A method for preparing nicotinamide adenine dinucleotide phosphate by enzymatic method

Abstract

The present application relates to a kind of methods for preparing nicotinamide adenine dinucleotide phosphate by enzyme, the method includes the following steps: the first step reaction: in reaction system I, with NMN, and adenosine triphosphate or adenosine triphosphate salt as substrate, under the catalysis of NMNAT, NAD and pyrophosphate are generated;Optionally, the pyrophosphate is hydrolyzed under the catalysis of IPP1, and the reaction is promoted to proceed in positive direction;Second step reaction: in reaction system II, the NAD generated in the first step reaction is generated under the catalysis of MfnK with the participation of phosphoryl donor, NADP is generated.The method provided by the present application has moderate substrate cost, short process route, high atom economy, less byproduct;Through preferred enzyme source and synthesis condition, synthesis time is short, enzyme catalysis efficiency is high, substrate tolerance is strong, synthesis conversion rate is high, can satisfy high concentration substrate high-efficiency catalysis synthesis NADP.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to an enzymatic method for preparing nicotinamide adenine dinucleotide phosphate. Background Technology

[0002] Nicotinamide adenine dinucleotide phosphate (NADP, also known as oxidized coenzyme II) is a crucial nucleotide coenzyme. It is a phosphorylated derivative of the ribonucleotide ring linked to adenine in nicotinamide adenine dinucleotide (NAD, also known as oxidized coenzyme II) at the 2'-position. It is an indispensable hydrogen carrier in biological oxidation processes, transferring protons, electrons, and energy through redox reactions. It participates in various anabolic reactions, such as the synthesis of lipids, fatty acids, and nucleotides. It can activate multi-enzyme systems, promote the synthesis and metabolism of nucleic acids, proteins, and polysaccharides, enhance substance transport and regulation, and improve metabolic function. Studies have shown that NADP can promote metabolism, energy metabolism, resist cellular aging, and has antioxidant effects. NADP is produced by the phosphorylation of NAD through NAD kinase.

[0003] In the asymmetric synthesis of chiral drugs and chiral intermediates, almost 50% of the reactions involve the enzymatic catalysis of coenzymes, and coenzymes need to be added in production to meet the requirements of reaction rate.

[0004] Currently, there are various methods for preparing NADP, including chemical synthesis, bio-enzyme catalysis, and yeast fermentation extraction. Chemical synthesis methods suffer from severe pollution and demanding conditions inherent in chemical engineering, coupled with low yields, resulting in high synthesis costs. Yeast fermentation extraction is limited in industrial-scale production due to the extremely low NADP content in yeast. Bio-enzyme catalysis, with its inherent advantages of high efficiency and environmental friendliness, is gradually becoming the mainstream method for industrial NADP preparation; however, existing bio-enzyme catalysis methods also have some drawbacks. For example:

[0005] Reference 1 discloses an enzymatically catalytic method for the preparation of oxidized coenzyme II. Using nicotinamide nucleoside (NR) and disodium adenosine triphosphate (ATP-Na2) as raw materials, a one-pot reaction is carried out in the presence of four enzymes: nicotinamide nucleoside kinase (NRK), inorganic pyrophosphatase, NAD kinase, and polyphosphokinase, in a buffer solution with a pH of 4.0–8.5 and a temperature of 10℃–40℃ to obtain oxidized coenzyme II. The method disclosed in Reference 1 suffers from problems such as a long process route, the use of multiple enzyme types, long synthesis reaction time, low conversion rate, and low NADP product concentration, which cannot meet the requirements for industrial-scale NADP production.

[0006] Reference 2 discloses a method for preparing NADP coenzyme, comprising: first, obtaining nicotinamide adenine dinucleotide; second, phosphorylating nicotinamide adenine dinucleotide with ATP to generate NADP; the first step of synthesis uses nicotinamide ribose as a starting substrate to synthesize nicotinamide adenine dinucleotide with the participation of nicotinamide ribokinase, nicotinamide mononucleotide adenosine transferase, and adenosine triphosphate: first, the starting substrate nicotinamide ribose reacts with adenosine triphosphate under the catalysis of nicotinamide ribokinase to obtain nicotinamide mononucleotide; second, nicotinamide mononucleotide reacts with another adenosine triphosphate under the action of nicotinamide mononucleotide adenosine transferase to generate nicotinamide adenine dinucleotide. However, in the method disclosed in Reference 2, the conversion rate of the first step is only up to 60%, and the second step uses ATP as a phosphoryl group donor, which is costly and therefore cannot meet the requirements for industrial production of NADP.

[0007] Reference 3 discloses an enzymatic method for preparing nicotinamide adenine dinucleotide phosphate (NADP). Using adenosine, sodium hexametaphosphate, and ATP as substrates, NADP is synthesized via four enzyme complexes: adenine phosphoribosyltransferase (APRT), nicotinamide phosphoribosyltransferase (NmPRT), nicotinamide mononucleotide adenosyltransferase (NMNAT), and polyphosphate-dependent NAD kinase (ppnk). The process flow diagram is shown below. Figure 1 As shown. The method disclosed in Reference 3 also suffers from a long process route, involves many enzymes, generates adenine as an equivalent byproduct, has poor atom economy, a low conversion rate of 90%-96%, and a low NADP product concentration of 34g / L-37g / L, resulting in low economics for industrial production.

[0008] Therefore, there is a need to develop a NADP synthesis method with a shorter process route and higher cost-effectiveness to meet the market demand for large-scale NADP production.

[0009] References:

[0010] Reference 1: CN102605027B;

[0011] Reference 2: CN108998484A;

[0012] Reference 3: CN110643587B. Summary of the Invention

[0013] The problem the invention aims to solve

[0014] In view of the problems existing in the above-mentioned prior art, such as the long process route, low yield, and poor economic practicality of NADP synthesis methods, the purpose of this invention is to develop a synthesis process for NADP that has a short process route, moderate substrate cost, high atom economy, few by-products, high conversion rate, short synthesis time, and high product concentration, and meets the requirements for industrial production of NADP.

[0015] Solution for solving the problem

[0016] This invention provides an enzymatic method for preparing nicotinamide adenine dinucleotide phosphate, characterized in that the method comprises the following steps:

[0017] Step 1: In reaction system I, using nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) or ATP salt as substrates, nicotinamide nucleoside adenosine yltransferase (NMNAT) catalyzes the generation of nicotinamide adenine dinucleotide (NAD) and pyrophosphate; optionally, the pyrophosphate is hydrolyzed under the catalysis of inorganic pyrophosphatase (IPP1) to promote the forward reaction.

[0018] Step 2: In reaction system II, the nicotinamide adenine dinucleotide generated in step 1 is converted into nicotinamide adenine dinucleotide phosphate (NADP) with the participation of a phosphoryl donor and under the catalysis of ATP / polyphosphate-dependent NAD kinase (MfnK).

[0019] Furthermore, the nicotinamide nucleoside adenosyltransferase is derived from Methanocaldococcus infernus, the inorganic pyrophosphatase is derived from Saccharomyces cerevisiae S288C strain, and the ATP / polyphosphate-dependent NAD kinase is derived from Micrococcus flavus.

