Recombinant strain for producing beta-nicotinamide mononucleotide and application thereof

Abstract

The present application relates to a kind of production β-nicotinamide mononucleotide recombinant strain and its application.The recombinant strain can overexpress nicotinamide ribosyltransferase, and contain at least one promoter for expressing nicotinamide ribosyltransferase.The recombinant strain provided by the present application can be used to produce β-nicotinamide mononucleotide, with the advantages of high β-nicotinamide mononucleotide yield.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a recombinant strain for producing β-nicotinamide mononucleotide and application thereof. BACKGROUND

[0002] Nicotinamide mononucleotide (NMN, CAS: 1094-61-7) has a molecular formula of C11H15N2O8P and a molecular weight of 334.22. NMN is a nucleotide widely existing in organisms and nature, and has two different forms of α and β; β-NMN is the active form of NMN, and therefore, unless specifically indicated as α-NMN, NMN generally refers to β-NMN.

[0003] In mammals, NMN is a key precursor for the synthesis of nicotinamide adenine dinucleotide (NAD + ). There are three pathways for the synthesis of NAD + in mammals, which are de novo synthesis pathway, Preiss-Handler pathway and salvage synthesis pathway; among them, the salvage synthesis pathway is the main synthesis pathway of NAD + . In the salvage synthesis pathway, nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP) are precursors, and NMN is synthesized by nicotinamide phosphoribosyltransferase (Nampt, EC 2.4.2.12) catalysis, and NMN is converted into NAD + under the catalysis of nicotinamide mononucleotide adenyltransferase (Nmnat). Therefore, NMN is a key precursor for the synthesis of NAD + in vivo, and NMN exerts corresponding physiological functions by being converted into NAD + . Increasing the content of NAD + in vivo can improve mitochondrial energy production and redox metabolism, activate NAD + -dependent enzymes, regulate cell survival and death, prolong the lifespan of mammals, etc. NMN is an important intermediate in the synthesis pathway of NAD + , and also exerts physiological activities by being converted into NAD Figure 1 . As shown in the following formula, NAD +The in vivo synthesis pathways of NAD mainly have two kinds: de novo synthesis pathway and salvage synthesis pathway. The de novo synthesis pathway takes tryptophan (Trp) as the precursor, and first generates quinolinic acid (QA) through five-step reaction. The generated QA generates nicotinic acid mononucleotide (NAMN) under the catalysis of quinolinic acid phosphoribosyltransferase (QPRT). QPRT is the rate-limiting enzyme of the whole reaction, and the reaction depends on adenosine triphosphate (ATP), and needs to consume magnesium ions and PRPP. NAMN generates nicotinic acid adenine dinucleotide (NAAD) through the catalysis of nicotinamide mononucleotide adenyltransferase (NMNAT), and finally NAAD generates NAD under the catalysis of NAD synthetase (NADS) + . The salvage synthesis pathway of NAD + takes NAM as the precursor, and synthesizes NAD + through two-step reaction. NMN is an important intermediate substance in the salvage synthesis pathway of NAD + . First, NAM is synthesized into NMN through the catalysis of nicotinamide phosphoribosyltransferase (Nampt, EC 2.4.2.12), and this step of reaction needs to consume ATP and PRPP; second, NMN is synthesized into NAD + through the catalysis of NMN consuming enzyme (NMNATs), and one ATP is consumed. Recent researches find that increasing the content of NMN in the body of patients has effects on improving diabetes, increasing the sensitivity of islets to blood glucose, restoring the systolic function of heart, improving the cognitive impairment caused by Alzheimer's disease, improving depression, protecting the secondary brain injury caused by cerebral hemorrhage, improving the survival rate of Parkinson's patients, and improving aging diseases.

[0004] Currently, the preparation methods of NMN are divided into chemical synthesis method, enzyme synthesis method and microbial fermentation synthesis method, wherein the chemical synthesis method is mainly used. The raw materials of the chemical synthesis method can be nicotinamide, nicotinamide riboside, coenzyme I or tetraacetyl ribose. The individual raw materials of these chemical synthesis methods are expensive and difficult to obtain; the route is long, and the separation of isomers and impurities is difficult; the environmental protection investment cost is large; and most importantly, the raw materials prepared by the chemical process are not recommended by the principle of new food raw material application in China. Therefore, the chemical synthesis method limits the possibility of large-scale production of NMN. There are two technical routes for the enzyme synthesis method. One is to use D-5-phosphoribosyl and nicotinamide as raw materials to synthesize β-NMN through the whole cell catalysis of immobilized phosphoribosyl pyrophosphate synthetase (PRPPS) and nicotinamide phosphoribosyl transferase (Nampt); the other is to use ATP and nicotinamide riboside as raw materials to generate NMN under the action of nicotinamide riboside kinase (NRK). The D-5-phosphoribosyl and ATP in the raw materials of the two methods are expensive, and the enzyme activity of the existing Nampt and NRK enzymes is low, so that the enzyme synthesis method has problems of long time consumption, high cost and low yield, which limits the large-scale application. The biological fermentation method for producing NMN mainly uses the biological synthesis pathway of the microbial strain itself to produce NMN, which can overcome the limitation of expensive raw materials in the enzyme production, but the existing microbial fermentation synthesis method has defects of low β-nicotinamide mononucleotide yield and low production efficiency.

