WO2025213622A1 - 一种烟酰胺核糖激酶突变体在大肠杆菌中的高效异源表达及其应用 - Google Patents

一种烟酰胺核糖激酶突变体在大肠杆菌中的高效异源表达及其应用

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Publication number
WO2025213622A1
WO2025213622A1 PCT/CN2024/107280 CN2024107280W WO2025213622A1 WO 2025213622 A1 WO2025213622 A1 WO 2025213622A1 CN 2024107280 W CN2024107280 W CN 2024107280W WO 2025213622 A1 WO2025213622 A1 WO 2025213622A1
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Prior art keywords
nicotinamide
ribokinase
nicotinamide ribokinase
mutant
seq
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English (en)
French (fr)
Inventor
龚劲松
毛歆安
马歇尔乔治·罗
史劲松
许正宏
苏畅
李恒
徐国强
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Jiangnan University
Seragon Biosciences Inc
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Jiangnan University
Seragon Biosciences Inc
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Publication of WO2025213622A1 publication Critical patent/WO2025213622A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01022—Ribosylnicotinamide kinase (2.7.1.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention belongs to the field of enzyme engineering technology and more specifically relates to the efficient heterologous expression of a nicotinamide ribokinase mutant in Escherichia coli and its application.
  • ⁇ -Nicotinamide mononucleotide is a bioactive nucleotide widely found in humans and animals.
  • NAD + nicotinamide adenine dinucleotide
  • ⁇ -NMN significantly increases NAD + levels in the body. It has important applications in preventing and improving aging-related symptoms and chronic diseases, and can be developed into dietary supplements or skincare products.
  • current ⁇ -NMN production cannot meet the growing demand.
  • ⁇ -NMN was mainly produced on a large scale through chemical synthesis using adenosine monophosphate (AMP), tetraacetyl ribose (TAR) and nicotinamide (NAM) as raw materials.
  • AMP adenosine monophosphate
  • TAR tetraacetyl ribose
  • NAM nicotinamide
  • NMN nicotinamide
  • PRPP phosphoribosyl pyrophosphate
  • NAM nicotinamide
  • PRPP phosphoribosyl pyrophosphate
  • Nampt phosphoribosyltransferase
  • NMN nicotinamide riboside
  • NR nicotinamide riboside
  • Nrk nicotinamide riboside kinase
  • the enzyme activity of existing Nrk after heterologous expression in host bacteria such as Escherichia coli is generally not high, and the nicotinamide ribokinase enzyme activity in the fermentation broth obtained through high-density fermentation processes is only about 100 U/mL, which still cannot meet the needs of efficient enzymatic synthesis of ⁇ -NMN.
  • the present invention addresses the problems existing in the above-mentioned prior art and provides a nicotinamide ribokinase that can be expressed in Escherichia coli. It also provides a mutant of nicotinamide ribokinase with improved enzyme activity relative to the parent, as well as a method and application for the efficient heterologous expression of nicotinamide ribokinase, which is conducive to the efficient biosynthesis of ⁇ -nicotinamide mononucleotide.
  • the first object of the present invention is to provide a nicotinamide ribokinase.
  • the second object of the present invention is to provide a gene encoding the nicotinamide ribokinase.
  • the third object of the present invention is to provide a mutant of the nicotinamide ribokinase.
  • the fourth object of the present invention is to provide a gene encoding the mutant.
  • the fifth object of the present invention is to provide a recombinant plasmid for expressing nicotinamide ribokinase.
  • the sixth object of the present invention is to provide a recombinant bacterium containing the recombinant plasmid.
  • a seventh object of the present invention is to provide a method for increasing the activity of nicotinamide ribokinase.
  • the eighth object of the present invention is to provide a method for efficiently heterologously expressing nicotinamide ribokinase.
  • a ninth object of the present invention is to provide use of the nicotinamide ribokinase, the gene, the mutant, the recombinant plasmid or the recombinant bacterium in the biosynthesis of ⁇ -nicotinamide mononucleotide or in the preparation of a product for the biosynthesis of ⁇ -nicotinamide mononucleotide.
  • the tenth object of the present invention is to provide a method for biosynthesizing ⁇ -nicotinamide mononucleotide.
  • the present invention provides a nicotinamide ribokinase, which can be expressed in Escherichia coli, and the amino acid sequence of the nicotinamide ribokinase is shown in SEQ ID NO.2.
  • the present invention also provides a gene encoding the nicotinamide ribokinase.
  • nucleotide sequence of the gene is shown as SEQ ID NO.1.
  • the present invention also provides a recombinant plasmid for expressing the nicotinamide ribokinase, and the vector used to construct the recombinant plasmid is pET-28a, pET-32a, pRSFDuet-1 or pET-3b.
  • the vector used to construct the recombinant plasmid is pRSFDuet-1.
  • the nicotinamide ribokinase enzyme activity expressed by the obtained recombinant bacterium is relatively higher.
  • the present invention also provides a mutant of the nicotinamide ribokinase, which is obtained by mutating any one or several amino acids at positions 45, 88 or 189 of the nicotinamide ribokinase shown in SEQ ID NO.2.
  • the mutation is:
  • isoleucine (I) at position 88 to any one of glycine (G), alanine (A), valine (V), isoleucine (I), serine (S), threonine (T), cysteine (C), methionine (M), asparagine (N), glutamine (Q), arginine (R), and histidine (H);
  • positions 45, 88, and 189 may be mutated to threonine (T), arginine (R), and alanine (A), respectively.
  • the gene sequence encoding the mutant is not listed here. Given the known amino acid sequence and the gene sequence encoding the parent nicotinamide ribokinase, the gene sequence encoding the mutant can be determined based on the codon correspondence between amino acids and bases known in the art. In addition to mutating the parent, the corresponding mutant can also be obtained through methods such as artificial synthesis based on the mutant sequence. Therefore, the present invention seeks protection for the gene encoding the nicotinamide ribokinase mutant. Using this gene, the corresponding mutant can be obtained through recombinant expression.
  • the present invention also provides a recombinant plasmid for expressing nicotinamide ribokinase.
  • the recombinant plasmid contains a gene encoding the nicotinamide ribokinase or a mutant thereof of the present invention; or on the basis of containing the gene encoding the mutant of the present invention, it also contains a ribosome binding site sequence and/or other promoter sequence between the original promoter sequence of the recombinant plasmid and the gene; the ribosome binding site sequence is shown as SEQ ID NO.12 or SEQ ID NO.13, and the other promoter sequence is shown as SEQ ID NO.17 or SEQ ID NO.21.
  • the vector used to construct the recombinant plasmid is pET-28a, pET-32a, pRSFDuet-1 or pET-3b.
  • the vector used to construct the recombinant plasmid is pRSFDuet-1.
  • the present invention also provides a recombinant bacterium containing the recombinant plasmid.
  • the recombinant bacteria is based on Escherichia coli, Bacillus subtilis, Bacillus cereus, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus fragilis, Bacillus clausii, alkalophilic Bacillus, Bacillus thuringiensis, Saccharomyces cerevisiae, Pichia pastoris or Kluyveromyces lactis as the starting strain.
  • Escherichia coli is used as the starting strain.
  • the present invention also provides a method for improving the activity of nicotinamide ribokinase enzyme, the method comprising: mutating any one or more of amino acids at positions 45, 88, or 189 of the nicotinamide ribokinase of the present invention;
  • the gene sequence encoding the nicotinamide ribokinase of the present invention is replaced with the gene sequence encoding the nicotinamide ribokinase mutant of the present invention.
  • the present invention also provides a method for efficiently heterologously expressing nicotinamide ribokinase, the method comprising: when constructing a recombinant plasmid for expressing the nicotinamide ribokinase or a mutant of the nicotinamide ribokinase of the present invention, the recombinant plasmid is modified as follows: a ribosome binding site sequence and/or other promoter sequences are inserted between the original promoter sequence and the gene sequence of the recombinant plasmid; the ribosome binding site sequence is as shown in SEQ ID NO.12 or SEQ ID NO.13, and the other promoter sequence is as shown in SEQ ID NO.17 or SEQ ID NO.21.
  • the ribosome binding site sequence shown in SEQ ID NO.12 or SEQ ID NO.13 and/or SEQ ID The use of the promoter sequence shown in SEQ ID NO. 17 or SEQ ID NO. 21 in increasing the enzymatic activity (or heterologous expression) of the nicotinamide ribokinase of the present invention should also be within the scope of protection of the present invention.
