The present invention relates to a novel recombinant microorganism for producing a nicotinamide derivative (NAm derivative) such as nicotinamide mononucleotide (NMN), and a novel production method for producing a NAm derivative, and is used for these. Regarding new vectors to be produced.
Background technology
[0002]
Nicotinamide adenine dinucleotide (NAD) is a nucleotide derived from ribose and nicotinamide. It is known that NAD functions as a coenzyme in various redox reactions in the living body and plays a central role in aerobic respiration (oxidative phosphorylation). In vivo, NAD can take on two states: oxidized (NAD +) and reduced (NADH). In the present specification, the term "NAD" shall comprehensively refer to both the oxidized form (NAD +) and the reduced form (NADH) unless otherwise specified.
[0003]
There are multiple biosynthetic pathways for NAD, but in mammalian cells, the main pathway is to use nicotinamide (NAm) as a starting material and then synthesize NAD through a two-step enzymatic reaction. In the first step, NAm taken up into cells is nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT: NMN synthase) in the presence of 5-phosphoribosyl-1-pyrrolinic acid (PRPP). And converted to pyrophosphate (P-Pi). In the subsequent second step, the NMN obtained in the previous step is converted to NAD by nicotinamide / nicotinic acid mononucleotide adenylyl transferase (NMNAT) in the presence of adenosine triphosphate (ATP).
[0004]
Here, it is known that NMN, which is a precursor of NAD in the above biosynthetic pathway, has various functions such as activation of mitochondria and activation of sirtuin gene, which is a so-called longevity gene. Especially in the living body, it is considered that NAD also decreases due to the decrease in NMN production ability with aging, the mitochondrial activity decreases, and the cell nucleus damage progresses. Furthermore, it has been reported that NMN is involved in aging-related diseases such as insulin resistance, diabetes, cancer and Alzheimer's disease. Therefore, NMN is attracting attention as various research tools, synthetic intermediates of NAD, and medicinal ingredients.
[0005]
Further, NMN can be used not only as a synthetic intermediate for NAD but also as a synthetic intermediate for various nicotinamide derivatives (NAm derivatives) such as nicotinamide riboside (NR) and nicotinic acid mononucleotide (NaMN). It is expected.
[0006]
[Chemical 1]

[0007]
Conventionally, as a method for synthesizing NMN, a method by organic synthesis, a method by decomposition of NAD, a synthetic biological method using a microorganism, and the like are known.
[0008]
The method by organic synthesis is a method of synthesizing NMN from D-ribose through several steps (Patent Document 1: US Patent Application Publication No. 2018/0291054). However, in this method, since several steps of synthesis steps are required, synthesis is time-consuming and costly.
[0009]
The method by decomposing NAD is a method for obtaining NMN by directly decomposing NAD biosynthesized in yeast with an enzyme without isolating it (Patent Document 2: International Publication No. 2017/200050). However, in this method, the productivity of NMN per cell is very poor.
[0010]
Synthetic biological techniques using microorganisms are the first step of the major biosynthetic system of NAD in mammals, namely, from NAm and PRPP to NMN by NAMPT by gene recombination of host microorganisms such as Escherichia coli. This is a method for constructing a recombinant microorganism that expresses an enzyme similar to the enzyme that catalyzes conversion (NAMPT: NMN synthase), and synthesizing NMN using the obtained recombinant microorganism (Patent Document 3: International Publication No. 1). 2015/069860; Non-Patent Document 1: Mariescu et al., Scientific reports, August 16, 2018, Vol.8, No.1, pp.12278). However, in this method, it takes a long time to produce NMN, and the amount of NMN obtained is small, so that practical productivity is not obtained.
Prior art literature
Patent documents
[0011]
Patent Document 1: US Patent Application Publication No. 2018/0291054
Patent Document 2: International Publication No. 2017/200050
Patent Document 3: International Publication No. 2015/069860
Non-patent literature
[0012]
Non-Patent Document 1: Mariescu et al., Scientific reports, August 16, 2018, Vol.8, No.1, pp.12278
Outline of the invention
Problems to be solved by the invention
[0013]
From the above background, it is required to efficiently synthesize a nicotinamide derivative (NAm derivative) such as nicotinamide mononucleotide (NMN).