[0005] Escherichia coli is paid attention by genetic engineering experts due to the advantages of clear genetic background, simple technical operation, simple culture condition and economic large-scale fermentation. However, Escherichia coli does not have nicotinamide phosphoribosyl transferase, and cannot synthesize NMN from NAM. In addition, the introduction of exogenous genes may lead to unstable growth state of the strain, which limits the further application. SUMMARY

[0006] In view of the problems existing in the prior art, the present application provides a recombinant strain for producing β-nicotinamide mononucleotide and application thereof. The recombinant strain provided by the present application can be used for producing β-nicotinamide mononucleotide, and has the advantages of high β-nicotinamide mononucleotide yield.

[0007] The technical solution of the present application to solve the above technical problems is as follows:

[0008] The present application provides a recombinant strain for producing β-nicotinamide mononucleotide, wherein the recombinant strain is Escherichia coli, and the recombinant strain expresses a nicotinamide ribosyl transferase gene.

[0009] Preferably, the recombinant strain overexpresses the nicotinamide riboside transferase gene. The present application has no special restrictions on the method for overexpressing the nicotinamide riboside transferase gene. The vector containing the nicotinamide riboside transferase gene can be constructed, and the nicotinamide riboside transferase gene can also be inserted into the genome of the strain. In order to meet the expression needs, the recombinant strain can also include elements related to the expression of the nicotinamide riboside transferase gene, such as a basic backbone, a resistance gene marker, an operon, and the like.

[0010] The nicotinamide riboside transferase gene can be selected from one or more of the following: H. sapiens-derived HsnadV (NP_005737.1), Chitinophaga pinensis-derived CpnadV (WP_012788281.1), H. ducreyi-derived HdnadV (NP_957670.1), and M. ruber-derived RnnadV (ADD29592.1) genes.

[0011] Preferably, the sequences of the HsnadV, CpnadV, HdnadV, and RnnadV genes are optimized, respectively. The optimized nucleotide sequence of HsnadV is shown in SEQ ID NO: 1, the optimized nucleotide sequence of CpnadV is shown in SEQ ID NO: 2, the optimized nucleotide sequence of HdnadV is shown in SEQ ID NO: 3, and the optimized nucleotide sequence of RnnadV is shown in SEQ ID NO: 4.

[0012] The beneficial effects of adopting the above technical solutions include: the present application screens many nicotinamide riboside transferases of different sources, and it is unexpectedly found that the four nicotinamide riboside transferases described above can be used to produce β-nicotinamide mononucleotide, especially the Chitinophaga pinensis-derived CpnadV can obtain higher yield of β-nicotinamide mononucleotide and stable growth state. The yield of β-nicotinamide mononucleotide can be further improved after optimizing the above-mentioned nicotinamide riboside transferases.

[0013] In order to further improve the yield of NMN, a suitable regulatory sequence can be selected. Taking the construction of an expression vector containing a nicotinamide riboside transferase gene as an example, the vector contains at least one promoter for expressing the nicotinamide riboside transferase. Preferably, the vector contains at least one promoter for overexpressing the nicotinamide riboside transferase. For example: the promoter can be selected from one or a combination of several of the following: ppheL, pyhjX, pompG, umuD, pygdI, placI, pbhsA, phemP, parfA, phokD, ptonB, ppdhR, and pcysK.

[0014] Preferably, the recombinant strain is named Escherichia coli nadVcp24, and the preservation number is CGMCC No. 25415. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Haidian District, Beijing, China, on July 27, 2022. The recombinant strain is used for the production of NMN, which can further improve the yield of NMN and stabilize the growth state.

[0015] The present application provides a preparation method of the above-mentioned recombinant strain, comprising the following steps: expressing nicotinamide riboside transferase gene in Escherichia coli. For example, a vector overexpressing nicotinamide riboside transferase gene can be constructed, and the vector is transformed into Escherichia coli.

