S-adenosylmethionine synthetase mutant and application thereof
By mutating the gene of S-adenosylmethionine synthase in Escherichia coli, the MATM1 mutant was obtained, which solved the problem of low activity of S-adenosylmethionine synthase and achieved efficient production of S-adenosylmethionine, with a significant increase in yield and conversion rate.
Patent Information
- Application Number
- CN202611499158.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-24
- Publication Date
- 2026-11-03
AI Technical Summary
Existing S-adenosylmethionine synthases have low activity or severe substrate inhibition, resulting in low S-adenosylmethionine conversion rates and making it difficult to achieve economical production.
By using genetic engineering, the aspartic acid at position 226 of S-adenosylmethionine synthase from Escherichia coli was mutated to glutamine, resulting in the mutant MATM1, which improves its enzyme activity and conversion rate.
The MATM1 mutant exhibited 2.77-fold increased enzyme activity, achieved a conversion rate of 86.80%, and yielded 4.15 g/L, significantly enhancing the synthesis efficiency of S-adenosylmethionine.
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Abstract
Description
Technical Field
[0001] This invention relates to an S-adenosylmethionine synthase mutant and its applications, belonging to the field of bioenzyme engineering technology. Background Technology
[0002] S-adenosylmethionine (SAM) is an important metabolic intermediate widely found in somatic cells, playing a crucial role in various biochemical reactions such as transmethylation and transsulfation, and is essential for maintaining cell membrane integrity and fluidity. Furthermore, SAM is an intermediate product of multiple metabolic pathways, participating in key processes such as methionine synthesis, and is also a major methyl donor and precursor of glutathione. Intracellularly, SAM has potential therapeutic effects on various diseases, such as liver disease, arthritis, and depression, through transmethylation, transsulfation, and transaminopropylation reactions. Currently, SAM is widely used in the treatment of liver disease, arthritis, and depression, with good efficacy and low side effects.
[0003] S-adenosylmethionine synthetase (MAT or SAMsynthetase) is a key metabolic enzyme widely distributed in organisms, responsible for catalyzing the reaction of methionine (methionine) with adenosine triphosphate (ATP) to synthesize S-adenosylmethionine (SAM / AdoMet). SAM is the most important methyl donor in cells, participating in DNA methylation, histone modification, RNA processing, phospholipid synthesis, and the biosynthesis of various neurotransmitters (such as adrenaline and melatonin), and has a profound impact on gene expression regulation, cell proliferation and differentiation, and liver detoxification function. Clinically, SAM itself is used as a drug or dietary supplement for adjuvant treatment of liver disease, osteoarthritis, and depression, while targeted inhibitors of MAT (such as MAT2A inhibitors) are becoming an emerging hot topic in anti-tumor drug development. In-depth research on SAM synthetase not only helps to reveal the core mechanisms of the methylation metabolic network but also provides important molecular targets for the treatment of metabolic diseases and cancer.
[0004] S-adenosylmethionine (SAM) can be converted from adenosine triphosphate (ATP) and methionine, but problems such as low activity or substrate inhibition exist. Therefore, improving the conversion rate or enzyme activity of SAM can achieve rapid synthesis of SAM. Developing a high-conversion-rate SAM synthase will facilitate the economical and low-cost production of SAM, thereby reducing its price and providing a more economical supply of SAM-related products. Summary of the Invention
[0005] Objective of the Invention: The technical problem to be solved by the present invention is to provide an S-adenosylmethionine synthase mutant, addressing the shortcomings of existing technologies. The present invention uses genetic engineering to mutate the aspartic acid at position 226 of the wild-type parental S-adenosylmethionine synthase to glutamine, obtaining the S-adenosylmethionine synthase mutant (N226Q), named MAT. M1 This mutant can increase the production of S-adenosylmethionine.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0007] The wild-type strain of the S-adenosylmethionine synthase mutant of this invention is derived from *Escherichia coli*. The S-adenosylmethionine synthase mutant is formed by mutating the aspartic acid N at position 226 of the amino acid sequence shown in SEQ ID NO. 1 to glutamine Q.
[0008] The nucleotide sequence of the gene encoding the S-adenosylmethionine synthase mutant is shown in SEQ ID NO. 2.
[0009] The present invention also provides an expression vector and recombinant cell for the above-mentioned mutant encoding gene.
[0010] The recombinant cell expression vector is plasmid PET-28a; the recombinant cells use bacteria or fungi as expression hosts.
