Nicotinamide riboside transporter mutant

Abstract

The present invention addresses the problem of providing a nicotinamide mononucleotide (NMN) transport protein that can efficiently recover NMN that has been synthesized using a fermentative production method from an extracellular environment.　Provided is a PnuC mutant that has a mutation (an 81A mutation) that replaces the amino acid at the position that corresponds to the 81st position in the amino acid sequence of SEQ ID NO:1 with alanine in PnuC derived from Bacillus mycoides or an ortholog protein from another species of genus Bacillus or Paenibacillus (including different strains of the same species).

Description

Nicotinamide riboside transporter mutants

[0001] The present invention relates to a nicotinamide riboside transporter PnuC mutant, a polynucleotide encoding the same, and a method for producing nicotinamide mononucleotide using the same.

[0002] Nicotinamide mononucleotide (NMN) is a precursor of nicotinamide adenine dinucleotide (NAD), a compound essential for mitochondrial energy production in eukaryotes. It has been reported that NMN supplementation can effectively increase NAD levels in vivo (Non-Patent Document 1).

[0003] It has also been reported that improving NAD levels in the body activates the sirtuin gene, also known as the longevity gene (Non-Patent Document 2), and NMN, a precursor of NAD, is attracting attention as an active ingredient in pharmaceuticals, cosmetics, functional foods, etc. from the perspective of improving vitality and anti-aging.

[0004] Known methods for producing NMN include extracting NAD from yeast and decomposing it to obtain NMN, chemical synthesis, and fermentation production, with synthesis of NMN from NAD being the most commonly used method. However, extraction from yeast and chemical synthesis have the drawback of requiring many steps and being expensive.

[0005] REVOLLO, Javier R. and GRIMM, Andrew A., Journal of Biological Chemistry, 2004, 279.49: 50754-50763.North BJ et al., EMBO J. 2014 Jul 1;33(13):1438-53.

[0006] The present inventors focused on fermentation-based NMN synthesis methods. However, they found that simply overexpressing NMN synthase not only failed to efficiently extract NMN from outside the cells, but also led to cell death due to excessive intracellular accumulation of NMN, even though the amount of NMN produced increased.

[0007] Recently, a nicotinamide riboside transporter, PnuC, derived from Bacillus mycoides, was reported to have excellent NMN secretion efficiency (Shoji S., et al., Metab Eng. 2021;65:167-177., doi:10.1016 / j.ymben.2020.11.008). Although attempts were made to synthesize NMN using this PnuC, it was found that there was still room for improvement in the NMN secretion efficiency.

[0008] An object of the present invention is to provide an NMN transport protein that enables efficient recovery of NMN synthesized based on fermentation production methods from the extracellular environment.

[0009] As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that when specific mutations are introduced into specific positions in the nicotinamide riboside transporter PnuC, the NMN secretion efficiency is significantly improved compared to before the mutations are introduced. The present invention is based on this novel finding and provides the following: [1] A nicotinamide riboside transporter PnuC mutant (PnuC mutant) shown in any one of (a) to (c) below: (a) (i) a PnuC mutant having a mutation in the amino acid sequence of SEQ ID NO: 1 in which the amino acid at position 81 is substituted with alanine (81A mutation), or (ii) a PnuC mutant derived from a bacterium of the genus Bacillus or Paenibacillus having a mutation in which the amino acid at the position corresponding to position 81 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (81A mutation); (b) a PnuC mutant having an amino acid sequence of the PnuC mutant of (a) that includes one or more amino acid deletions, substitutions, and / or additions in addition to the 81A mutation; or (c) a PnuC mutant having an amino acid sequence that has 90% or more sequence identity to the amino acid sequence of the PnuC mutant of (a) excluding the 81A mutation. [2] A polynucleotide encoding the PnuC mutant of [1]. [3] A vector having a nucleotide sequence encoding the PnuC mutant of [1]. [4] The vector according to [3], further comprising an overexpression promoter sequence. [5] A cell oversecreting nicotinamide mononucleotide, comprising the polynucleotide according to [2] and / or the vector according to [3] or [4]. [6] A method for producing nicotinamide mononucleotide, comprising: a culturing step of culturing the cell according to [5] in a medium; a mixing step of mixing the cell with niacin and / or its glycoside; and a recovery step of recovering nicotinamide mononucleotide from the medium containing the cell. This specification incorporates the disclosure of Japanese Patent Application No. 2024-081833, from which the present application claims priority.

[0010] The PnuC mutant of the present invention allows NMN to be secreted extracellularly with high efficiency.

[0011] The polynucleotides and vectors of the present invention allow the PnuC mutants of the present invention to be introduced into cells.

[0012] The cells of the present invention are capable of secreting excess NMN synthesized within the cells to the outside of the cells.

[0013] According to the production method of the present invention, NMN can be obtained efficiently.

[0014] 1. PnuC Mutants, Polynucleotides, and Vectors 1-1. Overview A first aspect of the present invention is a nicotinamide riboside transporter PnuC mutant, a polynucleotide encoding the mutant, and a vector comprising the nucleotide sequence. The PnuC mutant of the present invention has an 81A mutation, which improves the efficiency of extracellular secretion of nicotinamide mononucleotide. By incorporating the polynucleotides and vectors of the present invention into cells, cells that oversecrete nicotinamide mononucleotide can be prepared.

[0015] 1-2. Definitions Terms used herein are defined below. "Nicotinamide adenine dinucleotide (NAD)" is 3-(aminocarbonyl)-1-[5-O-[(5'-adenylyloxy)oxylatophosphinyl]-β-D-ribofuranosyl]pyridinium. NAD has a structure in which ribonucleotides, phosphorylated ribose, and nicotinamide are bound, and is also known as β-diphosphopyridine nucleotide, coenzyme I, and other names. NAD can exist in two forms in vivo: oxidized (NAD+) and reduced (NADH), both of which are encompassed herein. In vivo, NAD is produced by the conversion of nicotinic acid adenine dinucleotide to its amide form or by the addition of an adenine nucleotide to nicotinamide mononucleotide.

[0016] Nicotinamide mononucleotide (NMN) is 1-deoxy-1-(3-carbamoylpyridinio)-5-O-oxylatophosphonyl-β-D-ribofuranose (molecular weight: 334.2192). NMN is a synthetic intermediate of NAD and consists of nicotinamide and phosphorylated ribose. It is also known as nicotinamide ribonucleotide, nicotinamide nucleotide, or nicotinamide ribotide. Because NMN does not penetrate the cell membrane, its transport to and from the extracellular environment is usually via transporters. NMN is biosynthesized from extracellular nicotinamide (NAM), nicotinamide riboside (NR), and nicotinic acid (NA) as donors of the nicotine moiety. Three pathways for NMN synthesis are known, primarily from these three precursor compounds. In particular, the pathway from NAM uses phosphoribosyl pyrophosphate (PRPP) as the donor of the phosphorylated ribose moiety.

[0017] In the NMN synthesis pathway from NAM, NMN is synthesized from PRPP, which is produced from NAM and glucose, etc. In the NMN synthesis pathway from NA, nicotinic acid mononucleotide (NaMN) is synthesized from NA, and NMN is synthesized by converting the carboxyl group in the nicotinic acid moiety to an amide group. In the NMN synthesis pathway from NR, NMN is synthesized by introducing a phosphate group into extracellular NR. Note that NAM obtained by decomposition of NR can be converted to NA, and NMN can be synthesized via the NMN synthesis pathway from NA, but this synthetic pathway is not included in the NMN synthesis pathway from NAM in this specification.

[0018] "Nicotinamide riboside transporter PnuC" refers to a multi-transmembrane protein belonging to the nicotinamide ribonucleoside uptake permease (PnuC) family that is responsible for the intracellular uptake of nicotinamide riboside (NR) through the cell membrane. Since PnuC also has the activity of secreting NMN extracellularly, PnuC in this specification particularly refers to a protein that has the activity of secreting NMN extracellularly. PnuC orthologous proteins are widely found in Gram-negative and Gram-positive bacteria. For example, PnuC from Bacillus mycoides, a bacterium of the genus Bacillus, is exemplified by the amino acid sequence of SEQ ID NO: 1.

[0019] "Bacillus bacteria" are bacteria belonging to the genus Bacillus. Bacillus bacteria are gram-positive rod-shaped bacteria that are aerobic to facultative anaerobic and are found primarily in soil. The specific species of Bacillus bacteria used herein are not particularly limited, and examples include Bacillus mycoides, Bacillus cereus, Bacillus tovonensis, Bacillus wiedmannii, Bacillus thuringiensis, Bacillus nitratireducens, Bacillus anthracis, Bacillus cihuensis, Bacillus salipaludis, Bacillus sp. JJ722, Bacillus massiliigorillae, Bacillus sp. SRB_331, Bacillus sp. SRB_28, Bacillus sp. TH25, Bacillus sp. HNR-4, Bacillus toyonensis, Bacillus pacificus, and Bacillus tropicus.

[0020] "Paenibacillus bacteria" are bacteria belonging to the genus Paenibacillus. Paenibacillus bacteria are Gram-positive bacteria that exhibit facultative anaerobic properties and are found in a wide range of habitats, including soil and water. Paenibacillus bacteria are a group of bacteria that were separated from the Bacillaceae family during the reclassification of the genus Bacillus and reclassified as the genus Paenibacillus. Specific species of Paenibacillus bacteria as used herein are not particularly limited, but examples include Paenibacillus agilis.

