A recombinant engineering strain for synthesizing oxidized nicotinamide adenine dinucleotide and its application

Abstract

The present invention provides a recombinant engineering strain for synthesizing oxidized nicotinamide adenine dinucleotide and an application thereof. The engineering strain contains a recombinant vector shown in SEQ ID NO.5; the recombinant vector contains a gene fragment 1 shown in SEQ ID NO.1 and a gene fragment 2 shown in SEQ ID NO.2, so that the engineering strain contains nicotinamide mononucleotide adenyltransferase and pyrophosphohydrolase after metabolism, and reduces the molar ratio of raw materials ATP to NMN in the synthesis of oxidized nicotinamide adenine dinucleotide, improves the comprehensive utilization rate of the raw materials, and reduces the difficulty of subsequent purification; at the same time, the pH range of the reaction is reduced, which can significantly prevent the color of the reaction solution from deepening.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme catalysis, and in particular to a recombinant engineering strain for synthesizing oxidized nicotinamide adenine dinucleotide and an application thereof. Background Art

[0002] Oxidized nicotinamide adenine dinucleotide (NAD + ) is what is commonly referred to as the oxidized state of coenzyme I. It is the electron carrier of most redox reactions in the cells of all organisms. It participates in the metabolism of sugars, fats, amino acids, etc. in organisms. It is also a commonly used coenzyme in industrial enzyme catalysis. It can be synthesized in vitro by the precursor nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) enzyme.

[0003] In the patent document with application number CN201310752930.8 and subject name Nicotinamide mononucleotide adenylyltransferase mutant and its encoding gene and application and the application document with application number CN201210434517.2 and subject name Method for synthesizing nicotinamide adenine dinucleotide (NAD) using immobilized enzyme, a method derived from Methanocaldococcus jannaschii Nicotinamide mononucleotide adenylyltransferase of DSM 2661; in the application document with application number CN201610127030.8 and subject name A nicotinamide mononucleotide adenylyltransferase gene and its application, a gene derived from Methanothermobacter sp. Nicotinamide mononucleotide adenylyltransferase of CaT2; application number CN202110853020.3, subject name NAD + The application document of the preparation method and application of NMNAT discloses that five nicotinamide mononucleotide adenylyltransferases are derived from Escherichia albertii , Salmonella enterica , Methanotorris igneus and Methanococcus vannielii ; The application document with application number CN202311389935.9 and subject name A method for synthesizing nicotinamide adenine dinucleotide, mutants and applications thereof discloses a method derived from Sugiyamaella lignohabitans nicotinamide mononucleotide adenylyltransferase; however, no solution was given to the problem of pyrophosphate inhibition during the catalytic process.

[0004] The application document with application number CN202310400406.8 and subject name A method for preparing nicotinamide adenine dinucleotide phosphate by enzymatic method discloses a method derived from Methanocaldococcus infernus Nicotinamide mononucleotide adenylyltransferase, and also discloses the use of Saccharomyces cerevisiaeS288C's pyrophosphate hydrolase is used to solve the problem of pyrophosphate inhibition. In the application document, the pH of the catalytic conditions is 7.0-10.0, and the initial molar ratio of ATP to NMN (nicotinamide mononucleotide) is required to be 1:1-1.5. Under these conditions, the raw materials are not fully utilized, and a large amount of raw materials will remain in the reaction solution, resulting in a waste of raw materials. In addition, the residual substrate is difficult to remove during subsequent purification, making purification difficult. Summary of the invention

[0005] In view of this, the present invention provides a recombinant engineering strain for the synthesis of oxidized nicotinamide adenine dinucleotide and its application, wherein the engineering strain uses a recombinant vector containing SEQ ID NO.5; the recombinant vector contains gene fragment 1 shown in SEQ ID NO.1 and gene fragment 2 shown in SEQ ID NO.2, so that the engineering strain contains nicotinamide mononucleotide adenyltransferase and pyrophosphohydrolase after metabolism, which reduces the molar ratio of raw material ATP to NMN in the synthesis of oxidized nicotinamide adenine dinucleotide, improves the comprehensive utilization rate of raw materials, and reduces the difficulty of subsequent purification; at the same time, the pH range of the reaction is reduced, which can significantly prevent the color of the reaction solution from deepening.

