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CN-122029287-A - Method for producing NMN and method for producing NAD

CN122029287ACN 122029287 ACN122029287 ACN 122029287ACN-122029287-A

Abstract

Disclosed are a transformed microorganism capable of synthesizing NMN or NAD in high yield, and a method for producing NMN or NAD using the transformed microorganism. The present invention provides a method for producing NMN by synthesizing NMN in bacterial cells, wherein NMN is synthesized from Nam and PRPP by NAMPT having a region comprising an amino acid sequence having a sequence similarity of 90% -100% to the amino acid sequence shown in SEQ ID No.2 and an amino acid sequence having a sequence similarity of 0% -100% to the amino acid sequence shown in SEQ ID No. 3 on the C-terminal side of the amino acid sequence, or an amino acid sequence having a sequence similarity of 90% -100% to the amino acid sequence shown in SEQ ID No. 5 and an amino acid sequence having a sequence similarity of 90% -100% to the amino acid sequence shown in SEQ ID No.6 on the C-terminal side of the amino acid sequence.

Inventors

  • Huang Laiqitai
  • MIZUGUCHI MITSUHIRO
  • Chuan Qishou
  • KOMIYAMA JUN

Assignees

  • 东方酵母工业株式会社

Dates

Publication Date
20260512
Application Date
20241024
Priority Date
20231024

Claims (10)

