WO2023202281A1 - Method for biosynthesis of nmn - Google Patents

Method for biosynthesis of nmn Download PDF

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WO2023202281A1
WO2023202281A1 PCT/CN2023/081819 CN2023081819W WO2023202281A1 WO 2023202281 A1 WO2023202281 A1 WO 2023202281A1 CN 2023081819 W CN2023081819 W CN 2023081819W WO 2023202281 A1 WO2023202281 A1 WO 2023202281A1
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nucleotide sequence
gene
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华君
裴亮
潘月
刘伟
刘超
魏万涛
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四川盈嘉合生科技有限公司
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
    • C12Y603/01005—NAD+ synthase (6.3.1.5)
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Definitions

  • the present invention relates to the field of bioengineering technology, and in particular to a method for biosynthesizing NMN.
  • NAD is the substrate of three types of NAD-consuming enzymes and plays a vital role in the physiological processes of various cells.
  • the bioenergetic state of NAD even determines the life and death of cells.
  • NAD metabolism plays an important role in health and disease states and has gradually attracted people's attention. Therefore, enzymes involved in NAD biosynthesis and metabolism have also become hot drug targets in disease treatment.
  • NMN nicotinamide mononucleotide
  • NMN nicotinamide mononucleotide
  • Nampt nicotinamide phosphate ribose transferase
  • NMN outside the cell needs to be dephosphorylated and converted into nicotinamide ribose (NR) before it can enter the liver cells.
  • NR nicotinamide ribose
  • nicotinamide ribose is rephosphorylated by nicotinamide riboside kinase to generate NMN, and then NMN and ATP combines to produce NAD + .
  • NMN synthesis is mainly carried out through chemical catalysis and enzymatic catalysis.
  • the traditional method for preparing ⁇ -nicotinamide mononucleotide in vitro is chemical synthesis. Dangerous chemicals and a large amount of organic solvents are required to catalyze NMN, which damages the environment and is complicated to operate. It has many reaction steps, many intermediate products, and low yield. The efficiency is low and product purification is difficult.
  • Tanimori et al. used acetyl-protected ribose to condensate with nicotinamide under the catalysis of TMSOTf; for example, Palmarisa et al.
  • the company further improved the process, using nicotinamide, pyrophosphate or its salts and AMP as raw materials, and reacted under the catalysis of nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase to obtain nicotinamide mononuclear Glycoside;
  • the advantage of this process is that it uses phosphoribosyl pyrophosphate as raw material, which reduces the cost.
  • Shangke Bio reported that using D-5-ribose phosphate, ATP and nicotinamide as raw materials, it achieved efficient biocatalytic synthesis of beta by immobilizing active cells containing phosphoribosyl pyrophosphate synthase and nicotinamide phosphoribosyltransferase. -Nicotinamide mononucleotide. Immobilized cells or immobilized enzymes can be reused multiple times, which facilitates purification and reduces production costs. However, the synthesis of the required raw material ATP is relatively expensive, which also increases the production cost of NMN.
  • NMN nicotinamide phosphoribosyltransferase and 5'-phosphoribosyl pyrophosphate (PRPP) synthase in E.
  • PRPP 5'-phosphoribosyl pyrophosphate
  • NMN synthesis involves cellular energy metabolism, and in the future, high-density fermentation can be used to increase the product yield per unit volume.
  • the present invention provides a method for biosynthesizing NMN by recombinant E. coli.
  • the present invention provides the application of any of the following items in preparing NMN:
  • deletion of the nicotinamide nucleotide amidase encoding gene pncC deletion of the nicotinamide mononucleotide adenosyltransferase gene nadR, non-expression of the nicotinamide mononucleotide adenosyltransferase gene nadD and overexpression of NAD + synthase encoding gene nadE;
  • the present invention also provides nicotinic acid phosphoribosyltransferase mutants, which include Q54L and/or D453G site mutations.
  • the nicotinic acid phosphoribosyltransferase mutant has:
  • the present invention also provides nucleic acid molecules encoding the nicotinic acid phosphoribosyltransferase mutants.
  • the nucleic acid molecule is the NadV mu gene, having:
  • nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III), preferably including at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the nucleotide sequence.
  • the invention also provides a combination element, including the overexpressed nucleic acid molecule, the overexpressed ribose phosphate diphosphate kinase encoding gene Prs and the overexpressed ribokinase encoding gene RbsK;
  • the ribose phosphate diphosphate kinase encoding gene Prs has:
  • the ribokinase encoding gene RbsK has:
  • the combination elements further include any of the following:
  • (I) does not include the nicotinamide nucleotide amidase encoding gene pncC, does not include the nicotinamide mononucleotide adenosyltransferase gene nadR;
  • the nicotinamide nucleotide amidase encoding gene pncC has:
  • the nicotinamide mononucleotide adenosyltransferase gene nadR has:
  • the nicotinamide mononucleotide adenosyltransferase gene nadD has:
  • the NAD + synthase encoding gene nadE has:
  • the combination elements include:
  • the method of deleting the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR includes gene knockout; and/or
  • the method of not expressing nicotinamide mononucleotide adenosyltransferase gene nadD includes temperature-sensitively controlling the expression of nicotinamide mononucleotide adenosyltransferase gene nadD through CIts protein and PR/PL promoter.
  • the invention also provides expression vectors, including:
  • the expression vector also includes one or more of pet-28 ⁇ vector, pGEX4T3 vector, pCas9 vector or pTargetF vector.
  • the invention also provides genetically engineered bacteria, which include
  • deletion of the nicotinamide nucleotide amidase encoding gene pncC deletion of the nicotinamide mononucleotide adenosyltransferase gene nadR, non-expression of the nicotinamide mononucleotide adenosyltransferase gene nadD and overexpression of NAD + synthase encoding gene nadE;
  • the gene pncC encoding nicotinamide nucleotide amidase has:
  • the nicotinamide mononucleotide adenosyltransferase gene nadR has:
  • the nicotinamide mononucleotide adenosyltransferase gene nadD has:
  • the NAD + synthase encoding gene nadE has:
  • the niacin phosphoribosyltransferase mutant encoding gene NadV mu has:
  • nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III), preferably including at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the nucleotide sequence.
  • the ribose phosphate diphosphate kinase encoding gene Prs has:
  • the ribokinase encoding gene RbsK has:
  • the invention also provides a method for constructing the genetically engineered bacterium, which includes obtaining the genetically engineered bacterium based on an Escherichia coli host through any of the following:
  • the E. coli host is selected from one or more of JM109, BL21(DE3), Top10, DH5 ⁇ , Rosetta or Rosetta-gamipLysS.
  • the construction method includes:
  • the method of deleting the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR includes gene knockout; and/or
  • the method of not expressing nicotinamide mononucleotide adenosyltransferase gene nadD includes temperature-sensitively controlling the expression of nicotinamide mononucleotide adenosyltransferase gene nadD through CIts protein and PR/PL promoter.
  • the construction method includes:
  • Escherichia coli JM109 knock out the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR gene; and/or
  • CIts protein and PR/PL promoter temperature-sensitively control the expression of the nicotinamide mononucleotide adenyltransferase gene nadD gene; and/or
  • the genetically engineered bacterium is obtained by overexpressing the NadV mu , the ribose phosphate diphosphate kinase encoding gene Prs, and the ribokinase encoding gene RbsK based on the pGEX4T3 vector.
  • the present invention also provides the application of any of the following items in preparing NMN:
  • the present invention also provides a preparation method of NMN, which is based on any of the following items:
  • the preparation method includes the following steps:
  • Step 1 Take the genetically engineered bacteria or the genetically engineered bacteria obtained by the construction method, culture, express, and centrifuge to obtain bacterial cells;
  • Step 2 Using nicotinamide as a raw material and the bacteria described in step 1 as a catalyst, the NMN is catalytically prepared.
  • step 1 includes:
  • the genetically engineered bacteria or the genetically engineered bacteria obtained by the construction method inoculate it into the culture medium, culture it at 37°C and 200rpm until the OD 600 is 0.7, cool it for 10min, add 1mmol/LIPTG, and then inoculate it at 22°C and 200rpm. Expression was induced for 24 hours; the bacterial cells were collected by centrifugation to obtain the bacterial cells.
  • the culture medium includes PYA8 culture medium.
  • composition of the PYA8 medium is (w/v): 0.1% soy peptone, 1% glucose, 1.61% disodium hydrogen phosphate, 0.136% potassium dihydrogen phosphate, 0.05% sodium chloride, 0.5% yeast extract, 1% Sodium acetate, the rest is water, pH 7.0 ⁇ 7.2.
  • the catalyzed reaction system includes: nicotinamide 5 ⁇ 20mmol/L, ribose 10 ⁇ 20mmol/L, ATP 5 ⁇ 10mmol/L, NAD + 5 ⁇ 10mmol/L, Na 2 HPO 4 /NaH 2 PO 4 50mmol/L, sodium acetate 10mmol/L, calcium chloride 1mmol/L, the rest is water, pH 5 ⁇ 8.5; add the genetically engineered bacteria to make the bacterial concentration reach 5 ⁇ 100g/L, 15 ⁇ 22°C, 50-200rpm, protected from light, react for 10-25h to obtain the NMN.
  • the catalyzed reaction system includes: nicotinamide 15mmol/L, ribose 20mmol/L, ATP 5mmol/L, NAD + 10mmol/L, Na 2 HPO 4 /NaH 2 PO 4 50mmol /L, sodium acetate 10mmol/L, calcium chloride 1mmol/L, the rest is water, pH 8.0; add the genetically engineered bacteria to make the bacterial concentration reach 50g/L, 20°C, 100rpm, react in the dark for 20h, obtain The NMN.
  • the present invention uses specific recombinant Escherichia coli whole cells as catalysts for catalytic transformation, and the method is simple; the reaction substrates are nicotinamide and ribose, and the production cost is low; the present invention constructs an NMN biosynthetic pathway that relies on nicotinamide and ribose, so that Relevant enzymes can be expressed efficiently, improving NMN production.
  • the method of the present invention is used for biotransformation, and the functional gene for degrading NMN in Escherichia coli is deleted, so that the produced NMN is not easily degraded, NMN is retained efficiently, and the yield is increased.
  • the NMN yield reaches 2.88g/L.
  • the present invention conducts directed evolution of NadV, the gene encoding the synthetic nicotinic acid phosphoribosyltransferase, so as to optimize the catalytic efficiency of the enzyme and increase the NMN output to 2.88g/L.
  • Figure 1 shows the plasmid map of the gene editing vector pCas9
  • Figure 2 shows the map of pTargetF plasmid
  • Figure 3 shows pncC gene knockout verification
  • Figure 4 shows nadR gene knockout verification
  • Figure 5 shows the verification of temperature-sensitive expression of nadD gene
  • Figure 6 shows the map of pGEX4T3-NadV-Prs-RbsK plasmid.
  • the invention discloses a method for biosynthesizing NMN by recombinant Escherichia coli. Persons skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.
  • the present invention reduces or knocks out the functional gene for degrading NMN in Escherichia coli bacteria, so that the produced NMN is not easily degraded.
  • Gene editing technology was used to knock out the genes of these pathways, and by replacing the promoter with a temperature-sensitive mutant, an engineered strain that could efficiently retain NMN in the bacteria was constructed.
  • the present invention constructs a biosynthetic pathway of NMN that relies on nicotinamide and ribose in this strain. Then, through error-prone PCR screening technology, a NadV mutant was screened out, which can produce NMN more efficiently in this strain system.
  • One of the technical solutions provided by the present invention is a strain that can synthesize and accumulate NMN (nicotinamide) in large quantities.