[0020] Preferably,

[0021] The nicotinamide nucleoside adenosyltransferase is selected from any one of the following (a1) to (a4):

[0022] (a1) A polypeptide containing the amino acid sequence shown in SEQ ID NO.1 and having nicotinamide nucleoside adenosyltransferase activity;

[0023] (a2) A polypeptide having an amino acid sequence as shown in SEQ ID NO.1 with one or more amino acids substituted, deleted or added, and having nicotinamide nucleoside adenosyltransferase activity;

[0024] (a3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (a1) or (a2);

[0025] (a4) A polypeptide encoded by a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3, and having nicotinamide nucleoside adenosyltransferase activity;

[0026] The inorganic pyrophosphatase is selected from any one of the following (b1) to (b4):

[0027] (b1) A polypeptide containing the amino acid sequence shown in SEQ ID NO.4 and having inorganic pyrophosphatase activity;

[0028] (b2) A polypeptide having an amino acid sequence as shown in SEQ ID NO.4 with one or more amino acids substituted, deleted or added, and having inorganic pyrophosphatase activity;

[0029] (b3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (b1) or (b2);

[0030] (b4) A polypeptide encoded by a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO.5 and having inorganic pyrophosphatase activity;

[0031] The ATP / polyphosphate-dependent NAD kinase is selected from any one of the following (c1) to (c4):

[0032] (c1) A polypeptide containing the amino acid sequence shown in SEQ ID NO.6 and having ATP / polyphosphate-dependent NAD kinase activity;

[0033] (c2) A polypeptide having an amino acid sequence as shown in SEQ ID NO.6 with one or more amino acids substituted, deleted or added, and having ATP / polyphosphate-dependent NAD kinase activity;

[0034] (c3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (c1) or (c2);

[0035] (c4) A polypeptide encoded by a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.8, and having ATP / polyphosphate-dependent NAD kinase activity.

[0036] Furthermore, the nicotinamide nucleoside adenosyltransferase, the inorganic pyrophosphatase, and the ATP / polyphosphate-dependent NAD kinase participate in the reaction in the form of pure enzymes, crude enzymes, host cell lysates expressing them, or host cells expressing them.

[0037] Preferably, the nicotinamide nucleoside adenosine transferase, the inorganic pyrophosphatase, and the ATP / polyphosphate-dependent NAD kinase participate in the reaction in the form of host cells expressing them.

[0038] Optionally, in reaction system I or reaction system II, the wet weight concentrations of the host cells expressing the nicotinamide nucleoside adenosine transferase, the host cells expressing the inorganic pyrophosphatase, and the host cells expressing the ATP / polyphosphate-dependent NAD kinase are each independently 5 g / L to 50 g / L.

[0039] Furthermore, the first and second reactions are carried out in a reaction medium, which is a phosphate buffer solution, a Tris-HCl buffer solution, or pure water;

[0040] Optionally, the concentration of the phosphate buffer solution is 20 mM to 100 mM.

[0041] Furthermore, in the reaction system I, the initial concentration of the nicotinamide mononucleotide is 50 mM to 400 mM;

[0042] Preferably, in the reaction system I, the initial molar ratio of the nicotinamide mononucleotide to adenosine triphosphate or adenosine triphosphate salt is 1:1 to 1:1.5.

[0043] Furthermore, the reaction system I also contains divalent metal ions;

[0044] Preferably, the divalent metal ions include magnesium ions and / or manganese ions;

[0045] Preferably, in the reaction system I, the concentration of magnesium ions is 1 mM to 60 mM, and the concentration of manganese ions is 0.5 mM to 40 mM.

[0046] Furthermore, the reaction system I also contains Triton X-100;

[0047] Preferably, in the reaction system I, the concentration of Triton X-100 is 0.1% (V / V) to 0.2% (V / V).

[0048] Furthermore, the pH of the reaction system I is 7.0 to 10.0, the reaction temperature of the first step reaction is 25°C to 42°C, and the reaction time of the first step reaction is 1.0 h to 6.0 h.

[0049] Furthermore, in the reaction system II, the phosphoryl donor is an inorganic phosphate, preferably a polyphosphate or polyphosphate.

[0050] Preferably, based on the initial amount of the nicotinamide mononucleotide in reaction system I, the molar ratio of the nicotinamide mononucleotide to the phosphoryl donor in reaction system II is 1:0.6 to 1:1.5;

[0051] Preferably, in the second step of the reaction, the phosphoryl donor is added to the reaction system by liquid addition, and the addition time is controlled to be 1.0 h to 8.0 h.

[0052] Furthermore, the pH of the reaction system II is 5.0 to 8.0, the reaction temperature of the second step reaction is 30°C to 60°C, and the reaction time of the second step reaction is 1.5h to 15.0h.

[0053] The effects of the invention

[0054] By implementing the above technical solution, the present invention has achieved the following technical effects:

[0055] First, the enzymatic method for preparing nicotinamide adenine dinucleotide phosphate (NADP) provided by this invention uses NMN as a starting substrate and synthesizes NADP via the catalysis of three enzymes: NMNAT, IPP1, and MfnK. This method offers moderate substrate cost, a short process route, high atom economy, and few byproducts. Furthermore, this invention optimizes the enzyme source and synthesis conditions, resulting in short synthesis time, high enzyme catalytic efficiency, strong substrate tolerance, and high conversion rate, meeting the requirements for efficient NADP synthesis with high-concentration substrates. In addition, the method provided by this invention can use pure water as the reaction medium, simplifying the synthesis system. The NADP synthesis solution has a high NADP concentration and low impurity content, effectively reducing the subsequent extraction cost of NADP. Therefore, the enzymes and synthesis process selected in this invention meet the requirements for industrial-scale NADP production and have industrialization value. Attached Figure Description

[0056] Figure 1 This is a process flow diagram of an enzymatic method for preparing nicotinamide adenine dinucleotide phosphate disclosed in Chinese patent document CN110643587B.

[0057] Figure 2 A synthetic route diagram of nicotinamide adenine dinucleotide phosphate provided by the present invention. Detailed Implementation

[0058] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0059] In this invention, the range of values ​​represented by “value A ~ value B” or “value A - value B” refers to the range that includes the endpoint values ​​A and B.

[0060] In this invention, the word "may" has both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the case where the event occurs and the case where the event does not occur.

[0061] In this invention, the terms "a", "an", or "the" may refer to "one", "one or more", "at least one", or "one or more".

[0062] In this invention, the term "about" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned testing apparatus or method. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified with "about". Here, "about" generally means that the actual value is within ±10%, ±5%, ±1%, or ±0.5% of a particular value or range.

[0063] In this invention, the terms "peptide" and "protein" are used interchangeably to refer to a string of at least two amino acid residues linked together by covalent bonds (e.g., peptide bonds), which can be recombinant peptides, natural peptides, or synthetic peptides. Peptides can be linear, branched, or cyclic, and can contain modified amino acids, including modifications such as acetylation, formylation, phosphorylation, sulfonation, ubiquitination, glycosylation, amidation of the C-terminal amino acid, or any other operation, and can be separated by non-amino acid segments.

