Protein with NAMPT activity and method for producing NMN

Abstract

A protein according to the present invention is a protein which is disclosed in any one among [1]-[3], and which has nicotinamide phosphoribosyltransferase activity. [1] A protein comprising an amino acid sequence represented by SEQ ID NO: 3 or 5. [2] A mutated protein comprising an amino acid sequence represented by SEQ ID NO: 3 or 5, wherein 1-20 amino acids are missing, substituted, inserted, or added to the amino acid sequence. [3] A homologous protein comprising an amino acid sequence with at least 90% identity to an amino acid sequence represented by SEQ ID NO: 3 or 5.

Description

[Technical Field]

[0001] The present invention relates to a protein having nicotinamide phosphoribosyltransferase (NAMPT) activity and a method for producing nicotinamide mononucleotide (NMN) using the protein. [Background technology]

[0002] Nicotinamide mononucleotide (NMN) is a precursor of nicotinamide adenine dinucleotide (NAD), which is used as an electron carrier in mammals. It is known to have many functions, such as activating mitochondria and sirtuin genes, and is therefore expected to be a supplement (Non-patent Document 1, Non-patent Document 2).

[0003] Known methods for producing NMN include chemical synthesis (Patent Document 1), enzymatic decomposition of NAD (Non-Patent Document 3), and extraction from yeast (Patent Document 2). However, these methods have issues such as low productivity and high production costs, and a cheaper and more efficient production method has been desired.

[0004] A more efficient method for producing NMN is known, which involves intracellular condensation of nicotinamide (NAM) with phosphoribosyl pyrophosphate (PRPP), which has been produced enzymatically or biologically (Non-Patent Documents 4, 5, 6, and Patent Document 3). Nicotinamide phosphoribosyltransferase (NAMPT) is used in this condensation reaction between nicotinamide and PRPP. NAMPT is also known to be involved in NMN synthesis in humans (Non-Patent Document 7), and is considered to be a key enzyme in NMN synthesis.

[0005] An example of NAMPT is NAMPT derived from Haemophilus ducreyi, as disclosed in Non-Patent Document 4. Non-Patent Document 5 also discloses Napmt derived from Shewanella oneidensis, Sphingopyxis sp. C-1, Chitinophaga pinensis, Homo sapiens, Sus scrofa, Mus musculus, Boleophthalmus pectinirostris, Rhinopithecus roxellana, Pteropus alecto, and Xanthomonas translucens, and discloses that, among these, high NMN productivity is achieved when Napmt derived from Sphingopyxis sp. C-1 or Chitinophaga pinensis is used. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2016 / 160524 [Patent Document 2] International Publication No. 2017 / 022768 [Patent Document 3] International Publication No. 2019 / 065876 [Non-patent literature]

[0007] [Non-Patent Document 1] Michael S. Bonkowski et al, “Slowing aging by design: the rise of NAD+ and sirtuin-activating compounds”, Nature Reviews Molecular Cell Biology 17 (2016) 679-690 [Non-patent document 2] Rui-Xiong Huang et al., “Nicotinamide mononucleotide attenuates glucocorticoid-induced osteogenic inhibition by regulating the SIRT1 / PGC-1α signaling pathway”, Molecular Medicine Reports 22 (2020) 145-154

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

[0008] As mentioned above, there are many known methods for producing NMN from nicotinamide by expressing heterologous NAMPT in microorganisms such as E. coli, but the productivity is not sufficient. To produce NMN more efficiently, it is necessary to discover NAMPT that is highly active and can produce NMN with high efficiency.

[0009] An object of the present invention is to provide a protein with improved nicotinamide phosphoribosyltransferase (NAMPT) activity and a method for producing nicotinamide mononucleotide using the protein. [Means for solving the problem]

[0010] The present inventors have conducted extensive research to provide a novel protein with NAMPT activity, and have found that NAMPT derived from Bisgaardia hudsonensis and Chitinophaga rupis has higher NMN-producing activity than the previously known NAMPT derived from Haemophilus ducreyi and Shewanella oneidensis, leading to the completion of the present invention.

[0011] The present invention relates to the following 1 to 8. 1. A protein having nicotinamide phosphoribosyltransferase activity, which is described in any one of [1] to [3] below: [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 5 [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or 5, in which 1 to 20 amino acids are deleted, substituted, inserted, or added. [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 3 or 5. 2. DNA encoding the protein described in 1 above. 3. The DNA according to 2 above, which is a DNA according to any one of [4] to [6] below. [4] DNA consisting of the base sequence represented by SEQ ID NO: 2 or 4 [5] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 2 or 4. [6] DNA consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 or 4 4. A recombinant DNA containing the DNA described in 2 or 3 above. 5. A transformant obtained by transforming a host cell with the recombinant DNA described in 4 above. 6. The transformant according to 5 above, which has enhanced productivity of nicotinamide mononucleotide (NMN) compared to the host cell. 7. The transformant according to claim 5, wherein the host cell is Escherichia coli. 8. A method for producing nicotinamide mononucleotide (NMN), comprising producing NMN using the transformant according to any one of 5 to 7 above. 9. A method for producing nicotinamide mononucleotide (NMN), which comprises producing NMN using the protein described in 1 above. [Effects of the Invention]

