A recombinant Escherichia coli strain producing nicotinamide ribokinase and its construction method and application

Abstract

The present invention discloses a recombinant Escherichia coli producing nicotinamide ribokinase and a construction method and application thereof, belonging to the field of biotechnology. The present invention constructs a nicotinamide ribokinase, and its amino acid sequence is shown in SEQ ID NO.7. The present invention utilizes genetic engineering technology to construct a recombinant Escherichia coli NMN-HS01 expressing the above-mentioned nicotinamide ribokinase, and the nicotinamide ribokinase expressed by the recombinant bacteria or the whole cell of the recombinant bacteria is used for catalytic reaction, which can realize the efficient production of β-nicotinamide mononucleotide. The present invention also provides a method for producing β-nicotinamide mononucleotide, the catalytic reaction is carried out at normal temperature and pressure, and is environmentally friendly; no toxic and harmful organic solvents are used, which reduces environmental pollution; the entire catalytic process is a one-step reaction, which has the advantages of high conversion rate, concise purification steps and simple process, can better save production costs, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a recombinant Escherichia coli producing nicotinamide ribokinase, a construction method and application thereof. Background Art

[0002] β-nicotinamide mononucleotide, as a substance naturally present in the human body, plays a key role in the production of cellular energy. β-nicotinamide mononucleotide is one of the important precursors for the synthesis of nicotinamide adenine dinucleotide in mammals. Nicotinamide adenine dinucleotide plays a vital role in maintaining human health. It is mainly involved in electron transfer reactions. However, with age, the total content of nicotinamide adenine dinucleotide will gradually decrease. Since nicotinamide adenine dinucleotide cannot be directly supplemented externally, the role of β-nicotinamide mononucleotide is particularly important. It can enter the human blood, increase the content of β-nicotinamide mononucleotide in tissues, and then quickly convert into nicotinamide adenine dinucleotide in organs to play its important physiological functions.

[0003] The mitochondrial aging theory states that the accumulation of DNA mutations and mitochondrial dysfunction leads to the production of reactive oxygen species and free radicals, which ultimately cause aging. However, by restoring the content of nicotinamide adenine dinucleotide, the aging process can be delayed or even reversed. Since the direct ingestion of nicotinamide adenine dinucleotide is not well absorbed, people have turned to oral β-nicotinamide mononucleotide as an alternative. β-nicotinamide mononucleotide can easily cross the cell membrane through an active transport mechanism and then be effectively converted into nicotinamide adenine dinucleotide in the cell. β-nicotinamide mononucleotide can not only promote mitochondrial metabolism, but also improve mitochondrial metabolic disorders. Studies have found that by increasing the content of β-nicotinamide mononucleotide in the body, the normal morphology of mitochondria can be restored, their oxygen consumption rate can be increased, and the recovery of mitochondrial function can be promoted.

[0004] Common methods for synthesizing β-nicotinamide mononucleotide are chemical synthesis and biosynthesis. The chemical synthesis method is relatively complex, with many steps, requires the use of organic reagents for protection and deprotection, is environmentally unfriendly, isomer separation is difficult, and the product yield is low. Compared with chemical synthesis, the advantages of synthesizing β-nicotinamide mononucleotide using biological methods, i.e. whole-cell methods, include the following: (1) the reaction is carried out in one step, with few side reactions and relatively simple separation and purification; (2) green synthesis, high atomic utilization rate, and strong economy; (3) wastewater treatment after product purification is simple, which is conducive to large-scale application. Therefore, in-depth research on the biosynthesis of β-nicotinamide mononucleotide is of great significance to the industrial development of biosynthetic β-nicotinamide mononucleotide in order to improve synthesis efficiency and save production costs. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant Escherichia coli that produces nicotinamide ribokinase and a construction method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The recombinant Escherichia coli whole cell or the expressed nicotinamide ribokinase of the present invention can efficiently produce β-nicotinamide mononucleotide.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a nicotinamide ribokinase, and the amino acid sequence of the nicotinamide ribokinase is shown as SEQ ID NO.7.

[0008] The present invention also provides a gene encoding the nicotinamide ribokinase, and the nucleotide sequence of the gene encoding the nicotinamide ribokinase is shown in SEQ ID NO.6.

[0009] The present invention also provides the use of the nicotinamide ribokinase or the encoding gene in constructing a recombinant Escherichia coli producing nicotinamide ribokinase.

[0010] The present invention also provides a method for constructing a recombinant Escherichia coli NMN-HS01 that produces nicotinamide ribokinase, comprising the following steps:

[0011] Connecting the coding gene to an expression vector to construct a recombinant plasmid;

[0012] The recombinant plasmid is transformed into competent Escherichia coli, and after screening and verification, the recombinant Escherichia coli NMN-HS01 is obtained.