Means to solve problems
[0014]
In view of the above problems, the present inventors have verified a conventional synthetic biological production system of NMN using a microorganism, and as a result of diligent studies to improve it, an enzyme (NAMPT) that is a key to the biosynthesis of NMN. As a result, it was found that the production efficiency of NMN is remarkably improved by recombinantly introducing a specific enzyme having excellent activity into a host microorganism and expressing it to enhance the synthesis efficiency of NMN. In addition, a protein (niacin transporter) that promotes the uptake of NAm, which is a reaction product of NMN synthesis, and NA, which is a derivative thereof (NAm and NA may be collectively referred to as "niacin") into host microbial cells. ) And / or, as a protein (NAm derivative transporter) that promotes the excretion of NAm derivatives such as NMN from host microbial cells, a specific protein having excellent activity is recombinantly introduced into the host microorganism and expressed. , It was found that the production efficiency of NMN is further improved by enhancing the efficiency of uptake of NAm into host microbial cells. In addition, as one or two enzymes in a series of enzymes constituting the biosynthesis system of PRPP, which is another reaction product of NMN synthesis, a specific enzyme having excellent activity is recombinantly introduced into a host microorganism. It was found that the production efficiency of NMN is further improved by expressing it and enhancing the synthesis efficiency of PRPP. In addition, it has been found that if such an NMN system is used, not only NMN but also other NAm derivatives such as NAD, NR, and NaMN can be produced with high efficiency. Various inventions have been made based on these new findings.
[0015]
That is, the present invention relates to the following.
[1] A microorganism for producing a nicotinamide derivative.
The microorganism expresses a nicotinamide phosphoribosyltransferase (NAMPT) that produces a nicotinamide mononucleotide from nicotinamide and 5-phosphoribosyl-1-pyrrolinic acid, and / or carries a nucleic acid encoding the amino acid sequence of the NAMPT. As well as being transformed by the vector
A recombinant microorganism having a conversion efficiency of nicotinamide to nicotinamide mononucleotide by NAME is 5 times or more that of human NAPPT.
[2] The recombinant microorganism of [1], wherein the NAMPT comprises a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6.
[3] The microorganism further expresses a niacin transporter that promotes intracellular uptake of nicotinic acid and / or nicotinamide, and / or is characterized by a vector carrying a nucleic acid encoding the amino acid sequence of the niacin transporter. As well as being converted
The recombinant microorganism of [1] or [2], wherein the niacin transporter increases the intracellular uptake efficiency of nicotinic acid and / or nicotinamide by the host microorganism by 1.1 times or more.
[4] The recombinant microorganism of [3], wherein the niacin transporter comprises a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 12.
[5] The microorganism further expresses a nicotinamide derivative transporter that promotes extracellular excretion of the nicotinamide derivative, and / or is characterized by a vector carrying a nucleic acid encoding the amino acid sequence of the nicotinamide derivative transporter. As well as being converted
The recombinant microorganism of [1] to [4], wherein the nicotinamide derivative transporter increases the extracellular excretion efficiency of the nicotinamide derivative by the host microorganism by a factor of 3 or more.
[6] The recombinant microorganism of [5], wherein the nicotinamide derivative transporter comprises a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 15.
[7] The microorganism further expresses one or two or more enzymes that promote the pathway for synthesizing 5-phosphoribosyl-1-pyrrophosphate from glucose-6-phosphate and / or the one or more. The recombinant microorganisms of [1] to [6], which are further transformed with a vector carrying a nucleic acid encoding the amino acid sequence of the enzyme of.
[8] The one or more enzymes are phosphoglucose isomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconolacate dehydrogenase, ribose-5-phosphate isomerase, and phosphoribosyl. The recombinant microorganism of [7], which is one or more enzymes selected from the group consisting of pyrophosphate synthase.
[9] The phosphoglucose isomerase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 18.
The glucose-6-phosphate dehydrogenase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 21.
The 6-phosphogluconolactonase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 24.
The 6-phosphogluconate dehydrogenase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 27.
The ribose-5-phosphate isomerase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 30 or 33.
The recombinant microorganism of [8], wherein the phosphoribosylpyrrophosphate synthase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 36.
