NRK-containing fusion protein and genetically engineered bacterium thereof

Abstract

The invention discloses a fusion protein containing NRK and a genetically engineered bacterium thereof. The fusion protein comprises: nicotinamide ribokinase; a dissolution promoting label, wherein the nicotinamide ribokinase is connected with the dissolution promoting label; the genetically engineered bacterium carries a nucleic acid molecule encoding the fusion protein or carries an expression vector of the nucleic acid molecule, or expresses the fusion protein. The NRK in the fusion protein has the advantages of strong stability, high activity and the like, when the fusion protein is used for preparing the NMN, the substrate conversion efficiency and the NMN production final concentration can be improved, the substrate conversion efficiency can be stabilized at 90% or above, and the concentration of the prepared NMN can reach 60 g / L or above.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology. Specifically, the present invention relates to a fusion protein containing NRK and a genetically engineered bacterium thereof. More specifically, the present invention relates to a fusion protein, a nucleic acid molecule, an expression vector, a genetically engineered bacterium, a method for preparing NMN, and a method for improving the activity or stability of nicotinamide ribokinase. Background Art

[0002] β-Nicotinamide mononuclotide (NMN) is a derivative of nicotinic acid. Niacin is an essential vitamin for the human body. Niacin exists in the body in the form of nicotinamide, and β-nicotinamide mononucleotide is an important intermediate in the synthesis pathway of nicotinamide adenine dinucleotide (NAD+) in mammals. It plays a role in many biological redox reactions, including energy metabolism, gene stability, neurotransmission, metabolism, etc.

[0003] At present, the mainstream production method of β-nicotinamide mononucleotide is to use nicotinamide riboside chloride (NRCl) as raw material, and react with nicotinamide ribokinase (NR kinase, NRK) and ATP to obtain NMN, with low enzyme cost and simple process operation. However, during the use of nicotinamide ribokinase, there are problems such as poor stability (inclusion bodies are easily generated after fermentation), low conversion rate and substrate concentration, which limits the application of the one-step reaction method catalyzed by bio-enzymes in the industrial production of NMN.

[0004] Therefore, there is an urgent need to develop a high-nicotinamide ribokinase with high stability and high activity. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a fusion protein containing NRK and a genetically engineered bacterium thereof, wherein the fusion protein containing NRK can be prepared by using the genetically engineered bacterium, and the NRK in the fusion protein has the advantages of strong stability or high activity.

[0006] The present invention is accomplished based on the following findings of the inventors:

[0007] During the preparation of NRK, NRK can be expressed in a soluble form, but inclusion bodies often appear during the shake flask and fermentation process, which is unstable during use, affecting the efficiency of subsequent preparation of NMN; and during the preparation of NMN, the activity of nicotinamide ribokinase is low, affecting the concentration of the conversion substrate. At present, when using nicotinamide ribokinase to prepare NMN, the conversion rate can reach more than 90%, but the concentration of NMN is less than 30g / L.

[0008] However, in the process of preparing NRK, the inventors tried to add a lytic tag to the DNA expressing the NRK enzyme for fusion expression and found that connecting a specific lytic tag in a specific way can not only promote the correct folding of the fusion protein and improve the stability of NRK, but also unexpectedly found that it can also improve the activity of NRK. Specifically, when using NRK in the above-mentioned fusion protein as a catalyst to prepare NMN, in order to reduce the cost of raw materials during industrial production, the inventors used crude nicotinamide riboside or its salt (the content of nicotinamide riboside or its salt is 50%-75%). When the substrate concentration is about 200mM (the concentration of nicotinamide riboside or its salt is 60g / L), the substrate conversion efficiency is stable at more than 90% (the conversion rate is 93%-100%), and the concentration of the prepared NMN can reach more than 60g / L.

[0009] Therefore, in the first aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: nicotinamide ribokinase; a solubility-promoting tag, and the nicotinamide ribokinase and the solubility-promoting tag are connected. The NRK in the fusion protein of the present invention has the advantages of strong stability and high activity.

[0010] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase.

[0011] According to an embodiment of the present invention, the nicotinamide ribokinase is derived from Haemophilus influenzae.

[0012] According to an embodiment of the present invention, the nicotinamide ribokinase has an amino acid sequence as shown in SEQ ID NO:1.

[0013] According to an embodiment of the present invention, the lytic tag is selected from at least one of TrxA, SUMO and MBP.

[0014] According to an embodiment of the present invention, the TrxA has an amino acid sequence as shown in SEQ ID NO:2.

[0015] According to an embodiment of the present invention, the SUMO has an amino acid sequence as shown in SEQ ID NO:3.

[0016] According to an embodiment of the present invention, the MBP has an amino acid sequence as shown in SEQ ID NO:4.

[0017] According to an embodiment of the present invention, the fusion protein further comprises an ATP cycle enzyme.

[0018] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, and the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the ATP cycle enzyme.

[0019] According to an embodiment of the present invention, the ATP cycle enzyme is polyphosphate kinase.

[0020] According to an embodiment of the present invention, the polyphosphate kinase is PPK2.

[0021] According to an embodiment of the present invention, the PPK2 is derived from acidophilic sulfur bacteria.

[0022] According to an embodiment of the present invention, the PPK2 has an amino acid sequence as shown in SEQ ID NO: 5. At the same time, PPK2 polyphosphate kinase is used to regenerate ATP, thereby reducing the amount of ATP used and further reducing production costs.

[0023] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide.

[0024] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the ATP cycle enzyme.

[0025] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:6.

[0026] In a second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect.

[0027] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0028] In the third aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect.

[0029] According to an embodiment of the present invention, the expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0030] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0031] According to an embodiment of the present invention, the expression vector is pET28a(+).

[0032] In a fourth aspect of the present invention, the present invention provides a genetically engineered bacterium. According to an embodiment of the present invention, the genetically engineered bacterium carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or expresses the fusion protein described in the first aspect.

[0033] According to an embodiment of the present invention, the genetically engineered bacteria is obtained by introducing the expression vector described in the third aspect into a host bacteria.

[0034] According to an embodiment of the present invention, the host bacteria is Escherichia coli.

[0035] In the fifth aspect of the present invention, the present invention proposes a method for preparing NMN. According to an embodiment of the present invention, the method comprises: using the fusion protein described in the first aspect or the genetically engineered bacteria described in the fourth aspect to catalyze the reaction of nicotinamide ribose or its salt and ATP or its salt to obtain NMN. The method of the present invention has a simple preparation process and is conducive to improving the production efficiency of NMN and reducing the production cost of NMN.