[0006] The technical solution of the present invention is as follows: A recombinant vector, the gene sequence of the recombinant vector is shown as SEQ ID NO.5.

[0007] Preferably, the above-mentioned recombinant vector is prepared by inserting the nucleic acid sequence shown in SEQ ID NO.1 between the Nco I and Hind III restriction sites of the expression vector, and inserting the nucleic acid sequence shown in SEQ ID NO.2 between the NdeI and XhoI of the expression vector to obtain the recombinant vector.

[0008] Preferably, the expression vector is pETDuet-1 or pCDFDuet-1 or pACYCDuet-1 or pRSFDuet-1.

[0009] An engineered strain containing the recombinant vector shown in SEQ ID NO.5, the preparation process is as follows: (1) Strain construction After the recombinant vector is sequenced correctly, it is transformed into Escherichia coli BL21 (DE3) by heat shock method; take an appropriate amount of the bacterial solution after transformation and incubation to apply to LB resistance plate containing 100μg / ml ampicillin, and culture at 37℃ for 14-20 hours; seal with sealing film and store at 4℃ for future use; (2) Strain screening 8 to 10 single colonies were selected and inoculated into 3 ml of LB liquid culture medium containing 100 µg / ml ampicillin, respectively, and cultured at 220 rpm and 37°C for 4 to 5 hours to obtain bacterial solution A; 1 ml of the cultured bacterial solution A was taken, examined under a microscope, and then transferred to 50 ml of LB liquid culture medium containing 100 µg / ml ampicillin, and cultured at 220 rpm and 37°C for 4 to 5 hours to obtain bacterial solution B, examined under a microscope, and then IPTG was added to a final concentration of 0.1 mM, and expression was induced at 220 rpm and 28°C for 14 to 20 hours to obtain bacterial solution C; examined under a microscope and SDS-PAGE; The single colonies with the lowest percentage of small dots and wires in microscopic examination of bacterial solution A, B and C at three time points and normal SDS-PAGE detection were selected as the strains for subsequent enzyme preparation; the quality of BL21 (DE3) varies greatly depending on the manufacturer, so single colonies with reliable quality were obtained by liquid microscopic examination of the above three time points; (3) Seed preparation for seed bank Take 1.5 ml of the bacterial solution A corresponding to the single colony in step (2) and transfer it to 200 ml of LB liquid medium containing 100 μg / ml ampicillin. Incubate at 37°C for 6 to 7 hours until the OD 600 The pH value was 1.5-2.2, then transferred to a centrifuge cup under sterile conditions, placed in an ice water bath for 20 minutes, centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, 50 ml of 1 / 2 LB liquid culture medium containing 20% ​​glycerol pre-cooled with ice water was added, the suspension was evenly resuspended, and the suspension was divided into cryopreservation tubes; the divided strains were allowed to stand at 4°C for 1 hour, and then stored in a -80°C refrigerator as seeds.

[0010] Preferably, the Escherichia coli is Escherichia coli BL21 (DE3) or JM109 (DE3) or an Escherichia coli derived from these two host bacteria and having DE3 function.

[0011] The application of the above-mentioned engineered strain in the synthesis of oxidized nicotinamide adenine dinucleotide is to use wet bacteria or a double enzyme solution prepared from wet bacteria as a catalyst, NMN and ATP as substrates, and react at 28-32°C and pH 6.3-6.8 to obtain NAD + , NAD + The generation rate is >96%; among them, the initial molar ratio of NMN to ATP is 0.95-0.98.

[0012] Preferably, the preparation process of wet cells is as follows: Shake flask fermentation, take 300ul of the preserved seeds and inoculate them into 400ml of culture medium. The composition of the culture medium is: Na 2 HPO 4 100mM, NH 4Cl 50mM, MgSO 4 5mM, FeCl 3 0.1mM, peptone 20g / L, yeast powder 10g / L, glycerol 20g / L, defoamer 0.02%, culture at 37℃, 220rpm for 6-7 hours. 600 When the pH is 6.0-8.0, cool to 28°C, add IPTG to a final concentration of 0.1-0.2 mM, induce expression for 16-24 hours, then centrifuge at 5000 rpm for 20 min to collect the wet cells; the wet cells can be stored at -20°C for more than five years.