[0016] Specifically, the following methods can be used: synthesizing nicotinamide riboside transferase gene, connecting nicotinamide riboside transferase gene with expression vector (such as pET30a) through enzyme digestion, transformation and other operations, and obtaining recombinant Escherichia coli that can express nicotinamide riboside transferase after sequencing verification. At least one promoter is connected in series on the expression vector to make the nicotinamide riboside transferase gene more efficiently expressed.

[0017] The beneficial effects of adopting the above technical solutions include: the recombinant strain for producing β-nicotinamide mononucleotide is successfully prepared by using the above method.

[0018] The present application provides the application of the above-mentioned recombinant strain in the production of β-nicotinamide mononucleotide.

[0019] The present application provides a production method of β-nicotinamide mononucleotide, which uses the above-mentioned recombinant strain to ferment and produce β-nicotinamide mononucleotide.

[0020] The beneficial effects of adopting the above technical solutions include: the recombinant strain is used to ferment and produce β-nicotinamide mononucleotide, which has low raw material price, easy raw material acquisition, low cost, short time, high yield and high production efficiency, and can be applied on a large scale.

[0021] Further, the conditions of fermentation culture include 37℃ and 250rpm culture. The above-mentioned conditions are conducive to the fermentation culture of the recombinant strain.

[0022] For the vector containing lac operon, the OD 600 IPTG can be added to induce for 10h after reaching 0.4-0.6. The expression of nicotinamide riboside transferase gene can be induced by IPTG.

[0023] The method for producing the above can further include a centrifugation step to obtain the bacterial cells and the supernatant by centrifugation. The recombinant strain provided by the present application can extract β-nicotinamide mononucleotide from the bacterial cells and the supernatant after fermentation culture.

[0024] Further, the fermentation production is carried out using LB medium containing nicotinamide and glucose. The addition amount of nicotinamide can be 0.1% (mass / volume percentage), and the addition amount of glucose can be 1% (mass / volume percentage).

[0025] The beneficial effects of the above technical solutions include: the use of the above-mentioned addition amount is beneficial to improve the yield of β-nicotinamide mononucleotide. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a schematic diagram of the synthesis pathway of NAD+, wherein NA: nicotinic acid; NAPRT: nicotinic acid phosphoribosyltransferase; NAMN: nicotinic acid mononucleotide; NMNAT: nicotinamide mononucleotide adenosine transferase; NAAD: nicotinic acid adenine dinucleotide; NADS: NAD synthetase; NAD+: nicotinamide adenine dinucleotide; Trp: tryptophan; N-formylkin: N-formylkynurenine; L-kin: L-kynurenine; 3-HAA: 5-hydroxy-2-aminobenzoic acid; QA: quinolinic acid; ACMS: α-amino-β-carboxymuconate-ε-semialdehyde; ACMSD: α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase; TCA cycle: tricarboxylic acid cycle; NAM: nicotinamide; NR: nicotinamide riboside; NMN: nicotinamide mononucleotide.

[0027] Figure 2 is the result of gene expression protein of nadV-cp, nadV-hd, nadV-hs, and nadV-rn in Example 1.

[0028] Figure 3 is a standard curve for detecting the content of NMN.

[0029] Figure 4 is the result of detecting the content of NMN in the supernatant and the bacterial suspension of the four strains with Nampt genes, A is the result of detecting the content of NMN in the supernatant of the four strains with Nampt genes, and B is the result of detecting the content of NMN in the bacterial suspension of the four strains with Nampt genes.

[0030] Figure 5 is the result of gene transcriptome screening of Escherichia coli under fermentation conditions.

[0031] Figure 6 is the result of gene protein expression of nadVcp01-nadVcp13 in Example 3.

[0032] Figure 7 The results of detecting NMN content in the bacterial suspension and supernatant of recombinant E. coli nadVcp01 to recombinant E. coli nadVcp13 with single tandem promoter are shown, wherein A represents the results of detecting NMN content in the bacterial suspension, and B represents the results of detecting NMN content in the supernatant.

[0033] Figure 8 The results of expressing proteins from the genes nadVcp14 to nadVcp26 from right to left in Example 5 are shown.

[0034] Figure 9 The results of detecting NMN content in the bacterial suspension and supernatant of recombinant E. coli nadVcp14 to recombinant E. coli nadVcp26 with double tandem promoter are shown, wherein A represents the results of detecting NMN content in the bacterial suspension, and B represents the results of detecting NMN content in the supernatant. DETAILED DESCRIPTION

[0035] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and are not intended to limit the scope of the present application.