[0012] According to the present invention, a protein having improved nicotinamide phosphoribosyltransferase activity is provided, and an efficient method for producing nicotinamide mononucleotides using the protein can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0013] 1. Protein of the Present Invention The protein of the present invention is a protein having nicotinamide phosphoribosyltransferase activity and described in any one of [1] to [3] below. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 5 [2] A mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or 5, in which 1 to 20 amino acids are deleted, substituted, inserted, or added. [3] A homologous protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 3 or 5.

[0014] Here, the protein consisting of the amino acid sequence represented by SEQ ID NO: 3 is a protein having nicotinamide phosphoribosyltransferase activity derived from Bisgaardia hudsonensis, and the protein consisting of the amino acid sequence represented by SEQ ID NO: 5 is a protein having nicotinamide phosphoribosyltransferase activity derived from Chitinophaga rupis.

[0015] Nicotinamide phosphoribosyltransferase (NAMPT) is an enzyme that condenses nicotinamide (NAM) with phosphoribosylpyrophosphate (5-phospho-α-D-ribose 1-diphosphate, PRPP) to produce β-nicotinamide mononucleotide (compound name: [(2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methylhydrogen phosphate; β-NMN).

[0016] Nicotinamide phosphoribosyltransferase (NAMPT) activity refers to the activity of synthesizing β-NMN from NAM and PRPP, or the activity of synthesizing β-NMN using NAM, PRPP, ATP, and water molecules as substrates.

[0017] A mutant protein is a protein obtained by artificially deleting or substituting amino acid residues in a parent protein, or by artificially inserting or adding amino acid residues into the protein. In the mutant protein of [2], "deleted, substituted, inserted, or added amino acids" means that 1 to 20 amino acids have been deleted, substituted, inserted, or added at any position in the amino acid sequence represented by SEQ ID NO: 3 or 5, and may include, for example, deletion, substitution, insertion, or addition of 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid.

[0018] The amino acids to be substituted, inserted, or added may be natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0019] Examples of amino acids that can be substituted for each other are shown below. Amino acids in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine D group: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3-hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

[0020] Homologous proteins are proteins found in organisms existing in nature, and refer to a group of proteins that are derived from the same protein in evolutionary origin. Homologous proteins are similar in structure and function to each other. The amino acid sequence of the homologous protein [3] desirably has an identity of 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more, more preferably 96% or more, 97% or more, or 98% or more, and even more preferably 99% or more, to the amino acid sequence represented by SEQ ID NO: 3 or 5.

[0021] The identity of amino acid sequences or nucleotide sequences can be determined using the algorithm BLAST by Karlin and Altschul [Pro. Nat. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)]. Based on this algorithm, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing a nucleotide sequence using BLASTN based on BLAST, parameters are, for example, score = 100 and word length = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, parameters are, for example, score = 50 and word length = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0022] The mutant protein of [2] or the homologous protein of [3] above, NAMPT The activity can be confirmed, for example, by the following method. First, a recombinant DNA having a DNA encoding a protein whose activity is to be confirmed is prepared by the method described below. Next, the recombinant DNA is subjected to the following steps: NAMPTA microorganism lacking NAMPT activity or β-NMN production activity, for example, Escherichia coli W3110 strain lacking DNA encoding nicotinamidase (pncA gene), DNA encoding nicotinamide mononucleotide amidase (pncC gene), DNA encoding acid phosphatase (aphA gene), DNA encoding 5'-nucleotidase / UDP-hydrolase (ushA gene), and DNA encoding nicotinamide riboside phosphorylase (deoD gene), is transformed and cultured. Nicotinamide is added to the medium to produce β-NMN. Finally, β-NMN is detected in the culture supernatant using HPLC, as described below. The presence of NAMPT activity can be confirmed by detecting β-NMN.

[0023] 2. DNA of the present invention The DNA of the present invention is a DNA that encodes the protein of the present invention, that is, the protein of [1] above, the mutant protein of [2], or the homologous protein of [3].

[0024] Specific examples of the DNA of the present invention include DNAs described in any one of [4] to [6] below. [4] DNA consisting of the base sequence represented by SEQ ID NO: 2 or 4 [5] A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 2 or 4. [6] DNA consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 or 4

[0025] Here, the DNA represented by sequence number 2 is a codon-optimized DNA for expression in Escherichia coli of the base sequence (sequence number 1) of the gene encoding the nicotinamide phosphoribosyltransferase derived from Visgardia fuzonensis represented by sequence number 3, and the DNA represented by sequence number 4 is a gene encoding the nicotinamide phosphoribosyltransferase derived from Chitinophaga lapis represented by sequence number 5.

