Semi-synthetic method for NMN involving adenosine

Abstract

Provided is a method for semi-synthesizing NMN involving adenosine, which includes (A) a step of reacting adenosine, phosphate, and sugars that can be metabolized by yeast cells with the catalytic action of yeast cells to produce ATP in the same reaction system, and (B) a step of enzymatic phosphorylation of NR, which corresponds to reacting NR and ATP with the catalytic action of NRK to produce NMN and ADP, thereby achieving efficient synthesis of NMN in the process of producing and circulating ATP, simplifying the process and reducing emissions.

Description

[Technical Field]

[0001] The present invention relates to the technical field of β-nicotinamide mononucleotide (NMN) synthesis, and in particular to semi-synthetic methods for NMN involving adenosine. [Background technology]

[0002] β-Nicotinamide mononucleotide (NMN) is the direct precursor for synthesizing nicotinamide adenine dinucleotide (NAD) in the body. Supplementing with NMN is the most effective way to increase the body's NAD content level and has broad, profound, and significant health implications for promoting normal metabolism. Because NAD levels decrease in elderly people and they are unable to obtain sufficient NMN from food, NMN is expected to become a widely applied supplement.

[0003] Currently, there are four conventional NMN synthesis techniques: fermentation, chemical synthesis, semi-synthesis, and full-enzyme synthesis. Fermentation requires the creation of a microbial strain capable of producing NMN, which is then mass-cultured and propagated to synthesize NMN in the bacterial cells. Because the basic activity of the key enzyme (NAMPT, nicotinamide phosphoribosyltransferase) responsible for catalytically synthesizing NMN in various species, including lower unicellular organisms, is generally low, it is extremely difficult to create a bacterial strain capable of efficiently expressing NMN. Furthermore, the NMN synthesis pathway is long and involves multiple enzymes and natural degrading enzymes, making efficient large-scale NMN production via fermentation extremely difficult, resulting in high process costs and a lack of market competitiveness. Chemical synthesis uses basic raw materials such as nicotinamide (or nicotinic acid), tetraacetyl ribose, and triphenylphosphine oxide to first synthesize nicotinamide riboside (NR) through chemical methods, and then phosphorylates NR to obtain NMN. The main problem with this method is that the second step, the chemical phosphorylation step, is flammable, explosive, and highly toxic. Large-scale industrialization would pose serious challenges to environmental protection and production safety supervision and management. Other issues include the presence of chemical enantiomer impurities, residual toxic raw materials, and residual solvents, creating persistent safety concerns for consumers regarding the long-term use of the product on the human body. The semi-synthetic method involves chemically synthesizing NR and then enzymatically phosphorylating it to obtain NMN. This method combines the advantages and disadvantages of the chemical and enzymatic methods. The main problems are the risk of residual solvents and toxic components in the chemical method, and the high cost of the enzymatic phosphorylation step, which requires expensive adenosine triphosphate (ATP). The fully enzymatic method uses nicotinamide, ribose, and ATP (adenosine triphosphate) as basic materials and sequentially catalyzes the formation of NMN through a series of enzymes. This method has the advantages of being environmentally friendly and safe, but has the drawbacks of the expression, purification and immobilization of various enzymes, the difficulty of large-scale production in conventional techniques, and the high cost of the enzymes.