  • the present invention also seeks to protect the use of the nicotinamide ribokinase, the gene encoding the nicotinamide ribokinase, the mutant of the nicotinamide ribokinase, the gene encoding the mutant of the nicotinamide ribokinase, the recombinant plasmid or the recombinant bacteria in the biosynthesis of ⁇ -nicotinamide mononucleotide or in the preparation of products for the biosynthesis of ⁇ -nicotinamide mononucleotide.
  • the present invention also provides a method for biosynthesizing ⁇ -nicotinamide mononucleotide, comprising: replacing the nicotinamide ribokinase used in the biosynthesis of ⁇ -nicotinamide mononucleotide with a mutant of the nicotinamide ribokinase described in the present invention, or replacing the recombinant bacteria expressing nicotinamide ribokinase with the recombinant bacteria described in the present invention.
  • the method comprises: using nicotinamide riboside (NR) and ATP as substrates, and using the mutant of the nicotinamide ribokinase or the recombinant bacteria of the present invention to catalyze the synthesis of ⁇ -nicotinamide mononucleotide.
  • NR nicotinamide riboside
  • ATP nicotinamide riboside
  • the method comprises: activating the recombinant bacteria of the present invention to obtain a seed solution; inoculating the obtained seed solution into a fermentation tank, culturing at 35-37° C. until the OD600 reaches 28-32, cooling to 24-26° C., adding IPTG at a final concentration of 0.4-0.6 mM to induce expression for 10-12 hours, and then conducting fed-batch fermentation; during the fed-batch fermentation, controlling the pH of the culture system to 6.5-7.5 and the fermentation tank speed to 350-450 rpm; as the bacteria continue to grow, glucose is gradually consumed, and when its concentration is lower than 2 g/L, feeding is performed to utilize the glucose to catalyze the substrate to produce ⁇ -nicotinamide mononucleotide.
  • the method comprises: activating the recombinant bacteria of the present invention to obtain a seed solution; inoculating the obtained seed solution into a fermentation tank culture medium at an inoculum rate of 7.5% (v/v), culturing at 37°C until the OD600 reaches 30, cooling to 25°C, adding 0.5 mM IPTG to induce expression for 12 hours, and then conducting fed-batch fermentation; during the fed-batch fermentation process, controlling the pH of the culture system to 7.0 and the fermentation tank speed to 400 rpm; and performing fed-batch feeding when the glucose concentration is lower than 2 g/L, using the recombinant bacteria to catalyze the substrate to produce ⁇ -nicotinamide mononucleotide.
  • the recombinant bacteria of the present invention when used for whole-cell catalysis, when the first batch of substrate is detected to be exhausted, the second batch of substrate is added, and so on; when the third batch of substrate is added, the yield of ⁇ -nicotinamide mononucleotide is the highest.
  • the present invention provides a nicotinamide ribokinase capable of heterologously expressing in Escherichia coli, and also provides a mutant of the nicotinamide ribokinase, wherein the nicotinamide ribokinase activity of the mutant is significantly improved compared to that of the parent.
  • the present invention also provides a method for efficiently heterologously expressing nicotinamide ribokinase, namely, when constructing a recombinant plasmid, by introducing a ribosome binding site and inserting a specific promoter sequence into the plasmid, the enzymatic activity of the expressed nicotinamide ribokinase is increased compared to a control.
  • the present invention provides a method for expanding the culture of recombinant bacteria expressing the nicotinamide ribokinase, thereby further increasing the enzymatic activity of the expressed nicotinamide ribokinase, reaching a maximum of 208.3 U/mL.
  • the invention is conducive to the efficient biosynthesis of ⁇ -nicotinamide mononucleotide and is suitable for industrial production and application.
  • FIG1 shows the results of enzyme activity determination of nicotinamide ribokinase expressed by different recombinant bacteria constructed in Example 1.
  • FIG2 is the SDS-PAGE analysis results of nicotinamide ribokinase expressed by different recombinant bacteria constructed in Example 1.
  • Figure 3 shows the enzyme activity determination results of nicotinamide ribokinase expressed by recombinant bacteria containing different RBS sequences; control is the enzyme activity of nicotinamide ribokinase expressed by the control bacteria E. coli BL21 (DE3)/pRSFDuet-Nrk.
  • Figure 4 shows the enzyme activity determination results of nicotinamide ribokinase expressed by recombinant bacteria containing different dual promoter sequences
  • control is the enzyme activity of nicotinamide ribokinase expressed by the control bacteria E. coli BL21 (DE3)/pRSFDuet-35000-Nrk
  • P1 to P6 are the enzyme activities of nicotinamide ribokinase expressed by recombinant bacteria constructed using dnaKJ, alsR, ssrA, skp, gapA and aceE promoters, respectively.
  • FIG5 shows the relative enzyme activity determination results of nicotinamide ribokinase expressed by recombinant bacteria with different mutants.
  • FIG6 shows the relative enzyme activity determination results of nicotinamide ribokinase expressed by recombinant bacteria obtained by saturation mutation of position 45D of nicotinamide ribokinase according to the present invention.
  • FIG7 shows the relative enzyme activity assay results of nicotinamide ribokinase expressed by recombinant bacteria obtained by saturation mutation of position 88 of the nicotinamide ribokinase according to the present invention.
  • FIG8 shows the results of relative enzyme activity determination of nicotinamide ribokinase expressed by recombinant bacteria obtained by saturation mutation of position 189E of the nicotinamide ribokinase according to the present invention.
  • FIG9 shows the results of the relative enzyme activity assay of nicotinamide ribokinase expressed by the combined mutant NRK-TRA recombinant bacteria.
  • FIG10 shows the changes in OD600 during the amplification culture of the recombinant bacteria of the combined mutant NRK-TRA.
  • FIG11 shows the changes in the enzyme activity of nicotinamide ribokinase during the amplification culture of the recombinant bacteria of the combined mutant NRK-TRA.
  • FIG12 shows the optimum temperature of the nicotinamide ribokinase mutant NRK-TRA.
  • FIG13 shows the optimal pH of the nicotinamide ribokinase mutant NRK-TRA.
  • the present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
  • the present invention uses the gene encoding nicotinamide ribokinase (Nrk) from Kluyveromyces lactis as a parent and, through optimization design, obtains a nicotinamide ribokinase capable of expression in Escherichia coli.
  • Nrk nicotinamide ribokinase
  • the gene sequence of the optimized nicotinamide ribokinase is shown below (SEQ ID NO. 1):
  • amino acid sequence of the protein encoded by the optimized nicotinamide ribokinase gene is shown below (SEQ ID NO. 2):
  • Tianlin Biotechnology (Shanghai) Co., Ltd. was commissioned to artificially synthesize the gene sequence shown in SEQ ID NO. 1 and ligate it into pET-28a, pET-32a, pRSFDuet-1, and pET-3b vectors, respectively. After transformation, screening, and verification, the recombinant plasmids verified to be correct by sequencing were saved. The saved recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and verified, resulting in four recombinant strains expressing the gene sequence shown in SEQ ID NO. 1, which were each saved separately.
  • a single colony of the recombinant bacteria was selected and inoculated into 10 mL of LB medium containing 50 ⁇ g/mL kanamycin for 10 hours for activation.
  • the activated bacterial solution was then inoculated into 30 mL of LB medium at a 1% (v/v) inoculum and cultured at 37°C and 220 rpm until an OD600 of 0.6-0.8 was reached.
  • IPTG was then added to a final concentration of 0.5 mM and fermented for 12 hours.
  • the resulting bacterial solution was centrifuged at 4°C, and the pellet was thoroughly vortexed and resuspended in 10 mL of PBS. This process was repeated twice to obtain a cell suspension.
  • the nicotinamide ribokinase enzyme activity (or simply, enzyme activity) in the cell suspension was determined and analyzed by SDS-PAGE.
  • the nicotinamide ribokinase activity detection method is as follows:
  • Enzyme activity is calculated based on the amount of ⁇ -NMN produced. Nicotinamide ribokinase activity is defined as the amount of enzyme protein required to produce 1 ⁇ mol of ⁇ -NMN per minute under the above reaction conditions.
  • FIG. 1 The results of the enzyme activity determination of nicotinamide ribokinase expressed by different recombinant bacteria constructed in Example 1 of the present invention are shown in Figure 1.
  • the present invention uses the pRSFDuet-1 vector to construct a recombinant plasmid (pRSFDuet-Nrk), and the recombinant bacteria E. coli BL21 (DE3) / pRSFDuet-Nrk constructed therefrom have the highest enzyme activity of nicotinamide ribokinase, which is 2.14 U/mL.