[10] The nicotinamide derivative is selected from the group consisting of nicotinamide mononucleotide, nicotinamide adenine dinucleotide, nicotinamide riboside, nicotinic acid mononucleotide, nicotinamide adenine dinucleotide phosphate, and nicotinic acid adenine dinucleotide. The recombinant microorganisms of [1] to [9].
[11] The recombinant microorganisms of [1] to [10], wherein the recombinant microorganism is Escherichia coli or yeast.
[12] A method for producing a nicotinamide derivative, which comprises supplying nicotinamide to the recombinant microorganisms [1] to [11] and recovering the nicotinamide derivative produced by the microorganism. ..
[13] The method of [12] further comprising purifying the recovered nicotinamide derivative.
[14] A vector carrying a nucleic acid encoding the amino acid sequence of nicotinamide phosphoribosyltransferase (NAMPT), which produces a nicotinamide mononucleotide from nicotinamide and 5-phosphoribosyl-1-pyrrophosphate, wherein the nucleic acid is a sequence. A vector containing a base sequence having 80% or more sequence identity with the base sequence set forth in No. 2 or SEQ ID NO: 5.
[15] A vector carrying a nucleic acid encoding an amino acid sequence of a niacin transporter that promotes intracellular uptake of nicotinic acid and / or nicotinamide, wherein the nucleic acid is the base set forth in SEQ ID NO: 8 or SEQ ID NO: 11. A vector containing a base sequence having 80% or more sequence identity with the sequence.
[16] A vector carrying a nucleic acid encoding an amino acid sequence of a nicotine amide derivative transporter that promotes extracellular excretion of a nicotine amide derivative, wherein the nucleic acid is 80% or more of the base sequence set forth in SEQ ID NO: 14. Nucleic acid containing a base sequence having sequence identity Tar.
The invention's effect
[0016]
According to the present invention, it is possible to efficiently synthesize NAm derivatives such as NMN.
A brief description of the drawing
[0017]
FIG. 1 is a diagram schematically showing an example of a synthetic biological production system for a NAm derivative according to the present invention.
Embodiment for carrying out the invention
[0018]
Hereinafter, the present invention will be described in detail according to a specific embodiment. However, the present invention is not bound by the embodiments disclosed below, and can be implemented in any embodiment as long as the gist of the present invention is not deviated.
[0019]
The patent gazettes, patent application publication gazettes, and non-patentable documents cited in this disclosure shall be incorporated into this disclosure for all purposes by reference in their entirety.
[0020]
Further, in the present disclosure, "nucleic acid" includes ribonucleic acid, deoxyribonucleic acid, or a modified form of any nucleic acid, and also includes either single-stranded or double-stranded. Further, the nucleic acid (gene) in the present disclosure is an arbitrary method known to those skilled in the art using a database of a public institution known to those skilled in the art or a primer or probe prepared based on the base sequence disclosed in the present specification. Can be prepared with. For example, it can be easily obtained as a cDNA of the gene by using various PCR or other DNA amplification techniques known to those skilled in the art. Alternatively, those skilled in the art can synthesize nucleic acids using existing techniques as appropriate based on the sequence information disclosed herein.
[0021]
Further, in the present disclosure, "encoding" a protein or polypeptide by a nucleic acid or gene means expressing such a protein or polypeptide in a state having its activity. Further, "encoding" in the present disclosure includes both encoding the protein according to the present invention as a continuous structural sequence (exon) and encoding the protein via an appropriate intervening sequence (intron). included.
[0022]
In addition, in the present disclosure, genetic engineering techniques such as cloning of nucleic acids or genes, design and preparation of vectors, transformation into cells, expression of proteins or polypeptides are described, for example, in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, etc. can be referred to.
[0023]
[I. Overview]
First, the outline of the present invention will be described.
[0024]
As described above, as an attempt to synthetically produce nicotinamide mononucleotide (NMN) using a microorganism, the nicotinamide adenine dinucleotide in the above-mentioned mammal is obtained by gene recombination of a host microorganism such as Escherichia coli. A method has been proposed in which a recombinant microorganism expressing an enzyme similar to that of the main biosynthetic system of NAD) is constructed, and NMN is synthesized using the obtained recombinant microorganism (Patent Document 3: International Publication No. 2015 /). No. 069860; Non-Patent Document 1: Mariescu et al., Scientific reports, August 16, 2018, Vol.8, No.1, pp.12278). However, such a conventional method has a problem that it takes a long time to produce NMN and the amount of NMN obtained is low, so that practical productivity cannot be obtained.