[0013] Preferably, the expression vector is pET-28a.

[0014] Preferably, the competent Escherichia coli is competent Escherichia coli BL21 (DE3).

[0015] The present invention also provides recombinant Escherichia coli NMN-HS01 constructed by the construction method.

[0016] The present invention also provides the use of the nicotinamide ribokinase, the encoding gene or the recombinant Escherichia coli NMN-HS01 in the synthesis of β-nicotinamide mononucleotide.

[0017] The present invention also provides a method for producing β-nicotinamide mononucleotide using an enzyme, comprising the following steps:

[0018] Nicotinamide ribose chloride and disodium triphosphate are used as substrates, and the nicotinamide ribokinase is used to carry out a catalytic reaction to obtain the β-nicotinamide mononucleotide.

[0019] The present invention also provides a method for producing β-nicotinamide mononucleotide using whole cells, comprising the following steps:

[0020] Nicotinamide ribose chloride and disodium triphosphate are used as substrates, and the whole cells of the recombinant Escherichia coli NMN-HS01 are used to carry out a catalytic reaction to obtain the β-nicotinamide mononucleotide.

[0021] The present invention discloses the following technical effects:

[0022] The present invention constructs a nicotinamide ribokinase, and its amino acid sequence is shown in SEQ ID NO. 7. The present invention uses genetic engineering technology to construct a recombinant Escherichia coli NMN-HS01 expressing the above nicotinamide ribokinase, and the nicotinamide ribokinase expressed by the recombinant bacteria is used to produce β-nicotinamide mononucleotide, and the conversion rate can reach up to 87.64%; the whole cell of the recombinant bacteria is used to produce β-nicotinamide mononucleotide, and the conversion rate can reach up to 92.37%.

[0023] The present invention discloses a method for producing β-nicotinamide mononucleotide by using an enzyme method or a whole-cell method. The entire catalytic reaction is carried out at room temperature and pressure, which is environmentally friendly; no toxic or harmful organic solvents are used, thereby reducing environmental pollution; the entire catalytic process is a one-step reaction, which has the advantages of high conversion rate, concise purification steps and simple procedures, thereby better saving production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Schematic diagram of the process for constructing recombinant Escherichia coli NMN-HS01;

[0026] Figure 2 This is a diagram of the process of synthesizing β-nicotinamide mononucleotide catalyzed by nicotinamide ribokinase produced by recombinant Escherichia coli NMN-HS01;

[0027] Figure 3 This is the peak diagram of the liquid chromatography of β-nicotinamide mononucleotide standard;

[0028] Figure 4 This is the liquid chromatography peak diagram of the reaction of nicotinamide riboside chloride and disodium triphosphate catalyzed by recombinant Escherichia coli NMN-HS01. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] In the following examples or comparative examples: Escherichia coli DH5α and Escherichia coli BL21 (DE3) were purchased from Quanshijin Biotechnology Co., Ltd.; the vector pET-28a-(+) was purchased from Novagen Co., Ltd.

[0035] The components and concentrations in LB medium are: yeast extract 5 g / L, tryptone 10 g / L, and sodium chloride 10 g / L.

[0036] The specific determination method of enzyme activity is as follows: prepare 5 mL of 50 mmol / L nicotinamide ribose chloride and 50 mmol / L disodium triphosphate with Tris-HCl buffer, add 20 mmol / L magnesium chloride, adjust the pH to 7.5 with Tris, add 1 mL of enzyme solution, react at 37°C for 1 h, draw 1 mL of reaction solution, heat in a 100°C water bath for 5 min, detect the concentration of β-nicotinamide mononucleotide by high performance liquid chromatography, and the amount of enzyme required to catalyze the reaction of 250 μmol nicotinamide ribose chloride to generate 1 μmol β-nicotinamide mononucleotide within 1 h at 37°C is defined as one enzyme activity unit (U).