[0036] The present application provides a recombinant E. coli with high yield of β-nicotinamide mononucleotide (β-NMN), and a method for producing β-nicotinamide mononucleotide by using the recombinant E. coli. The recombinant E. coli has the following characteristics: (1) nicotinamide and glucose added in the fermentation medium can be converted to NMN by the recombinant E. coli; (2) nicotinamide phosphoribosyltransferase is overexpressed by using a plasmid; and (3) one or more promoters that can efficiently express the nicotinamide phosphoribosyltransferase gene are added in front of the nicotinamide phosphoribosyltransferase gene.

[0037] Specifically, the method can include the following steps:

[0038] (1) Screening and overexpressing efficient nicotinamide phosphoribosyltransferase gene to produce NMN

[0039] E. coli has no nicotinamide phosphoribosyltransferase and cannot synthesize NMN from NAM. Therefore, the present application introduces an exogenous nicotinamide phosphoribosyltransferase to enable E. coli cells to accumulate NMN from nicotinamide as a substrate. However, different organisms have complex and different metabolic regulation processes, and it is unknown whether the exogenous nicotinamide phosphoribosyltransferase gene can be normally or overexpressed in E. coli, whether the nicotinamide phosphoribosyltransferase gene expression can increase the yield of beta-nicotinamide mononucleotide, and whether NMN can be stably produced. The inventors have conducted a large amount of screening work on nicotinamide phosphoribosyltransferase genes in the early stage, and unexpectedly found that not all nicotinamide phosphoribosyltransferase genes can be used for the production of beta-NMN. The following four nicotinamide phosphoribosyltransferase genes can be expressed in E. coli after optimization: HsnadV (NP_005737.1) from Homo sapiens, CpnadV (WP_012788281.1) from Chitinophaga pinensis, HdnadV (NP_957670.1) from Haemophilus ducreyi, and RnnadV (ADD29592.1) from Meiothermus ruber. The nucleotide sequence of the optimized HsnadV is shown in SEQ ID NO: 1, the nucleotide sequence of the optimized CpnadV is shown in SEQ ID NO: 2, the nucleotide sequence of the optimized HdnadV is shown in SEQ ID NO: 3, and the nucleotide sequence of the optimized RnnadV is shown in SEQ ID NO: 4.

[0040] (2) Tandem high-efficiency promoters to increase the content of nicotinamide phosphoribosyltransferase

[0041] To further improve the yield of beta-NMN, the present application selects 13 genes whose transcriptional amounts in E. coli are increased under fermentation conditions compared to ordinary culture conditions through transcriptome analysis. The promoters of these 13 genes (ppheL, pyhjX, pompG, umuD, pygdI, placI, pbhsA, phemP, parfA, phokD, ptonB, ppdhR, and pcysK) are inserted into a gene vector containing Nampt, and the most efficient promoter vector is selected to increase the enzyme synthesis amount of Nampt, thereby increasing the synthesis efficiency of NMN and the yield of NMN.

[0042] (3) Tandem multiple high-efficiency promoters to increase the content of nicotinamide phosphoribosyltransferase

[0043] In order to further improve the yield of beta-NMN, 13 high-efficiency promoters (ppheL, pyhjX, pompG, pumuD, pygdI, placI, pbhsA, phemP, parfA, phokD, ptonB, ppdhR, pcysK) are inserted into the preferred promoter vector of step (2) respectively, the highest efficiency tandem double promoter vector is screened out, and the enzyme synthesis amount of Nampt is further improved by the above method, and the synthesis efficiency of NMN and the yield of NMN are increased.

[0044] The present application can produce nicotinamide mononucleotide efficiently and stably by the above method, increase the yield of NMN, solve the problem that the existing E. coli cannot produce beta-NMN, and the problems of low efficiency, low production, expensive raw materials and instability in the existing microbial fermentation production of nicotinamide mononucleotide, and achieve the green and low-cost production requirements of nicotinamide mononucleotide.

[0045] In the present application, if not specially stated, reagents, experimental materials and the like are conventional reagents, experimental materials in the art, which can be obtained by purchase or prepared by conventional methods in the art. If not specially stated, the experimental methods used in the present application are conventional experimental methods in the art.

[0046] LB medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.4.

[0047] LB solid medium: add agar powder (concentration 15-20 g / L) to the LB medium.

[0048] In the examples, the formula of the fermentation medium includes: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, glucose 10 g / L, nicotinamide 10 g / L.

[0049] In the examples, the addition amount of IPTG can be 1 mmol.