[0004] Of the four conventional methods for synthesizing NMN, the semi-synthetic method is currently the mainstream. This method can use nicotinamide (or nicotinic acid) and tetraacetyl ribose as starting materials. NR is first synthesized chemically, and then NMN is produced using NR and ATP with the catalysis of a specific kinase, or NMN is produced directly using NR as a starting material through an enzymatic reaction. The core step of the semi-synthetic method is the enzymatic phosphorylation of NR, in which a phosphate group is donated from ATP to NR to form NMN, which then becomes adenosine diphosphate (ADP). The reaction equation is NR + ATP → NMN + ADP, and the enzyme that catalyzes this reaction is nicotinamide riboside kinase (NRK). To reduce the amount of ATP used, ADP and polyphosphate (e.g., sodium pyrophosphate, sodium tripolyphosphate, or sodium hexametaphosphate) are generally converted into ATP through an enzymatic reaction, enabling repeated use of ATP. The reaction is ADP + PPi (pyrophosphate) → ATP + Pi (phosphate), and the enzyme that catalyzes this reaction is adenylate phosphotransferase (PPK2). These two steps of the enzymatic reaction (NR phosphorylation and ATP regeneration) can be carried out separately or together. There are two main difficulties with this process. First, a large amount of phosphate accumulates during the reaction, interfering with further reactions, making the process of separating and removing the phosphate difficult and affecting the ATP recovery rate. Second, the reaction system involves two enzymes, which requires a large amount of enzyme, which is costly. Many enzymes inevitably result in impurities, and there are high levels of decomposition side reactions of NR, NMN, ATP, ADP, etc. In addition, the reaction system contains nicotinamide, ribose, ADP, AMP, NR, adenosine, adenine, and phosphate, which are produced by side reactions, making the components in the system complex and difficult to control. This makes the NMN product purification process difficult, costly, and difficult to control the stability of product quality. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for semi-synthesizing NMN involving adenosine, which simplifies the purification process of NMN products and reduces costs compared to conventional semi-synthetic methods. An object of the present invention is to provide a method for semi-synthesizing NMN involving adenosine that combines the advantages of chemical and enzymatic methods, ensures the synthesis efficiency of NMN products, and can reduce emissions, resulting in correspondingly lower production and environmental costs. Compared to conventional semi-synthetic methods, the present invention uses inexpensive adenosine instead of ATP, and introduces yeast cells into the reaction to convert adenosine to ATP in accordance with energy metabolism. This, combined with the conventional NR phosphorylation process, enables repeated use of ATP, eliminating the ATP recovery process, and uses the phosphate formed in the conventional NR phosphorylation process as a reactant, eliminating the phosphate removal process. One object of the present invention is to provide a method for semi-synthesizing NMN involving adenosine, which simplifies the NMN product purification process through the involvement of adenosine. An object of the present invention is to provide a method for semi-synthesizing NMN involving adenosine, which, compared to conventional semi-synthetic methods, uses inexpensive adenosine instead of ATP, and introduces yeast cells into the reaction to convert adenosine to ATP according to energy metabolism. This method combines with the conventional NR phosphorylation process to achieve repeated use of ATP, thereby reducing the molar amount of adenosine used. Furthermore, since the price of adenosine is much lower than that of ATP, the raw material cost of the corresponding NMN product is significantly reduced, resulting in lower production costs. Compared to conventional semi-synthetic methods, the present invention uses inexpensive adenosine instead of ATP, and introduces yeast cells into the reaction to convert adenosine to ATP according to energy metabolism. This method combines the conventional NR phosphorylation process to achieve repeated use of ATP, and the phosphate formed in the conventional NR phosphorylation process can be used as a reactant. In other words, after separating and purifying the NMN product, other reactants and products can be reused, reducing emissions. One objective of the present invention is to provide an NMN semi-synthetic method involving adenosine, which makes the production of the corresponding NMN product more environmentally friendly and reduces environmental costs. One object of the present invention is to provide a method for the semi-synthesis of NMN involving adenosine that is simpler, easier to implement, and less costly than conventional semi-synthetic methods, because it uses NR, phosphate, adenosine, and sucrose as raw materials, NRK, and yeast cells as catalysts, and integrates ATP production, NR phosphorylation, and ATP utilization within a single reaction system to complete the efficient synthesis of NMN, thereby achieving both the benefits of chemical methods in ensuring the synthesis efficiency of NMN products and the benefits of enzymatic methods in which various reactants (NR, phosphate, adenosine, sucrose, etc.) are essentially completely consumed, reducing excretion. [Means for solving the problem]