  • the engineered strain is in the E. coli host and lacks the nicotinamide nucleotide amidase encoding gene pncC and nicotinamide mononucleotide adenosine transfer.
  • nadD and nadR While expressing the enzyme genes nadD and nadR, overexpress the NAD + synthase encoding gene nadE to increase the retention of NMN; express the nicotinic acid phosphoribosyltransferase mutant encoding gene NadV mu , ribose phosphate diphosphate kinase encoding gene Prs and ribose
  • the kinase encoding gene RbsK constructs the NMN biosynthetic pathway that relies on nicotinamide and ribose;
  • the method of deleting and expressing the nadD gene is to temperature-sensitively control the expression of the nadD gene through CIts protein and PR/PL promoter;
  • nadE gene was expressed through the pET-28a vector
  • NadV mu , Prs, and RbsK genes were overexpressed through plasmids;
  • pGEX4T3 vector is used to overexpress NadV mu , Prs, and RbsK genes;
  • nucleotide sequence of the wild-type NadV gene is shown in the sequence list SEQ ID NO: 1;
  • nucleotide sequence of the pncC gene is shown in the sequence list SEQ ID NO: 2;
  • nucleotide sequence of the nadD gene is shown in the sequence list SEQ ID NO: 3;
  • nucleotide sequence of the nadR gene is shown in the sequence list SEQ ID NO: 4;
  • nucleotide sequence of the nadE gene is shown in the sequence list SEQ ID NO: 5;
  • nucleotide sequence of the Prs gene is shown in the sequence list SEQ ID NO: 6;
  • nucleotide sequence of the RbsK gene is shown in the sequence list SEQ ID NO: 7;
  • nucleotide sequence of the gene NadV mu encoding the nicotinic acid phosphoribosyltransferase mutant is shown in the sequence list SEQ ID NO: 11; the amino acid sequence of the nicotinic acid phosphoribosyltransferase mutant is shown in SEQ ID NO: 12 ;
  • E. coli host is selected from JM109, BL21 (DE3), Top 10, DH5 ⁇ , Rosetta, Rosetta-gami pLysS, etc.;
  • the E. coli host is E. coli JM109.
  • the second technical solution provided by the present invention is the construction method of the above-mentioned genetically engineered bacteria, specifically as follows:
  • the Escherichia coli gene editing vector pCas9 was used to edit the genome of the Escherichia coli host cell to construct a knockout strain of pncC and nadR genes (JM109 ⁇ pncC ⁇ nadR). Since the nadD pathway cannot be knocked out, the expression of nadD was controlled through ts temperature-sensitive expression. Construct an engineering strain that expresses nadD in a temperature-sensitive manner (JM109 ⁇ pncC ⁇ nadRnadD(ts)); continue to construct the nadE gene induced by IPTG in the genome; as a chassis cell, this strain can efficiently preserve NMN synthesized by E. coli without being degraded;
  • an expression vector that can efficiently express NadV mu , Prs and RbsK under IPTG induction was constructed, and the expression vector was expressed in the strain obtained in step (1).
  • the third technical solution provided by the present invention is the application of the above-mentioned engineering bacteria in the production of NMN;
  • the reaction system includes: nicotinamide 5 ⁇ 20mmol/L, ribose 10 ⁇ 20mmol/L, ATP 5 ⁇ 10mmol/L, NAD + 5 ⁇ 10mmol/L, Na 2 HPO 4 /NaH 2 PO 4 50mmol/L, sodium acetate 10mmol /L, calcium chloride 1mmol/L, the rest is water pH 5 ⁇ 8.5; add production bacteria to make the bacterial concentration reach 5 ⁇ 100g/L, react at 15 ⁇ 22°C, 50 ⁇ 200rpm, protected from light for 10 ⁇ 25h;
  • NMN output reached 1.31 to 2.88g/L
  • bacterial culture method is as follows:
  • composition of the PYA8 culture medium is (w/v): 0.1% soy peptone, 1% glucose, 1.61% disodium hydrogen phosphate, 0.136% potassium dihydrogen phosphate, 0.05% sodium chloride, 0.5% yeast extract.
  • Paste, 1% sodium acetate, the rest is water, pH 7.0 ⁇ 7.2;
  • NMN bioconversion conditions are: bacterial cell concentration 50g/L, nicotinamide 15mmol/L, ribose 20mmol/L, ATP 5mmol/L, NAD + 10mmol/L, pH 8.0, 20°C, 100rpm, protected from light The reaction was carried out for 20 hours; the NMN output reached 8.36mmol/L.
  • the present invention has the following advantages:
  • the present invention uses specific recombinant Escherichia coli whole cells as catalysts to catalyze transformation, and the method is simple; the reaction substrates are nicotinamide and ribose, and the production cost is low; the present invention constructs an NMN biosynthetic pathway that relies on nicotinamide and ribose, so that Relevant enzymes can be expressed efficiently, improving NMN production.
  • the method of the present invention is used for biotransformation, and the functional gene for degrading NMN in Escherichia coli is deleted, so that the produced NMN is not easily degraded, NMN is retained efficiently, and the yield is increased.
  • the NMN yield reaches 2.88g/L.
  • the present invention conducts directed evolution of NadV, the gene encoding the synthetic nicotinic acid phosphoribosyltransferase, so as to optimize the catalytic efficiency of the enzyme and increase the NMN output to 2.88g/L.
  • the raw materials and reagents used in the method for biosynthesizing NMN by recombinant Escherichia coli provided by the present invention can all be purchased from the market.
  • the E. coli gene editing vector pCas9 was used to edit the genome of E. coli host cell JM109 to construct a knockout strain of pncC and nadR genes (JM109 ⁇ pncC ⁇ nadR). Since the nadD pathway cannot be knocked out, an engineering strain (JM109 ⁇ pncC ⁇ nadR) that expresses nadD in a temperature-sensitive manner was constructed. nadD(ts)); continue to construct the nadE gene induced by IPTG in the genome; as a chassis cell, this strain can efficiently preserve NMN synthesized by E. coli without being degraded;
  • the E. coli gene editing vector pCas9 was transformed into JM109 competent cells to obtain a chassis that stably expresses the CAS9 protein.
  • pTargetF as a vector, insert sgRNA sequence 1 and donor DNA fragment into it to obtain a recombinant plasmid. Transform the recombinant plasmid into JM109 competent cells expressing CAS9 protein to obtain a knockout strain containing the plasmid. After screening, the knockout strain JM109 ⁇ pncC was obtained. .
  • nucleotide sequence of pncC is shown as SEQ ID NO: 2, and the protein ID: AAN81705.1 was obtained from the NCBI database.
  • the pTargetF plasmid is shown in Figure 2.
  • the target sequence is introduced between the promoter and the gRNA scaffold sequence on the pTargetF plasmid, and the target sequence and the gRNA scaffold sequence are connected to form sgRNA sequence 1, and the pTargetF-1 plasmid is obtained.
  • primers for the upstream and downstream homology arms of pncC based on the sequences of about 300 bp upstream and downstream of pncC, and introduced XhoI and PshA I enzyme digestion respectively. site. Obtain the upstream and downstream homology arms through PCR, and then use F3 and R2 primers to obtain the donor DNA fragment through fusion PCR.
  • the sequence is shown in SEQ ID NO: 8.
  • the pTargetF-1 plasmid and donor DNA were double-digested, and the fragments were recovered and then ligated.
  • the JM109 competent cells expressing CAS9 protein obtained in step 1 were transformed, and then coated with chloramphenicol/spectinomycin double antibody plates for screening.
  • the primers were used to perform colony PCR detection on the single clone grown. The results are shown in Figure 3 (the original length is 1312bp (negative), the knockout part is 472bp, and the successful knockout is 840bp (positive)). Positive clones with successful pncC knockout were selected. , and then used chloramphenicol monoresistant medium for continuous subculture to obtain the JM109 ⁇ pncC strain with pTargetF plasmid removed.
  • pTargetF as the vector, insert sgRNA sequence 2 and the donor DNA fragment into it to obtain the recombinant plasmid.
  • the recombinant plasmid is transformed into JM109 ⁇ pncC competent cells to obtain a knockout strain containing the plasmid. After screening, the knockout strain JM109 ⁇ pncC ⁇ nadR is obtained.
  • nucleotide sequence of nadR is shown as SEQ ID NO: 4, and the protein ID: ACI72736.1 was obtained from the NCBI database.
  • step 2 select the gene knockout target, design primers to insert the target sequence into the pTargetF vector to form sgRNA sequence 2, and obtain the pTargetF-2 plasmid.
  • Design primers for the upstream and downstream homology arms of the nadR gene introduce XhoI and PshAI restriction sites, and obtain the two homology arms, and obtain the donor DNA fragment through fusion PCR.
  • the sequence is shown in SEQ ID NO: 9.
  • the pTargetF-2 plasmid and donor DNA were then double-digested, and the fragments were recovered and then ligated.
  • the JM109 ⁇ pncC competent cells obtained in step 2 were transformed and then coated on a chloramphenicol/spectinomycin double-antibody plate.
  • sgRNA sequence 3 and the donor DNA fragment were inserted into it to obtain a recombinant plasmid.
  • the recombinant plasmid was transformed into JM109 ⁇ pncC ⁇ nadR competent cells, and the strain JM109 ⁇ pncC ⁇ nadRnadD(ts) was obtained after screening.
  • nucleotide sequence of nadD is shown as SEQ ID NO: 3, protein ID: CTV94997.1, derived from E.coli.
  • the gene was inserted into the target site, and primers were designed to insert the target sequence into the pTargetF vector to form sgRNA sequence 3 and obtain the pTargetF-3 plasmid.
  • Double-digest the pTargetF-3 plasmid and donor DNA recover the fragments and ligate them, transform the JM109 ⁇ pncC ⁇ nadR competent cells obtained in step 3, and then coat the chloramphenicol/spectinomycin double antibody plate.
  • nucleotide sequence of nadE is shown in SEQ ID NO: 5, protein ID: WP_003037081.1, derived from Francisella tularensis.
  • the fully synthesized nadE gene sequence introduces an EcoR I restriction site at the 5' end and an NcoI restriction site at the 3' end, namely: 5'-CCGGAATTC-nadE-CCATGGATG-3', a total of 765 bp.
  • the pET-28a vector was used to construct a recombinant plasmid. Both the gene fragment and the pET-28a plasmid were subjected to NcoI/EcoR I double enzyme digestion, and the digested fragments were recovered and ligated with T4 ligase.
  • an expression vector that can efficiently express NadV, Prs and RbsK under IPTG induction was constructed, and the expression vector was expressed in the strain obtained in step (1).
  • pGEX-4T-3 vector to construct a recombinant plasmid, obtain the CDS sequences of NadV, Prs and RbsK from the NCBI database (SEQ ID NO: 1, 6, 7), insert the lac operator and tac promoter elements in the middle, and insert them at both ends. BamHII and NotI restriction sites were designed, and a total sequence of 3330 bp was fully synthesized. Then, the synthetic sequence and the pGEX-4T-3 plasmid were double-digested respectively, and the digested fragments were recovered and ligated to construct the pGEX4T3-NadV-Prs-RbsK plasmid, as shown in Figure 6.
  • NadV nicotinic acid phosphoribosyltransferase
  • Prs ribose phosphate diphosphate kinase encoding gene Prs
  • RbsK ribokinase encoding gene
  • the present invention uses error-prone PCR technology to conduct evolutionary screening of NadV, and selects strains whose growth rate decreases in LB culture medium as screening targets.
  • Example 1 using the pGEX4T3-NadV-Prs-RbsK plasmid constructed in Example 1 as a template, design primers at both ends of NadV, introduce BamHI and SgrAI restriction sites respectively, and perform mutation using error-prone PCR.
  • the PCR product and template plasmid were double digested with BamHI/SgrAI, the fragments were recovered and ligated, and then transformed into JM109 ⁇ pncC ⁇ nadRnadD(ts)-nadE competent cells.
  • the nucleotide sequence of the nicotinic acid phosphoribosyltransferase mutant encoding gene NadV mu obtained after point mutation is shown in the sequence list SEQ ID NO: 11, and the amino acid sequence of the nicotinic acid phosphoribosyltransferase mutant is shown in SEQ ID NO: 12 Show.
  • Chromatographic column Phenomenex LunaC18 column (4.6mm ⁇ 250mm 5 ⁇ m);
  • Mobile phase A is an aqueous solution of 0.25% sodium dihydrogen phosphate and 0.2 ⁇ phosphoric acid
  • NMN production yield of the recombinant bacteria JM109 ⁇ pncC ⁇ nadR nadD(ts)-nadE, pGEX-NadV mu -Prs-RbsK constructed by the present invention can reach 8.36mmol/L (approximately 2.88g/L), And the modified nicotinic acid phosphoribosyltransferase mutant can further increase the production of NMN.