[0025]
The present invention is a NAm derivative synthesis system based on a conventional NMN synthetic biological production system using a microorganism, and is a microorganism that uses various enzymes and / or transporter proteins involved in the synthesis and / or transport of NAm derivatives. By introducing and expressing it in NMN, the production efficiency of NAm derivatives such as NMN is improved.
[0026]
An example of a synthetic biological production system for a NAm derivative according to one aspect of the present invention will be described more specifically with reference to FIG. However, FIG. 1 is merely an example, and the present invention is not limited to the synthetic system shown in FIG.
[0027]
The synthetic biological NAm derivative synthesis system shown in FIG. 1 contains a NAm derivative synthesis system consisting of a series of enzyme groups in a host microbial cell. As shown in FIG. 1, the main reaction pathway of the NMN synthesis system is a reaction pathway that converts NAm and PRPP into NMN and P-Pi.
[0028]
NAm, which is one of the main reaction pathways of the NAm derivative synthesis system, is taken up into the cell from the outside of the host microorganism. Similarly, NA and NAm taken up from outside the cell of the host microorganism into the cell are mutually converted by nicotine amidase.
[0029]
PRPP, which is the other reaction product of the main reaction pathway of the NAm derivative synthesis system, is synthesized from glucose (Glu) taken into the cell from the outside of the host microorganism via the following series of reaction pathways.
-Phosphorylation of glucose (Glu) to glucose-6-phosphate (G6P) by hexokinase (HK).
-Conversion of fructose-6-phosphate (F6P) to glucose-6-phosphate (G6P) by phosphoglucose isomerase (PGI).
-Conversion of G6P to 6-phosphoglucono-1,5-lactone (6PGL) by glucose-6-phosphate dehydrogenase (GPD).
-Conversion of 6PGL to 6-phosphogluconolacic acid (6PG) by 6-phosphogluconolactonase (PGL).
-Conversion of 6PG to ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (PGD).
-Conversion of Ru5P to ribose-5-phosphate (R5P) by ribose-5-phosphate isomerase (RPI).
-Conversion of R5P to 5-phosphoribosyl-1-pyrrolinic acid (RRPP) by phosphoribosylpyrophosphate synthase (PRS).
[0030]
The NMN produced by the above main reaction is converted to NAD by NMDAT, to nicotinamide mononucleotide (NaMN) by nicotinamide nucleotide amidase (NANA), and nicotinamide riboside by nicotinamide mononucleotide-5-nucleotidase (NMNN). It is converted to each and is appropriately excreted to the outside of the cell.
[0031]
According to one aspect of the present invention, as a key enzyme for biosynthesis of NMN (NAMPT: NMN synthase), a specific enzyme having excellent activity is recombinantly introduced into a host microorganism and expressed to synthesize NMN. By enhancing the efficiency, the production efficiency of the NAm derivative is improved.
[0032]
According to a preferred embodiment of the present invention, the NAm derivative synthesis system has excellent activity as a transport protein (niacin transporter) that promotes the uptake of NAm and / or NA (niacin) into host microbial cells. The specific protein possessed by the host microorganism is recombinantly introduced into the host microorganism and expressed to enhance the efficiency of uptake of niacin into the host microorganism cells, thereby further improving the production efficiency of the NAm derivative.
[0033]
According to another preferred embodiment of the present invention, a series of enzyme groups (GPI, GPD, PGL, etc.) constituting the biosynthesis system of PRPP, which is another reaction product of NMN synthesis, with respect to the NAm derivative synthesis system. Production of NAm derivatives by recombinantly introducing and expressing a specific enzyme having excellent activity as one or two enzymes of PGD, RPI, and PRS) into a host microorganism to enhance the synthesis efficiency of PRPP. Further improve efficiency.
[0034]
According to another preferred embodiment of the present invention, a specific activity as a transporter protein (NAm derivative transporter) that promotes excretion of a NAm derivative from a host microbial cell is excellent with respect to the NAm derivative synthesis system. By recombinantly introducing the protein into the host microorganism and expressing it, and enhancing the excretion efficiency of the produced NAm derivative from the host microbial cells, the production efficiency of the NAm derivative is further improved.