  • FIG. 2 The results of SDS-PAGE analysis of the nicotinamide ribokinase expressed by the different recombinant bacteria constructed in Example 1 of the present invention are shown in Figure 2.
  • 1 to 4 in Figure 2 correspond to the recombinant bacteria constructed using the pET-32a(+), pET-28a(+), pRSFDuet-1, and pET-3b vectors, respectively.
  • the supernatants of the different recombinant bacteria all have a specific protein band at 27.3 kDa, indicating that the nicotinamide ribokinase gene shown in SEQ ID NO. 1 has been successfully expressed intracellularly in E. coli BL21(DE3).
  • the band in the supernatant of the recombinant bacteria E. coli BL21(DE3)/pRSFDuet-Nrk is thicker, indicating that the soluble expression level of nicotinamide ribokinase in the recombinant bacteria E. coli BL21(DE3)/pRSFDuet-Nrk is high.
  • the present invention utilizes the following different methods.
  • the present invention designs 13 different RBS sequences with translation initiation rates of 5000, 7500, 10000, 12500, 15000, 17500, 20000, 25000, 30000, 35000, 40000, 80000, and 100000 a.u., respectively. These sequences are designated R1 to R13, and their sequences (SEQ ID NOs. 3 to 15) are shown in Table 1. Also provided are primers for amplifying linearized plasmids carrying different RBSs (R1 to R13) using reverse polymerase chain reaction, as shown in Table 2.
  • the present invention used the recombinant plasmid pRSFDuet-Nrk constructed in Example 1 as a DNA template and the primers shown in Table 2 to obtain 13 linearized plasmids carrying different RBSs by reverse polymerase chain reaction and constructed corresponding recombinant bacteria.
  • the constructed recombinant bacteria containing different RBS sequences were induced to express using the method described in Example 1, and the enzyme activity of the expressed nicotinamide ribokinase was measured.
  • the reverse polymerase chain reaction was performed in a 50 ⁇ L reaction system, adding 20 ⁇ L of ddH2O, 1 ⁇ L of template, 25 ⁇ L of 2 ⁇ Phanta Max Master Mix, and 2 ⁇ L each of upstream and downstream primers.
  • the reaction conditions were as follows: pre-denaturation at 95°C for 3 minutes, followed by 34 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 2.5 minutes; the final extension was at 72°C for 5 minutes.
  • the methylated template plasmid pRSFDuet-Nrk was then digested with the Dpn I restriction enzyme and transformed into E. coli BL21(DE3).
  • the resulting transformants were sequenced and screened for positive results, yielding recombinant strains expressing nicotinamide ribokinase containing different RBS sequences. Expression of the resulting recombinant strains was induced using the method described in Example 1, and the enzymatic activity of the expressed nicotinamide ribokinase was measured.
  • Control represents the enzyme activity of nicotinamide ribokinase expressed in the control strain E. coli BL21(DE3)/pRSFDuet-Nrk
  • R1 to R13 represent the enzyme activities of nicotinamide ribokinase expressed in recombinant strains R1 to R13, respectively.
  • Figure 3 compared to the control strain, the enzyme activity of nicotinamide ribokinase expressed in recombinant strains R10 (E.
  • the effective expression of proteins is closely related to their transcription efficiency. Promoters are the key to promoting high-level gene expression and Important regulatory elements for recombinant protein production.
  • the present invention discovered that, based on the constructed pRSFDuet-35000-Nrk recombinant plasmid (the recombinant plasmid contained in the R10 recombinant bacteria), by inserting different promoter sequences in tandem with the original T7 promoter sequence in the recombinant plasmid, the enzymatic activity of the expressed nicotinamide ribokinase can be altered. In other words, the enzymatic activity of the nicotinamide ribokinase described in the present invention can be enhanced by introducing a specific promoter sequence.
  • the present invention inserts the promoter sequence into the constructed pRSFDuet-35000-Nrk recombinant plasmid by enzyme digestion followed by ligation.
  • the reverse polymerase chain reaction was performed in a 50 ⁇ L reaction system, adding 20 ⁇ L of ddH2O, 1 ⁇ L of template, 25 ⁇ L of 2 ⁇ Phanta Max Master Mix, and 2 ⁇ L each of upstream and downstream primers. Reaction conditions were as follows: pre-denaturation at 95°C for 3 minutes, followed by 34 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 2.5 minutes; the final extension was at 72°C for 5 minutes.
  • the methylated template plasmid pRSFDuet-35000-Nrk was digested with Dpn I restriction enzyme and transformed into E. coli BL21(DE3).
  • the resulting transformants were sequenced and screened for successful constructs, resulting in recombinant strains harboring different dual promoters. Expression of the resulting recombinant strains was induced using the method described in Example 1, and the enzymatic activity of the expressed nicotinamide ribokinase was measured.
  • Table 3 Specific primers used to amplify promoter sequences and primers required for linearization of templates Note: The capital letters in the primers represent the promoter sequence, and the lowercase letters represent the homology arms.
  • control is the enzyme activity of nicotinamide ribokinase expressed by the control bacteria E. coli BL21 (DE3) / pRSFDuet-35000-Nrk
  • P1 is the enzyme activity of nicotinamide ribokinase expressed by the recombinant bacteria E.
  • P2 is the enzyme activity of nicotinamide ribokinase expressed by the alsR promoter.
  • P3 is the enzyme activity of nicotinamide ribokinase obtained by expressing the recombinant bacteria E.coli BL21(DE3)/pRSFDuet-35000-P ssrA -Nrk using the ssrA promoter;
  • P4 is the enzyme activity of nicotinamide ribokinase obtained by expressing the recombinant bacteria E.coli BL21(DE3)/pRSFDuet-35000-P skp -Nrk using the skp promoter;
  • P5 is the enzyme activity of nicotinamide ribokinase obtained by expressing the recombinant bacteria E.coli BL21(DE3)/pRSFDuet-35000-P gapA
  • the nicotinamide ribokinase enzyme activity obtained by expressing the same nicotinamide ribokinase gene using different dual promoters is different.
  • the nicotinamide ribokinase enzyme activity of the recombinant bacteria obtained by expressing the alsR promoter (shown in SEQ ID NO.17) or the aceE promoter (shown in SEQ ID NO.21) in series with the T7 promoter is the highest.
  • the nicotinamide ribokinase enzyme activity of the recombinant bacteria obtained by expressing the alsR promoter in series with the T7 promoter is the highest, at 5.56 U/mL, which is 30% higher than that of the control bacteria, indicating that the introduction of the alsR promoter and its series connection with the T7 promoter can significantly improve the enzyme activity of the expressed nicotinamide ribokinase.
  • the present invention uses the recombinant plasmid pRSFDuet-35000-P alsR -Nrk as a DNA template and uses site-directed mutagenesis primers containing mutation sites (as shown in Table 4) to obtain a series of mutants through reverse polymerase chain reaction.
  • PCR reverse polymerase chain reaction
  • the reaction conditions were as follows: pre-denaturation at 95°C for 3 minutes, followed by 34 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 2.5 minutes, followed by a final extension at 72°C for 5 minutes.
  • the methylated template plasmid pRSFDuet-35000-P alsR -Nrk was then digested with the Dpn I restriction enzyme and transformed into E. coli BL21(DE3). The resulting transformants were sequenced and screened for successful constructs, yielding different nicotinamide ribokinase mutant strains.
  • the present invention obtained plasmids containing the gene sequences of the nicotinamide ribokinase mutants D45P, I88Q, and E189G as shown in SEQ ID NO. 2.
  • the D45P mutant is a mutant of nicotinamide ribokinase with position 45 D mutated to P, and the same applies to the remaining mutants.
  • recombinant bacteria (or mutant strains) expressing the mutants were constructed by transforming the plasmids containing the mutant gene sequences into Escherichia coli BL21(DE3).
  • the present invention induced expression of the mutant recombinant bacteria and measured the nicotinamide ribokinase enzyme activity.
  • the relative enzyme activity of all mutant strains was calculated using E. coli BL21(DE3)/pRSFDuet-35000-P alsR -Nrk as a control.
  • the relative enzyme activity results of the nicotinamide ribokinase expressed by the recombinant bacteria of the different mutants are shown in Figure 5.
  • Figure 5 compared with the control bacteria, the enzyme activities of nicotinamide ribokinase expressed by the recombinant bacteria of mutants D45P, I88Q and E189G were all improved.