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Abstract

The present invention relates to the technical field of bioengineering, and particularly, to a method for biosynthesis of NMN utilizing a genetically engineered bacterium. The genetically engineered bacterium is an Escherichia coli host with deletions of nicotinamide nucleotide amidase encoding gene pncC and nicotinamide mononucleotide adenylyltransferase gene nadR, absence of nicotinamide mononucleotide adenylyltransferase gene nadD expression and overexpression of NAD+ synthase encoding gene nadE, and/or overexpression of nicotinate phosphoribosyltransferase mutant encoding gene NadVmu, overexpression of ribose-phosphate diphosphokinase encoding gene Prs and overexpression of ribokinase encoding gene RbsK. By using the method for genetic transformation, functional genes for degrading nicotinamide-mononucleotide (NMN) in E. coli are knocked out, such that the generated NMN is unlikely to be degraded and efficiently reserved, thus elevating the maximum NMN yield to 2.88 g/L.

Description

一种生物合成NMN的方法A method for biosynthesizing NMN
本申请要求于2022年04月19日提交中国专利局、申请号为202210408487.1、发明名称为“一种生物合成NMN的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims priority to the Chinese patent application submitted to the China Patent Office on April 19, 2022, with the application number 202210408487.1 and the invention title "A method of biosynthesizing NMN", the entire content of which is incorporated into this application by reference. .
技术领域Technical field
本发明涉及生物工程技术领域，特别涉及一种生物合成NMN的方法。The present invention relates to the field of bioengineering technology, and in particular to a method for biosynthesizing NMN.
背景技术Background technique
作为生命体内200多种还原反应的辅酶，NAD是3类NAD消耗酶的底物，在多种细胞的生理过程中起着至关重要的作用，NAD的生物能量状态甚至决定了细胞的生死存亡。同时，NAD的代谢在健康和疾病状态中起着重要作用，逐渐引起人们的关注，因此参与NAD生物合成和代谢的酶也成为了疾病治疗中具有关注热度的药物靶标。As a coenzyme for more than 200 reduction reactions in life, NAD is the substrate of three types of NAD-consuming enzymes and plays a vital role in the physiological processes of various cells. The bioenergetic state of NAD even determines the life and death of cells. At the same time, NAD metabolism plays an important role in health and disease states and has gradually attracted people's attention. Therefore, enzymes involved in NAD biosynthesis and metabolism have also become hot drug targets in disease treatment.
NMN(nicotinamide mononucleotide)，即烟酰胺单核苷酸，是一种自然存在的生物活性核苷酸，NMN作为细胞内参与NAD合成的重要底物，在人体细胞能量生成中扮演重要角色。NMN是人体内原本存在的物质，但随着年龄的增长而减少。在哺乳动物体内，NMN主要由烟酰胺在烟酰胺磷酸核糖转移酶(Nicotinamide phosphate ribose transferase，Nampt)的催化作用下生成，随后NMN在烟酰胺单核苷酸腺苷转移酶的催化下生成NAD+。在细胞外的NMN则需要脱去磷酸转化为烟酰胺核糖(NR)才能进入肝细胞内部，进入胞内后，烟酰胺核糖在烟酰胺核苷激酶的作用下再磷酸化生成NMN，随后NMN和ATP结合生成NAD+。NMN (nicotinamide mononucleotide), nicotinamide mononucleotide, is a naturally occurring biologically active nucleotide. As an important substrate involved in NAD synthesis within cells, NMN plays an important role in the energy generation of human cells. NMN is a substance that originally exists in the human body, but it decreases with age. In mammals, NMN is mainly generated from nicotinamide under the catalysis of nicotinamide phosphate ribose transferase (Nampt), and then NMN generates NAD + under the catalysis of nicotinamide mononucleotide adenosyltransferase. . NMN outside the cell needs to be dephosphorylated and converted into nicotinamide ribose (NR) before it can enter the liver cells. After entering the cell, nicotinamide ribose is rephosphorylated by nicotinamide riboside kinase to generate NMN, and then NMN and ATP combines to produce NAD + .
目前NMN合成主要通过化学催化与酶催化进行。传统的体外制备β-烟酰胺单核苷酸的方法为化学合成法，催化NMN时需要用到危险化学品和大量的有机溶剂，会破坏环境且操作复杂、反应步骤多、中间产物多、得率低、产品纯化困难。比如，Tanimori等人用乙酰基保护的核糖与烟酰胺在TMSOTf的催化下发生缩合反应；又比如Palmarisa等人使用硅烷化试剂对烟酰胺进行硅烷化，然后与乙酰核糖在TMSOTf的催化下进行反应。这些化学合成方法存在成本高、收率低而且化学试剂污染大等问题。At present, NMN synthesis is mainly carried out through chemical catalysis and enzymatic catalysis. The traditional method for preparing β-nicotinamide mononucleotide in vitro is chemical synthesis. Dangerous chemicals and a large amount of organic solvents are required to catalyze NMN, which damages the environment and is complicated to operate. It has many reaction steps, many intermediate products, and low yield. The efficiency is low and product purification is difficult. For example, Tanimori et al. used acetyl-protected ribose to condensate with nicotinamide under the catalysis of TMSOTf; for example, Palmarisa et al. used a silanization reagent to silanize nicotinamide, and then reacted with acetyl ribose under the catalysis of TMSOTf. . These chemical synthesis methods have problems such as high cost, low yield and large pollution of chemical reagents.
至于酶催化，所需原料ATP比较昂贵，生产NMN的成本很高。早在1994年，Jeck等利用二磷酸吡啶核苷酸为原料，在焦磷酸化酶的催化水解下生成烟酰胺单核苷酸。2016年，深圳邦泰公司利用烟酰胺、ATP和核糖为底物，在烟酰胺磷酸核糖转移酶、核糖磷酸焦磷酸激酶以及核糖激酶的催化下生成烟酰胺单核苷酸。2017年，该公司进一步改进工艺，以烟酰胺、焦磷酸或其盐和AMP为原料，在烟酰胺磷酸核糖转移酶和腺膘呤磷酸核糖转移酶的催化作用下发生反应，获得烟酰胺单核苷酸；该工艺优势在于使用磷酸核糖焦磷酸为原料，降低了成本。2018年，尚科生物报道以D-5-磷酸核糖、ATP和烟酰胺为原料，通过固定化含有磷酸核糖焦磷酸合成酶和烟酰胺磷酸核糖转移酶的活性细胞，实现了高效生物催化合成β-烟酰胺单核苷酸。固定化细胞或固定化酶可以重复多次使用，利于纯化，以降低生产成本，但是合成所需原料ATP相对比较昂贵，也增加了NMN的生产成本。As for enzyme catalysis, the required raw material ATP is relatively expensive, and the cost of producing NMN is high. As early as 1994, Jeck et al. used pyridine diphosphate as raw material and hydrolyzed it under the catalysis of pyrophosphorylase to generate nicotinamide mononucleotide. In 2016, Shenzhen Bangtai Company used nicotinamide, ATP and ribose as substrates to generate nicotinamide mononucleotide under the catalysis of nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphate kinase and ribokinase. In 2017, the company further improved the process, using nicotinamide, pyrophosphate or its salts and AMP as raw materials, and reacted under the catalysis of nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase to obtain nicotinamide mononuclear Glycoside; The advantage of this process is that it uses phosphoribosyl pyrophosphate as raw material, which reduces the cost. In 2018, Shangke Bio reported that using D-5-ribose phosphate, ATP and nicotinamide as raw materials, it achieved efficient biocatalytic synthesis of beta by immobilizing active cells containing phosphoribosyl pyrophosphate synthase and nicotinamide phosphoribosyltransferase. -Nicotinamide mononucleotide. Immobilized cells or immobilized enzymes can be reused multiple times, which facilitates purification and reduces production costs. However, the synthesis of the required raw material ATP is relatively expensive, which also increases the production cost of NMN.
因此考虑到食品安全因素以及化学催化与酶催化的环境污染问题和局限性，目前多使用生物合成法制备NMN，即通过构建工程菌使相关酶在大肠杆菌中重组表达，再进行发酵生产。最早由Marinescu等构建基因工程菌发酵合成NMN，该团队将烟酰胺磷酸核糖转移酶和5’-磷酸核糖焦磷酸(Phosphoribosyl pyrophosphate，PRPP)合成酶在大肠杆菌中重组表达，以尼克酰胺和乳糖为底物进行发酵生产，但最终NMN产量比较低，仅为15.4mg/L。随后该团队还开发了分子筛色谱法分离NMN的方法， 但是从色谱图发现，还是有大量尼克酰胺没有转化为NMN。分析可知，NMN合成涉及细胞能量代谢，未来可以通过高密度发酵提高单位体积的产物得率。生物合成的NMN产量比较低，且合成出NMN后有较多的途径分解NMN，最终导致NMN在微生物体内无法保存较高的含量，因此常规的生物合成手段无法解决高效发酵生产NMN的问题。Therefore, considering food safety factors and environmental pollution issues and limitations of chemical catalysis and enzymatic catalysis, biosynthetic methods are currently used to prepare NMN, that is, by constructing engineering bacteria to recombinantly express relevant enzymes in E. coli, and then perform fermentation production. NMN was first synthesized by fermentation of genetically engineered bacteria constructed by Marinescu et al. The team recombinantly expressed nicotinamide phosphoribosyltransferase and 5'-phosphoribosyl pyrophosphate (PRPP) synthase in E. coli, using nicotinamide and lactose as The substrate was fermented for production, but the final NMN yield was relatively low, only 15.4 mg/L. Subsequently, the team also developed a method to separate NMN using molecular sieve chromatography. However, it was found from the chromatogram that there was still a large amount of nicotinamide that was not converted into NMN. The analysis shows that NMN synthesis involves cellular energy metabolism, and in the future, high-density fermentation can be used to increase the product yield per unit volume. The yield of biosynthesized NMN is relatively low, and there are many ways to decompose NMN after synthesis, which ultimately results in the inability of NMN to retain high content in microorganisms. Therefore, conventional biosynthetic methods cannot solve the problem of efficient fermentation to produce NMN.
发明内容Contents of the invention
有鉴于此，本发明提供一种重组大肠杆菌生物合成NMN的方法。In view of this, the present invention provides a method for biosynthesizing NMN by recombinant E. coli.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
本发明提供了以下任意项在制备NMN中的应用：The present invention provides the application of any of the following items in preparing NMN:
(I)、缺失对烟酰胺核苷酸酰胺酶编码基因pncC、缺失烟酰胺单核苷酸腺苷转移酶基因nadR、不表达烟酰胺单核苷酸腺苷转移酶基因nadD和过表达NAD+合酶编码基因nadE；和/或(I), deletion of the nicotinamide nucleotide amidase encoding gene pncC, deletion of the nicotinamide mononucleotide adenosyltransferase gene nadR, non-expression of the nicotinamide mononucleotide adenosyltransferase gene nadD and overexpression of NAD + synthase encoding gene nadE; and/or
(II)、过表达烟酸磷酸核糖转移酶突变体编码基因NadVmu、过表达核糖磷酸二磷酸激酶编码基因Prs和过表达核糖激酶编码基因RbsK。(II), overexpression of the nicotinic acid phosphoribosyltransferase mutant encoding gene NadV mu , overexpression of the ribose phosphate diphosphate kinase encoding gene Prs and overexpression of the ribokinase encoding gene RbsK.
本发明还提供了烟酸磷酸核糖转移酶突变体，其包括Q54L和/或D453G位点突变。The present invention also provides nicotinic acid phosphoribosyltransferase mutants, which include Q54L and/or D453G site mutations.
在本发明的一些具体实施方案中，所述烟酸磷酸核糖转移酶突变体具有：In some embodiments of the invention, the nicotinic acid phosphoribosyltransferase mutant has:
(I)、如SEQ ID NO:12所示的氨基酸序列；或(I), the amino acid sequence shown in SEQ ID NO:12; or
(II)、在如(I)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或(II), a sequence in which one or more amino acids are substituted, deleted, added and/or substituted based on the amino acid sequence shown in (I); or
(III)、与如(I)或(II)所示的氨基酸序列具有至少90％序列同源性的氨基酸序列，优选的，包括具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性的氨基酸序列。(III), an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in (I) or (II), preferably including at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence.
本发明还提供了编码所述烟酸磷酸核糖转移酶突变体的核酸分子。The present invention also provides nucleic acid molecules encoding the nicotinic acid phosphoribosyltransferase mutants.
在本发明的一些具体实施方案中，所述核酸分子为NadVmu基因，具有：In some specific embodiments of the invention, the nucleic acid molecule is the NadV mu gene, having:
(I)、如SEQ ID NO:11所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:11; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列，优选的，包括具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III), preferably including at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the nucleotide sequence.
本发明还提供了组合元件，包括过表达的所述的核酸分子、过表达的核糖磷酸二磷酸激酶编码基因Prs和过表达的核糖激酶编码基因RbsK；The invention also provides a combination element, including the overexpressed nucleic acid molecule, the overexpressed ribose phosphate diphosphate kinase encoding gene Prs and the overexpressed ribokinase encoding gene RbsK;
所述核糖磷酸二磷酸激酶编码基因Prs具有：The ribose phosphate diphosphate kinase encoding gene Prs has:
(I)、如SEQ ID NO:6所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:6; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或 (III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
所述核糖激酶编码基因RbsK具有：The ribokinase encoding gene RbsK has:
(I)、如SEQ ID NO:7所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:7; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III).
在本发明的一些具体实施方案中，所述组合元件还包括如下任意项：In some specific embodiments of the invention, the combination elements further include any of the following:
(I)、不包括对烟酰胺核苷酸酰胺酶编码基因pncC、不包括烟酰胺单核苷酸腺苷转移酶基因nadR；和(I), does not include the nicotinamide nucleotide amidase encoding gene pncC, does not include the nicotinamide mononucleotide adenosyltransferase gene nadR; and
(II)、不表达烟酰胺单核苷酸腺苷转移酶基因nadD；和(II), does not express nicotinamide mononucleotide adenosyltransferase gene nadD; and
(III)、过表达NAD+合酶编码基因nadE；(III), overexpression of the NAD + synthase encoding gene nadE;
所述对烟酰胺核苷酸酰胺酶编码基因pncC具有：The nicotinamide nucleotide amidase encoding gene pncC has:
(I)、如SEQ ID NO:2所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:2; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
所述烟酰胺单核苷酸腺苷转移酶基因nadR具有：The nicotinamide mononucleotide adenosyltransferase gene nadR has:
(I)、如SEQ ID NO:4所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:4; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
所述烟酰胺单核苷酸腺苷转移酶基因nadD具有：The nicotinamide mononucleotide adenosyltransferase gene nadD has:
(I)、如SEQ ID NO:3所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:3; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列； (IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
所述NAD+合酶编码基因nadE具有：The NAD + synthase encoding gene nadE has:
(I)、如SEQ ID NO:5所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:5; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III).
在本发明的一些具体实施方案中，所述组合元件包括：In some embodiments of the invention, the combination elements include:
(I)、所述缺失对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadR的方式包括基因敲除；和/或(1) The method of deleting the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR includes gene knockout; and/or
(II)、所述不表达烟酰胺单核苷酸腺苷转移酶基因nadD的方式包括通过CIts蛋白及PR/PL启动子温敏控制烟酰胺单核苷酸腺苷转移酶基因nadD表达。(II) The method of not expressing nicotinamide mononucleotide adenosyltransferase gene nadD includes temperature-sensitively controlling the expression of nicotinamide mononucleotide adenosyltransferase gene nadD through CIts protein and PR/PL promoter.