[0013] Preferably, wet cells are used to prepare NAD + , the process is as follows: In a 1000ml three-necked flask, add 300ml of purified water, stir mechanically, then add NMN, ATP and magnesium chloride hexahydrate, the pH after dissolution is 2.5-2.8, adjust the pH to 6.3-6.8 with 30% NaOH, adjust the temperature to 28-32°C, then add 10.0g of wet bacteria, start the reaction, and use 6M HCl to control the pH value to no higher than 7.0; react for 8 hours, HPLC detection, NAD + When the generation rate is greater than 96%, the reaction can be terminated.

[0014] Preferably, the preparation process of the double enzyme solution is as follows: Take 20g of wet bacteria, add 100ml of 20mM pH7.0 PBS buffer, break it once at 300bar and twice at 900bar with a high-pressure homogenizer; add 1ml of 10% polyimide, stir for 10 minutes, centrifuge at 10000rpm for 20 minutes, take the supernatant, which is the double enzyme solution, and store it at 4℃.

[0015] Preferably, a double enzyme solution is used to catalyze the preparation of NAD + The process is as follows: In a 1000ml three-necked flask, add 300ml of purified water, stir mechanically, add NMN, ATP and magnesium chloride hexahydrate, the pH after dissolution is 2.5-2.8, adjust the pH to 6.3-6.8 with 30% NaOH, adjust the temperature to 28-32°C, then add the double enzyme solution, start the reaction, and use 6M HCl to control the pH value not higher than 7.0; react for 8 hours, HPLC detection, NAD + When the generation rate is greater than 96%, the reaction is terminated.

[0016] Compared with the prior art, the beneficial effects of the present invention are: In the present invention, a recombinant vector containing gene fragment 1 shown in SEQ ID NO.1 and gene fragment 2 shown in SEQ ID NO.2 and an expression vector is used to construct an engineered strain obtained by transferring into Escherichia coli, and its metabolites contain nicotinamide mononucleotide adenyltransferase and pyrophosphate hydrolase. In the process of catalytic synthesis of oxidized nicotinamide adenine dinucleotide, while ensuring a high generation rate, the molar ratio of raw materials ATP to NMN is reduced, the utilization rate of raw materials is effectively improved, and the problem of raw material waste is avoided. In addition, the residual substrate after the reaction is completed does not precipitate during crystallization, does not affect purification, and reduces the difficulty of subsequent purification; and solves the problem of pyrophosphate inhibition during the synthesis process; in addition, the catalytic reaction can be carried out under the condition of pH 6.3-6.8, which effectively prevents the problem of darkening of the reaction solution. After the catalytic reaction, NAD + The generation rate is >96%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : is a map of the recombinant vector in Example 1. In the map, SEQ01 is SEQ ID NO.1, SEQ02 is SEQ ID NO.2, and pETDuet-SEQ01-SEQ02 is the recombinant vector.

[0018] Figure 2 This is the structure of the multiple cloning region of the recombinant vector constructed in Example 1; in the figure, SEQ01 is SEQ ID NO.1, SEQ02 is SEQ ID NO.2, and pETDuet-SEQ01-SEQ02 is the recombinant vector.

[0019] Figure 3 The SDS-PAGE electrophoresis results of the four single colonies selected in Example 2 after induced expression, wherein 1, 2, 3, and 4 are the selected colony numbers, respectively. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0021] Example 1 Preparation of recombinant vector Combination Figure 1 - Figure 2 , a recombinant vector, the preparation process is: The nucleic acid sequence shown in SEQ ID NO.1 and the nucleic acid sequence shown in SEQ ID NO.2 were synthesized by Qingke gene; The nucleic acid sequence shown in SEQ ID NO.1 was inserted between the Nco I and Hind III restriction sites of pETDuet-1, and the nucleic acid sequence shown in SEQ ID NO.2 was inserted between the Nde I and XhoI of the expression vector; the gene sequence of the obtained recombinant vector is shown in SEQ ID NO.5.