  • mutants D45P, I88Q, and E189G were used as DNA templates to perform saturation mutagenesis on them, respectively.
  • position 45D of the nicotinamide ribokinase shown in SEQ ID NO.2 was mutated to G/A/V/L/I/P/F/Y/W/S/T/C/M/N/Q/E/K/H/R
  • position 88I of the nicotinamide ribokinase shown in SEQ ID NO.2 was mutated to G/A/V/L/H/P/F/Y/W/S/T/C/M/N/Q/D/E/K/R
  • position 189E of the nicotinamide ribokinase shown in SEQ ID NO.2 was mutated to G/A/V/L/H/P/F/Y/W/S/T/C/M/N/Q/D/I/K/R.
  • the present invention obtained plasmids containing the aforementioned gene sequences saturated with different site mutations.
  • Recombinant bacteria expressing the mutants were constructed by transforming the plasmids containing the mutant gene sequences into E. coli BL21 (DE3).
  • the reverse PCR reaction was performed in a 50 ⁇ L reaction system, adding 20 ⁇ L of ddH2O, 1 ⁇ L of template, 25 ⁇ L of 2 ⁇ Phanta Max Master Mix, and 2 ⁇ L each of upstream and downstream primers.
  • the reaction conditions were: 34 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 2.5 minutes, followed by a final extension at 72°C for 5 minutes.
  • the methylated template plasmids D45P, I88Q, or E189G were then digested with the Dpn I restriction enzyme and transformed into E. coli BL21(DE3).
  • the resulting transformants were sequenced and verified, and positive transformants were screened to obtain different nicotinamide ribokinase mutant strains.
  • the present invention induced expression of the mutant recombinant bacteria and measured their nicotinamide ribokinase enzyme activities. Relative enzyme activities were calculated using E. coli BL21(DE3)/pRSFDuet-35000-P alsR -Nrk as a control.
  • the relative enzyme activity assay results of the nicotinamide ribokinase expressed by the recombinant strains obtained by saturation mutation of position 189 E of the nicotinamide ribokinase shown in SEQ ID NO. 2 are shown in Figure 8 .
  • the glutamic acid (E) at position 189 is mutated to glycine (G), alanine (A), or glutamine (Q)
  • the enzyme activity of the nicotinamide ribokinase of the mutant strains is increased compared to the control.
  • the present invention also carried out a combined mutation of the nicotinamide ribokinase shown in SEQ ID NO.2 to obtain a mutant strain capable of expressing the combined mutant NRK-TRA (D45T-I88R-E189A).
  • the expressed nicotinamide ribokinase mutant was purified by nickel column affinity chromatography and its relative enzyme activity was measured.
  • Example 3 Scale-up of Nicotinamide Ribokinase Recombinant Bacteria in a 5 L Fermenter
  • the present invention used a 5L fermentor to perform batch fermentation on the recombinant bacteria of the combined mutant NRK-TRA to further improve the enzyme activity of NRK-TRA.
  • the recombinant bacterial strain was activated and a seed solution was obtained by culturing; the seed solution was inoculated into a fermentation tank culture medium at an inoculum size of 7.5% (v/v), cultured at 37° C. until the OD 600 reached 30, then cooled to 25° C., and IPTG was added at a final concentration of 0.5 mM to induce expression for 12 hours; during the fed-batch fermentation process, the pH of the culture system was controlled at 7.0, and the fermentation tank speed was controlled at 400 rpm.
  • the present invention used whole-cell catalysis to determine the relative enzyme activity of nicotinamide ribokinase at different temperatures and pH.
  • the activity of the recombinant nicotinamide ribokinase mutant NRK-TRA was measured at temperatures ranging from 30°C to 75°C.
  • the relative enzyme activity at different reaction temperatures was calculated using the enzyme activity at 55°C as a control.
  • the results are shown in Figure 12.