  1. 1. A method for producing nicotinamide mononucleotide, which is a method for synthesizing nicotinamide mononucleotide in a bacterial cell, wherein, Nicotinamide mononucleotide is synthesized from nicotinamide and phosphoribosyl pyrophosphate by nicotinamide phosphoribosyl transferase, The nicotinamide riboside phosphate transferase has a region consisting of: (a1) A polypeptide having an amino acid sequence represented by SEQ ID NO. 2 and an amino acid sequence represented by SEQ ID NO. 3 on the C-terminal side of the amino acid sequence; (a2) A polypeptide having a nicotinamide riboside transferase activity, which has an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No.2, and an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 3 on the C-terminal side of the amino acid sequence; (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 on the C-terminal side of the amino acid sequence, or (A4) A polypeptide having a nicotinamide riboside transferase activity, which has an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 5, and an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 6 on the C-terminal side of the amino acid sequence.
  2. 2. The method for producing nicotinamide mononucleotide according to claim 1, wherein, The bacteria were cultured in a solution containing glucose and nicotinamide.
  3. 3. A method for producing nicotinamide adenine dinucleotide, which comprises synthesizing nicotinamide adenine dinucleotide in a bacterial cell of a bacterium, Nicotinamide mononucleotide is synthesized from nicotinamide and phosphoribosyl pyrophosphate by nicotinamide phosphoribosyl transferase, Synthesizing nicotinamide adenine dinucleotide from the obtained nicotinamide mononucleotide and ATP by nicotinamide mononucleotide adenyltransferase, The nicotinamide riboside phosphate transferase has a region consisting of: (a1) A polypeptide having an amino acid sequence represented by SEQ ID NO. 2 and an amino acid sequence represented by SEQ ID NO. 3 on the C-terminal side of the amino acid sequence; (a2) A polypeptide having a nicotinamide riboside transferase activity, which has an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No.2, and an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 3 on the C-terminal side of the amino acid sequence; (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 on the C-terminal side of the amino acid sequence, or (A4) A polypeptide having a nicotinamide riboside transferase activity, which has an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 5, and an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 6 on the C-terminal side of the amino acid sequence, The nicotinamide mononucleotide adenylate transferase is introduced into the bacterium encoding a modified nadR gene encoding a polypeptide having a nicotinamide riboside kinase domain and a nicotinamide mononucleotide adenylate transferase domain but not a DNA binding domain, The nicotinamide riboside kinase domain is (B1) A polypeptide comprising the amino acid sequence of 230 th to 410 th amino acids of SEQ ID NO. 7, or (B2) A polypeptide having a nicotinamide riboside kinase activity and having a sequence similarity of 90% or more to an amino acid sequence including amino acids 230 to 410 of SEQ ID NO.7, Said nicotinamide mononucleotide glands the nucleotide transferase domain is (B3) A polypeptide comprising the amino acid sequence comprising amino acids 63 to 229 of SEQ ID NO. 7, or (B4) A polypeptide having a nicotinamide mononucleotide adenyltransferase activity, which has a sequence similarity of 90% or more to the amino acid sequence including amino acids 63 to 229 of SEQ ID NO. 7.
  4. 4. The method for producing nicotinamide adenine dinucleotide according to claim 3, wherein, The bacterium expresses exogenous or endogenous transporter genes, and the synthesized nicotinamide adenine dinucleotide is secreted outside the bacterium.
  5. 5. The method for producing nicotinamide adenine dinucleotide according to claim 4, wherein, The transporter is (C1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 8; (c2) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 8 and having a secretion ability of nicotinamide adenine dinucleotide; (c3) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 9; (c4) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 9 and having a secretion ability of nicotinamide adenine dinucleotide; (c5) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 10; (c6) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 10 and having a secretion ability of nicotinamide adenine dinucleotide; (c7) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 11; (c8) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 11 and having a secretion ability of nicotinamide adenine dinucleotide; (c9) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 12; (c10) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 12 and having a secretion ability of nicotinamide adenine dinucleotide; (c11) A polypeptide comprising the amino acid sequence of SEQ ID NO. 13; (c12) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 13 and having a secretion ability of nicotinamide adenine dinucleotide; (c13) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 14; (c14) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 14 and having a secretion ability of nicotinamide adenine dinucleotide; (c15) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 15; (c16) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 15 and having a secretion ability of nicotinamide adenine dinucleotide; (c17) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 16; (c18) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 16 and having a nicotinamide adenine dinucleotide secretion ability; (c19) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 17; (c20) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 17 and having a secretion ability of nicotinamide adenine dinucleotide; (c21) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 18; (c22) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 18 and having a secretion ability of nicotinamide adenine dinucleotide; (c23) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 19; (c24) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence represented by SEQ ID NO. 19 and having a secretion ability of nicotinamide adenine dinucleotide; (c25) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 20; (c26) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 20 and having a secretion ability of nicotinamide adenine dinucleotide; (c27) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 21; (c28) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 21 and having a secretion ability of nicotinamide adenine dinucleotide; (c29) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 22; (c30) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 22 and having a secretion ability of nicotinamide adenine dinucleotide; (c31) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 23; (c32) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 23 and having a secretion ability of nicotinamide adenine dinucleotide; (c33) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 24; (c34) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 24 and having a secretion ability of nicotinamide adenine dinucleotide; (c35) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 25; (c36) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 25 and having a secretion ability of nicotinamide adenine dinucleotide; (c37) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 26; (c38) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 26 and having a secretion ability of nicotinamide adenine dinucleotide; (c39) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 27; (c40) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 27 and having a secretion ability of nicotinamide adenine dinucleotide; (c41) A polypeptide comprising the amino acid sequence shown in SEQ ID No. 28, or (C42) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 28 and having a secretion ability of nicotinamide adenine dinucleotide.