[0021] Bacteria are classified into subspecies, pathovar, and strain, which are lower than species.

[0022] "Niacin" is a general term for vitamin B3, which consists of nicotinamide (NAM) and nicotinic acid (NA).

[0023] The term "glycoside" refers to a compound in which a sugar and another compound are linked via a glycosidic bond. In particular, the term "glycoside" as used herein refers to a compound in which a monosaccharide and another compound are linked via a glycosidic bond. For example, the glycoside of niacin includes compounds in which NAM and a monosaccharide are linked and compounds in which NA and a monosaccharide are linked, but preferably nicotinamide riboside (NR), which is a compound in which NAM and ribose are linked.

[0024] As used herein, "significant" refers to statistical significance. Statistically significant refers to a significant difference between the measured value of a test subject and the control value when the difference between the two is statistically processed. For example, the risk rate (significance level) of the obtained value is small, specifically, less than 5% (p<0.05), less than 1% (p<0.01), or less than 0.1% (p<0.001). The "p (value)" shown here indicates the probability that a test statistic will take that value by chance in a distribution based on the null hypothesis in a statistical test. Therefore, the smaller the "p", the lower the probability that the test statistic will take that value, meaning that the null hypothesis is more likely to be rejected. The statistical processing test method can be any known test method capable of determining the presence or absence of significance, and is not particularly limited. For example, Student's t-test, paired Student's t-test, Welch's t-test, Wilcoxon rank sum test, analysis of variance, Tukey post-hoc test, etc. can be used, but are not particularly limited.

[0025] 1-3. PnuC Mutants The nicotinamide riboside transporter PnuC mutants of the present invention (hereinafter often abbreviated as "PnuC mutants") are, for example, (a) PnuC mutants that have a mutation in Bacillus mycoides-derived PnuC or its orthologous protein in other Bacillus or Paenibacillus species (including other strains of the same species) in which the amino acid at the position corresponding to position 81 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with alanine (81A mutation) in the PnuC derived from Bacillus mycoides or its orthologous protein in other Bacillus or Paenibacillus species (including other strains of the same species).

[0026] The PnuC mutant derived from Bacillus mycoides (i) has a mutation in the amino acid sequence shown in SEQ ID NO: 1 in which the amino acid at position 81 is substituted with alanine (81A mutation).

[0027] Since the amino acid at position 81 in the amino acid sequence shown in SEQ ID NO:1 is isoleucine (I), this mutant has an I81A mutation and consists of the amino acid sequence shown in SEQ ID NO:2.

[0028] Furthermore, the PnuC mutant derived from an orthologous protein of another species of the genus Bacillus or Paenibacillus (including other strains of the same species) is (ii) a PnuC mutant derived from a bacterium of the genus Bacillus or Paenibacillus having a mutation in which the amino acid at the position corresponding to position 81 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with alanine (81A mutation).

[0029] A "position corresponding to" a particular position in a reference sequence refers to a position in a test sequence that is aligned to a particular position in the reference sequence when a test sequence having a certain level of sequence identity to the reference sequence is aligned with the reference sequence.

[0030] The amino acid at the position corresponding to position 81 in the amino acid sequence shown in SEQ ID NO: 1 in the amino acid sequence of an orthologous protein refers to the amino acid in the amino acid sequence of the orthologous protein that is aligned at position 81 in the amino acid sequence shown in SEQ ID NO: 1 when the amino acid sequence of the orthologous protein is aligned to the amino acid sequence shown in SEQ ID NO: 1. Specifically, when the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence of the orthologous protein are aligned, if a gap of n1 amino acids (n1 is an integer of 0 or more) is introduced on the orthologous protein side from the N-terminus to the amino acid at position 81 in the amino acid sequence shown in SEQ ID NO: 1 and a gap of n2 amino acids (n2 is an integer of 0 or more) is introduced on the SEQ ID NO: 1 side, the 81-n1+n2 amino acid from the N-terminus of the amino acid sequence of the orthologous protein will be the amino acid at the position corresponding to position 81 in the amino acid sequence shown in SEQ ID NO: 1.

[0031] The alignment method is not particularly limited, and can be performed using, for example, a program exemplified below for sequence identity, or other programs such as the Parallel Editor (Multiple Alignment) in Genetyx, a program included in ClustalW.

[0032] The orthologous protein may be a PnuC derived from a bacterium of the genus Bacillus or Paenibacillus, and its specific amino acid sequence is not particularly limited. These PnuCs have NMN secretion activity equivalent to or greater than that of a PnuC consisting of the amino acid sequence set forth in SEQ ID NO: 1, for example. "Equal to or greater than" NMN secretion activity means that the NMN secretion activity is not significantly lower than that of a PnuC consisting of the amino acid sequence set forth in SEQ ID NO: 1, and / or that the NMN secretion activity is 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the NMN secretion activity of a PnuC consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0033] The method for measuring the NMN secretion activity is not particularly limited. For example, it can be measured using the method for measuring the amount of NMN exemplified in the recovery step of the production method of the third embodiment.

[0034] There are no particular limitations on the sequence identity of the amino acid sequence of an orthologous protein to the amino acid sequence set forth in SEQ ID NO: 1. The sequence identity of the amino acid sequence of an orthologous protein to the amino acid sequence set forth in SEQ ID NO: 1 may be, for example, 60% or more, 70% or more, 80% or more, 81% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0035] For example, an orthologous protein can be a protein having an amino acid sequence that has 89% or more, 90% or more, 95% or more, 97% or more, or 98% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Alternatively, for example, a protein can be used that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an amino acid sequence that is identical to SEQ ID NO: 1 at the following positions: 82 and 83, 81 to 83, 82 to 90, 78 to 88, 75 to 88, 78 to 96, 75 to 96, 78 to 97, 75 to 97, and 60 to 111 in the amino acid sequence set forth in SEQ ID NO: 1.

[0036] The 81A mutation is a mutation in which the amino acid at the position corresponding to position 81 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine. The type of original amino acid in an orthologous protein having the 81A mutation is not particularly limited as long as it is other than alanine. Examples include isoleucine, glycine, leucine, valine, etc., and for example, an orthologous protein in which the original amino acid is isoleucine can be preferably used. An 81A mutation in which the original amino acid is isoleucine is specifically referred to as an I81A mutation.

[0037] The species of Bacillus bacterium from which the original PnuC of the PnuC mutant is derived is not particularly limited. For example, PnuC derived from Bacillus mycoides, Bacillus cereus, Bacillus tovonensis, Bacillus wiedmannii, Bacillus thuringiensis, Bacillus nitratireducens, Bacillus anthracis, Bacillus cihuensis, Bacillus salipaludis, or Bacillus sp. JJ722, and particularly PnuC derived from Bacillus mycoides, can be preferably used.

[0038] The species of Paenibacillus bacteria from which the original PnuC of the PnuC mutant is derived is not particularly limited. For example, PnuC derived from Paenibacillus agilis can be preferably used.

[0039] Furthermore, the PnuC mutant of the present invention may be, for example, (b) a PnuC mutant consisting of an amino acid sequence containing one or more amino acid deletions, substitutions, and / or additions in addition to the 81A mutation in the amino acid sequence of the PnuC mutant of (a), wherein the amino acid at the position corresponding to position 81 in the amino acid sequence set forth in SEQ ID NO: 1 remains alanine.

[0040] Here, as used herein, "multiple" refers to 2 to 10, for example, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In an orthologous protein, for example, deletions, substitutions, and / or additions of 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acids may be included.

[0041] Here, the amino acid substitution is preferably a conservative amino acid substitution. Generally, if the amino acid substitution is conservative, a protein having an amino acid sequence after the substitution is likely to have substantially the same structure or properties as the amino acid sequence before the substitution. Conservative amino acids refer to the relationship between amino acids classified in the same amino acid group. Known amino acid groups include nonpolar amino acids (glycine, alanine, phenylalanine, valine, leucine, isoleucine, methionine, proline, tryptophan), polar amino acids (amino acids other than nonpolar amino acids), charged amino acids (acidic amino acids (aspartic acid, glutamic acid) and basic amino acids (arginine, histidine, lysine)), uncharged amino acids (amino acids other than charged amino acids), aromatic amino acids (phenylalanine, tryptophan, tyrosine), branched-chain amino acids (leucine, isoleucine, valine), and aliphatic amino acids (glycine, alanine, leucine, isoleucine, valine).

[0042] Furthermore, the PnuC mutant of the present invention may be, for example, (c) a PnuC mutant consisting of an amino acid sequence that has 90% or more sequence identity to the amino acid sequence of the PnuC mutant of (a) other than the 81A mutation, in which the amino acid at the position corresponding to position 81 in the amino acid sequence set forth in SEQ ID NO: 1 remains alanine.

[0043] As used herein, the term "sequence identity" of an amino acid sequence refers to the percentage (%) of identical amino acids in one polypeptide relative to the total number of amino acids in the other polypeptide when the amino acid sequences of two polypeptides are aligned, with gaps introduced, if necessary, into one of the amino acid sequences to maximize the degree of amino acid identity between the two. This percentage of amino acid identity can be easily determined using known programs such as the homology search program BLAST (Basic local alignment search tool; Altschul, S. F. et al., J. Mol. Biol., 215, 403-410, 1990). The degree of sequence identity here is not particularly limited, as long as it is 90% or greater. For example, it may be 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater. In the case of orthologous proteins, the degree of sequence identity may be, for example, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater.