[0037] Example 1

[0038] A variety of nicotinamide ribokinase genes were searched from NCBI, and after comparison, a gene (KPH72847.1) from Haemophilus influenzae was obtained. After codon optimization, the gene NRK1 was synthesized. According to the sequences of the NRK1 gene and the pET-28a-(+) plasmid, primers were designed using primer premier 5.0. The forward primer was 5'-CGGATCCGGATCCATGGGCTTTACCACCG-3' (SEQ ID NO.1), and the reverse primer was 5'-TGCTCGAGCTCGAGCTGACTAATACCCTTGATCG-3' (SEQ ID NO.2). By PCR amplification technology, a gene clone fragment NRK-HI01 (SEQ ID NO.5) containing BamH I and Xho I restriction sites was obtained. The above gene clone fragment NRK-HI01 was double-digested (BamH I and Xho I restriction site) was connected to the pET-28a-(+) plasmid to obtain the recombinant plasmid pET-28a-(+)-NRK-HI01, and the recombinant plasmid was introduced into BL21(DE3) competent cells, the plasmid was extracted, and the plasmid was used as a template to mutate the 8th amino acid using a forward primer (SEQ ID NO.3: 5'-AGCCGTGAATTTCGCCCAGCGCTGCGCAT-3') and a reverse primer (SEQ ID NO.4: 5'-CTGGGCGAAATTCACGGCTGGTGGTAAAGC-3'), so that the codon encoding glycine (GGC) was mutated to a codon encoding serine (AGC), and the mutated gene NMN-HS01 (SEQ ID NO.6), introduced into DMT competent medium to extract plasmid and sequence verification, after verification, the plasmid was introduced into BL21 (DE3) competent medium, and the recombinant nicotinamide ribokinase-producing Escherichia coli NMN-HS01 was screened on a kanamycin-resistant plate. The schematic diagram of the process for constructing recombinant Escherichia coli NMN-HS01 is shown in Figure 1 shown.

[0039] The nucleotide sequence of NRK-HI01 (SEQ ID NO.5):

[0040] GGATCCATGGGCTTTACCACC GGC

[0041] Nucleotide sequence of NMN-HS01 (SEQ ID NO.6):

[0042] GGATCCATGGGCTTTACCACC AGC

[0043] Example 2

[0044] The recombinant E. coli NMN-HS01 was inoculated into 50 mL LB medium containing 100 μg / mL kanamycin sulfate and cultured at 37°C, 220 rpm for 12 h until the absorbance OD 600 4.0, to obtain seed solution; take 1 mL of seed solution to 50 mL of LB medium, and culture at 37°C, 220 rpm for 6 h until the absorbance OD 600 The pH value was 4.0, and IPTG inducer with a concentration of 62.5 μg / mL was added. The fermentation was induced at 25°C for 12 h. Centrifugation was performed at 8000 rpm at 4°C for 15 min. The supernatant was removed, and Tris-HCl buffer with a pH of 7.5 was added to prepare the bacteria into a 0.1 g / mL whole-cell suspension with an enzyme activity of 180 U / mL.

[0045] Example 3

[0046] The recombinant E. coli NMN-HS01 was inoculated into 50 mL LB medium containing 100 μg / mL kanamycin sulfate and cultured at 37°C, 220 rpm for 12 h until the absorbance OD 600 4.0, to obtain seed solution; take 1 mL of seed solution to 50 mL of LB medium, and culture at 37°C, 220 rpm for 6 h until the absorbance OD 600 The concentration of the induction agent was 4.0, and the concentration of IPTG inducer was 62.5 μg / mL. The fermentation was induced at 25°C for 12 hours, and the supernatant was removed at 8000 rpm at 4°C for 15 minutes. The Tris-HCl buffer with a pH of 7.5 was added to prepare the bacteria into a 0.1 g / mL whole cell suspension with an enzyme activity of 179 U / mL. An ice-water bath was added, and the supernatant was obtained by centrifugation, i.e., the crude enzyme solution with an enzyme activity of 174 U / mL.

[0047] The amino acid sequence of nicotinamide ribokinase expressed by recombinant E. coli NMN-HS01 (SEQ ID NO.7) is as follows:

[0048] GSMGFTTSREFRPALRMRAKYNAKYLVTKSEREKYFHLSYNKHTQFLRYQEQIMSKAKEKKVGVIFGKFYPVHTGHINMIYEAFSKVDELHVIVCSDTERDLKLFY DSKMKRMPTVQDRLRWMQQIFKYQKNQIFIHHLVEDGIPSYPNGWQSWSEAVKTLFHEKHFEPSIVFSSEPQDKAPYEKYLGLEVSLVDPDRTFFNVSATKIRTTPF QYWKFIPKEVRPFFAKTVAILGGESSGKSVLVNKLAAVFNTTSAWEYGREFVFEKLGGNEQAMQYSDYPQMALGHQRYIDYAVRHAHKIAFIDTDFITTQAFCIQY EGKAHPFLDSMIKEYPFDVTILLKNNTEWVDDGLRSLGSQKQRQQFQQLLKKLLDKYKVPYIEIESPSYLERYNQVKAVIEKVLNEEEISELQNTTFPIKGISQLE.

[0049] Example 4

[0050] A mixed solution of 50 mmol / L nicotinamide ribose chloride and 50 mmol / L disodium triphosphate was prepared using a Tris-HCl buffer at pH = 7.5, and 20 mmol / L magnesium chloride was added. The pH of the substrate solution was adjusted to 7.5 with a Tris solution, and the whole cell suspension prepared in Example 2 was added to make the enzyme activity in the reaction system 30 U / mL. The mixture was stirred at 37° C. for 4 h to catalyze the synthesis of β-nicotinamide mononucleotide (see the catalytic synthesis process). Figure 2 ), the conversion rate was 92.37%.