[0026] An example of a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 is a DNA having the nucleotide sequence represented by SEQ ID NO: 2 or 1, and an example of a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 5 is a DNA having the nucleotide sequence represented by SEQ ID NO: 4. An example of a DNA encoding the mutant protein of 1[2] above or the homologous protein of [3] above is the DNA of [4] or [5] above.

[0027] Regarding the DNA of [5], hybridization refers to the process in which DNA hybridizes to a DNA having a specific base sequence or a part of the DNA. Therefore, the base sequence of the DNA having the specific base sequence or the DNA that hybridizes to a part of the DNA may be a DNA of a length that is useful as a probe for Northern or Southern blot analysis or can be used as an oligonucleotide primer for PCR analysis.

[0028] DNA used as a probe can be at least 100 bases long, preferably at least 200 bases long, and more preferably at least 500 bases long, and DNA used as a primer can be at least 10 bases long, preferably at least 15 bases long.

[0029] Methods for DNA hybridization experiments are well known, and hybridization conditions can be determined and experiments can be performed according to numerous standard textbooks, such as Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Methods for General and Molecular Bacteriology (ASM Press, 1994), and Immunology Methods Manual (Academic Press, 1997).

[0030] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with commercially available hybridization kits, such as the Random Primed DNA Labeling Kit (Roche Diagnostics), which uses a random primed probe to prepare probes and hybridize them under stringent conditions.

[0031] The above-mentioned stringent conditions include, for example, incubating a DNA-immobilized filter and probe DNA overnight at 42°C in a solution containing 50% formamide, 5x SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA, followed by washing the filter in a 0.2x SSC solution at approximately 65°C.

[0032] The various conditions described above can also be achieved by adding or changing blocking reagents used to suppress background in hybridization experiments. The addition of blocking reagents described above may be accompanied by changes in hybridization conditions to suit the conditions.

[0033] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA consisting of a base sequence that has an identity of 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more, more preferably 96% or more, 97% or more, or 98% or more, and even more preferably 99% or more to the base sequence represented by SEQ ID NO: 2 or 4, when calculated based on the above parameters using, for example, a program such as BLAST or FASTA.

[0034] The DNA of the present invention can be obtained, for example, by Southern hybridization to a chromosomal DNA library of a microorganism, preferably the genus Visgardia or Chitinophaga, more preferably Visgardia fuzonensis or Chitinophaga lapis, using a probe that can be designed based on the base sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 5, or by PCR [PCR Protocols, Academic Press (1990)] using primer DNA that can be designed based on the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 5 and the above chromosomal DNA library as a template.

[0035] The DNA of the present invention can also be obtained by, for example, using DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 5 (e.g., DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 or 4), introducing mutations into a portion of the nucleotide sequence encoding 1 to 20 consecutive or non-consecutive amino acid residues located on the DNA by site-directed mutagenesis as described in, for example, Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press (2012)) and Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), and substituting the nucleotide sequence with a nucleotide sequence encoding other amino acid residues. Alternatively, the DNA of the present invention can be obtained using a PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.), etc.

[0036] For example, DNA encoding a mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 3 or 5 as described in 1[2] above can be obtained, for example, by subjecting DNA consisting of the base sequence represented by SEQ ID NO: 2 or 4 as a template to error-prone PCR or the like.

[0037] Alternatively, DNA encoding a mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 3 or 5 in 1[2] above can be obtained by site-specific mutagenesis using PCR, using a pair of PCR primers each having a base sequence at its 5' end designed to introduce the desired mutation (deletion, substitution, insertion or addition) [Gene, 77, 51 (1989)].

[0038] Alternatively, the DNA of the present invention can be obtained by following the instructions provided with a commercially available site-directed mutagenesis kit, such as the PrimeSTAR® Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), which can introduce a mutation (deletion, substitution, insertion, or addition) at the desired site.

[0039] That is, first, a pair of mutagenesis primers is designed, with a 15-base overlap at the 5' end, using a plasmid containing a nucleotide sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition) as a template. The overlapping portion contains the desired mutation. Next, PCR is performed using the mutagenesis primers and a plasmid containing the nucleotide sequence into which the desired mutation is to be introduced as a template. The resulting amplified fragment is transformed into Escherichia coli, yielding a plasmid containing the nucleotide sequence into which the desired mutation has been introduced.

[0040] The DNA encoding a homologous protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 3 or 5 described in 1[3] above can be obtained, for example, by the following method. Specifically, for example, various gene sequence databases are searched for nucleotide sequences having 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more, more preferably 96% or more, 97% or more, or 98% or more, and even more preferably 99% or more identity to the nucleotide sequence represented by SEQ ID NO: 2 or 4, and DNA encoding the homologous protein can be obtained by a method similar to the method for obtaining DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 5 described above, using probe DNA or primer DNA that can be designed based on the nucleotide sequence or amino acid sequence obtained by the search, and a microorganism containing the DNA.

