A method for the synthesis of beta-nicotinamide mononucleotides using adenylate phosphoribosyltransferase

Abstract

The application discloses a method for synthesizing beta-nicotinamide mononucleotide by using adenine phosphoribosyl transferase, taking nicotinamide and 5-phosphoribosyl-1-pyrophosphate as substrates, and condensing the substrates into beta-nicotinamide mononucleotide by an adenine phosphoribosyl transferase catalytic system, wherein the catalytic system is composed of adenine phosphoribosyl transferase and a PRPP synthesis system. The method for synthesizing nicotinamide mononucleotide by using adenine phosphoribosyl transferase as a catalyst has the advantages of simple reaction steps, mild reaction conditions, high catalytic rate, environmental friendliness, simple enzyme expression and purification, and the like, and has a good application and development prospect in the field of industrial synthesis of beta-nicotinamide mononucleotide and related products.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme engineering technology, specifically relating to a method for synthesizing β-nicotinamide mononucleotide using adenine phosphoribosyltransferase. It is a method that uses adenine phosphoribosyltransferase as a catalyst to stereoselectively catalyze the condensation of nicotinamide with 5-phosphoribose-1-pyrophosphate to obtain β-nicotinamide mononucleotide. Background Technology

[0002] Nicotinamide mononucleotide (NMN) is a B vitamin derivative and a naturally occurring bioactive nucleotide. It is a precursor to nicotinamide adenine dinucleotide (NAD, also known as coenzyme I) and an important intermediate metabolite in cells. NMN has two isomers, α and β, with the β isomer being the active form (hereinafter referred to as NMN). Since NAD has difficulty directly entering cells, NMN, as a direct precursor to NAD synthesis, is well-tolerated. Recently, NMN has become known for its potential anti-aging and life-extending properties (Tsubota K. The first human clinical study for NMN has started in Japan. NPJAging Mech Dis. 2016; 2:16021). Regarding safety, an increasing number of NMN-related clinical trials have been approved (such as NCT03151239, UMIN 0 0 0 0 3 0 6 0 9, UMIN 0 0 0 0 2 1 3 0 9, and UMIN000025739). A Phase II clinical trial by Igarashi et al. showed that elderly men who took NMN continuously for 6 or 12 weeks did not experience any adverse reactions (Igarashi M, Nakagawa-Nagahama Y, Miura M, et al. Chronicnicotinamide mononucleotide supplementation elevates blood nicotinamide adenine dinucleotide levels and alters muscle function in healthy oldermen. NPJ Aging. 2022; 8(1):5.). Therefore, NMN has become the preferred supplement for NAD. However, the content of natural NMN in the body is extremely low and cannot be obtained through separation and extraction. Currently, the traditional method for preparing NMN is through chemical synthesis. However, due to the difficulty in controlling stereoselectivity and the low yield, the cost of producing high-purity NMN is high, making products with NMN as the main active ingredient very expensive. Compared with chemical synthesis, the bio-enzymatic synthesis of NMN has the advantages of high efficiency and environmental friendliness, with no organic solvent residue and no chiral issues. Currently, the nicotinamide phosphoribosyltransferases that catalyze NMN synthesis are limited by their large molecular weight, complex structure, and difficulty in directional evolution. Novel and highly efficient phosphoribosyltransferases suitable for NMN synthesis are still urgently needed for development.

[0003] Adenine phosphoribosyl transferase (APRT) belongs to the 6-oxopurine phosphoribosyl transferase (PRTase) family. Its reported biological function is to catalyze the condensation of adenine with 5-phosphoribose-1-pyrophosphate (PRPP) to generate adenine monophosphate (AMP), making it a key enzyme in the adenine salvage synthesis pathway. Summary of the Invention

[0004] The purpose of this invention is to provide a method for synthesizing β-nicotinamide mononucleotide using adenine phosphoribosyltransferase. This method utilizes the stereoselective catalysis of adenine phosphoribosyltransferase to condense nicotinamide (NAM) with 5-phosphoribose-1-pyrophosphate (PRPP) to obtain NMN, representing an extension of the substrate spectrum of adenine phosphoribosyltransferase. This method offers advantages such as easy preparation of the enzyme catalyst, high operability, simple reaction steps, and high conversion efficiency.

[0005] The present invention provides a method for synthesizing NMN using adenine phosphoribosyltransferase, which uses NAM and PRPP as substrates and obtains NMN through an enzyme catalytic system, wherein the enzyme catalytic system consists of adenine phosphoribosyltransferase and a PRPP synthesis system.