[0036] According to an embodiment of the present invention, before the catalytic reaction, magnesium ions and polyphosphate are mixed with the nicotinamide riboside or a salt thereof and ATP or a salt thereof.

[0037] According to an embodiment of the present invention, the nicotinamide riboside or a salt thereof is NRCl.

[0038] According to an embodiment of the present invention, the nicotinamide riboside or its salt is selected from NRCl with a purity of 75%.

[0039] According to an embodiment of the present invention, the mass ratio of ATP: NRCl with a purity of 75% is 1: (3-7).

[0040] According to an embodiment of the present invention, the magnesium ions are derived from MgCl 2 ﹒ 6H 2 O.

[0041] According to an embodiment of the present invention, the polyphosphate is sodium hexametaphosphate.

[0042] According to an embodiment of the present invention, the ATP: NRCl with a purity of 75%: sodium hexametaphosphate: MgCl 2 ﹒ 6H 2 The mass ratio of O is 1:(3~7):(1~1.5):(0.1~0.5).

[0043] According to an embodiment of the present invention, the catalytic reaction is carried out at 25-40° C. and a pH value of 5-7.

[0044] According to an embodiment of the present invention, the reaction time of the catalytic reaction is 0.5 to 3 hours.

[0045] According to an embodiment of the present invention, in the overall system of the catalytic reaction, the added concentration of the fusion protein or genetically engineered bacteria is 5 to 15 g / L.

[0046] In the sixth aspect of the present invention, the present invention proposes a method for improving the activity or stability of nicotinamide ribokinase. According to an embodiment of the present invention, the method comprises: connecting a lytic tag to the nicotinamide ribokinase. Thus, the stability and activity of NRK can be improved, and when the above NRK is used to prepare NMN, the concentration and conversion efficiency of nicotinamide ribokinase conversion substrate can be increased.

[0047] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase.

[0048] According to an embodiment of the present invention, the nicotinamide ribokinase is derived from Haemophilus influenzae.

[0049] According to an embodiment of the present invention, the nicotinamide ribokinase has an amino acid sequence as shown in SEQ ID NO:1.

[0050] According to an embodiment of the present invention, the lytic tag is selected from at least one of TrxA, SUMO and MBP.

[0051] According to an embodiment of the present invention, the TrxA has an amino acid sequence as shown in SEQ ID NO:2.

[0052] According to an embodiment of the present invention, the SUMO has an amino acid sequence as shown in SEQ ID NO:3.

[0053] According to an embodiment of the present invention, the MBP has an amino acid sequence as shown in SEQ ID NO:4.

[0054] According to an embodiment of the present invention, an ATP cycle enzyme is further included.

[0055] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, and the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the ATP cycle enzyme.

[0056] According to an embodiment of the present invention, the ATP cycle enzyme is polyphosphate kinase.

[0057] According to an embodiment of the present invention, the polyphosphate kinase is PPK2.

[0058] According to an embodiment of the present invention, the PPK2 is derived from acidophilic sulfur bacteria.

[0059] According to an embodiment of the present invention, the PPK2 has an amino acid sequence as shown in SEQ ID NO:5.

[0060] According to an embodiment of the present invention, it further includes a connecting peptide.

[0061] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the ATP cycle enzyme.

[0062] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:6.

[0063] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0065] Figure 1This is a protein gel image verifying that the target gene carried by each expression plasmid constructed in Example 1 of the present invention can be normally expressed in bacteria; wherein, band 1 is the MBP-NRK-ppk2 fusion protein obtained by expressing the E. coli BL21 (DE3)-pET28a-III-MBP-NRK-PPK2 plasmid in the strain; band 2 is the GST-NRK-ppk2 fusion protein obtained by expressing the E. coli BL21 (DE3)-pET28a-III-GST-NRK-PPK2 plasmid in the strain, band 3 is the SUMO-NRK-ppk2 fusion protein obtained by expressing the E. coli BL21 (DE3)-pET28a-III-SUMO-NRK-PPK2 plasmid in the strain, and band 4 is the E. coli Band 5 is the NusA-NRK-ppk2 fusion protein expressed by E. coli BL21(DE3)-pET28a-III-TrxA-NRK-PPK2 plasmid in the strain, Band 6 is the NRK-ppk2 fusion protein expressed by E. coli BL21(DE3)-pET28a-II-NRK-PPK2 plasmid in the strain, Band 7 is the DsbA-NRK-ppk2 fusion protein expressed by E. coli BL21(DE3)-pET28a-III-DsbA-NRK-PPK2 plasmid in the strain.

[0066] Figure 2This is a protein gel image verifying that the target genes carried by each expression plasmid constructed in Comparative Example 1 of the present invention can be normally expressed in bacteria; wherein, band 8 is the NRK-ppk2-TrxA fusion protein obtained by expressing the E. coli BL21 (DE3) -pET28a-III-NRK-PPK2-TrxA plasmid in the strain, band 9 is the NRK-ppk2-NusA fusion protein obtained by expressing the E. coli BL21 (DE3) -pET28a-III-NRK-PPK2-NusA plasmid in the strain, band 10 is the NRK-ppk2-MBP fusion protein obtained by expressing the E. coli BL21 (DE3) -pET28a-III-NRK-PPK2-MBP plasmid in the strain, and band 11 is the E. coli NRK-ppk2-DsbA fusion protein obtained by expressing BL21(DE3)-pET28a-III-NRK-PPK2-DsbA plasmid in the strain; Band 12 is the NRK-ppk2-GST fusion protein obtained by expressing E. coli BL21(DE3)-pET28a-III-NRK-PPK2-GST plasmid in the strain, Band 13 is the NRK-ppk2-SUMO fusion protein obtained by expressing E. coli BL21(DE3)-pET28a-III-NRK-PPK2-SUMO plasmid in the strain, and Band 14 is the NRK-ppk2 fusion protein obtained by expressing E. coli BL21(DE3)-pET28a-II-NRK-PPK2 plasmid in the strain. DETAILED DESCRIPTION

[0067] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0068] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0069] Detailed description of the invention

[0070] Definitions and general terms

[0071] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0072] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0073] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0074] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0075] The present invention proposes a fusion protein, a nucleic acid molecule, an expression vector, a genetically engineered bacterium, a method for preparing NMN, and a method for improving the activity or stability of nicotinamide ribokinase, which will be described in detail below.