[0044] The PnuC mutant of the present invention has improved NMN secretion activity compared to the original PnuC before the 81A mutation was introduced. "Improved NMN secretion activity" refers to an increase in the amount of NMN secreted compared to the original PnuC, and the degree of this increase is not particularly limited. For example, the amount of NMN secreted may be significantly increased, and the amount of NMN secreted may be increased by 1.08 times or more, 1.09 times or more, 1.1 times or more, 1.11 times or more, 1.12 times or more, 1.13 times or more, 1.14 times or more, 1.15 times or more, 1.16 times or more, 1.17 times or more, 1.18 times or more, 1.185 times or more, 1.19 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.45 times or more, or 1.46 times or more compared to the original NMN secretion activity.

[0045] The polynucleotide of the present invention is a polynucleotide that encodes a PnuC mutant.

[0046] Since the PnuC mutant has been described above, a detailed description thereof will be omitted here. The nucleotide sequence encoding the PnuC mutant is not particularly limited, as long as it is a nucleotide sequence that allows the PnuC mutant to be biosynthesized as a result of translation in a host cell. Whether or not a PnuC mutant is biosynthesized in a host cell can be confirmed by any method known in the art.

[0047] Specific examples of base sequences encoding the amino acid sequence shown in SEQ ID NO: 2 include the base sequence shown in SEQ ID NO: 3 (the codon consisting of bases at positions 241 to 243 from the 5' end corresponds to I at position 81 from the N-terminus in SEQ ID NO: 2); a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 3; a base sequence having 85% or more, 90% or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, or 99.5% or more sequence identity to the base sequence shown in SEQ ID NO: 3; and a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 3.

[0048] As used herein, "sequence identity" with respect to nucleotide sequences refers to a numerical value indicating the percentage of sites with the same base type within the comparison range of two nucleotide sequences. Even when the lengths of the two nucleotide sequences are different, nucleotide sequence identity can be calculated by aligning the sequences to maximize the degree of base identity within the comparison range. A representative algorithm for such analysis is BLAST, although not limited thereto. BLAST is available in a variety of software and web services. For example, nucleotide sequence identity can be easily calculated using the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ) or the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). In addition to BLAST, there are also algorithms such as FASTA, which can be used as long as they can calculate reasonable identity; the method used is not particularly limited.

[0049] The term "highly stringent conditions" refers to environmental conditions that make it difficult for nonspecific hybridization to occur. Under highly stringent conditions, a nucleic acid having a target nucleotide sequence can form a hybrid, but a nucleic acid having a nonspecific nucleotide sequence cannot substantially form a hybrid. Generally, highly stringent conditions refer to conditions of low salt concentration and high temperature. A low salt concentration refers to, for example, 15 mM to 750 mM, preferably 15 mM to 500 mM, 15 mM to 300 mM, or 15 mM to 200 mM. Furthermore, a high temperature refers to, for example, 50 to 68°C or 55 to 70°C. A specific example of highly stringent conditions is washing after hybridization at 65°C with 0.1×SSC and 0.1% SDS.

[0050] The nucleotide sequence of the polynucleotide can be codon-optimized for expression in the host cell to be used, if necessary. The specific method for optimizing the codons is not particularly limited, and any method known in the art can be used.

[0051] The type of host cell is not particularly limited, and examples include the species exemplified for the cells of the second aspect (for example, bacteria of the genus Escherichia such as E. coli).

[0052] The polynucleotide of this embodiment may contain additional sequences as needed. The specific sequences contained in the polynucleotide of this embodiment are not particularly limited, but may include, for example, a signal peptide sequence, an additional gene, and any other structural elements as described below for vectors.

[0053] An intervening sequence may be further included between the additional sequence and the PnuC mutant peptide. The specific intervening sequence is not particularly limited, and for example, any intervening sequence known in the art may be used.

[0054] The type of additional gene contained in the polynucleotide of this embodiment is not particularly limited. It may be a sequence related to the activity of the PnuC mutant or the production, degradation, and / or secretion of NMN, or it may be a sequence unrelated to these. Examples of sequences related to the production, degradation, and / or secretion of NMN include base sequences used in the additional treatments described for the cell of the second embodiment (such as overexpression of the NMN synthesis pathway and / or disruption of the NMN degradation pathway).

[0055] The genetic engineering techniques used herein, such as cloning of nucleic acids or genes, designing and constructing vectors, transforming cells, and expressing proteins or polypeptides, are not particularly limited. For example, any technique known in the art can be used. For specific examples, see Sambrook, J. et al., 1989.

[0056] The enzymes, cloning hosts, etc. used for genetic manipulation are not particularly limited, and for example, commercially available enzymes can be used according to the protocols provided by the manufacturers. The specific enzymes used can be selected appropriately depending on the purpose, the type of host, and the type of vector, and are not particularly limited.

[0057] The type of polynucleotide is not particularly limited. For example, it may be DNA, RNA, or a combination thereof. Preferably, DNA is used.

[0058] The nucleotide encoding the PnuC mutant integrated into the genome of the host is encompassed by the polynucleotide of this embodiment if it is derived from an exogenous gene (e.g., a vector having a nucleotide sequence encoding the PnuC mutant). However, even if it is an endogenous gene expression vector, an endogenous gene expression vector that originates from an exogenous gene expression vector, such as the progeny of a transgenic insect, is also encompassed by the exogenous gene expression vector of the present invention.

[0059] The vector of the present invention is a vector having a nucleotide sequence encoding a PnuC mutant.

[0060] The type of vector of the present invention is not particularly limited. Examples include autonomously replicating vectors such as plasmids (bacterial plasmids, yeast plasmids, etc.) or bacmids, viral vectors (phages such as lambda phage, retroviruses, baculoviruses, vaccinia viruses, adenoviruses, etc.), vectors capable of homologous recombination into chromosomes, or combinations thereof. The vector may also be a shuttle vector capable of replicating in other bacteria. Furthermore, the vector of the present invention may be an expression vector capable of expressing a PnuC mutant gene, or a vector (e.g., a recombinant vector) that cannot express a PnuC mutant gene by itself.

[0061] The specific type of vector is not particularly limited and can be appropriately selected depending on the type of host cell, etc. For example, examples of plasmid vectors when the host is Escherichia coli include pUC, pET, and pBAD, and examples of plasmid vectors when the host is yeast include pPink-HC, pPink-LC, pPinkα-HC, pPCIZ, pPCIZα, pPCI6, pPCI6α, pFLD1, pFLD1α, pGAPZ, pGAPZα, pPIC9K, pPIC9, pD912, and pD915.

[0062] The vector of this embodiment can contain optional constituent elements such as a promoter, an enhancer, a multicloning site, a 5'UTR, a 3'UTR, a signal peptide sequence, a terminator, a marker gene, an insulator, an inverted terminal repeat of a transposon, an enhancer, a replication origin, a ribosome binding site, etc. Each constituent element will be specifically described below.

[0063] The arrangement, number, and type of each component element are not particularly limited, and can be appropriately selected according to the purpose, for example, based on any information known in the art.

[0064] (1) Promoter The promoter is a structural element that plays a central role when the vector of this embodiment is used as an expression vector.

[0065] As used herein, a "promoter" refers to a gene expression regulatory region capable of controlling the expression of a gene or the like located downstream (3' end). The promoter used in the present invention is particularly an overexpression promoter. An "overexpression promoter" refers to a promoter capable of overexpressing a gene under its control. Overexpression refers to expression at a level greater than the expression level of one copy of the gene. The specific expression level is not particularly limited, but may be, for example, 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 75 times or more, 100 times or more, 150 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, or 1000 times or more of the expression level of one copy of the gene.

[0066] When classified based on the timing of expression, promoters are classified into constitutively active promoters, inducible promoters, and time-specific active promoters. Constitutively active promoters can constitutively express a target gene or the like in cells. Inducible promoters can induce the expression of a target gene or the like in cells at any time based on an inducing stimulus. The promoter that controls the PnuC mutant gene of the present invention may be either a constitutively active promoter or an inducible promoter.

[0067] Examples of promoters that can be used in E. coli host cells include the T7 promoter, T5 promoter, lac promoter, trp promoter, PL promoter, PR promoter, and tac promoter, and examples of promoters that can be used in yeast host cells include the AOX1 promoter, TEF1 promoter, ADE2 promoter, CYC1 promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, and AOX1 promoter.

[0068] When a promoter is included, the PnuC mutant gene is linked to the promoter in an expressible state. As used herein, the term "expressible state" refers to the ability to place a PnuC mutant gene, etc. under the control of a promoter so that it can be expressed, or the PnuC mutant gene, etc. is placed under the control of a promoter so that it can be expressed.

[0069] (2) Expression Induction System The expression induction system is a selective constituent element of the vector of this embodiment, and is an element for inducing expression regardless of the properties of the promoter itself.