[0006] The specific steps are as follows: using NAM and PRPP as raw materials, and Mg... 2+ As a cofactor, NMN is obtained through condensation catalyzed by adenine phosphoribosyltransferase in the enzyme catalysis system, as shown in the following reaction formula:

[0007]

[0008] In the reaction process, NAM and PRPP are used as substrates. However, since PRPP is expensive and easily decomposed, synthesizing PRPP through a PRPP synthesis system can significantly reduce the reaction cost.

[0009] In this invention, the PRPP synthesis system comprises: ribokinase and phosphoribosylpyrokinase as key enzymes, ATP and D-ribose as substrates, and Mg... 2+ PRPP synthesis system as a cofactor.

[0010] The enzyme catalytic system of this invention comprises adenine phosphoribosyltransferase, ribokinase, phosphoribosyl pyrokinase, and magnesium chloride. In this enzyme catalytic system, adenine phosphoribosyltransferase, ribokinase, and phosphoribosyl pyrokinase are all purified free enzymes. ATP, D-ribose, NAM, and PRPP are added to this enzyme reaction system, and under controlled pH and temperature conditions, magnesium chloride is added... 2+As a cofactor, ATP phosphorylates D-ribose to 5-phosphate ribose under the catalysis of ribokinase, and then phosphorylates 5-phosphate ribose to PRPP under the catalysis of phosphoribosyl pyrokinase. Finally, adenine phosphoribosyltransferase condenses NAM with PRPP to generate NMN. The reaction formula is as follows:

[0011]

[0012] Specifically, the nucleotide sequence of adenine phosphoribosyltransferase was obtained from GenBank, numbered M14040.1, belonging to Escherichia coli (strain K12) adenine phosphoribosyltransferase. The encoding, after codon optimization, is shown in the sequence listing as EcAPRT-DNA (SEQ. No. 1), and was obtained by whole-genome synthesis by a biotechnology company.

[0013] Specifically, the amino acid sequence of adenine phosphoribosyltransferase is shown in the sequence listing as EcAPRT-AA (SEQ. No. 2).

[0014] As those skilled in the art will know, the nucleotide sequence of the adenine phosphoribosyltransferase gene of the present invention may also be any other nucleotide sequence that encodes the amino acid sequence shown in the sequence listing EcAPRT-AA (SEQ. No. 2).

[0015] Any nucleotide sequence obtained by substituting, inserting, or replacing one or more nucleotides into the nucleotide sequence shown in EcAPRT-DNA, provided that it has more than 90% homology with the nucleotide, is within the scope of protection of this invention.

[0016] Any amino acid sequence shown in EcAPRT-AA that has one or more amino acids deleted, inserted, or replaced and has NMN synthesis activity is still within the scope of protection of this invention.

[0017] Any isoenzyme of adenine phosphoribosyltransferase from any other source that has NMN synthetic activity is within the scope of protection of this invention.

[0018] Specifically, the ribokinase sequence is derived from GenBank, number 948260. After codon optimization, it is Escherichia coli (strain K12) ribokinase, as shown in the sequence listing RBKS-DNA (SEQ. No. 3), and was obtained by whole-genome synthesis by a biotechnology company.

[0019] Specifically, the amino acid sequence of ribokinase is shown in the sequence listing as RBKS-AA (SEQ. No. 4).

[0020] Specifically, the nucleotide sequence of the phosphoribosyl pyrophosphate kinase is obtained from GenBank, number: 885993, which is Mycobacterium tuberculosis phosphoribosyl pyrophosphate kinase. After codon optimization, it is shown as MtPRS-DNA (SEQ. No. 5) in the sequence listing and was obtained by whole-genome synthesis by a biotechnology company.

[0021] Specifically, the amino acid sequence of phosphoribosyl pyrophosphokinase is shown in the sequence listing as MtPRS-AA (SEQ. No. 6).

[0022] Preferably, in the catalytic system, the amount of ribokinase added is 0.05-3.0 mg / ml; the amount of phosphoribosylpyrokinase added is 0.1-6.0 mg / ml; and the amount of adenine phosphoribosyltransferase added is 0.5-5.0 mg / ml.

