SRKNAYHGSS VGSGSLGGMT AMHAQGGLPI PGIVHIDQPN WWAEGGSMSR

[0096] 201 EEFGVSRAKQ LEVAILEHGE DKVAAFIAEP IQGAGGVIIP PETYWPEIQR

[0097] 251 ICDKYDILLI ADEVICGFGR TGNWFGSQTL GIKPHIMTIA KGLSSGYQPI

[0098] 301 GGSIVCDEVA EVIGSGEFNH GYTYSGHPVA AAVALENLRI LEEENVLDHV [[ID=,16]]

[0099] 351 RDVAMPALHE MWHGLADHPL VGETTITGMM GSLALTPHKD SRAKFAMDAG

[0100] 401 TAGFMCRERC FANNLVMRHV YDRMVISPPL IITPDEIAEI GRRARTALDE

[0101] 451 CYVQLKDGDM LKPAA

[0102] SEQ ID NO. 4: Mutant ω-TA12-T136H Protein Sequence<,

[0103] 1 MPAITNHLPT SELQALDAAH HMHPFTAGGE LAAKGARVIT RANGVFLHDS

[0104] 51 EGNEILDGMA GLWCVNIGYG RGELADVAAR QMRELPYYNT FFQTTHVPAI

[0105] 101ALTQKIAELA PGDLNHVFFA GSGSEANDTN LRMVRHYWAI KGKPDKHIVI

[0106] 151 SRKNAYHGSS VGSGSLGGMT AMHAQGGLPI PGIVHIDQPN WWAEGGSMSR

[0107] 201 EEFGVSRAKQ LEVAILEHGE DKVAAFIAEP IQGAGGVIIP PETYWPEIQR

[0108] 251 ICDKYDILLI ADEVICGFGR TGNWFGSQTL GIKPHIMTIA KGLSSGYQPI

[0109] 301 GGSIVCDEVA EVIGSGEFNH GYTYSGHPVA AAVALENLRI LEEENVLDHV

[0110] 351 RDVAMPALHE MWHGLADHPL VGETTITGMM GSLALTPHKD SRAKFAMDAG

[0111] 401 TAGFMCRERC FANNLVMRHV YDRMVISPPL IITPDEIAEI GRRARTALDE

[0112] 451 CYVQLKDGDM LKPAA

Claims

1. A mutant of ω-transaminase TA12, having an amino acid substitution at any position selected from 26, 74 and / or 136 or any combination thereof, relative to the wild-type ω-transaminase TA12 shown in SEQ ID NO:

1.

2. The ω-transaminase TA12 mutant according to claim 1, which, relative to the wild-type ω-transaminase TA12 shown in SEQ ID NO: 1, has any one of the amino acid substitutions T26S, L74M and T136H or any combination thereof.

3. A nucleic acid molecule encoding an ω-transaminase TA12 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 epitope 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 TA12 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 TA12 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 pyridoxal coenzyme 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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