Recombinant escherichia coli for producing nicotinamide mononucleotide as well as construction method and application of recombinant escherichia coli

Abstract

The invention provides recombinant escherichia coli for producing nicotinamide mononucleotide as well as a construction method and application of the recombinant escherichia coli. The invention provides recombinant escherichia coli for producing nicotinamide mononucleotide. The recombinant escherichia coli is used for simultaneously expressing NMN synthetase, ribose phosphate pyrophosphate kinase, nicotinamide riboside transporter, L-aspartate oxidase, quinolinic acid synthase and quinolinic acid phosphoribose transferase. The recombinant escherichia coli provided by the invention can be used for directly synthesizing nicotinamide mononucleotide by taking cheap compounds such as glucose as substrates, so that the synthesis yield of the nicotinamide mononucleotide is increased, the use of nicotinamide as the substrate is avoided, and the production cost of the nicotinamide mononucleotide is reduced.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a recombinant Escherichia coli for producing nicotinamide mononucleotide, a construction method thereof, and an application thereof. Background Art

[0002] Nicotinamide mononucleotide (NMN) is a B vitamin derivative widely found in natural foods such as vegetables, meat, and shrimp. As an important precursor of NAD+ in mammals, NMN has many biological benefits.

[0003] At present, the production methods of NMN mainly include chemical synthesis, enzymatic catalysis and fermentation. The chemical synthesis method is mature, but it has problems such as complicated synthesis route, harsh reaction conditions, difficulty in chiral separation of products, and the use of organic solvents, which leads to high product prices. The enzymatic catalysis method is safe and environmentally friendly, and the product has a single optical rotation, but it has problems such as high raw material costs, low enzyme activity and conversion rate. The fermentation method uses microorganisms to directly ferment to produce NMN. It has attracted widespread attention due to its mild reaction conditions, less pollution to the environment, and less restrictions on raw materials. The powerful productivity of microbial cell factories can maintain a stable production supply of NMN and greatly reduce production costs.

[0004] To meet market demand for NMN and achieve high-level production, researchers have developed recombinant Escherichia coli that overexpress nicotinamide phosphoribosyltransferase, the NMN transporter, and 5-phosphoribosyl-1-pyrophosphate synthetase, as well as knocking out some competing metabolic pathways, to produce NMN through fermentation. The fermented NMN yield reached 16.2 g / L (Huang et al., 2022). However, current fermentation methods for NMN production primarily use nicotinamide (NAM) as a precursor. On the one hand, NAM is primarily obtained from petrochemical raw materials; on the other hand, NAM is relatively expensive compared to bulk chemicals such as glucose and glycerol. Therefore, the development of recombinant microorganisms that can directly utilize simple carbon sources such as glucose and glycerol to produce NMN is worthy of attention.

[0005] Escherichia coli, with its clear genetic background, abundant genetic manipulation tools, and rapid growth, has become an efficient, economical, and flexible cell factory in synthetic biology research. Currently, the de novo NMN production in E. coli is low, at only 153 mg / L (Wang et al., 2024). Improving the de novo NMN production in E. coli through genetic engineering and other means is a hot topic in this field. Summary of the Invention

[0006] The present invention provides a recombinant Escherichia coli for producing nicotinamide mononucleotide, which improves the yield of nicotinamide mononucleotide de novo synthesis.

[0007] In a first aspect, the present invention provides a recombinant Escherichia coli for producing nicotinamide mononucleotide, wherein the recombinant Escherichia coli simultaneously expresses NMN synthase, ribose phosphate pyrophosphokinase, nicotinamide riboside transporter, L-aspartate oxidase, quinolinate synthase, and quinolinate phosphoribosyltransferase.

[0008] The recombinant Escherichia coli described above, wherein the NMN synthase (FtnadE) is derived from Francisella tularens is used to catalyze the synthesis of NMN from β-nicotinic acid mononucleotide (NaMN), and the NMN synthase (FtnadE) is specifically selected from any one of A11) to A14):

[0009] A11) a protein having the amino acid sequence of SEQ ID NO: 1;

[0010] A12) a protein having NMN synthase activity obtained by substituting and / or deleting and / or adding amino acid residues of the protein shown in A11);

[0011] A13) a protein having 80% or greater identity with the protein shown in A11) and having NMN synthase activity;

[0012] A14) A fusion protein obtained by fusing a protein tag to the carboxyl terminus and / or amino terminus of any of the proteins shown in A11) to A13).