  6. 6. The method for producing nicotinamide adenine dinucleotide according to claim 4, wherein, The transporter is an MFS transporter having a SLDQ motif or a SLEQ motif in the first transmembrane region.
  7. 7. A transformed microorganism which is a transformant of a bacterium into which a gene for an exogenous nicotinamide riboside transferase has been introduced, wherein, The nicotinamide riboside phosphate transferase has a region consisting of: (a1) A polypeptide having an amino acid sequence represented by SEQ ID NO. 2 and an amino acid sequence represented by SEQ ID NO. 3 on the C-terminal side of the amino acid sequence; (a2) A polypeptide having a nicotinamide riboside transferase activity, which has an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No.2, and an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 3 on the C-terminal side of the amino acid sequence; (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 on the C-terminal side of the amino acid sequence, or (A4) A polypeptide having a nicotinamide riboside transferase activity, which has an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 5, and an amino acid sequence having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID No. 6 on the C-terminal side of the amino acid sequence.
  8. 8. The transformed microorganism according to claim 7, wherein, Further introducing a modified nadR gene, The modified nadR gene encodes a polypeptide having a nicotinamide riboside kinase domain and a nicotinamide mononucleotide adenylate transferase domain but not a DNA binding domain, The nicotinamide riboside kinase domain is (B1) A polypeptide comprising the amino acid sequence of 230 th to 410 th amino acids of SEQ ID NO. 7, or (B2) A polypeptide having a nicotinamide riboside kinase activity and having a sequence similarity of 90% or more to the amino acid sequence including the 230 th to 410 th amino acids of SEQ ID NO. 7, Said nicotinamide mononucleotide glands the nucleotide transferase domain is (B3) A polypeptide comprising the amino acid sequence comprising amino acids 63 to 229 of SEQ ID NO. 7, or (B4) A polypeptide having a nicotinamide mononucleotide adenyltransferase activity, which has a sequence similarity of 90% or more to the amino acid sequence including amino acids 63 to 229 of SEQ ID NO. 7.
  9. 9. The transformed microorganism according to claim 7 or 8, wherein, Further introducing exogenous or endogenous transporter genes, The transporter is (C1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 8; (c2) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 8 and having a secretion ability of nicotinamide adenine dinucleotide; (c3) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 9; (c4) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 9 and having a secretion ability of nicotinamide adenine dinucleotide; (c5) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 10; (c6) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 10 and having a secretion ability of nicotinamide adenine dinucleotide; (c7) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 11; (c8) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 11 and having a secretion ability of nicotinamide adenine dinucleotide; (c9) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 12; (c10) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 12 and having a secretion ability of nicotinamide adenine dinucleotide; (c11) A polypeptide comprising the amino acid sequence of SEQ ID NO. 13; (c12) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 13 and having a secretion ability of nicotinamide adenine dinucleotide; (c13) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 14; (c14) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 14 and having a secretion ability of nicotinamide adenine dinucleotide; (c15) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 15; (c16) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 15 and having a nicotinamide adenine dinucleotide secretion ability; (c17) A polypeptide comprising the amino acid sequence of SEQ ID NO. 16; (c18) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 16 and having a secretion ability of nicotinamide adenine dinucleotide; (c19) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 17; (c20) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 17 and having a secretion ability of nicotinamide adenine dinucleotide; (c21) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 18; (c22) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 18 and having a secretion ability of nicotinamide adenine dinucleotide; (c23) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 19; (c24) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence represented by SEQ ID NO. 19 and having a secretion ability of nicotinamide adenine dinucleotide; (c25) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 20; (c26) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 20 and having a nicotinamide adenine dinucleotide secretion ability; (c27) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 21; (c28) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 21 and having a secretion ability of nicotinamide adenine dinucleotide; (c29) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 22; (c30) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 22 and having a secretion ability of nicotinamide adenine dinucleotide; (c31) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 23; (c32) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 23 and having a secretion ability of nicotinamide adenine dinucleotide; (c33) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 24; (c34) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 24 and having a secretion ability of nicotinamide adenine dinucleotide; (c35) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 25; (c36) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 25 and having a secretion ability of nicotinamide adenine dinucleotide; (c37) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 26; (c38) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 26 and having a secretion ability of nicotinamide adenine dinucleotide; (c39) A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 27; (c40) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 27 and having a secretion ability of nicotinamide adenine dinucleotide; (c41) A polypeptide comprising the amino acid sequence shown in SEQ ID No. 28, or (C42) A polypeptide having a sequence similarity of 90% or more to the amino acid sequence shown in SEQ ID NO. 28 and having a secretion ability of nicotinamide adenine dinucleotide.
  10. 10. The transformed microorganism according to claim 7 or 8, wherein, Further introducing exogenous or endogenous transporter genes, The transporter is an MFS transporter having a SLDQ motif or a SLEQ motif in the first transmembrane region.