  • the activity of the recombinant nicotinamide ribokinase mutant NRK-TRA was measured in buffer solutions with different pH values (pH 6-11). The relative activity of the enzyme at different pH values was calculated using the activity at pH 7.2 as a control. The results are shown in Figure 13. As shown in Figure 13, the nicotinamide ribokinase mutant exhibited the highest activity at pH 7.2 and was better able to maintain its activity under neutral conditions.
  • the present invention explored the conditions for batch transformation using the recombinant bacteria of the whole-cell catalyst combination mutant NRK-TRA as the experimental object under the optimal reaction temperature and pH conditions explored in Example 4.
  • the bacterial liquid obtained from high-density fermentation was centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was discarded; 30 mL of PBS buffer was added to resuspend the bacteria, and the mixture was centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was discarded. This was repeated twice to wash the bacteria (cells); 15 mL of PBS buffer was added to the bacterial pellet, the bacteria were resuspended, and then ice-bathed for 10 minutes to obtain the whole-cell catalyst.

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Abstract

提供了一种烟酰胺核糖激酶突变体在大肠杆菌中的高效异源表达及其应用。提供了一种能够在大肠杆菌中异源表达的烟酰胺核糖激酶及其突变体，所述突变体的烟酰胺核糖激酶活性较亲本有明显提升。在所述烟酰胺核糖激酶及其突变体的基础上，还提供了一种高效异源表达所述烟酰胺核糖激酶的方法，即在构建重组质粒时，通过在质粒中引入核糖体结合位点、插入特定的启动子序列等，使表达所得烟酰胺核糖激酶的酶活力较对照更高。此外，还提供了培养表达所述烟酰胺核糖激酶的重组菌的方法，使表达所得烟酰胺核糖激酶的酶活最高为208.3U/mL。有利于β-烟酰胺单核苷酸的高效生物合成，适合工业化生产和应用。

Description

一种烟酰胺核糖激酶突变体在大肠杆菌中的高效异源表达及其应用 技术领域
本发明属于酶工程技术领域。更具体地，涉及一种烟酰胺核糖激酶突变体在大肠杆菌中的高效异源表达及其应用。
背景技术
β-烟酰胺单核苷酸(β-NMN)是广泛存在于人类和动物体内的一种生物活性核苷酸。作为烟酰胺腺嘌呤二核苷酸(NAD+)的直接前体，β-NMN对提高体内NAD+水平具有显著作用，在预防和改善衰老相关的症状和慢性疾病方面具有重要应用，可开发为膳食补充剂或护肤品等。但当前β-NMN的产量并不能满足其日益增长的需求。
2010年以前，β-NMN主要以单磷酸腺苷(AMP)、四乙酰核糖(TAR)和烟酰胺(NAM)为原料，通过化学合成的方式大规模生产。但由于有机溶剂的使用量增加、环境污染和纯化等难题，这种方法最终被终止。结合生物催化和微生物代谢工程生物合成β-NMN具有绿色、环保的优点。随着对β-NMN代谢途径的分析以及代谢和酶工程技术的发展，生物催化已成为大规模生产β-NMN的主要方法。
通过大肠杆菌代谢工程方法合成NMN通常是以烟酰胺(NAM)和磷酸戊糖途径的中间体磷酸核糖基焦磷酸(PRPP)作为底物，经过磷酸核糖糖基转移酶(Nampt，EC 2.4.2.12)催化生成。但是以葡萄糖、木糖作为原料很难达到细胞内PRPP的高水平积累，最终导致NMN的产量并不理想。另一种NMN的生物合成方法是以烟酰胺核糖(NR)和ATP作为底物，经过烟酰胺核糖激酶(nicotinamide riboside kinase，Nrk)催化生成β-NMN。但现有Nrk在大肠杆菌等宿主菌中异源表达后的酶活力普遍不高，通过高密度发酵工艺等所得发酵液中的烟酰胺核糖激酶酶活也仅在100U/mL左右，仍不能很好的满足酶法高效合成β-NMN的需要。
发明内容
本发明针对上述现有技术中存在的问题，提供了一种能够在大肠杆菌中表达的烟酰胺核糖激酶，同时还提供了酶活相对亲本提高的烟酰胺核糖激酶的突变体，以及高效异源表达烟酰胺核糖激酶的方法和应用，有利于β-烟酰胺单核苷酸的高效生物合成。
本发明的第一个目的是提供一种烟酰胺核糖激酶。
本发明的第二个目的是提供一种编码所述烟酰胺核糖激酶的基因。
本发明的第三个目的是提供所述烟酰胺核糖激酶的突变体。
本发明的第四个目的是提供一种编码所述突变体的基因。
本发明的第五个目的是提供一种表达烟酰胺核糖激酶的重组质粒。
本发明的第六个目的是提供一种含有所述重组质粒的重组菌。
本发明的第七个目的是提供一种提高烟酰胺核糖激酶酶活力的方法。
本发明的第八个目的是提供一种高效异源表达烟酰胺核糖激酶的方法。
本发明的第九个目的是提供所述烟酰胺核糖激酶、所述基因、所述突变体、所述重组质粒或所述重组菌在生物合成β-烟酰胺单核苷酸或在制备用于生物合成β-烟酰胺单核苷酸的产品中的应用。
本发明的第十个目的是提供一种生物合成β-烟酰胺单核苷酸的方法。
本发明上述目的通过以下技术方案实现：
本发明提供了一种烟酰胺核糖激酶，所述烟酰胺核糖激酶可以在大肠杆菌中表达，所述烟酰胺核糖激酶的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了编码所述烟酰胺核糖激酶的基因。
作为其中的一种可选实施方式，所述基因的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种表达所述烟酰胺核糖激酶的重组质粒，构建所述重组质粒所用载体为pET-28a、pET-32a、pRSFDuet-1或pET-3b。
优选地，构建所述重组质粒所用载体为pRSFDuet-1，利用该载体构建得到的重组质粒转化宿主菌后，所得重组菌表达所得的烟酰胺核糖激酶的酶活力相对更高。
本发明还提供了所述烟酰胺核糖激酶的突变体，所述突变体是对SEQ ID NO.2所示烟酰胺核糖激酶的第45、88或189位氨基酸中的任意一个或几个氨基酸进行突变得到。
具体地，所述突变为：
将第45位天冬氨酸(D)突变为甘氨酸(G)、丙氨酸(A)、缬氨酸(V)、脯氨酸(P)、苯丙氨酸(F)、酪氨酸(Y)、丝氨酸(S)、苏氨酸(T)、半胱氨酸(C)、蛋氨酸(M)、天冬酰胺(N)、谷氨酰胺(Q)、谷氨酸(E)、赖氨酸(K)、精氨酸(R)中的任意一种；
将第88位异亮氨酸(I)突变为甘氨酸(G)、丙氨酸(A)、缬氨酸(V)、异亮氨酸(I)、丝氨酸(S)、苏氨酸(T)、半胱氨酸(C)、蛋氨酸(M)、天冬酰胺(N)、谷氨酰胺(Q)、精氨酸(R)、组氨酸(H)中的任意一种；
将第189位谷氨酸(E)突变为甘氨酸(G)、丙氨酸(A)、谷氨酰胺(Q)中的任意一种；
或将第45、88和189位分别突变为苏氨酸(T)、精氨酸(R)和丙氨酸(A)。
如上，各突变体的具体氨基酸序列不再列出。
另编码所述突变体的基因序列也不再列出。在氨基酸序列已知以及编码亲本烟酰胺核糖激酶的基因序列已知的基础上，按本领域公知氨基酸与碱基的密码子对应关系即可确定编码所述突变体的基因序列。除对亲本进行突变外，也可以依据突变体的序列，通过人工合成等方法获得相应突变体。因此，本发明请求保护编码所述烟酰胺核糖激酶突变体的基因。利用所述基因，可通过重组表达获得相应的突变体。
本发明还提供了一种表达烟酰胺核糖激酶的重组质粒。
具体地，所述重组质粒中含有编码本发明所述烟酰胺核糖激酶或其突变体的基因；或在含有编码本发明所述突变体的基因的基础上，在所述重组质粒的原有启动子序列与所述基因间还含有核糖体结合位点序列和/或其它启动子序列；所述核糖体结合位点序列如SEQ ID NO.12或SEQ ID NO.13所示，所述其它启动子序列如SEQ ID NO.17或SEQ ID NO.21所示。
可选地，构建所述重组质粒所用载体为pET-28a、pET-32a、pRSFDuet-1或pET-3b。