[0013] The recombinant Escherichia coli as described above, wherein the ribose phosphate pyrophosphokinase (BaPRS) is derived from Bacillus myloliquefaciens and is used to catalyze the synthesis of 5-phosphoribosyl-1α-pyrophosphate (PRPP), and is specifically selected from any one of A21) to A24):

[0014] A21) a protein having the amino acid sequence of SEQ ID NO: 3;

[0015] A22) a protein having ribose phosphate pyrophosphokinase activity obtained by substituting and / or deleting and / or adding amino acid residues of the protein shown in A21);

[0016] A23) a protein having 80% or greater identity with the protein shown in A21) and having ribose phosphate pyrophosphokinase activity;

[0017] A24) A fusion protein obtained by fusing a protein tag to the carboxyl terminus and / or amino terminus of any of the proteins shown in A21) to A23).

[0018] The recombinant Escherichia coli as described above, wherein the nicotinamide riboside transporter (BmpnuC) is derived from Bacillus mycoides and is used to transport the synthesized NMN from the cytoplasm to the extracellular space, is specifically selected from any one of A31) to A34):

[0019] A31) a protein having an amino acid sequence of SEQ ID NO: 5;

[0020] A32) a protein having nicotinamide riboside transporter activity obtained by substituting and / or deleting and / or adding amino acid residues of the protein shown in A31);

[0021] A33) a protein having greater than 80% identity with the protein shown in A31) and having nicotinamide riboside transporter activity;

[0022] A34) A fusion protein obtained by fusing a protein tag to the carboxyl terminus and / or amino terminus of any of the proteins shown in A31) to A33).

[0023] The recombinant Escherichia coli as described above, wherein the L-aspartate oxidase (NadB) is derived from Escherichia coli and is used to catalyze the synthesis of iminoaspartic acid from aspartic acid, and is specifically selected from any one of A41) to A44):

[0024] A41) a protein having an amino acid sequence of SEQ ID NO: 7;

[0025] A42) a protein having L-aspartate oxidase activity obtained by substituting and / or deleting and / or adding amino acid residues of the protein shown in A41);

[0026] A43) a protein having 80% or greater identity with the protein shown in A41) and having L-aspartate oxidase activity;

[0027] A44) A fusion protein obtained by fusing a protein tag to the carboxyl terminus and / or amino terminus of any one of the proteins shown in A41) to A43).

[0028] The recombinant Escherichia coli described above, wherein the quinolinic acid synthase (NadA) is derived from Escherichia coli and is used to catalyze the synthesis of quinolinic acid from iminoaspartic acid, is specifically selected from any one of A51) to A54):

[0029] A51) a protein having an amino acid sequence of SEQ ID NO: 9;

[0030] A52) a protein having quinolinate synthase activity obtained by substituting and / or deleting and / or adding amino acid residues of the protein shown in A51);

[0031] A53) a protein having 80% or greater identity with the protein shown in A51) and having quinolinate synthase activity;

[0032] A54) A fusion protein obtained by fusing a protein tag to the carboxyl terminus and / or amino terminus of any of the proteins shown in A51) to A53).

[0033] As described above, the recombinant Escherichia coli, wherein the quinolinate phosphoribosyltransferase (NadC) is also derived from Escherichia coli and is used to catalyze the synthesis of β-nicotinic acid mononucleotide (NaMN) from quinolinic acid, is specifically selected from any one of A61) to A64):

[0034] A61) a protein having an amino acid sequence of SEQ ID NO: 11;

[0035] A62) A protein having quinolinate phosphoribosyltransferase activity obtained by substituting and / or deleting and / or adding amino acid residues of the protein shown in A61);

[0036] A63) a protein having 80% or greater identity with the protein shown in A61) and having quinolinate phosphoribosyltransferase activity;

[0037] A64) A fusion protein obtained by fusing a protein tag to the carboxyl terminus and / or amino terminus of any one of the proteins shown in A61) to A63).

[0038] The recombinant E. coli described above, wherein expressing the protein in the recombinant E. coli comprises introducing a gene encoding the protein into the E. coli, so that the gene is expressed in the E. coli to synthesize the protein. Furthermore, introducing the gene encoding the protein into the E. coli comprises constructing a recombinant expression vector comprising the gene encoding the protein.

[0039] Furthermore, the gene encoding the NMN synthase is selected from any one of B11)-B12):

[0040] B11) the nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0041] B12) A DNA molecule that is more than 80% identical to the DNA molecule defined in B11) and encodes the same protein.

