Method for producing NMN and method for producing nad

Abstract

The present invention provides transformed microorganisms capable of synthesizing NMN or NAD at a high yield and a method for producing NMN or NAD using the transformed microorganisms. The present invention is a method for synthesizing NMN in bacterial cells, wherein NMN is synthesized from Nam and PRPP by NAMPT and the NAMPT has a region comprising: a polypeptide having an amino acid sequence that has 90-100% sequence similarity with an amino acid sequence represented by SEQ ID NO: 2 and an amino acid sequence that has 0-100% sequence similarity with an amino acid sequence represented by SEQ ID NO: 3 on the C-terminal side of that amino acid sequence; or a polypeptide having an amino acid sequence that has 90-100% sequence similarity with an amino acid sequence represented by SEQ ID NO: 5 and an amino acid sequence that has 90-100% sequence similarity with an amino acid sequence represented by SEQ ID NO: 6 on the C-terminal side of that amino acid sequence.

Description

Methods for producing NMN and NAD

[0001] The present invention relates to a method for synthesizing nicotinamide mononucleotide (NMN) and a method for synthesizing nicotinamide adenine dinucleotide (NAD) using a microorganism. This application claims priority to Japanese Patent Application No. 2023-182293, filed on October 24, 2023, the contents of which are incorporated herein by reference.

[0002] With the recent development of genetic engineering technology, production methods using microorganisms as reaction systems for synthetic reactions are increasingly being used for industrial mass production. While chemical synthesis often requires organic solvents, high-temperature and high-pressure reaction conditions, heavy metal catalysts, and the like, by utilizing the metabolic pathways possessed by microorganisms, useful organic compounds can be synthesized relatively inexpensively under mild conditions of room temperature and atmospheric pressure. For example, NAD is a coenzyme for many oxidoreductases, and NMN is an intermediate metabolic product in the biosynthesis of NAD, and various biological functions have been reported. Because both are compounds in high demand, there is a need to develop microorganisms that can produce them in large quantities.

[0003] In the synthesis of organic compounds using microorganisms, various methods are used to modify the metabolic pathways inherent in the microorganisms, such as introducing enzyme genes to supplement the metabolic pathways that are deficient in the microorganisms, or enhancing the metabolic efficiency (flux) of the microorganisms by increasing the expression level of the enzyme genes or introducing more active foreign genes or mutant forms thereof. Several pathways for the synthesis of NAD and NMN are known. For example, in mammalian cells, NMN is synthesized from nicotinamide (Nam) and a phosphorylated ribose donor such as phosphoribosyl pyrophosphate (PRPP) by nicotinamide phosphoribosyltransferase (NAMPT: NMN nucleosidase; hereinafter, sometimes referred to as NAMPT in this specification). NAD is synthesized from NMN and adenosine triphosphate (ATP) by nicotinamide mononucleotide adenylyltransferase (hereinafter, sometimes referred to as NMN-AT in this specification). Therefore, Patent Document 1 describes that the efficiency of NMN synthesis can be increased by transforming a microorganism used for synthesizing NMN or NAD by introducing a highly active NAMPT gene into the microorganism.

[0004] Microbial NAMPT, also known as NadV, is a protein having significant homology to mammalian NAMPT and is encoded by the nadV gene. For example, Non-Patent Document 1 discloses that by introducing the gene for NAMPT derived from Chitinophagaea pinensis, which has high NMN synthesis activity, into Escherichia coli, a transformant capable of selectively and efficiently producing NMN from glucose and Nam can be obtained.

[0005] In bacteria such as Escherichia coli, Salmonella typhimurium, and Haemophilus ducreyi, the reaction of synthesizing NAD from NMN is known to be catalyzed by the NadR protein, the product of the nadR gene (Non-Patent Document 2). These NadR proteins are composed of a domain that converts NMN to NAD (NMN-AT function), a DNA-binding domain that functions as a transcriptional regulator (Non-Patent Document 3), and a domain that functions as a nicotinamide riboside kinase that synthesizes NMN from nicotinamide riboside (hereinafter sometimes referred to as NR) (Non-Patent Document 4). The NadR protein is thought to bind to DNA in an NAD-dependent manner and regulate the expression of genes involved in the synthesis of the nicotinamide skeleton (Non-Patent Document 3). However, the role of the DNA-binding domain in enzymatic function and NAD synthesis efficiency remains unknown.

[0006] International Publication No. 2020 / 129997

[0007] Shoji, et al., Metabolic Engineering, 2021, vol.65, p.167-177.Raffaelli et al., Journal of Bacteriology, 1999, vol. 181(17), p.5509-5511.Penfound and Foster, Journal of Bacteriology, 1999, vol.181(2), p.648-655.Kurnasov et al., Journal of Bacteriology, 2002, vol.184(24), p.6906-6917.Pao et al., MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 1998, vol.62(1), p.1-34.Drew et al., Chemical Reviews, 2021, vol.121, p.5289-5335.

[0008] An object of the present invention is to provide a transformed microorganism capable of synthesizing NMN or NAD in high yield, and a method for producing NMN or NAD using the transformed microorganism.

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that the amount of NMN accumulated in Escherichia coli cells can be significantly increased by expressing NadV derived from Sandaracinus amylolyticus (SaNadV) or NadV derived from Convivina intestine (CiNadV) in Escherichia coli, and that the amount of NAD accumulated in Escherichia coli cells can be significantly increased by expressing NadR lacking the DNA-binding domain, thereby completing the present invention.