[0011] This invention provides the application of S-adenosylmethionine synthase mutants and recombinant cells in the preparation of S-adenosylmethionine.
[0012] The method described above uses L-methionine and ATP as substrates and S-adenosylmethionine synthase mutants or recombinant cells as catalysts to synthesize S-adenosylmethionine.
[0013] The initial molar ratio of L-methionine to ATP is 1:1-1.5.
[0014] The catalytic reaction satisfies one or more of the following conditions:
[0015] (1) The amount of catalyst added is 0.1-10 g / L, and the substrate concentration is 1-50 mM;
[0016] (2) Magnesium chloride 30 mmol / L and potassium chloride 100 mmol / L are coenzyme factors;
[0017] (3) The reaction temperature is 20-50 ℃, preferably 25-40 ℃, and most preferably 37 ℃; the reaction pH is 5.0-9.0, preferably 7.0-8.0, and most preferably pH 8.0;
[0018] (4) The reaction time is 1-6h, preferably 3-6h, and most preferably 6h.
[0019] The present invention also provides a method for synthesizing S-adenosylmethionine, comprising the following steps:
[0020] (1) Culturing and expressing the S-adenosylmethionine synthase mutant to obtain S-adenosylmethionine synthase;
[0021] (2) The S-adenosylmethionine synthase is mixed with L-methionine and ATP to form a reaction system; the molar ratio of L-methionine to ATP is 1:1-1.5; and 30 mmol / L magnesium chloride and 100 mmol / L potassium chloride are added as coenzyme factors.
[0022] (3) S-adenosylmethionine is synthesized by reacting at a reaction temperature of 20-50 ℃ (preferably 25-40 ℃, most preferably 37 ℃) and a reaction pH of 5.0-9.0 (preferably 7.0-8.0, most preferably pH 8.0) for 1-6 h (preferably 3-6 h, most preferably 6 h).
[0023] Using the above synthesis method, the optimal yield of S-adenosylmethionine in this invention reaches over 4.15 g / L, and the substrate conversion rate of L-methionine reaches 86.80%.
[0024] The S-adenosylmethionine synthase mutant obtained by the method of this invention has an enzyme activity that is 2.77 times higher than that of wild-type WT S-adenosylmethionine synthase.
[0025] Beneficial effects:
[0026] (1) In this invention, S-adenosylmethionine synthase (MAT) from Escherichia coli is modified by genetic engineering. By mutating the aspartic acid N at position 266 of the enzyme to glutamine Q, a mutant of S-adenosylmethionine synthase with improved conversion rate and enzyme activity is finally obtained, which can achieve efficient synthesis of SAM at the optimal temperature.
[0027] (2) MAT M1 Enzyme activity was measured under conditions where both substrate ATP and L-Met concentrations were 10 mM. MAT M1 Its enzyme activity is more than 2.77 times that of WT.
[0028] (3) MAT M1 The reaction was carried out in an 8L system with both substrate ATP and L-Met concentrations of 12mM, and the yield reached 4.15g / L with a conversion rate of 86.80%, which is 4 times the conversion rate of WT under the same conditions. Attached Figure Description
[0029] Figure 1 The relative enzyme activity is for a single-point mutant.
[0030] Figure 2 This is a gel electrophoresis image. Lanes 1 and 2 contain the supernatant after BL21 fermentation, lysis, and centrifugation containing the empty PET-28a plasmid. Lane 3 contains the supernatant after BL21 fermentation, lysis, and centrifugation containing the PET-28a-MAT recombinant plasmid. Lane 4 contains the supernatant after PET-28a-MAT recombinant plasmid. M1 The supernatant after fermentation, disruption, and centrifugation of the recombinant plasmid BL21.
[0031] Figure 3 For variant MAT M1 Conversion rate under 40 mL system conditions.
[0032] Figure 4 For variant MAT M1 Conversion rate under 400 mL system conditions.
[0033] Figure 5 For variant MAT M1 Conversion rate under 8L system conditions. Detailed Implementation
[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0035] The calculation methods involved in the following embodiments:
[0036] Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of SAM within 1 min, which is one enzyme activity unit (U).
[0037] Enzyme activity assay: 1 g / L of enzyme was incubated at 30°C for 5 min in 200 mmol / L PB buffer (pH 8.0) containing 10 mmol / L ATP and L-Met in a total volume of 500 μL.