[0022] The nucleic acid sequence shown in SEQ ID NO.1 is the nucleic acid sequence of nicotinamide nucleotide adenylate transferase encoded by NMNat, and the amino acid sequence is shown in SEQ ID NO.3. The nicotinamide nucleotide adenylate transferase is derived from Pseudomonas aeruginosa ( Pseudomonas aeruginosa ); The nucleic acid sequence shown in SEQ ID NO.2 is the nucleic acid sequence of the pyrophosphohydrolase encoded by PPase, and the amino acid sequence is shown in SEQ ID NO.4. The pyrophosphohydrolase is derived from Escherichia coli ( Escherichia coli K12); SEQ ID NO.1 ATGGGCAAACGTATCGGCCTGTTCGGCGGCACTTTCGACCCCGTGCATATCGGACATATGCGCAGCGCCGTGGAAATGGCCGAGCAGTTCGCCCTCGACGAACTGCGCCTGCTGCCCAATGCGCGGCCGCCGCACCGGGAAACCCCGCAGGTGTCGGCCGCGCAGCGGTTGGCGATGGTCGAGCGGGCGGTGGCGGGAATCGAGCGATTGACGGTCGATCCCCGCGAACTGCAGCGCGACAAGCCGTCCTACACCATCGACACCCTGGAGACCGTGCGGGCGGAACTGGCGGCCGACGACCAGTTGTTCATGCTGATCGGCTGGGATGCCTTCTGCGGCTTGCCGACGTGGCACCGCTGGGAGGCGTTGCTGGATCACTGCCACATCGTCGCCTTGCAGCGTCCGGATGCCGACAGCGAGCCGCCGGAGTCGCTGCGCGACCTGCTGGCGGCGCGGAGCGTGGCCGACCCGCAGGCCCTGAAAGGGCCGGGCGGACAGATCACCTTCGTCTGGCAGACGCCGCTGGCGGTGTCCGCCACCCAGATCCGCGCGCTGCTGGGTGCCGGGCGCTCCGTGCGTTTCCTGGTGCCGGACGCGGTATTGAACTACATCGAGGCGCACCACCTGTATCGTGCGCCACACTGA SEQ ID NO.2 CATATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCACATGAGCTTACTCAATGTTCCAGCAGGTAAGGATCTGCCAGAGGACATCTATGTTGTTATTGAGATCCCTGCTAACGCAGATCCAATCAAATACGAAATCGACAAAGAAAGCGGTGCTCTGTTCGTTGACCGTTTCATGTCAACAGCTATGTTCTATCCTTGCAACTACGGTTACATCAACCACACCCTGTCTCTGGACGGAGATCCTGTTGACGTACTGGTTCCAACTCCGTACCCATTACAGCCGGGTTCTGTGATCCGTTGCCGTCCAGTTGGTGTTCTGAAAATGACTGACGAAGCAGGAGAAGATGCGAAACTGATTGCTGTTCCGCACACCAAACTGAGCAAAGAATACGATCACATTAAAGACGTAAACGATCTGCCAGAGCTGCTGAAAGCGCAGATCGCTCACTTCTTCGAGCACTACAAAGACCTCGAAAAAGGCAAGTGGGTGAAAGTTGAAGGTTGGGAAAACGCAGAAGCCGCTAAAGCTGAAATCGTTGCCTCCTTCGAGCGTGCAAAGAATAAATAACTCGAG SEQ ID NO.3 MGKRIGLFGGTFDPVHIGHMRSAVEMAEQFALDELRLLPNARPPHRETPQVSAAQRLAMVERAVAGIERLTVDPRELQRDKPSYTIDTLETVRAELAADDQLFMLIGWDAFCGLPTWHRWEALLDHCHIVALQRPDADTEPPESLRDLLAARSVADPQALKGPGGQITFVWQTPLAVSATQIRALLGAGRSVRFLVPDAVLNYIEAHHLYRAPH SEQ ID NO.4 MGSSHHHHHHSSGLVPRGSHMSLLNVPAGKDLPEDIYVVIEIPANADPIKYEIDKESGALFVDRFMSTAMFYPCNYGYINHTLSLDGDPVDVLVPTPYPLQPGSVIRCRPVGVLKMTDEAGEDAKLIAVPHTKLSKEYDHIKDVNDLPELLKAQIAHFFEHYKDLEKGKWVKVEGWENAEAAKAEIVASFERAKNK* SEQ ID No.5 Example 2 Construction of engineered strains A strain containing the recombinant vector engineering strain shown in SEQ ID NO.5 described in Example 1, the preparation process is as follows: (1) Strain construction After the