[0064] In this invention, the term "amino acid" can include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers. The amino acids, their abbreviations, and English abbreviations in this invention are shown below:

[0065] Histidine (His, H); Serine (S); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Threonine (Thr, T); Phenylalanine (Phe, F); Aspartic acid (Asp, D); Tyrosine (Tyr, Y); Leucine (Leu, L); Isoleucine (Ile, I); Arginine (Arg, R); Alanine (Ala, A); Valine (Val, V); Tryptophan (Trp, W); Methionine (Met, M); Asparagine (Asn, N); Cysteine ​​(Cys, C); Lysine (Lys, K); Proline (Pro, P).

[0066] In this invention, amino acid deletion may refer to the deletion of 1, 2 or 3 or more amino acids from the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0067] In this invention, amino acid addition can refer to adding 1, 2 or 3 or more amino acids at any position at the C-terminus, N-terminus or between the C-terminus and N-terminus of the amino acid sequence, as long as the modified sequence completely or partially retains the activity of the original amino acid sequence.

[0068] In this invention, amino acid substitution refers to the replacement of an amino acid at a certain position in an amino acid sequence with another amino acid, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence. Amino acid substitution can be conserved amino acid substitution, meaning that compared to the original amino acid sequence, several amino acids are replaced by amino acids with similar or related properties to form a peptide (conserved variant peptide). For example, these conserved variant peptides can be generated based on the following amino acid substitutions: substitution of Ala with Val, Leu, or Ile; substitution of Arg with Lys, Gln, Asn, or His; substitution of Asn with Gln, His, Lys, or Arg; substitution of Asp with Glu or Asn; substitution of Cys with Ser or Ala; substitution of Gln with Asn or Glu; substitution of Glu with Asp or Gln; substitution of Gly with Ala; substitution of His with Asn, Lys, Gln, or Arg; substitution of Cys with Leu, Met, Ala, Val, Phe, or leucine. Substitutions include: Ile substitutions; substitutions of Leu with Ile, Met, Ala, Val, Phe, or leucine; substitutions of Lys with Asn, Gln, or Arg; substitutions of Met with Ile, Leu, or Phe; substitutions of Phe with Leu, Val, Ile, Ala, or Tyr; substitutions of Pro with Ala; substitutions of Ser with Thr; substitutions of Thr with Ser or Val; substitutions of Trp with Phe or Tyr; substitutions of Tyr with Trp, Phe, Thr, or Ser; and substitutions of Val with Phe, Ala, Met, Ile, Leu, or leucine. Amino acid substitutions can also be non-conserved amino acid substitutions.

[0069] In this invention, "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0070] In this invention, the term "codon optimization" refers to the configuration of the nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism in order to improve gene expression and translation efficiency in the host cell or organism.

[0071] In this invention, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, etc.

[0072] In this invention, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0073] In this invention, "wet weight of cells" refers to the mass of cells under normal living conditions.

[0074] In this invention, "pure enzyme" refers to a single enzyme substance, such as a single enzyme protein. "Crude enzyme" refers to a collection of enzyme-containing substances that have undergone appropriate purification during the enzyme production process, such as removing cell debris and other impurities through centrifugation, but still contain small amounts of other macromolecular or small molecule impurities.

[0075] In this invention, "cell disruption fluid" refers to a liquid mixture in which the original intact cell structure is disrupted by external force, such as ultrasound or grinding. For example, the cell disruption fluid may contain cell fragments, cell contents, and solvents such as water or cell culture medium.

[0076] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0077] The technical solution of the present invention will be described in detail below:

[0078] This invention provides an enzymatic method for preparing nicotinamide adenine dinucleotide phosphate, which mainly adopts the following technical route:

[0079] In the first step of synthesis, nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) or ATP salt are used as substrates to generate nicotinamide adenine dinucleotide (NAD) and pyrophosphate under the catalysis of nicotinamide nucleoside adenosine yltransferase (NMNAT). At the same time, inorganic pyrophosphatase (IPP1) is added to hydrolyze pyrophosphate to promote the forward reaction.

[0080] In step 2, the NAD synthesized in step 1 is converted to nicotinamide adenine dinucleotide phosphate (NADP) via ATP / polyphosphate-dependent NAD kinase (MfnK) catalysis with the participation of a phosphoryl group donor. The specific technical route is as follows: Figure 2 As shown.

[0081] In some embodiments, the enzymatic method for preparing nicotinamide adenine dinucleotide phosphate provided by the present invention includes the following steps:

[0082] Step 1: In reaction system I, using nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) or ATP salt as substrates, nicotinamide nucleoside adenosine yltransferase (NMNAT) catalyzes the generation of nicotinamide adenine dinucleotide (NAD) and pyrophosphate; optionally, the pyrophosphate is hydrolyzed under the catalysis of inorganic pyrophosphatase (IPP1) to promote the forward reaction.

[0083] Step 2: In reaction system II, the nicotinamide adenine dinucleotide generated in step 1 is converted into nicotinamide adenine dinucleotide phosphate (NADP) with the participation of a phosphoryl donor and under the catalysis of ATP / polyphosphate-dependent NAD kinase (MfnK).

[0084] In some embodiments, the adenosine triphosphate salt includes adenosine triphosphate disodium salt.

[0085] In some embodiments, the nicotinamide-nucleotide adenylyltransferase (also known as nicotinamide mononucleotide adenylyltransferase) (NMNAT) is derived from *Methanocaldococcus infernus*, NCBI accession number WP_013099845.

[0086] In some implementations, the inorganic diphosphatase (IPP1) is derived from Saccharomyces cerevisiae S288C strain, NCBI accession number NP_009565.

[0087] In some embodiments, the ATP / polyphosphate-dependent NAD kinase (also known as poly(P) / ATP-NAD kinase) (MfnK) is derived from Micrococcus flavus, NCBI accession number WP_135030347.

[0088] In some embodiments, the nicotinamide nucleoside adenosyltransferase is selected from any one of (a1) to (a4):

[0089] (a1) A polypeptide containing the amino acid sequence shown in SEQ ID NO.1 and having nicotinamide nucleoside adenosyltransferase activity;

[0090] (a2) A polypeptide having an amino acid sequence as shown in SEQ ID NO.1 with one or more amino acids substituted, deleted or added, and having nicotinamide nucleoside adenosyltransferase activity;

[0091] (a3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (a1) or (a2);

[0092] (a4) A polypeptide encoded by a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3, and having nicotinamide nucleoside adenosyltransferase activity.

[0093] In some preferred embodiments, the amino acid sequence of the nicotinamide nucleoside adenosyltransferase is shown in SEQ ID NO. 1.

[0094] In some preferred embodiments, the encoding nucleotide sequence of the nicotinamide nucleoside adenosyltransferase is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0095] In some embodiments, the inorganic pyrophosphatase is selected from any one of the following (b1) to (b4):

[0096] (b1) A polypeptide containing the amino acid sequence shown in SEQ ID NO.4 and having inorganic pyrophosphatase activity;

[0097] (b2) A polypeptide having an amino acid sequence as shown in SEQ ID NO.4 with one or more amino acids substituted, deleted or added, and having inorganic pyrophosphatase activity;

[0098] (b3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (b1) or (b2);

[0099] (b4) A polypeptide encoded by a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO.5 and having inorganic pyrophosphatase activity.

[0100] In some preferred embodiments, the amino acid sequence of the inorganic pyrophosphatase is shown in SEQ ID NO.4.