[0041] The identity of the nucleotide sequence or amino acid sequence can be determined by the same method as in 1 above. Furthermore, whether the mutant protein or homologous protein encoded by the DNA of the present invention has NAPRT activity can be confirmed by the same method as in 1 above.

[0042] The DNA of the present invention obtained by the above method can be used as is or cleaved with an appropriate restriction enzyme or the like, and then inserted into a vector by standard methods. The resulting recombinant DNA can then be introduced into host cells, and the base sequence of the DNA can be determined by a commonly used base sequence analysis method, such as the dideoxy method [Proc. Nat. Acad. Sci., USA, 74, 5463 (1977)], or by analysis using a base sequence analyzer such as the Applied Biosystems 3500 Genetic Analyzer or the Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific).

[0043] Examples of vectors that can be used to determine the base sequence of the DNA of the present invention include pBluescriptII KS(+), pPCR-Script Amp SK(+) (both manufactured by Agilent Technologies), pT7Blue (manufactured by Merck Millipore), pCRII (manufactured by Thermo Fisher Scientific), pCR-TRAP (manufactured by Gene Hunter), and pDIRECT [Nucleic Acids Res., 18, 6069 (1990)].

[0044] The host cell may be any cell that can be transformed with the vector and grow, and examples thereof include Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm-, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio Inc.), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, Escherichia coli NM522, etc.

[0045] Any method for introducing DNA into host cells can be used to introduce the recombinant DNA obtained by incorporating the DNA of the present invention into host cells, such as the calcium ion method [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (JP 63-248394 A), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0046] If the result of determining the base sequence shows that the obtained DNA is a partial length, full-length DNA can be obtained by Southern hybridization or the like against a chromosomal DNA library using the partial length DNA as a probe.

[0047] Furthermore, the desired DNA can be prepared by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd., based on the determined DNA base sequence.

[0048] 3. Recombinant DNA of the Present Invention The recombinant DNA of the present invention contains the DNA of the present invention. The recombinant DNA of the present invention is DNA that can autonomously replicate in a host cell, and is DNA that has been incorporated into an expression vector containing a promoter at a position where the DNA of the present invention described above in 2 can be transcribed.

[0049] DNA that can be integrated into a chromosome in a host cell and that contains the DNA of the present invention is also a recombinant DNA of the present invention.

[0050] When the recombinant DNA is capable of being integrated into a chromosome, it may or may not contain a promoter.

[0051] When a prokaryote such as a bacterium is used as a host cell, the recombinant DNA of the present invention is preferably a recombinant DNA comprising a promoter, a ribosome binding sequence, the DNA of the present invention described above in 2, and a transcription termination sequence. It may further comprise a gene that controls the promoter.

[0052] Here, it is preferable to adjust the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon to an appropriate distance, for example, 6 to 18 bases.

[0053] In addition, in the recombinant DNA of the present invention, a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.

[0054] The expression vector is not particularly limited as long as it is a suitable nucleic acid molecule for introducing, amplifying, and expressing the target DNA into a host, and not only plasmids but also, for example, artificial chromosomes, vectors using transposons, and cosmids may be used.

[0055] When a microorganism belonging to the genus Escherichia is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of the expression vector include pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio Inc.), pET21a, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE-60, pQE80L (all manufactured by Qiagen), pET-3, pBluescriptII SK(+), pBluescriptII KS(-) (all manufactured by Agilent Technologies), pKYP10 (Japanese Patent Application Laid-Open No. 110600 / 1983), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, p6378-6385], pPE167 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pPAC31 (WO98 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (JP-A-63-233798), etc.

[0056] When using the above expression vectors, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples of promoters that can be used include promoters derived from Escherichia coli or phages, such as the trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter.Artificially designed and modified promoters such as a promoter consisting of two trp promoters in tandem, the tac promoter, the trc promoter, the lacT5 promoter, the lacT7 promoter, and the letI promoter can also be used.

[0057] When a coryneform bacterium is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of expression vectors include pCG1 (Japanese Patent Publication No. 57-134500), pCG2 (Japanese Patent Publication No. 58-35197), pCG4 (Japanese Patent Publication No. 57-183799), pCG11 (Japanese Patent Publication No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Publication No. 58-105999), pCE51, pCE52, and pCE53 [all Molecular and General Genetics, 196, 175 (1984)].

[0058] When using the above expression vector, any promoter that functions in the cells of coryneform bacteria can be used, but for example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, p674-679 (2000)] can be used.

[0059] When a yeast strain is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0060] When using the above expression vector, any promoter that functions in the cells of a yeast strain may be used, and examples include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0061] The recombinant DNA of the present invention can be produced, for example, by treating the DNA fragment prepared by method 2 above with a restriction enzyme and inserting it downstream of the promoter of the appropriate expression vector described above.

[0062] Here, the expression level of the protein encoded by the DNA of the present invention can be improved by substituting bases in the base sequence of the DNA of the present invention so that the codons are optimal for expression in the host cell. Information on codon usage in host cells is available through public databases.