[0050] The vector pET30a, the competent cells of E. coli DH5a and the competent cells of E. coli BL21 (DE3) are purchased from Solabio Company.

[0051] The information of the constructed vectors and strains in the present application is shown in Table 1.

[0052] Table 1 Information of constructed vectors and strains

[0053]

[0054]

[0055] In the present application, nicotinamide mononucleotide (NMN) is β-nicotinamide mononucleotide (β-NMN) unless otherwise specified as α-nicotinamide mononucleotide (i.e. α-NMN).

[0056] The following is described by specific examples.

[0057] Example 1 Preparation of recombinant E. coli nadV

[0058] The present application expresses HsnadV (NP_005737.1) from Homo sapiens, CpnadV (WP_012788281.1) from Chitinophaga pinensis, HdnadV (NP_957670.1) from Haemophilus ducreyi, and RnnadV (ADD29592.1) from Meiothermus ruber, respectively. Specifically, the genes HsnadV, CpnadV, HdnadV, and RnnadV (Huada Gene) optimized for E. coli were synthesized, the nucleotide sequence of the optimized HsnadV is shown in SEQ ID NO: 1, the nucleotide sequence of the optimized CpnadV is shown in SEQ ID NO: 2, the nucleotide sequence of the optimized HdnadV is shown in SEQ ID NO: 3, and the nucleotide sequence of the optimized RnnadV is shown in SEQ ID NO: 4. The synthesized product was digested with restriction enzymes NdeI and NotI, and then ligated with the vector pET30a digested with the same restriction enzymes NdeI and NotI, and transformed into E. coli competent cells DH5α, and cultured overnight on a kanamycin-resistant LB plate (containing kanamycin 50 mg / L) inverted, and a single colony was picked for sequencing verification, and the recombinants with completely correct gene sequences were obtained, and expression vectors pNA01 to pNA04 were successfully constructed. Four kinds of single colonies with correct sequences were inoculated into kanamycin-resistant LB test tubes (containing kanamycin 50 mg / L), and cultured at 37°C, 250 rpm for 8 hours, then transferred into new kanamycin-resistant LB medium (containing kanamycin 50 mg / L), and cultured at 37°C, 250 rpm for 4 hours, and the OD600 of the culture was measured, and the cells were collected by centrifugation at 4°C, 5000 rpm for 5 minutes, and then resuspended in LB medium (containing kanamycin 50 mg / L) to a final OD600 of 0.1, and then induced with IPTG at a final concentration of 0.1 mmol / L at 37°C, 250 rpm for 10 hours, and the protein expression level was detected by SDS-PAGE, and the results are shown in FIG. 1. 600 When the OD600 reached 0.4, IPTG was added, and the culture was induced at 37°C, 250 rpm for 10 hours, and the protein expression level was detected by SDS-PAGE, and the results are shown in FIG. 2. Figure 2Marker, nadV-cp, nadV-hd, nadV-hs, nadV-rn, respectively, from right to left, as shown, which can prove that the four genes are all high-efficiency soluble expression. That is, the recombinant E. coli nadV-hs, the recombinant E. coli nadV-cp, the recombinant E. coli nadV-hd, and the recombinant E. coli nadV-rn are successfully obtained.

[0059] Example 2 Screening of high-efficiency recombinant E. coli nadV

[0060] (1) From the plate, the recombinant E. coli hadV-hs, the recombinant E. coli nadV-cp, the recombinant E. coli nadV-hd, and the recombinant E. coli nadV-rn were picked and inoculated into 10 mL kanamycin-resistant test tubes (LB medium containing kanamycin 50 mg / L), 37℃, 250 rpm, cultured for 8 hours, then transferred to new kanamycin-resistant fermentation medium (kanamycin 50 mg / L), 37℃, 250 rpm, cultured for 4 hours, OD 600 When the OD reached 0.4, IPTG was added, and the culture was induced at 37℃, 250 rpm for 10 hours, centrifuged, and the bacterial bodies and supernatant of the four kinds of recombinant E. coli were collected, respectively.

[0061] (2) After the bacterial bodies were resuspended with 10 mL pH 7.0 PBS (1 mM) buffer, the cells were broken by ultrasonic at 50% power (ultrasonic for 3 s, pause for 5 s, for 4 min), and the broken liquid was centrifuged at 10000 rpm for 10 min. The supernatant obtained by centrifugation was the bacterial suspension.