[0101] In some preferred embodiments, the encoding nucleotide sequence of the inorganic pyrophosphatase is shown in SEQ ID NO.5.

[0102] In some embodiments, the ATP / polyphosphate-dependent NAD kinase is selected from any one of the following (c1) to (c4):

[0103] (c1) A polypeptide containing the amino acid sequence shown in SEQ ID NO.6 and having ATP / polyphosphate-dependent NAD kinase activity;

[0104] (c2) A polypeptide having an amino acid sequence as shown in SEQ ID NO.6 with one or more amino acids substituted, deleted or added, and having ATP / polyphosphate-dependent NAD kinase activity;

[0105] (c3) A polypeptide encoded by a polynucleotide encoding the amino acid sequence shown in (c1) or (c2);

[0106] (c4) A polypeptide encoded by a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.8, and having ATP / polyphosphate-dependent NAD kinase activity.

[0107] In some preferred embodiments, the amino acid sequence of the ATP / polyphosphate-dependent NAD kinase is shown in SEQ ID NO. 6.

[0108] In some preferred embodiments, the encoding nucleotide sequence of the ATP / polyphosphate-dependent NAD kinase is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0109] This invention utilizes a rational combination of three enzymes: nicotinamide nucleoside adenosine transferase, inorganic pyrophosphatase, and ATP / polyphosphate-dependent NAD kinase, to enzymatically catalyze the synthesis of NADP from NMN.

[0110] This invention does not particularly limit the form of the enzymes involved in the reaction. For example, nicotinamide nucleoside adenosine transferase, inorganic pyrophosphatase, and ATP / polyphosphate-dependent NAD kinase can participate in the reaction as pure enzymes, crude enzyme solutions, host cells expressing them, or fermentation broth containing host cells expressing them. Host cells can be collected by centrifugation or filtration for host cell-catalyzed transformation.

[0111] The enzymatic method for preparing nicotinamide adenine dinucleotide phosphate according to the present invention, which includes a two-step reaction, requires the combined use of enzymes. The encoding nucleotide sequences of nicotinamide nucleoside adenylate transferase and inorganic pyrophosphatase can be ligated into different expression vectors and introduced into host cells for individual expression, or the encoding nucleotide sequences of nicotinamide nucleoside adenylate transferase and inorganic pyrophosphatase can be ligated into the same expression vector and introduced into host cells for co-expression. The co-expression strategy is preferred.

[0112] In some embodiments, the nicotinamide nucleoside adenylate transferase, the inorganic pyrophosphatase, and the ATP / polyphosphate-dependent NAD kinase participate in the reaction in the form of pure enzymes, crude enzymes, host cell lysates expressing them, or host cells expressing them. In some preferred embodiments, the nicotinamide nucleoside adenylate transferase, the inorganic pyrophosphatase, and the ATP / polyphosphate-dependent NAD kinase participate in the reaction in the form of host cells expressing them. Further, in reaction system I or reaction system II, the wet weight concentrations of the host cells expressing the nicotinamide nucleoside adenosyltransferase, the host cells expressing the inorganic pyrophosphatase, and the host cells expressing the ATP / polyphosphate-dependent NAD kinase are each independently 5 g / L to 50 g / L; for example, they can be independently about 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L, etc.; preferably, they are independently 15 g / L to 40 g / L.

[0113] reaction medium

[0114] In some embodiments, the first and second reactions are carried out in a reaction medium, which is a phosphate buffer solution, a Tris-HCl buffer solution, or pure water, preferably pure water.

[0115] In some specific embodiments, when the phosphate buffer solution is used as a reaction medium, its concentration is 20mM to 100mM; for example, it can be about 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, etc.

[0116] Step 1 reaction

[0117] In some embodiments, the first step of the reaction includes the following specific steps: dissolving the reaction substrate nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) or ATP salt in a reaction medium; adding divalent metal ions such as magnesium ions, manganese ions, or a mixture of magnesium and manganese ions; adjusting the pH of the reaction solution; adding nicotinamide nucleoside adenosyltransferase (NMNAT) and inorganic pyrophosphatase (IPP1); adding Triton X-100; controlling the reaction temperature and pH of the synthesis system to obtain reaction system I, and synthesizing nicotinamide adenine dinucleotide (NAD). The reaction progress can be monitored by high-performance liquid chromatography (HPLC) during the reaction, and the NMN conversion rate at the end of the synthesis is 98.0%-100.0%.

[0118] To control the substrate concentration and thus improve synthesis efficiency, in some embodiments, the initial concentration of the nicotinamide mononucleotide in reaction system I is 50 mM to 400 mM; for example, it can be about 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM or 400 mM, etc.; preferably 150 mM to 300 mM.

[0119] Furthermore, in some embodiments, in the reaction system I, the initial molar ratio of the nicotinamide mononucleotide to adenosine triphosphate or adenosine triphosphate salt is 1:1 to 1:1.5; for example, it can be about 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5, etc.; preferably 1:1.1 to 1:1.3.

[0120] To facilitate the reaction, in some embodiments, the reaction system I further includes divalent metal ions.

[0121] In some specific implementations, the divalent metal ions include magnesium ions and / or manganese ions.

[0122] In some preferred embodiments, the reaction system I includes both magnesium ions and manganese ions.

[0123] The present invention does not impose any particular limitation on the method of adding divalent metal ions. For example, they can be added in the form of chlorides, sulfates, sulfate hydrates, nitrates, or nitrate hydrates.

[0124] In some embodiments, the concentration of magnesium ions in the reaction system I is 1 mM to 60 mM, for example, it can be about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM or 60 mM, preferably 5 mM to 40 mM; the concentration of manganese ions is 0.5 mM to 40 mM, for example, it can be about 0.5 mM, 1.5 mM, 2.5 mM, 3.5 mM, 5.5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM or 40 mM, preferably 2.5 mM to 20 mM.

[0125] In some embodiments, the reaction system I further comprises Triton X-100.

[0126] In some specific embodiments, the concentration of Triton X-100 in the reaction system I is 0.1% (V / V) to 0.2% (V / V), for example, it can be about 0.1% (V / V), 0.12% (V / V), 0.14% (V / V), 0.16% (V / V), 0.18% (V / V), or 0.20% (V / V), etc.

[0127] In some embodiments, the pH of the reaction system I is 7.0 to 10.0, for example, it can be about 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0, etc., preferably 7.5 to 9.0. The present invention does not particularly limit the substance used to adjust the pH, for example, it can be 5M sodium hydroxide.

[0128] In some embodiments, the reaction temperature of the first step reaction is 25°C to 42°C, for example, it can be about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C or 42°C, preferably 30°C to 37°C.

[0129] Depending on the different starting substrate concentrations and enzyme dosages, in some embodiments, the reaction time of the first step reaction is 1.0 h to 6.0 h, for example, it can be about 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h, 4.5 h, 5.0 h, 5.5 h or 6.0 h, preferably 2.0 h to 5.0 h.