[0010] That is, the present invention encompasses the following aspects: [1] A method for synthesizing nicotinamide mononucleotide in bacterial cells, comprising synthesizing nicotinamide mononucleotide from nicotinamide and phosphoribosyl pyrophosphate using nicotinamide phosphoribosyltransferase, wherein the nicotinamide phosphoribosyltransferase is: (a1) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence represented by SEQ ID NO: 3, (a2) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 3, and having nicotinamide phosphoribosyltransferase activity, (a3) ​​a polypeptide having an amino acid sequence represented by SEQ ID NO: 5 and, at the C-terminal side of the amino acid sequence, an amino acid sequence represented by SEQ ID NO: 6, or (a4) A method for producing nicotinamide mononucleotide, comprising: (a) an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 5; and (b) an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 6 at the C-terminal side of the amino acid sequence; and (c) a polypeptide having nicotinamide phosphoribosyltransferase activity. [2] The method for producing nicotinamide mononucleotide according to [1], comprising culturing the bacterium in a solution containing glucose and nicotinamide. [3] A method for synthesizing nicotinamide adenine dinucleotide in bacterial cells, comprising: synthesizing nicotinamide mononucleotide from nicotinamide and phosphoribosyl pyrophosphate using nicotinamide phosphoribosyltransferase; and synthesizing nicotinamide adenine dinucleotide from the resulting nicotinamide mononucleotide and ATP using nicotinamide mononucleotide adenylyltransferase, wherein the nicotinamide phosphoribosyltransferase is capable of producing: (a1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 and, at the C-terminal side of the amino acid sequence, the amino acid sequence represented by SEQ ID NO: 3;(a2) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 3, and having nicotinamide phosphoribosyltransferase activity; (a3) ​​a polypeptide having an amino acid sequence represented by SEQ ID NO: 5 and, at the C-terminal side of the amino acid sequence, an amino acid sequence represented by SEQ ID NO: 6; or (a4) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 5 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 6, and having nicotinamide phosphoribosyltransferase activity; the nicotinamide mononucleotide adenylyltransferase is encoded by a modified nadR gene introduced into the bacterium, and the modified nadR gene encodes a polypeptide having a nicotinamide riboside kinase domain and a nicotinamide mononucleotide adenylyltransferase domain but no DNA-binding domain, the nicotinamide riboside kinase domain is: (b1) a polypeptide consisting of an amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7, or (b2) a polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7 and having nicotinamide riboside kinase activity, and the nicotinamide mononucleotide adenylyltransferase domain is: (b3) a polypeptide consisting of an amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7, or (b4) A method for producing nicotinamide adenine dinucleotide, which is a polypeptide having 90% or more sequence similarity with the amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7 and having nicotinamide mononucleotide adenylyltransferase activity.[4] The method for producing nicotinamide adenine dinucleotide according to [3], wherein the bacterium expresses an exogenous or endogenous transporter gene, and the synthesized nicotinamide adenine dinucleotide is secreted outside the bacterium. [5] The transporter is selected from the group consisting of: (c1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8; (c2) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 8 and having the ability to secrete nicotinamide adenine dinucleotide; (c3) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9; (c4) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 9 and having the ability to secrete nicotinamide adenine dinucleotide; (c5) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10; (c6) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 10 and having the ability to secrete nicotinamide adenine dinucleotide; (c7) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11; (c8) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 11 and having the ability to secrete nicotinamide adenine dinucleotide; (c9) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 12; (c10) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 12 and having the ability to secrete nicotinamide adenine dinucleotide; (c11) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13; (c12) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 13 and having the ability to secrete nicotinamide adenine dinucleotide; (c13) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14; (c14) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 14 and having the ability to secrete nicotinamide adenine dinucleotide; (c15) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 15; (c16) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 15 and having the ability to secrete nicotinamide adenine dinucleotide;(c17) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16, (c18) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 16 and having the ability to secrete nicotinamide adenine dinucleotide, (c19) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17, (c20) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 17 and having the ability to secrete nicotinamide adenine dinucleotide, (c21) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 18, (c22) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 18 and having the ability to secrete nicotinamide adenine dinucleotide, (c23) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 19, (c24) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 19 and having the ability to secrete nicotinamide adenine dinucleotide, (c25) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 20, (c26) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:20 and having the ability to secrete nicotinamide adenine dinucleotide, (c27) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:21, (c28) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:21 and having the ability to secrete nicotinamide adenine dinucleotide, (c29) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:22, (c30) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:22 and having the ability to secrete nicotinamide adenine dinucleotide, (c31) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:23, (c32) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:23 and having the ability to secrete nicotinamide adenine dinucleotide, (c33) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:24, (c34) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 24 and having the ability to secrete nicotinamide adenine dinucleotide;(c35) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:25; (c36) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:25 and having the ability to secrete nicotinamide adenine dinucleotide; (c37) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:26; (c38) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:26 and having the ability to secrete nicotinamide adenine dinucleotide; (c39) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:27; (c40) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:27 and having the ability to secrete nicotinamide adenine dinucleotide; (c41) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:28; or (c42) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:28 and having the ability to secrete nicotinamide adenine dinucleotide. [6] The method for producing nicotinamide adenine dinucleotide according to [4], wherein the transporter is an MFS transporter having an SLDQ motif or an SLEQ motif in the first transmembrane domain. [7] A bacterial transformant into which an exogenous nicotinamide phosphoribosyltransferase gene has been introduced, wherein the nicotinamide phosphoribosyltransferase is: (a1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 and, at the C-terminal side of the amino acid sequence, the amino acid sequence represented by SEQ ID NO: 3, (a2) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 3, and having nicotinamide phosphoribosyltransferase activity, (a3) ​​a polypeptide having the amino acid sequence represented by SEQ ID NO: 5 and the amino acid sequence represented by SEQ ID NO: 6, or(a4) A transformed microorganism having a region consisting of an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 5, and an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 6 at the C-terminal side of the amino acid sequence, and a polypeptide having nicotinamide phosphoribosyltransferase activity. [8] The transformed microorganism of [7] above, further comprising a modified nadR gene introduced therein, wherein the modified nadR gene encodes a polypeptide modified to have a nicotinamide riboside kinase domain and a nicotinamide mononucleotide adenylyltransferase domain but no DNA-binding domain, wherein the nicotinamide riboside kinase domain is: (b1) a polypeptide consisting of an amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7, or (b2) a polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7 and having nicotinamide riboside kinase activity, and wherein the nicotinamide mononucleotide adenylyltransferase domain is: (b3) a polypeptide consisting of an amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7, or (b4) a polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7 and having nicotinamide mononucleotide adenylyltransferase activity. [9] Furthermore, an exogenous or endogenous transporter gene is introduced, and the transporter is: (c1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8; (c2) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 8 and having the ability to secrete nicotinamide adenine dinucleotide; (c3) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9; (c4) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 9 and having the ability to secrete nicotinamide adenine dinucleotide; (c5) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10;(c6) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 10 and having the ability to secrete nicotinamide adenine dinucleotide, (c7) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11, (c8) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 11 and having the ability to secrete nicotinamide adenine dinucleotide, (c9) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 12, (c10) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 12 and having the ability to secrete nicotinamide adenine dinucleotide, (c11) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13, (c12) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 13 and having the ability to secrete nicotinamide adenine dinucleotide, (c13) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14, (c14) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 14 and having the ability to secrete nicotinamide adenine dinucleotide, (c15) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 15, (c16) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 15 and having the ability to secrete nicotinamide adenine dinucleotide, (c17) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16, (c18) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 16 and having the ability to secrete nicotinamide adenine dinucleotide, (c19) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17, (c20) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 17 and having the ability to secrete nicotinamide adenine dinucleotide, (c21) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 18, (c22) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 18 and having the ability to secrete nicotinamide adenine dinucleotide, (c23) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 19,(c24) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 19 and having the ability to secrete nicotinamide adenine dinucleotide, (c25) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 20, (c26) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 20 and having the ability to secrete nicotinamide adenine dinucleotide, (c27) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 21, (c28) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 21 and having the ability to secrete nicotinamide adenine dinucleotide, (c29) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22, (c30) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 22 and having the ability to secrete nicotinamide adenine dinucleotide, (c31) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 23, (c32) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:23 and having the ability to secrete nicotinamide adenine dinucleotide, (c33) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:24, (c34) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:24 and having the ability to secrete nicotinamide adenine dinucleotide, (c35) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:25, (c36) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:25 and having the ability to secrete nicotinamide adenine dinucleotide, (c37) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:26, (c38) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:26 and having the ability to secrete nicotinamide adenine dinucleotide, (c39) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:27, (c40) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 27 and having the ability to secrete nicotinamide adenine dinucleotide; (c41) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 28; or(c42) The transformed microorganism according to the above [7] or [8], which is a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 28 and having the ability to secrete nicotinamide adenine dinucleotide.

[10] The transformed microorganism according to the above [7] or [8], which further comprises an exogenous or endogenous transporter gene introduced therein, and the transporter is an MFS transporter having an SLDQ motif or SLEQ motif in the first transmembrane domain.

[0011] The present invention provides a transformant capable of producing NMN and NAD in large quantities. Furthermore, NMN and NAD can be produced in high yields simply by culturing the transformant.

[0012] In the present invention and the present specification, unless otherwise specified, "NAD" refers to the oxidized form (NAD ＋ ) and reduced form (NADH).

[0013] In the present invention and the present specification, the term "transformed microorganism" refers to a microorganism into which a desired gene has been introduced so that the structural protein encoded by the gene can be expressed. Furthermore, unless otherwise specified, the term "host" refers to a microorganism into which a desired foreign gene has been introduced.

[0014] <Method for producing NMN and transformed microorganism used therefor> The method for producing NMN in this embodiment is a method for synthesizing NMN within bacterial cells, in which NMN is synthesized from Nam and PRPP using NAMPT, and is characterized by using an enzyme consisting of the NAMPT catalytic domain of Sa-NadV or an enzyme consisting of the NAMPT catalytic domain of Ci-NadV as NAMPT.

[0015] NadV is a protein having two conserved domains, the N-terminal and the C-terminal, and by having both the N-terminal and the C-terminal domains, NadV has NAMPT activity.

[0016] Sa_NadV is a protein consisting of 463 amino acids in total, and its amino acid sequence is registered in the protein database UniProt (https: / / www.uniprot.org) (A0A0F6W0P6). The region from amino acids 8 to 105 of Sa_NadV is the N-terminal domain, and the region from amino acids 176 to 419 is the C-terminal domain.

[0017] Ci_NadV is a protein consisting of 462 amino acids in total, and the amino acid sequence of this full-length protein is registered in the UniProt database (A0A2U1D921). The region from amino acids 3 to 98 of Ci_NadV constitutes the N-terminal domain, and the region from amino acids 171 to 442 constitutes the C-terminal domain.

[0018]

[0019] Specifically, the NAMPT used in the method for producing NMN of this embodiment is a protein having a region consisting of any one of the polypeptides (a1) to (a4) below. (a1) A polypeptide having an amino acid sequence represented by SEQ ID NO: 2 and, at the C-terminal side of said amino acid sequence, an amino acid sequence represented by SEQ ID NO: 3. (a2) A polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 2 and, at the C-terminal side of said amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 3, and having NAMPT activity. (a3) ​​A polypeptide having an amino acid sequence represented by SEQ ID NO: 5 and, at the C-terminal side of said amino acid sequence, an amino acid sequence represented by SEQ ID NO: 6. (a4) A polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 5 and, at the C-terminal side of said amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 6, and having NAMPT activity.

[0020] In the present invention and this specification, the term "polypeptide" refers to a polymer of 20 or more amino acids bonded together by peptide bonds, and the term "peptide" refers to a polymer of 2 to 19 amino acids bonded together by peptide bonds.

[0021] Generally, one or more amino acids can be deleted, substituted, or added to a protein (polypeptide) having some physiological activity without impairing the physiological activity. That is, one or more amino acids can be deleted, substituted, or added to both Sa_NadV and Ci_NadV without losing NAMPT activity.

[0022] In the present invention and this specification, "amino acid deletion in a polypeptide" means that some of the amino acid residues constituting the polypeptide are lost (removed). In the present invention and this specification, "amino acid substitution in a polypeptide" means that an amino acid residue constituting the polypeptide is changed to another amino acid residue. In the present invention and this specification, "amino acid addition in a polypeptide" means that a new amino acid residue is inserted into the polypeptide.