[0038] Conversion rate is defined as the percentage or fraction of reactants (substrate) converted into the product SAM under certain conditions. Conversion rate = Amount of substrate converted ÷ Amount of initial substrate × 100%.
[0039] Product SAM yield (g / L) = molar concentration × 398.44 g / mol (molecular weight of SAM)
[0040] Conversion rate determination method 1: 2 g / L of enzyme was incubated in 40 mL of pure water containing 10 mmol / L ATP, 10 mmol / L L-Met, 30 mmol / L magnesium chloride, 100 mmol / L potassium chloride, with the pH adjusted to 8.0 by sodium hydroxide, at 30 °C for 1-6 h.
[0041] Method 2 for conversion determination: 2 g / L of enzyme was incubated in 400 mL of pure water containing 10 mmol / L ATP, 10 mmol / L L-Met, 30 mmol / L magnesium chloride, 100 mmol / L potassium chloride, with the pH adjusted to 8.0 by sodium hydroxide, at 30°C for 1-6 h.
[0042] Method 3 for conversion determination: 2 g / L of enzyme was incubated in 8 L of pure water containing 12 mmol / L ATP, 12 mmol / L L-Met, 30 mmol / L magnesium chloride, 100 mmol / L potassium chloride, with the pH adjusted to 8.0 by sodium hydroxide, at 30°C for 1-6 h.
[0043] The determination of the products: ATP, L-Met and SAM were determined using a C18 column, 85% mobile phase A (40 mM potassium dihydrogen phosphate and 1 mM sodium heptanesulfonate (pH 6.0)) and 15% mobile phase B (methanol), at a flow rate of 0.8 ml / min, and using a UV detector.
[0044] Raw materials used in the examples:
[0045] The culture media involved in the following examples are as follows:
[0046] LB medium: 10 g / L tryptone (Thermo Scientific™ Oxoid), 5 g / L yeast extract (Thermo Scientific™ Oxoid), 10 g / L sodium chloride (NaCl) (China National Pharmaceutical Group), pH 7.4; LB liquid medium with 2% agar added becomes LB solid medium.
[0047] TB medium: tryptone 12 g / L (Thermo Scientific™ Oxoid), yeast extract 24 g / L (Thermo Scientific™ Oxoid), K2HPO4 12.54 g / L, KH2PO4 2.31 g / L (Sinopharm), glycerol 5 g / L (Sinopharm).
[0048] The solutions involved in the examples are as follows:
[0049] PB buffer: 2.299 g Na2HPO4 (China National Pharmaceutical Group), 0.455 g NaH2PO4 (China National Pharmaceutical Group), bring to a final volume of 1L.
[0050] Example 1: S-adenosylmethionine synthase and its mutant (MAT) M1 Construction of )
[0051] (1) Construction of recombinant plasmids containing the MAT gene
[0052] The amino acid sequence of S-adenosylmethionine synthase from Escherichia coli was selected as the wild type. The amino acid sequence of the S-adenosylmethionine synthase is shown in SEQ ID NO. 1, and the nucleotide sequence encoding the S-adenosylmethionine synthase is shown in SEQ ID NO. 2. The nucleotide sequence SEQ ID NO. 2 was synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0053] NcoI and XhoI on PET-28a were selected as insertion sites for the target gene, and the target gene was amplified. The sequences of primers F are shown in Table 1, ensuring the target gene contained restriction enzyme sites. PET-28a was double-digested and purified to obtain a linear plasmid fragment. The target gene with restriction enzyme sites was ligated to the linear plasmid fragment via homologous recombination. The system used for homologous recombination contained: 1 μL of restriction-digested linear plasmid, 1 μL of the target fragment, 2 μL of buffer, 1 μL of homologous recombination enzyme (ClonExpress Multis One Step cloning Kit, Nanjing Novizan Biotechnology Co., Ltd.), and 5 μL of H2O. Homologous recombination was performed at 37℃ for 30 min. The ligation product was then stored at -20℃.
[0054] The ligation product was transformed into competent Escherichia coli DE3 (BL21) cells and cultured at 37°C for 1 h. The bacterial culture was then spread onto LB agar plates containing kanamycin-resistant bacteria and cultured overnight. Single colonies were picked, amplified, and plasmids were extracted. Colony PCR was performed using universal primers. Plasmids with positive PCR results were sequenced for verification. Finally, the correct S-adenosylmethionine synthase gene recombinant plasmid PET-28a-MAT and recombinant strain E. coli DE3 (BL21) / PET-28a-MAT were obtained.