[0130] Step 2 reaction

[0131] In some embodiments, the second step reaction includes the following specific steps: After the first step synthesis reaction is completed, the pH of the first step synthesis solution and the temperature of the reaction system are adjusted, ATP / polyphosphate-dependent NAD kinase (MfnK) is added, a phosphoryl donor is fed in, and the temperature and pH of the reaction system are controlled to obtain reaction system II, synthesizing nicotinamide adenine dinucleotide phosphate (NADP). The reaction progress can be monitored by high-performance liquid chromatography (HPLC) during the reaction. The NAD conversion rate at the end of the synthesis is 98.0%-99.5%. The total synthesis conversion rate is 96.0%-99.5%.

[0132] To synthesize nicotinamide adenine dinucleotide phosphate from the nicotinamide adenine dinucleotide obtained in step 1, a phosphoryl donor needs to be added to reaction system I after the reaction is completed.

[0133] In some embodiments, the phosphoryl donor in reaction system II is an inorganic phosphate.

[0134] In some specific embodiments, the phosphoryl donor is a polyphosphate or polyphosphate, such as sodium trimetaphosphate, sodium hexametaphosphate, sodium polyphosphate, sodium tetraphosphate, potassium metaphosphate, or potassium triphosphate, preferably sodium tetraphosphate.

[0135] In some embodiments, based on the initial amount of the nicotinamide mononucleotide in reaction system I, the molar ratio of the nicotinamide mononucleotide to the phosphoryl donor in reaction system II is 1:0.6 to 1:1.5, for example, it can be about 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5, preferably 1:0.8 to 1:1.1. Furthermore, the amount of phosphoryl donor in reaction system II refers to the total amount of phosphoryl donor added to reaction system I at the end of step 1.

[0136] In some specific embodiments, in the second step of the reaction, the phosphoryl donor can be prepared as a mother liquor in advance and added to the reaction system by liquid feeding. The feeding rate can be controlled as uniform feeding or variable feeding, preferably uniform feeding. The uniform feeding time is controlled to be 1.0 h to 8.0 h, for example, it can be about 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h, 4.5 h, 5.0 h, 5.5 h, 6.0 h, 6.5 h, 7.0 h, 7.5 h and 8.0 h, etc., preferably 1.5 h to 2.5 h.

[0137] In some embodiments, the pH of reaction system II is 5.0 to 8.0, for example, it can be about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0, preferably 5.5 to 7.0. The present invention does not particularly limit the substance used to adjust the pH; for example, it can be phosphoric acid.

[0138] In some embodiments, the reaction temperature of the second step reaction is 30°C to 60°C, for example, it can be about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 45°C, 50°C, 55°C or 60°C, preferably 37°C to 55°C.

[0139] Depending on the starting substrate concentration and enzyme dosage, in some embodiments, the reaction time of the second step reaction is 1.5 h to 15.0 h, for example, it can be about 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h, 4.5 h, 5.0 h, 5.5 h, 6.0 h, 6.5 h, 7.0 h, 8.0 h, 9.0 h, 10.0 h, 11.0 h, 12.0 h, 13.0 h, 14.0 h, 15.0 h, etc., preferably 2.5 h to 10 h.

[0140] The amino acid sequence and nucleotide sequence involved in this invention

[0141] Amino acid sequence of NMNAT (SEQ ID NO. 1): MRGLLVGRFQPFHNGHLNV VLSIMKEVDELIIVVGSAEKSHSLDNPFTAGERILMISKTLRKYNFPFYVIPI KDIEFNSLWVSYVESLTPKFDVVYSGNPLVRVLFKERNYKVKRPQMFNR KELSGEEIRRRMLSGEPWENLVPKEVAEVIEEIDGVNRLRSLLESDKL*.

[0142] Nucleotide sequence encoding NMNAT (SEQ ID NO.2): ATGAGAGGCTTATTGG TTGGAAGATTTCAACCCTTTCATAATGGACATTTAAATGTTGTCTTAAGTATAATGAAGGAGGTTGATGAGTTAATTATAGTTGTTGGTAGTGCTGAGAAAAGCCATAGCTTAGACAATCCCTTCACAGCTGGAGAAAGGATATTAATGATCTCTAAAACCTTGAGAAAATATAATTTCCCTTTTTATGTCATTCCAATAAAGGATATTGAATTTAACTCCCTCTGGGTTTCTTATGTAGAATCTCTAACTCCTAAGTTTGATGTGGTCTATTCTGGAAATCCATTAGTTAGGGTTCTATTTAAAGAGAGAAACTATAAGGTTAAGAGGCCACAGATGTTTAATAGGAAAGAGCTTTCTGGAGAGGAGATAAGGAGAAGAATGCTCTCAGGAGAGCCTTGGGAAAACCTAGTCCCTAAGGAAGTGGCTGAGGTTATAGAGGAGATTGATGGGGTTAATAGATTAAGAAGCCTCTTAGAGAGTGATAAGTTATGA.

[0143] Codon-optimized nucleotide sequence encoding NMNAT (SEQ ID NO.3): ATGCGT GGCCTGCTGGTTGGCCGTTTCCAGCCGTTCCACAACGGCCACCTGAACGTTGTTCTGAGCATCATGAAAGAAGTTGATGAACTGATCATCGTTGTTGGCAGCGCGGAAAAAAGCCACAGCCTGGATAACCCGTTCACCGCGGGCGAACGTATCCTGATGATCAGCAAAACCCTGCGTAAATACAACTTCCCGTTCTACGTTATCCCGATCAAAGATATCGAATTCAACAGCCTGTGGGTTAGCTACGTTGAAAGCCTGACCCCGAAATTCGATGTTGTTTACAGCGGCAACCCGCTGGTTCGTGTTCTGTTCAAAGAACGTAACTACAAAGTTAAACGTCCGCAGATGTTCAACCGTAAAGAACTGAGCGGCGAAGAAATCCGTCGTCGTATGCTGAGCGGTGAACCGTGGGAAAACCTGGTTCCGAAAGAAGTTGCGGAAGTTATCGAAGAAATCGATGGCGTTAACCGTCTGCGTAGCCTGCTGGAAAGCGATAAACTG.

[0144] Amino acid sequence of IPP1 (SEQ ID NO.4): MTYTTRQIGAKNTLEYKVYIEK DGKPVSAFHDIPLYADKENNIFNMVVEIPRWTNAKLEITKEETLNPIIQDTKKGKLRFVRNCFPHHGYIHNYGAFPQTWEDPNVSHPETKAVGDNDPIDVLEIGETIAYTGQVKQVKALGIMALLDEGETDWKVIAIDINDPLAPKLNDIEDVEKYFPGLLRATNEWFRIYKIPDGKPENQFAFSGEAKNKKYALDIIKETHDSWKQLIAGKSSDSKGIDLTNVTLPDTPTYSKAASDAIPPASPKADAPIDKSIDKWFFISGSV*.