[0023] The sequence similarity between two amino acid sequences can be determined by globally aligning the sequences using the Needleman-Wunsch algorithm, a well-known technique. The amino acid substitution score can be defined using the common matrix BLOSUM62. The similarity between two amino acid sequences is calculated as a percentage, which is the proportion of similar amino acids in the resulting alignment. Similar amino acids are defined as those with positive scores in the substitution score matrix.

[0024] The polypeptide (a1) is not particularly limited as long as it has the N-terminal domain of Sa_NadV (SEQ ID NO: 2) (hereinafter sometimes referred to as the "Sa_NadV N-terminal domain") and the C-terminal domain of Sa_NadV (SEQ ID NO: 3) (hereinafter sometimes referred to as the "Sa_NadV C-terminal domain"). The polypeptide (a1) may be a polypeptide in which the Sa_NadV N-terminal domain and the Sa_NadV C-terminal domain are directly linked, or both domains may be linked via a linker consisting of a peptide or polypeptide of one or more amino acids. Furthermore, the polypeptide (a1) may have a peptide or polypeptide linked to the N-terminus of the Sa_NadV N-terminal domain or the C-terminus of the Sa_NadV C-terminal domain. An example of the polypeptide (a1) is the full-length Sa_NadV protein.

[0025] The sequence similarity of the amino acid sequence of (a2) to the amino acid sequence represented by SEQ ID NO: 2 is not particularly limited as long as it is 90% or more but less than 100%, but is preferably 95% or more but less than 100%, and more preferably 98% or more but less than 100%. Furthermore, the sequence similarity of the amino acid sequence of (a2) to the amino acid sequence represented by SEQ ID NO: 2 may be 70% or more, or may be 80% or more.

[0026] The sequence similarity of the amino acid sequence of (a2) to the amino acid sequence represented by SEQ ID NO: 3 is not particularly limited as long as it is 90% or more but less than 100%, but is preferably 95% or more but less than 100%, and more preferably 98% or more but less than 100%. Furthermore, the sequence similarity of the amino acid sequence of (a2) to the amino acid sequence represented by SEQ ID NO: 3 may be 70% or more, or may be 80% or more.

[0027] The polypeptide (a2) may be a polypeptide having an amino acid sequence having 90% to less than 100%, preferably 95% to less than 100%, and more preferably 98% to less than 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence at the C-terminus of the amino acid sequence having 90% to less than 100%, preferably 95% to less than 100%, and more preferably 98% to less than 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 3, and having NAMPT activity.

[0028] The sequence identity between two amino acid sequences is determined by aligning the two amino acid sequences with gaps at the insertion and deletion sites so that the most corresponding amino acid residues are identical, and calculating the percentage of identical amino acid residues relative to the entire amino acid sequence excluding gaps in the resulting alignment. The sequence identity between amino acid sequences can be determined using various homology search software known in the art. For example, the sequence identity value of amino acid sequences can be calculated based on the alignment obtained using the known homology search software BLASTP.

[0029] The polypeptide (a2) is not particularly limited as long as it has an N-terminal domain (hereinafter sometimes referred to as the "mutant Sa_NadV N-terminal domain") consisting of an amino acid sequence with 90% or more sequence similarity to the Sa_NadV N-terminal domain (SEQ ID NO: 2) and a C-terminal domain (hereinafter sometimes referred to as the "mutant Sa_NadV C-terminal domain") consisting of an amino acid sequence with 90% or more sequence similarity to the Sa_NadV C-terminal domain (SEQ ID NO: 3), and has NAMPT activity. The polypeptide (a2) may be a polypeptide in which the mutant Sa_NadV N-terminal domain and the mutant Sa_NadV C-terminal domain are directly linked, or both domains may be linked via a linker consisting of a peptide or polypeptide of one or more amino acids. Furthermore, the polypeptide (a2) may have a peptide or polypeptide linked to the N-terminus of the mutant Sa_NadV N-terminal domain or the C-terminus of the mutant Sa_NadV C-terminal domain. Examples of the polypeptide (a2) include a polypeptide consisting of an amino acid sequence having 90% or more sequence similarity to the amino acid sequence of the full-length protein of Sa-NadV (SEQ ID NO: 1) and having NAMPT activity.

[0030] The polypeptide (a3) ​​is not particularly limited as long as it has the N-terminal domain of Ci_NadV (SEQ ID NO: 5) (hereinafter sometimes referred to as the "Ci_NadV N-terminal domain") and the C-terminal domain of Ci_NadV (SEQ ID NO: 6) (hereinafter sometimes referred to as the "Ci_NadV C-terminal domain"). The polypeptide (a3) ​​may be a polypeptide in which the Ci_NadV N-terminal domain and the Ci_NadV C-terminal domain are directly linked, or both domains may be linked via a linker consisting of a peptide or polypeptide of one or more amino acids. Furthermore, the polypeptide (a3) ​​may have a peptide or polypeptide linked to the N-terminus of the Ci_NadV N-terminal domain or the C-terminus of the Ci_NadV C-terminal domain. An example of the polypeptide (a3) ​​is the full-length Ci_NadV protein.

[0031] The sequence similarity of the amino acid sequence of (a4) to the amino acid sequence represented by SEQ ID NO: 5 is not particularly limited as long as it is 90% or more but less than 100%, but is preferably 95% or more but less than 100%, and more preferably 98% or more but less than 100%. Furthermore, the sequence similarity of the amino acid sequence of (a4) to the amino acid sequence represented by SEQ ID NO: 5 may be 70% or more, or may be 80% or more.

[0032] The sequence similarity of the amino acid sequence of (a4) to the amino acid sequence represented by SEQ ID NO: 6 is not particularly limited as long as it is 90% or more but less than 100%, but is preferably 95% or more but less than 100%, and more preferably 98% or more but less than 100%. Furthermore, the sequence similarity of the amino acid sequence of (a4) to the amino acid sequence represented by SEQ ID NO: 6 may be 70% or more, or may be 80% or more.

[0033] The polypeptide (a4) may be a polypeptide having an amino acid sequence having 90% to less than 100%, preferably 95% to less than 100%, and more preferably 98% to less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and an amino acid sequence at the C-terminus of the amino acid sequence having 90% to less than 100%, preferably 95% to less than 100%, and more preferably 98% to less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, and having NAMPT activity.

[0034] The polypeptide (a4) is not particularly limited as long as it has an N-terminal domain (hereinafter sometimes referred to as the "mutant Ci_NadV N-terminal domain") consisting of an amino acid sequence with 90% or more sequence similarity to the Ci_NadV N-terminal domain (SEQ ID NO: 5) and a C-terminal domain (hereinafter sometimes referred to as the "mutant Ci_NadV C-terminal domain") consisting of an amino acid sequence with 90% or more sequence similarity to the Ci_NadV C-terminal domain (SEQ ID NO: 6), and has NAMPT activity. The polypeptide (a4) may be a polypeptide in which the mutant Ci_NadV N-terminal domain and the mutant Ci_NadV C-terminal domain are directly linked, or both domains may be linked via a linker consisting of a peptide or polypeptide of one or more amino acids. Furthermore, the polypeptide (a4) may have a peptide or polypeptide linked to the N-terminus of the mutant Ci_NadV N-terminal domain or the C-terminus of the mutant Ci_NadV C-terminal domain. Examples of the polypeptide (a4) include a polypeptide consisting of an amino acid sequence having 90% or more sequence similarity to the amino acid sequence of the full-length protein of Ci_NadV (SEQ ID NO: 1) and having NAMPT activity.

[0035] The polypeptides (a1) to (a4) may have various functional domains not found in Sa-NadV or Ci-NadV, as long as they retain NAMPT activity in host cells. Examples of such functional domains include various signal peptides and various tag peptides. These signal peptides and tag peptides can be appropriately selected from various signal peptides and tag peptides commonly used in the production of recombinant proteins. Examples of such tag peptides include tags commonly used in the expression or purification of recombinant proteins, such as His tags, HA (hemagglutinin) tags, Myc tags, and Flag tags.

[0036] In the method for producing NMN in this embodiment, NMN is synthesized by a transformed microorganism (hereinafter sometimes referred to as a "NAMPT-introduced microorganism") in which a gene encoding a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) has been introduced into a host bacterium. The polynucleotide encoding the protein having a NAMPT catalytic domain consisting of the polypeptides (a1) to (a4) is not particularly limited, but it is preferable to select degenerate codons that are frequently used in the host. Modifying the nucleotide sequence encoding the polypeptide of interest to codons frequently used in the host without changing the amino acid sequence of the polypeptide can be achieved by known gene sequence mutation techniques or artificial gene synthesis methods.