[0042] Furthermore, the gene encoding the ribose phosphate pyrophosphokinase is selected from any one of B21)-B22):

[0043] B21) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 4;

[0044] B22) is a DNA molecule that is more than 80% identical to the DNA molecule defined in B21) and encodes the same protein.

[0045] Furthermore, the gene encoding the nicotinamide riboside transporter is selected from any one of B31)-B32):

[0046] B31) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 6;

[0047] B32) is a DNA molecule that is more than 80% identical to the DNA molecule defined in B31) and encodes the same protein.

[0048] Furthermore, the gene encoding the L-aspartate oxidase is selected from any one of B41)-B42):

[0049] B41) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 8;

[0050] B42) is a DNA molecule that is more than 80% identical to the DNA molecule defined in B41) and encodes the same protein.

[0051] Furthermore, the gene encoding quinolinic acid synthase is selected from any one of B51)-B52):

[0052] B51) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 10;

[0053] B52) is a DNA molecule that is more than 80% identical to the DNA molecule defined in B51) and encodes the same protein.

[0054] Furthermore, the gene encoding quinolinate phosphoribosyltransferase is selected from any one of B61)-B62):

[0055] B61) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 12;

[0056] B62) is a DNA molecule that is more than 80% identical to the DNA molecule defined in B61) and encodes the same protein.

[0057] In the present invention, greater than 80% identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0058] Furthermore, the recombinant expression vector comprising the above-mentioned protein encoding gene is preferably pRSF-FtnadE_NadC-BaPRS and pET-NadA-NadB_BmpnuC, wherein pRSF-FtnadE_NadC-BaPRS is firstly amplified using primers nadE-F and nadE-R on a pET28a recombinant vector (pET28a-FtnadE) comprising the FtnadE encoding gene, and linearized using primers RSF-fan1-F and RSF-fan1-R to obtain a linearized fragment, thereby constructing the pRSF-FtnadE vector through a one-step cloning method, and then the Escherichia coli is cloned using primers nadC-F and nadC-Ba-R. coliK-12MG1655 genome, the amplification product obtained by amplifying the pET28a recombinant vector (pET28a-BaPRS) including the BaPRS coding gene using primers BaPRS-F and primer dC_BaPRS-R, and the linearized fragment obtained by linearizing the pRSF-FtnadE vector using primers RSF-fan2-F and primer RSF-fan2-R were used to construct a recombinant vector by a one-step cloning method; pET-NadA-NadB_BmpnuC was firstly amplified by amplifying the Escherichia coli K-12MG1655 genome using primers nadA-F and primer nadA_dB-R, and the linearized fragment obtained by linearizing the Escherichia coli vector using primers nadB-F and primer dA_nadB-R. The pET-NadA-NadB vector was constructed by a one-step cloning method using the amplification product obtained by amplifying the K-12MG1655 genome and the linearized fragment obtained by linearizing pETDuet using primers ET-fan1-F and primers ET-fan1-R. Subsequently, the amplification product obtained by amplifying the pET28a recombinant vector (pET28a-BmpnuC) including the BmpnuC encoding gene using primers BmpnuC-F and primers BmpnuC-R and the linearized fragment obtained by linearizing the pET-NadA-NadB vector using primers ET-fan2-F and primers ET-fan2-R were used to construct a recombinant vector by a one-step cloning method.

[0059] The nucleotide sequences of the above primers are shown in Tables 1-4.

[0060] The recombinant Escherichia coli described above has pncC, ushA, nadR, and pncA genes in the recombinant Escherichia coli genome knocked out, wherein the GenBank number of the pncC gene is AAB60061.1, the GenBank number of the ushA gene is AAB40234.1, the GenBank number of the nadR gene is AAA97286.1, and the GenBank number of the pncA gene is AAC74838.2.

[0061] The above-mentioned gene knockout method can be performed according to conventional technical means in the field, such as CRISPR-Cas9 gene editing technology.

[0062] The recombinant E. coli described above is constructed using E. coli BL21 (DE3) as the starting strain.

[0063] A second aspect of the present invention provides a recombinant expression vector comprising at least two of the genes encoding NMN synthase, ribose phosphate pyrophosphokinase, nicotinamide riboside transporter, L-aspartate oxidase, quinolinate synthase, and quinolinate phosphoribosyltransferase.