[0023] In the catalytic system, the substrate NAM was added at a concentration of 0.5-30 mM; PRPP was added at a concentration of 0.75-45 mM; and the cofactor Mg... 2+ The addition amount is 0.1-20 mM. In the PRPP synthesis system, the addition amount of substrate ATP is 1.0-30 mM; the addition amount of D-ribose is 0.5-10 mM.

[0024] Preferably, in the enzyme catalytic system, the reaction temperature is 25-40℃ and the reaction time is 3-20h; more preferably, the temperature is 30-37℃ and the time is 4-14h.

[0025] Preferably, the pH of the reaction is controlled at 6 to 9, with sodium hydroxide used to control the decrease of pH and formic acid used to control the increase of pH.

[0026] The beneficial effects of this invention are mainly reflected in the following aspects: It provides a method for obtaining NMN by catalyzing the condensation of NAM and PRPP using adenine phosphoribosyltransferase as a biocatalyst, which has not been reported before; the adenine phosphoribosyltransferase described in this invention has a small molecular weight, is easy to purify, and the heterologous expression level in E. coli can reach 15 mg / L of culture medium. It can be purified in one step by nickel affinity chromatography to obtain free enzyme with a purity of more than 95%. At the same time, the reaction is carried out at a near-neutral pH and at room temperature, without the need for transition metals and organic solvents to participate in the reaction. Therefore, it has the characteristics of high catalytic reaction rate, high heterologous expression level, and easy purification. It also has the advantages of mild reaction conditions and environmental friendliness in biocatalysis, and has good prospects for industrial application development. Attached Figure Description

[0027] Figure 1 This is an SDS-PAGE image of purified adenine phosphoribosyltransferase.

[0028] Figure 2 This is a high-performance liquid chromatogram of the NAM blank control.

[0029] Figure 3 The high-performance liquid chromatogram of the reaction solution after 6.0 h of reaction in the adenine phosphoribosyltransferase catalytic system. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] Unless otherwise specified, the experimental methods used in this invention are conventional methods.

[0032] The plasmid extraction kit used in this invention was purchased from Tiangen Biotech (Beijing) Co., Ltd.; E. coli DH5α, E. coli BL21(DE3), etc. were purchased from Sangon Biotech (Shanghai) Co., Ltd.; and the pre-stained protein Maker was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.

[0033] The commonly used reagents used in this invention, including the substrates, were purchased from Aladdin Chemical Reagent Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd., and Sinopharm Chemical Reagent Co., Ltd.

[0034] Example 1: Expression of adenine phosphoribosyltransferase

[0035] The sequence encoding the adenine phosphoribosyltransferase gene (GenBank: M14040.1) of *Escherichia coli* (strain K12) was codon-optimized (sequence as shown in SEQ ID NO.1), synthesized by Sangon Biotech (Shanghai) Co., Ltd., and ligated into the pET-28a(+) vector to construct the *EcAPRT-pET-28a(+)* plasmid. After sequence verification, the plasmid was heat-transformed into *E. coli* BL21(DE3) competent cells to obtain engineered bacteria expressing adenine phosphoribosyltransferase. Single colonies were picked from LB agar plates containing 50 μg / ml kanamycin and inoculated into LB liquid medium containing 50 μg / ml kanamycin. After culturing at 37°C with shaking at 200 rpm for 12 h, the culture was transferred to 3L of liquid LB medium for expansion culture and continued to be cultured at 37°C with shaking at 200 rpm for 8 h. When the optical density OD of the culture medium reached a certain level, the culture was considered complete. 600 When the concentration reaches 0.6, the temperature is lowered to 23℃, and IPTG solution with a final concentration of 0.5mM is added to induce expression for 16h. The culture medium is centrifuged at 8000rpm for 10min, the supernatant culture medium is discarded, and the cells are stored at -20℃ for later use.

[0036] Example 2: Expression of ribokinase

[0037] The sequence encoding the Escherichia coli (strain K12) ribokinase gene (GenBank: 948260), after codon optimization (sequence as shown in SEQ ID NO.3), was fully synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the pET-28a(+) vector to construct the RBKS-pET-28a(+) plasmid. After sequence verification, the plasmid was heat-transformed into E. coli BL21(DE3) competent cells to obtain engineered bacteria expressing adenine phosphoribosyltransferase. Single colonies were picked from LB agar plates containing 50 μg / ml kanamycin and inoculated into LB liquid medium containing 50 μg / ml kanamycin. After culturing at 37°C with shaking at 200 rpm for 12 h, the culture was transferred to 1.5 L of liquid LB medium for expansion culture and continued to be cultured at 37°C with shaking at 200 rpm for 8 h. When the optical density OD of the culture medium reached a certain level, the culture was considered complete. 600 When the concentration reaches 0.6, the temperature is lowered to 16℃, and IPTG solution with a final concentration of 0.2mM is added to induce expression for 16h. The culture medium is centrifuged at 8000rpm for 10min, the supernatant culture medium is discarded, and the cells are stored at -20℃ for later use.