[0055] Table 1. Nucleotide sequences of primers
[0056]
[0057] (2) Construction of adenosylmethionine synthase mutant
[0058] Using PET-28a-MAT plasmid as a template, full plasmid PCR was performed using the primer pairs in Table 2 for the five mutant sites. Mutants with increased enzyme activity relative to WT were screened at single sites, yielding three positive mutants (E56N, I303V, and N226Q) with relative enzyme activities of 171.58%, 164.66%, and 227.51%, respectively. Figure 1 As shown. Among them, MAT M1 That is, N226Q, which is obtained by introducing the N226Q site into wild-type MAT through whole-plasmid PCR. The recombinant plasmid MAT... M1 Digested with DpnI (Nanjing Novizan Biotechnology Co., Ltd.) for approximately 2 hours, the bacteria were then transformed into competent E. coli BL21(DE3) cells using the heat shock method. Single colonies were selected and cultured overnight in LB medium at 37°C and 200 rpm. Plasmids were extracted from the single-clone cultures and sent to a sequencing company for sequencing. The recombinant strain containing the successfully mutated mutant was identified by the sequencing results: E. coli BL21(DE3) / PET-28a-MAT. M1 .
[0059] (3) Induction of target protein expression
[0060] The correctly sequenced monoclonal strain E. coli DE3 (BL21) / PET-28a-MAT was activated by shaking in LB medium for 12 h, and then inoculated into fresh TB medium and cultured at 37°C and 200 rpm until OD. 600 The concentration of the inducing agent IPTG was 0.8, and the cells were cultured at 16°C and 200 rpm for 20 h. After culture, the precipitate was collected by centrifugation. The precipitate was then processed by a cell disruptor and centrifuged again to obtain the supernatant, which yielded a crude enzyme solution. The crude enzyme solution was centrifuged and then subjected to gel electrophoresis to detect its protein expression. Lanes 1 and 2 contain the supernatant of BL21 cells containing the empty PET-28a plasmid after fermentation, disruption, and centrifugation. Lane 3 contains the supernatant of BL21 cells containing the PET-28a-MAT recombinant plasmid after fermentation, disruption, and centrifugation. Lane 4 contains the mutant PET-28a-MAT obtained by inducing expression according to the above method. M1 Crude enzyme solution. Gel electrophoresis pattern as shown. Figure 2 As shown, the results indicate that no protein was expressed in the empty vector cells, and the molecular weight of the protein MAT expressed by the recombinant PET-28a-MAT was 40 kDa.
[0061] Table 2 Primers for mutant construction
[0062]
[0063] Example 2: Mutant MATM1 Scale-up and optimization of the conversion system
[0064] The specific mode is as follows:
[0065] (1) Using conversion determination method 1, in a 40 mL reaction system, with a final concentration of 10 mmol / L ATP, 10 mmol / L L-Met, 30 mmol / L magnesium chloride, 100 mmol / L potassium chloride, and sodium hydroxide adjusted to pH 8.0, MAT enzyme solution was added to make a final concentration of 2 g / L. The mixture was incubated at 30 °C for 4 h, and samples were taken at 2 h and 4 h respectively. The MAT enzyme solution was detected by the product determination method, and its conversion rate was calculated.
[0066] (2) Using conversion determination method 2, in a 400 mL reaction system, with a final concentration of 10 mmol / L ATP, 10 mmol / L L-Met, 30 mmol / L magnesium chloride, 100 mmol / L potassium chloride, and sodium hydroxide adjusted to pH 8.0, MAT enzyme solution was added to make a final concentration of 2 g / L. The mixture was incubated at 30 °C for 6 h, and samples were taken every 1 h. The MAT enzyme solution was tested by the product determination method, and its conversion rate was calculated.
[0067] (3) Using conversion determination method 3, in an 8L reaction system, with a final concentration of 12 mmol / L ATP, 12 mmol / L L-Met, 30 mmol / L magnesium chloride, 100 mmol / L potassium chloride, and sodium hydroxide adjusted to pH 8.0, MAT enzyme solution was added to a final concentration of 2 g / L, and incubated at 30℃ for 6 h. Samples were taken every 1 h, and the MAT enzyme solution was detected by the product determination method, and its conversion rate was calculated.
[0068] The experimental results are as follows:
[0069] In a 40 mL reaction system, the results are as follows Figure 3 As shown, the wild-type WT substrate conversion rate was 26.54% after 2 hours of reaction and 24.97% after 4 hours of reaction; while MAT... M1 The substrate conversion rate was 52.5% after 2 hours of reaction and 67.53% after 4 hours of reaction, with a final yield of 2.69 g / L.