[0062] (3) The content of nicotinamide mononucleotide in the supernatant of step (1) and the bacterial suspension of step (2) was detected by fluorescence method. The detection method includes the following steps:

[0063] First, a standard curve for NMN was prepared, including the following steps: accurately weigh 2.2 mg of NMN, dissolve in 323.53 ul of pure water, fully dissolve and mix to prepare a 20 mM NMN standard solution, then gradually dilute to various concentrations, and freeze at -20℃.

[0064] Secondly, 69uL of the sample to be detected was added to the 96-well plate, 7uL of 20% acetophenone dimethyl sulfoxide and 27.7uL of 2mol / L potassium hydroxide were added; incubate on ice for 2 min; add 125uL of 88% formic acid; incubate at 37℃ for 10 min; under the excitation light of 382 nm wavelength, measure the emission light at 445 nm wavelength with ultraviolet spectrophotometer.

[0065] Figure 3The standard curve for NMN content detection, wherein the abscissa is the fluorescence intensity (unitless), and the ordinate is the NMN content (umol / L). The NMN content of the sample to be detected is calculated according to the standard curve and the emission light detection result of the above-mentioned sample to be detected. Figure 4 The NMN content detection results of the supernatant and bacterial suspension of the four Nampt gene strains, wherein Control refers to the ordinary BL21 strain without any gene transfection. It can be seen that the NMN content produced by nadV-hd is the most in the supernatant and bacterial suspension. However, it was found in further research that the data of nadV-hd in the bacterial suspension showed a large fluctuation, and it was guessed that the growth state of the BL21 transduced with HdnadV was unstable, so this strain is not suitable for development as a fermentation strain. Therefore, the recombinant Escherichia coli nadV-cp was selected as the subsequent research strain.

[0066] Example 3 Preparation of single tandem promoter recombinant Escherichia coli

[0067] The nadV-cp strain was cultured in kanamycin-resistant LB medium (kanamycin 50 mg / L) and kanamycin-resistant fermentation medium (kanamycin 50 mg / L) at 37°C, 250 rpm for 4 h, and the OD 600 When the OD reached 0.4, IPTG was added, and the induction was continued for 10 hours. The bacterial cells fermented in the two media were subjected to transcriptome analysis, and the genes with up-regulated gene expression in the fermentation medium compared with the LB medium were screened. The species identification method of transcriptome analysis used Sanger sequencing method, the RNA purity and concentration detection used NanoDrop2000 method, the RNA integrity detection used agarose gel electrophoresis method, and the RIN value detection used Agilent2100 method. The reference genome selected was Escherichia coli BL21(DE3). The experimental results are shown in Figure 5 The gene information with up-regulated gene expression in the fermentation medium compared with the LB medium is shown in Table 2, and there are 13 genes in total.

[0068] Table 2 List of genes with up-regulated transcription

[0069]

[0070] The gene sequences of 13 promoters were found on the NCBI website according to the GeneID in the above table. The promoters of the 13 genes were fully synthesized to obtain gene fragments ppheL, pyhjX, pompG, umuD, pygdI, placI, pbhsA, phemP, parfA, phokD,ptonB, ppdhR and pcysK. The 13 fragments were connected to the pNA02 vector respectively by overlap extension PCR method. The operation method of overlap extension PCR can refer to the literature: G-C Marinescu, Popescu R-G, Stoian G, et al. beta-nicotinamide mononucleotide (NMN) production in Escherichia coli [J]. SCIENTIFIC REPORTS, 2018, 8.

[0071] The primers for connecting the promoters with the vector pNA02 are shown in Table 3, wherein p27 and p28 are vector primers, and recombinant vectors pNA05 to pNA17 are obtained.

[0072] Table 3 Primers for connecting promoters with vector pNA02 (first promoter connection primers)

[0073]

[0074]

[0075] The recombinant vectors pNA05 to pNA17 were transformed into Escherichia coli competent cells BL21 (DE3) respectively, and were cultured at 37°C on kanamycin-resistant LB plates (containing kanamycin 50 mg / L) overnight, and single colonies were picked for sequencing verification to obtain recombinants with completely correct gene sequences, and expression vectors pNA05 to pNA17 were successfully constructed. The single colonies with correct sequencing were inoculated into kanamycin-resistant LB test tubes (containing kanamycin 50 mg / L), and were cultured at 37°C, 250 rpm for 8 hours, then transferred into new kanamycin-resistant LB medium, and were cultured at 37°C, 250 rpm for 4 hours, and the OD 600 was 0.4, IPTG was added, and the culture was induced at 37°C, 250 rpm for 10 hours, and the protein expression amount was detected by SDS-PAGE, and the results are shown in Figure 6 Figure, from right to left are the gene protein expression gels of nadVcp01-nadVcp13, and from the figure it can be seen that all the proteins are soluble, that is, the single tandem promoter recombinant Escherichia coli nadVcp01 to nadVcp13 are obtained.