[0076] Fusion Protein

[0077] In the first aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein includes: nicotinamide ribokinase; a solubility-promoting tag, and the nicotinamide ribokinase and the solubility-promoting tag are connected. The NRK in the fusion protein of the present invention has the advantages of strong stability or high activity. When the fusion protein is used to prepare NMN, the substrate conversion efficiency and the production concentration of NMN can be improved, especially the substrate conversion efficiency is stable at more than 90%, and the concentration of the prepared NMN can reach more than 60g / L.

[0078] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase. Thus, the stability and activity of NRK can be further improved, and the fusion protein obtained by this connection method is used to prepare NMN, which can further improve the final concentration and substrate conversion rate of NMN.

[0079] According to an embodiment of the present invention, the nicotinamide ribokinase is derived from Haemophilus influenzae.

[0080] According to an embodiment of the present invention, the nicotinamide ribokinase has an amino acid sequence as shown in SEQ ID NO:1.

[0081] MGFTTGREFHPALRMRAKYNAKYLGTKSEREKYFHLAYNKHTQFLRYQEQIMSKTKEKKVGVIFGKFYPVHTGHINMIYEAFSKVDELHVIVCSDTVRDLKLFYDS KMKRMPTVQDRLRWMQQIFKYQKNQIFIHHLVEDGIPSYPNGWQSWSEAVKTLFHEKHFEPSIVFSSEPQDKAPYEKYLGLEVSLVDPDRTFFNVSATKIRTTPFQ YWKFIPKEARPFFAKTVAILGGESSGKSVLVNKLAAVFNTTSAWEYGREFVFEKLGGDEQAMQYSDYPQMALGHQRYIDYAVRHSHKIAFIDTDFITTQAFCIQYE GKAHPFLDSMIKEYPFDVTILLKNNTEWVDDGLRSLGSQKQRQQFQQLLKKLLDKYKVPYIEIESPSYLDRYNQVKAVIEKVLNEEEISELQNTTFPIKGTSQ(SEQ ID NO:1).

[0082] According to an embodiment of the present invention, the solubility-promoting tag is selected from at least one of TrxA, SUMO and MBP. Thus, the stability of nicotinamide ribokinase can be further improved, the soluble expression of nicotinamide ribokinase can be promoted, and the activity of nicotinamide ribokinase can be increased.

[0083] According to an embodiment of the present invention, the TrxA has an amino acid sequence as shown in SEQ ID NO:2.

[0084] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLA (SEQ ID NO: 2).

[0085] According to an embodiment of the present invention, the SUMO has an amino acid sequence as shown in SEQ ID NO: 3. Therefore, the fusion protein not only has high soluble expression and strong stability, but also has high activity of nicotinamide ribokinase.

[0086] HRYRKSRGRSSTSFFGVLQKQKEAACDKFQLPSASTEFEEIRIFAFLRDLLPRGIVLSFYVWFTNFRVLNWTFCLVFFRTARPAFDAPQKKKKTFLTTQLQQATKKSTCLTKRREVRPSTSTRSSARTTPSSSSRSRS THPGSTPTATVPDSPCRWCASVSTDSPSTRTTLRPRWRWRRATPSRFTSSRLVALHKITLSLLLLQLHTNEKEKNTRKTTKAKTQQQQHEESNKPGPKNRLNMLLVQL.ILNASVCVNNKNICRQENGKSILTT(SEQ ID NO:3).

[0087] According to an embodiment of the present invention, the MBP has an amino acid sequence as shown in SEQ ID NO:4.

[0088] MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQS GLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYE NGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT(SEQ ID NO:4).

[0089] According to an embodiment of the present invention, the fusion protein further comprises an ATP cycle enzyme.

[0090] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, and the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the ATP cycle enzyme, thereby further improving the stability or activity of NRK.

[0091] According to an embodiment of the present invention, the ATP cycle enzyme is polyphosphate kinase.

[0092] According to an embodiment of the present invention, the polyphosphate kinase is PPK2.

[0093] According to an embodiment of the present invention, the PPK2 is derived from acidophilic sulfur bacteria.

[0094] According to an embodiment of the present invention, the PPK2 has an amino acid sequence as shown in SEQ ID NO: 5. Thus, the advantages of NRK such as strong stability or high activity can be further improved. At the same time, the use of PPK2 for ATP regeneration can reduce the amount of ATP used and further reduce production costs.

[0095] MTKCKEEKITEKQRYKEELHQLQIELVKLQRHIISKNEKILVILEGRDTAGKDGTIKRITQHLSPRETRVVALNKPSDREESQWYFQRYTHYLPSAAEIVFFNRSWYNRAGVEKVMGFCTDAQYEEFFSEVT DYEGMLVRSGIHFFKIYLDISRNEQKKRLEARKDDPLKQWKTSPIDAQAVKHWKDYSSARNAMFARTHTSFTPWTIVTADDKKVARLQIIKWLLSHLDYAGKDVSTLLPDQTIIFGYDPLCLENGMIKA(SEQ ID NO:5).

[0096] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide.

[0097] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the ATP cycle enzyme.

[0098] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:6.

[0099] KESGSSVSSEQLAQFRSLD (SEQ ID NO: 6).

[0100] Nucleic acid molecules, expression vectors and genetically engineered bacteria

[0101] In the second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. The nucleic acid molecule of the present invention can encode the fusion protein described in the first aspect.

[0102] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0103] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that any one of the complementary double strands is actually included. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0104] In the third aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect. The expression vector may include an optional control sequence, and the control sequence is operably connected to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in a host. The expression vector thus constructed can effectively express the fusion protein of the first aspect of the present invention.

[0105] When the nucleic acid molecule is connected to the expression vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. The nucleic acid molecule can be operably connected to the control element.

[0106] In this article, the term "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the nucleic acid molecules described above can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large amount of protein encoded by the nucleic acid molecules.

[0107] According to an embodiment of the present invention, the expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0108] According to an embodiment of the present invention, the expression vector can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or a viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, pET28a (+), etc.

[0109] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0110] According to an embodiment of the present invention, the expression vector is pET28a(+).

[0111] In the fourth aspect of the present invention, the present invention proposes a genetically engineered bacterium. According to an embodiment of the present invention, the genetically engineered bacterium includes: carrying the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expressing the fusion protein described in the first aspect. The genetically engineered bacterium of the present invention is cultured under appropriate conditions to express the fusion protein described in the first aspect. As can be seen from the above, the NRK in the fusion protein has the advantages of strong stability or high activity.