[0145] Nucleotide sequence encoding IPP1 (SEQ ID NO.5): ATGACCTACACTACCAGA CAAATTGGTGCCAAGAACACCTTGGAATACAAAGTTTATATCGAAAAGGATGGTAAGCCAGTTTCTGCCTTCCACGACATTCCCTTGTACGCTGACAAGGAAAACAACATTTTCAACATGGTTGTTGAAATTCCACGTTGGACCAACGCCAAGTTAGAAATCACCAAGGAAGAAACTTTGAACCCAATCATCCAAGACACCAAGAAGGGCAAGTTGAGATTTGTTAGAAACTGTTTCCCTCACCATGGTTACATTCACAACTATGGTGCTTTCCCACAAACTTGGGAAGACCCAAACGTAAGCCACCCAGAAACTAAGGCAGTTGGTGACAACGATCCAATTGATGTGTTGGAAATTGGTGAAACTATTGCTTACACTGGTCAAGTCAAGCAAGTTAAGGCTCTAGGTATCATGGCTTTATTGGATGAAGGTGAGACCGATTGGAAAGTTATTGCCATTGATATTAACGATCCATTAGCCCCAAAATTGAACGACATTGAGGATGTTGAGAAATACTTCCCAGGTCTGTTGAGGGCTACTAACGAATGGTTCAGAATTTACAAAATCCCAGATGGTAAGCCAGAAAACCAATTTGCCTTCTCCGGTGAAGCTAAGAACAAGAAGTACGCTTTGGATATCATCAAGGAAACACATGACTCCTGGAAACAATTAATTGCTGGTAAGTCTTCTGACAGCAAGGGTATTGATTTGACCAATGTTACTTTGCCTGACACCCCAACCTACTCCAAGGCTGCCTCTGATGCCATCCCACCAGCTTCTCCAAAGGCAGATGCTCCAATTGACAAGTCTATTGACAAGTGGTTCTTCATCTCCGGTTCTGTTTAA.

[0146] Amino acid sequence of MfnK (SEQ ID NO. 6): MSETPARRVLVLAHTGRQDAIDAALQAIRILDEEGLDSVMPAADVAAVRAAVGEDPGFRPLSLGEDCALSDVSLGLVLGGDGSVLRAADLVRGHGIPLLAVNLGHVGFLAESERTDLHRTVKAIADEAYVVIERMALDVVVRVDGREAARTWALNEASVEKSNRERMLEVVVSVDDSPLTAFGCDGVVLGTPTGSTAYAFSAGGPVVWPGVEALLFVPISAHALFARPLVVGPHSTIAVDIMTRTRETGVLWCDGRRTVDLPPNARVEVTRSTEPVRLARLSPVPFADRLVRKFRLPTEGWRGPVTDEDVRAREATAVAEVETVEPRHAGPRPDVVPPPTSALPVLTPEDLERYRSRDGRGGDRTS*.

[0147]

[0148]

[0149] The nucleotide sequence (SEQ ID NO.9) of primer IPP1-F 5'-3' for amplifying the target fragment of IPP1 using Saccharomyces cerevisiae genomic DNA as a template is: ATGACCTACACTACCAGAC.

[0150] The nucleotide sequence (SEQ ID NO.10) of primer IPP1-R 5'-3' for amplifying the target fragment of IPP1 using Saccharomyces cerevisiae genomic DNA as a template is: TTAAACAGAACCGGAGATGAAG.

[0151] The nucleotide sequence of RBS (SEQ ID NO.11): GAAGGAGATATACAT.

[0152] The nucleotide sequence of primer pET28a-NMNAT-linear-F 5'-3' (SEQ ID NO.12): AAGCTTGGCGGCCGCACTCGAG.

[0153] The nucleotide sequence of primer pET28a-NMNAT-linear-R 5'-3' (SEQ ID NO.13): CAGTTTATCGCTTTCCAG.

[0154] The nucleotide sequence (SEQ ID NO.14) of primer IPP1-F 5'-3' for amplifying the target fragment of IPP1 using pEASY-Blunt E1-IPP1 plasmid as a template is: CTGGAAAGCGATAAACTGTGAGAAGGAGATATACATATGACCTACACTACCAG.

[0155] The nucleotide sequence (SEQ ID NO.15) of primer IPP1-R 5'-3' for amplifying the target fragment of IPP1 using pEASY-Blunt E1-IPP1 plasmid as a template is: GAGTGCGGCCGCAAGCTTATGTATATCTCCTTCTCAAACAGAACCGGAGATGAAG.

[0156] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0157] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0158] The seed culture medium was LB medium, with a formula of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl. The fermentation medium formula was: 10 g / L yeast extract, 5 g / L ammonium sulfate, 10 g / L NaCl, 2 g / L dipotassium hydrogen phosphate, and 1 g / L magnesium sulfate heptahydrate, sterilized at 121℃ for 20 min by moist heat. A 600 g / L glucose supplementation solution was prepared separately and sterilized at 115℃ for 20 min by moist heat.

[0159] Example 1: Preparation of enzymes for NADP production

[0160] The nucleotide sequence of nicotinamide nucleoside adenosine yltransferase (NMNAT) after codon optimization (for Escherichia coli) is shown in SEQ ID NO.3. The whole genome was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the pET28a vector through NdeⅠ and HindⅢ restriction sites. After successful ligation and correct sequencing, the recombinant plasmid pET28a-NMNAT was transformed into E. coli BL21(DE3) strain and verified by colony PCR, thus obtaining the NMNAT expression strain.

[0161] The inorganic pyrophosphatase (IPP1) nucleotide sequence is shown in SEQ ID NO. 5. Genomic DNA was extracted from *Saccharomyces cerevisiae* S288C strain and used as a template. Conventional PCR techniques were employed, using TaKaRa high-fidelity DNA polymerase (TAPR) to... The IPP1 gene fragment was amplified by PCR using Max DNA Polymerase. The primer sequences were as follows: IPP1-F 5'-3' (SEQ ID NO. 9) and IPP1-R 5'-3' (SEQ ID NO. 10). A full-gold assay kit was used to amplify the prokaryotic expression vector. The Blunt E1 Expression Kit ligates the IPP1 fragment into the pEASY-Blunt E1 vector. The ligation product is the pEASY-Blunt E1-IPP1 recombinant plasmid. After being transformed into a positive clone of E. coli BL21(DE3) and sequenced to confirm its correctness, the IPP1 expression strain is obtained.

[0162] The nucleotide sequence of ATP / polyphosphate-dependent NAD kinase (MfnK) after codon optimization (for E. coli) is shown in SEQ ID NO.8. The whole genome was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the pET28a vector through NdeⅠ and HindⅢ restriction sites. After successful ligation and correct sequencing, the recombinant plasmid pET28a-MfnK was transformed into E. coli BL21(DE3) strain and verified by colony PCR, thus obtaining the MfnK expression strain.