[0070] The specific type of expression induction system is not particularly limited. Any expression induction system known in the art can be used. Specific expression induction systems include, for example, lactose operon-based induction systems (e.g., pET system), tetracycline resistance operon-based induction systems (e.g., Tet-on system), GAL4 protein-based methods (e.g., GAL4 / UAS system), and gene recombinase-based methods (e.g., Cre-lox system, Flp-FRT system), etc.

[0071] (3) Multicloning Site A multicloning site is an optional component of the vector of this embodiment. There are no particular limitations on the base sequence that constitutes it or the type and number of restriction enzyme sites that it contains. For example, it can be selected appropriately depending on the purpose based on any information known in the art. There are also no particular limitations on the number or location of multicloning sites in a vector. For example, to simplify introduction into a vector, it can be placed near the region where a PnuC mutant gene or the like is integrated. Furthermore, for example, if the vector contains a promoter, it can be placed within the regulatory region of the promoter.

[0072] (4) 5'UTR and 3'UTR The 5'UTR and 3'UTR are optional structural elements of the vector of this embodiment, and are polynucleotides consisting of untranslated regions that do not themselves encode proteins, fragments thereof, or functional nucleic acids. They are composed of nucleotide sequences located upstream of the start codon (5' end) and downstream of the stop codon (3' end) of the mRNA coding region of the gene of interest, respectively. Specifically, for example, the 3'UTR can contain a poly(A) signal.

[0073] (5) Terminator The terminator is an optional constituent element of the vector of this embodiment, and is composed of a nucleotide sequence that can terminate the transcription of the PnuC mutant gene or the like when it is expressed.

[0074] There are no particular limitations on the specific terminator. For example, examples of terminators that can be used in Escherichia coli host cells include the T7 terminator, the fd phage terminator, the T4 terminator, the tetracycline resistance gene terminator, and the Escherichia coli trpA gene terminator. Examples of terminators that can be used in yeast host cells include the PGK1 terminator, the CYC1 terminator, and the DIT1 terminator.

[0075] (6) Selection Marker Gene The selection marker gene is a selective component of the vector of this embodiment, and is a polynucleotide consisting of a nucleotide sequence encoding a protein also called a selection marker. Selection marker proteins include enzymes (e.g., factors encoded by drug resistance genes and temperature-sensitive replication initiators such as RepA), nutrient genes (e.g., biosynthetic genes for leucine, uracil, adenine, histidine, lysine, or tryptophan), fluorescent or luminescent proteins (e.g., luciferase, β-galactosidase, β-glucuronidase (GUS), or GFP), pigment synthesis proteins, or luminescent proteins.

[0076] The selection marker gene is used to confirm that the vector has been properly introduced into the host cell or properly integrated into the genome (in the case of a vector that does not have the ability to self-replicate). The specific selection marker gene to be used is not particularly limited. For example, it can be appropriately selected and used depending on the type of host cell to be used. Specifically, for example, a selection marker based on a drug resistance gene that can be used in E. coli host cells is Kan R (kanamycin resistance gene), Sm R (streptomycin resistance gene), Amp R (ampicillin resistance gene), Tet R (tetracycline resistance gene), Cm R (chloramphenicol resistance gene), Em R (erythromycin resistance gene), Neo R (neomycin resistance gene), Spc R (spectinomycin resistance gene), Hyg R (hygromycin resistance gene), Gm R (gentamicin resistance gene), Rif R (rifampicillin resistance gene), Zeocin R (Zeocin resistance gene), Blasticidin R(blasticidin resistance gene), and the like. Examples of selectable markers that can be used in yeast host cells include URA3, TRP1, SUP4, ADE2, HIS3, LEU2, LYS2, KANMX, AUR1-C, CYH2, CAN1, PDR4, and hphMX.

[0077] (7) Insulator: An insulator is an optional structural element when the vector of this embodiment is used as a vector for introduction into eukaryotic cells. An insulator is a base sequence that can stably control the transcription of the gene sandwiched between the insulator sequences without being affected by the chromatin of the surrounding chromosomes. Examples of insulators include the chicken cHS4 sequence and the Drosophila gypsy sequence.

[0078] (8) Inverted terminal repeat sequence of transposon "Inverted terminal repeat sequence of transposon" is an optional structural element that can be included when the vector of this embodiment is a vector capable of homologous recombination. Inverted terminal repeat sequences are usually used in pairs. The transposon to be used is not particularly limited, but examples include piggyBac, mariner, and minos (Shimizu, K. et al., 2000, Insect Mol. Biol., 9, 277-281; ​​Wang W. et al., 2000, Insect Mol. Biol. 9(2):145-55).

[0079] 2. Nicotinamide Mononucleotide Oversecreting Cells 2-1. Overview A second aspect of the present invention is a nicotinamide mononucleotide oversecreting cell. The cells of the present invention contain a polynucleotide and / or a vector and oversecrete nicotinamide mononucleotide. The cells of the present invention can be used in a method for producing nicotinamide mononucleotide.

[0080] The cell of the present invention comprises a polynucleotide and / or a vector. The polynucleotide and vector are as described in the first embodiment.

[0081] The type of cell in this embodiment is not particularly limited. For example, either eukaryotic cells and / or prokaryotic cells, such as fungi including yeast, can be used. Preferably, the cells of the present invention are prokaryotic cells (bacteria). Specific bacteria used are not particularly limited, but examples include bacteria of the genus Escherichia, Corynebacterium, Staphylococcus, Bacillus, Pseudomonas, Proteus, and Actinomyces. For example, bacteria of the genus Escherichia, such as Escherichia coli (E. coli), and bacteria of the genus Corynebacterium can be preferably used. Examples of fungi include yeast and filamentous fungi. Specific examples of yeast include yeasts of the genus Saccharomyces, Candida, Yarrowia, Pichia, and Kluyveromyces.

[0082] Many strains of E. coli are known, and any of them can be used. Specific examples include E. coli K-12 and B strains, and their derivatives BW25113, W3110, JM109, CJ236, HB101, DH5, DH5α, HST04, HST08, TH2, BMH71-18mutS, C600, BL21, BL21(DE3), XL1-Blue, and K802. Multiple types of cells can be used in combination as the cells of the present invention.

[0083] The cell of this embodiment may contain both or either of the polynucleotide and the vector described in Aspect 1. For example, the cell may contain the polynucleotide described in Aspect 1 in its genomic DNA and may further contain the expression vector described in Aspect 1. Furthermore, the cell of this embodiment may contain two or more types of PnuC mutants.

[0084] The method for introducing polynucleotides and vectors into cells is not particularly limited. Any method known in the art can be used, for example, as described in Sambrook, J. et al., 1989. When incorporating polynucleotides into the genomic DNA of cells, they can be incorporated by genome editing or genetic recombination. Specific methods include, for example, electroporation, calcium phosphate, lipofection, spheroplast, lithium acetate, and viral infection-based methods.

[0085] The cells of this embodiment may be subjected to additional treatments as necessary, such as genetic modification treatments such as disruption of endogenous genes or overexpression of additional genes.

[0086] Disrupting a gene refers to reducing the amount of a protein encoded by the gene and / or reducing its activity compared to the wild-type. For example, this can be achieved by knockout and / or knockdown, or by frameshift mutation or missense mutation of an amino acid important for activity. The degree of reduction is not particularly limited, but may be, for example, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, or less than 1% of the wild-type.

[0087] In the cell of this embodiment, preferably, one or more genes encoding NMN production-inhibitory proteins are disrupted. "NMN production-inhibitory proteins" refer to proteins that have the activity of consuming NMN to synthesize other compounds or inhibiting the synthesis of NMN's raw material compounds, thereby reducing the amount of NMN.

[0088] For example, one or more protein genes that reduce NMN production can be disrupted in a pathway selected from the group consisting of the NAD synthesis pathway from NMN, the NMN synthesis pathway from nicotinic acid (NA), the NMN synthesis pathway from nicotinamide riboside (NR), and the NMN synthesis pathway from nicotinamide (NAM) (including the phosphoribosyl pyrophosphate (PRPP) synthesis pathway). Preferably, one or more genes for NMN production-inhibitory proteins are disrupted in each of these pathways.

[0089] The specific protein whose gene is disrupted is not particularly limited. For example, a protein in the NAD synthesis pathway from NMN includes NMN adenylyltransferase. An example of an NMN production-inhibitory protein in the NMN synthesis pathway from NA includes NMN amidohydrolase, which consumes NMN to produce nicotinic acid mononucleotide (NaMN). For example, an NMN production-inhibitory protein in the NMN synthesis pathway from NR includes UDP-sugar hydrolase, which consumes NMN to produce NR, purine nucleoside phosphorylase, which consumes NR to produce NAM, and nicotinamidase, which consumes NAM to produce NA. For example, the gene for UDP-sugar hydrolase can be suitably disrupted. For example, an example of an NMN production-inhibitory protein in the NMN synthesis pathway from NAM includes a DNA-binding transcriptional repressor that suppresses the synthesis of PRPP. When the host is Escherichia coli, for example, NadR as an NMN adenylyltransferase, UshA as a UDP-sugar hydrolase, PncC as an NMN amidohydrolase, PurR as a DNA-binding transcriptional repressor, pncA as a nicotinamidase, and deoD as a purine nucleoside phosphorylase can be disrupted.

[0090] Preferably, two or more, three or more, or four or more proteins in the NMN degradation pathway are disrupted. Specifically, for example, NMN adenylyltransferase, UDP-sugar hydrolase, NMN amidohydrolase, and DNA-binding transcriptional repressor, or orthologs thereof, can all be disrupted.