[0112] Moreover, the traditional genetically engineered bacteria in the art often have plasmid instability during the passage process, resulting in a small number of passages, which can generally be passed on for 5 generations. However, the inventors unexpectedly discovered during the experiment that compared with traditional genetically engineered bacteria or mutant engineered bacteria (such as patent CN113755353B), the genetically engineered bacteria of the present invention have the advantages of strong stability and high number of passages. It can be stably passed on for 15-20 generations, thereby saving costs for the subsequent industrial preparation of NMN. Therefore, after fusing the lytic tag and NRK, the inventors found that not only the stability of NRK can be improved, but also the activity of NRK and the number of passages of genetically engineered bacteria can be further improved.

[0113] Herein, the "suitable conditions" refer to conditions suitable for the expression of the fusion protein of the present invention. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the fusion protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host bacteria state, suitable host bacteria density, suitable bacterial culture environment, and suitable genetic engineering bacteria culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the fusion protein according to the specific environment of the laboratory.

[0114] According to an embodiment of the present invention, the genetically engineered bacteria is obtained by introducing the expression vector into a host bacteria.

[0115] According to an embodiment of the present invention, the host bacteria is Escherichia coli.

[0116] Methods for preparing NMN

[0117] In the fifth aspect of the present invention, the present invention proposes a method for preparing NMN. According to an embodiment of the present invention, the method comprises: using the fusion protein described in the first aspect or the genetically engineered bacteria described in the fourth aspect to catalyze the reaction of nicotinamide riboside or its salt and ATP or its salt to obtain NMN. As can be seen from the foregoing, the above-mentioned fusion protein of the present invention has the advantages of high stability and high activity, especially on the basis of using the same amount of fusion protein or genetically engineered bacteria, due to the high activity of the fusion protein of the present invention, the substrate conversion rate, the production efficiency of NMN or the final concentration of NMN can be further improved, thereby effectively reducing the production cost of NMN.

[0118] According to an embodiment of the present invention, before the catalytic reaction, magnesium ions and polyphosphate are mixed with the nicotinamide riboside or a salt thereof and ATP or a salt thereof.

[0119] According to an embodiment of the present invention, the nicotinamide riboside or a salt thereof is NRCl.

[0120] According to an embodiment of the present invention, the nicotinamide riboside or its salt is selected from NRCl with a purity of 75%. NRCl with a purity of 75% is lower in price than NRCl with a purity of 99%. Therefore, the method of the present application can reduce the production cost of preparing NMN.

[0121] According to an embodiment of the present invention, the mass ratio of ATP: NRCl with a purity of 75% is 1: (3-7).

[0122] According to an embodiment of the present invention, the magnesium ions are derived from MgCl 2 ﹒ 6H 2 O.

[0123] According to an embodiment of the present invention, the polyphosphate is sodium hexametaphosphate.

[0124] According to an embodiment of the present invention, the ATP: NRCl with a purity of 75%: sodium hexametaphosphate: MgCl 2 ﹒ 6H 2 The mass ratio of O is 1:(3~7):(1~1.5):(0.1~0.5).

[0125] According to an embodiment of the present invention, the catalytic reaction is carried out at 25-40° C. and a pH value of 5-7.

[0126] According to an embodiment of the present invention, the reaction time of the catalytic reaction is 0.5 to 3 h, for example, 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3 h or any range of values ​​with two of them as endpoint values, exemplarily 0.5 h to 2.5 h, 1 h to 3 h, etc.

[0127] According to an embodiment of the present invention, in the overall system of the catalytic reaction, the added concentration of the fusion protein or genetically engineered bacteria is 5 to 15 g / L.

[0128] Method for increasing the activity or stability of nicotinamide ribokinase

[0129] In a sixth aspect of the present invention, the present invention provides a method for improving the activity or stability of nicotinamide ribokinase. According to an embodiment of the present invention, the method comprises: connecting a solubility-promoting tag to the nicotinamide ribokinase.

[0130] When genetically engineered bacteria are used to prepare nicotinamide ribokinase, nicotinamide ribokinase can be expressed soluble in the process, but the conversion efficiency is unstable and inclusion bodies often occur. The inventors of the present invention have found that by fusing a solubility-promoting tag with nicotinamide ribokinase, not only can the correct folding of nicotinamide ribokinase be promoted, but also the stability of nicotinamide ribokinase can be improved.

[0131] Moreover, when preparing NMN by the above method, the inventor unexpectedly discovered that fusing the lytic tag and nicotinamide ribokinase can improve the activity of nicotinamide ribokinase, and when the same amount of fusion protein or genetically engineered bacteria is used, the substrate conversion rate, the production efficiency of NMN and / or the final concentration of NMN can be further improved, thereby effectively reducing the production cost of NMN. Exemplarily, when preparing NMN by the above method, the substrate conversion efficiency is stable at more than 90%, and the concentration of the prepared NMN can reach more than 60g / L.

[0132] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase.

[0133] According to an embodiment of the present invention, the nicotinamide ribokinase is derived from Haemophilus influenzae.

[0134] According to an embodiment of the present invention, the nicotinamide ribokinase has an amino acid sequence as shown in SEQ ID NO:1.

[0135] According to an embodiment of the present invention, the lytic tag is selected from at least one of TrxA, SUMO and MBP.

[0136] According to an embodiment of the present invention, the TrxA has an amino acid sequence as shown in SEQ ID NO:2.

[0137] According to an embodiment of the present invention, the SUMO has an amino acid sequence as shown in SEQ ID NO:3.

[0138] According to an embodiment of the present invention, the MBP has an amino acid sequence as shown in SEQ ID NO:4.

[0139] According to an embodiment of the present invention, an ATP cycle enzyme is further included, that is, the ATP cycle enzyme is connected to nicotinamide ribokinase.

[0140] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, and the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the ATP cycle enzyme.

[0141] According to an embodiment of the present invention, the ATP cycle enzyme is polyphosphate kinase.

[0142] According to an embodiment of the present invention, the polyphosphate kinase is PPK2.

[0143] According to an embodiment of the present invention, the PPK2 is derived from acidophilic sulfur bacteria.

[0144] According to an embodiment of the present invention, the PPK2 has an amino acid sequence as shown in SEQ ID NO:5.

[0145] According to an embodiment of the present invention, a connecting peptide is further included, that is, the connecting peptide is connected to nicotinamide ribokinase.