[0038] Example 3 Expression of phosphoribosylpyrokinase

[0039] The sequence encoding the Mycobacterium tuberculosis phosphoribosyl pyrophosphate kinase gene (GenBank: 885993), after codon optimization (sequence as shown in SEQ ID NO. 5), was fully synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the pET-28a(+) vector to construct the MtPRS-pET-28a(+) plasmid. After sequence verification, it was heat-transformed into E. coli BL21(DE3) competent cells to obtain engineered bacteria expressing adenine phosphoribosyltransferase. Single colonies were picked from LB agar plates containing 50 μg / ml kanamycin and inoculated into LB liquid medium containing 50 μg / ml kanamycin. After culturing at 37℃ with shaking at 200 rpm for 12 h, the culture was transferred to 1.5 L of liquid LB medium for expansion culture and continued to be cultured at 37℃ with shaking at 200 rpm for 8 h. When the optical density OD of the culture medium reached a certain level, the culture was considered complete. 600 When the concentration reaches 0.6, the temperature is lowered to 16℃, and IPTG solution with a final concentration of 0.4mM is added to induce expression for 16h. The culture medium is centrifuged at 8000rpm for 10min, the supernatant culture medium is discarded, and the cells are stored at -20℃ for later use.

[0040] Example 4: Purification of adenine phosphoribosyltransferase, ribokinase, and phosphoribosylpyrokinase

[0041] Three g of engineered bacteria expressing adenine phosphoribosyltransferase or ribokinase / phosphoribophosphate pyrokinase were resuspended in 20 ml of lysis buffer (10 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1% Tween-20, pH 8.0). After shaking and mixing, lysozyme (1 mg / ml) was added. The mixture was incubated on ice for 40 min, then sonicated three times for 3 min each time, with a 15 min interval between each time. The mixture was then centrifuged at 14000 rpm for 15 min. The resulting supernatant was the crude enzyme solution. Ni-IDA protein purification magnetic beads were used as the purification material. A single use consisted of 5 ml of 10% magnetic bead suspension and 10 ml of lysis buffer to equilibrate the Ni-IDA beads. After magnetic separation, crude enzyme solution was added, and the mixture was incubated at 4°C for 1 h. Unadsorbed protein was removed by elution with lysis buffer (50 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1% Tween-20, pH 8.0). Finally, the target protein was collected by elution with elution buffer (500 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1% Tween-20, pH 8.0). 7.4) Dialyze the target protein to remove salt and imidazole. SDS-PAGE analysis showed that the purified yields of adenine phosphoribosyltransferase, ribokinase, and phosphoribosylpyrokinase were 10 mg / L, 20 mg / L, and 8 mg / L of culture medium, respectively, with a purity of over 95%. Figure 1 ).

[0042] Example 3: Synthesis of NMN by adenine phosphoribosyltransferase

[0043] The adenine phosphoribosyltransferase, ribokinase, and phosphoribosyl pyrokinase purified enzymes obtained in Example 2 were added to the reaction system at a concentration of 1.0 mg / ml. A buffer solution of 50 mM pH 7.4 Kpi was used. Then, 0.8 mM NAM, 0.8 mM PRPP, 1.0 mM ATP, 0.8 mM D-ribose, and 10 mM MgCl2 were added to a final concentration. The reaction was carried out at 37°C with constant shaking (666 rpm) for 6.0 h. An equal volume of methanol was added to terminate the reaction. After centrifugation at 14000 rpm for 15 min, the supernatant was injected into high-performance liquid chromatography (HPLC) for analysis of substrate and product quantities. The HPLC analysis method was as follows: Shimadzu LC-20AT high-performance liquid chromatography; Extend-C18 column (4.6*250mm); column temperature 30℃; flow rate 0.7ml / min; detection wavelength 245nm; mobile phase: 10% water (100mM sodium phosphate), 90% methanol. The conversion rate and yield of NMN catalyzed by adenine phosphoribosyltransferase were calculated using the concentration curves of NAM and NMN standards. The conversion rate was calculated to be 50.4%, and the yield 45.1%. The corresponding HPLC chromatograms are attached. Figure 2 and Figure 3 The fluorescence analysis method was as follows: 10 μl of DMSO (containing 20% ​​acetophenone), 10 μl of 2MKOH, and 25 μl of reaction supernatant were mixed, incubated on ice for 2 min, and then 45 μl of 88% formic acid was added. The mixture was incubated at 37°C for 10 min. Subsequently, 40 μl of the reaction solution was added to a 384-well black plate, and fluorescence was detected at an excitation wavelength of 382 nm and an emission wavelength of 445 nm. The yield of NMN catalyzed by xanthine-guanine phosphoribosyltransferase was calculated using the NMN standard concentration curve. The calculated yield reached 40.1%, consistent with the results obtained by HPLC.