本发明还提供了表达载体，包括：The invention also provides expression vectors, including:
(I)、所述核酸分子；或(I), the nucleic acid molecule; or
(II)、所述组合元件。(II) The combination component.
在本发明的一些具体实施方案中，所述表达载体还包括pet-28α载体、pGEX4T3载体、pCas9载体或pTargetF载体中的一种或多种。In some specific embodiments of the invention, the expression vector also includes one or more of pet-28α vector, pGEX4T3 vector, pCas9 vector or pTargetF vector.
本发明还提供了基因工程菌，其包括The invention also provides genetically engineered bacteria, which include
(I)、缺失对烟酰胺核苷酸酰胺酶编码基因pncC、缺失烟酰胺单核苷酸腺苷转移酶基因nadR、不表达烟酰胺单核苷酸腺苷转移酶基因nadD和过表达NAD+合酶编码基因nadE；和/或(I), deletion of the nicotinamide nucleotide amidase encoding gene pncC, deletion of the nicotinamide mononucleotide adenosyltransferase gene nadR, non-expression of the nicotinamide mononucleotide adenosyltransferase gene nadD and overexpression of NAD + synthase encoding gene nadE; and/or
(II)、过表达烟酸磷酸核糖转移酶突变体编码基因NadVmu、过表达核糖磷酸二磷酸激酶编码基因Prs和过表达核糖激酶编码基因RbsK；或 or
(III)、过表达的所述核酸分子；或(III), the overexpressed nucleic acid molecule; or
(IV)、所述组合元件；或(IV), the combination element; or
(V)、所述表达载体。(V), the expression vector.
在本发明的一些具体实施方案中，所述对烟酰胺核苷酸酰胺酶编码基因pncC具有：In some specific embodiments of the invention, the gene pncC encoding nicotinamide nucleotide amidase has:
(I)、如SEQ ID NO:2所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:2; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
所述烟酰胺单核苷酸腺苷转移酶基因nadR具有：The nicotinamide mononucleotide adenosyltransferase gene nadR has:
(I)、如SEQ ID NO:4所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:4; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷 酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), the nucleotide sequence shown in (I) or (II) with one or more nucleosides substituted, deleted or added or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
所述烟酰胺单核苷酸腺苷转移酶基因nadD具有：The nicotinamide mononucleotide adenosyltransferase gene nadD has:
(I)、如SEQ ID NO:3所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:3; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
所述NAD+合酶编码基因nadE具有：The NAD + synthase encoding gene nadE has:
(I)、如SEQ ID NO:5所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:5; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III).
所述烟酸磷酸核糖转移酶突变体编码基因NadVmu具有：The niacin phosphoribosyltransferase mutant encoding gene NadV mu has:
(I)、如SEQ ID NO:11所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:11; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列，优选的，包括具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III), preferably including at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the nucleotide sequence.
所述核糖磷酸二磷酸激酶编码基因Prs具有：The ribose phosphate diphosphate kinase encoding gene Prs has:
(I)、如SEQ ID NO:6所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:6; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
所述核糖激酶编码基因RbsK具有：The ribokinase encoding gene RbsK has:
(I)、如SEQ ID NO:7所示的核苷酸序列；或 (1), the nucleotide sequence shown in SEQ ID NO:7; or
(II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
(III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
(IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III).
本发明还提供了所述基因工程菌的构建方法，其包括基于大肠杆菌宿主，通过如下任意项获得所述基因工程菌：The invention also provides a method for constructing the genetically engineered bacterium, which includes obtaining the genetically engineered bacterium based on an Escherichia coli host through any of the following:
(I)、缺失所述对烟酰胺核苷酸酰胺酶编码基因pncC、缺失烟酰胺单核苷酸腺苷转移酶基因nadR、不表达烟酰胺单核苷酸腺苷转移酶基因nadD和过表达所述NAD+合酶编码基因nadE；和/或(1) Deletion of the nicotinamide nucleotide amidase encoding gene pncC, deletion of the nicotinamide mononucleotide adenosyltransferase gene nadR, non-expression and overexpression of the nicotinamide mononucleotide adenosyltransferase gene nadD The NAD + synthase encoding gene nadE; and/or
(II)、过表达所述核酸分子、过表达核糖磷酸二磷酸激酶编码基因Prs和过表达核糖激酶编码基因RbsK。(II) Overexpressing the nucleic acid molecule, overexpressing the ribose phosphate diphosphate kinase encoding gene Prs and overexpressing the ribokinase encoding gene RbsK.
在本发明的一些具体实施方案中，所述大肠杆菌宿主选自JM109、BL21(DE3)、Top10、DH5α、Rosetta或Rosetta-gamipLysS中的一种或多种。In some specific embodiments of the invention, the E. coli host is selected from one or more of JM109, BL21(DE3), Top10, DH5α, Rosetta or Rosetta-gamipLysS.
在本发明的一些具体实施方案中，所述构建方法包括：In some specific embodiments of the invention, the construction method includes:
(I)、所述缺失对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadR的方式包括基因敲除；和/或(1) The method of deleting the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR includes gene knockout; and/or
(II)、所述不表达烟酰胺单核苷酸腺苷转移酶基因nadD的方式包括通过CIts蛋白及PR/PL启动子温敏控制烟酰胺单核苷酸腺苷转移酶基因nadD表达。(II) The method of not expressing nicotinamide mononucleotide adenosyltransferase gene nadD includes temperature-sensitively controlling the expression of nicotinamide mononucleotide adenosyltransferase gene nadD through CIts protein and PR/PL promoter.
在本发明的一些具体实施方案中，所述构建方法包括：In some specific embodiments of the invention, the construction method includes:
以大肠杆菌JM109为宿主，敲除所述对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadR基因；和/或Using Escherichia coli JM109 as the host, knock out the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR gene; and/or
CIts蛋白及PR/PL启动子温敏控制所述烟酰胺单核苷酸腺苷转移酶基因nadD基因表达；和/或CIts protein and PR/PL promoter temperature-sensitively control the expression of the nicotinamide mononucleotide adenyltransferase gene nadD gene; and/or
基于pET-28a载体过表达对NAD+合酶编码基因nadE基因；和/或Overexpression of the NAD + synthase encoding gene nadE gene based on pET-28a vector; and/or
基于pGEX4T3载体过表达所述NadVmu、核糖磷酸二磷酸激酶编码基因Prs、核糖激酶编码基因RbsK，获得所述基因工程菌。The genetically engineered bacterium is obtained by overexpressing the NadV mu , the ribose phosphate diphosphate kinase encoding gene Prs, and the ribokinase encoding gene RbsK based on the pGEX4T3 vector.
本发明还提供了以下任意项在制备NMN中的应用：The present invention also provides the application of any of the following items in preparing NMN:
(I)、所述基因工程菌；和/或(I), the genetically engineered bacteria; and/or
(II)、所述构建方法获得的基因工程菌。(II) Genetically engineered bacteria obtained by the construction method.
本发明还提供了NMN的制备方法，基于如下任意项制备NMN：The present invention also provides a preparation method of NMN, which is based on any of the following items:
(I)、所述基因工程菌；和/或(I), the genetically engineered bacteria; and/or
(II)、所述构建方法获得的基因工程菌。(II) Genetically engineered bacteria obtained by the construction method.
在本发明的一些具体实施方案中，所述制备方法，包括如下步骤：In some specific embodiments of the invention, the preparation method includes the following steps:
步骤1、取所述基因工程菌或所述构建方法获得的基因工程菌，培养、表达、离心得到菌体；Step 1. Take the genetically engineered bacteria or the genetically engineered bacteria obtained by the construction method, culture, express, and centrifuge to obtain bacterial cells;
步骤2、以烟酰胺为原料，步骤1所述菌体为催化剂，催化制得所述NMN。Step 2: Using nicotinamide as a raw material and the bacteria described in step 1 as a catalyst, the NMN is catalytically prepared.
在本发明的一些具体实施方案中，所述步骤1包括：In some specific embodiments of the invention, step 1 includes:
取所述基因工程菌或所述构建方法获得的基因工程菌，接种于培养基中，37℃、200rpm培养至OD600为0.7，冷却10min，加入1mmol/LIPTG后，于22℃、200rpm条件下诱导表达24h；离心收集菌体获得所述菌体。 Take the genetically engineered bacteria or the genetically engineered bacteria obtained by the construction method, inoculate it into the culture medium, culture it at 37°C and 200rpm until the OD 600 is 0.7, cool it for 10min, add 1mmol/LIPTG, and then inoculate it at 22°C and 200rpm. Expression was induced for 24 hours; the bacterial cells were collected by centrifugation to obtain the bacterial cells.
在本发明的一些具体实施方案中，所述培养基包括PYA8培养基。In some specific embodiments of the invention, the culture medium includes PYA8 culture medium.
所述PYA8培养基组成为(w/v)：0.1％大豆蛋白胨，1％葡萄糖，1.61％磷酸氢二钠，0.136％磷酸二氢钾，0.05％氯化钠，0.5％酵母浸膏，1％醋酸钠，其余为水，pH7.0～7.2。The composition of the PYA8 medium is (w/v): 0.1% soy peptone, 1% glucose, 1.61% disodium hydrogen phosphate, 0.136% potassium dihydrogen phosphate, 0.05% sodium chloride, 0.5% yeast extract, 1% Sodium acetate, the rest is water, pH 7.0~7.2.
在本发明的一些具体实施方案中，所述催化的反应体系包括：烟酰胺5～20mmol/L、核糖10～20mmol/L、ATP 5～10mmol/L、NAD+5～10mmol/L、Na2HPO4/NaH2PO450mmol/L、醋酸钠10mmol/L、氯化钙1mmol/L，其余为水，pH5～8.5；加入所述基因工程菌使菌体浓度达到5～100g/L，15～22℃，50～200rpm，避光反应10～25h，获得所述NMN。In some specific embodiments of the invention, the catalyzed reaction system includes: nicotinamide 5~20mmol/L, ribose 10~20mmol/L, ATP 5~10mmol/L, NAD + 5~10mmol/L, Na 2 HPO 4 /NaH 2 PO 4 50mmol/L, sodium acetate 10mmol/L, calcium chloride 1mmol/L, the rest is water, pH 5~8.5; add the genetically engineered bacteria to make the bacterial concentration reach 5~100g/L, 15 ~22°C, 50-200rpm, protected from light, react for 10-25h to obtain the NMN.
在本发明的一些具体实施方案中，所述催化的反应体系包括：烟酰胺15mmol/L、核糖20mmol/L、ATP 5mmol/L、NAD+10mmol/L、Na2HPO4/NaH2PO450mmol/L、醋酸钠10mmol/L、氯化钙1mmol/L，其余为水，pH8.0；加入所述基因工程菌使菌体浓度达到50g/L，20℃，100rpm，避光反应20h，获得所述NMN。In some specific embodiments of the invention, the catalyzed reaction system includes: nicotinamide 15mmol/L, ribose 20mmol/L, ATP 5mmol/L, NAD + 10mmol/L, Na 2 HPO 4 /NaH 2 PO 4 50mmol /L, sodium acetate 10mmol/L, calcium chloride 1mmol/L, the rest is water, pH 8.0; add the genetically engineered bacteria to make the bacterial concentration reach 50g/L, 20°C, 100rpm, react in the dark for 20h, obtain The NMN.
本发明包括且不限于取得如下有益效果：The present invention includes but is not limited to achieving the following beneficial effects:
1、本发明采用特定的重组大肠杆菌全细胞为催化剂催化转化，方法简单；反应底物为烟酰胺和核糖，生产成本较低；本发明构建了依赖烟酰胺和核糖的NMN生物合成途径，使相关酶能够高效表达，提高了NMN产量。1. The present invention uses specific recombinant Escherichia coli whole cells as catalysts for catalytic transformation, and the method is simple; the reaction substrates are nicotinamide and ribose, and the production cost is low; the present invention constructs an NMN biosynthetic pathway that relies on nicotinamide and ribose, so that Relevant enzymes can be expressed efficiently, improving NMN production.
2、采用本发明方法进行生物转化，敲除了大肠杆菌菌体内降解NMN的功能基因，使产生的NMN不容易被降解，高效保留NMN，提高了产量，NMN产量达到2.88g/L。2. The method of the present invention is used for biotransformation, and the functional gene for degrading NMN in Escherichia coli is deleted, so that the produced NMN is not easily degraded, NMN is retained efficiently, and the yield is increased. The NMN yield reaches 2.88g/L.
3、本发明对NadV即编码合成烟酸磷酸核糖转移酶的基因进行了定向进化，使该酶的催化效率达到最佳，可将NMN产量提高到2.88g/L。3. The present invention conducts directed evolution of NadV, the gene encoding the synthetic nicotinic acid phosphoribosyltransferase, so as to optimize the catalytic efficiency of the enzyme and increase the NMN output to 2.88g/L.
附图说明Description of the drawings
图1示基因编辑载体pCas9质粒图谱；Figure 1 shows the plasmid map of the gene editing vector pCas9;
图2示pTargetF质粒图谱；Figure 2 shows the map of pTargetF plasmid;
图3示pncC基因敲除验证；Figure 3 shows pncC gene knockout verification;
图4示nadR基因敲除验证；Figure 4 shows nadR gene knockout verification;
图5示温敏表达nadD基因验证；Figure 5 shows the verification of temperature-sensitive expression of nadD gene;
图6示pGEX4T3-NadV-Prs-RbsK质粒图谱。Figure 6 shows the map of pGEX4T3-NadV-Prs-RbsK plasmid.
具体实施方式Detailed ways
本发明公开了一种重组大肠杆菌生物合成NMN的方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a method for biosynthesizing NMN by recombinant Escherichia coli. Persons skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.
本发明通过降低或敲除了大肠杆菌菌体内降解NMN的功能基因，使产生的NMN不容易被降解。利用基因编辑技术对这些途径的基因进行敲除，并通过启动子温敏突变体的替换从而构建出了一株可以在菌体内高效保留NMN的工程菌株。另外本发明在该菌株中构建了一条依赖于烟酰胺、核糖的NMN的生物合成途径。然后通过易错PCR筛选技术，筛选出了一个NadV的突变体，该突变体可以在该菌株体系中更高效的生成NMN。The present invention reduces or knocks out the functional gene for degrading NMN in Escherichia coli bacteria, so that the produced NMN is not easily degraded. Gene editing technology was used to knock out the genes of these pathways, and by replacing the promoter with a temperature-sensitive mutant, an engineered strain that could efficiently retain NMN in the bacteria was constructed. In addition, the present invention constructs a biosynthetic pathway of NMN that relies on nicotinamide and ribose in this strain. Then, through error-prone PCR screening technology, a NadV mutant was screened out, which can produce NMN more efficiently in this strain system.
本发明提供的技术方案之一，是一株可以大量合成并积累NMN(nicotinamide  mononucleotide，即烟酰胺单核苷酸)的大肠杆菌基因工程菌，所述工程菌是在大肠杆菌宿主中，缺失对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadD、nadR的表达的同时，过表达NAD+合酶编码基因nadE来提高NMN的保留量；表达烟酸磷酸核糖转移酶突变体编码基因NadVmu、核糖磷酸二磷酸激酶编码基因Prs以及核糖激酶编码基因RbsK来构建依赖烟酰胺和核糖的NMN生物合成途径；One of the technical solutions provided by the present invention is a strain that can synthesize and accumulate NMN (nicotinamide) in large quantities. E. coli genetically engineered strain of nicotinamide mononucleotide (nicotinamide mononucleotide). The engineered strain is in the E. coli host and lacks the nicotinamide nucleotide amidase encoding gene pncC and nicotinamide mononucleotide adenosine transfer. While expressing the enzyme genes nadD and nadR, overexpress the NAD + synthase encoding gene nadE to increase the retention of NMN; express the nicotinic acid phosphoribosyltransferase mutant encoding gene NadV mu , ribose phosphate diphosphate kinase encoding gene Prs and ribose The kinase encoding gene RbsK constructs the NMN biosynthetic pathway that relies on nicotinamide and ribose;
进一步地，pncC、nadR基因缺失表达的方式为基因敲除；Furthermore, the method of deletion and expression of pncC and nadR genes is gene knockout;
进一步地，nadD基因缺失表达的方式为通过CIts蛋白及PR/PL启动子温敏控制nadD基因的表达；Furthermore, the method of deleting and expressing the nadD gene is to temperature-sensitively control the expression of the nadD gene through CIts protein and PR/PL promoter;