[0037] A NAMPT-introduced microorganism can be obtained by introducing into a host bacterium an expression vector incorporating a gene encoding a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) in a state in which the NAMPT can be expressed in the host cell. Such an expression vector can be produced by known gene sequence mutation techniques or artificial gene synthesis methods. For example, the expression vector can be produced by incorporating, from upstream, an expression cassette consisting of DNA having a promoter sequence, a polynucleotide encoding a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4), and DNA having a terminator sequence into a commonly used plasmid. Specific examples of such promoters include the T7 promoter, lambda PR promoter, lac promoter, tac promoter, and pBAD / AraC promoter. The polynucleotide encoding a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) or an expression cassette containing the same into an expression vector can be incorporated using known genetic engineering techniques, or a commercially available expression vector construction kit can be used.

[0038] An expression vector for expressing a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) is preferably an expression vector into which a drug resistance gene, etc. is incorporated in addition to an expression cassette for a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4). Examples of such drug resistance genes include an ampicillin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, and a chloramphenicol resistance gene.

[0039] The expression vector into which the expression cassette for a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) is inserted is not particularly limited, and may be, for example, a commonly used vector such as a plasmid or phage. Specific examples include pET, pSTV29, pMW218, pUC18, and pBR322.

[0040] The host bacterium is not particularly limited, and a person skilled in the art can appropriately select and use a bacterium that can be expected to have a similar effect. The bacterium may be a gram-negative bacterium or a gram-positive bacterium. Examples of gram-negative bacteria include Escherichia bacteria such as Escherichia coli. Examples of Gram-positive bacteria include Bacillus bacteria such as Bacillus subtilis, and coryneform bacteria such as Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium tuberculostearicum, Corynebacterium sp. p3-SID1194, Corynebacterium genitalium, Corynebacterium pseudotuberculosis, and Corynebacterium kroppenstedtii. The bacterium used as a host in the method for producing NMN of this embodiment is preferably appropriately selected from bacteria of the genus Escherichia that are commonly used for culture, more preferably Escherichia coli, and even more preferably E. coli K12 and strains derived therefrom, or E. coli B and strains derived therefrom.

[0041] The host bacterium may be a natural bacterium, i.e., a bacterium that has not been artificially genetically modified, or may be a bacterium that has been genetically modified other than by the introduction of an expression cassette for a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4).

[0042] The method for producing a transformed microorganism using an expression vector for expressing a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) is not particularly limited, and can be any method commonly used for producing transformants, such as electroporation, calcium chloride, and PEG (polyethylene glycol) methods.

[0043] The NAMPT-introduced microorganism can be cultured in the same manner as the host bacterium. The NAMPT-introduced microorganism can be cultured, for example, at 18 to 37°C in a medium containing nutrients necessary for growth, such as a carbon source, a nitrogen source, and minerals. As the culture medium, media commonly used for culturing Escherichia coli, such as LB (Lysogeny Broth) medium, minimal salts (M9) medium, TB (Terrific Broth) medium, SOB medium, SOC medium, 2xYT medium, and NZCYM liquid medium, can be used with appropriate modifications as necessary.

[0044] In the method for producing NMN in this embodiment, NMN is synthesized from Nam and PRPP within the cells of the NAMPT-introduced microorganism by the introduced NAMPT (a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4)). Nam and PRPP may be taken up by the NAMPT-introduced microorganism as they are, or Nam and PRPP synthesized within the NAMPT-introduced microorganism may be used. For example, PRPP is synthesized from glucose taken up by the transformed microorganism from an external source, using the metabolic pathway of the transformed microorganism.

[0045] For example, NMN can be produced by culturing a NAMPT-introduced microorganism in a solution containing Nam and glucose under conditions that allow the introduced NAMPT to be expressed. The solution may be a culture medium, but is preferably a buffer containing glucose and Nam, as this makes it easier to purify the produced NMN.

[0046] In a transformed microorganism into which an expression cassette incorporating a promoter that does not have the ability to control the timing of expression, etc., NAMPT (a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4)) encoded by a polynucleotide incorporated downstream of the promoter is constitutively expressed. On the other hand, in a transformed microorganism into which a so-called expression-inducible promoter that induces expression in response to a specific compound, temperature condition, or the like is incorporated, an induction treatment appropriate for the respective expression induction conditions is carried out to express in the transformant a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4).

[0047] <Method for Producing NAD and Transformed Microorganism Used Therefor> The method for producing NAD in this embodiment is a method for synthesizing NAD within bacterial cells, which involves synthesizing NMN from Nam and PRPP using NAMPT, and then synthesizing NAD from the resulting NMN and ATP using NMN-AT. This method is characterized by using, as NAMPT, a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) above, and further using NMN-AT encoded by a nadR gene modified to lack the DNA-binding domain. This modified nadR gene encodes a polypeptide that has been modified to lack the DNA-binding domain but has the nicotinamide riboside kinase domain and the nicotinamide mononucleotide adenyltransferase domain. Herein, the polypeptide encoded by this modified nadR gene is sometimes referred to as a NadR DNA-binding domain-deleted mutant.

[0048] The NMN-AT used in the NAD production method of this embodiment is a mutant of NadR lacking the DNA-binding domain. Compared to expressing the full-length NadR protein or only the NMN-AT domain, NAD productivity is dramatically increased. When a mutant in which only the nicotinamide riboside kinase domain is deleted from the full-length NadR protein is expressed, such an effect of improving NAD productivity cannot be obtained.

[0049] E. coli NadR (Ec_NadR) is a protein consisting of 410 amino acids, and its full-length amino acid sequence has been registered in the UniProt database (P272786). The region from amino acids 7 to 62 of Ec_NadR is a DNA-binding (HTH cro / C1-type) domain, the region from amino acids 63 to 229 is a nicotinamide mononucleotide adenylyltransferase (NMN-AT) domain, and the region from amino acids 230 to 410 is a nicotinamide riboside kinase (Ribosylnicotinamide kinase) domain.

[0050]

[0051] When the NMN-AT used in the NAD production method of this embodiment is a DNA-binding domain-deleted mutant of Ec_NadR, the nicotinamide riboside kinase domain is (b1) or (b2) below, and the NMN-AT domain is (b3) or (b4) below. Examples of the DNA-binding domain include (b5) or (b6) below.

[0052] (b1) A polypeptide consisting of an amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7. (b2) A polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7 and having nicotinamide riboside kinase activity.

[0053] (b3) A polypeptide consisting of an amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7. (b4) A polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7 and having NMN-AT activity.

[0054] (b5) A polypeptide consisting of an amino acid sequence represented by the amino acid sequence consisting of amino acids 7 to 62 of SEQ ID NO: 7. (b6) A polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 7 to 62 of SEQ ID NO: 7 and having DNA binding activity.

[0055] The sequence similarity of the amino acid sequence of the polypeptide (b2) to the amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7 is not particularly limited as long as it is 90% or more but less than 100%, but is preferably 95% or more but less than 100%, and more preferably 98% or more but less than 100%. Furthermore, the sequence similarity of the amino acid sequence of the polypeptide (b2) to the amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7 may be 70% or more, or may be 80% or more.

[0056] The sequence similarity of the amino acid sequence of the polypeptide (b4) to the amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7 is not particularly limited as long as it is 90% or more but less than 100%, but is preferably 95% or more but less than 100%, and more preferably 98% or more but less than 100%. Furthermore, the sequence similarity of the amino acid sequence of the polypeptide (b4) to the amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7 may be 70% or more, or may be 80% or more.

[0057] The sequence similarity of the amino acid sequence of the polypeptide (b6) to the amino acid sequence consisting of amino acids 7 to 62 of SEQ ID NO: 7 is not particularly limited as long as it is 90% or more but less than 100%, but is preferably 95% or more but less than 100%, and more preferably 98% or more but less than 100%. Furthermore, the sequence similarity of the amino acid sequence of the polypeptide (b6) to the amino acid sequence consisting of amino acids 7 to 62 of SEQ ID NO: 7 may be 70% or more, or may be 80% or more.

[0058] The polypeptide (b2) may be a polypeptide that has 90% to less than 100%, preferably 95% to less than 100%, and more preferably 98% to less than 100%, sequence identity with the amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO: 7, and that has nicotinamide riboside kinase activity.

[0059] The polypeptide (b4) may be a polypeptide having 90% to less than 100%, preferably 95% to less than 100%, more preferably 98% to less than 100%, sequence identity with the amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7, and having NMN-AT activity.

[0060] The polypeptide (b6) may be a polypeptide that has 90% to less than 100%, preferably 95% to less than 100%, and more preferably 98% to less than 100%, sequence identity with the amino acid sequence consisting of amino acids 7 to 62 of SEQ ID NO: 7, and that has DNA binding activity.

[0061] A similar effect of improving the NAD yield can be obtained by using a DNA-binding domain-deleted mutant of NadR derived from bacteria such as Salmonella typhimurium or Haemophilus ducreyi instead of the DNA-binding domain-deleted mutant of Ec_NadR from Escherichia coli.