recombinant vector sequencing is completely correct, it is transformed into Escherichia coli BL21 (DE3) by chemical heat shock method; take an appropriate amount of the bacterial solution after transformation and incubation to apply to LB resistance plate containing 100μg / ml ampicillin, and culture at 37℃ for 18 hours; seal with sealing film and store at 4℃ for future use; (2) Strain screening 10 single colonies were selected and inoculated into 3 ml of LB liquid culture medium containing 100 µg / ml ampicillin (10 LB liquid culture mediums), and cultured at 220 rpm and 37°C for 5 hours to obtain bacterial solution A; 1 ml of the cultured bacterial solution A was taken, and after microscopic examination, it was transferred to 50 ml of LB liquid culture medium containing 100 µg / ml ampicillin, and cultured at 220 rpm and 37°C for 5 hours to obtain bacterial solution B, which was examined under a microscope, and then IPTG was added to a final concentration of 0.1 mM, and expression was induced at 220 rpm and 28°C for 18 hours to obtain bacterial solution C, which was examined under a microscope and SDS-PAGE; The single colonies with the lowest percentage of small dots and wires in microscopic examination of bacterial solution A, B and C at three time points and normal SDS-PAGE detection were selected as the strains for subsequent enzyme preparation; the quality of BL21 (DE3) varies greatly depending on the manufacturer, so single colonies with reliable quality were obtained by liquid microscopic examination of the above three time points; Figure 3 The SDS-PAGE electrophoresis results of 4 single colonies selected from 10 single colonies after induced expression, where 1, 2, 3, and 4 are the selected colony numbers respectively; (3) Seed preparation for seed bank Take 1.5 ml of the bacterial solution A corresponding to the single colony in step (2) and transfer it to 200 ml of LB liquid medium containing 100 μg / ml ampicillin. Incubate at 37°C for 6.5 hours until the OD 600 The suspension was set to 2.0, and then aseptically transferred into a centrifuge cup, placed in an ice water bath for 20 minutes, centrifuged at 4000 rpm for 5 minutes, the supernatant discarded, 50 ml of 1 / 2 LB liquid culture medium containing 20% ​​glycerol pre-cooled with ice water was added, the suspension was evenly resuspended, and the suspension was divided into cryopreservation tubes; the divided strains were allowed to stand at 4°C for 1 hour, and then stored in a -80°C refrigerator as seeds.