[0091] In the cell of this embodiment, preferably, a gene encoding one or more NMN synthesis-enhancing proteins is overexpressed. "NMN synthesis-enhancing proteins" refer to proteins that have the activity of increasing the amount of NMN produced.

[0092] For example, an NMN synthesis-promoting protein in one or more pathways selected from the group consisting of the pathway for NMN synthesis from NA, the pathway for NMN synthesis from NR, the pathway for NMN synthesis from NAM, and the phosphoribosyl pyrophosphate (PRPP) synthesis pathway can be overexpressed. Preferably, an NMN synthesis-promoting protein in the pathway for NMN synthesis from NAM and / or the PRPP synthesis pathway can be overexpressed.

[0093] The specific proteins whose genes are overexpressed are not particularly limited, but include niacin transporters that promote the uptake of NAM and NA into cells, nicotinamide phosphoribosyltransferase (NAMPT) that synthesizes NMN from NAM in the NMN synthesis pathway from NA, phosphoribosylpyrophosphate synthase (PRPS) that synthesizes PRPP from ribose-5-phosphate in the PRPP synthesis pathway, nicotinamide riboside kinases (e.g., FtNadE) that synthesize NMN from NR and NaMN, and nicotinic acid phosphoribosyltransferases (e.g., PncB) that synthesize NaMN from NA. For example, NAMPT and / or PRPS can be suitably overexpressed.

[0094] The specific type and origin of each protein are not particularly limited. For example, they may be endogenous proteins of the host or mutants thereof, or proteins or mutants thereof derived from other species. The species of origin is not particularly limited. Examples include eukaryotes (vertebrates such as mammals, primates, reptiles, and fish, arthropods such as insects, plants, fungi, etc.), prokaryotes (eubacteria, archaea), and viruses (DNA viruses, reverse transcription viruses, RNA viruses, etc.).

[0095] Specifically, NAMPT derived from a bacteriophage can be used, for example. The type of bacteriophage is not particularly limited. For example, NAMPT derived from Vibrio Phage (Vibrio Phage KVP40, etc.) can be preferably used. PRPS derived from bacteria can be used, for example. The type of bacteria is not particularly limited, but PRPS derived from Bacillus bacteria such as Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus caldolyticus can be used. For example, PRPS derived from Bacillus amyloliquefaciens or its L135I mutant, or a corresponding mutant in its homologous protein can be preferably used.

[0096] The structure of the vector when overexpressing multiple NMN synthesis-promoting proteins in the NMN synthesis pathway is not particularly limited, but it is preferable that the genes for two or more proteins are carried on the same vector. It is preferable that the expression of the NMN synthesis-promoting protein is inducible. Therefore, it is preferable that the expression vector encoding the NMN synthesis-promoting protein contains an expression-inducible promoter or an expression induction system.

[0097] The cells of this embodiment oversecrete NMN. Oversecreting NMN refers to an increase in the amount of NMN secreted into the extracellular environment when a PnuC mutant is used compared to when a PnuC without the 81A mutation is used. For example, it can be determined that the cells are oversecreting NMN if there is a significant increase compared to when a PnuC without the 81A mutation is used, or if the amount of NMN secreted is increased by a certain amount or more compared to when a PnuC without the 81A mutation is used. When the amount of NMN secreted is increased by a certain amount or more compared to when PnuC without the 81A mutation is used, the degree of increase is not particularly limited, but it may be, for example, 1.08-fold or more, 1.09-fold or more, 1.1-fold or more, 1.11-fold or more, 1.12-fold or more, 1.13-fold or more, 1.14-fold or more, 1.15-fold or more, 1.16-fold or more, 1.17-fold or more, 1.18-fold or more, 1.185-fold or more, 1.19-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.45-fold or more, or 1.46-fold or more of the amount of NMN secreted compared to when PnuC without the 81A mutation is used.

[0098] The method for confirming the amount of secretion is not particularly limited. For example, it can be confirmed by any method known in the art. For example, it can be measured using the method for measuring the amount of NMN exemplified in the recovery step of the production method of the third embodiment.

[0099] 3. Method for Producing Nicotinamide Mononucleotide 3-1. Overview A third aspect of the present invention is a method for producing nicotinamide mononucleotide. The method of this aspect includes a culturing step, a mixing step, and a recovery step as essential steps, and includes a preparation step and a purification step as optional steps. According to the method of this aspect, nicotinamide mononucleotide can be produced with high efficiency.

[0100] 3-2. Steps 3-2-1. Preparation step The "preparation step" is an optional step in which the cells described in the second aspect are prepared. The method for preparing the cells is not particularly limited. For example, the cells described in the second aspect may be produced by gene transfer or the like, or the obtained cells described in the second aspect may be used.

[0101] The cell configuration and production method are similar to those described in Aspect 2. Even when the cells described in Aspect 2 are obtained, additional treatments (e.g., disruption of endogenous genes or overexpression of NMN synthesis-promoting proteins described in Aspect 2) can be performed depending on the purpose.

[0102] In this step, any treatment necessary for the subsequent culture step can be performed. The specific treatment content can be appropriately selected depending on the purpose and is not particularly limited. For example, if the obtained cells are frozen cells, a cell waking treatment or the like can be performed.

[0103] The "culturing step" is an essential step, in which the cells described in the second aspect are cultured in a medium. When the preparation step is performed, this step can be performed simultaneously with or after the preparation step.

[0104] The culture conditions and culture method can be appropriately selected depending on the purpose and the type of cells used, and are not particularly limited.

[0105] The medium may be, for example, a basal medium, a complex medium, a defined medium, or a special medium, and may be a solid medium or a liquid medium. For example, any medium containing nutrients, inorganic salts, buffers, etc., generally known in the art may be used.

[0106] Specific nutrient sources are not particularly limited, but examples include sugars such as glucose, sucrose, maltose, etc.; organic acids such as lactic acid, acetic acid, citric acid (citric acid monohydrate, etc.), propionic acid, etc.; alcohols such as methanol, ethanol, glycerol, etc.; hydrocarbons such as paraffin; oils such as soybean oil, rapeseed oil, etc.; carbon sources such as mixtures thereof; nitrogen sources such as urea, yeast extract, meat extract, peptone, casein, corn steep liquor, etc.; phosphate compounds such as ATP, amino acids, vitamins, etc.

[0107] Specific inorganic salts are not particularly limited, but examples include phosphorus sources such as phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, ammonium salts such as ammonium sulfate and ammonium phosphate, nitrogen sources such as nitrates, and metal salts such as magnesium sulfate.

[0108] The specific buffer is not particularly limited, but for example, phosphate buffer, phosphate buffered saline (PBS), citrate buffer, Good's buffer, or a combination thereof can be used.

[0109] As the medium, known media can be used as is or with modifications. When the cells are bacteria such as E. coli, known media include, for example, LB medium, M9 medium, TB medium, SOB medium, SOC medium, 2X YT medium, NZCYM medium, and modified media thereof, and for example, LB medium or modified media thereof can be preferably used. When the cells are yeast, known media include, for example, yeast nitrogen base medium.

[0110] The medium may contain additional additives as needed. For example, if the cells contain a selection marker, antibiotics or the like may be added to select the cells of interest.

[0111] The pH of the medium is not particularly limited as long as it allows the cells to grow, and can be, for example, 3 to 10, 4 to 9, 5 to 9, 6 to 9, 6 to 8, 7 to 8, etc.

[0112] The temperature is not particularly limited as long as the cells used can grow at that temperature. For example, the cells can be cultured at 20°C to 40°C (e.g., 37°C).

[0113] The atmospheric conditions during the reaction are not particularly limited. Depending on the cells used, the reaction can be carried out, for example, in an aerobic atmosphere, a low-oxygen atmosphere, an anaerobic atmosphere, etc. When Escherichia coli is used, this step can usually be carried out in an aerobic atmosphere.

[0114] In this step, shaking or stirring may be performed as appropriate. In this case, the speed is not particularly limited. For example, it can be 100 rpm to 500 rpm (e.g., 300 rpm).

[0115] The culture time is not particularly limited. For example, it can be 30 minutes or more, 1 hour or more, 2 hours or more, 5 hours or more, 6 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 16 hours or more, 20 hours or more, or 24 hours or more. It can also be, for example, 1 week or less, 5 days or less, 2 days or less, 36 hours or less, 30 hours or less, or 24 hours or less.

[0116] The culture method is not particularly limited, and for example, any method known in the art (batch method, continuous batch method, flow microreactor method, loop reactor method, single-use method, or a combination thereof) can be used.

[0117] This step can be carried out multiple times. In this case, the medium composition and culture conditions may be the same for all cultures, or may be different for one or more cultures. When multiple cultures are carried out, the purpose of each culture is not particularly limited. For example, expansion culture and / or subculture may be carried out to increase the number of cells, or selective culture may be carried out to select the target cells. Furthermore, the medium composition and culture conditions when the induction step and mixing step described below are carried out may be the same as or different from the medium composition and culture conditions for the other cultures.

[0118] The medium can be replaced and / or renewed as needed. The composition and culture conditions of the medium before and after the replacement may be the same or different. The frequency of the replacement and / or renewal is not particularly limited. The medium may also be replaced and / or renewed by continuous perfusion.