[0062] In the method for producing NAD in this embodiment, NAD is synthesized by a transformed microorganism (hereinafter sometimes referred to as an "NAD-producing microorganism") into which a gene encoding a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) and a modified nadR gene have been introduced into a host bacterium. The NAD-producing microorganism can be produced in the same manner as the NAMPT-introduced microorganism described above, except for the introduction of the modified nadR gene.

[0063] The modified nadR gene is not particularly limited, but it is preferable to select a degenerate codon that is frequently used in the host. An expression vector incorporating the modified nadR gene in a state that allows it to be expressed in a host cell and its introduction into the host can be produced in the same manner as in the above-mentioned NAMPT-introduced microorganism.

[0064] Either the expression vector for the modified nadR gene or the expression vector for the protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) may be introduced into the host first, or they may be introduced into the host simultaneously. Alternatively, an expression vector incorporating both an expression cassette containing the modified nadR gene and an expression cassette containing a gene encoding a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) may be introduced into the host.

[0065] The NAD-producing microorganism used in this embodiment preferably contains, in addition to a gene encoding a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4) and a modified nadR gene, an exogenous or endogenous transporter gene incorporated so as to be expressible. When the exogenous transporter gene is expressed or the endogenous transporter is expressed in greater amounts than in the host bacterium, NAD synthesized within the NAD-producing microorganism is secreted outside the microorganism. This allows relatively pure NAD to be easily obtained simply by recovering the culture medium.

[0066] The exogenous or endogenous transporter gene is not particularly limited as long as it is a transporter that can secrete NAD in the NAD-producing microorganism, and examples thereof include transporters known to use NAD as a substrate and multidrug efflux transporters (MFS transporters) classified as part of the MFS superfamily. MFS transporters are monomeric proteins that can transport small solutes in response to a proton gradient, and many of them consist of 400 to 600 amino acids and have 12 transmembrane α-helix domains (TMs) connected by hydrophilic loops (Non-Patent Documents 5 and 6). MFS transporters are particularly preferred as transporters for which the NAD-producing microorganism used in this embodiment may have an exogenous or endogenous gene.

[0067] Specific examples of transporters other than the MFS transporter for which the NAD-producing microorganism used in this embodiment may have an exogenous or endogenous gene include the region from bases 82 to 366 (SEQ ID NO: 8) of yeast Yia6p / Ndt1p (Todisco et al., J. Biol. Chem., 2006, vol. 281(3), pp. 1524-1531), a transporter known to use NAD as a substrate.Specific examples of MFS transporters that the NAD-producing microorganism used in this embodiment may have an exogenous or endogenous gene for include Ntt4 from Chlamydia trachomatis, which is an MFS transporter known to use NAD as a substrate (Haferkamp et al., Nature, 2004, vol. 432(7017) , p.622-625.) (SEQ ID NO: 13), the 52-252 region of MdfA, an MFS-type multidrug efflux transporter of Escherichia coli (SEQ ID NO: 10), the 19-398 region of HsrA, an MFS transporter of Escherichia coli (SEQ ID NO: 11), the 54-450 region of the MFS transporter of Corynebacterium stasionis, a coryneform bacterium (referred to as Cs_Mfs1) (SEQ ID NO: 9), the 2-288 region of the MFS transporter of Corynebacterium stasionis (Cs_Mfs2) (SEQ ID NO: 13), the 26-422 region of the MFS transporter of Corynebacterium tuberculostearicum (Ctu_Mfs) (SEQ ID NO: 15), the MFS transporter of Corynebacterium sp. p3-SID1194 (Cs_Mfs1) (SEQ ID NO: 16), the 26-422 region of the MFS transporter of Corynebacterium tuberculostearicum (Ctu_Mfs) (SEQ ID NO: 16), the 26-422 region of the MFS transporter of Corynebacterium sp. p3-SID1194 (Cs_Mfs2) (SEQ ID NO: 16), the 26-422 region of the MFS transporter of Corynebacterium tuberculostearicum (Ctu_Mfs ... Examples of such a region include a region from 18 to 414 of the MFS transporter (Cspp_Mfs) (SEQ ID NO: 17), a region from 25 to 421 of the MFS transporter of Corynebacterium genitalium (Cge_Mfs) (SEQ ID NO: 19), a region from 54 to 450 of the MFS transporter of Corynebacterium glutamicum (Cgl_Mfs1) (SEQ ID NO: 21), a region from 50 to 445 of the MFS transporter of Corynebacterium glutamicum (Cgl_Mfs2) (SEQ ID NO: 23), a region from 47 to 443 of the MFS transporter of Corynebacterium pseudotuberculosis (Cpt_Mfs2) (SEQ ID NO: 25), and a region from 68 to 463 of the MFS transporter of Corynebacterium kloppenstettii (Ckr_Mfs) (SEQ ID NO: 27).

[0068] As the MFS transporter, which may have a foreign or endogenous gene in the NAD-producing microorganism used in this embodiment, an MFS transporter having an SLDQ motif or an SLEQ motif in the first transmembrane domain is particularly preferred because of its high efficiency of extracellular secretion of NAD. Among MFS transporters, an MFS transporter having an SLGQ motif in the first transmembrane domain has high NAD secretion efficiency. Furthermore, among MFS transporters having an SLGQ motif, an MFS transporter in which the G (glycine residue) is substituted with an acidic amino acid residue, D (aspartic acid residue) or E (glutamic acid residue), has even higher NAD secretion efficiency.

[0069] In the case of an exogenous or endogenous transporter expressed in an NAD-producing microorganism, the first S (serine), the second L (leucine), and the fourth Q (glutamine) in the SLDQ motif or SLEQ motif of a natural MFS transporter can be substituted with amino acids similar in function and structure, respectively, while still retaining the NAD secretion ability. Examples of amino acids similar to S include T (threonine), etc., examples of amino acids similar to L include V (valine) and I (isoleucine), etc., and examples of amino acids similar to Q include N (asparagine), etc. In addition to MFS transporters having an SLDQ motif or SLEQ motif, exogenous or endogenous transporters expressed in an NAD-producing microorganism may also be MFS transporters having any of the TLDQ motif, SIDQ motif, SVDQ motif, SLDN motif, TLEQ motif, SIEQ motif, SVEQ motif, and SLEN motif.

[0070] Many MFS transporters have conserved domain structures. Data on the domain structures conserved in MFS transporters is compiled in the "MFS Superfamily" (accession number: cl28910) of the NCBI Conserved Domain Database (CDD: https: / / www.ncbi.nlm.nih.gov / cdd). When RPS-BLAST is performed using the amino acid sequence information of a protein as the target with an E-value of 0.01 or less, if a cl28910 domain model is found, the protein can be said to be an MFS transporter. RPS-BLAST can be performed using a known program such as CD-Search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi).

[0071] The MFS transporter having an SLDQ motif or SLEQ motif in the first transmembrane domain may be a natural MFS transporter such as Cgl_Mfs2, Cpt_Mfs2, or Ckr_Mfs2, or may be a mutant in which the SLGQ motif in the natural MFS transporter is replaced with an SLDQ motif or SLEQ motif. Examples of such mutants include Cs_Mfs1 represented by SEQ ID NO: 14. Ｇ６４Ｅ , Ctu_Mfs represented by SEQ ID NO: 16 Ｇ３６Ｅ , Cspp_Mfs represented by SEQ ID NO: 18 Ｇ２８Ｅ , Cge_Mfs represented by SEQ ID NO: 20 Ｇ３５Ｅ , Cgl_Mfs1 represented by SEQ ID NO: 22 Ｇ６４Ｅ In Tables 3 to 7, the motifs are shown in reversed black and white.

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] The exogenous or endogenous transporters to be expressed in the NAD-producing microorganism may be any of the transporters listed in Table 3, as well as transporters into which mutations have been introduced to the extent that the transporter activity is not impaired.

[0078] Specific examples of exogenous or endogenous transporters to be expressed in NAD-producing microorganisms include the following polypeptides (c1) to (c42): (c1) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8. (c2) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 8 and having the ability to secrete NAD. (c3) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9. (c4) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 9 and having the ability to secrete NAD. (c5) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10. (c6) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 10 and having the ability to secrete NAD. (c7) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11. (c8) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 11 and having the ability to secrete NAD. (c9) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 12. (c10) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 12 and having NAD secretion ability. (c11) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13. (c12) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 13 and having NAD secretion ability. (c13) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14. (c14) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 14 and having NAD secretion ability. (c15) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 15. (c16) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 15 and having NAD secretion ability. (c17) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16. (c18) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 16 and having NAD secretion ability. (c19) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 17.(c20) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 17 and having NAD secretion ability. (c21) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 18. (c22) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 18 and having NAD secretion ability. (c23) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 19. (c24) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 19 and having NAD secretion ability. (c25) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 20. (c26) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 20 and having NAD secretion ability. (c27) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 21. (c28) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 21 and having NAD secretion ability. (c29) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22. (c30) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:22 and having NAD secretion ability. (c31) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:23. (c32) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:23 and having NAD secretion ability. (c33) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:24. (c34) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:24 and having NAD secretion ability. (c35) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:25. (c36) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:25 and having NAD secretion ability. (c37) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:26. (c38) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:26 and having NAD secretion ability. (c39) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO:27.(c40) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 27 and having NAD secretion ability. (c41) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 28. (c42) A polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 28 and having NAD secretion ability.