优选地，构建所述重组质粒所用载体为pRSFDuet-1。
本发明还提供了一种含有上述重组质粒的重组菌。
可选地，所述重组菌是以大肠杆菌、枯草芽孢杆菌、蜡样芽孢杆菌、蜡状芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌、脆弱杆菌、克劳氏芽孢杆菌、嗜碱芽孢杆菌、苏云金芽孢杆菌、酿酒酵母、毕赤酵母或乳酸克鲁维酵母为出发菌株。
在本发明具体的实施例中是以大肠杆菌为出发菌株。
本发明还提供了一种提高烟酰胺核糖激酶酶活力的方法，所述方法为：对本发明所述烟酰胺核糖激酶的第45、88或189位氨基酸中的任意一个或几个氨基酸进行突变；
或将编码本发明所述烟酰胺核糖激酶的基因序列替换为编码本发明所述烟酰胺核糖激酶突变体的基因序列。
本发明还提供了一种高效异源表达烟酰胺核糖激酶的方法，所述方法为：在构建用于表达本发明所述烟酰胺核糖激酶或所述烟酰胺核糖激酶的突变体的重组质粒时，对重组质粒进行如下改造：在重组质粒的原有启动子序列与基因序列之间插入核糖体结合位点序列和/或其它启动子序列；所述核糖体结合位点序列如SEQ ID NO.12或SEQ ID NO.13所示，所述其它启动子序列如SEQ ID NO.17或SEQ ID NO.21所示。
SEQ ID NO.12或SEQ ID NO.13所示的核糖体结合位点序列和/或SEQ ID  NO.17或SEQ ID NO.21所示启动子序列在提高本发明所述烟酰胺核糖激酶的酶活(或说异源表达)中的应用也应在本发明的保护范围之内。
本发明还请求保护所述烟酰胺核糖激酶、编码所述烟酰胺核糖激酶的基因、所述烟酰胺核糖激酶的突变体、编码所述烟酰胺核糖激酶突变体的基因、所述重组质粒或所述重组菌在生物合成β-烟酰胺单核苷酸或在制备用于生物合成β-烟酰胺单核苷酸的产品中的应用。
本发明还提供了一种生物合成β-烟酰胺单核苷酸的方法，所述方法为：将生物合成β-烟酰胺单核苷酸所用烟酰胺核糖激酶替换为本发明所述烟酰胺核糖激酶的突变体，或将所用表达烟酰胺核糖激酶的重组菌替换为本发明所述重组菌。
具体地，所述方法为：以烟酰胺核糖(NR)和ATP作为底物，用本发明所述烟酰胺核糖激酶的突变体或所述重组菌催化合成β-烟酰胺单核苷酸。
作为其中一种具体的实施方式，所述方法为：活化本发明所述重组菌，得种子液；将所得种子液接入发酵罐中，35～37℃培养至OD600达到28～32时，降温至24～26℃并加入终浓度为0.4～0.6mM的IPTG诱导表达10～12h后进行补料发酵；补料发酵过程中，控制培养体系pH为6.5～7.5，控制发酵罐转速为350～450rpm；随着菌体的不断生长，葡萄糖被逐步消耗，当其浓度低于2g/L时，进行流加补料，利用其催化底物生成β-烟酰胺单核苷酸。
具体地，所述方法为：活化本发明所述重组菌，得种子液；将所得种子液以7.5％(v/v)的接种量接入发酵罐培养基中，37℃培养至OD600达到30时，降温至25℃，加入0.5mM的IPTG诱导表达12h后进行补料发酵；在补料发酵过程中，控制培养体系pH为7.0，控制发酵罐转速为400rpm；当葡萄糖浓度低于2g/L时，进行流加补料，利用所述重组菌催化底物生成β-烟酰胺单核苷酸。
此外，利用本发明所述重组菌进行全细胞催化时，当检测到第一批底物耗尽时，加入第二批底物，以此类推；加入第三批底物时，β-烟酰胺单核苷酸的产量最高。
本发明具有以下有益效果：
本发明提供了一种能够在大肠杆菌中异源表达的烟酰胺核糖激酶，同时还提供了所述烟酰胺核糖激酶的突变体，所述突变体的烟酰胺核糖激酶活性相对亲本有了明显提升。在所述烟酰胺核糖激酶及其突变体的基础上，本发明还提供了一种高效异源表达烟酰胺核糖激酶的方法，即在构建重组质粒时，通过在质粒中引入核糖体结合位点、插入特定的启动子序列等，使表达所得烟酰胺核糖激酶的酶活力较对照更高。此外，本发明还提供了扩大培养表达所述烟酰胺核糖激酶的重组菌的方法，使其表达所得的烟酰胺核糖激酶酶活进一步提高，最高为208.3U/mL。
本发明有利于β-烟酰胺单核苷酸的高效生物合成，适合工业化生产和应用。
附图说明
图1为实施例1中构建的不同重组菌表达所得烟酰胺核糖激酶的酶活力测定结果。
图2为实施例1中构建的不同重组菌表达所得烟酰胺核糖激酶的SDS-PAGE分析结果。
图3为含不同RBS序列的重组菌表达得到的烟酰胺核糖激酶的酶活力测定结果；control为对照菌E.coli BL21(DE3)/pRSFDuet-Nrk表达所得烟酰胺核糖激酶的酶活。
图4为含不同双启动子序列的重组菌表达得到的烟酰胺核糖激酶的酶活力测定结果；control为对照菌E.coli BL21(DE3)/pRSFDuet-35000-Nrk表达所得烟酰胺核糖激酶的酶活；P1～P6依次为利用dnaKJ、alsR、ssrA、skp、gapA和aceE启动子构建所得重组菌表达所得烟酰胺核糖激酶的酶活。
图5为不同突变体的重组菌表达得到的烟酰胺核糖激酶的相对酶活力测定结果。
图6为本发明所述烟酰胺核糖激酶的第45位D进行饱和突变所得各重组菌表达得到的烟酰胺核糖激酶的相对酶活力测定结果。
图7为本发明所述烟酰胺核糖激酶的第88位I进行饱和突变所得各重组菌表达得到的烟酰胺核糖激酶的相对酶活力测定结果。
图8为本发明所述烟酰胺核糖激酶的第189位E进行饱和突变所得各重组菌表达得到的烟酰胺核糖激酶的相对酶活力的测定结果。
图9为组合突变体NRK-TRA重组菌表达得到的烟酰胺核糖激酶的比酶活测定结果。
图10为组合突变体NRK-TRA的重组菌的放大培养过程中，OD600的变化情况。
图11为组合突变体NRK-TRA的重组菌的放大培养过程中，烟酰胺核糖激酶的酶活力变化情况。
图12为烟酰胺核糖激酶突变体NRK-TRA的最适温度。
图13为烟酰胺核糖激酶突变体NRK-TRA的最适pH。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1 烟酰胺核糖激酶的重组表达
本发明以来源于Kluyveromyces lactis的编码烟酰胺核糖激酶(Nrk)的基因为亲本，通过优化设计得到了一个能够在大肠杆菌中表达的烟酰胺核糖激酶。优化设计得到的烟酰胺核糖激酶的基因序列如下(SEQ ID NO.1)所示：
优化设计得到的烟酰胺核糖激酶基因所编码的蛋白的氨基酸序列如下(SEQ ID NO.2)所示：
委托天霖生物科技(上海)有限公司人工合成SEQ ID NO.1所示基因序列并将其分别连接至pET-28a、pET-32a、pRSFDuet-1和pET-3b载体中，经转化、筛选及验证等过程后，保存经测序验证正确的重组质粒。将保存的重组质粒分别转化至大肠杆菌BL21(DE3)中并验证，获得表达SEQ ID NO.1所示基因序列的4种重组菌，分别保存。
分别挑取上述保存的重组菌的单菌落接种于10mL含50μg/mL卡那霉素的LB培养基中培养10h进行活化，活化菌液以1％(v/v)接种量接种于30mL LB培养基中，37℃、220rpm培养至OD600为0.6～0.8时，加入终浓度为0.5mM的IPTG发酵培养12h。发酵培养结束后，将所得菌液4℃离心，沉淀加10mL的PBS溶液充分振荡后重悬，重复2次后得细胞液，测定细胞液中烟酰胺核糖激酶的酶活力(或简称酶活)并进行烟酰胺核糖激酶的SDS-PAGE分析。
烟酰胺核糖激酶活力检测方法如下：
(1)先配制1.2mol/L烟酰胺核糖(NR)、1.2mol/L ATP和0.6mol/L MgCl2，各取50μL，加入ddH2O 800μL和细胞液50μL配制得到全细胞催化体系(1mL)；37℃、1500rpm振荡反应10min，反应结束后加100μL 25％ HCl终止反应；
(2)使用C18柱(5μm，250×4.6mm)进行HPLC检测；流动相主要包括两部分，流动相A是甲醇、水、冰醋酸以及四丁基氢氧化铵体积比为3：97：1：0.60配制得到，流动相B是100％甲醇；洗脱程序为：0min，100％ A；5min，100％ A；25min，40％ A；30min，20％ A；45min，100％ A；每组实验测3次取平均值；
通过产物β-NMN的生成量计算酶活力。烟酰胺核糖激酶的酶活力定义：在上述反应条件下，1个酶活力单位(U)定义为每分钟产生1μmolβ-NMN所需的酶蛋白量。
本发明实施例1中构建的不同重组菌表达所得烟酰胺核糖激酶的酶活力测定结果如图1所示。由图1可知，利用不同载体进行烟酰胺核糖激酶的表达会对其表达所得烟酰胺核糖激酶的酶活力有一定影响。本发明利用pRSFDuet-1载体构建得到重组质粒(pRSFDuet-Nrk)，再构建得到的重组菌E.coli BL21(DE3)/pRSFDuet-Nrk表达所得烟酰胺核糖激酶的酶活最高，为2.14U/mL。
本发明实施例1中构建的不同重组菌表达所得烟酰胺核糖激酶的SDS-PAGE分析结果如图2所示，图2中的1～4分别对应利用pET-32a(+)、pET-28a(+)、pRSFDuet-1、pET-3b载体构建所得重组菌。由图2可知，不同重组菌的上清液在27.3kDa处均有特异蛋白条带，表明SEQ ID NO.1所示烟酰胺核糖激酶基因在E.coli BL21(DE3)中成功实现了胞内表达。其中，重组菌E.coli BL21(DE3)/pRSFDuet-Nrk的上清液中的条带较粗，表明重组菌E.coli BL21(DE3)/pRSFDuet-Nrk中烟酰胺核糖激酶的可溶性表达水平较高。
