A halophilic adenosine kinase mutant and its encoding gene and application

Abstract

The present invention discloses a halophilic adenosine kinase mutant, its encoding gene, and its application. By rationally modifying adenosine kinase (EC 2.7.1.20) derived from Candida parapolymorpha (ATCC 26012), the amino acid sequence of which is shown in SEQ ID NO: 1, the halophilic adenosine kinase mutant exhibits higher catalytic activity and salt tolerance than the wild-type adenosine kinase.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a halophilic adenosine kinase mutant derived from Candida parapolymorpha (strain ATCC 26012) subjected to salt-tolerance modification, a coding gene thereof, and an application thereof. Background Art

[0002] During the production of adenosine monophosphate (AMP), the substrate acetyl phosphate is added in a fed-batch manner to react and generate acetic acid as a byproduct. Alkali is then added to maintain a stable pH. This acid-base neutralization process causes the salt concentration in the reaction solution to gradually increase. The inhibitory effect on adenosine kinase (ADOK) (EC: 2.7.1.20) activity also increases with increasing salt concentration. When the salt concentration exceeds 300mM, the activity is significantly inhibited, and at 1200mM, the activity decreases by more than 50%. To adapt to the salt environment of the reaction, we need to find an efficient and salt-tolerant adenosine kinase.

[0003] Halophilic enzymes are generally derived from halophilic bacteria. Their main characteristic is their strict dependence on a certain salt ion concentration in the system. They can maintain their structural stability in high-salt environments and are resistant to denaturation under high temperature, pH, and the presence of organic solvents. Therefore, they have important application value in catalysis in high-salt, aqueous / organic, and non-aqueous media. Based on a summary of the structures and characteristics of existing halophilic enzymes in the Protein Data Bank (PDB), it is believed that halophilic enzymes differ from non-halophilic enzymes in that they have significantly more salt bridges and hydrogen bonds, contain some special salt ion binding sites, often exist in the form of oligomers, and have a significantly increased content of surface acidic amino acids. Research directions for the development of halophilic enzymes are to modify halophilic enzymes based on rational design to improve specific enzyme activity and stability. Summary of the Invention

[0004] To address the technical problems existing in the prior art, the present invention provides a halophilic adenosine kinase mutant, its encoding gene, and its application. This invention improves the enzyme activity and salt tolerance of adenosine kinase by rationally modifying the amino acid sequence of SEQ ID NO: 1, derived from Candida parapolymorpha (strain ATCC 26012).

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] A halophilic adenosine kinase mutant M1 has an amino acid sequence as shown in SEQ ID NO: 2. The amino acid sequence of the halophilic adenosine kinase mutant M1 is obtained by mutating lysine at position 40 of the adenosine kinase with a sequence as SEQ ID NO: 1 to aspartic acid; glutamine at position 63 to glutamate; asparagine at position 122 to aspartic acid; histidine at position 137 to glutamate; serine at position 244 to aspartic acid; lysine at position 288 to glutamate; serine at position 334 to aspartic acid; and serine at position 343 to glutamate.

[0007] A halophilic adenosine kinase mutant M2 has an amino acid sequence as shown in SEQ ID NO: 3. The amino acid sequence of the halophilic adenosine kinase mutant M2 is obtained by mutating lysine at position 40 to aspartic acid, glutamine at position 63 to glutamic acid, asparagine at position 122 to aspartic acid, histidine at position 137 to glutamic acid, serine at position 244 to aspartic acid, lysine at position 288 to glutamic acid, serine at position 334 to aspartic acid, serine at position 343 to glutamic acid, lysine at position 88 to glutamic acid, lysine at position 98 to aspartic acid, lysine at position 116 to glutamic acid, asparagine at position 183 to aspartic acid, serine at position 240 to glutamic acid, and serine at position 316 to glutamic acid in the adenosine kinase sequence as shown in SEQ ID NO: 1.

[0008] A halophilic adenosine kinase mutant M3, the amino acid sequence of the halophilic adenosine kinase mutant M3 is shown in SEQ ID NO: 4, the amino acid sequence of the halophilic adenosine kinase mutant M3 is shown in SEQ ID NO: The adenosine kinase of NO: 1 is mutated by mutating lysine at position 40 to aspartic acid; glutamine at position 63 to glutamate; asparagine at position 122 to aspartic acid; histidine at position 137 to glutamate; serine at position 244 to aspartic acid; lysine at position 288 to glutamate; serine at position 334 to aspartic acid; serine at position 343 to glutamate; lysine at position 88 to glutamate; lysine at position 98 to aspartic acid; lysine at position 116 to glutamate; asparagine at position 183 to aspartic acid; serine at position 240 to glutamate; serine at position 316 to glutamate; lysine at position 86 to glutamate; lysine at position 145 to aspartic acid; lysine at position 311 to aspartic acid; and lysine at position 325 to glutamate.