进一步地，nadE基因通过pET-28a载体进行表达；Further, the nadE gene was expressed through the pET-28a vector;
进一步地，NadVmu、Prs、RbsK基因通过质粒过表达；Further, NadV mu , Prs, and RbsK genes were overexpressed through plasmids;
优选地，采用pGEX4T3载体对NadVmu、Prs、RbsK基因进行过表达；Preferably, pGEX4T3 vector is used to overexpress NadV mu , Prs, and RbsK genes;
进一步地，野生型NadV基因的核苷酸序列如序列表SEQ ID NO：1所示；Further, the nucleotide sequence of the wild-type NadV gene is shown in the sequence list SEQ ID NO: 1;
进一步地，pncC基因的核苷酸序列如序列表SEQ ID NO：2所示；Further, the nucleotide sequence of the pncC gene is shown in the sequence list SEQ ID NO: 2;
进一步地，nadD基因的核苷酸序列如序列表SEQ ID NO：3所示；Further, the nucleotide sequence of the nadD gene is shown in the sequence list SEQ ID NO: 3;
进一步地，nadR基因的核苷酸序列如序列表SEQ ID NO：4所示；Further, the nucleotide sequence of the nadR gene is shown in the sequence list SEQ ID NO: 4;
进一步地，nadE基因的核苷酸序列如序列表SEQ ID NO：5所示；Further, the nucleotide sequence of the nadE gene is shown in the sequence list SEQ ID NO: 5;
进一步地，Prs基因的核苷酸序列如序列表SEQ ID NO：6所示；Further, the nucleotide sequence of the Prs gene is shown in the sequence list SEQ ID NO: 6;
进一步地，RbsK基因的核苷酸序列如序列表SEQ ID NO：7所示；Further, the nucleotide sequence of the RbsK gene is shown in the sequence list SEQ ID NO: 7;
进一步地，烟酸磷酸核糖转移酶突变体编码基因NadVmu的核苷酸序列如序列表SEQ ID NO：11所示；烟酸磷酸核糖转移酶突变体的氨基酸序列如SEQ ID NO：12所示；Further, the nucleotide sequence of the gene NadV mu encoding the nicotinic acid phosphoribosyltransferase mutant is shown in the sequence list SEQ ID NO: 11; the amino acid sequence of the nicotinic acid phosphoribosyltransferase mutant is shown in SEQ ID NO: 12 ;
进一步地，所述大肠杆菌宿主选自JM109、BL21(DE3)、Top 10、DH5α、Rosetta、Rosetta-gami pLysS等；Further, the E. coli host is selected from JM109, BL21 (DE3), Top 10, DH5α, Rosetta, Rosetta-gami pLysS, etc.;
优选地，所述大肠杆菌宿主为大肠杆菌JM109。Preferably, the E. coli host is E. coli JM109.
本发明提供的技术方案之二，是上述基因工程菌的构建方法，具体如下：The second technical solution provided by the present invention is the construction method of the above-mentioned genetically engineered bacteria, specifically as follows:
(1)构建高NMN保留量的工程菌株(1) Construct an engineering strain with high NMN retention
利用大肠杆菌基因编辑载体pCas9对大肠杆菌宿主细胞基因组进行基因编辑，构建pncC、nadR基因的敲除菌株(JM109ΔpncCΔnadR)，因nadD途径无法敲除，所以通过ts温敏表达对nadD的表达进行控制，构建温敏表达nadD的工程菌株(JM109ΔpncCΔnadRnadD(ts))；继续在基因组构建受到IPTG诱导的nadE基因；该菌株作为底盘细胞可以高效的保存大肠杆菌合成的NMN不被降解；The Escherichia coli gene editing vector pCas9 was used to edit the genome of the Escherichia coli host cell to construct a knockout strain of pncC and nadR genes (JM109ΔpncCΔnadR). Since the nadD pathway cannot be knocked out, the expression of nadD was controlled through ts temperature-sensitive expression. Construct an engineering strain that expresses nadD in a temperature-sensitive manner (JM109ΔpncCΔnadRnadD(ts)); continue to construct the nadE gene induced by IPTG in the genome; as a chassis cell, this strain can efficiently preserve NMN synthesized by E. coli without being degraded;
(2)构建依赖烟酰胺和核糖的NMN生物合成途径(2) Construct a nicotinamide- and ribose-dependent NMN biosynthetic pathway
利用pGEX4T3载体为骨架，构建可在IPTG诱导情况下高效表达NadVmu、Prs以及RbsK的表达载体，并将所述表达载体在步骤(1)所得菌株中表达。Using the pGEX4T3 vector as the backbone, an expression vector that can efficiently express NadV mu , Prs and RbsK under IPTG induction was constructed, and the expression vector was expressed in the strain obtained in step (1).
本发明提供的技术方案之三，是上述工程菌在生产NMN中的应用；The third technical solution provided by the present invention is the application of the above-mentioned engineering bacteria in the production of NMN;
进一步地，采用上述工程菌发酵生产NMN的方法如下：Further, the method for producing NMN by fermentation using the above-mentioned engineering bacteria is as follows:
反应体系包括：烟酰胺5～20mmol/L、核糖10～20mmol/L、ATP 5～10mmol/L、NAD+5～10mmol/L、Na2HPO4/NaH2PO450mmol/L、醋酸钠10mmol/L、氯化钙1mmol/L，其余为水pH5～8.5；加入生产菌使菌体浓度达到5～100g/L，在15～22℃，50～200rpm，避光下反应10～25h；The reaction system includes: nicotinamide 5～20mmol/L, ribose 10～20mmol/L, ATP 5～10mmol/L, NAD + 5～10mmol/L, Na 2 HPO 4 /NaH 2 PO 4 50mmol/L, sodium acetate 10mmol /L, calcium chloride 1mmol/L, the rest is water pH 5~8.5; add production bacteria to make the bacterial concentration reach 5~100g/L, react at 15~22℃, 50~200rpm, protected from light for 10~25h;
10～25h后，NMN产量达到1.31～2.88g/L；After 10 to 25 hours, NMN output reached 1.31 to 2.88g/L;
进一步地，菌体培养方法如下： Further, the bacterial culture method is as follows:
将工程菌种子液按1％的接种量接种于PYA8培养基中，37℃、200rpm培养6h(OD600为0.7左右)，自来水冷却10min，加1mmol/LIPTG后，22℃、200rpm条件下诱导表达24h；离心收集菌体；Inoculate the engineering bacterial seed liquid into PYA8 medium at an inoculation amount of 1%, culture it at 37°C and 200rpm for 6 hours (OD 600 is about 0.7), cool it with tap water for 10 minutes, add 1mmol/LIPTG, and induce expression at 22°C and 200rpm. 24h; centrifuge to collect bacteria;
更进一步地，所述PYA8培养基组成为(w/v)：0.1％大豆蛋白胨，1％葡萄糖，1.61％磷酸氢二钠，0.136％磷酸二氢钾，0.05％氯化钠，0.5％酵母浸膏，1％醋酸钠，其余为水，pH 7.0～7.2；Furthermore, the composition of the PYA8 culture medium is (w/v): 0.1% soy peptone, 1% glucose, 1.61% disodium hydrogen phosphate, 0.136% potassium dihydrogen phosphate, 0.05% sodium chloride, 0.5% yeast extract. Paste, 1% sodium acetate, the rest is water, pH 7.0~7.2;
优选地，NMN生物转化条件为：菌体浓度为50g/L，烟酰胺15mmol/L、核糖20mmol/L、ATP 5mmol/L、NAD+10mmol/L，pH8.0，20℃，100rpm，避光下反应20h；NMN产量达到8.36mmol/L。Preferably, NMN bioconversion conditions are: bacterial cell concentration 50g/L, nicotinamide 15mmol/L, ribose 20mmol/L, ATP 5mmol/L, NAD + 10mmol/L, pH 8.0, 20°C, 100rpm, protected from light The reaction was carried out for 20 hours; the NMN output reached 8.36mmol/L.
有益效果：与现有技术相比，本发明具有以下优点：Beneficial effects: Compared with the existing technology, the present invention has the following advantages:
1、本发明采用特定的重组大肠杆菌全细胞为催化剂催化转化，方法简单；反应底物为烟酰胺和核糖，生产成本较低；本发明构建了依赖烟酰胺和核糖的NMN生物合成途径，使相关酶能够高效表达，提高了NMN产量。1. The present invention uses specific recombinant Escherichia coli whole cells as catalysts to catalyze transformation, and the method is simple; the reaction substrates are nicotinamide and ribose, and the production cost is low; the present invention constructs an NMN biosynthetic pathway that relies on nicotinamide and ribose, so that Relevant enzymes can be expressed efficiently, improving NMN production.
2、采用本发明方法进行生物转化，敲除了大肠杆菌菌体内降解NMN的功能基因，使产生的NMN不容易被降解，高效保留NMN，提高了产量，NMN产量达到2.88g/L。2. The method of the present invention is used for biotransformation, and the functional gene for degrading NMN in Escherichia coli is deleted, so that the produced NMN is not easily degraded, NMN is retained efficiently, and the yield is increased. The NMN yield reaches 2.88g/L.
3、本发明对NadV即编码合成烟酸磷酸核糖转移酶的基因进行了定向进化，使该酶的催化效率达到最佳，可将NMN产量提高到2.88g/L。3. The present invention conducts directed evolution of NadV, the gene encoding the synthetic nicotinic acid phosphoribosyltransferase, so as to optimize the catalytic efficiency of the enzyme and increase the NMN output to 2.88g/L.
本发明提供的一种重组大肠杆菌生物合成NMN的方法中所用原料及试剂均可由市场购得。The raw materials and reagents used in the method for biosynthesizing NMN by recombinant Escherichia coli provided by the present invention can all be purchased from the market.
下面结合实施例，进一步阐述本发明：The present invention will be further described below in conjunction with the examples:
实施例1 NMN生产基因工程菌株的构建Example 1 Construction of genetically engineered strains for NMN production
(1)构建高NMN保留量的工程菌株(1) Construct an engineering strain with high NMN retention
利用大肠杆菌基因编辑载体pCas9对大肠杆菌宿主细胞JM109基因组进行基因编辑，构建pncC、nadR基因的敲除菌株(JM109ΔpncCΔnadR)，因nadD途径无法敲除，所以再构建温敏表达nadD的工程菌株(JM109ΔpncCΔnadR nadD(ts))；继续在基因组构建受到IPTG诱导的nadE基因；该菌株作为底盘细胞可以高效的保存大肠杆菌合成的NMN不被降解；The E. coli gene editing vector pCas9 was used to edit the genome of E. coli host cell JM109 to construct a knockout strain of pncC and nadR genes (JM109ΔpncCΔnadR). Since the nadD pathway cannot be knocked out, an engineering strain (JM109ΔpncCΔnadR) that expresses nadD in a temperature-sensitive manner was constructed. nadD(ts)); continue to construct the nadE gene induced by IPTG in the genome; as a chassis cell, this strain can efficiently preserve NMN synthesized by E. coli without being degraded;
具体的构建步骤如下：The specific construction steps are as follows:
①底盘构建①Chassis construction
如图1所示，将大肠杆菌基因编辑载体pCas9转化入JM109感受态细胞，获得稳定表达CAS9蛋白的底盘。As shown in Figure 1, the E. coli gene editing vector pCas9 was transformed into JM109 competent cells to obtain a chassis that stably expresses the CAS9 protein.
②pncC基因进行敲除②pncC gene knockout
采用pTargetF为载体，在其中插入sgRNA序列1和供体DNA片段，得到重组质粒，将重组质粒转化至表达CAS9蛋白的JM109感受态细胞，得到包含质粒的敲除菌株，经筛选得到敲除菌株JM109ΔpncC。Use pTargetF as a vector, insert sgRNA sequence 1 and donor DNA fragment into it to obtain a recombinant plasmid. Transform the recombinant plasmid into JM109 competent cells expressing CAS9 protein to obtain a knockout strain containing the plasmid. After screening, the knockout strain JM109ΔpncC was obtained. .
其中，pncC的核苷酸序列如SEQ ID NO：2所示，蛋白ID：AAN81705.1，通过NCBI数据库获取得到。pTargetF质粒如图2所示。Among them, the nucleotide sequence of pncC is shown as SEQ ID NO: 2, and the protein ID: AAN81705.1 was obtained from the NCBI database. The pTargetF plasmid is shown in Figure 2.
首先，利用CRISPR网站(http://crispr.dbcls.jp/)进行分析，选取评分较高且靠近起始密码子的序列为基因敲除靶点，设计靶点序列引物，通过PCR方式将敲除靶点序列引入至pTargetF质粒上的启动子和gRNAscaffold序列之间，使靶点序列和gRNA scaffold序列连接形成sgRNA序列1，获得pTargetF-1质粒。进一步根据pncC上下游各300bp左右的序列设计pncC上、下游同源臂引物，分别引入XhoI和PshA I酶切 位点。通过PCR获得上下游同源臂，再用F3和R2引物，通过融合PCR方式获得供体DNA片段，序列如SEQ ID NO：8所示。First, use the CRISPR website (http://crispr.dbcls.jp/) for analysis, select sequences with higher scores and close to the start codon as gene knockout targets, design target sequence primers, and knockout the genes by PCR. The target sequence is introduced between the promoter and the gRNA scaffold sequence on the pTargetF plasmid, and the target sequence and the gRNA scaffold sequence are connected to form sgRNA sequence 1, and the pTargetF-1 plasmid is obtained. Further, we designed primers for the upstream and downstream homology arms of pncC based on the sequences of about 300 bp upstream and downstream of pncC, and introduced XhoI and PshA I enzyme digestion respectively. site. Obtain the upstream and downstream homology arms through PCR, and then use F3 and R2 primers to obtain the donor DNA fragment through fusion PCR. The sequence is shown in SEQ ID NO: 8.
然后将pTargetF-1质粒和供体DNA进行双酶切，回收片段之后连接，转化步骤①获得的表达CAS9蛋白的JM109感受态细胞，再涂布氯霉素/壮观霉素双抗平板，以筛选引物对长出的单克隆进行菌落PCR检测，结果如图3所示(原长度1312bp(阴性)，敲除部分472bp，成功敲除后840bp(阳性))，筛选出成功敲除pncC的阳性克隆，再用氯霉素单抗性培养基进行连续传代培养，得到去除pTargetF质粒的JM109ΔpncC菌株。Then, the pTargetF-1 plasmid and donor DNA were double-digested, and the fragments were recovered and then ligated. The JM109 competent cells expressing CAS9 protein obtained in step ① were transformed, and then coated with chloramphenicol/spectinomycin double antibody plates for screening. The primers were used to perform colony PCR detection on the single clone grown. The results are shown in Figure 3 (the original length is 1312bp (negative), the knockout part is 472bp, and the successful knockout is 840bp (positive)). Positive clones with successful pncC knockout were selected. , and then used chloramphenicol monoresistant medium for continuous subculture to obtain the JM109ΔpncC strain with pTargetF plasmid removed.
相关引物或序列见表1记载：Relevant primers or sequences are listed in Table 1:
表1 pncC基因敲除相关序列
Table 1 pncC gene knockout related sequences
③nadR基因敲除③nadR gene knockout
采用pTargetF为载体，在其中插入sgRNA序列2和供体DNA片段，得到重组质粒，将重组质粒转化至JM109ΔpncC感受态细胞，得到包含质粒的敲除菌株，经筛选得到敲除菌株JM109ΔpncCΔnadR。Use pTargetF as the vector, insert sgRNA sequence 2 and the donor DNA fragment into it to obtain the recombinant plasmid. The recombinant plasmid is transformed into JM109ΔpncC competent cells to obtain a knockout strain containing the plasmid. After screening, the knockout strain JM109ΔpncCΔnadR is obtained.
其中，nadR的核苷酸序列如SEQ ID NO：4所示，蛋白ID：ACI72736.1，通过NCBI数据库获取得到。Among them, the nucleotide sequence of nadR is shown as SEQ ID NO: 4, and the protein ID: ACI72736.1 was obtained from the NCBI database.
与步骤②相同，同样的选择基因敲除靶点，设计引物将靶点序列插入pTargetF载体，形成sgRNA序列2，获得pTargetF-2质粒。设计nadR基因上、下游同源臂引物，引入XhoI和PshAI酶切位点，获取两段同源臂后，通过融合PCR获得供体DNA片段，序列如SEQ ID NO：9所示。然后将pTargetF-2质粒和供体DNA进行双酶切，回收片段之后连接，转化步骤②获得的JM109ΔpncC的感受态细胞，再涂布氯霉素/壮观霉素双抗平板。以筛选引物对长出的单克隆进行菌落PCR检测，结果如图4所示(原长度1723bp(阴性)，敲除部分1098bp，成功敲除后625bp(阳性))，筛选出成功敲除nadR的阳性单克隆，再用氯霉素单抗性培养基进行连续传代培养，得到去除pTargetF质粒的JM109ΔpncCΔnadR菌株。Same as step ②, select the gene knockout target, design primers to insert the target sequence into the pTargetF vector to form sgRNA sequence 2, and obtain the pTargetF-2 plasmid. Design primers for the upstream and downstream homology arms of the nadR gene, introduce XhoI and PshAI restriction sites, and obtain the two homology arms, and obtain the donor DNA fragment through fusion PCR. The sequence is shown in SEQ ID NO: 9. The pTargetF-2 plasmid and donor DNA were then double-digested, and the fragments were recovered and then ligated. The JM109ΔpncC competent cells obtained in step ② were transformed and then coated on a chloramphenicol/spectinomycin double-antibody plate. Use screening primers to perform colony PCR detection on the grown single clone. The results are shown in Figure 4 (original length 1723bp (negative), knockout part 1098bp, successful knockout 625bp (positive)), and the successful knockout of nadR was screened out. The positive single clone was then continuously subcultured in chloramphenicol-resistant medium to obtain the JM109ΔpncCΔnadR strain with the pTargetF plasmid removed.
相关引物或序列见表2记载：Relevant primers or sequences are listed in Table 2:
表2 nadR基因敲除相关序列