[0079] Examples of polypeptides that have 90% or more sequence similarity to the amino acid sequence represented by any one of SEQ ID NOs: 12, 15, 17, 19, and 21 and have NAD secretion ability include polypeptides consisting of amino acid sequences in which the SLGQ motif in these amino acid sequences has been replaced with a TLGQ motif, a SIGQ motif, a SVGQ motif, a SLGN motif, a TLDQ motif, a SIDQ motif, a SVDQ motif, or a SLDN motif.

[0080] Examples of polypeptides that have 90% or more sequence similarity to the amino acid sequence represented by any one of SEQ ID NOs: 14, 16, 18, 20, and 22 and have NAD secretion ability include polypeptides consisting of amino acid sequences in which the SLEQ motif in these amino acid sequences has been replaced with a TLEQ motif, SIEQ motif, SVEQ motif, SLEN motif, SLDQ motif, TLDQ motif, SIDQ motif, SVDQ motif, or SLDN motif.

[0081] Examples of polypeptides that have 90% or more sequence similarity to the amino acid sequence represented by any one of SEQ ID NOs: 23, 25, and 27 and have NAD secretion ability include polypeptides consisting of amino acid sequences in which the SLDQ motif in these amino acid sequences has been replaced with a TLDQ motif, a SIDQ motif, a SVDQ motif, or a SLDN motif, a SLEQ motif, a TLEQ motif, a SIEQ motif, a SVEQ motif, or a SLEN motif.

[0082] The polynucleotide encoding the exogenous or endogenous transporter is not particularly limited, but it is preferable to select degenerate codons that are frequently used in the host. An expression vector incorporating a gene encoding an exogenous or endogenous transporter in a state capable of being expressed in host cells and introducing the vector into the host can also be produced in the same manner as in the above-mentioned NAMPT-introduced microorganism.

[0083] The NAD-producing microorganism used in this embodiment can be cultured in a medium containing nutrients necessary for growth, such as a carbon source, a nitrogen source, and minerals, at 18 to 37° C., similar to the NAMPT-introduced microorganism. The same medium as described above can be used as the culture medium.

[0084] In the method for producing NAD in this embodiment, NMN is synthesized from Nam and PRPP within the cells of the NAD-producing microorganism by the introduced NAMPT (a protein having a NAMPT catalytic domain consisting of any one of the polypeptides (a1) to (a4)). NAD is synthesized from the resulting NMN and ATP by the NMN-AT encoded by the introduced modified nadR gene. Most of the synthesized NAD is accumulated within the cells, but if an exogenous or endogenous transporter has also been introduced into the NAD-producing microorganism, NAD is secreted outside the cells. Nam, PRPP, and ATP may be taken up by the NAD-producing microorganism as they are, or those synthesized within the NAD-producing microorganism may be used. For example, PRPP is synthesized from glucose taken up by the NAD-producing microorganism from an external source using the metabolic pathway of the NAD-producing microorganism.

[0085] For example, NAD can be produced by culturing an NAD-producing microorganism in a solution containing Nam, glucose, and ATP under conditions in which the introduced NAMPT, the DNA-binding domain-deficient mutant of NadR, and an exogenous or endogenous transporter are expressed. The solution may be a culture medium, but is preferably a buffer containing glucose, Nam, and ATP, because this makes it easier to purify the produced NAD.

[0086] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0087] [Example 1] In the synthesis of NMN, the reaction of synthesizing NMN from PRPP and Nam is rate-limiting in many organisms. Therefore, Shoji et al. (Non-Patent Document 1) selected highly active NAMPTs that catalyze this reaction. However, in substance production in microorganisms, not only the enzyme reaction rate (Vmax) but also K Ｍ The more important indicator is the actual amount of the produced substance that is accumulated, taking into consideration factors such as the product inhibition and the like. Therefore, we selected a new microbial NadV that can significantly accumulate NMN when actually expressed in E. coli.

[0088] As candidates, Cp_NadV derived from Chitinophagaea pinensis, which was shown to have high NMN synthesis activity in the study by Shoji et al., as well as NadVs derived from five other microorganisms were selected from public databases: NadV (Mr_NadV) derived from Meiothermus ruber, Ci_NadV, NadV (Fd_NadV) derived from Fructobacillus durionis, Sa_NadV, and NadV (Af_NadV) derived from Acidithiobacillus ferrivorans.

[0089] DNA fragments were artificially synthesized in which the codons of the gene sequences encoding these NadVs were optimized for E. coli, and these were then introduced into the pET-11a vector to create expression vectors. The resulting expression vectors were transformed into E. coli BL21 (DE3) strain and cultured overnight with shaking at 25°C in LB medium containing 0.1 mM IPTG. The cells were harvested and the OD ６００ The cells were suspended in expression buffer A (30 g / L glucose, 44 mM potassium phosphate, 2 g / L nicotinamide) so that the value became 20, and cultured with shaking at 20°C for 4 hours.

[0090] After cultivation, the bacterial cells were collected, and the culture medium was heated at 90°C for 5 minutes to extract the components within the bacterial cells into a buffer. The amount of NMN in the buffer was measured using a commercially available measurement kit, "CycLex Nampt Colorimetric Assay Kit Ver. 2" (manufactured by MBL). Specifically, the amount of NAD in the buffer was measured by an enzymatic method, and then all of the NMN in the buffer was converted to NAD. The amount of NAD was then measured in the same manner as above to obtain the total amount of NAD and NMN in the buffer. The difference between these two values ​​was taken as the amount of NMN in the buffer.

[0091] The results of measuring the amount of NMN in each transformed microorganism are shown in Table 8. We were able to find Ci_NadV and Sa_NadV that accumulate NMN at higher concentrations in the bacterial cells when expressed in E. coli than Cp_NadV, which was found to be highly active in the study by Shoji et al. We found that NMN can be industrially produced by extracting it from these bacterial cells.

[0092]

[0093] [Example 2] The NadR protein, which catalyzes the reaction of synthesizing NAD from NMN in Escherichia coli, has, in addition to the catalytic domain, a function as a transcriptional regulator (Non-Patent Document 3) and a function as a nicotinamide riboside kinase that synthesizes NMN from nicotinamide riboside (Non-Patent Document 4). Therefore, the following experiment was carried out to clarify which domain of nadR is important for creating Escherichia coli that highly produces NAD.

[0094] DNA fragments encoding the full-length NadR protein (SEQ ID NO: 7), a deletion of the nicotinamide riboside kinase domain (a protein consisting of the amino acid sequence of SEQ ID NO: 7, with the region from positions 230 to 410 deleted), a deletion of the DNA-binding domain (a protein consisting of the amino acid sequence of SEQ ID NO: 7, with the region from positions 7 to 62 deleted), and a deletion of both the nicotinamide riboside kinase domain and the DNA-binding domain (a protein consisting of the amino acid sequence of SEQ ID NO: 7, with the region from positions 7 to 62 deleted and the region from positions 230 to 410 deleted). These synthetic DNA fragments were introduced into the pSTV29 vector, and the resulting expression vector was introduced into the transformed Escherichia coli expressing Sa_NadV prepared in Example 1 for transformation.

[0095] The obtained strain was cultured overnight at 25°C with shaking in LB medium containing 0.1 mM IPTG. The cells were collected and ６００ The cells were suspended in expression buffer B (30 g / L glucose, 44 mM potassium phosphate, 2 g / L nicotinamide, 2 g / L adenosine) so that the value became 20, and cultured with shaking at 20°C for 24 hours.

[0096] After the cultivation, the cells were collected and the culture medium was heated at 90°C for 5 minutes to extract the components in the cells into a buffer. The amounts of NMN and NAD in the buffer were measured using a commercially available measurement kit, "CycLex Nampt Colorimetric Assay Kit Ver. 2" (MBL).

[0097]

[0098] The results of measuring the amounts of NAD and NMN in each transformed microorganism are shown in Table 9. As a result, the amount of NAD accumulated in the cells was significantly increased by co-expressing a mutant in which only the DNA-binding domain (transcription factor domain) was deleted with Sa_NadV. It was demonstrated that NAD can be industrially produced by extracting NAD from these cells.

[0099] [Example 3] Because the condensation reaction of NMN and ATP by NadR is a reversible reaction, it was thought that NAD production could be improved by secreting accumulated NAD extracellularly. Furthermore, secreting NAD extracellularly would be industrially useful in NAD production, as it would allow the elimination of an extraction step. However, a transporter capable of excreting high amounts of accumulated NAD from the bacterial cells during cultivation has not yet been discovered, and so a search for such a transporter was undertaken.