[0163] Construction of a nicotinamide nucleoside adenosine transferase (NMNAT) and inorganic pyrophosphatase (IPP1) co-expression strain: The IPP1 fragment was ligated to the pET28a-NMNAT recombinant plasmid via a ribosome binding site (RBS) sequence following the NMNAT stop codon sequence, forming the pET28a-NMNAT-IPP1 co-expression recombinant plasmid. The RBS nucleotide sequence connecting the NMNAT and IPP1 fragments is shown in SEQ ID NO. 11. The specific procedure involved using standard PCR techniques with TaKaRa high-fidelity DNA polymerase (...). Using Max DNA Polymerase, the recombinant plasmid pET28a-NMNAT was used as a template for PCR with upstream and downstream linearization primers to obtain a linearized vector. The upstream and downstream linearization primer sequences were as follows: pET28a-NMNAT-linear-F 5'-3' (SEQ ID NO. 12) and pET28a-NMNAT-linear-R 5'-3' (SEQ ID NO. 13). Using pEASY-Blunt E1-IPP1 plasmid as a template, the IPP1 target fragment was obtained by PCR with upstream and downstream primers for the IPP1 fragment. The upstream and downstream primer sequences for IPP1 were as follows: IPP1-F 5'-3' (SEQ ID NO. 14) and IPP1-R 5'-3' (SEQ ID NO. 15). The pET28a-NMNAT linearized vector and the IPP1 fragment were then cloned using the Beyotime Seamless Cloning Kit. The ligation product, pET28a-NMNAT-IPP1 co-expression recombinant plasmid, is obtained by ligation using a kit. The ligation product is transformed into E. coli BL21(DE3) strain and verified by sequencing to be correct, thus obtaining the NMNAT and IPP1 co-expression strain, hereinafter referred to as the NMNAT-IPP1 co-expression strain. NMNAT and IPP1 can be co-expressed in one strain through induced expression.

[0164] The above-mentioned expression strains were inoculated into seed culture medium under aseptic conditions and cultured to the logarithmic growth phase. Then, they were transferred to a 2L fermenter containing 1L of fermentation medium. During fermentation, the pH of the fermentation broth was controlled to 6.5-7.0 with ammonia water / ammonia gas. Sugar was added sequentially according to the fermentation progress to control the residual sugar level to no more than 5g / L. After 7 hours of culture, a final concentration of 0.1mM IPTG was added, and the mixture was induced at 30℃ for 15 hours. Fermentation was then terminated, and the wet weight was measured. The cells were collected by centrifugation or the fermentation broth was used directly for subsequent whole-cell catalytic synthesis of NADP.

[0165] Example 2: Preparation of NADP

[0166] Step 1 Synthesis: Using a 1000mL fermenter as the reaction vessel, add 6.75g of substrate NMN, 10.25g of ATP, 0.099g of MgSO4·7H2O, 0.034g of MnSO4·H2O, and 0.8mL of Triton X-100 to 250mL of deionized water. Start stirring at 300r / min to dissolve the substrate. Adjust the pH to 7.0 with 5M sodium hydroxide solution. Add 2.0g of wet cells of the NMNAT-PPA1 co-expression strain collected by centrifugation to the reaction solution. Add water to make up to 400mL. Control the reaction temperature at 25℃ and maintain the pH of the reaction system at 7.0. Start the first step of the timed reaction. Monitor the reaction progress with HPLC. After 1.0h of reaction, the NMN conversion rate is 99.2%, and the first step of the synthesis is ended.

[0167] Step 2 Synthesis: Adjust the pH of the Step 1 synthesis solution to 5.0 in situ with phosphate, add 2.0 g of wet cells of the MfnK expression strain, weigh 5.64 g of sodium tetrapolyphosphate and prepare a 1 M stock solution for later use. Control the reaction system temperature at 30℃, start adding the sodium tetrapolyphosphate stock solution. Add the sodium tetrapolyphosphate stock solution to the reaction system at a constant rate using a peristaltic pump, controlling the addition time to 1.0 h. Maintain the reaction system temperature and pH, start the timing for Step 2 synthesis, monitor the reaction progress with HPLC, and after 1.5 h of reaction, the NAD conversion rate is 99.4%. Step 2 synthesis ends, the NADP product concentration is 35.5 g / L, and the total NMN conversion rate is 98.6%.

[0168] Example 3: Preparation of NADP

[0169] Step 1 Synthesis: Using a 1000mL fermenter as the reaction vessel, add 40.51g of substrate NMN, 92.22g of ATP, 5.945g of MgSO4·7H2O, 2.704g of MnSO4·H2O, and 0.8mL of Triton X-100 to 250mL of deionized water. Start stirring at 300r / min to dissolve the substrate. Adjust the pH to 10.0 with 5M sodium hydroxide solution. Add 20.0g of wet cells of the NMNAT-PPA1 co-expression strain collected by centrifugation to the reaction solution. Add water to make up to 400mL. Control the reaction temperature at 42℃ and maintain the pH of the reaction system at 10.0. Start the first step of the timed reaction. Monitor the reaction progress with HPLC. After 5.0h of reaction, the NMN conversion rate is 98.1%, and the first step of the synthesis is ended.

[0170] Step 2 Synthesis: The pH of the synthesis solution from Step 1 was adjusted to 8.0 in situ with phosphoric acid. 20.0 g of wet cells of the MfnK expression strain was added. 84.57 g of sodium tetrapolyphosphate was weighed and prepared as a 1M stock solution for later use. The reaction system temperature was controlled at 60℃. The sodium tetrapolyphosphate stock solution was added to the reaction system at a constant rate using a peristaltic pump. The addition time was controlled at 3.0 h. The reaction system temperature and pH were maintained. The timing of Step 2 synthesis was started. The reaction process was monitored by HPLC. After 10.0 h of reaction, the NAD conversion rate was 98.2%. Step 2 synthesis was then terminated. The NADP product concentration was 148.1 g / L, and the total NMN conversion rate was 96.3%.

[0171] Example 4: Preparation of NADP

[0172] Step 1 Synthesis: Using a 1000mL fermenter as the reaction vessel, add 20.26g of substrate NMN, 33.81g of ATP, 0.495g of MgSO4·7H2O, 0.169g of MnSO4·H2O, and 0.8mL of Triton X-100 to 250mL of deionized water. Start stirring at 300r / min to dissolve the substrate. Adjust the pH to 7.5 with 5M sodium hydroxide solution. Add 31mL of NMNAT-PPA1 co-expression bacterial fermentation broth (fermentation broth cell wet weight 196g / L) to the reaction solution. Add water to make up to 400mL. Control the reaction temperature at 30℃ and maintain the pH of the reaction system at 7.5. Start the first step of the timed reaction. Monitor the reaction progress with HPLC. After 4.5h of reaction, the NMN conversion rate is 98.8%, and the first step of the synthesis is completed.

[0173] Step 2 Synthesis: The pH of the synthesis solution from Step 1 was adjusted to 5.5 in situ with phosphoric acid. 33 ml of MfnK expression bacteria fermentation broth (wet weight of fermentation broth cells 182 g / L) was added. 22.55 g of sodium tetrapolyphosphate was weighed and prepared as a 1 M stock solution for later use. The reaction system temperature was controlled at 37℃. The sodium tetrapolyphosphate stock solution was added to the reaction system at a constant rate using a peristaltic pump. The addition time was controlled at 3.0 h. The reaction system temperature and pH were maintained. The timing of Step 2 synthesis was started. The reaction process was monitored by HPLC. After 7.0 h of reaction, the NAD conversion rate was 98.5%. Step 2 synthesis was then terminated. The NADP product concentration was 90.2 g / L, and the total NMN conversion rate was 97.3%.