[0146] According to an embodiment of the present invention, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the ATP cycle enzyme.

[0147] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:6.

[0148] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0149] The nucleotide sequences of the NRK (amino acid sequence as shown in SEQ ID NO: 1), PPK2 (amino acid sequence as shown in SEQ ID NO: 5), connecting peptide (amino acid sequence as shown in SEQ ID NO: 6) and lytic tag (amino acid sequence as shown in SEQ ID NO: 2 to 4 and 11 to 13) of the present invention can be obtained according to their amino acid sequences using conventional methods or conventional software (for example, the online program NCBI, (website: https: / / www.ncbi.nlm.nih.gov / , GeneOptimizer online program, etc.).

[0150] Example 1: Construction of engineered bacteria producing nicotinamide ribokinase

[0151] 1. Construction of Nicotinamide Ribokinase (NRK)-producing Engineering Bacteria

[0152] Referring to patent CN115637262A, the DNA sequences encoding the lytic tag, NRK and PPK2 were connected to the pET28a vector to obtain the plasmid pET28a. The specific preparation method is as follows:

[0153] 1.1 The DNA sequence of NRK (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 12) was connected to the restriction sites BamHI and EcoRI at the head and tail respectively by PCR, and then connected to the plasmid pET28a by restriction ligation to obtain the recombinant plasmid pET28a-I. The primers were designed as follows:

[0154] NRK-BamHI-F:CGCGGATCCATGGGTTTTACCACAGGACG (SEQ ID NO:7);

[0155] NRK-EcoRI-R:GGCCTTAAGAATTTGGGAGGTGCCTTTTATTGGAA (SEQ ID NO: 8).

[0156] 1.2 The PPK2 gene (amino acid sequence as shown in SEQ ID NO: 5) was connected to the restriction sites ECOR I and HindIII at the head and tail by PCR, and then connected to the recombinant plasmid pET28a-I by restriction ligation to obtain plasmid pET28a-II. The primers were designed as follows:

[0157] Ppk-EcoRI-F:CCGGAATTCATGGGACAGGAGAAATTG (SEQ ID NO:9);

[0158] Ppk-HindIII-R: CCCAAGCTTCTCTGGCTGTTCCAACG (SEQ ID NO: 10).

[0159] 1.3 N-terminal lysis-promoting tag of NRK in E.coli BL21 (DE3)

[0160] The N-terminus of NRK is fused with a lytic tag, namely MBP (amino acid sequence as shown in SEQ ID NO: 4), TrxA (amino acid sequence as shown in SEQ ID NO: 2), SUMO (amino acid sequence as shown in SEQ ID NO: 3), NusA (amino acid sequence as shown in SEQ ID NO: 11), DsbA (amino acid sequence as shown in SEQ ID NO: 12) or GST (amino acid sequence as shown in SEQ ID NO: 13). Specifically, different lytic tag genes are connected between BamHI and Nhel restriction sites by PCR, and then connected to the recombinant plasmid pET28a-II by restriction ligation to obtain plasmid pET28a-III.

[0161] MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVRVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILREDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEIKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTVDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDEPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDLAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA (SEQ ID NO:11);

[0162] MKKIWLALAGLVLAFSASAAQYEDGKQYTTLEKPVAGAPQVLEFFSFFCPHCYQFEEVLHISDNVKKKLPEGVKMTKYHVNFMGDGDLGKDLTQAWAVAMALGVEDKVTVPLFEGVQKTQTIRSASDIRDVFINAGIGGEEYDAAWNSSFVVKSLVAQQEKAAADVQLRGVPAMFVNGKYQLNPQGMDTSNMDVFVQQYADTVKYLSEKK(SEQ ID NO:12);

[0163] ARTRFQSRRGERRREVEEGRGQRDRRWGRSMGQPCPRARHVFSCAWRRWEPSTSWCPSISPPVNISSPLTSPVTYVFSLLNPLRLLMILLHSFAIPFFFLPSFFSDGDRLLIRTIGLSMQPFGQVPAFEDGALMLHGELPSYYLFLDLFICSGEIFRWKETNIEMIGFQFNTLFLTKLPMERGDSCGLQIFHDVLHMFPFPMRTWSRFTRKFRSWIPNLLQIRRIANFQKKQFTVFVGLI.KYKQIRMLIWNLLKFLLNPFIFLPTRLGKEDKSWFIIIYKRRLFHPISSYPSYLEWIGNHDPKSELNEPGDYGLESWIEHDLNSNYGGNREQIMHRNIRIEGNNTLNKYMPNVSNLDFEPNTKWMTQLSGCTGFQSSQNPAHLELHFSQVCNLLWLHTSAILQRFVISKIPILWIVVKSKDARQMTLKATEPGNFVTD.CSEKLQVGPWLRWAKASFASSNNFHIVKCPSSMSLHHCCYYIFSLSSLKIMKSESRAIARYVSRKYKSSGADLLKEGGLEESAMVDVWLEVESHQFDPAIGPIFFQSFIVPMIGGVPDQTVINTNLEKLCKVLDIYEARLSKTKYLAGDFFSLADLSHVPLLYYFMGSPHASVVNSRPHVKAWWEAVSSRPACKKVTSAMPGSA.EVSVGGHGEERKIKESASVVGLTICVCVWVPGCEAFVLVPVYDTWLCLGWDGGNMRCIHLFLCLLWCFICILVIQL(SEQ ID NO:13)。

[0164] 1.4 The genes fused in step 1.2 and step 1.3 were connected to plasmid pET28a to obtain recombinant plasmids pET28a-II-NRK-PPK2, pET28a-III-MBP-NRK-PPK2, pET28a-III-TrxA-NRK-PPK2, pET28a-III-SUMO-NRK-PPK2, pET28a-III-NusA-NRK-PPK2, pET28a-III-DsbA-NRK-PPK2, and pET28a-III-GST-NRK-PPK2, and the recombinant plasmids were transformed into Escherichia coli to obtain recombinant E. coli BL21 (DE3)-pET28a-II-NRK-PPK2, E. coli BL21 (DE3)-pET28a-III-MBP-NRK-PPK2, and E. coli BL21(DE3)-pET28a-III-TrxA-NRK-PPK2, E.coli BL21(DE3)-pET28a-III-SUMO-NRK-PPK2, E.coli BL21(DE3)-pET28a-III-NusA-NRK-PPK2, E.coli BL21(DE3)-pET28a-III-DsbA-NRK-PPK2, E.coli BL21(DE3)-pET28a-III-GST-NRK-PPK2, stored at -80°C.