[0035]
In particular, in the present invention, for the NAm derivative synthesis system, the specific NAMPT (NMN synthase), niacin transporter, NAm derivative transporter, and PRPP synthase (GPI, GPD, PGL, PGD, RPI, and By introducing a plurality, preferably all of the PRS) into the recombinant microorganism, the production efficiency of the final NAm derivative will be significantly improved.
[0036]
The NAm derivative produced by the synthetic system of the present invention is not limited to these, but in addition to NMN, NAD, nicotinamide riboside (NR), nicotinamide mononucleotide (NaMN), and nicotinamide adeninedi. Nucleotide phosphate (NADP), nicotinamide adenine dinucleotide and the like can be mentioned.
[0037]
In the following chapters, the case of synthesizing NMN mainly by the synthetic system of the present invention will be described, but the case of synthesizing NAm derivatives other than NMN will also be briefly described below.
[0038]
For example, the synthesis of NAD by the synthetic system of the present invention is a nicotinamide / nicotinic acid mononucleotide adenylyl transferase that further converts NMN into NAD for a host microorganism incorporating a series of genes for NMN synthesis. NMDAT) can be achieved by recombinantly introducing and expressing it by the same procedure as described above, and enhancing the conversion efficiency from NMN to NAD.
[0039]
Further, in the synthesis of NADP by the synthetic system of the present invention, the above-mentioned NMNAT and NAD + kinase that converts NAD into NADP are further applied to the host microorganism incorporating a series of genes for NMN synthesis. It can be achieved by recombinantly introducing and expressing by the same procedure as in the above, and enhancing the conversion efficiency from NMN to NAD and the conversion efficiency from NAD to NADP.
[0040]
In addition, the synthesis of NR by the synthetic system of the present invention is a nicotinamide mononucleotide-5-nucleotidase (NMNN) that further converts NMN to NR for a host microorganism incorporating a series of genes for NMN synthesis. Can be achieved by recombinantly introducing and expressing by the same procedure as described above, and enhancing the conversion efficiency from NMN to NR.
[0041]
Further, in the synthesis of Namn by the synthetic system of the present invention, nicotinamide nucleotide amidase (NANA), which further converts NMN to Namn, is applied to the host microorganism incorporating a series of genes for NMN synthesis in the same manner as described above. It can be achieved by recombinantly introducing and expressing it according to the procedure of the above procedure to enhance the conversion efficiency from NMN to NaMN.
[0042]
According to the method using the NAm derivative synthesis system of the present invention having the above configuration, the production efficiency of NAm derivatives such as NMN is significantly improved as compared with the conventional synthetic biological method for producing NMN. Can be done. For example, according to the results shown in Examples described later, the method using the NAm derivative synthesis system of the present invention has an amount of NMN more than 10 times that of the production amount of NMN by the conventional synthetic biological method. Can be seen to be produced. Further, in the method using the synthesis system of NAm derivative of the present invention, by-products are reduced and the selectivity of NAm derivative such as NMN is also excellent.
[0043]
[II. enzyme]
Next, the enzymes involved in the production of the NAm derivative used in the present invention will be described.
[0044]
In the present disclosure, the "homogeneity" of two amino acid sequences is the ratio at which the same or similar amino acid residues appear at each corresponding portion when both amino acid sequences are aligned, and the "sameness" of the two amino acid sequences. "Sex" is the ratio at which the same amino acid residue appears at each corresponding site when both amino acid sequences are aligned.
[0045]
The "homology" and "identity" of the two amino acid sequences are, for example, the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably. Is version 5.00 or later) Needl The e-program can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453).
[0046]
Examples of similar amino acids include amino acids belonging to the same group in the classification based on the structure, characteristics, type of side chain, etc. described below.
Aromatic amino acids: F, H, W, Y;
Aliphatic amino acids: I, L, V;
Hydrophobic amino acids: A, C, F, H, I, K, L, M, T, V, W, Y;
Charged amino acids: D, E, H, K, R, etc .:
Positively charged amino acids: H, K, R;
Load-bearing amino acids: D, E;
Polar amino acids: C, D, E, H, K, N, Q, R, S, T, W, Y;
Small amino acids: A, C, D, G, N, P, S, T, V, etc .:
Ultra-small amino acids: A, C, G, S;
Amino acids with aliphatic side chains: G, A, V, L, I;
Amino acids with aromatic side chains: F, Y, W;
Amino acids with sulfur-containing side chains: C, M;
Amino acids with aliphatic hydroxyl side chains: S, T;
Amino acids with basic side chains: K, R, H;
Acidic amino acids and their amide derivatives: D, E, N, Q.