[0174] Example 5: Preparation of NADP

[0175] Step 1 Synthesis: Using a 1000mL fermenter as the reaction vessel, add 33.76g of substrate NMN, 66.60g of ATP, 3.963g of MgSO4·7H2O, 1.352g of MnSO4·H2O, and 0.8mL of Triton X-100 to 250mL of deionized water. Start stirring at 300r / min to dissolve the substrate. Adjust the pH to 9.0 with 5M sodium hydroxide solution. Add 82ml of NMNAT-PPA1 co-expression bacterial fermentation broth (fermentation broth cell wet weight 196g / L) to the reaction solution. Add water to make up to 400mL. Control the reaction temperature at 37℃ and maintain the pH of the reaction system at 9.0. Start the first step of the timed reaction. Monitor the reaction progress with HPLC. After 4.0h of reaction, the NMN conversion rate is 98.1%, and the first step of the synthesis is completed.

[0176] Step 2 Synthesis: The pH of the Step 1 synthesis solution was adjusted to 7.0 in situ with phosphoric acid. 88 ml of MfnK expression bacteria fermentation broth (fermentation broth wet weight 182 g / L) was added. 51.68 g of sodium tetrapolyphosphate was weighed and prepared as a 1 M stock solution for later use. The reaction system temperature was controlled at 55℃. The sodium tetrapolyphosphate stock solution was added to the reaction system at a constant rate using a peristaltic pump. The addition time was controlled at 5.0 h. The reaction system temperature and pH were maintained. Step 2 synthesis was started. The reaction process was monitored by HPLC. After 8.0 h of reaction, the NAD conversion rate was 98.4%. Step 2 synthesis was ended. The NADP product concentration was 120.0 g / L, and the total NMN conversion rate was 96.5%.

[0177] Example 6: Preparation of NADP

[0178] Step 1 Synthesis: Using a 1000mL fermenter as the reaction vessel, add 27.00g of substrate NMN, 49.18g of ATP, 1.982g of MgSO4·7H2O, 0.676g of MnSO4·H2O, and 0.8mL of Triton X-100 to 250mL of deionized water. Start stirring at 300r / min to dissolve the substrate. Adjust the pH to 8.0 with 5M sodium hydroxide solution. Add 51ml of NMNAT-PPA1 co-expression bacterial fermentation broth (196g / L wet weight of fermentation broth cells) to the reaction solution. Add water to make up to 400mL. Control the reaction temperature at 37℃ and maintain the pH of the reaction system at 8.0. Start the first step of the timed reaction. Monitor the reaction progress with HPLC. After 3.0h of reaction, the NMN conversion rate is 99.4%, and the first step of the synthesis is completed.

[0179] Step 2 Synthesis: The pH of the synthesis solution from Step 1 was adjusted to 6.0 in situ with phosphoric acid. 55 ml of MfnK expression bacteria fermentation broth (wet weight of fermentation broth cells 182 g / L) was added. 37.59 g of sodium tetrapolyphosphate was weighed and prepared as a 1 M stock solution for later use. The reaction system temperature was controlled at 45℃. The sodium tetrapolyphosphate stock solution was added to the reaction system at a constant rate using a peristaltic pump. The addition time was controlled at 4 h. The reaction system temperature and pH were maintained. The timing of Step 2 synthesis was started. The reaction process was monitored by HPLC. After 6.0 h of reaction, the NAD conversion rate was 99.3%. Step 2 synthesis was then completed. The NADP product concentration was 109.61 g / L, and the total NMN conversion rate was 98.7%.

Claims

1. A method for the enzymatic preparation of nicotinamide adenine dinucleotide phosphate, characterized in that, The method includes the following steps: Step 1: In reaction system I, using nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) or ATP salt as substrates, nicotinamide nucleoside adenosine yltransferase (NMNAT) catalyzes the generation of nicotinamide adenine dinucleotide (NAD) and pyrophosphate; the pyrophosphate is then hydrolyzed under the catalysis of inorganic pyrophosphatase (IPP1) to promote the forward reaction. Step 2: In reaction system II, the nicotinamide adenine dinucleotide generated in step 1 is converted into nicotinamide adenine dinucleotide phosphate (NADP) with the participation of a phosphoryl donor and under the catalysis of ATP / polyphosphate-dependent NAD kinase (MfnK). The amino acid sequence of the nicotinamide nucleoside adenosyltransferase is shown in SEQ ID NO.1; the amino acid sequence of the inorganic pyrophosphatase is shown in SEQ ID NO.4; and the amino acid sequence of the ATP / polyphosphate-dependent NAD kinase is shown in SEQ ID NO.

6. The nicotinamide nucleoside adenosyltransferase, the inorganic pyrophosphatase, and the ATP / polyphosphate-dependent NAD kinase participate in the reaction in the form of host cells expressing them, wherein the encoding nucleotide sequences of nicotinamide nucleoside adenosyltransferase and inorganic pyrophosphatase are ligated into the same expression vector and introduced into host cells for co-expression. In the reaction system I, the initial concentration of the nicotinamide mononucleotide is 50 mM to 400 mM.

2. The method according to claim 1, characterized in that, In reaction system I or reaction system II, the wet weight concentrations of the host cells expressing the nicotinamide nucleoside adenosine transferase, the host cells expressing the inorganic pyrophosphatase, and the host cells expressing the ATP / polyphosphate-dependent NAD kinase are independently 5 g / L to 50 g / L.

3. The method according to claim 1, characterized in that, The first and second reactions are carried out in a reaction medium, which is a phosphate buffer solution, a Tris-HCl buffer solution, or pure water.

4. The method according to claim 3, characterized in that, The concentration of the phosphate buffer solution is 20 mM to 100 mM.

5. The method according to claim 1, characterized in that, In the reaction system I, the initial molar ratio of the nicotinamide mononucleotide to adenosine triphosphate or adenosine triphosphate salt is 1:1 to 1:1.

5.

6. The method according to claim 1, characterized in that, The reaction system I also contains divalent metal ions.

7. The method according to claim 6, characterized in that, The divalent metal ions include magnesium ions and / or manganese ions.

8. The method according to claim 7, characterized in that, In the reaction system I, the concentration of magnesium ions is 1 mM to 60 mM, and the concentration of manganese ions is 0.5 mM to 40 mM.

9. The method according to claim 1, characterized in that, The reaction system I also contains Triton X-100.

10. The method according to claim 9, characterized in that, In the reaction system I, the concentration of Triton X-100 is 0.1% (V / V) to 0.2% (V / V).

11. The method according to claim 1, characterized in that, The pH of the reaction system I is 7.0~10.0, the reaction temperature of the first step reaction is 25℃~42℃, and the reaction time of the first step reaction is 1.0h~6.0h.

12. The method according to claim 1, characterized in that, In the reaction system II, the phosphoryl group donor is an inorganic phosphate.

13. The method according to claim 1, characterized in that, In the reaction system II, the phosphoryl group donor is a polyphosphate or a polyphosphate.

14. The method according to claim 1, characterized in that, Based on the initial amount of the nicotinamide mononucleotide in reaction system I, the molar ratio of the nicotinamide mononucleotide to the phosphoryl donor in reaction system II is 1:0.6 to 1:1.

5.

15. The method according to claim 1, characterized in that, In the second step of the reaction, the phosphoryl donor is added to the reaction system by liquid addition, and the addition time is controlled to be 1.0 h to 8.0 h.

16. The method according to claim 1, characterized in that, The pH of reaction system II is 5.0~8.0, the reaction temperature of the second step reaction is 30℃~60℃, and the reaction time of the second step reaction is 1.5h~15.0h.

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