[0063] 4. Transformant of the Present Invention The transformant of the present invention is a transformant obtained by transforming a host cell with the recombinant DNA described in 3 above, which contains the DNA of the present invention described in 2 above. The transformant of the present invention is preferably a transformant in which productivity of nicotinamide mononucleotide (NMN) is enhanced compared to the host cell.

[0064] The host cell into which the recombinant DNA of the present invention is introduced may be any of prokaryotes, yeast, animal cells, insect cells, plant cells, etc., but is preferably a prokaryote or yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and particularly preferably Escherichia coli.

[0065] These are Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, and Escherichia coli XL2-Blue(XL2-Blue Explanation) Escherichia coli BL21(DE3)pLysS(メルクリリッยยา), Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm―, Escherichia coli coli JM109, Escherichia coli HB101, Escherichia coliCJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (いずれもカラバイाಽ), Escherichia coli W, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli ATCC9637, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, and Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicumExamples of suitable yeast strains include prokaryotes such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and Candida utilis.

[0066] As the host cell, preferably, a bred strain in which nicotinamide or β-nicotinamide mononucleotide degrading activity has been artificially weakened, a bred strain in which productivity of nicotinamide, which is a substrate for the target β-nicotinamide mononucleotide, has been artificially imparted or enhanced, or a bred strain that has been subjected to both.

[0067] Methods for artificially attenuating the nicotinamide or β-nicotinamide mononucleotide degrading activity of cells, particularly microorganisms, used as host cells include methods for attenuating or blocking at least one enzyme having nicotinamide or β-nicotinamide mononucleotide degrading activity.

[0068] Methods for artificially imparting or enhancing the ability (productivity) of cells used as host cells, particularly microorganisms, to produce nicotinamide include (a) a method for alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway that produces the target nicotinamide, (b) a method for enhancing the expression of at least one of the enzymes involved in the biosynthetic pathway that produces the target nicotinamide, (c) a method for increasing the copy number of at least one of the enzyme genes involved in the biosynthetic pathway that produces the target nicotinamide, and (d) a method for weakening or blocking at least one of the metabolic pathways that branch off from the biosynthetic pathway that produces the target nicotinamide to a metabolite other than the target substance, and the above-mentioned known methods can be used alone or in combination.

[0069] Specific examples of the above-mentioned method for weakening nicotinamide or β-nicotinamide mononucleotide decomposition activity and imparting or enhancing productivity of nicotinamide as a substrate include known methods such as various genetic manipulation methods (WO 2018 / 211028).

[0070] Specifically, as described in Non-Patent Document 5, for example, in order to further improve NMN productivity using E. coli as a host cell, the nicotinamide uptake system NiaP, the NMN efflux system PnuC, and the PRPP synthase PrsA may be enhanced.

[0071] Furthermore, as disclosed by William B. Black et al. (Microbial Cell Factories 19 (2020) 150), the NMN degradation system pncC may be disrupted, the NMN synthesis system NRK1 may be enhanced, or the NAD repressor nadR may be disrupted, with the primary purpose being to block the NMN degradation system.

[0072] Grose et al. (Journal of Bacteriology 187 (2005) 4521-4530) reported that in Salmonella, aphA is the NMN decomposition system and pncA is the system that decomposes the substrate nicotinamide. It is predicted that disruption of these enzymes, which are also present in E. coli, will improve NMN productivity.

[0073] Furthermore, Chakwan Siew et al. (Journal of Biological Chemistry 286 (2011) 40365-40375) reported on pncC, an NMN decomposition system, and Yang Liu et al. (Microbial Biotechnology 14 (2021) 2581-2591) mentioned the disruption of purR, a regulator of purine nucleic acids, and the disruption of the AMP-decomposing enzyme amn in order to improve PRPP supply. Patent Document 3 discloses that in addition to the breeding mentioned above, ushA, deoD, rihA, rihB, and rihC have been disrupted to suppress various types of degradation, and therefore it is expected that the disruption of these enzymes will also lead to improved NMN productivity.

[0074] Methods for introducing the recombinant DNA of the above 3 into host cells as an autonomously replicable plasmid include, for example, the above-mentioned calcium ion method, the protoplast method, the electroporation method, the spheroplast method [Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)], and the lithium acetate method [J. Bacteriol., 153, 163 (1983)].

[0075] Methods for integrating recombinant DNA into the chromosome of a host cell include, for example, homologous recombination. Examples of homologous recombination include a method using a plasmid for homologous recombination, which can be prepared by ligating a plasmid DNA carrying a drug resistance gene that cannot autonomously replicate in the host cell to be introduced. Examples of methods using homologous recombination that are frequently used in Escherichia coli include a method in which recombinant DNA is introduced using the homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0076] Furthermore, E. coli in which a target region on the chromosomal DNA of a host cell has been replaced with recombinant DNA can be obtained using a selection method that utilizes the fact that E. coli becomes sensitive to sucrose due to Bacillus subtilis levansucrase incorporated into the chromosome together with recombinant DNA, or a selection method that utilizes the fact that E. coli becomes sensitive to streptomycin when a wild-type rpsL gene is incorporated into E. coli that has a mutant rpsL gene that confers streptomycin resistance [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)].