[0076] Example 4 Selection of high-efficiency single tandem promoter recombinant E. coli

[0077] (1) The recombinant E. coli nadVcp01 to nadVcp13 were picked from the plates and inoculated into 10 mL kanamycin-resistant test tubes (LB containing kanamycin 50 mg / L) and cultured at 37°C, 250 rpm for 8 hours. Then, the culture was transferred into new kanamycin-resistant fermentation medium (containing kanamycin 50 mg / L) and cultured at 37°C, 250 rpm for 4 hours. The OD 600 When the OD reached 0.4, IPTG was added and the culture was induced at 37°C, 250 rpm for 10 hours. The cells and supernatant in the fermentation broth of the 13 recombinant E. coli were collected by centrifugation.

[0078] (2) The cells were resuspended in 10 mL PBS (1 mM) buffer at pH 7.0, and the cells were broken by ultrasonic treatment at 50% power (ultrasonic treatment for 3 s, pause for 5 s, for 6 min). The broken cells were centrifuged at 10,000 rpm for 10 min, and the supernatant obtained by centrifugation was the bacterial suspension.

[0079] (3) The content of nicotinamide mononucleotide in the supernatant of step (1) and the bacterial suspension of step (2) was detected by fluorescence method. The detection method was the same as in Example 2.

[0080] The results of detecting the content of NMN by fluorescence method are shown in Table 4 and Figure 7 . Figure 7 The results of detecting the content of NMN in the bacterial suspension and supernatant of the recombinant E. coli nadVcp01 to nadVcp13 are shown in Table 4. A is the result of detecting the content of NMN in the supernatant of step (1) by fluorescence method, and B is the result of detecting the content of NMN in the bacterial suspension of step (2) by fluorescence method. Control is the ordinary BL21 strain without any gene transfection.

[0081] As can be seen from Figure 7 , the content of NMN in the bacterial suspension of nadVcp09 is the highest, and the content of NMN in the supernatant of nadVcp11 is the highest. Because the content of NMN in the cells is significantly higher than that in the supernatant, the strain with the highest content of NMN in the cells is selected as the strain for further study, and therefore, nadVcp09 is selected as the strain for further study.

[0082] Table 4 Detection results of NMN content in the bacterial suspension and supernatant of nadVcp01 to nadVcp13

[0083]

[0084]

[0085] Example 5 Preparation of Dual Tandem Promoter Recombinant Escherichia coli

[0086] The gene fragments ppheL, pyhjX, pompG, pumuD, pygdI, placI, pbhsA, phemP, parfA, phokD, ptonB, ppdhR, and pcysK were respectively ligated into the pNA13 vector by overlap extension PCR. The primers used for ligation are shown in Table 5, where p55 and p56 are vector primers, to obtain recombinant vectors pNA18 to pNA30, respectively.

[0087] Table 5 Second promoter connection primers

[0088]

[0089] The recombinant vectors pNA18 to pNA30 were transformed into E. coli competent cells BL21 (DE3) and inverted and cultured overnight on kanamycin-resistant LB plates (containing 50 mg / L kanamycin). Single colonies were selected for sequencing verification, and recombinants with completely correct gene sequences were obtained. Expression vectors pNA18 to pNA30 were successfully constructed. Single colonies with correct sequencing were inoculated into kanamycin-resistant LB tubes (containing 50 mg / L kanamycin) and cultured at 37°C and 250 rpm for 8 hours. After that, they were transferred to new kanamycin-resistant LB medium and cultured at 37°C and 250 rpm for 4 hours. The OD 600 When the pH reached 0.4, IPTG was added and the culture was induced at 37 °C and 250 rpm for 10 h. The protein expression was detected by SDS-PAGE. Figure 8 As shown, the results showed that all proteins of the genes nadVcp14 to nadVcp26 were solublely expressed, that is, the recombinant Escherichia coli nadVcp14 to the recombinant Escherichia coli nadVcp26 of the double tandem promoter were obtained.

[0090] Example 6 Selection of efficient dual tandem promoter recombinant Escherichia coli

[0091] (1) Single clones of recombinant E. coli nadVcp14 and nadVcp26 expressing dual tandem promoters were picked from the plate and placed in a 10 mL kanamycin-resistant test tube (LB, containing 50 mg / L kanamycin). After culturing at 37°C and 250 rpm for 8 hours, the cells were transferred to a new kanamycin-resistant fermentation medium (containing 50 mg / L kanamycin, 1% glucose, and 0.1% NAM) and cultured at 37°C and 250 rpm for 4 hours. The OD 600When the OD600 reached 0.4, IPTG was added, and the culture was induced at 37°C, 250 rpm for 10 hours. The bacterial cells and supernatant in 10 mL of fermentation broth were collected by centrifugation.