[0119] During this process, some components may be added individually. In this case, the components to be added are not particularly limited, but for example, nutrient sources (glucose, ATP, etc.) can be added.

[0120] The "induction step" is an optional step of inducing gene expression in the cells according to the second aspect. This step can be carried out simultaneously with or after the culturing step.

[0121] This step can be carried out when the cells contain an inducible promoter or an inducible system, and the expression of a gene under its control is necessary or useful for the production of NMN.

[0122] Any induction method can be used as long as it can induce the expression of the desired inducible promoter or expression induction system, and an appropriate stimulus can be selected depending on the type of promoter or induction system. Specific examples of suitable stimuli include inducers, light, heat, etc. Specifically, in the case of an induction system based on a lactose operon, such as the pET system, allolactose or its analogs (allolactose, isopropyl-β-thiogalactopyranoside (IPTG), etc.) can be added as an inducer. In the case of an induction system based on a tetracycline resistance operon, such as the Tet-on system, tetracycline or its derivatives (doxycycline, etc.) can be added as an inducer. The concentration of the inducer is not particularly limited as long as it can induce the expression of the desired gene.

[0123] The cell density at which this step is performed is not particularly limited. For example, this step can be performed at the cell density described later in the mixing step.

[0124] 3-2-4. Mixing Step The "mixing step" is an essential step in which cells are mixed with niacin and / or its glycosides. This step can be performed simultaneously with or after the culturing step. If an induction step is performed, it can be performed simultaneously with or after the culturing step.

[0125] The compound to be mixed in this step is not particularly limited as long as it is a compound that can serve as a raw material for NMN synthesis. For example, it may be selected based on the NMN synthetic pathway overexpressed in the cells used in the method of this embodiment, or a raw material compound for an NMN synthetic pathway that is not overexpressed may be used. Preferably, a raw material compound for an NMN synthetic pathway overexpressed in the cells used is used. For example, if an NMN synthesis-promoting protein in the NMN synthetic pathway from nicotinamide (NAM) is overexpressed, it can be mixed with NAM in this step. Furthermore, for example, if multiple proteins in the NMN synthetic pathway are overexpressed, it can be mixed with two or more selected from the group consisting of nicotinamide, nicotinic acid, and nicotinamide riboside, depending on the pathway. Even when a raw material compound for an overexpressed NMN synthetic pathway is used, other raw material compounds can be used additionally.

[0126] The form of niacin and / or its glycosides is not particularly limited as long as the cells used can use niacin and / or its glycosides as raw materials for NMN synthesis. Niacin and / or its glycosides may be pure substances, may be a mixture with other substances, or may be in the form of a salt or prodrug.

[0127] The compounds may be mixed in one batch, multiple batches, or consecutively. When cells are mixed with two or more compounds, the compounds may be mixed simultaneously or separately.

[0128] The method of mixing is not particularly limited as long as niacin and / or its glycosides are available to the cells. For example, niacin and / or its glycosides may be added directly to the medium, a medium containing them may be added, or the medium may be replaced in whole or in part with a medium containing these compounds.

[0129] When this step is started simultaneously with the culturing step, this step can be carried out simultaneously with the culturing step by using a medium containing these compounds as the medium in the culturing step.

[0130] The concentration of niacin and / or its glycoside is not particularly limited. It is preferable that the concentration is sufficient for the cells used to produce NMN. The specific final concentration is not particularly limited, but can be, for example, 0.01 mM or more, 0.1 mM or more, 1 mM or more, 2 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 8.5 mM or more, 8.6 mM or more, 8.7 mM or more, 8.8 mM or more, 8.9 mM or more, 8.98 mM or more, 9 mM or more, 10 mM or more, 11 mM or more, 15 mM or more, 16 mM or more, 19 mM or more, 20 mM or more, 20.5 mM or more, 20.6 mM or more, 20.9 mM or more, or 20.98 mM or more. The upper limit is not particularly limited, and the final concentration can be, for example, 10 M or less, 1 M or less, 100 mM or less, 50 mM or less, 30 mM or less, 25 mM or less, 24 mM or less, 23 mM or less, 22 mM or less, 21 mM or less, or 20.98 mM or less. For example, the final concentration can be 1 mM or more and 50 mM or less, 5 mM or more and 30 mM or less, 6 mM or more and 25 mM or less, 8 mM or more and 22 mM or less, 8.9 mM or more and 21 mM or less, or 8.98 mM or more and 20.98 mM or less.

[0131] The concentration of niacin and / or its glycosides may be determined based on the number of cells. A specific final concentration is, for example, 1 × 10 8 For a cell density of cells / mL, the concentration can be 0.01 μM or more, 0.1 μM or more, 1 μM or more, 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, 210 μM or more, 225 μM or more, 240 μM or more, 250 μM or more, 255 μM or more, 259 μM or more, 260 μM or more, 261 μM or more, 261.5 μM or more, 262 μM or more, 265 μM or more, 270 μM or more, 280 μM or more, 290 μM or more, 300 μM or more, 310 μM or more, 340 μM or more, 350 μM or more, 360 μM or more, 370 μM or more, 371 μM or more, 372 μM or more, 373 μM or more, or 374 μM or more. There is no particular upper limit, but for example, 1 × 10 8The final concentration can be set to 100M or less, 50M or less, 10M or less, 1M or less, 500mM or less, 100mM or less, 50mM or less, 10mM or less, 1mM or less, 900µM or less, 600µM or less, 500µM or less, 450µM or less, 400µM or less, 390µM or less, 380µM or less, 379µM or less, 378µM or less, 377µM or less, 376µM or less, 375µM or less, or 374.5µM or less, relative to the cell density of cells / mL. For example, 1x10 8 The final concentration relative to the cell density of cells / mL can be 150 μM to 600 μM, 200 μM to 500 μM, 250 μM to 400 μM, 260 μM to 377 μM, 261.5 μM to 374.5 μM, etc.

[0132] In addition, when multiple molecules of niacin and / or its glycosides are produced from one molecule, the above concentration is converted to the concentration of the niacin and / or its glycosides produced.

[0133] When mixing is performed in multiple batches, the concentration may be the same for each batch or may be different for one or more batches. When mixing is performed continuously, the concentration may be constant or may change during the process.

[0134] The cell density when performing this step is not particularly limited. For example, 8 Cells / mL or more, 0.1×10 8 Cells / mL or more, 0.5×10 8 Cells / mL or more, 1×10 8 Cells / mL or more, 5×10 8 Cells / mL or more, 10×10 8 Cells / mL or more, 15×10 8 Cells / mL or more, 20×10 8 Cells / mL or more, 21×10 8 Cells / mL or more, 22×10 8 Cells / mL or more, 23×10 8 Cells / mL or more, 24×10 8 Cells / mL or more, 25×10 8 Cells / mL or more, 30×10 8 Cells / mL or more, 40×10 8 Cells / mL or more, 50×108 Cells / mL or more, 60×10 8 Cells / mL or more, 70×10 8 Cells / mL or more, 80×10 8 The concentration can be, for example, 1000 x 10 cells / mL or more. 8 Cells / mL or less, 500×10 8 Cells / mL or less, 200×10 8 Cells / mL or less, 100×10 8 Cells / mL or less, 90×10 8 Cells / mL or less, 88×10 8 Cells / mL or less, 85×10 8 Cells / mL or less, 81×10 8 Cells / mL or less, 80×10 8 Cells / mL or less, 79×10 8 Cells / mL or less, 75×10 8 Cells / mL or less, 70×10 8 Cells / mL or less, 50×10 8 Cells / mL or less, 40×10 8 Cells / mL or less, 32×10 8 Cells / mL or less, 30×10 8 Cells / mL or less, 25×10 8 Cells / mL or less, 24×10 8 For example, the cell density during this step can be 10 × 10 cells / mL or less. 8 cells / mL or more 100×10 8 Cells / mL or less, 15×10 8 cells / mL or more 90×10 8 Cells / mL or less, 20×10 8 cells / mL or more 85×10 8 Cells / mL or less, 24×10 8 cells / mL or more 80×10 8 Cells / mL or less, 24×10 8 cells / mL or more 40×10 8 Cells / mL or less, 24×10 8 cells / mL or more 32×10 8 It can be less than cells / mL, etc.

[0135] The cell density can be measured by any method. For example, it may be an actual measurement value or a value calculated from an index such as an OD value. For example, when the cells are Escherichia coli, the cell density can be calculated based on the OD600 value. Specifically, for example, [cell density (cells / mL)] = [OD600 value] × 8 × 10 8 It can be calculated using the formula:

[0136] The timing of carrying out this step is not particularly limited. For example, this step may be carried out when the progress of the culture meets certain conditions, or when a certain amount of time has elapsed since the start of the culture. The specific conditions for carrying out this step are not particularly limited, but for example, this step can be carried out when the cell density reaches the above-mentioned range. Furthermore, the time from the start of the culture at which this step is carried out is not particularly limited.

[0137] When mixing is performed in multiple batches, the time between mixing is not particularly limited, and may be, for example, the time exemplified in relation to the culture conditions.

[0138] In this step, the liquid component may be fluidized by stirring, shaking, or the like.

[0139] The "recovery step" is an essential step in which nicotinamide mononucleotide (NMN) is recovered from the culture medium. This step can be performed simultaneously with or after the mixing step.