[0077] Whether the transformant obtained by the above method has the DNA of the present invention described in 2 above can be confirmed, for example, by culturing the transformant in a medium and comparing the amount of target NMN produced and accumulated in the culture with that of the parent strain (host cell). Alternatively, it can be confirmed by preparing an extract containing the protein of the present invention from the culture, adding the extract and two different types of nicotinamide to an aqueous medium, and comparing the amount of NMN produced and accumulated in the aqueous medium with that of the parent strain.

[0078] Examples of such a transformant of the present invention include the transformant described below in the Examples.

[0079] 5. Method for producing nicotinamide mononucleotide of the present invention The method for producing nicotinamide mononucleotide (NMN) of the present invention includes a fermentation method for producing NMN, characterized by culturing a microorganism capable of producing the protein of the present invention described above in 1 in a medium and producing NMN in the culture. The production method may include, for example, producing NMN in the culture, allowing it to accumulate, and collecting NMN from the culture.

[0080] The microorganism used in the fermentation method for producing NMN is preferably the transformant of the present invention described above in 4. Furthermore, it is preferable that the transformant has the ability to produce nicotinamide, which is a substrate for NMN, and / or that the decomposition activity of nicotinamide or NMN has been artificially weakened.

[0081] The transformant in item 4 above can be cultured according to a conventional method used for culturing microorganisms.

[0082] The medium for culturing the transformant may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the transformant and allows the transformant to be cultured efficiently. Any substance that can be utilized by the transformant may be used, for example, sugars such as glucose, fructose, sucrose, molasses containing these, starch or starch hydrolysates, organic acids such as acetic acid or propionic acid, or alcohols such as glycerol, ethanol or propanol.

[0083] Examples of nitrogen sources that can be used include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation bacteria, and digested products thereof.

[0084] Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0085] In the method for producing NMN by fermentation, if the transformant used does not have the ability to produce the substrate nicotinamide, the nicotinamide is added to the medium during cultivation.

[0086] Furthermore, in the method for producing NMN by fermentation, if the transformant used does not have the ability to produce the substrate nicotinamide, instead of adding nicotinamide to the medium during cultivation, nicotinamide may be supplied to the transformant of the present invention by co-culturing a microorganism capable of producing nicotinamide with the transformant of the present invention.

[0087] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture. The culture temperature is usually 15 to 40°C, and the culture time is usually 5 hours to 7 days. The pH of the culture solution during cultivation is usually maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0088] Furthermore, antibiotics such as ampicillin or tetracycline may be added to the medium as needed during culture. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added to the medium when culturing a microorganism transformed with an expression vector using the lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using the trp promoter.

[0089] By culturing as described above, NMN can be produced in the culture, thereby producing NMN. Specifically, for example, NMN can be produced by producing and accumulating NMN in the culture, and then collecting NMN from the culture.

[0090] The obtained NMN can be analyzed by conventional methods such as high-performance liquid chromatography (HPLC). NMN can be collected from the above-mentioned culture or from a processed product of the culture by conventional methods using activated carbon, ion exchange resin, etc. When NMN accumulates within the bacterial cells, for example, the bacterial cells can be disrupted by ultrasound, removed by centrifugation, and the resulting supernatant can be used to collect NMN using activated carbon, ion exchange resin, etc.

[0091] The method for producing NMN of the present invention may also be a method for producing NMN using the protein of the present invention. NMN can be produced by reacting the protein of the present invention with its substrate, nicotinamide.

[0092] Examples of the present invention will be described below, but the present invention is not limited to these examples. [Example]

[0093] [Analysis example] Analysis and quantification of NMN In the examples, analysis and quantification of NMN were performed using an analyzer SPD-20AV (manufactured by Shimadzu Corporation). (Analysis conditions) Column: Shodex Asahipak MN2P-50 4E 4.6Φ, 250 mm, 5 μm (Showa Denko) Column temperature: 30℃ Mobile phase: 50 mM ammonium formate (pH 4.0):acetonitrile = 60:40 (v / v) Flow rate: 0.6ml / min Detection wavelength: 260 nm

[0094] [Example 1] Construction of microorganisms expressing various NAMPTs (1) Obtaining DNA encoding NAMPT Using the DNA listed in the "Template" column of Table 1 as a template, PCR was performed using the "Primer Set" listed in Table 1 to amplify the target DNA fragment. [Table 1]

[0095] The DNA represented by SEQ ID NO:2 is a codon-optimized DNA for expression in Escherichia coli of the nucleotide sequence (SEQ ID NO:1) encoding the nicotinamide phosphoribosyltransferase nadV (hereinafter referred to as "BhnadV") derived from Bisgaardia hudsonensis (SEQ ID NO:3). Chromosomal DNA from Chitinophaga rupis was prepared by standard methods. CrnadV (SEQ ID NO:4) encodes the nadV (SEQ ID NO:5) derived from Chitinophaga rupis (hereinafter referred to as "CrnadV"). The GenBank accession numbers for the genomic DNA of Bisgaardia fuzonensis strain M327 / 99 / 2 are CP016605.1 and for the genomic DNA of Chitinophaga rupis strain DSM 21039 are NZ_FOBB01000001.1.