[0064] Furthermore, the recombinant expression vector comprising the above-mentioned protein encoding gene is preferably pRSF-FtnadE_NadC-BaPRS and pET-NadA-NadB_BmpnuC, wherein pRSF-FtnadE_NadC-BaPRS is firstly amplified using primers nadE-F and nadE-R on a pET28a recombinant vector (pET28a-FtnadE) comprising the FtnadE encoding gene, and linearized using primers RSF-fan1-F and RSF-fan1-R to obtain a linearized fragment, thereby constructing the pRSF-FtnadE vector through a one-step cloning method, and then the Escherichia coli is cloned using primers nadC-F and nadC-Ba-R. coliK-12MG1655 genome, the amplification product obtained by amplifying the pET28a recombinant vector (pET28a-BaPRS) including the BaPRS coding gene using primers BaPRS-F and primer dC_BaPRS-R, and the linearized fragment obtained by linearizing the pRSF-FtnadE vector using primers RSF-fan2-F and primer RSF-fan2-R were used to construct a recombinant vector by a one-step cloning method; pET-NadA-NadB_BmpnuC was firstly amplified by amplifying the Escherichia coli K-12MG1655 genome using primers nadA-F and primer nadA_dB-R, and the linearized fragment obtained by linearizing the Escherichia coli vector using primers nadB-F and primer dA_nadB-R. The pET-NadA-NadB vector was constructed by a one-step cloning method using the amplification product obtained by amplifying the K-12MG1655 genome and the linearized fragment obtained by linearizing pETDuet using primers ET-fan1-F and primers ET-fan1-R. Subsequently, the amplification product obtained by amplifying the pET28a recombinant vector (pET28a-BmpnuC) including the BmpnuC encoding gene using primers BmpnuC-F and primers BmpnuC-R and the linearized fragment obtained by linearizing the pET-NadA-NadB vector using primers ET-fan2-F and primers ET-fan2-R were used to construct a recombinant vector by a one-step cloning method.

[0065] The nucleotide sequences of the above primers are shown in Tables 1-4.

[0066] The third aspect of the present invention provides a method for constructing any of the above-mentioned recombinant Escherichia coli, wherein the coding genes corresponding to NMN synthase, ribose phosphate pyrophosphokinase, nicotinamide riboside transporter, L-aspartate oxidase, quinolinate synthase and quinolinate phosphoribosyltransferase are introduced into Escherichia coli to obtain the recombinant Escherichia coli.

[0067] A fourth aspect of the present invention provides a product for producing nicotinamide mononucleotide, comprising any of the above-mentioned recombinant Escherichia coli.

[0068] The product as described above may be a bacterial agent.

[0069] A fifth aspect of the present invention provides use of any of the above-mentioned recombinant Escherichia coli or the above-mentioned product in the production of nicotinamide mononucleotide or its derivatives.

[0070] A sixth aspect of the present invention provides a method for producing nicotinamide mononucleotide, comprising: using a culture medium containing glucose as a fermentation medium, inoculating any of the above-mentioned recombinant Escherichia coli into the fermentation medium for fermentation, and obtaining nicotinamide mononucleotide from the fermentation product.

[0071] In the method as described above, the fermentation medium comprises at least yeast extract, tryptone, K2HPO4, KH2PO4 and glucose.

[0072] Furthermore, the concentration of the yeast extract is 24 g / L, the concentration of tryptone is 12 g / L, the concentration of K2HPO4 is 12.54 g / L, the concentration of KH2PO4 is 2.31 g / L, and the concentration of glucose is 20 g / L.

[0073] In the method described above, an inducer is added during the fermentation process to induce gene expression, the induction temperature is 28-37°C, and the concentration of the inducer is 0.2-0.6 mM; further, the induction temperature is 37°C, and the inducer concentration is 0.6 mM.

[0074] The recombinant Escherichia coli provided by the present invention can synthesize NMN by fermentation using glucose as a substrate, which not only increases the synthesis yield of NMN, but also avoids the use of NAM as a substrate, thereby reducing the production cost of NMN. While reducing costs, it also reduces the operational difficulty of the subsequent purification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Schematic diagram of the synthesis of nicotinamide mononucleotide provided in one embodiment of the present invention;

[0076] Figure 2 NMN fermentation yield of different engineered bacteria;

[0077] Figure 3 NMN fermentation yield under different culture media;

[0078] Figure 4 NMN fermentation yield under different induction conditions;

[0079] Figure 5Scale-up verification for 5L fermenter;

[0080] Figure 6 is the plasmid map of pRSF-FtnadE_NadC-BaPRS;

[0081] Figure 7 This is the plasmid map of pRSF-FtnadE-BaPRS_NadC;

[0082] Figure 8 is the plasmid map of pET-NadA-NadB_BmpnuC;

[0083] Figure 9 This is the plasmid map of pET-NadA_BmpnuC-NadB.