[0047]
(1) Enzyme that catalyzes the synthesis of NMN from NAm and PRPP (NMN synthase):
As the NMN synthase (NAMPT) that catalyzes the synthesis of NMN from NAm and PRPP, various enzymes derived from various microorganisms are conventionally known. As a result, such a known enzyme can be appropriately selected and used.
[0048]
In the present invention, it is preferable to use a specific enzyme having excellent activity as an NMN synthase (NAMPT). This is because low activity of NAMPT reduces the selectivity of NMN production from the substrates NAm and PRPP. In addition, it takes time to produce NMN, and the production amount may decrease due to the influence of decomposition and conversion of NMN. Such a highly active NMN enzyme may be appropriately referred to as "the NMN synthase of the present invention" below. However, the NMN synthase (NAMPT) that can be used in the present invention is not limited to the following.
[0049]
Specifically, in the NMN synthase (NAMPT) of the present invention, the conversion efficiency from NAm to NMN (NAMPT conversion efficiency) is usually 5 times or more, particularly 7 times or more, as compared with the NAMET conversion efficiency of human NAMPT. Further, it is preferably 9 times or more. As shown in the column of [Examples] described later, according to the study by the present inventors, the cells expressing NAMPT having a conversion efficiency of 2 times or 3 times are the cells expressing human NAMPT. Similarly, the production of NMN was not confirmed, but in the cells expressing NAMPT having a conversion efficiency of 6 times, a significant increase in the production of NMN was confirmed as compared with the cells expressing human NAMPT.
[0050]
Specific examples of the method for measuring the NAMPT conversion efficiency of NAMPT include the following methods. That is, a plasmid in which a gene for expressing NAMPT is inserted into pRSFDuet-1 is transformed into an Escherichia coli (hereinafter, appropriately referred to as "E. Coli") BL21 (DE3) strain to prepare a construct. Then, inoculate the cells into a test tube containing 5 mL of LB medium and incubate at 37 ° C. at 200 rpm for 12 hours, and then put the culture solution in a 500 mL triangular flask containing 200 mL of LB medium so that the OD600 becomes 0.03. Inoculate and incubate at 37 ° C. and 200 rpm, and when OD600 reaches 0.4, add isopropyl-β-thiogalactopyranoside to a final concentration of 0.1 mM and at 25 ° C. and 200 rpm. Incubate for 16 hours. Transfer 30 mL of the culture solution to a 50 mL conical tube, centrifuge at 3000 g for 5 minutes, and collect the cells. Further, 1 × PBS is added, the cells are washed, and the cells are centrifuged at 3000 g for 5 minutes to collect the cells. This operation is performed twice. The recovered cells are suspended in 15 mL of Cell Lysis Buffer (MBL), and a cell decomposition solution (Lysate) is prepared by a generally recommended method. The OD595 of the bacterial cell decomposition solution is measured using a protein assay Bradford reagent (manufactured by Wako Pure Chemical Industries, Ltd.), and the bacterial cell decomposition solution is diluted with water so that the OD595 becomes 0.1. Using this diluted solution as a NAMPT solution, CycLexR NAMPT Colorimetric Assay Kit Ver. NAMPT activity is measured according to 2 (MBL) One-Step Assay Method. For the measurement, a SpectraMaxR iD3 multimode microplate reader (manufactured by Molecular Device Co., Ltd.) or the like can be used. In the present invention, the absorbance obtained by measuring the absorbance at 450 nm at 30 ° C. every 5 minutes for up to 60 minutes and selecting the absorbance at three points having the maximum slope is defined as the NAMET conversion efficiency.

The scope of the claims
[Claim 1]
A microorganism for producing nicotinamide derivatives,
The microorganism expresses a nicotinamide phosphoribosyltransferase (NAMPT) that produces a nicotinamide mononucleotide from nicotinamide and 5-phosphoribosyl-1-pyrrolinic acid, and / or carries a nucleic acid encoding the amino acid sequence of the NAMPT. As well as being transformed by the vector
A recombinant microorganism having a conversion efficiency of nicotinamide to nicotinamide mononucleotide by NAME is 5 times or more that of human NAPPT.