[0100] Specifically, the candidates were Ntt4 of Chlamydia purpura and Yia6p of yeast, which are nucleic acid-nucleic acid antiporters known to take up NAD into cells; MdfA and HsrA of Escherichia coli, which are MFS transporters with broad substrate specificity; and Cs_Mfs1 and Cs_Mfs2, which are MFS transporters of Corynebacterium stassionis, a coryneform bacterium with a proven track record as a nucleic acid producer.

[0101] DNA fragments encoding transporters consisting of the amino acid sequences listed in Table 3 were obtained by artificial synthesis or by PCR cloning from the genomes of the respective microorganisms. These were introduced into the pMW218 vector, and the resulting expression vector was transformed into Escherichia coli transformed with the Sa-NadV and NadR DNA-binding domain-deleted variants prepared in Example 2. For comparison, Escherichia coli transformed with only the expression vector for the NadR DNA-binding domain-deleted variant was also prepared.

[0102] The obtained strain was cultured overnight at 25°C with shaking in LB medium containing 0.1 mM IPTG. The cells were collected and ６００ The cells were suspended in expression buffer B (30 g / L glucose, 44 mM potassium phosphate, 2 g / L nicotinamide, 2 g / L adenosine) so that the value became 20, and cultured with shaking at 20°C for 48 hours.

[0103] The culture supernatants were collected, and the NAD content in each culture supernatant was measured in the same manner as in Example 2.

[0104]

[0105] The results of measuring the amount of NAD in the culture supernatant of each transformed microorganism are shown in Table 10. As a result, it was confirmed that the amount of NAD secreted into the culture supernatant was significantly increased in the transformed microorganisms (conditions 4 to 9) into which the selected transporters had been introduced, compared to the transformed microorganism under condition 3. This indicates that these transporters possess the activity of secreting NAD extracellularly. It was demonstrated that NAD can be industrially produced with high efficiency by purifying these culture supernatants.

[0106] [Example 4] By introducing specific mutations into transporters, it is possible to increase the activity and enhance the productivity of useful substances. For Cs_Mfs1, which showed a significant effect of improving the amount of NAD secreted outside the cells in Example 3, it was examined whether it was possible to further increase the NAD secretion ability by mutation.

[0107] Specifically, a mutation has been reported that enhances the activity of MFS transporters by enhancing interaction with the RxxQG motif conserved in their sequences (Kise et al., Abstracts of the Annual Meeting of the Agricultural Chemical Society of Japan, 2023, 2B04-09, ISSN 2186-7976 2023). Based on this report, it was suggested that the G64E mutation in Cs_Mfs1 may contribute to improving NAD excretion capacity, and we therefore investigated its effect.

[0108] PCR was performed using the Cs_Mfs1 expression vector prepared in Example 3 as a template to obtain a DNA fragment in which the G64E mutation was introduced into Cs_Mfs1 (a fragment containing a base sequence encoding the amino acid sequence (SEQ ID NO: 14) in which the 54th glycine in the amino acid sequence represented by SEQ ID NO: 12 was replaced with glutamic acid). The obtained DNA fragment was cyclized using an "In Fusion Cloning kit" (manufactured by TaKaRa) and then introduced into Escherichia coli. From the obtained clones, clones in which the mutation had been correctly inserted were selected to obtain Cs_Mfs1 (Cs_Mfs1) carrying the G64E mutation. Ｇ６４Ｅ ) expression vector was obtained.

[0109] The obtained expression vector was introduced into the transformed Escherichia coli strain expressing Sa_NadV and a deletion of the DNA-binding domain of nadR prepared in Example 2. The obtained strain was cultured in the same manner as in Example 3, and the amount of NAD in the culture supernatant was measured in the same manner as in Example 3.

[0110]

[0111] The results of measuring the amount of NAD in the culture supernatant of each transformed microorganism are shown in Table 11, along with the results of Condition 8 in Example 3. Ｇ６４Ｅ Under condition 10, in which Cs_Mfs1 was expressed, the amount of NAD in the culture supernatant increased significantly compared to condition 8 in Example 3. This result indicated that the introduction of the G64E mutation improved the NAD secretion activity of Cs_Mfs1. It was demonstrated that more efficient industrial production of NAD is possible by purifying this culture supernatant.

[0112] [Example 5] Cs_Mfs1 used in Example 4 Ｇ６４Ｅ is an MFS transporter in which the SLGQ motif in the first transmembrane domain is replaced with a SLEQ motif. Therefore, we investigated the NAD secretion activity of other MFS transporters when G in the SLGQ motif was replaced with E, an acidic amino acid.

[0113] Specifically, DNA fragments having nucleotide sequences encoding transporters containing the amino acid sequences represented by SEQ ID NOs: 16, 18, 20, and 22 shown in Tables 4 to 6 were introduced into the pMW218 vector, respectively, to obtain expression vectors for these transporters. PCR was performed using the obtained expression vectors as templates, and cyclization was performed using an "In Fusion Cloning kit" (manufactured by TaKaRa). As a result, an expression vector encoding a transporter containing the amino acid sequence represented by SEQ ID NO: 15 was obtained from the expression vector for a transporter containing the amino acid sequence represented by SEQ ID NO: 16; an expression vector encoding a transporter containing the amino acid sequence represented by SEQ ID NO: 17 was obtained from the expression vector for a transporter containing the amino acid sequence represented by SEQ ID NO: 18; an expression vector encoding a transporter containing the amino acid sequence represented by SEQ ID NO: 19 was obtained from the expression vector for a transporter containing the amino acid sequence represented by SEQ ID NO: 20; and an expression vector encoding a transporter containing the amino acid sequence represented by SEQ ID NO: 21 was obtained from the expression vector for a transporter containing the amino acid sequence represented by SEQ ID NO: 22.

[0114] The obtained expression vector was introduced into the transformed Escherichia coli strain expressing Sa_NadV and a deletion of the DNA-binding domain of nadR prepared in Example 2. The obtained strain was cultured in the same manner as in Example 3, and the amount of NAD in the culture supernatant was measured in the same manner as in Example 3.

[0115]

[0116] The amount of NAD in the culture supernatant under each condition is shown in Table 12 as a relative value, with the amount of NAD in the culture supernatant under condition 3 (comparison) set to 1.0. The values ​​in Table 12 are the average value ± standard deviation of three independent trials. For all MFS transporters, the amount of NAD in the culture supernatant was higher when the native MFS transporter had an SLGQ motif, but the G had been replaced with the acidic amino acid E to form an SLEQ motif.

[0117] Example 6 The NAD secretion activity of a native MFS transporter having an SLDQ motif in the first transmembrane domain was examined. Furthermore, the effect of substituting the acidic amino acid D in the motif with the neutral amino acid G on the NAD secretion activity was also examined.

[0118] DNA fragments having nucleotide sequences encoding transporters containing the amino acid sequences represented by SEQ ID NOs: 23, 25, and 27 shown in Tables 6 and 7 were each introduced into the pMW218 vector to obtain expression vectors for these transporters. PCR was performed using the obtained expression vectors as templates, and cyclization was performed using an "In Fusion Cloning kit" (manufactured by TaKaRa). As a result, an expression vector encoding a transporter containing the amino acid sequence represented by SEQ ID NO: 24 was obtained from the expression vector for a transporter containing the amino acid sequence represented by SEQ ID NO: 23; an expression vector encoding a transporter containing the amino acid sequence represented by SEQ ID NO: 26 was obtained from the expression vector for a transporter containing the amino acid sequence represented by SEQ ID NO: 25; and an expression vector encoding a transporter containing the amino acid sequence represented by SEQ ID NO: 28 was obtained from the expression vector for a transporter containing the amino acid sequence represented by SEQ ID NO: 27.

[0119] The obtained expression vector was introduced into the transformed Escherichia coli strain expressing Sa_NadV and a deletion of the DNA-binding domain of nadR prepared in Example 2. The obtained strain was cultured in the same manner as in Example 3, and the amount of NAD in the culture supernatant was measured in the same manner as in Example 3.

[0120]

[0121] The amount of NAD in the culture supernatant under each condition is shown in Table 13 as a relative value, with the amount of NAD in the culture supernatant under condition 3 (comparison) set to 1.0. The values ​​in Table 13 are the mean value ± standard deviation of three independent trials. For all MFS transporters, the culture supernatant of an MFS transporter whose native form had an SLDQ motif had a higher amount of NAD than that of an MFS transporter in which D was replaced with G to form an SLGQ motif.

Claims

1. A method for synthesizing nicotinamide mononucleotide within a bacterial cell, comprising synthesizing nicotinamide mononucleotide from nicotinamide and phosphoribosyl pyrophosphate using nicotinamide phosphoribosyltransferase, wherein the nicotinamide phosphoribosyltransferase is: (a1) a polypeptide having an amino acid sequence represented by SEQ ID NO:2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence represented by SEQ ID NO:3; (a2) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:3, and having nicotinamide phosphoribosyltransferase activity; (a3) ​​a polypeptide having an amino acid sequence represented by SEQ ID NO:5 and, at the C-terminal side of the amino acid sequence, an amino acid sequence represented by SEQ ID NO:6; or (a4) a method for producing nicotinamide mononucleotide, the method comprising the steps of: (a) an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:5; and (b) an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:6 at the C-terminal side of the amino acid sequence; and (c) a polypeptide having nicotinamide phosphoribosyltransferase activity.