[0023] Example 3 Preparation of wet cells The preparation process of wet bacteria is as follows: For shake flask fermentation, 300ul of the seeds preserved in Example 2 were inoculated into 400ml of culture medium, the composition of which was: Na 2 HPO 4 100mM, NH 4 Cl 50mM, MgSO 4 5mM, FeCl 3 0.1mM, peptone 20g / L, yeast powder 10g / L, glycerol 20g / L, defoamer 0.02%, cultured at 37℃, 220rpm for 6.5 hours. 600 When the pH value is 7.0, cool down to 28°C, add IPTG to a final concentration of 0.2 mM, induce expression for 20 hours, and then centrifuge at 5000 rpm for 20 min to collect the wet cells; the wet cells can be stored at -20°C for more than five years.

[0024] Example 4 Application of wet bacteria in the synthesis of oxidized nicotinamide adenine dinucleotide Preparation of NAD using wet bacteria + , the process is as follows: In a 1000ml three-necked flask, 300ml of purified water was added, and mechanical stirring was performed. Then, 102g (300mmol) of NMN, 160g (290mmol) of ATP, and 10.0g of magnesium chloride hexahydrate were added. After dissolution, the pH was 2.7. The pH was adjusted to 6.3 with 30% NaOH. The consumption of NaOH solution was 140ml. The temperature was adjusted to 30°C. Then, 10.0g of wet bacteria prepared in Example 3 was added to start the reaction. During the reaction, the pH value was controlled to be no higher than 6.8 with 6M HCl solution. The reaction was continued for 8 hours. HPLC detection showed that NAD + When the generation rate is greater than 96%, the reaction can be terminated.

[0025] Example 5 Preparation of double enzyme solution The preparation process of double enzyme solution is as follows: Take 20 g of the wet bacteria prepared in Example 3, add 100 ml of 20 mM pH 7.0 PBS buffer, and crush once at 300 bar and twice at 900 bar using a high-pressure homogenizer; add 1 ml of 10% polyimide, stir for 10 minutes, centrifuge at 10000 rpm for 20 minutes, take the supernatant, which is the dual enzyme solution, and store the dual enzyme solution at 4°C.

[0026] Example 6 Application of dual enzyme solution in the synthesis of oxidized nicotinamide adenine dinucleotide Preparation of NAD using dual enzyme solution + The process is as follows: In a 1000ml three-necked flask, 300ml of purified water was added, and the mixture was stirred mechanically. 102g (300mmol) of NMN, 160g (290mmol) of ATP, and 10.0g of magnesium chloride hexahydrate were added. After dissolution, the pH was 2.6. A 30% NaOH solution was used to adjust the pH to 6.3. The consumption of the NaOH solution was 139.5ml. The temperature was adjusted to 30°C. Then 40ml of the double enzyme solution prepared in Example 5 was added to start the reaction. The pH value was controlled to be no higher than 6.8 with 6M HCl. The reaction was performed for 8 hours. HPLC detection showed that NAD + When the generation rate is greater than 96%, the reaction is terminated.

[0027] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A recombinant vector, characterized in that: The gene sequence of the recombinant vector is shown in SEQ ID NO.

5.

2. The recombinant vector according to claim 1, characterized in that The preparation process is as follows: inserting the nucleic acid sequence shown in SEQ ID NO.1 between the Nco I and Hind III restriction sites of the expression vector, and inserting the nucleic acid sequence shown in SEQ ID NO.2 between the Nde I and XhoI of the expression vector to obtain a recombinant vector.

3. The recombinant vector according to claim 2, characterized in that The expression vector is pETDuet-1 or pCDFDuet-1 or pACYCDuet-1 or pRSFDuet-1.

4. An engineered strain comprising the recombinant vector shown in SEQ ID NO.5, characterized in that: The preparation process is as follows: (1) Strain construction After the recombinant vector sequencing is completely correct, transform Escherichia coli BL21 (DE3) by heat shock method; take an appropriate amount of the incubated bacterial solution to apply to LB resistance plate containing 100μg / ml ampicillin, and culture at 37℃ for 14-20 hours; seal with sealing film and store at 4℃ for future use; (2) Strain screening 8 to 10 single colonies were selected and inoculated into 3 ml of LB liquid culture medium containing 100 µg / ml ampicillin, respectively, and cultured at 220 rpm and 37°C for 4 to 5 hours to obtain bacterial solution A; 1 ml of the cultured bacterial solution A was taken, examined under a microscope, and then transferred to 50 ml of LB liquid culture medium containing 100 µg / ml ampicillin, and cultured at 220 rpm and 37°C for 4 to 5 hours to obtain bacterial solution B, examined under a microscope, and then IPTG was added to a final concentration of 0.1 mM, and expression was induced at 220 rpm and 28°C for 14 to 20 hours to obtain bacterial solution C; examined under a microscope and SDS-PAGE; The single colonies with the lowest percentage of small dots and wires in microscopic examination of bacterial solution A, B and C at three time points and normal SDS-PAGE detection were selected as the strains for subsequent enzyme preparation; the quality of BL21 (DE3) varies greatly depending on the manufacturer, so single colonies with reliable quality were obtained by liquid microscopic examination of the above three time points; (3) Seed preparation for seed bank Take 1.5 ml of the bacterial solution A corresponding to the single colony in step (2) and transfer it to 200 ml of LB liquid medium containing 100 μg / ml ampicillin. Incubate at 37°C for 6 to 7 hours until the OD 600 The pH value was 1.5-2.2, then transferred to a centrifuge cup under sterile conditions, placed in an ice water bath for 20 minutes, centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, 50 ml of 1 / 2LB liquid culture medium containing 20% ​​glycerol pre-cooled with ice water was added, the suspension was evenly resuspended, and the suspension was divided into cryopreservation tubes; the divided strains were allowed to stand at 4°C for 1 hour, and then transferred to a -80°C refrigerator for storage as seeds.