[0009] The nucleotide sequence encoding the above-mentioned halophilic adenosine kinase mutant is also within the protection scope of the present invention.

[0010] Specifically, the gene encoding the halophilic adenosine kinase mutant M1 is shown in SEQ ID NO: 6; the gene encoding the halophilic adenosine kinase mutant M2 is shown in SEQ ID NO: 7; and the gene encoding the halophilic adenosine kinase mutant M3 is shown in SEQ ID NO: 8.

[0011] The recombinant vector and recombinant strain containing the above-mentioned halophilic adenosine kinase mutant are also within the protection scope of the present invention.

[0012] Specifically, the present invention provides a recombinant vector comprising the gene encoding the above-mentioned halophilic adenosine kinase mutant M1 and a recombinant strain comprising the recombinant vector, or a recombinant vector comprising the gene encoding the above-mentioned halophilic adenosine kinase mutant M2 and a recombinant strain comprising the recombinant vector, or a recombinant vector comprising the gene encoding the above-mentioned halophilic adenosine kinase mutant M3 and a recombinant strain comprising the recombinant vector.

[0013] The application of the aforementioned halophilic adenosine kinase mutant, recombinant vector, and recombinant strain in the synthesis of adenosine monophosphate is specifically to synthesize adenosine monophosphate using adenosine, adenosine triphosphate, and acetyl phosphate as substrates and the halophilic adenosine kinase mutant as a catalytic enzyme.

[0014] Compared to wild-type adenosine kinase (WT), the halophilic adenosine kinase mutants M1, M2, and M3 of the present invention exhibit similar activities to wild-type adenosine kinase (WT) in salt-free solutions. In high-salt solutions, the halophilic adenosine kinase mutants M1 and M2 exhibit higher catalytic activity and salt tolerance. In particular, the halophilic adenosine kinase mutants M2 and M3 retain over 50% of their enzyme activity in a 2000 mM KAc solution compared to WT. Furthermore, potassium acetate exhibits less inhibitory effects on enzyme activity than other acetates. Therefore, potassium hydroxide, an alkaline reagent, was selected to neutralize the acetic acid generated in the reaction system to form potassium acetate, maintaining a constant pH value. This allows for better application in the preparation of adenosine monophosphate (AMP). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is shown that the recombinant vector contains the adenosine kinase WT encoding gene (ADOK);

[0016] Figure 2 is a diagram showing the predicted structure of adenosine kinase WT protein;

[0017] Figure 3 is the conserved and nonconserved sequence model of adenosine kinase WT;

[0018] Figure 4 It is a protein electrostatic potential energy diagram showing adenosine kinase WT and adenosine kinase mutant M2. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail below with reference to specific examples. The examples will facilitate understanding of the present invention, but the scope of protection of the present invention is not limited to the following examples. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0020] Example 1

[0021] To generate the adenosine kinase mutants of the present invention, a rational engineering approach was employed. This approach integrates modern computational biology, using computer protein modeling, molecular dynamics simulations, high-throughput DNA sequencing, and gene synthesis techniques to develop new, rapid, and efficient de novo enzyme synthesis technologies. Guided by a specific target reaction, transition state models are established based on the reaction's catalytic mechanism. Protein and gene sequences with specific catalytic properties are then constructed to catalyze a range of non-natural reactions that are not catalyzed by natural enzymes.

[0022] The present invention uses AlphaFold2 to predict the protein structure of adenosine kinase WT, and its protein structure prediction model is as follows: Figure 2 As shown, AlphaFold2 is a new protein structure prediction tool developed based on deep learning algorithms. It can predict tertiary structure through primary amino acid sequence. This protein structure prediction tool is completely open to external use.

[0023] The conserved sequence positions of enzyme proteins were determined by computer simulation and multiple sequence alignment, such as Figure 3 As shown, the mutation site is selected as a non-conservative sequence, and a group of specific sites (located on the surface of the enzyme protein, evenly distributed in the structural space, positively charged or uncharged) are anchored in the non-conservative sequence mutation region, and gradually mutated into negatively charged amino acids to maximize the distribution of negative charge on the protein surface. At the same time, the increase in surface salt bridges and hydrogen bonds is taken into account to maintain its structural stability in a high-salt environment, and finally the halophilic adenosine kinase mutants M1, M2, and M3 are screened. Figure 4 The electrostatic potential energy diagram of adenosine kinase WT and halophilic adenosine kinase mutant M2 is shown. The negative charge distribution on the surface of M2 is significantly higher than that of WT (the darker the color on the protein surface, the more positive charge), and the mutation site is more conducive to the interaction of salt bridges and hydrogen bonds, thereby giving ADOK salt tolerance.