[0165] 2. Fermentation to produce enzymes

[0166] 2.1 For the different recombinant E. coli and the recombinant E. coli without a lytic tag obtained in step 1 of this example, single colonies were selected and inoculated into LB medium, and shake flasks were cultured at 37° C. for 8 h.

[0167] 2.2 Expand the first-level shake flask to a second-level shake flask, culture at 37°C for 3-7 hours, with an inoculation size of 2-3%.

[0168] 2.3 The secondary shake flask is inoculated with a culture medium containing protein chen, yeast powder, glycerol, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, chloride, anhydrous sodium sulfate, magnesium sulfate, and defoaming agent, and cultured at 37°C for 6-10 hours. Ammonia water and glycerol are supplemented during the culture process, and the inoculation amount is 2-5%.

[0169] 2.4 When the fermentation broth OD 600 =20-30, add IPTG (final IPTG concentration is 1 mM) and culture at 25°C. During the culture process, the dissolved oxygen is maintained at 25-40% and the glycerol concentration is about 2 g / L until the fermentation is completed.

[0170] 2.5 After the fermentation, the fermentation liquid was centrifuged at 7000 rpm for 10 min, the precipitate was collected and subjected to cell wall breaking treatment such as ultrasound or homogenization, and the cell wall-broken bacterial solution (containing NRK and PPK2) was obtained by centrifugation for later use. The expressed fusion protein was then identified by SDS-PAGE. The identification results are shown in Figure 1 The results showed that the fusion proteins in each recombinant E. coli were successfully expressed; and compared with NRK-ppk2, NusA-NRK-ppk2, DsbA-NRK-ppk2 and GST-NRK-ppk2, the expression levels of MBP-NRK-ppk2 (band 1), TrxA-NRK-ppk2 (band 4) and SUMO-NRK-ppk2 (band 3) fusion proteins were increased. In addition, the quality and enzyme activity of the enzymes produced in each bacterial solution were determined, and the results are shown in Table 1.

[0171] Table 1: Content of inclusion bodies (insoluble enzyme) obtained from different recombinant E. coli

[0172] Bacteria Insoluble enzyme content (%) E.coli BL21(DE3)-pET28a-II-NRK-ppk2 27 E.coli BL21(DE3)-pET28a-III-MBP-NRK-ppk2 13 E.coli BL21(DE3)-pET28a-III-TrxA-NRK-ppk2 19 E.coli BL21(DE3)-pET28a-III-SUMO-NRK-ppk2 5 E.coli BL21(DE3)-pET28a-III-NusA-NRK-ppk2 8 E.coli BL21(DE3)-pET28a-III-DsbA-NRK-ppk2 3 E.coli BL21(DE3)-pET28a-III-GST-NRK-ppk2 9

[0173] As shown in Table 1, after the solubilizing tag was connected to the N-terminus of NRK, the content of inclusion bodies was significantly reduced, and the inclusion bodies obtained were all less than 20%.

[0174] Example 2: Preparation of β-nicotinamide mononucleotide

[0175] The preparation steps of β-nicotinamide mononucleotide (NMN) are as follows:

[0176] The reaction system was added with (75%) NRCl 80 g / L, sodium hexametaphosphate 20 g / L, MgCl 2 ﹒ 6H 2 O 5g / L and ATP 16g / L, the pH value was adjusted to 6-7, the temperature was 30°C, and the bacterial solution prepared in Example 1 after removing the inclusion bodies (the bacterial solution contained NRK and PPK2) was added in an amount of 10g / L. The content of β-nicotinamide mononucleotide was detected every 0.5h. When NRCl was consumed, the reaction was terminated (about 1h), and hydrochloric acid was added to adjust the pH to 2-3 and then quenched. Among them, the yield of NMN was monitored by HPLC, and the conversion rate of NR to NMN was calculated, and the final concentration of β-nicotinamide mononucleotide after the reaction was calculated. The final concentration of NMN prepared by the fusion protein obtained from different recombinant Escherichia coli and the substrate conversion rate results are shown in Table 2.

[0177] Table 2: Final concentration and substrate conversion rate of NMN prepared by fusion proteins obtained from different recombinant E. coli

[0178]

[0179]

[0180] As shown in Table 2, compared with other solubilizing tags, the fusion protein obtained by connecting MBP, TrxA and SUMO to the N-terminus of NRK as a catalyst can shorten the reaction time (1-3h), increase the substrate conversion rate (maintained above 93%) and the final concentration of NMN. Among them, the fusion protein with the SUMO tag can completely convert NRCl after 1h of reaction. After 1h of reaction, the substrate conversion rate of E.coliBL21(DE3)-pET28a-III-SUMO-NRK-PPK2 reached a maximum of 100%, and the final concentration of β-nicotinamide mononucleotide was 68.63g / L.

[0181] Example 3: Verification of bacterial strain stability

[0182] 1. Spread the E. coli BL21 (DE3) -pET28a-III-SUMO-NRK-ppk2 prepared in Example 1 on an LB plate and culture it in a 37 ° C biochemical incubator for 12-16 hours. A single colony grows on the plate and a single colony is picked as the first generation of bacteria; the first generation of bacteria is spread on an LB solid medium and cultured under the same culture conditions for 12-16 hours, and a single colony is picked as the second generation of bacteria; repeat the subculture until the 25th generation of bacteria is obtained.

[0183] Each strain obtained by subculture was amplified by shaking flasks, centrifuged to collect the cells, and then ultrasonically disrupted with PB buffer for the preparation of NMN. The reaction system is as follows:

[0184] Add (75%) NRCl 80g / L, sodium hexametaphosphate 20g / L, MgCl 2 ﹒ 6H 2 O 5g / L and ATP 16g / L, adjust the pH to 6-7, the temperature is 30°C, add the ultrasonically broken bacterial solution, and the addition amount is 10g / L. Samples are taken every 0.5h to detect the content of β-nicotinamide mononucleotide. When NRCl is consumed, the reaction ends (about 1h), and hydrochloric acid is added to adjust the pH to 2-3 and then quench. Among them, HPLC is used to monitor the production of NMN, and the conversion rate of NR to NMN is calculated. The experimental data are shown in Table 3.