[0070] In a 400 mL reaction system, the results are as follows Figure 4 As shown, the wild-type WT substrate conversion rate was 19.95% after 1 hour of reaction, and the product growth was almost negligible over the following 6 hours, with the conversion rate remaining relatively stable throughout, reaching a final conversion rate of 23.2% at 6 hours; MAT M1The conversion rate can reach 46.00% after 1 hour of reaction, and then slowly increases. The final conversion rate can reach 83.00% after 6 hours, and the final yield can reach 3.31 g / L.
[0071] In an 8L reaction system, the results are as follows: Figure 5 As shown, increasing the concentration and ratio of substrates ATP and L-Met resulted in a substrate conversion rate of 20.4% for wild-type WT after 1 hour of reaction. Subsequently, product growth was minimal over the next 6 hours, with the conversion rate remaining relatively stable, reaching a final conversion rate of 22.3% at 6 hours. MAT M1 The conversion rate can reach 62.4% after 1 hour of reaction, and then slowly increases. The final conversion rate can reach 86.80% after 6 hours, and the final yield can reach 4.15 g / L.
[0072] In summary, this invention provides an S-adenosylmethionine synthase mutant, MAT M1 The enzyme activity was 2.77 times that of WT. For the optimized 8L catalytic system, the mutant MAT... M1 The yield of S-adenosylmethionine can reach 4.15 g / L under the conditions of substrate L-Met and ATP concentration of 12 mM, with a conversion rate of 86.80%. With a slight increase in substrate concentration, the product conversion rate increases slightly. This indicates that the modified enzyme weakens the inhibitory effect of the substrate on its conversion ability, allowing the substrate concentration to bind more fully with the enzyme substrate. It is expected that the substrate concentration can be further increased while maintaining the conversion rate, thereby increasing the product yield.
[0073] This invention provides an S-adenosylmethionine synthase mutant and its applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.
Claims
1. An S-adenosylmethionine synthase mutant, characterized in that, The mutant is formed by mutating aspartic acid N at position 226 of the amino acid sequence shown in SEQ ID NO 1 to glutamine Q.
2. The encoding gene of the S-adenosylmethionine synthase mutant according to claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.
2.
3. An expression carrier, characterized in that, It contains the gene described in claim 2.
4. A recombinant cell, characterized in that, It contains the gene of claim 2, or carries the expression vector of claim 3.
5. The use of the S-adenosylmethionine synthase mutant of claim 1, or the recombinant cells of claim 4, in the preparation of S-adenosylmethionine.
6. The application according to claim 5, characterized in that, Using L-methionine and ATP as substrates, and the S-adenosylmethionine synthase mutant described in claim 1 or the recombinant cell described in claim 4 as a catalyst, S-adenosylmethionine is synthesized.
7. The application according to claim 6, characterized in that, The initial molar ratio of L-methionine to ATP is 1:1-1.
5.
8. The application according to claim 6, characterized in that, The catalytic reaction satisfies one or more of the following conditions: (1) The catalyst is 0.1-10 g / L based on the content of S-adenosylmethionine synthase in the system, and the substrate concentration is 1-50 mM; (2) Magnesium chloride and potassium chloride are coenzyme factors; preferably, magnesium chloride is 30 mmol / L and potassium chloride is 100 mmol / L. (3) The reaction temperature is 20-50 ℃, and the reaction pH is 5.0-9.0; (4) The reaction time is 1-6 hours.
9. The application according to claim 8, characterized in that, The yield of S-adenosylmethionine reached over 4.15 g / L, and the substrate conversion rate of L-methionine exceeded 80%.
10. A method for synthesizing S-adenosylmethionine, characterized in that, Includes the following steps: (1) Cultivate and express the S-adenosylmethionine synthase mutant according to claim 1 to obtain S-adenosylmethionine synthase; (2) The S-adenosylmethionine synthase is mixed with L-methionine and ATP to form a reaction system; the molar ratio of L-methionine to ATP is 1:1-1.5; and 30 mmol / L magnesium chloride and 100 mmol / L potassium chloride are added as coenzyme factors. (3) S-adenosylmethionine was synthesized by reacting at a reaction temperature of 20-50 ℃ and a reaction pH of 5.0-9.0 for 1-6 h.