5. The engineered strain according to claim 4, characterized in that The Escherichia coli is Escherichia coli BL21 (DE3) or JM109 (DE3) or Escherichia coli derived from these two host bacteria and having DE3 function.

6. Use of the engineered strain according to claim 4 in the synthesis of oxidized nicotinamide adenine dinucleotide, characterized in that: Using wet bacteria or double enzyme solution prepared from wet bacteria as catalyst, NMN and ATP as substrates, react at 28-32°C and pH 6.3-6.8 to obtain NAD + , NAD + The generation rate is >96%; among them, the initial molar ratio of NMN to ATP is 0.95-0.

98.

7. Use of the engineered strain according to claim 6 in the synthesis of oxidized nicotinamide adenine dinucleotide, characterized in that: The preparation process of wet bacteria is as follows: For shake flask fermentation, 300ul of the preserved seeds were inoculated into 400ml of culture medium. The composition of the culture medium was: Na2HPO4 100mM, NH4Cl 50mM, MgSO4 5mM, FeCl3 0.1mM, peptone 20g / L, yeast powder 10g / L, glycerol 20g / L, defoamer 0.02%. The culture was carried out at 37℃ and 220rpm for 6-7 hours. When the OD 600 When the pH value is 6.0-8.0, cool down to 28°C, add IPTG to a final concentration of 0.1-0.2 mM, induce expression for 16-24 hours, and then centrifuge at 5000 rpm for 20 min to collect the wet cells; the wet cells are stored at -20°C.

8. Use of the engineered strain according to claim 7 in the synthesis of oxidized nicotinamide adenine dinucleotide, characterized in that: Preparation of NAD using wet bacteria + , the process is as follows: In a 1000ml three-necked flask, add 300ml of purified water, stir mechanically, then add NMN, ATP and magnesium chloride hexahydrate. After dissolution, the pH is 2.5-2.

8. Use 30% NaOH to adjust the pH to 6.3-6.8, adjust the temperature to 28-32°C, then add 10.0g of wet bacteria, start the reaction, and use 6M HCl to control the pH value to no higher than 7.0; react for 8 hours, and detect by HPLC. NAD + When the generation rate is greater than 96%, the reaction can be terminated.

9. Use of the engineered strain according to claim 6 in the synthesis of oxidized nicotinamide adenine dinucleotide, characterized in that: The preparation process of double enzyme solution is as follows: Take 20g of wet bacteria, add 100ml of 20mM pH7.0 PBS buffer, break it once at 300bar and twice at 900bar with a high-pressure homogenizer; add 1ml of 10% polyimide, stir for 10 minutes, centrifuge at 10000rpm for 20 minutes, take the supernatant, which is the double enzyme solution, and store it at 4℃.

10. Use of the engineered strain according to claim 9 in the synthesis of oxidized nicotinamide adenine dinucleotide, characterized in that: Preparation of NAD using dual enzyme solution + The process is as follows: In a 1000ml three-necked flask, add 300ml of purified water, stir mechanically, add NMN, ATP and magnesium chloride hexahydrate, after dissolution, the pH is 2.5-2.8, use 30% NaOH to adjust the pH to 6.3-6.8, adjust the temperature to 28-32℃, then add the double enzyme solution, start the reaction, and use 6M HCl to control the pH value not higher than 7.0; react for 8 hours, HPLC detection, NAD + When the generation rate is greater than 96%, the reaction is terminated.

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