[0024] The amino acid sequence of adenosine kinase mutant M1 is shown in SEQ ID NO: 2, which has the following mutations: K40D, Q63E, N122D, H137E, S244D, K288E, S334D and S343E.

[0025] The amino acid sequence of adenosine kinase mutant M2 is shown in SEQ ID NO: 3, which has the following mutations: K40D, Q63E, N122D, H137E, S244D, K288E, S334D, S343E, K88E, K98D, K116E, N183D, S240E and S316E.

[0026] The amino acid sequence of adenosine kinase mutant M3 is shown in SEQ ID NO: 4, which contains the following mutations: K40D, Q63E, N122D, H137E, S244D, K288E, S334D, S343E, K88E, K98D, K116E, N183D, S240E, S316E, K86E, K145D, K311D and K325E.

[0027] Construction of halophilic adenosine kinase recombinant strain:

[0028] Adenosine kinase WT was synthesized from the genome of Candida parapolymorpha (strain ATCC 26012) (synthesized by GENEWIZ). The amino acid sequence is shown in SEQ ID NO: 1, and the encoding gene is shown in SEQ ID NO: 5.

[0029] The target genes of adenosine kinase mutants M1, M2, and M3 were synthesized by whole gene synthesis (synthesized by GENEWIZ). The target genes and vectors were cleaved by the corresponding restriction endonucleases ( NdI and XOt ) enzyme digestion treatment; the target gene was connected to the vector to obtain a recombinant expression plasmid, and the recombinant plasmid was transformed into Escherichia coli competent cells by the calcium chloride method (according to the molecular cloning manual).

[0030] According to the amino acid sequence of the above-mentioned adenosine kinase, the corresponding base sequence was artificially synthesized, wherein the nucleotide sequence encoding SEQ ID NO: 2 is shown as SEQ ID NO: 6; the nucleotide sequence encoding SEQ ID NO. 3 is shown as SEQ ID NO: 7; and the nucleotide sequence encoding SEQ ID NO. 4 is shown as SEQ ID NO: 8, and the expression vector was amplified and constructed.

[0031] Specifically, the corresponding base sequence was artificially synthesized based on the amino acid sequence of the aforementioned adenosine kinase mutant, and upstream primer F (5'-CGCCATATGATGGCTTTCGATCTTGT-3') and downstream primer R (5'-CCGCTCGAGCTAATTACTGTATGTTTGC-3') were designed and synthesized to obtain the adenosine kinase and mutant genes as templates for PCR amplification. The PCR reaction system included: 25 μL 2× PCR buffer, 10 μL 2 mM dNTPs, 1.5 μL each of 10 pmol / μL primer F and primer R, 1 μL template DNA, 1 μL high-fidelity polymerase, and finally, the volume was adjusted to 50 μL with sterile double-distilled water. The PCR reaction parameters were: 94°C melting for 2 minutes, 98°C denaturation for 10 seconds, 55°C annealing for 30 seconds, 68°C extension for 1 minute, set for 30 cycles, and 68°C fill for 5 minutes, and then stored at 4°C.

[0032] Adenosine kinase WT or its mutants M1, M2, and M3 were inserted into the PET-28a expression vector and transformed into Escherichia coli BL21 (DE3) cells.

[0033] Recombinant E. coli were cultured in LB medium (final concentration: 50 mg / L Kan) for 12 hours at 37°C and 200 rpm. The cells were then transferred to 1 L of TB medium (final concentration: 50 mg / L Kan, 1% (v / v) inoculum). Induction with 0.2 mM isopropyl β-d-1-mercaptogalactopyranoside (IPTG) was performed to an OD600 of 0.6-0.8 at 30°C and 150 rpm for 16 hours. Cells were harvested by centrifugation when the OD600 reached 5 or higher and used in the catalytic reaction.