[0006] According to one aspect of the present invention, the method for semi-synthesizing NMN involving adenosine according to the present invention includes the following steps in the same reaction system: (A) reacting adenosine, phosphate, and sugars that can be metabolized by yeast cells catalyzed by the yeast cells to produce ATP; (B) A corresponding enzymatic phosphorylation step of NR, which reacts with NR and ATP to generate NMN and ADP, catalyzed by NRK. In one embodiment, in the reaction system of the NMN semi-synthesis method involving adenosine, the NR raw material is at least one selected from a commercially available pure NR product, a solid containing NR, and a liquid containing NR. In one embodiment, in the reaction system of the NMN semi-synthesis method involving adenosine, the sugar that can be metabolized by yeast cells is at least one selected from glucose, sucrose, starch, and glycerin. In one embodiment, in the reaction system of the NMN semi-synthesis method involving adenosine, the NRK enzyme is present in at least one initial form of a liquid enzyme form and an immobilized enzyme form. In one embodiment, in the reaction system of the NMN semi-synthesis method involving adenosine, the yeast cells are yeast cells capable of oxidative phosphorylation metabolism. In one embodiment, in the reaction system of the method for semi-synthesizing NMN involving adenosine, the yeast cells are at least one species selected from Pichia yeast and Saccharomyces cerevisiae. In one embodiment, a metal ion is further added to the reaction system of the NMN semi-synthesis method involving adenosine. In one embodiment, in the reaction system of the NMN semi-synthesis method involving adenosine, the added metal ion is at least one selected from magnesium ions and manganese ions. In one embodiment, in the reaction system of the NMN semi-synthesis method involving adenosine, the molar ratio of adenosine to NR ranges from 0.01 to 1. In one embodiment, in the reaction system of the NMN semi-synthesis method involving adenosine, the molar ratio of NR to phosphate ranges from 1 to 20. In one embodiment, in the reaction system of the method for semi-synthesizing NMN involving adenosine, the yeast cells are frozen and preserved wet yeast. In one embodiment, at least one organic reagent selected from toluene, n-butanol, and Tween 20 is further added to the reaction system of the NMN semi-synthesis method involving adenosine. In one embodiment, step (A) is performed before step (B), thereby providing ATP to the reaction of step (B), thereby forming a reaction state in which step (A) and step (B) mutually promote each other in the same reaction system. In one embodiment, the method for semi-synthesizing NMN involving adenosine further comprises the step of regenerating ATP by the action of yeast cells using ADP and phosphate. DETAILED DESCRIPTION OF THE INVENTION

[0007] The following description is intended to disclose the present invention and enable those skilled in the art to realize the present invention. The preferred embodiments in the following description are merely illustrative, and other obvious modifications may occur to those skilled in the art. The basic principles of the present invention defined in the following description may be applied to other embodiments, modifications, improvements, equivalents, and other technical means without departing from the spirit and scope of the present invention. It should be understood by those skilled in the art that in the disclosure of the present invention, the use of terms such as "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," etc., to indicate direction or positional relationships is merely for the purpose of facilitating and simplifying the description of the present invention, and does not indicate or suggest that the devices or elements shown must have a particular orientation, be configured, or operate in a particular orientation, and therefore such terms should not be construed as limiting the present invention. It should be noted that the term "one" should be understood to mean "at least one" or "one or more," i.e., in one embodiment, the number of an element may be one, and in another embodiment, the number of the element may be multiple, and the term "one" should not be understood to limit the number.

[0008] Compared to conventional semi-synthetic methods, the present invention uses inexpensive adenosine instead of ATP, and introduces yeast cells into the reaction to convert adenosine to ATP in accordance with energy metabolism. This method, combined with the conventional NR phosphorylation process, enables repeated use of ATP, and provides a method for semi-synthesizing NMN involving adenosine, in which phosphate formed in the conventional NR phosphorylation process is used as a reactant.

[0009] Specifically, the above-mentioned adenosine-related NMN semisynthesis method uses NR, phosphate, adenosine, and sugars that can be metabolized by yeast cells (e.g., glucose, sucrose, glycerin, etc.) as raw materials, NRK and yeast cells as catalysts, and integrates ATP production, NR phosphorylation, and ATP utilization within a single reaction system to efficiently complete NMN synthesis, with the reaction equation being NR + sucrose + adenosine + phosphate + O2 → NMN + ATP + CO2 + HO. In this reaction system, yeast cells provide energy through the oxidation of sugars during oxidative phosphorylation metabolism, promoting the binding of phosphate and adenosine to produce adenylic acid (AMP), which then produces ADP and ATP. ATP is converted to ADP through NR phosphorylation and then automatically converted back to ATP, which continues to participate in the reaction. That is, adenosine, AMP, ADP, etc. in the reaction system can all be rapidly converted into ATP, which can participate in the phosphorylation of NR. Compared to conventional semi-synthetic methods, the phosphate formed in the phosphorylation process of NR can be used as a reactant, eliminating the phosphate removal process, and ATP can be reused, eliminating the ATP recovery process. In this way, the involvement of adenosine simplifies the purification process of NMN products.