[0096] PCR was performed using the expression vector pTrc99A (E Amann, B Ochs, KJ Abel 1988 Gene 30;69(2):301-315) as a template and a DNA fragment consisting of the base sequences represented by SEQ ID NOs: 17 and 18 as a primer set to obtain a vector fragment of approximately 4.0 kb. Forward primer: 5'-tgcctggcggcagtagcg-3' (SEQ ID NO: 17) Reverse primer: 5'-ggtctgtttcctgtgtgaaat-3' (SEQ ID NO: 18) The base sequences represented by SEQ ID NOs: 13, 15 and 18, and SEQ ID NOs: 14, 16 and 17 each contain a complementary sequence at the 3' end.

[0097] The resulting amplified DNA fragment containing BhnadV or CrnadV was ligated to a vector fragment using In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing various NAMPTs, pBhnadV and pCrnadV.

[0098] (2) Construction of co-expression plasmid Plasmids for expressing NAMPT were constructed as follows: each of the genes encoding NAMPT obtained in (1) above and a gene encoding Escherichia coli ribose-phosphate diphosphokinase (PrsA) represented by SEQ ID NO: 12 were placed under the trc promoter. The GenBank accession number for the genomic DNA of Escherichia coli BL21 strain is CP053602.1.

[0099] PCR was performed using the expression vector pTrc99A as a template and a primer set consisting of DNA with the nucleotide sequences shown in SEQ ID NOs: 19 and 18 to obtain a trc promoter fragment of approximately 250 bp. PCR was performed using chromosomal DNA of Escherichia coli BL21 strain prepared by a standard method as a template and a primer set consisting of DNA with the nucleotide sequences shown in SEQ ID NOs: 20 and 21 to obtain a prsA fragment of approximately 950 bp. Forward primer: 5'-gcgcgaattgatctggtttgacagcttatcatcg-3' (SEQ ID NO: 19) Reverse primer: 5'-ggtctgtttcctgtgtgaaat-3' (SEQ ID NO: 18) Forward primer: 5'-tttcacacaggaaacagaccatgaagctttttgctggtaacg-3' (SEQ ID NO: 20) Reverse primer: 5'-tttcacacaggaaacagaccatgaagctttttgctggtaacg-3' (SEQ ID NO: 21)

[0100] Using the trc promoter fragment and prsA fragment obtained above as templates, PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 19 and 21 as a primer set to obtain a DNA fragment of approximately 1200 bp (hereinafter referred to as the Ptrc-prsA fragment).

[0101] PCR was performed using pBhnadV and pCrnadV constructed in (1) above as templates and DNA consisting of the base sequences represented by sequence numbers 22 and 23 as primer sets, to obtain vector fragments of approximately 5.5 kb each (hereinafter referred to as pBhnadV fragment and pCrnadV fragment). Forward primer: 5'-gttttgacagcttatcatcgac-3' (SEQ ID NO: 22) Reverse primer: 5'-cagatcaattcgcgctaactc-3' (SEQ ID NO: 23)

[0102] The Ptrc-prsA fragment obtained above was ligated to the pBhnadV fragment or pCrnadV fragment using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct pPrsA-BhnadV and pPrsA-CrnadV, plasmids that co-express various NAMPTs and PrsAs.

[0103] (3) Construction of E. coli carrying the co-expression plasmid The Escherichia coli W3110 strain was transformed with the various co-expression plasmids obtained in (2) above to construct E. coli strains carrying the various plasmids, which were designated W3110 / pPrsA-BhnadV and W3110 / pPrsA-CrnadV, respectively.

[0104] [Comparative Example 1] Construction of a microorganism expressing known NAMPT (1) Obtaining the NAMPT sequence Using the DNA shown in the "Template" column of Table 2 as a template, PCR was performed using the "Primer Set" shown in Table 2 to amplify the target DNA fragment. [Table 2]

[0105] Chromosomal DNA of Shewanella oneidensis was prepared by standard methods. The DNA represented by SEQ ID NO: 6 encodes the nicotinamide phosphoribosyltransferase nadV (hereinafter referred to as "SonadV") derived from Shewanella oneidensis (SEQ ID NO: 7). The DNA represented by SEQ ID NO: 9 is a codon-optimized DNA for expression in Escherichia coli of the nucleotide sequence (SEQ ID NO: 8) encoding the nicotinamide phosphoribosyltransferase nadV (hereinafter referred to as "HdnadV") derived from Haemophilus ducreyi (SEQ ID NO: 10). It was prepared by artificial synthesis. The nucleotide sequences represented by SEQ ID NOs: 24, 26, and 18, and SEQ ID NOs: 25, 27, and 17 each contain complementary sequences at their 5' ends. The GenBank accession number for the genomic DNA of the Shewanella oneidensis MR-1 strain is CP053946.1, and the GenBank accession number for the genomic DNA of the Haemophilus douculei FDAARGOS 297 strain is CP022037.2.