[0185] Table 3: Transformation efficiency results of different passages of strains producing SUMO-NRK-ppk2 fusion protein

[0186] strain Average conversion rate of 75% NRCl (%) Original genetically modified strains 100％ 1st-5th generation strains 99.8％ 6th-10th generation strains 99.5％ 11-15 generation strains 99.3％ 16-20 generation strains 99.6％ 21-25 generation strains 98.6％

[0187] As shown in Table 3, the pBET28a-III-SUMO-NRK-PPK2 plasmid was passaged in E. coli for up to 20 times, and NMN was still stably obtained at a conversion rate of 99%. After 25 passages, NMN was still obtained at a conversion rate of more than 98%.

[0188] 2. The E. coli BL21 (DE3) -pET28a-III-TrxA-NRK-ppk2 strains and E. coli BL21 (DE3) -pET28a-III-TrxA-NRK-ppk2 strains prepared in Example 1 were subcultured according to the above method, and then each strain obtained by subculture was used to prepare NMN, and the conversion rate of NR to NMN was calculated to verify the subculture stability. The results showed that after 20 subcultures of E. coli BL21 (DE3) -pET28a-III-TrxA-NRK-ppk2 strains and E. coli BL21 (DE3) -pET28a-III-TrxA-NRK-ppk2 strains, the produced enzymes could maintain a conversion rate of more than 98%.

[0189] Embodiment 4:

[0190] Prepare a fusion protein connected between NRK and a lytic tag, prepare the recombinant plasmid pET28a-I-NRK according to the method of step 1 in Example 1, and then connect each lytic tag to pET28a-I-NRK by enzyme ligation to obtain recombinant plasmids pET28a-II-MBP-NRK, pET28a-II-TrxA-NRK, pET28a-II-SUMO-NRK, pET28a-II-NusA-NRK, pET28a-II-DsbA-NRK, and pET28a-II-GST-NRK, and then transform the recombinant plasmids into Escherichia coli to obtain recombinant Escherichia coli E. coli BL21 (DE3) -pET28a-I-NRK, E. coli BL21 (DE3) -pET28a-II-MBP-NRK, E. coli BL21 (DE3) -pET28a-II-TrxA NRK, E.coli BL21 (DE3) -pET28a-II-SUMO-NRK, E.coli BL21 (DE3) -pET28a-II-NusA-NRK, E.coli BL21 (DE3) -pET28a-II-DsbA-NRK, E.coli BL21 (DE3) -pET28a-II-GST-NRK. The enzyme was fermented and produced according to the method of step 2 of Example 1, and then the expressed fusion protein was identified by SDS-PAGE. It was found that the fusion protein in each recombinant Escherichia coli was successfully expressed. In addition, the quality of the produced enzyme and the enzyme activity were determined (since there was no ATP cycle enzyme, the equivalent of ATP added during the enzyme activity determination was the same as the equivalent of the reactant NRCl). As shown in Table 4, after the N-terminus of the NRK single enzyme was connected to the solubilizing tag, the content of the inclusion body was significantly reduced, and the inclusion bodies obtained were all less than 11%.

[0191] The enzymes produced by the recombinant E. coli prepared in this example were used to prepare NMN, and the conversion rate of NR to NMN and the final concentration of β-nicotinamide mononucleotide after the reaction were calculated. The results showed that simply adding a lytic tag to the N-terminus of NRK could also increase the activity of the NRK enzyme. In particular, compared with E. coli BL21(DE3)-pET28a-I-NRK, E. coli BL21(DE3)-pET28a-II-NusA-NRK, E. coli BL21(DE3)-pET28a-II-DsbA-NRK, and E. coli BL21(DE3)-pET28a-II-GST-NRK, the fusion proteins containing NRK expressed by E. coli BL21(DE3)-pET28a-II-MBP-NRK, E. coli BL21(DE3)-pET28a-II-TrxA-NRK, and E. coli BL21(DE3)-pET28a-II-SUMO-NRK as catalysts can increase the substrate conversion rate and the final concentration of NMN.

[0192] Table 4: Content of inclusion bodies (insoluble enzyme) obtained from different recombinant E. coli

[0193] Bacteria Insoluble enzyme content (%) E.coli BL21(DE3)-pET28a-I-NRK 18 E.coli BL21(DE3)-pET28a-II-MBP-NRK 11 E.coli BL21(DE3)-pET28a-II-TrxA-NRK 8 E.coli BL21(DE3)-pET28a-II-SUMO-NRK 2 E.coli BL21(DE3)-pET28a-II-NusA-NRK 5 E.coli BL21(DE3)-pET28a-II-DsbA-NRK 3 E.coli BL21(DE3)-pET28a-II-GST-NRK 6

[0194] Comparative Example 1:

[0195] Prepare fusion protein of NKR-PPK2-lytic tag, prepare recombinant plasmids pET28a-III-NRK-PPK2-MBP, pET28a-III-NRK-PPK2-TrxA, pET28a-III-NRK-PPK2-SUMO, pET28a-III-NRK-PPK2-NusA, pET28a-III-NRK-PPK2-DsbA, pET28a-III-NRK-PPK2-GST according to the method of step 1 of Example 1, and then transform the recombinant plasmids into Escherichia coli respectively to obtain recombinant Escherichia coli E. coli BL21 (DE3)-pET28a-III-NRK-PPK2-MBP, E. coli BL21 (DE3)-pET28a-III-NRK-PPK2-TrxA, E. coli BL21(DE3)-pET28a-III-NRK-PPK2-SUMO, E. coli BL21(DE3)-pET28a-III-NRK-PPK2-NusA, E. coli BL21(DE3)-pET28a-III-NRK-PPK2-DsbA, E. coli BL21(DE3)-pET28a-III-NRK-PPK2-GST, stored at -80°C. Then SDS-PAGE was used to identify the expressed fusion protein. The identification results are shown in Figure 2 The results showed that the fusion proteins in each recombinant E. coli were successfully expressed; and compared with the fusion protein prepared in Example 1, the expression level of the fusion protein prepared by the method of this comparative example was reduced, for example, the expression level of the fusion protein prepared by using NusA, MBP, DsbA, and GST was significantly reduced compared with the fusion protein in Example 1. The enzyme was fermented and produced according to the method of step 2 of Example 1, and then the quality and enzyme activity of the produced enzyme were determined. The quality results of the enzyme are shown in Table 5.

[0196] Then, referring to the method of Example 2, NMN was prepared using the enzymes produced by the recombinant E. coli in each group prepared in this comparative example, and the conversion rate of NMN prepared from NR and the final concentration of β-nicotinamide mononucleotide after the reaction were calculated. The final concentration of NMN prepared by the fusion proteins obtained from different recombinant E. coli and the substrate conversion rate results are shown in Table 6.