[0010] Furthermore, in one embodiment of the present invention, the above-mentioned method for semi-synthesizing NMN involving adenosine uses NR, phosphate, adenosine, sucrose, and magnesium ions as raw materials, NRK (not limited to liquid enzyme or immobilized enzyme) and yeast cells as catalysts, and reacts them in an aqueous solution at a neutral initial pH with stirring and exposure to air. In this way, ATP production, NR phosphorylation, and ATP utilization occur within a single reaction system, and various reactants (NR, phosphate, adenosine, sucrose, etc.) are essentially completely consumed. The corresponding reaction system is simple, easy to implement, low-cost, and environmentally friendly, resulting in lower environmental costs.

[0011] In another embodiment of the present invention, the above-mentioned method for semi-synthesizing NMN involving adenosine uses NR and adenosine as substrates, and produces NMN in a one-pot method using yeast and nicotinamide riboside kinase. For example, to a 1 L reaction system, NRC to a final concentration of 100 mM, adenosine to a final concentration of 50 mM, dipotassium hydrogen phosphate to a final concentration of 330 mM, potassium dihydrogen phosphate to a final concentration of 70 mM, sucrose to a final concentration of 120 mM, magnesium chloride to a final concentration of 50 mM, manganese chloride to a final concentration of 5 mM, 300 g of yeast, and 500 mg of freeze-dried powder of nicotinamide riboside kinase crude enzyme were added in that order and thoroughly stirred to dissolve. After that, the reaction temperature was controlled to 37°C and the mixture was stirred at 300 rpm to carry out the reaction. The concentration of NMN was detected by high performance liquid chromatography during the reaction process and the reaction was completed within 6 hours, yielding 29.84 g of NMN, for a reaction yield of 89.3%.

[0012] In another embodiment of the present invention, the adenosine-related NMN semisynthesis method uses NR and adenosine as substrates, and produces NMN in a one-pot process using budding yeast and magnetically immobilized nicotinamide riboside kinase enzyme. Illustratively, adenosine (final concentration: 50 mM), dipotassium hydrogen phosphate (final concentration: 330 mM), potassium dihydrogen phosphate (final concentration: 70 mM), sucrose (final concentration: 120 mM), magnesium chloride (final concentration: 50 mM), manganese chloride (final concentration: 5 mM), and 300 g of wet budding yeast are added in that order to a 1 L reaction system, and the mixture is thoroughly stirred to dissolve. The reaction temperature is then controlled at 37°C, and the mixture is allowed to stand for 1 hour for fermentation. To the yeast fermentation broth, NRC (final concentration: 100 mM) and 300 g of magnetically immobilized nicotinamide riboside kinase enzyme were added, and the mixture was stirred at 300 rpm to react. The reaction temperature was controlled at 37°C, and the reaction pH was controlled at 6.0 with 3 M sodium hydroxide using an automatic titrator. The NMN concentration was detected by high-performance liquid chromatography during the reaction, and the reaction was completed within 2 hours, yielding 31.58 g of NMN, with a reaction conversion rate of 94.5%.

[0013] To further illustrate the present invention, the method for semi-synthesizing NMN involving adenosine described in the present invention can be carried out in the same reaction system by: (A) reacting adenosine, phosphate, and sugars that can be metabolized by yeast cells catalyzed by the yeast cells to produce ATP; (B) A corresponding enzymatic phosphorylation step of NR, which reacts with NR and ATP to generate NMN and ADP, catalyzed by NRK. In the reaction system of the above-mentioned NMN semi-synthesis method involving adenosine, the NR raw material is at least one of commercially available pure NR, commercially available pure nicotinamide chloride (NRC), a solid containing NR, a solid containing NRC, a liquid containing NR, and a liquid containing NRC.

[0014] Furthermore, in the reaction system of the above-mentioned NMN semi-synthesis method involving adenosine, sugars that can be metabolized by yeast cells include, but are not limited to, a single sugar or a mixture of sugars selected from glucose, sucrose, starch, and glycerin. In particular, in the reaction system of the above-mentioned NMN semi-synthetic method involving adenosine, the NRK enzyme may be a liquid enzyme or an immobilized enzyme, and the present invention is not limited thereto. Furthermore, in the reaction system of the above-mentioned NMN semi-synthesis method involving adenosine, the yeast cells are various yeast cells capable of oxidative phosphorylation metabolism, such as Pichia yeast or Saccharomyces cerevisiae.