Table 2 NadR gene knockout related sequences

④温敏表达nadD基因的菌株构建④Construction of strains expressing nadD gene in a temperature-sensitive manner
采用pTargetF为载体，在其中插入sgRNA序列3和供体DNA片段，得到重组质粒，将重组质粒转化至JM109ΔpncCΔnadR感受态细胞，经筛选得到菌株JM109ΔpncCΔnadRnadD(ts)。Using pTargetF as the vector, sgRNA sequence 3 and the donor DNA fragment were inserted into it to obtain a recombinant plasmid. The recombinant plasmid was transformed into JM109ΔpncCΔnadR competent cells, and the strain JM109ΔpncCΔnadRnadD(ts) was obtained after screening.
nadD的核苷酸序列如SEQ ID NO：3所示，蛋白ID：CTV94997.1，来源于E.coli。The nucleotide sequence of nadD is shown as SEQ ID NO: 3, protein ID: CTV94997.1, derived from E.coli.
根据nadD上游区域序列分析基因插入靶点，设计引物将靶点序列插入pTargetF载体，形成sgRNA序列3，获得pTargetF-3质粒。选择nadD基因的上游部分序列和基因前段部分序列为两段同源臂，中间插入CIts蛋白及PR/PL启动子序列(SEQ ID NO：10)，两端引入XhoI和PshAI酶切位点，共约2.2kb进行全合成，得到供体DNA片段。Based on the sequence analysis of the nadD upstream region, the gene was inserted into the target site, and primers were designed to insert the target sequence into the pTargetF vector to form sgRNA sequence 3 and obtain the pTargetF-3 plasmid. Select the upstream part of the nadD gene and the front part of the gene as two homology arms, insert the CIts protein and PR/PL promoter sequence (SEQ ID NO: 10) in the middle, and introduce XhoI and PshAI restriction sites at both ends, a total of about 2.2 kb for total synthesis to obtain donor DNA fragments.
将pTargetF-3质粒和供体DNA进行双酶切，回收片段之后连接，转化步骤③获得的JM109ΔpncCΔnadR感受态细胞，再涂布氯霉素/壮观霉素双抗平板。以筛选引物对长出的单克隆进行菌落PCR，结果如图5所示(成功插入DNA的单克隆可以通过PCR检测到1kb左右的条带(阳性)，未成功插入的无法检测到条带(阴性))，筛选出改造成功的阳性单克隆，再用氯霉素单抗性培养基进行连续传代培养，得到去除pTargetF质粒的JM109ΔpncCΔnadRnadD(ts)，使nadD成为温敏表达型蛋白。Double-digest the pTargetF-3 plasmid and donor DNA, recover the fragments and ligate them, transform the JM109ΔpncCΔnadR competent cells obtained in step ③, and then coat the chloramphenicol/spectinomycin double antibody plate. Use the screening primers to perform colony PCR on the grown single clones, and the results are shown in Figure 5 (single clones that have successfully inserted DNA can detect a band of about 1 kb (positive) by PCR, while those that have not successfully inserted cannot detect the band ( negative)), screen out the successfully transformed positive single clones, and then use chloramphenicol-resistant medium for continuous subculture to obtain JM109ΔpncCΔnadRnadD(ts) with the pTargetF plasmid removed, making nadD a temperature-sensitive expression protein.
相关引物或序列见表3记载：Relevant primers or sequences are listed in Table 3:
表3温敏表达nadD基因的菌株构建相关序列

Table 3 Relevant sequences for construction of strains expressing nadD gene in a temperature-sensitive manner