[0197] Table 5: Content of inclusion bodies (insoluble enzyme) obtained by different recombinant E. coli

[0198] Bacteria Insoluble enzyme content (%) E.coli BL21(DE3)-pET28a-III-NRK-ppk2-MBP 29 E.coli BL21(DE3)-pET28a-III-NRK-ppk2-TrxA 22 E.coli BL21(DE3)-pET28a-III-NRK-ppk2-SUMO 11 E.coli BL21(DE3)-pET28a-III-NRK-ppk2-NusA 9 E.coli BL21(DE3)-pET28a-III-NRK-ppk2-DsbA 17 E.coli BL21(DE3)-pET28a-III-NRK-ppk2-GST 13

[0199] It can be seen from Table 5 that changing the connection mode of the solubility-promoting tag will reduce the stability of the NRK enzyme in the fusion protein.

[0200] Table 6: Final concentration and substrate conversion rate of NMN prepared by fusion proteins obtained from different recombinant E. coli

[0201]

[0202]

[0203]

[0204] Comparing Table 6 with Table 2, it was found that after changing the connection mode of the solubilizing tag, the highest conversion rate was only 84.66%. Among them, the highest conversion rate of the fusion protein with the SUMO tag was reduced from 100% to 76.64%; the highest conversion rate of the fusion protein with the TrxA tag was reduced from 97.7% to 84.66%; the highest conversion rate of the fusion protein with the MBP tag was reduced from 93.7% to 26.33%.

[0205] Therefore, when adding a solubility-promoting tag, the connection method is also a technical means that can produce unexpected effects.

[0206] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0207] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that include: Nicotinamide ribokinase; A solubility-promoting tag, wherein the nicotinamide ribokinase is connected to the solubility-promoting tag.

2. The fusion protein according to claim 1, characterized in that The C-terminus of the solubility-promoting tag is connected to the N-terminus of the nicotinamide ribokinase; Optionally, the nicotinamide ribokinase is derived from Haemophilus influenzae; Optionally, the nicotinamide ribokinase has an amino acid sequence as shown in SEQ ID NO: 1; Optionally, the lytic tag is selected from at least one of TrxA, SUMO and MBP; Optionally, the TrxA has an amino acid sequence as shown in SEQ ID NO: 2; Optionally, the SUMO has an amino acid sequence as shown in SEQ ID NO: 3; Optionally, the MBP has the amino acid sequence shown in SEQ ID NO:

4.

3. The fusion protein according to claim 2, characterized in that further including ATP cycle enzymes; Optionally, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, and the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the ATP cycle enzyme; Optionally, the ATP cycle enzyme is polyphosphate kinase; Optionally, the polyphosphate kinase is PPK2; Optionally, the PPK2 is derived from an acidosulfur bacterium; Optionally, the PPK2 has an amino acid sequence as shown in SEQ ID NO: 5; Optionally, the fusion protein further comprises a connecting peptide; Optionally, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the ATP cycle enzyme; Optionally, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:

6.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 3; Optionally, the nucleic acid molecule is DNA.

5. An expression vector, characterized in that: comprising the nucleic acid molecule of claim 4; Optionally, the expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector; Optionally, the expression vector is a plasmid expression vector; Preferably, the expression vector is pET28a(+).

6. A genetically engineered bacterium, characterized in that: include: Carrying the nucleic acid molecule of claim 4 or the expression vector of claim 5; or Expressing the fusion protein according to any one of claims 1 to 3; Optionally, the genetically engineered bacteria is obtained by introducing the expression vector of claim 5 into a host bacteria; Optionally, the host bacteria is Escherichia coli.

7. A method for preparing NMN, characterized in that: include: The fusion protein according to any one of claims 1 to 3 or the genetically engineered bacteria according to claim 6 is used to catalyze the reaction of nicotinamide riboside or its salt and ATP or its salt to obtain NMN.

8. The method according to claim 7, characterized in that Prior to the catalytic reaction, magnesium ions and polyphosphate are mixed with the nicotinamide riboside or its salt and ATP or its salt; Optionally, the nicotinamide riboside or a salt thereof is NRCl; Preferably, the nicotinamide riboside or its salt is selected from NRCl with a purity of 75%; Optionally, the mass ratio of ATP: NRCl with a purity of 75% is 1: (3-7); Optionally, the magnesium ions are derived from MgCl2﹒6H2O; Optionally, the polyphosphate is sodium hexametaphosphate; Optionally, the mass ratio of ATP: NRCl with a purity of 75%: sodium hexametaphosphate: MgCl2﹒6H2O is 1: (3-7): (1-1.5): (0.1-0.5); Optionally, the catalytic reaction is carried out at 25 to 40° C. and a pH value of 5 to 7; Optionally, the reaction time of the catalytic reaction is 0.5 to 3 hours; Optionally, in the total system of the catalytic reaction, the added concentration of the fusion protein or genetically engineered bacteria is 5 to 15 g / L.

9. A method for increasing the activity or stability of nicotinamide ribokinase, characterized in that: include: A solubility-promoting tag is attached to the nicotinamide ribokinase.

10. The method according to claim 9, characterized in that The C-terminus of the solubility-promoting tag is connected to the N-terminus of the nicotinamide ribokinase; Optionally, the nicotinamide ribokinase is derived from Haemophilus influenzae; Optionally, the nicotinamide ribokinase has an amino acid sequence as shown in SEQ ID NO: 1; Optionally, the lytic tag is selected from at least one of TrxA, SUMO and MBP; Optionally, the TrxA has an amino acid sequence as shown in SEQ ID NO: 2; Optionally, the SUMO has an amino acid sequence as shown in SEQ ID NO: 3; Optionally, the MBP has an amino acid sequence as shown in SEQ ID NO:4; Optionally, further comprising an ATP cycle enzyme; Optionally, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, and the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the ATP cycle enzyme; Optionally, the ATP cycle enzyme is polyphosphate kinase; Optionally, the polyphosphate kinase is PPK2; Optionally, the PPK2 is derived from an acidosulfur bacterium; Optionally, the PPK2 has an amino acid sequence as shown in SEQ ID NO: 5; Optionally, further comprising a connecting peptide; Optionally, the C-terminus of the lytic tag is connected to the N-terminus of the nicotinamide ribokinase, the C-terminus of the nicotinamide ribokinase is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the ATP cycle enzyme; Optionally, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:6.

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