[0051] Example 5

[0052] A mixed solution of 50 mmol / L nicotinamide ribose chloride and 50 mmol / L disodium triphosphate was prepared using a Tris-HCl buffer solution at pH = 7.5, and 20 mmol / L magnesium chloride was added. The pH of the substrate solution was adjusted to 7.5 using a Tris solution, and the crude enzyme solution prepared in Example 3 was added to make the enzyme activity in the reaction system 30 U / mL. The mixture was stirred at 37° C. for 4 h to catalyze the synthesis of β-nicotinamide mononucleotide (see the catalytic synthesis process). Figure 2 ), the conversion rate was 87.64%.

[0053] Example 6

[0054] A mixed solution of 50 mmol / L nicotinamide ribose chloride and 50 mmol / L disodium triphosphate was prepared using Tris-HCl buffer at pH = 7.5, and 20 mmol / L magnesium chloride was added. The pH of the substrate solution was adjusted to 7.5 using Tris solution, and the crude enzyme solution prepared in Example 3 was added to ensure that the pH of the reaction solution was maintained within 7.5 ± 0.05. When the pH of the reaction solution no longer changed, it indicated that the reaction was complete. The reaction was terminated by heating at 100° C. for 5 min. The reaction solution was diluted 5 times using Tris-HCl buffer, centrifuged to remove the precipitate, and the supernatant was filtered using a 0.22 μm filter membrane. The filtered supernatant was detected by high performance liquid chromatography (detection conditions: the chromatographic column was an amide column (4.6 mm×250 mm, 5 μm); the mobile phase was 0.05 M KH2PO4 (pH4.0): acetonitrile = 35:65; the flow rate was 0.9 mL / min, and the detection wavelength was 260 nm). The liquid chromatographic peak diagram of the reaction solution is as shown in the figure. Figure 4 As shown; 4 g / L β-nicotinamide mononucleotide standard solution was prepared using Tris-HCl solution, and the HPLC detection was also performed. The liquid chromatogram of the β-nicotinamide mononucleotide standard solution is shown in Figure 3 shown.

[0055] Comparative Example 1

[0056] Same as Example 5, except that the crude enzyme solution prepared in Example 3 is replaced by the crude enzyme solution prepared by the following method:

[0057] The NRK-HI01 gene was used to construct a recombinant Escherichia coli NRK-HI01, which was then fermented to produce nicotinamide ribokinase to obtain a crude enzyme solution with an enzyme activity of 65 U / mL. The construction method and enzyme production fermentation method refer to patent document CN107603936A.

[0058] The conversion rate of β-nicotinamide mononucleotide prepared in this comparative example was 72.41%.

[0059] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A nicotinamide ribokinase, characterized in that The amino acid sequence of the nicotinamide ribokinase is shown in SEQ ID NO.

7.

2. The gene encoding nicotinamide ribokinase according to claim 1, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

6.

3. Use of the coding gene as claimed in claim 2 in constructing a recombinant Escherichia coli producing nicotinamide ribokinase.

4. A recombinant Escherichia coli producing nicotinamide ribokinase ( Escherichia coli ) The method for constructing NMN-HS01, characterized in that: The following steps are involved: Connecting the coding gene according to claim 2 to an expression vector to construct a recombinant plasmid; The recombinant plasmid is transformed into competent Escherichia coli, and after screening and verification, the recombinant Escherichia coli NMN-HS01 is obtained.

5. The construction method according to claim 4, characterized in that: The expression vector is pET-28a.

6. The construction method according to claim 4, characterized in that: The competent Escherichia coli is competent Escherichia coli BL21 (DE3).

7. The recombinant Escherichia coli NMN-HS01 constructed according to the construction method as described in any one of claims 4 to 6.

8. Use of the nicotinamide ribokinase according to claim 1, the encoding gene according to claim 2 or the recombinant Escherichia coli NMN-HS01 according to claim 7 in the synthesis of β-nicotinamide mononucleotide.

9. A method for producing β-nicotinamide mononucleotide by enzymatic method, characterized in that: The following steps are involved: Nicotinamide riboside chloride and disodium triphosphate are used as substrates, and the nicotinamide ribokinase described in claim 1 is used to carry out a catalytic reaction to obtain the β-nicotinamide mononucleotide.

10. A method for producing β-nicotinamide mononucleotide by a whole-cell method, characterized in that: The following steps are involved: Nicotinamide ribose chloride and disodium triphosphate are used as substrates, and the whole cells of the recombinant Escherichia coli NMN-HS01 according to claim 7 are used to carry out a catalytic reaction to obtain the β-nicotinamide mononucleotide.

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