[0044] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for synthesizing β-nicotinamide mononucleotide using adenine phosphoribosyltransferase, characterized in that, β-Nicotinamide mononucleotide was obtained by catalysis using nicotinamide and 5-phosphate ribose-1-pyrophosphate as substrates via an enzyme catalytic system. The enzyme catalytic system consisted of adenine phosphoribosyltransferase and a PRPP synthesis system, and was achieved through the following steps: using nicotinamide and 5-phosphate ribose-1-pyrophosphate as raw materials, and Mg... 2+ As a cofactor, under the catalysis of adenine phosphoribosyltransferase in the enzyme catalysis system, the substrate is condensed to obtain β-nicotinamide mononucleotide, reaction formula: ； The reaction is based on the synthesis of PRPP. Through the PRPP synthesis system, ATP and D-ribose are used as substrates, and PRPP is synthesized through two phosphorylation reactions catalyzed by ribokinase and phosphoribosyl pyrokinase. Adenine phosphoribosyltransferase is derived from Escherichia coli (E. coli) Escherichia coli (strain K12) The nucleotide sequence encoding adenine phosphoribosyltransferase is shown in SEQ. No.1, and its amino acid sequence is shown in SEQ. No.

2.

2. The method according to claim 1, characterized in that, The PRPP synthesis system is as follows: ribokinase and phosphoribosylpyrokinase are used as key enzymes, ATP and D-ribose are used as substrates, and Mg is used as the substrate. 2+ As a cofactor, ATP phosphorylates D-ribose to ribose-5-phosphate under the catalysis of ribokinase, and then phosphorylates ribose-5-phosphate to PRPP under the catalysis of phosphoribosyl pyrokinase. PRPP, as a substrate of adenine phosphoribosyltransferase, condenses with NAM to generate NMN, as shown in the following reaction formula: 。 3. The method according to claim 1 or 2, characterized in that, The enzyme catalytic system includes adenine phosphoribosyltransferase, ribokinase, phosphoribosylpyrokinase, substrate, and cofactor Mg. 2+ In the enzyme catalytic system, adenine phosphoribosyltransferase, ribokinase, and phosphoribosylpyrokinase were all purified to be free pure enzymes.

4. The method according to claim 3, characterized in that, The ribokinase is derived from Escherichia coli (E. coli) Escherichia coli(strain K12) ribokinase.

5. The method according to claim 3, characterized in that, The phosphoribosylpyrokinase is derived from Mycobacterium tuberculosis ( ) Mycobacterium tuberculosis Phosphoribose pyrophosphate kinase.

6. The method according to claim 1, characterized in that, In the enzyme catalytic system, the amount of adenine phosphoribosyltransferase added is 0.5-5.0 mg / ml, the amount of ribokinase added is 0.05-3.0 mg / ml, and the amount of phosphoribosylpyrokinase added is 0.1-6.0 mg / ml.

7. The method according to claim 1, characterized in that, In the enzyme catalytic system, the substrate nicotinamide was added at a concentration of 0.5-30 mM, and 5-phosphoribose-1-pyrophosphate was added at a concentration of 0.75-45 mM; cofactor Mg 2+ The amount of added ATP in the PRPP synthesis system is 0.1-20 mM, the amount of added substrate ATP is 1.0-30 mM, the amount of added D-ribose is 0.5-10 mM, the reaction temperature is 25-40℃, and the reaction time is 3-20 h.

8. The method according to claim 1, characterized in that, The pH value of the reaction is controlled at 6-9.

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