Description

Method for producing NMN and method for producing NAD Technical Field The present invention relates to a method for synthesizing Nicotinamide Mononucleotide (NMN) and a method for synthesizing Nicotinamide Adenine Dinucleotide (NAD) by utilizing microorganisms. The present application claims priority based on japanese patent application No. 2023-182293 invented in japan at 10/24 of 2023, the contents of which are incorporated herein by reference. Background With recent development of genetic engineering techniques, a method of producing a microorganism as a reaction system for a synthesis reaction has been applied to industrial mass production. In chemical synthesis, an organic solvent, high-temperature and high-pressure reaction conditions, a heavy metal catalyst, and the like are often required, but by utilizing a metabolic pathway possessed by microorganisms, useful organic compounds can be synthesized relatively inexpensively under mild conditions of normal temperature and normal pressure. For example, NAD is a coenzyme for many oxidoreductases, NMN is an intermediate metabolite of NAD biosynthesis, and various biological functions are reported. Both are compounds that are in high demand, and therefore development of microorganisms capable of producing them in large quantities is demanded. In the synthesis of organic compounds by microorganisms, modification methods have been used, such as introduction of enzyme genes for supplementing deficient metabolic pathways in the microorganism for the synthesis, enhancement of the expression level of enzyme genes for improving metabolic efficiency (flux) of metabolic pathways of the microorganism, introduction of exogenous genes having higher activity, and variants thereof. Multiple synthetic pathways for NAD and NMN are known. For example, in mammalian cells, NMN is synthesized from a phosphorylated ribose donor such as nicotinamide (Nam) or phosphoribosyl pyrophosphate (PRPP) by nicotinamide phosphoribosyl transferase (NAMPT: NMN ribosidase; hereinafter, referred to as NAMPT in the present specification). NAD is synthesized from NMN and Adenosine Triphosphate (ATP) by nicotinamide mononucleotide adenyltransferase (hereinafter, referred to as NMN-AT in the present specification). For this reason, patent document 1 describes that the synthesis efficiency of NMN can be improved by introducing and transforming a gene of NAMPT having a high activity into a microorganism used for the synthesis of NMN and NAD. NAMPT of a microorganism, also called NadV, is a protein having significant homology to NAMPT of a mammal, encoded by nadV gene. For example, non-patent document 1 discloses that a transformant capable of selectively and efficiently producing NMN from glucose and Nam can be obtained by introducing a gene derived from NAMPT of a chitin-binding pine bacterium (Chitinophagaea pinensis) having a high NMN synthesis activity into escherichia coli (ESCHERICHIA COLI). It is known that in bacteria such as escherichia coli, salmonella (Salmonella typhimurium), and haemophilus ducreyi (Haemophilus ducreyi), a reaction of synthesizing NAD from NMN is catalyzed by nadR protein which is a product of nadR gene (non-patent document 2). These NadR proteins are composed of, in addition to a domain having a function of converting NMN into NAD (NMN-AT function), a DNA binding domain having a function as a transcription control factor (non-patent document 3) and a domain having a function as nicotinamide riboside kinase for synthesizing NMN from nicotinamide riboside (hereinafter, sometimes referred to as NR in the present specification) (non-patent document 4). It is considered that the NadR protein binds to DNA in an NAD-dependent manner, and controls expression of a gene involved in synthesis of nicotinamide skeleton (non-patent document 3). However, the effect of the DNA binding domain on the enzyme function and NAD synthesis efficiency is not clear. Prior art literature Patent literature Patent document 1 International publication No. 2020/129997 Non-patent literature Non-patent document 1 Shoji, et al Metabolic Engineering,2021, vol.65, p.167-177. Non-patent literature 2:Raffaelli et al, journal of Bacteriology,1999, vol.181 (17), p.5509-5511. Non-patent document 3:Penfound and Foster,Journal of Bacteriology,1999,vol.181 (2), p.648-655. Non-patent document 4, kurnasov et al, journal of Bacteriology,2002, vol.184 (24), p.6906-6917. Non-patent document 5 Pao et al MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS,1998, vol.62 (1), p.1-34. Non-patent document 6, drew et al, CHEMICAL REVIEWS,2021, vol.121, p.5289-5335. Disclosure of Invention Problems to be solved by the invention The purpose 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. Means for solving the problems As a result of intensive studies to solve the above problems, the present in