[0034] Example 2

[0035] Enzyme activity detection of wild-type adenosine kinase WT and halophilic adenosine kinase mutants M1, M2, and M3

[0036] Preparation of adenosine kinase extract: Resuspend the halophilic adenosine kinase mutants M1, M2, and M3 prepared in Example 1, as well as the synthesized adenosine kinase WT, in an aqueous solution to obtain a 30 g / L aqueous solution. Add 1 / 1000th of the volume (v / v) of Triton-X100 and stir at 200 rpm for 5 minutes at 4°C to prepare a standard reaction mixture (5 ml):

[0037] The reaction system consisted of 100 mmol / L Tris-HCl (pH 7.5), 5 mmol / L adenosine, 5 mmol / L ATP, 10 mmol / L acetyl phosphate, and 2 mmol / L MgCl2. 30 g / L adenosine kinase extract was added at 30°C. The reaction was allowed to stand for 5 minutes, then shaken. Samples were collected every 10 minutes, and the reaction was continued for 1 hour. The reaction was then quenched in a boiling water bath. Enzyme activity was defined as the amount of enzyme required to produce 1 μmol AMP per minute.

[0038] Table 1 Enzyme activity parameters (M1–M3) of WT and mutants

[0039]

[0040] Table 1 compares the activities of WT and mutant adenosine kinase in salt-free solution. The activities of M1 and M2 are similar to those of WT, demonstrating that the selected surface residue substitutions have minimal negative impact on enzyme activity. However, M3, which contains the most substituted residues, exhibits a 45% decrease in activity. This decrease is attributed to decreased stability in low-salt solution. We hypothesize that the structural changes in mutant M3 significantly impair its proper folding and expression under low ionic strength conditions.

[0041] Example 3

[0042] Detection of salt tolerance of halophilic adenosine kinase mutants

[0043] To test the effect of high-salt solution on the enzyme, four acetate solutions (KAc, NaAc, LiAc, and NH4Ac) were prepared with concentrations ranging from 300 to 2000 mmol / L. Each acetate solution was mixed into the reaction system (same as in Example 2), and then the crude enzyme was added to initiate the reaction. This simulated the actual catalytic environment during the catalytic process and tested the effect of a high-salt environment on the enzyme. The percentage difference in activity was calculated as the enzyme activity measured in each reaction system / the WT enzyme activity under optimal conditions, 30.1 ± 1.5 U / g.

[0044] Table 2 Activity differences of WT and mutant adenosine kinase at different concentrations of KAc solution

[0045]

[0046] Table 3 Activity differences of adenosine kinase WT and mutants at different concentrations of LiAc solution

[0047]

[0048] Table 4 Activity differences of adenosine kinase WT and mutants at different concentrations of NaAc solution

[0049]

[0050] Table 5 Activity differences of adenosine kinase WT and mutants at different concentrations of NH4Ac solution

[0051]

[0052] The experimental results are shown in Table 2. When the acetate concentration was high, the activity of the WT was significantly inhibited, and the three mutants showed higher activity than the WT under all conditions. Among the four solutions, KAc had the lowest inhibitory effect on all mutants. The activity of the mutants in KAc solution was 1.1-2.0 times higher than that in NaAc or NH4Ac solution. The activities of M2 and M3 remained above 50% in 1500mM KAc salt solution, while the activities decreased by approximately 10% and 30% in NaAc and NH4Ac solutions, respectively. The activities of M2 and M3 in 2000mM KAc solution were several times higher than those in other salt solutions. This shows that the modified halophilic adenosine kinase has higher catalytic activity and better salt tolerance.

[0053] Combining Tables 2-5, it can be concluded that the tolerance of halophilic adenosine kinase mutants to high salt environment is M3>M2>M1. However, from Table 1, it can be seen that the enzyme activity of mutant M3 in low salt environment is greatly reduced compared with WT, that is, the enzyme activity is not good in the low salt environment at the beginning of the reaction. Therefore, M2, which has a salt tolerance similar to M3, was selected as the most suitable halophilic adenosine mutant.

[0054] The present invention provides a strategy and method for modifying a halophilic adenosine kinase mutant. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A halophilic adenosine kinase mutant, characterized in that The amino acid sequence of the halophilic adenosine kinase mutant is shown in SEQ ID NO:

3.

2. A gene encoding the halophilic adenosine kinase mutant according to claim 1, characterized in that: The gene encoding the halophilic adenosine kinase mutant is shown in SEQ ID NO:

7.

3. A recombinant vector, characterized in that The recombinant vector comprises the gene encoding the halophilic adenosine kinase mutant according to claim 2.

4. A recombinant strain, characterized in that The recombinant strain comprises the recombinant vector according to claim 3.

5. Use of the halophilic adenosine kinase mutant according to claim 1, the recombinant vector according to claim 3, or the recombinant strain according to claim 4 in synthesizing adenosine monophosphate.

6. The use according to claim 5, characterized in that The application is to use adenosine, adenosine triphosphate and acetyl phosphate as substrates and the halophilic adenosine kinase mutant according to claim 1 as a catalytic enzyme to synthesize adenosine monophosphate.

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