实施例2 烟酰胺核糖激酶酶活的提升方案
为进一步提高在大肠杆菌中表达的烟酰胺核糖激酶的酶活，本发明利用了下述不同的方法。
1、利用核糖体结合位点(RBS)提高烟酰胺核糖激酶的酶活
本发明设计了翻译起始速率分别为5000、7500、10000、12500、15000、17500、20000、25000、30000、35000、40000、80000、100000a.u.的13条不同的RBS序列，依次命名为R1～R13，其序列(SEQ ID NO.3～15)如表1所示。同时提供了利用反向聚合酶链式反应扩增携带不同RBS(R1～R13)的线性化质粒所用引物，如表2所示。
表1不同翻译起始速率的RBS序列
表2反向聚合酶链式反应扩增携带不同RBS的线性化质粒所用引物


注：引物中的大写字母为不同的RBS序列，小写字母为同源臂。
本发明以实施例1中构建的重组质粒pRSFDuet-Nrk为DNA模板，利用表2所示引物，通过反向聚合酶链式反应得到了13个携带不同RBS的线性化质粒并构建得到了对应的重组菌，利用实施例1所述方法对构建得到的含不同RBS序列的重组菌进行了诱导表达并测定了其表达所得烟酰胺核糖激酶的酶活。
其中，所述反向聚合酶链式反应在50μL反应体系中进行，加入20μL的ddH2O、1μL的模板、25μL的2×Phanta Max Master Mix以及上、下游引物各2μL。反应条件为：在95℃预变性3min后开始循环：95℃变性15s，55℃退火15s，72℃延伸2.5min，共34个循环；72℃终延伸5min。然后用Dpn I限制酶消化甲基化的模板质粒pRSFDuet-Nrk，转化到E.coli BL21(DE3)中，对得到的转化子进行测序验证，筛选出构建成功的阳性转化子，得到含有不同RBS序列的表达所述烟酰胺核糖激酶的重组菌。利用实施例1所述方法对所得重组菌进行诱导表达并测定表达所得烟酰胺核糖激酶的酶活。
含不同RBS序列的重组菌表达得到的烟酰胺核糖激酶的酶活力测定结果如图3所示；其中，control为对照菌E.coli BL21(DE3)/pRSFDuet-Nrk表达所得烟酰胺核糖激酶的酶活；图中R1～R13依次为R1～R13重组菌表达所得烟酰胺核糖激酶的酶活。由图3可知，与对照菌相比，R10重组菌(E.coli BL21(DE3)/pRSFDuet-35000-Nrk)和R11重组菌(E.coli BL21(DE3)/pRSFDuet-40000-Nrk)表达所得烟酰胺核糖激酶的酶活显著提高，尤其是R10改造的重组菌，其酶活达到4.28U/mL，较对照菌提升了50％。
2、利用启动子序列提高烟酰胺核糖激酶的酶活
蛋白质的有效表达与其转录效率密切相关，启动子是促进高水平基因表达和 重组蛋白生产的重要调控元件。本发明发现，在构建所得的pRSFDuet-35000-Nrk重组质粒(R10重组菌中所含重组质粒)的基础上，通过在该重组质粒中插入不同的启动子序列与原有的T7启动子序列串联，可以改变表达所得烟酰胺核糖激酶的酶活，即可通过引入特定的启动子序列来提升本发明所述烟酰胺核糖激酶的酶活。
本发明引入的特定启动子序列如下所示：
本发明通过酶切后连接的方式，将上述启动子序列插入到了构建所得的pRSFDuet-35000-Nrk重组质粒中。
对于上述所有启动子，均以大肠杆菌基因组为DNA模板，通过特异性引物扩增得到上述的启动子片段(如表3所示)，扩增该结束后回收所有启动子片段。以质粒pRSFDuet-35000-Nrk为模板，采用反向聚合酶连反应获得线性化的质粒片段(线性化引物F：taatacgactcactataggggaattgtgagcg；线性化引物R：atttcctaatgcaggagtcgcataagggaga)。
其中，所述反向聚合酶链式反应在50Μl反应体系中进行，加入20μL的ddH2O、1μL的模板、25μL的2×Phanta Max Master Mix以及上、下游引物各2μL。反应条件为：在95℃预变性3min后开始循环：95℃变性15s，55℃退火15s，72℃延伸2.5min，共34个循环；72℃终延伸5min。用Dpn I限制酶消化甲基化的模板质粒pRSFDuet-35000-Nrk，转化到E.coli BL21(DE3)中，对得到的转化子进行测序验证，筛选出构建成功的阳性转化子，构建得到含有不同双启动子的重组菌。利用实施例1所述方法对所得重组菌进行诱导表达并测定表达所得烟酰胺核糖激酶的酶活。
表3扩增启动子序列所用特异性引物及线性化模板所需引物

注：引物中的大写字母为启动子序列部分，小写字母为同源臂。
含不同双启动子序列的重组菌表达得到的烟酰胺核糖激酶的酶活力测定结果如图4所示；其中，control为对照菌E.coli BL21(DE3)/pRSFDuet-35000-Nrk表达所得烟酰胺核糖激酶的酶活；P1为dnaKJ启动子重组菌E.coli BL21(DE3)/pRSFDuet-35000-PdnaKJ-Nrk表达所得烟酰胺核糖激酶的酶活；P2为alsR启动子 重组菌E.coli BL21(DE3)/pRSFDuet-35000-PalsR-Nrk表达所得烟酰胺核糖激酶的酶活；P3为ssrA启动子重组菌E.coli BL21(DE3)/pRSFDuet-35000-PssrA-Nrk表达所得烟酰胺核糖激酶的酶活；P4为skp启动子重组菌E.coli BL21(DE3)/pRSFDuet-35000-Pskp-Nrk表达所得烟酰胺核糖激酶的酶活；P5为gapA启动子重组菌E.coli BL21(DE3)/pRSFDuet-35000-PgapA-Nrk表达所得烟酰胺核糖激酶的酶活；P6为aceE启动子重组菌E.coli BL21(DE3)/pRSFDuet-35000-PaceE-Nrk表达所得烟酰胺核糖激酶的酶活。
由图4可知，利用不同双启动子表达相同的烟酰胺核糖激酶基因所得的烟酰胺核糖激酶的酶活不同。在本发明所使用的启动子中，将alsR启动子(SEQ ID NO.17所示)或aceE启动子(SEQ ID NO.21所示)与T7启动子串联后表达所得重组菌的烟酰胺核糖激酶的酶活力均有最高；其中，将alsR启动子与T7启动子串联后表达所得重组菌的烟酰胺核糖激酶的酶活力最高，为5.56U/mL，较对照菌提升了30％，表明引入alsR启动子，将其与T7启动子串联可以显著提高表达的烟酰胺核糖激酶的酶活力。
3、通过突变SEQ ID NO.2所示烟酰胺核糖激酶提高其酶活
本发明以重组质粒pRSFDuet-35000-PalsR-Nrk为DNA模板，通过使用含有突变位点的定点突变引物(如表4所示)，经反向聚合酶链式反应获得了一系列突变体。
表4定点突变引物

注：括号内数字代表突变的氨基酸位点。
其中，所述反向聚合酶链式反应在50μL反应体系中进行，加入20μL的ddH2O、1μL的模板、25μL的2×Phanta Max Master Mix以及上、下游引物各2μL。反应条件为：在95℃预变性3min后开始循环：95℃变性15s，55℃退火15s，72℃延伸2.5min，共34个循环；72℃终延伸5min。然后用Dpn I限制酶消化甲基化的模板质粒pRSFDuet-35000-PalsR-Nrk，转化到E.coli BL21(DE3)中，对得到的转化子进行测序验证，筛选出构建成功的阳性转化子，得到不同的烟酰胺核糖激酶的突变菌株。
通过反向聚合酶链式反应，本发明得到了包含SEQ ID NO.2所示烟酰胺核糖激酶的突变体D45P、I88Q和E189G的基因序列的质粒；其中，所述突变体D45P为将SEQ ID NO.2所示烟酰胺核糖激酶的第45位D突变为P，其余突变体以此类推。同时，通过将所述含突变体的基因序列的质粒转化至大肠杆菌E.coli BL21(DE3)，构建得到了表达所述突变体的重组菌(或说突变菌株)。利用实施例1所述方法，本发明对所述的突变体重组菌进行了诱导表达并测定了其烟酰胺核糖激酶的酶活力。为了更直观的观察所得突变菌株较出发菌株的酶活力变化，所有突变菌株均以E.coli BL21(DE3)/pRSFDuet-35000-PalsR-Nrk为对照，计算其相对酶活力。不同突变体的重组菌表达得到的烟酰胺核糖激酶的相对酶活力测定结果如图5所示。由图5可知，与对照菌相比，突变体D45P、I88Q和E189G的重组菌表达所得的烟酰胺核糖激酶的酶活均有所提升。
在上述结果的基础上，本发明以突变体D45P、I88Q和E189G作为DNA模板，分别对其进行了饱和突变。具体地，将SEQ ID NO.2所示烟酰胺核糖激酶的第45位D突变为G/A/V/L/I/P/F/Y/W/S/T/C/M/N/Q/E/K/H/R；将SEQ ID NO.2所示烟酰胺核糖激酶的第88位I突变为G/A/V/L/H/P/F/Y/W/S/T/C/M/N/Q/D/E/K/R；将SEQ ID NO.2所示烟酰胺核糖激酶的第189位E突变为G/A/V/L/H/P/F/Y/W/S/T/C/M/N/Q/D/I/K/R。