[0140] The form of NMN recovered in this step is not particularly limited. For example, it can be recovered in the form of a liquid and / or solid containing NMN.

[0141] The recovery method used in this step is not particularly limited as long as it can recover NMN from the culture solution. When the medium is solid, for example, the solid medium may be recovered as is, or the medium may be dissolved by heat or the like and recovered, or the surface of the medium may be washed with any liquid and the resulting washing solution may be recovered. When the medium is liquid, for example, the liquid medium may be recovered as is, or the liquid from which impurities such as bacterial cells have been removed may be recovered.

[0142] Methods for removing impurities such as bacterial cells are widely known in the art and are not particularly limited. Examples include centrifugation, membrane separation (including filtration using reverse osmosis membranes, nanofiltration membranes, microfiltration membranes, ultrafiltration membranes, microfiltration membranes, etc.), activated carbon treatment, methods using ion exchange resins, or combinations thereof. Methods normally used for replacing or renewing culture media can be used.

[0143] There are no particular limitations on when this step can be performed. For example, this step can be performed when the NMN concentration reaches a certain level or above, or when a certain amount of time has passed since the start of the culturing step and / or the start of the mixing step.

[0144] The time from the start of culture in this step is not particularly limited, and can be, for example, 30 minutes or more, 1 hour or more, 2 hours or more, 5 hours or more, 6 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 16 hours or more, 20 hours or more, or 24 hours or more. Also, the time can be, for example, 1 week or less, 5 days or less, 2 days or less, 36 hours or less, 30 hours or less, or 24 hours or less.

[0145] The time from the start of the mixing step in this step is not particularly limited, and can be, for example, 30 minutes or more, 1 hour or more, 2 hours or more, 5 hours or more, 6 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 16 hours or more, 18 hours or more, 19 hours or more, or 20 hours or more. Also, the time can be, for example, 1 week or less, 5 days or less, 2 days or less, 36 hours or less, 30 hours or less, 24 hours or less, 21 hours or less, 20 hours or less, or 19 hours or less.

[0146] The method for measuring the concentration of NMN is not particularly limited, and examples thereof include high performance liquid chromatography (HPLC), nuclear magnetic resonance analysis (NMR), mass spectrometry, and methods based on binding molecules such as antibodies.

[0147] This step can be carried out multiple times, and in such cases, the recovery method and impurity removal method used may be the same each time, or may be different in one or more steps.

[0148] 3-2-6. Purification step The "purification step" is an optional step in which the recovered nicotinamide mononucleotide (NMN) is purified. This step can be performed simultaneously with or after the recovery step.

[0149] As used herein, the term "purification" refers to reducing the content of one or more substances other than the target substance.

[0150] The purification method for NMN used in this step is not particularly limited. Any method used in purifying medium components or compounds can be used. Specific examples include the methods for removing impurities exemplified in the recovery step, as well as chromatographic methods such as affinity column chromatography and high-performance liquid chromatography (HPLC), vacuum concentration, membrane concentration, freeze-drying, solvent extraction, distillation, and recrystallization precipitation methods, or combinations thereof.

[0151] 3-3. Effects The method of this embodiment enables simple and highly efficient production of NMN. For example, compared to when wild-type PnuC is used, the amount of NMN obtained increases by 1.08-fold or more, 1.09-fold or more, 1.1-fold or more, 1.11-fold or more, 1.12-fold or more, 1.13-fold or more, 1.14-fold or more, 1.15-fold or more, 1.16-fold or more, 1.17-fold or more, 1.18-fold or more, 1.185-fold or more, 1.19-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.45-fold or more, or 1.46-fold or more.

[0152] In this method, NMN can be recovered from the extracellular environment without destroying the cells. Therefore, for example, by repeatedly performing the culturing and recovery steps, NMN can be continuously and easily recovered.

[0153] According to the method of this embodiment, the NMN secretion efficiency of cells is improved, so that a high cell survival rate can be maintained.

[0154] Example 1. Preparation of cells (Objective) To prepare cells capable of synthesizing NMN in excess and expressing various types of nicotinamide riboside transporter PnuC.

[0155] (Methods and Results) 1. Disruption of the NMN Degradation Pathway 1-1. Cells The cells used were Escherichia coli BW25113 strain (obtained from the National Institute of Genetics).

[0156] 1-2. Disruption of NMN adenylyltransferase nadR First, a plasmid vector pTH18cs1-nadR-UD for disrupting the endogenous nadR gene of the E. coli BW25113 strain was constructed by the following procedure.

[0157] A DNA fragment (SEQ ID NO: 4) containing the upstream and downstream sequences of the nadR gene was chemically synthesized using artificial nucleic acid synthesis (GenScript), amplified by PCR, and then digested with restriction enzymes XbaI (New England BioLabs) and HindIII (New England BioLabs) to prepare a nucleic acid fragment for insertion. The temperature-sensitive plasmid pTH18cs1 (National Institute of Genetics; GenBank accession number: AB019610 (as of May 9, 2024)) containing a chloramphenicol resistance gene was digested with XbaI and HindIII and then ligated with the nucleic acid fragment for insertion using Ligation high Ver. 2 (Toyobo).

[0158] The resulting plasmid vector, pTH18cs1-nadR-UD, was introduced into E. coli BW25113 by electroporation using an electroporator (Neppa Gene). Electroporation was performed according to the manufacturer's recommended protocol. The vector-introduced BW25113 strain was cultured on LB agar plates for selection of disrupted strains at 30°C for 1 day to obtain transformants. The LB agar plates were prepared using LB medium (Sigma-Aldrich) and agar (Nacalai Tesque), and the LB agar plates for selection of disrupted strains were further supplemented with 10 μg / mL chloramphenicol (Fujifilm Wako Pure Chemical Industries).

[0159] The resulting transformants were expanded in LB liquid medium for selection of disrupted strains by shaking overnight at 30°C. LB liquid medium for selection of disrupted strains contained 10 μg / mL of chloramphenicol. The culture medium obtained after expansion was cultured on LB agar plates for selection of disrupted strains at 42°C to select transformants in which the vector had been integrated into the genome.

[0160] To excise the target sequence in the selected transformants, the transformants were cultured overnight at 42°C in simple LB liquid medium without chloramphenicol and then cultured on simple LB agar plates without chloramphenicol for 1 day at 42°C. Colonies obtained from the simple LB agar plates were cultured on simple LB agar plates and on LB agar plates for selection of disrupted strains, and chloramphenicol-sensitive transformants that formed colonies only on the simple LB agar plates were selected as candidate strains for nadR gene disruption.

[0161] The genomic DNA sequence of the nadR gene disruption candidate strain was analyzed, and one strain lacking the initiation codon to termination codon of the nadR gene was isolated as the ΔnadR strain.

[0162] First, a plasmid vector pTH18cs1-pncC-UD was constructed for disrupting the endogenous pncC gene of E. coli strain BW25113. The plasmid was constructed in the same manner as pTH18cs1-nadR-UD, except that a DNA fragment (SEQ ID NO: 5) containing the upstream and downstream sequences of the pncC gene was used.

[0163] Next, the plasmid vector pTH18cs1-pncC-UD was introduced into the ΔnadR strain, and transformants were selected. One strain lacking the initiation codon to termination codon of the pncC gene was isolated as the ΔnadR / ΔpncC strain. The procedure for preparing the ΔnadR strain was the same as that for the ΔnadR strain, except that the ΔnadR strain was used as the target strain and pTH18cs1-pncC-UD was used as the plasmid vector.

[0164] 1-4. Disruption of UDP-sugar hydrolase UshA First, a plasmid vector, pTH18cs1-ushA-UD, was constructed for disrupting the endogenous UshA gene of E. coli strain BW25113. The plasmid was constructed in the same manner as pTH18cs1-nadR-UD, except that a DNA fragment (SEQ ID NO: 6) containing the upstream and downstream sequences of the UshA gene was used.

[0165] Next, the plasmid vector pTH18cs1-ushA-UD was introduced into the ΔnadR / ΔpncC strain, and transformants were selected. One strain lacking the UshA gene from the initiation codon to the termination codon was isolated as the ΔnadR / ΔpncC / ΔUshA strain. The procedure for preparing the ΔnadR strain was the same as that for the ΔnadR strain, except that the ΔnadR / ΔpncC strain was used as the target strain and pTH18cs1-ushA-UD was used as the plasmid vector.

[0166] 2. Preparation of an overexpression vector for the NMN synthesis pathway. A DNA fragment (SEQ ID NO: 7) containing a T5 promoter, a PRPS gene (L135I mutant) derived from Bacillus amyloliquefaciens, a NadV gene derived from Vibrio phage KVP40, and a terminator sequence derived from lambda phage was chemically synthesized using artificial nucleic acid synthesis (GenScript). The fragment was amplified by PCR and then digested with the restriction enzymes BglII (New England BioLabs) and EcoRI (New England BioLabs) to prepare an insert nucleic acid fragment. Plasmid pET-28a(+) (MERCK; Cat. No.: 69864), which contains a kanamycin resistance gene, was digested with BglII and EcoRI, and then ligated with the insert nucleic acid fragment using Ligation high Ver. 2 (Toyobo). This resulted in the overexpression plasmid vector pET-28a-PT5-BaPRS-VpNadV for NMN synthesis pathway proteins.