[0015] Preferably, metal ions such as magnesium ions and manganese ions may be further added to the reaction system of the above-mentioned NMN semi-synthesis method involving adenosine. Preferably, in the reaction system of the above-mentioned method for semi-synthesizing NMN involving adenosine, the molar ratio of adenosine to NR is in the range of 0.01 to 1. Preferably, in the reaction system of the above-mentioned method for semi-synthesizing NMN involving adenosine, the molar ratio of NR to phosphate is in the range of 1-20.

[0016] It should be noted that in the reaction system of the above-mentioned NMN semi-synthesis method involving adenosine, the yeast cells may be frozen and stored wet yeast. In particular, in the reaction system of the above-mentioned NMN semi-synthesis method involving adenosine, at least one organic reagent selected from toluene, n-butanol, and Tween 20 may be further added. It should be noted that in some embodiments of the present invention, step (A) is carried out before step (B) in the reaction process, thereby providing ATP to the reaction of step (B), so that step (A) and step (B) form a reaction state in which they mutually promote each other in the same reaction system. In particular, in these embodiments of the present invention, the adenosine-involving NMN semi-synthetic method further comprises the step of regenerating ATP by the action of yeast cells using ADP and phosphate.

[0017] As will be understood by those skilled in the art, the above examples are merely illustrative, and by combining features of different examples with each other, embodiments can be obtained that are easily conceivable based on the contents disclosed in the present invention but are not explicitly pointed out in the above description. It should be understood by those skilled in the art that the embodiments of the present invention shown in the above description are merely illustrative and do not limit the present invention. The objects of the present invention have already been fully and effectively achieved. The function and structural principles of the present invention have been shown and explained in the examples, and any variations or modifications can be made to the embodiments of the present invention without departing from the above principles.

Claims

1. In the same reaction system, adenosine, phosphate, sugars that can be metabolized by yeast cells, yeast cells, NR, and NRK are added and reacted in one pot, and the following steps are carried out: (A) reacting adenosine, phosphate, and sugars that can be metabolized by the yeast cells catalyzed by the yeast cells to produce ATP; (B) a phosphorylation step by NRK, which corresponds to the reaction of NR with ATP catalyzed by NRK to produce NMN and ADP.

2. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein in the reaction system of the method for semi-synthesizing NMN involving adenosine, the NR raw material is at least one selected from a commercially available pure NR product, a commercially available pure NRC product, a solid containing NR, a solid containing NRC, a liquid containing NR, and a liquid containing NRC.

3. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein in the reaction system of the method for semi-synthesizing NMN involving adenosine, the sugar that can be metabolized by yeast cells is at least one selected from glucose, sucrose, and starch.

4. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein in the reaction system of the method for semi-synthesizing NMN involving adenosine, the NRK enzyme is present in at least one initial form selected from the group consisting of a liquid enzyme form and an immobilized enzyme form.

5. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein the yeast cells in the reaction system of the method for semi-synthesizing NMN involving adenosine are yeast cells capable of oxidative phosphorylation metabolism.

6. 6. The method for semi-synthesizing NMN involving adenosine according to claim 5, wherein the yeast cells in the reaction system of the method for semi-synthesizing NMN involving adenosine are at least one species selected from Pichia yeast and Saccharomyces cerevisiae.

7. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein a metal ion is further added to the reaction system of the method for semi-synthesizing NMN involving adenosine.

8. 8. The method for semi-synthesizing NMN involving adenosine according to claim 7, wherein the metal ion added in the reaction system of the method for semi-synthesizing NMN involving adenosine is at least one selected from magnesium ions and manganese ions.

9. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein the molar ratio of NR to phosphate in the reaction system of the method for semi-synthesizing NMN involving adenosine is in the range of 1:1 to 20.

10. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein the yeast cells in the reaction system of the method for semi-synthesizing NMN involving adenosine are frozen and stored wet yeast cells.

11. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein at least one organic reagent selected from the group consisting of toluene, n-butanol, and Tween 20 is further added to the reaction system of the method for semi-synthesizing NMN involving adenosine.

12. 2. The method for semi-synthesizing NMN involving adenosine according to claim 1, wherein step (A) is carried out before step (B), thereby providing ATP for the reaction of step (B), thereby forming a reaction state in which step (A) and step (B) promote each other in the same reaction system.

13. 13. The method for semi-synthesis of NMN involving adenosine according to any one of claims 1 to 12, further comprising the step of regenerating ATP by the action of yeast cells using ADP and phosphate.