通过反向聚合酶链式反应，本发明得到了包含上述不同位点饱和突变后的基因序列的质粒，通过将所述含突变体的基因序列的质粒转化至大肠杆菌E.coli BL21(DE3)，构建得到了表达所述突变体的重组菌。其中，所述反向聚合酶链式反应在50μL反应体系中进行，加入20μL的ddH2O、1μL的模板、25μL的2×Phanta Max Master Mix以及上、下游引物各2μL。反应条件为：在95℃预变性3min后开始循环：95℃变性15s，55℃退火15s，72℃延伸2.5min，共34个循环；72℃终延伸5min。然后用Dpn I限制酶消化甲基化的模板质粒D45P、I88Q或E189G，转化到E.coli BL21(DE3)中，对得到的转化子进行测序验证，筛选出构建成功的阳性转化子，得到不同的烟酰胺核糖激酶的突变菌株。利用实施例1所述方法，本发明对所述的突变体重组菌进行了诱导表达并测定了其烟酰胺核糖激酶的酶活力，并以E.coli BL21(DE3)/pRSFDuet-35000-PalsR-Nrk为对照计算得到了其相对酶活力。
SEQ ID NO.2所示烟酰胺核糖激酶的第45位D进行饱和突变所得各重组菌表达得到的烟酰胺核糖激酶的相对酶活力测定结果如图6所示。由图6可知，当第45位天冬氨酸(D)突变为甘氨酸(G)、丙氨酸(A)、缬氨酸(V)、脯氨酸(P)、苯丙氨酸(F)、酪氨酸(Y)、丝氨酸(S)、苏氨酸(T)、半胱氨酸(C)、蛋氨酸(M)、天冬酰胺(N)、谷氨酰胺(Q)、谷氨酸(E)、 赖氨酸(K)或精氨酸(R)，突变株的烟酰胺核糖激酶的酶活相较于对照均有所提升。
SEQ ID NO.2所示烟酰胺核糖激酶的第88位I进行饱和突变所得各重组菌表达得到的烟酰胺核糖激酶的相对酶活力测定结果如图7所示。由图7可知，当第88位异亮氨酸(I)突变为甘氨酸(G)、丙氨酸(A)、缬氨酸(V)、异亮氨酸(I)、丝氨酸(S)、苏氨酸(T)、半胱氨酸(C)、蛋氨酸(M)、天冬酰胺(N)、谷氨酰胺(Q)、精氨酸(R)或组氨酸(H)，突变株的烟酰胺核糖激酶的酶活相较于对照均有所提升。
SEQ ID NO.2所示烟酰胺核糖激酶的第189位E进行饱和突变所得各重组菌表达得到的烟酰胺核糖激酶的相对酶活力测定结果如图8所示。由图8可知，当第189位谷氨酸(E)突变为甘氨酸(G)、丙氨酸(A)或谷氨酰胺(Q)，突变株的烟酰胺核糖激酶的酶活相较于对照均有所提升。
在突变体D45T、I88R和E189A的基础上，本发明还对SEQ ID NO.2所示烟酰胺核糖激酶进行了组合突变，得到了能够表达组合突变体NRK-TRA(D45T-I88R-E189A)的突变菌株，通过镍柱亲和层析方法对其表达所得烟酰胺核糖激酶突变体进行纯化后测定了其相对酶活力。
组合突变体NRK-TRA重组菌表达得到的烟酰胺核糖激酶(NRK-TRA)的比酶活测定结果如图9所示。由图9可知，组合突变体NRK-TRA重组菌表达得到的烟酰胺核糖激酶的相对酶活力为22.95U/mg，是对照菌的2.9倍。
实施例3 烟酰胺核糖激酶重组菌在5L发酵罐中的放大培养
为进一步评估烟酰胺核糖激酶突变体的产酶能力，基于恒速补料策略，本发明应用5L发酵罐对组合突变体NRK-TRA的重组菌进行了分批发酵，以进一步提高NRK-TRA的酶活力。
具体为：活化所述重组菌菌株，培养得种子液；以7.5％(v/v)的接种量将种子液接入发酵罐培养基中，37℃培养至OD600达到30时，降温至25℃，加入终浓度为0.5mM的IPTG诱导表达12h；在补料发酵过程中，控制培养体系pH为7.0，控制发酵罐转速为400rpm。
组合突变体NRK-TRA的重组菌的放大培养过程中，OD600和烟酰胺核糖激酶酶活力的变化情况分别如图10和图11所示。由图10和图11可知，随着菌体的不断生长，葡萄糖被逐步消耗，当其浓度低于2g/L时，进行流加补料。经过补料发酵，组合突变体NRK-TRA重组菌的OD600在培养19h最高达到119，此时的酶活力也达到最高，为208.3U/mL。此时重组菌也已经获得了生物量的积累，产酶的速率也维持在一个较为平稳的水平。在补料第19h后，菌株的OD600开始下降，酶活性也有所下降。
实施例4 烟酰胺核糖激酶的酶学性质表征
为探究温度和pH对烟酰胺核糖激酶酶活力的影响，本发明使用全细胞催化测定了不同温度和pH下，烟酰胺核糖激酶的相对酶活力。
在30～75℃范围内测定重组烟酰胺核糖激酶突变体NRK-TRA的活性，并以55℃时该酶的酶活力为对照，计算不同反应温度下的相对酶活力，结果如图12所示。由图12可知，随着温度升高，分子运动更加剧烈，底物与酶更易结合反应，因此酶活力逐渐升高，并在55℃时达到最高酶活力。在过高温度下，酶结构及活力往往会遭到破坏，反应生成的产物也更容易降解。在温度超过55℃时，酶活力开始逐渐下降。因此，该酶的最适反应温度为55℃。
在不同pH的缓冲液(pH 6～11)中测定重组烟酰胺核糖激酶突变体NRK-TRA的活性，并以pH为7.2时该酶的酶活力为对照，计算不同pH下该酶的相对酶活力，结果如图13所示。由图13可知，烟酰胺核糖激酶突变体在pH 7.2时表现出最高酶活力，在中性条件下能够更好地保持酶活。
实施例5 全细胞催化体系的优化
为探索生物转化实验的最适底物浓度，本发明在实施例4探索得到的最佳反应温度、pH条件下，以全细胞催化剂组合突变体NRK-TRA的重组菌为实验对象探索了批次转化的条件。
将高密度发酵所得菌液于4℃、8000rpm离心10min，弃上清液；加入30mL的PBS缓冲液重悬菌体，4℃、8000rpm离心10min，弃上清液，重复2次，目的是洗涤菌体(细胞)；向菌体沉淀中加入15mL的PBS缓冲液，重悬菌体后冰浴10min，得到全细胞催化剂。
配制0.4mol/L NR，0.5mol/L ATP，0.2mol/L MgCl2，各取50μL，加入ddH2O 800μL和全细胞催化剂50μL，37℃，1500rpm振荡，反应10min。反应结束后加入100μL 25％ HCl终止反应，4℃、8000rpm离心1min，分离反应液与细胞。
结果表明，加入第一批底物，使用HPLC检测到底物耗尽时，加入第二批底物继续反应，以此类推，当反应体系加入第三批底物时，此时β-NMN的产量达到最高，为15.16g/L。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种烟酰胺核糖激酶，其特征在于，所述烟酰胺核糖激酶的氨基酸序列如SEQ ID NO.2所示。
  2. 一种编码权利要求1所述烟酰胺核糖激酶的基因。
  3. 权利要求1所述烟酰胺核糖激酶的突变体，其特征在于，所述突变体是对SEQ ID NO.2所示烟酰胺核糖激酶的第45、88或189位氨基酸中的任意一个或几个氨基酸进行突变得到。
  4. 一种编码权利要求3所述突变体的基因。
  5. 一种表达烟酰胺核糖激酶的重组质粒，其特征在于，所述重组质粒中含有权利要求2或4所述基因；
    或所述重组质粒中含有权利要求2或4所述基因，且所述重组质粒的原有启动子序列与所述基因间含有核糖体结合位点序列和/或其它启动子序列；所述核糖体结合位点序列如SEQ ID NO.12或SEQ ID NO.13所示，所述其它启动子序列如SEQ ID NO.17或SEQ ID NO.21所示。
  6. 一种含有权利要求5所述重组质粒的重组菌。
  7. 一种提高烟酰胺核糖激酶酶活力的方法，其特征在于，所述方法为：对权利要求1所述烟酰胺核糖激酶的第45、88或189位氨基酸中的任意一个或几个氨基酸进行突变；
    或将编码权利要求1所述烟酰胺核糖激酶的基因序列替换为编码权利要求3所述烟酰胺核糖激酶突变体的基因序列。
  8. 一种高效异源表达烟酰胺核糖激酶的方法，其特征在于，在构建用于表达权利要求1所述烟酰胺核糖激酶或权利要求3所述突变体的重组质粒时，对重组质粒进行如下改造：在重组质粒的原有启动子序列与基因序列之间插入核糖体结合位点序列和/或其它启动子序列；所述核糖体结合位点序列如SEQ ID NO.12或SEQ ID NO.13所示，所述其它启动子序列如SEQ ID NO.17或SEQ ID NO.21所示。
  9. 权利要求1所述烟酰胺核糖激酶、权利要求2所述基因、权利要求3所述突变体、权利要求4所述基因、权利要求5所述重组质粒或权利要求6所述重组菌在生物合成β-烟酰胺单核苷酸或在制备用于生物合成β-烟酰胺单核苷酸的产品中的应用。
  10. 一种生物合成β-烟酰胺单核苷酸的方法，其特征在于，用权利要求3所述烟酰胺核糖激酶的突变体或权利要求6所述重组菌作为生物合成β-烟酰胺单核苷酸所需烟酰胺核糖激酶或重组菌。
PCT/CN2024/107280 2024-04-07 2024-07-24 一种烟酰胺核糖激酶突变体在大肠杆菌中的高效异源表达及其应用 Pending WO2025213622A1 (zh)

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