⑤IPTG诱导nadE基因的菌株构建⑤Construction of strain with IPTG-induced nadE gene
nadE的核苷酸序列如SEQ ID NO：5所示，蛋白ID：WP_003037081.1，来源于弗朗西斯氏菌(Francisella tularensis)。The nucleotide sequence of nadE is shown in SEQ ID NO: 5, protein ID: WP_003037081.1, derived from Francisella tularensis.
全合成nadE基因序列，5’端引入EcoR I酶切位点，3’端引入NcoI酶切位点，即：5’-CCGGAATTC-nadE-CCATGGATG-3’，共765bp。用pET-28a载体构建重组质粒，基因片段和pET-28a质粒均进行NcoI/EcoR I双酶切，回收酶切片段，用T4连接酶进行连接。转化入步骤④获得的JM109ΔpncCΔnadR nadD(ts)感受态细胞，在含有Kan+的LB平板上涂布，挑取转化子进行验证，得到JM109ΔpncCΔnadRnadD(ts)-nadE菌株。The fully synthesized nadE gene sequence introduces an EcoR I restriction site at the 5' end and an NcoI restriction site at the 3' end, namely: 5'-CCGGAATTC-nadE-CCATGGATG-3', a total of 765 bp. The pET-28a vector was used to construct a recombinant plasmid. Both the gene fragment and the pET-28a plasmid were subjected to NcoI/EcoR I double enzyme digestion, and the digested fragments were recovered and ligated with T4 ligase. Transform into the JM109ΔpncCΔnadR nadD(ts) competent cells obtained in step ④, spread on the LB plate containing Kan+, pick the transformants for verification, and obtain the JM109ΔpncCΔnadRnadD(ts)-nadE strain.
(2)构建依赖烟酰胺和核糖的NMN生物合成途径(2) Construct a nicotinamide- and ribose-dependent NMN biosynthetic pathway
利用pGEX-4T-3载体为骨架以及tac启动子控制元件，构建可在IPTG诱导情况下高效表达NadV、Prs以及RbsK的表达载体，并将所述表达载体在步骤(1)所得菌株中表达。Using the pGEX-4T-3 vector as the backbone and the tac promoter control element, an expression vector that can efficiently express NadV, Prs and RbsK under IPTG induction was constructed, and the expression vector was expressed in the strain obtained in step (1).
用pGEX-4T-3载体构建重组质粒，分别从NCBI数据库获取NadV、Prs以及RbsK的CDS序列(SEQ ID NO：1、6、7)，中间插入lac操纵子及tac启动子元件，两端分别设计BamHII和NotI酶切位点，共3330bp的序列进行全合成。然后将合成序列与pGEX-4T-3质粒分别进行双酶切，回收酶切片段，进行连接，构建pGEX4T3-NadV-Prs-RbsK质粒，如图6所示。将连接产物转化入JM109ΔpncCΔnadR nadD(ts)-nadE感受态细胞，在含有Kan+和Amp+双抗的LB平板上涂布，挑取转化子进行验证，获得重组菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX4T3-NadV-Prs-RbsK。Use the pGEX-4T-3 vector to construct a recombinant plasmid, obtain the CDS sequences of NadV, Prs and RbsK from the NCBI database (SEQ ID NO: 1, 6, 7), insert the lac operator and tac promoter elements in the middle, and insert them at both ends. BamHII and NotI restriction sites were designed, and a total sequence of 3330 bp was fully synthesized. Then, the synthetic sequence and the pGEX-4T-3 plasmid were double-digested respectively, and the digested fragments were recovered and ligated to construct the pGEX4T3-NadV-Prs-RbsK plasmid, as shown in Figure 6. Transform the ligation product into JM109ΔpncCΔnadR nadD(ts)-nadE competent cells, spread it on LB plates containing Kan+ and Amp+ double antibodies, pick the transformants for verification, and obtain the recombinant strain JM109ΔpncCΔnadR nadD(ts)-nadE, pGEX4T3- NadV-Prs-RbsK.
实施例2 NadV定向进化Example 2 NadV directed evolution
NadV(烟酸磷酸核糖转移酶)是决定NMN合成的关键基因，该基因的催化效率 以及催化的最适环境是配合其它两个酶(核糖磷酸二磷酸激酶编码基因Prs以及核糖激酶编码基因RbsK)催化的关键因素，而野生型NadV的催化效率达不到配适需求。NadV (nicotinic acid phosphoribosyltransferase) is a key gene that determines the synthesis of NMN. The catalytic efficiency of this gene And the optimal environment for catalysis is a key factor in cooperating with the catalysis of the other two enzymes (ribose phosphate diphosphate kinase encoding gene Prs and ribokinase encoding gene RbsK). However, the catalytic efficiency of wild-type NadV does not meet the adaptation requirements.
本发明利用易错PCR技术对NadV进行了进化筛选，选取在LB培养基中生长速度下降的菌株为筛选目标。The present invention uses error-prone PCR technology to conduct evolutionary screening of NadV, and selects strains whose growth rate decreases in LB culture medium as screening targets.
具体为：以实施例1构建的pGEX4T3-NadV-Prs-RbsK质粒为模板，设计NadV两端的引物，分别引入BamHI和SgrAI酶切位点，用易错PCR方式进行突变。将PCR产物和模板质粒进行BamHI/SgrAI双酶切，回收片段进行连接，再转化JM109ΔpncCΔnadRnadD(ts)-nadE感受态细胞。Specifically: using the pGEX4T3-NadV-Prs-RbsK plasmid constructed in Example 1 as a template, design primers at both ends of NadV, introduce BamHI and SgrAI restriction sites respectively, and perform mutation using error-prone PCR. The PCR product and template plasmid were double digested with BamHI/SgrAI, the fragments were recovered and ligated, and then transformed into JM109ΔpncCΔnadRnadD(ts)-nadE competent cells.
以在LB培养基中生长速度下降的菌株为筛选目标，筛选出菌株后，接种在96孔板的LB培养基中，37℃震荡培养4h，降温至22℃，加入IPTG诱导表达24h，得到菌液；然后在96孔板中加入反应体系进行反应，所述反应体系为4mmol/LNR(烟酰胺核糖)，2mmol/LATP，2mmol/L氯化钙，与菌液按体积比1:1混合，20℃反应1h，离心得到上清液。Use strains whose growth rate decreases in LB medium as the screening target. After screening out the strains, inoculate them into LB medium in a 96-well plate, culture with shaking at 37°C for 4 hours, cool to 22°C, add IPTG to induce expression for 24 hours, and obtain the bacteria. liquid; then add a reaction system to the 96-well plate for reaction. The reaction system is 4mmol/LNR (nicotinamide ribose), 2mmol/LATP, 2mmol/L calcium chloride, mixed with the bacterial liquid in a volume ratio of 1:1, React at 20°C for 1 hour and centrifuge to obtain the supernatant.
取上清液，用荧光酶标仪进行荧光检测(入射光382nm，发射光445nm)，荧光越强表明产NMN能力越强，最终筛选出6株荧光较强的突变体，对这些菌株测序发现其有2个共同的突变位点：Q54L和D453G。Take the supernatant and perform fluorescence detection with a fluorescent microplate reader (incident light 382nm, emitted light 445nm). The stronger the fluorescence, the stronger the ability to produce NMN. Finally, 6 mutants with stronger fluorescence were screened out, and these strains were sequenced and found that It has two common mutation sites: Q54L and D453G.
以pGEX4T3-NadV-Prs-RbsK质粒为模板，设计引物在这两处进行定点突变，将突变后的质粒转化回JM109ΔpncCΔnadR nadD(ts)-nadE感受态细胞，挑选单克隆验证，经测序后确认构建出工程菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX4T3-NadVmu-Prs-RbsK。Using the pGEX4T3-NadV-Prs-RbsK plasmid as a template, design primers for site-directed mutation at these two locations, transform the mutated plasmid back into JM109ΔpncCΔnadR nadD(ts)-nadE competent cells, select single clones for verification, and confirm the construction after sequencing The engineering bacteria JM109ΔpncCΔnadR nadD(ts)-nadE and pGEX4T3-NadV mu -Prs-RbsK were produced.
点突变引物：
Point mutation primers:
点突变后获得的烟酸磷酸核糖转移酶突变体编码基因NadVmu的核苷酸序列如序列表SEQ ID NO：11所示，烟酸磷酸核糖转移酶突变体氨基酸序列如SEQ ID NO：12所示。The nucleotide sequence of the nicotinic acid phosphoribosyltransferase mutant encoding gene NadV mu obtained after point mutation is shown in the sequence list SEQ ID NO: 11, and the amino acid sequence of the nicotinic acid phosphoribosyltransferase mutant is shown in SEQ ID NO: 12 Show.
实施例3 NMN生物合成Example 3 NMN biosynthesis
(1)菌株培养及表达(1) Strain culture and expression
实验菌株：Experimental strains:
实施例2构建的重组菌JM109ΔpncCΔnadRnadD(ts)-nadE，pGEX-NadVmu-Prs-RbsK；The recombinant bacteria JM109ΔpncCΔnadRnadD(ts)-nadE, pGEX-NadV mu -Prs-RbsK constructed in Example 2;
实施例1构建的重组菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX-NadV-Prs-RbsK。The recombinant bacteria JM109ΔpncCΔnadR nadD(ts)-nadE, pGEX-NadV-Prs-RbsK constructed in Example 1.
挑取重组大肠杆菌接种于20mL含卡那霉素50μg/mL、氨苄100μg/mL的LB培养，37℃、200rpm培养16h，然后按1％接种于PYA8培养基(0.1％大豆蛋白胨，1％葡萄糖，1.61％磷酸氢二钠，0.136％磷酸二氢钾，0.05％氯化钠，0.5％酵母浸膏，1％醋酸钠，其余为水，pH 7.0～7.2)中(500mL三角瓶)，37℃、200rpm培养6h(OD600(～0.7))，自来水冷却10min，加1mmol/LIPTG后，22℃、200rpm条件下诱导表达24h。然后离心(4℃，10000g，离心5min)收集菌体。Pick the recombinant E. coli and inoculate it into 20 mL of LB containing kanamycin 50 μg/mL and ampicillin 100 μg/mL, culture it at 37°C and 200 rpm for 16 hours, and then inoculate it into PYA8 medium (0.1% soy peptone, 1% glucose) at 1% , 1.61% disodium hydrogen phosphate, 0.136% potassium dihydrogen phosphate, 0.05% sodium chloride, 0.5% yeast extract, 1% sodium acetate, the rest is water, pH 7.0~7.2) (500mL Erlenmeyer flask), 37°C , culture at 200rpm for 6h (OD 600 (~0.7)), cool with tap water for 10min, add 1mmol/LIPTG, and induce expression for 24h at 22°C and 200rpm. Then centrifuge (4°C, 10000g, centrifuge for 5 minutes) to collect the bacterial cells.
(2)NMN生物转化(2) NMN biotransformation
使用上述收集菌体为催化剂进行生物转化，按表4称取原料配置反应液，溶解后 加入菌体，搅拌均匀，在对应条件下避光反应得到产物。Use the above collected bacterial cells as a catalyst to carry out biological transformation. Weigh the raw materials according to Table 4 to prepare the reaction solution. After dissolving Add the bacterial cells, stir evenly, and react in the dark under corresponding conditions to obtain the product.
(3)产物测定(3) Product determination
反应结束后，向反应液中加入1/2倍体积的双蒸水，置10℃，10min。在4℃、12000g条件下离心10min，收集上清，在向上清中加入5倍体积的丙酮，置4℃过夜，在4℃、12000g条件下离心10min，收集沉淀，得到初步纯化的反应物。再采用去离子水溶解沉淀，对反应产物进行高效液相色谱法(HPLC法)。After the reaction is completed, add 1/2 volume of double-distilled water to the reaction solution and set it at 10°C for 10 minutes. Centrifuge at 4°C and 12,000g for 10 minutes, collect the supernatant, add 5 times the volume of acetone to the supernatant, leave it at 4°C overnight, centrifuge at 4°C and 12,000g for 10 minutes, collect the precipitate, and obtain the initially purified reactant. Then use deionized water to dissolve the precipitate, and perform high-performance liquid chromatography (HPLC method) on the reaction product.
色谱条件如下：The chromatographic conditions are as follows:
检测前样品处理——用70％甲醇水溶液将样品稀释50倍，然后用孔径为0.22μm的微孔滤器过滤后装到样品瓶中；Sample treatment before detection - dilute the sample 50 times with 70% methanol aqueous solution, then filter it with a microporous filter with a pore size of 0.22 μm and put it into a sample bottle;
仪器设备——美国安捷伦公司1260高效液相色谱仪；Instruments and equipment—Agilent 1260 high performance liquid chromatograph;
色谱柱：Phenomenex LunaC18色谱柱(4.6mm×250mm 5μm)；Chromatographic column: Phenomenex LunaC18 column (4.6mm×250mm 5μm);
柱温：35℃；Column temperature: 35℃;
流动相：流动相为流动相A为0.25％磷酸二氢钠和0.2‰磷酸的水溶液；流动相B为甲醇，流动相A：流动相B＝1：1；Mobile phase: Mobile phase A is an aqueous solution of 0.25% sodium dihydrogen phosphate and 0.2‰ phosphoric acid; mobile phase B is methanol, mobile phase A: mobile phase B = 1:1;
进样量：5μL。Injection volume: 5μL.
流速：0.3mL/min。Flow rate: 0.3mL/min.
表4重组大肠杆菌催化体系
Table 4 Recombinant E. coli catalytic system
上述结果说明，由本发明构建的重组菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX-NadVmu-Prs-RbsK，生产NMN的产量能够达到8.36mmol/L(约为2.88g/L)， 且经过改造后的烟酸磷酸核糖转移酶突变体能够进一步提高NMN的产量。The above results show that the NMN production yield of the recombinant bacteria JM109ΔpncCΔnadR nadD(ts)-nadE, pGEX-NadV mu -Prs-RbsK constructed by the present invention can reach 8.36mmol/L (approximately 2.88g/L), And the modified nicotinic acid phosphoribosyltransferase mutant can further increase the production of NMN.
以上对本发明所提供的一种重组大肠杆菌生物合成NMN的方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。











The method for biosynthesizing NMN by recombinant E. coli provided by the present invention has been introduced in detail above. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.











Claims (21)