[0106] The resulting amplified DNA fragments containing SonadV or HdnadV were linked to the vector fragment prepared in Example 1(1) using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing various NAMPTs, pSonadV and pHdnadV.

[0107] (2) Construction of co-expression plasmid Plasmids for expressing the genes encoding various NAMPTs and the gene encoding Escherichia coli-derived ribose-phosphate diphosphokinase (PrsA) were constructed as follows, with the genes being placed under the trc promoter.

[0108] PCR was performed using the pSonadV and pHdnadV constructed in (1) above as templates and DNA consisting of the base sequences represented by sequence numbers 22 and 23 as a primer set, to obtain vector fragments of approximately 5.5 kb each (hereinafter referred to as the pSonadV fragment and the pHdnadV fragment).

[0109] The Ptrc-prsA fragment obtained in Example 1(2) was ligated with the pSonadV fragment or the pHdnadV fragment using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids pPrsA-SonadV and pPrsA-HdnadV that co-express various NAMPTs and PrsAs.

[0110] (3) Construction of E. coli carrying the co-expression plasmid The Escherichia coli W3110 strain was transformed with the co-expression plasmids obtained above to construct E. coli strains carrying various plasmids, which were designated W3110 / pPrsA-SonadV and W3110 / pPrsA-HdnadV, respectively.

[0111] [Example 2] Production of NMN The NMN productivity was evaluated for the W3110 / pPrsA-BhnadV strain and the W3110 / pPrsA-CrnadV strain constructed in Example 1. As positive controls, W3110 / pPrsA-SonadV and W3110 / pPrsA-HdnadV constructed in Comparative Example 1 were used.

[0112] Each strain was inoculated into a wide test tube containing 5 mL of LB medium (10 g / L bactotryptone (Difco), 5 g / L yeast extract (Difco), 10 g / L sodium chloride) containing 100 mg / L ampicillin, and cultured with shaking at 30°C for 20 hours. The resulting culture was inoculated at 1% into a wide test tube containing 5 mL of LB medium containing 100 mg / L ampicillin, and cultured with shaking at 30°C for 16 hours. Two hours after the start of culture, IPTG was added to a final concentration of 0.5 mM.

[0113] After the incubation, 5 mL of each culture medium was centrifuged, and the supernatant was removed to obtain wet bacterial cells. The entire amount of wet bacterial cells was suspended in a wide test tube containing 5 mL of reaction medium (potassium dihydrogen phosphate 3 g / L, disodium hydrogen phosphate 6.8 g / L, sodium chloride 0.5 g / L, ammonium chloride 1 g / L, glucose 20 g / L, magnesium sulfate heptahydrate 0.25 g / L, calcium chloride 14.7 mg / L, and NAM 2.6 g / L). The reaction was carried out by shaking at 30°C for 2 hours. After the incubation, the reaction solution was centrifuged, and the supernatant was subjected to NMN analysis. The test was performed three times independently. The results are shown in Table 3. [Table 3]

[0114] These results demonstrate that BhnadV and CrnadV have higher NMN-producing activity than the known NAMPTs SonadV and HdnadV. In particular, BhnadV produced NMN more than 10 times more efficiently than the known NAMPTs, demonstrating that NMN can be produced efficiently using this enzyme.

Claims

1. DNA encoding a protein having nicotinamide phosphoribosyltransferase activity and consisting of the base sequence represented by sequence number 2.

2. A recombinant DNA comprising the DNA of claim 1.

3. A transformant obtained by transforming a host cell with the recombinant DNA according to claim 2.

4. The transformant according to claim 3, which has enhanced productivity of nicotinamide mononucleotide (NMN) compared to the host cell.

5. The transformant according to claim 3 , wherein the host cell is Escherichia coli.

6. A method for producing nicotinamide mononucleotide (NMN), comprising producing NMN using the transformant according to claim 3.

7. A method for producing nicotinamide mononucleotide (NMN), which produces NMN using a protein having nicotinamide phosphoribosyltransferase activity and consisting of an amino acid sequence represented by SEQ ID NO: 3 or 5.

8. A method for producing nicotinamide mononucleotide (NMN), comprising transforming a host cell with DNA encoding a protein having nicotinamide phosphoribosyltransferase activity and comprising a base sequence represented by SEQ ID NO: 2 or 4, to obtain a transformant, which produces NMN.

9. The method described in claim 8, wherein the transformant has enhanced productivity of nicotinamide mononucleotide (NMN) compared to the host cell.

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