[0092] (2) The bacterial cells were resuspended in 10 mL of PBS (1 mM) buffer at pH 7.0, and the cells were broken by ultrasonic treatment at 50% power (ultrasonic treatment for 3 s, pause for 5 s, 6 min). The broken cell solution was centrifuged at 10,000 rpm for 10 min, and the supernatant obtained by centrifugation was the bacterial suspension.

[0093] (3) The content of nicotinamide mononucleotide in the supernatant of step (1) and the bacterial suspension of step (2) was detected by fluorescence method. The detection method was the same as that of Example 2.

[0094] The detection results are shown in Table 6 and Figure 9 A is the content of NMN in the bacterial suspension of step (2) detected by fluorescence method, and B is the content of NMN in the supernatant of step (1) detected by fluorescence method.

[0095] Among them, control is the ordinary BL21 strain without transfection of any gene. It can be seen from Figure 9 that the NMN content of nadVcp24 is the highest in the bacterial suspension and the supernatant. Therefore, nadVcp24 is selected as the optimal recombinant fermentation strain.

[0096] Table 6 Detection results of NMN content in bacterial suspension and supernatant of nadVcp14 to nadVcp26

[0097]

[0098] The fermentation strain nadVcp24 for producing β-NMN is Escherichia coli BL21 transfected with pET30a plasmid carrying arfA and tonB double promoter CpnadV (WP_012788281.1) from Chitinophaga pinensis. Further research found that this strain has the advantages of high β-NMN yield and stable growth state. On July 27, 2022, the strain was preserved in the General Microbiological Center of China Microbial Culture Collection Management Committee (CGMCC), located at No. 1, Beichen West Road, Haidian District, Beijing, China, with a postcode of 100101. The preservation name is Escherichia coli nadVcp24, and the preservation number is CGMCC No. 25415.

[0099] The fermentation method includes the following steps:

[0100] (1) Pick a nadVcp24 monoclonal clone and culture it in LB medium containing nicotinamide and glucose. The amount of nicotinamide added can be 0.1% (mass volume percentage) and the amount of glucose added can be 1% (mass volume percentage); ferment at 37°C and 250 rpm until the OD value of the strain reaches 0. 600 When the value was between 0.4 and 0.6, 1 mM IPTG was added for induction and the fermentation was continued at 37°C and 250 rpm for 10 hours. The cells were then collected by centrifugation.

[0101] (2) After resuspending the bacteria in 10 mL of PBS (1 mM) buffer at pH 7.0, the cells were disrupted by ultrasound at 50% power (ultrasound for 3 seconds, pause for 5 seconds, and continue for 6 minutes). The disrupted liquid was centrifuged at 10,000 rpm for 10 minutes. The supernatant obtained by centrifugation was the bacterial suspension.

[0102] (3) The fluorescence method in Example 2 was used to detect the NMN production in the bacterial suspension. After testing, the NMN production in the bacterial suspension obtained in step (2) can reach 66.8 mg / L.

[0103] The method for producing NMN provided by the present invention has a moderate substrate price, easy raw material acquisition, low cost, short time, high output, and high production efficiency. It can be applied on a large scale and has significant economic benefits.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant strain for producing β-nicotinamide mononucleotide, characterized in that: The recombinant strain is named Escherichia coli nadVcp24 ( Escherichia coli nadVcp24), with the deposit number being CGMCC No.25415.

2. Use of the recombinant strain according to claim 1 in the production of β-nicotinamide mononucleotide.

3. A method for producing β-nicotinamide mononucleotide, characterized in that: The recombinant strain according to claim 1 is used to ferment and produce β-nicotinamide mononucleotide.

4. The method for producing β-nicotinamide mononucleotide according to claim 3, wherein: The fermentation culture conditions include: culturing at 37° C. and 250 rpm; and fermentation production using LB culture medium containing nicotinamide and glucose.

5. The method for producing β-nicotinamide mononucleotide according to claim 3 or 4, characterized in that: The recombinant strain includes a vector for expressing the nicotinamide ribosyltransferase gene, the vector carrying the lac operon, and when OD 600 After reaching 0.4-0.6, IPTG is added for induction for 10 hours; the process also includes the steps of centrifugation, collection of bacteria and supernatant.

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