2. A method for producing nicotinamide mononucleotide as described in claim 1, wherein the bacterium is cultured in a solution containing glucose and nicotinamide.

3. A method for synthesizing nicotinamide adenine dinucleotide within a bacterial cell, comprising: synthesizing nicotinamide mononucleotide from nicotinamide and phosphoribosyl pyrophosphate using nicotinamide phosphoribosyltransferase; and synthesizing nicotinamide adenine dinucleotide from the resulting nicotinamide mononucleotide and ATP using nicotinamide mononucleotide adenylyltransferase, wherein the nicotinamide phosphoribosyltransferase is: (a1) a polypeptide having an amino acid sequence represented by SEQ ID NO:2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence represented by SEQ ID NO:3; (a2) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:3, and having nicotinamide phosphoribosyltransferase activity; (a3) ​​a polypeptide having an amino acid sequence represented by SEQ ID NO:5 and an amino acid sequence represented by SEQ ID NO:6, at the C-terminal side of the amino acid sequence; or (a4) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:5 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:6, and having nicotinamide phosphoribosyltransferase activity, wherein the nicotinamide mononucleotide adenylyltransferase is encoded by a modified nadR gene introduced into the bacterium, and the modified nadR gene encodes a polypeptide having a nicotinamide riboside kinase domain and a nicotinamide mononucleotide adenylyltransferase domain but no DNA binding domain, and the nicotinamide riboside kinase domain is: (b1) a polypeptide consisting of an amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO:7, or (b2) a polypeptide having 90% or more sequence similarity to an amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO:7, and having nicotinamide riboside kinase activity,and the nicotinamide mononucleotide adenylyltransferase domain is: (b3) a polypeptide consisting of an amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7; or (b4) a polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO: 7 and having nicotinamide mononucleotide adenylyltransferase activity.

4. A method for producing nicotinamide adenine dinucleotide as described in claim 3, wherein the bacterium expresses an exogenous or endogenous transporter gene and the synthesized nicotinamide adenine dinucleotide is secreted outside the bacterial cell.

5. The transporter is selected from the group consisting of: (c1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8; (c2) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 8 and having the ability to secrete nicotinamide adenine dinucleotide; (c3) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9; (c4) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 9 and having the ability to secrete nicotinamide adenine dinucleotide; (c5) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10; (c6) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 10 and having the ability to secrete nicotinamide adenine dinucleotide; (c7) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11; (c8) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 11 and having the ability to secrete nicotinamide adenine dinucleotide; (c9) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 12; (c10) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:12 and having the ability to secrete nicotinamide adenine dinucleotide; (c11) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:13; (c12) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:13 and having the ability to secrete nicotinamide adenine dinucleotide; (c13) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:14; (c14) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:14 and having the ability to secrete nicotinamide adenine dinucleotide; (c15) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:15; (c16) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:15 and having the ability to secrete nicotinamide adenine dinucleotide; (c17) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:16;(c18) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:16 and having the ability to secrete nicotinamide adenine dinucleotide; (c19) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:17; (c20) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:17 and having the ability to secrete nicotinamide adenine dinucleotide; (c21) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:18; (c22) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:18 and having the ability to secrete nicotinamide adenine dinucleotide; (c23) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:19; (c24) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:19 and having the ability to secrete nicotinamide adenine dinucleotide; (c25) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:20; (c26) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:20 and having the ability to secrete nicotinamide adenine dinucleotide; (c27) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:21; (c28) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:21 and having the ability to secrete nicotinamide adenine dinucleotide; (c29) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:22; (c30) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:22 and having the ability to secrete nicotinamide adenine dinucleotide; (c31) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:23; (c32) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:23 and having the ability to secrete nicotinamide adenine dinucleotide; (c33) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:24; (c34) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:24 and having the ability to secrete nicotinamide adenine dinucleotide; (c35) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:25;(c36) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:25 and having the ability to secrete nicotinamide adenine dinucleotide; (c37) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:26; (c38) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:26 and having the ability to secrete nicotinamide adenine dinucleotide; (c39) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:27; (c40) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:27 and having the ability to secrete nicotinamide adenine dinucleotide; (c41) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:28; or (c42) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:28 and having the ability to secrete nicotinamide adenine dinucleotide.

6. The method for producing nicotinamide adenine dinucleotide according to claim 4, wherein the transporter is an MFS transporter having an SLDQ motif or a SLEQ motif in the first transmembrane domain.

7. A transformed microorganism, which is a bacterial transformant into which an exogenous nicotinamide phosphoribosyltransferase gene has been introduced, wherein the nicotinamide phosphoribosyltransferase has a region consisting of: (a1) a polypeptide having an amino acid sequence represented by SEQ ID NO:2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence represented by SEQ ID NO:3; (a2) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:2 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:3, and having nicotinamide phosphoribosyltransferase activity; (a3) ​​a polypeptide having an amino acid sequence represented by SEQ ID NO:5 and, at the C-terminal side of the amino acid sequence, an amino acid sequence represented by SEQ ID NO:6; or (a4) a polypeptide having an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:5 and, at the C-terminal side of the amino acid sequence, an amino acid sequence having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:6, and having nicotinamide phosphoribosyltransferase activity.

8. The transformed microorganism according to claim 7, further comprising a modified nadR gene encoding a polypeptide having a nicotinamide riboside kinase domain and a nicotinamide mononucleotide adenylyltransferase domain but no DNA-binding domain, wherein the nicotinamide riboside kinase domain is: (b1) a polypeptide consisting of an amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO:7, or (b2) a polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 230 to 410 of SEQ ID NO:7 and having nicotinamide riboside kinase activity, and wherein the nicotinamide mononucleotide adenylyltransferase domain is: (b3) a polypeptide consisting of an amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO:7, or (b4) a polypeptide having 90% or more sequence similarity to the amino acid sequence consisting of amino acids 63 to 229 of SEQ ID NO:7 and having nicotinamide mononucleotide adenylyltransferase activity.

9. Furthermore, an exogenous or endogenous transporter gene is introduced, and the transporter is: (c1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8; (c2) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 8 and having the ability to secrete nicotinamide adenine dinucleotide; (c3) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9; (c4) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 9 and having the ability to secrete nicotinamide adenine dinucleotide; (c5) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10; (c6) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 10 and having the ability to secrete nicotinamide adenine dinucleotide; (c7) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11; (c8) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO: 11 and having the ability to secrete nicotinamide adenine dinucleotide; (c9) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 12; (c10) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:12 and having the ability to secrete nicotinamide adenine dinucleotide; (c11) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:13; (c12) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:13 and having the ability to secrete nicotinamide adenine dinucleotide; (c13) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:14; (c14) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:14 and having the ability to secrete nicotinamide adenine dinucleotide; (c15) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:15; (c16) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:15 and having the ability to secrete nicotinamide adenine dinucleotide; (c17) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:16;(c18) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:16 and having the ability to secrete nicotinamide adenine dinucleotide; (c19) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:17; (c20) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:17 and having the ability to secrete nicotinamide adenine dinucleotide; (c21) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:18; (c22) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:18 and having the ability to secrete nicotinamide adenine dinucleotide; (c23) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:19; (c24) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:19 and having the ability to secrete nicotinamide adenine dinucleotide; (c25) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:20; (c26) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:20 and having the ability to secrete nicotinamide adenine dinucleotide; (c27) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:21; (c28) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:21 and having the ability to secrete nicotinamide adenine dinucleotide; (c29) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:22; (c30) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:22 and having the ability to secrete nicotinamide adenine dinucleotide; (c31) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:23; (c32) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:23 and having the ability to secrete nicotinamide adenine dinucleotide; (c33) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:24; (c34) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:24 and having the ability to secrete nicotinamide adenine dinucleotide; (c35) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:25;(c36) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:25 and having the ability to secrete nicotinamide adenine dinucleotide; (c37) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:26; (c38) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:26 and having the ability to secrete nicotinamide adenine dinucleotide; (c39) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:27; (c40) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:27 and having the ability to secrete nicotinamide adenine dinucleotide; (c41) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:28; or (c42) a polypeptide having 90% or more sequence similarity to the amino acid sequence represented by SEQ ID NO:28 and having the ability to secrete nicotinamide adenine dinucleotide. The transformed microorganism according to claim 7 or 8, 10. The transformed microorganism according to claim 7 or 8, further comprising an exogenous or endogenous transporter gene introduced therein, the transporter being an MFS transporter having an SLDQ motif or a SLEQ motif in the first transmembrane domain.

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