  1. 以下任意项在制备NMN中的应用：Application of any of the following items in the preparation of NMN:
    (I)、缺失对烟酰胺核苷酸酰胺酶编码基因pncC、缺失烟酰胺单核苷酸腺苷转移酶基因nadR、不表达烟酰胺单核苷酸腺苷转移酶基因nadD和过表达NAD+合酶编码基因nadE；和/或(I), deletion of the nicotinamide nucleotide amidase encoding gene pncC, deletion of the nicotinamide mononucleotide adenosyltransferase gene nadR, non-expression of the nicotinamide mononucleotide adenosyltransferase gene nadD and overexpression of NAD + synthase encoding gene nadE; and/or
    (II)、过表达烟酸磷酸核糖转移酶突变体编码基因NadVmu、过表达核糖磷酸二磷酸激酶编码基因Prs和过表达核糖激酶编码基因RbsK。(II), overexpression of the nicotinic acid phosphoribosyltransferase mutant encoding gene NadV mu , overexpression of the ribose phosphate diphosphate kinase encoding gene Prs and overexpression of the ribokinase encoding gene RbsK.
  2. 烟酸磷酸核糖转移酶突变体，其特征在于，其包括Q54L和/或D453G位点突变。Nicotinic acid phosphoribosyltransferase mutant, characterized in that it includes Q54L and/or D453G site mutations.
  3. 如权利要求2所述的烟酸磷酸核糖转移酶突变体，其特征在于，所述烟酸磷酸核糖转移酶突变体具有：The nicotinic acid phosphoribosyltransferase mutant according to claim 2, wherein the nicotinic acid phosphoribosyltransferase mutant has:
    (I)、如SEQ ID NO:12所示的氨基酸序列；或(I), the amino acid sequence shown in SEQ ID NO:12; or
    (II)、在如(I)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或(II), a sequence in which one or more amino acids are substituted, deleted, added and/or substituted based on the amino acid sequence shown in (I); or
    (III)、与如(I)或(II)所示的氨基酸序列具有至少90％序列同源性的氨基酸序列，优选的，包括具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性的氨基酸序列。(III), an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in (I) or (II), preferably including at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence.
  4. 编码如权利要求2或3所述烟酸磷酸核糖转移酶突变体的核酸分子。Nucleic acid molecule encoding the nicotinic acid phosphoribosyltransferase mutant according to claim 2 or 3.
  5. 如权利要求4所述的核酸分子，其特征在于，其为NadVmu基因，具有：The nucleic acid molecule of claim 4, which is a NadV mu gene and has:
    (I)、如SEQ ID NO:11所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:11; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列，优选的，包括具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III), preferably including at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the nucleotide sequence.
  6. 组合元件，其特征在于，包括过表达的如权利要求4或5所述的核酸分子、过表达的核糖磷酸二磷酸激酶编码基因Prs和过表达的核糖激酶编码基因RbsK；The combination element is characterized in that it includes an overexpressed nucleic acid molecule as claimed in claim 4 or 5, an overexpressed ribose phosphate diphosphate kinase encoding gene Prs and an overexpressed ribokinase encoding gene RbsK;
    所述核糖磷酸二磷酸激酶编码基因Prs具有：The ribose phosphate diphosphate kinase encoding gene Prs has:
    (I)、如SEQ ID NO:6所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:6; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述核糖激酶编码基因RbsK具有：The ribokinase encoding gene RbsK has:
    (I)、如SEQ ID NO:7所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:7; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或 (II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III).
  7. 如权利要求6所述的组合元件，其特征在于，还包括如下任意项：The combined component of claim 6, further comprising any of the following:
    (I)、不包括对烟酰胺核苷酸酰胺酶编码基因pncC、不包括烟酰胺单核苷酸腺苷转移酶基因nadR；和(I), does not include the nicotinamide nucleotide amidase encoding gene pncC, does not include the nicotinamide mononucleotide adenosyltransferase gene nadR; and
    (II)、不表达烟酰胺单核苷酸腺苷转移酶基因nadD；和(II), does not express nicotinamide mononucleotide adenosyltransferase gene nadD; and
    (III)、过表达NAD+合酶编码基因nadE；(III), overexpression of the NAD + synthase encoding gene nadE;
    所述对烟酰胺核苷酸酰胺酶编码基因pncC具有：The nicotinamide nucleotide amidase encoding gene pncC has:
    (I)、如SEQ ID NO:2所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:2; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述烟酰胺单核苷酸腺苷转移酶基因nadR具有：The nicotinamide mononucleotide adenosyltransferase gene nadR has:
    (I)、如SEQ ID NO:4所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:4; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述烟酰胺单核苷酸腺苷转移酶基因nadD具有：The nicotinamide mononucleotide adenosyltransferase gene nadD has:
    (I)、如SEQ ID NO:3所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:3; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述NAD+合酶编码基因nadE具有：The NAD + synthase encoding gene nadE has:
    (I)、如SEQ ID NO:5所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:5; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核 苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) Nucleotides with the same or similar functions as the nucleotide sequences shown nucleotide sequence; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列。(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III).
  8. 如权利要求7所述的组合元件，其特征在于，包括：The combination component according to claim 7, characterized in that it includes:
    (I)、所述缺失对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadR的方式包括基因敲除；和/或(1) The method of deleting the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR includes gene knockout; and/or
    (II)、所述不表达烟酰胺单核苷酸腺苷转移酶基因nadD的方式包括通过CIts蛋白及PR/PL启动子温敏控制烟酰胺单核苷酸腺苷转移酶基因nadD表达。(II) The method of not expressing nicotinamide mononucleotide adenosyltransferase gene nadD includes temperature-sensitively controlling the expression of nicotinamide mononucleotide adenosyltransferase gene nadD through CIts protein and PR/PL promoter.
  9. 表达载体，其特征在于，包括：Expression vector, characterized by including:
    (I)、如权利要求4或5所述的核酸分子；或(1), the nucleic acid molecule as claimed in claim 4 or 5; or
    (II)、如权利要求6至8任一项所述的组合元件。(II) The combination element according to any one of claims 6 to 8.
  10. 如权利要求7所述的表达载体，其特征在于，还包括pet-28α载体、pGEX4T3载体、pCas9载体或pTargetF载体中的一种或多种。The expression vector according to claim 7, further comprising one or more of pet-28α vector, pGEX4T3 vector, pCas9 vector or pTargetF vector.
  11. 基因工程菌，其特征在于，包括Genetically engineered bacteria, characterized by including
    (I)、缺失对烟酰胺核苷酸酰胺酶编码基因pncC、缺失烟酰胺单核苷酸腺苷转移酶基因nadR、不表达烟酰胺单核苷酸腺苷转移酶基因nadD和过表达NAD+合酶编码基因nadE；和/或(I), deletion of the nicotinamide nucleotide amidase encoding gene pncC, deletion of the nicotinamide mononucleotide adenosyltransferase gene nadR, non-expression of the nicotinamide mononucleotide adenosyltransferase gene nadD and overexpression of NAD + synthase encoding gene nadE; and/or
    (II)、过表达烟酸磷酸核糖转移酶突变体编码基因NadVmu、过表达核糖磷酸二磷酸激酶编码基因Prs和过表达核糖激酶编码基因RbsK；或 or
    (III)、过表达的如权利要求4或5所述的核酸分子；或(III), overexpressed nucleic acid molecule according to claim 4 or 5; or
    (IV)、如权利要求6至8任一项所述的组合元件；或(IV), the combination element according to any one of claims 6 to 8; or
    (V)、如权利要求9或10所述的表达载体。(V). The expression vector according to claim 9 or 10.
  12. 如权利要求11所述的基因工程菌，其特征在于，所述对烟酰胺核苷酸酰胺酶编码基因pncC具有：The genetically engineered bacterium according to claim 11, wherein the nicotinamide nucleotide amidase encoding gene pncC has:
    (I)、如SEQ ID NO:2所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:2; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述烟酰胺单核苷酸腺苷转移酶基因nadR具有：The nicotinamide mononucleotide adenosyltransferase gene nadR has:
    (I)、如SEQ ID NO:4所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:4; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述烟酰胺单核苷酸腺苷转移酶基因nadD具有：The nicotinamide mononucleotide adenosyltransferase gene nadD has:
    (I)、如SEQ ID NO:3所示的核苷酸序列；或 (1), the nucleotide sequence shown in SEQ ID NO:3; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述NAD+合酶编码基因nadE具有：The NAD + synthase encoding gene nadE has:
    (I)、如SEQ ID NO:5所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:5; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述烟酸磷酸核糖转移酶突变体编码基因NadVmu具有：The niacin phosphoribosyltransferase mutant encoding gene NadV mu has:
    (I)、如SEQ ID NO:11所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:11; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列，优选的，包括具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III), preferably including at least 90%, 91%, 92% , a nucleotide sequence with 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology;
    所述核糖磷酸二磷酸激酶编码基因Prs具有：The ribose phosphate diphosphate kinase encoding gene Prs has:
    (I)、如SEQ ID NO:6所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:6; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核苷酸序列；(IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);
    所述核糖激酶编码基因RbsK具有：The ribokinase encoding gene RbsK has:
    (I)、如SEQ ID NO:7所示的核苷酸序列；或(I), the nucleotide sequence shown in SEQ ID NO:7; or
    (II)、与(I)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(I)所示的核苷酸序列不同的核苷酸序列；或(II), a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or
    (III)、与(I)或(II)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(I)或(II)所示的核苷酸序列功能相同或相似的核苷酸序列；或(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (I) or (II), and being identical to the nucleotide sequence represented by (I) or (II) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
    (IV)、与(I)、(II)或(III)所述核苷酸序列具有至少90％序列同源性的核 苷酸序列。(IV), a core having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III) nucleotide sequence.
  13. 如权利要求11或12所述基因工程菌的构建方法，其特征在于，其包括基于大肠杆菌宿主，通过如下任意项获得所述基因工程菌：The method for constructing genetically engineered bacteria according to claim 11 or 12, characterized in that it includes obtaining the genetically engineered bacteria based on an Escherichia coli host by any of the following:
    (I)、缺失所述对烟酰胺核苷酸酰胺酶编码基因pncC、缺失烟酰胺单核苷酸腺苷转移酶基因nadR、不表达烟酰胺单核苷酸腺苷转移酶基因nadD和过表达所述NAD+合酶编码基因nadE；和/或(1) Deletion of the nicotinamide nucleotide amidase encoding gene pncC, deletion of the nicotinamide mononucleotide adenosyltransferase gene nadR, non-expression and overexpression of the nicotinamide mononucleotide adenosyltransferase gene nadD The NAD + synthase encoding gene nadE; and/or
    (II)、过表达如权利要求4所述核酸分子、过表达核糖磷酸二磷酸激酶编码基因Prs和过表达核糖激酶编码基因RbsK。(II) Overexpressing the nucleic acid molecule according to claim 4, overexpressing the ribose phosphate diphosphate kinase encoding gene Prs and overexpressing the ribokinase encoding gene RbsK.
  14. 如权利要求13所述的构建方法，其特征在于，所述大肠杆菌宿主选自JM109、BL21(DE3)、Top10、DH5α、Rosetta或Rosetta-gami pLysS中的一种或多种。The construction method of claim 13, wherein the E. coli host is selected from one or more of JM109, BL21 (DE3), Top10, DH5α, Rosetta or Rosetta-gami pLysS.
  15. 如权利要求14所述的构建方法，其特征在于，包括：The construction method according to claim 14, characterized in that it includes:
    (I)、所述缺失对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadR的方式包括基因敲除；和/或(1) The method of deleting the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR includes gene knockout; and/or
    (II)、所述不表达烟酰胺单核苷酸腺苷转移酶基因nadD的方式包括通过CIts蛋白及PR/PL启动子温敏控制烟酰胺单核苷酸腺苷转移酶基因nadD表达。(II) The method of not expressing nicotinamide mononucleotide adenosyltransferase gene nadD includes temperature-sensitively controlling the expression of nicotinamide mononucleotide adenosyltransferase gene nadD through CIts protein and PR/PL promoter.
  16. 如权利要求13至15任一项所述的构建方法，其特征在于，包括：The construction method according to any one of claims 13 to 15, characterized in that it includes:
    以大肠杆菌JM109为宿主，敲除所述对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadR基因；和/或Using Escherichia coli JM109 as the host, knock out the nicotinamide nucleotide amidase encoding gene pncC and the nicotinamide mononucleotide adenosyltransferase gene nadR gene; and/or
    CIts蛋白及PR/PL启动子温敏控制所述烟酰胺单核苷酸腺苷转移酶基因nadD基因表达；和/或CIts protein and PR/PL promoter temperature-sensitively control the expression of the nicotinamide mononucleotide adenyltransferase gene nadD gene; and/or
    基于pET-28a载体过表达对NAD+合酶编码基因nadE基因；和/或Overexpression of the NAD + synthase encoding gene nadE gene based on pET-28a vector; and/or
    基于pGEX4T3载体过表达所述NadVmu、核糖磷酸二磷酸激酶编码基因Prs、核糖激酶编码基因RbsK，获得所述基因工程菌。The genetically engineered bacterium is obtained by overexpressing the NadV mu , the ribose phosphate diphosphate kinase encoding gene Prs, and the ribokinase encoding gene RbsK based on the pGEX4T3 vector.
  17. 以下任意项在制备NMN中的应用：Application of any of the following items in the preparation of NMN:
    (I)、如权利要求11或12所述的基因工程菌；和/或(1), the genetically engineered bacterium as claimed in claim 11 or 12; and/or
    (II)、如权利要求13至16任一项所述构建方法获得的基因工程菌。(II) Genetically engineered bacteria obtained by the construction method according to any one of claims 13 to 16.
  18. NMN的制备方法，其特征在于，基于如下任意项制备NMN：The preparation method of NMN is characterized in that NMN is prepared based on any of the following items:
    (I)、如权利要求11或12所述的基因工程菌；和/或(1), the genetically engineered bacterium as claimed in claim 11 or 12; and/or
    (II)、如权利要求13至16任一项所述构建方法获得的基因工程菌。(II) Genetically engineered bacteria obtained by the construction method according to any one of claims 13 to 16.
  19. 如权利要求18所述的制备方法，其特征在于，包括如下步骤：The preparation method according to claim 18, characterized in that it includes the following steps:
    步骤1、取如权利要求11或12所述的基因工程菌或如权利要求13至16任一项所述构建方法获得的基因工程菌，培养、表达、离心得到菌体；Step 1. Take the genetically engineered bacterium as claimed in claim 11 or 12 or the genetically engineered bacterium obtained by the construction method as described in any one of claims 13 to 16, culture, express, and centrifuge to obtain bacterial cells;
    步骤2、以烟酰胺为原料，步骤1所述菌体为催化剂，催化制得所述NMN。Step 2: Using nicotinamide as a raw material and the bacteria described in step 1 as a catalyst, the NMN is catalytically produced.
  20. 如权利要求19所述的制备方法，其特征在于，所述步骤1包括：The preparation method according to claim 19, characterized in that said step 1 includes:
    取如权利要求11或12所述的基因工程菌或如权利要求13至16任一项所述构建方法获得的基因工程菌，接种于培养基中，37℃、200rpm培养至OD600为0.7，冷却10min，加入1mmol/L IPTG后，于22℃、200rpm条件下诱导表达24h；离心收集菌体获得所述菌体。Take the genetically engineered bacterium as described in claim 11 or 12 or the genetically engineered bacterium obtained by the construction method as described in any one of claims 13 to 16, inoculate it into the culture medium, and culture it at 37°C and 200rpm until OD 600 is 0.7. Cool for 10 minutes, add 1 mmol/L IPTG, and induce expression for 24 hours at 22°C and 200 rpm; collect the cells by centrifugation to obtain the cells.
  21. 如权利要求19或20所述的制备方法，其特征在于，The preparation method according to claim 19 or 20, characterized in that,
    所述催化的反应体系包括：烟酰胺5～20mmol/L、核糖10～20mmol/L、ATP 5～10mmol/L、NAD+5～10mmol/L、Na2HPO4/NaH2PO450mmol/L、醋酸钠10mmol/L、氯化钙1mmol/L，其余为水pH5～8.5；加入所述基因工程菌使菌体浓度达到5～100g/L，15～22℃，50～200rpm，避光反应10～25h，获得所述NMN。 The catalyzed reaction system includes: nicotinamide 5~20mmol/L, ribose 10~20mmol/L, ATP 5~10mmol/L, NAD + 5~10mmol/L, Na 2 HPO 4 /NaH 2 PO 4 50mmol/L , sodium acetate 10mmol/L, calcium chloride 1mmol/L, and the rest is water pH 5-8.5; add the genetically engineered bacteria to make the bacterial concentration reach 5-100g/L, 15-22°C, 50-200rpm, avoid light reaction In 10-25h, the NMN is obtained.
PCT/CN2023/081819 2022-04-19 2023-03-16 Method for biosynthesis of nmn WO2023202281A1 (en)

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