[0167] 3. Preparation of PnuC overexpression vector 3-1. Preparation of wild-type PnuC expression vector A DNA fragment (SEQ ID NO: 8) containing a T5 promoter, a PnuC gene derived from Bacillus mycoides, and a terminator sequence derived from lambda phage was chemically synthesized using artificial nucleic acid synthesis (GenScript), amplified by PCR, and then digested with restriction enzymes AclI (New England BioLabs) and AgeI (New England BioLabs) to prepare a nucleic acid fragment for insertion. The plasmid pCDFDuet, which contains a streptomycin resistance gene, TM After digesting pCDFDuet-1 (MERCK; Cat. No. 71340) with AclI and AgeI, ligation with the nucleic acid fragment to be inserted was carried out using Ligation high Ver. 2 (Toyobo). This resulted in the overexpression of the wild-type PnuC plasmid vector pCDFDuet TM -1-PT5-PnuC-wt was obtained.

[0168] 3-2. Construction of mutant PnuC expression vector Mutant PnuC overexpression plasmid vector pCDFDuet TM -1-PT5-PnuC-mut was the same as pCDFDuet except that a DNA fragment containing a mutant PnuC gene with a substitution mutation at I81 was used instead of the PnuC gene. TM The nucleotide sequence of the DNA fragment containing the T5 promoter, the I81A mutant PnuC gene derived from Bacillus mycoides, and the terminator sequence derived from lambda phage is shown in SEQ ID NO:9.

[0169] 4. Generation of NMN-overproducing cells The NMN synthesis pathway protein overexpression plasmid vector pET-28a-PT5-BaPRS-VpNadV and PnuC expression vector (pCDFDuet TM -1-PT5-PnuC-wt or pCDFDuet TMThe vector (-1-PT5-PnuC-mut) was introduced into the ΔnadR / ΔpncC / ΔUshA strain by electroporation using an electroporator (Neppa Gene). Electroporation was performed according to the manufacturer's recommended protocol. The ΔnadR / ΔpncC / ΔUshA strain containing the vector was cultured on an LB agar plate for overexpression selection at 37°C for 1 day to obtain overexpression candidate strains. The LB agar plate for overexpression selection was prepared by adding 30 μg / mL kanamycin (Fujifilm Wako Pure Chemical Industries, Ltd.) and 25 μg / mL streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0170] The genomic DNA sequence of the overexpression candidate strain was analyzed, and the pET-28a-PT5-BaPRS-VpNadV and PnuC expression vector (pCDFDuet TM -1-PT5-PnuC-wt or pCDFDuet TM The strain into which pCDFDuet -1-PT5-PnuC-mut was introduced was used as a wild-type PnuC expression strain. TM -1-PT5-PnuC-wt), and one strain each expressing mutant PnuC (pCDFDuet TM -1-PT5-PnuC-mut) was isolated.

[0171] Example 2: Relationship between type of substitution mutation and amount of NMN secreted (Objective) To investigate the relationship between the type of amino acid substituted in PnuC and the amount of NMN secreted by mutant PnuC.

[0172] (Method) 1. Culturing The wild-type PnuC expression strain and various mutant PnuC expression strains obtained in Example 1 were cultured in test tubes to produce NMN.

[0173] First, the strain was cultured in 3 mL of LB medium for selection of overexpression strains (containing 30 μg / mL kanamycin and 25 μg / mL streptomycin) with shaking at 300 rpm at 37° C. for 8 hours.

[0174] Next, 30 μL of this culture was cultured in 3 mL of a production medium with shaking at 300 rpm at 37° C. for 24 hours. The production medium had the following composition: 6 g / L potassium dihydrogen phosphate, 16.4 g / L dipotassium hydrogen phosphate, 5 g / L ammonium sulfate, 1.1 g / L citric acid monohydrate, 1 g / L magnesium sulfate, 10 g / L yeast extract, and 20 g / L glucose.

[0175] During cultivation, cell density was measured using a spectrophotometer (Hitachi: U-2900), and when the absorbance at 600 nm (OD600) reached approximately 3.0 (4 to 5 hours into cultivation), isopropyl-β-thiogalactopyranoside was added to a final concentration of 0.1 mM to induce expression of the introduced gene, and nicotinamide was added to a final concentration of 3 g / L (approximately 8.98 mM). After 24 hours of cultivation, an appropriate amount of the culture medium was sampled and centrifuged at 15,000 rpm, and the supernatant was collected for measuring NMN concentration.

[0176] 2. Measurement of NMN production amount The collected supernatant was purified using an ultrafiltration spin column (Pharmafoods; molecular partitioning capacity: 10K), and then diluted 10 times with distilled water to prepare a measurement sample.

[0177] The NMN concentration in the measurement samples was measured using a high-performance liquid chromatograph (Shimadzu Corporation; LC-20 Prominence).

[0178] The HPLC analysis conditions were as follows: Column: COSMOSIL PBr Packed Column 3.0 mm x 150 mm (Nacalai Tesque) Mobile phase: Prepared by dissolving 1.26 g of ammonium formate (Fujifilm Wako Pure Chemical Industries) in 950 mL of distilled water and adding 50 mL of methanol (Fujifilm Wako Pure Chemical Industries) Flow rate: 0.4 mL / min Detection: UV detector λ = 260 nm Column temperature: 40°C Injection volume: 5 μL.

[0179] (Results) The results are shown in Table 1.

[0180]

[0181] As shown in Table 1, NMN was produced and secreted extracellularly in both the wild type and all of the mutants confirmed, and NMN was detected in the culture medium. However, differences in NMN concentration were observed depending on the type of amino acid substituted. Of these, only in the case of the mutation in which isoleucine at position 81 was substituted with alanine did NMN concentration increase compared to the wild type, reaching approximately 1.5 times that of the wild type.

[0182] This suggests that the I81A mutation in PnuC significantly improves the efficiency of NMN secretion into the culture medium.

[0183] Example 3: Verification of NMN secretion efficiency on a large scale (Objective) The efficiency of NMN production using a PnuC mutant with the I81A mutation was confirmed by culturing on a scale larger than that of a test tube.

[0184] (Method) The wild-type PnuC expression strain and the I81A mutant PnuC expression strain obtained in Example 1 were cultured in shake flasks (volume: 500 mL) to produce NMN.

[0185] First, the strain was cultured in 50 mL of LB medium for selection of overexpression strains with shaking at 150 rpm at 37°C for 16 hours.

[0186] Next, 40 mL of this culture solution was cultured in 2 L of production medium using a culture device (Marubishi Bioengine; Bioneer-Neo) with aeration of 4 L / min, pH 6.7, 600 rpm, and shaking at 37°C.

[0187] During the culture, when the absorbance at 600 nm (OD600) reached approximately 10 (4 to 5 hours after culture), isopropyl-β-thiogalactopyranoside was added to a final concentration of 0.1 mM to induce expression of the introduced gene, and nicotinamide was added to a final concentration of 7 g / L (approximately 20.94 mM). After 24 hours of culture, an appropriate amount of the culture medium was sampled and centrifuged at 15,000 rpm, and the supernatant was collected for NMN concentration measurement. NMN production was measured as in Example 2.

[0188] (Results) The results are shown in Table 2.

[0189]

[0190] As shown in Table 2, even under conditions using a relatively large shake flask, NMN was produced and secreted outside the cells, and NMN was detected in the culture medium, as in Example 2. Furthermore, even under these conditions, it was shown that the use of mutant PnuC with the I81A mutation improved the NMN secretion efficiency and increased the NMN concentration in the culture medium.

[0191] This suggests that the use of mutant PnuC with the 81A mutation can significantly increase the efficiency of NMN recovery from culture medium, even when mass-producing NMN on a large scale.All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A nicotinamide riboside transporter PnuC mutant (PnuC mutant) shown in any one of (a) to (c) below: (a) (i) a PnuC mutant having a mutation in the amino acid sequence shown in SEQ ID NO: 1, in which the amino acid at position 81 has been substituted with alanine (81A mutation), or (ii) a PnuC mutant derived from a Bacillus bacterium or a Paenibacillus bacterium, having a mutation in the amino acid at the position corresponding to position 81 in the amino acid sequence shown in SEQ ID NO: 1, in which the amino acid at position 81 has been substituted with alanine (81A mutation); (b) a PnuC mutant consisting of an amino acid sequence of the PnuC mutant of (a), which contains one or more amino acid deletions, substitutions and / or additions in addition to the 81A mutation; (c) a PnuC mutant consisting of an amino acid sequence that has 90% or more sequence identity to the amino acid sequence of the PnuC mutant of (a), excluding the 81A mutation.

2. A polynucleotide encoding the PnuC mutant of claim 1.

3. A vector having a base sequence encoding the PnuC mutant of claim 1.

4. The vector of claim 3, further comprising an overexpression promoter sequence.

5. A nicotinamide mononucleotide oversecreting cell comprising the polynucleotide of claim 2 and / or the vector of claim 3 or 4.

6. A method for producing nicotinamide mononucleotide, comprising: a culturing step of culturing the cells according to claim 5 in a culture medium; a mixing step of mixing the cells with niacin and / or its glycoside; and a recovery step of recovering nicotinamide mononucleotide from the culture medium for the cells.

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