[Claim 2]
The recombinant microorganism according to claim 1, wherein the NAMPT comprises a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6.
[Claim 3]
The microorganism is further transformed with a vector carrying a nucleic acid encoding a niacin transporter that promotes intracellular uptake of nicotinic acid and / or nicotinamide and / or the amino acid sequence of the niacin transporter. At the same time
The recombinant microorganism according to claim 1 or 2, wherein the niacin transporter increases the intracellular uptake efficiency of nicotinic acid and / or nicotinamide by a host microorganism by 1.1 times or more.
[Claim 4]
The recombinant microorganism according to claim 3, wherein the niacin transporter comprises a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 12.
[Claim 5]
The microorganism is further transformed with a vector carrying a nucleic acid encoding a nicotinamide derivative transporter that promotes extracellular excretion of the nicotinamide derivative and / or the amino acid sequence of the nicotinamide derivative transporter. At the same time
The recombinant microorganism according to any one of claims 1 to 4, wherein the nicotinamide derivative transporter increases the extracellular excretion efficiency of the nicotinamide derivative by a host microorganism by a factor of 3 or more.
[Claim 6]
The recombinant microorganism according to claim 5, wherein the nicotinamide derivative transporter comprises a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 15.
[Claim 7]
The microorganism further expresses one or more enzymes that promote the pathway for synthesizing 5-phosphoribosyl-1-pyrrolic acid from glucose-6-phosphate and / or the one or more enzymes. The recombinant microorganism according to any one of claims 1 to 6, further transformed with a vector carrying a nucleic acid encoding an amino acid sequence.
[Claim 8]
The one or more enzymes are phosphoglucose isomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconolate dehydrogenase, ribose-5-phosphate isomerase, and phosphoribosylpyrrophosphate synthase. The recombinant microorganism according to claim 7, which is one or more enzymes selected from the group consisting of.
[Claim 9]
The phosphoglucose isomerase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 18.
The glucose-6-phosphate dehydrogenase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 21.
The 6-phosphogluconolactonase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 24.
The 6-phosphogluconate dehydrogenase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 27.
The ribose-5-phosphate isomerase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 30 or 33.
The recombinant microorganism according to claim 8, wherein the phosphoribosylpyrrophosphate synthase is a polypeptide having 80% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 36.
[Claim 10]
The nicotinamide derivative is selected from the group consisting of nicotinamide mononucleotide, nicotinamide adenine dinucleotide, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide phosphate, and nicotinamide adenine dinucleotide. The recombinant microorganism according to any one of claims 1 to 9.
[Claim 11]
The recombinant microorganism according to any one of claims 1 to 10, wherein the recombinant microorganism is Escherichia coli or yeast.
[Claim 12]
A method for producing a nicotinamide derivative, wherein nicotinamide is supplied to the recombinant microorganism according to any one of claims 1 to 11 and the nicotinamide derivative produced by the microorganism is recovered. How to include.
[Claim 13]
The method according to claim 12, further comprising purifying the recovered nicotinamide derivative.
[Claim 14]
A vector carrying a nucleic acid encoding the amino acid sequence of nicotinamide phosphoribosyl transferase (NAMPT), which produces a nicotinamide mononucleotide from nicotinamide and 5-phosphoribosyl-1-pyrrophosphate, wherein the nucleic acid is SEQ ID NO: 2 or A vector containing a base sequence having 80% or more sequence identity with the base sequence set forth in SEQ ID NO: 5.
[Claim 15]
A vector carrying a nucleic acid encoding an amino acid sequence of a niacin transporter that promotes intracellular uptake of nicotinic acid and / or nicotinamide, wherein the nucleic acid is the base sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 11 and 80. A vector containing a base sequence having a sequence identity of% or more.
[Claim 16]
A vector carrying a nucleic acid encoding an amino acid sequence of a nicotine amide derivative transporter that promotes extracellular excretion of a nicotine amide derivative, wherein the nucleic acid has 80% or more sequence identity with the base sequence set forth in SEQ ID NO: 14. A vector containing a base sequence having.