[0084] Figure 2-4 The data are expressed as the average of two parallel experiments. DETAILED DESCRIPTION

[0085] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments 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 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 making creative efforts shall fall within the scope of protection of the present invention.

[0086] In the present invention, a vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing the protein encoded by the inserted polynucleotide, it is called an expression vector. A vector can be introduced into a host cell through transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell.

[0087] Example 1. Construction of recombinant Escherichia coli

[0088] 1. Preparation of recombinant expression vector

[0089] The genes encoding NMN synthase (FtnadE), ribose phosphate pyrophosphokinase (BaPRS), and nicotinamide riboside transporter (BmpnuC) were synthesized by Suzhou Jinweizhi Co., Ltd. and subcloned into the pET28a vector (containing the restriction sites NdeI and XhoI), designated pET28a-FtnadE, pET28a-BaPRS, and pET28a-BmpnuC, respectively. The genes encoding L-aspartate oxidase (NadB), quinolinate synthase (NadA), and quinolinate phosphoribosyltransferase (NadC) were amplified using the genome of Escherichiacoli K-12MG1655 as a template.

[0090] PCR amplification was performed using the primers listed in Table 1-4 to obtain a target fragment including the above-mentioned protein encoding gene, wherein the PCR system is as shown in Table 1-1.

[0091] Table 1-1 PCR system

[0092]

[0093]

[0094] The target fragment PCR amplification program is shown in Table 1-2.

[0095] Table 1-2 Target fragment PCR program

[0096] step Temperature (℃) Time(s) 1 95 300 2 98 10 3 55 15 4 72 100 Return to step 2, 28 cycles 5 72 300 6 12 ∞

[0097] The vector inverse PCR amplification program is shown in Table 1-3.

[0098] Table 1-3 Vector inverse PCR program

[0099] step Temperature (℃) Time(s) 1 95 300 2 98 10 3 55 15 4 72 360 Return to step 2, 25 cycles 5 72 300 6 12 ∞

[0100] The primers used for PCR amplification are shown in Tables 1-4.

[0101] Table 1-4 Amplification primer sequence list

[0102]

[0103]

[0104] After the target fragment PCR reaction is completed, 1% (w / v) agarose gel electrophoresis is performed, and the DNA fragment of the correct size is recovered from the gel. After the vector reverse PCR reaction is completed, 1μL Dpn I is added to the system, pipetted to mix, and reacted at 37°C for 2 hours. Then, 1% (w / v) agarose gel electrophoresis is performed, and the DNA fragment of the correct size is recovered from the gel. The successfully amplified target gene is ligated using a one-step cloning method using 2×CE Mix (Nanjing Novozymes). The one-step cloning system is shown in Table 1-5. The mixture is reacted at 50°C for 10 minutes, then immediately placed on ice for 5 minutes. The mixture is then transferred to E. coli JM109 competent cells, plated onto LB plates containing kanamycin (50μg / mL) or ampicillin (100μg / mL), and incubated inverted at 37°C for 12-14 hours. Two to three single colonies were selected and transferred to 5 mL of LB liquid medium containing spectinomycin (50 μg / mL). Culture was performed at 37°C, 220 rpm for 8 to 10 hours. Plasmids were extracted and sent to Qingke for sequencing. After two one-step cloning reactions, recombinant plasmids were successfully obtained and named pRSF-FtnadE_NadC-BaPRS, pRSF-FtnadE-BaPRS_NadC, pET-NadA-NadB_BmpnuC, and pET-NadA_BmpnuC-NadB (Tables 1-6).

[0105] Among them, pRSF-FtnadE_NadC-BaPRS was first constructed by amplifying the target fragment obtained by using primers nadE-F and nadE-R to amplify pET28a-FtnadE and linearizing the pRSFDuet using primers RSF-fan1-F and RSF-fan1-R to obtain a linearized fragment, and then a recombinant vector was constructed by a one-step cloning method using primers nadC-F and nadC_Ba-R to amplify the target fragment obtained by using primers BaPRS-F and dC_BaPRS-R to amplify pET28a-BaPRS and linearize the pRSF-FtnadE vector using primers RSF-fan2-F and RSF-fan2-R. The plasmid map is shown in Figure 2. Figure 6 shown.

[0106] pRSF-FtnadE-BaPRS_NadC was constructed by first amplifying the target fragment obtained by amplifying pET28a-FtnadE using primers nadE-F and primer nadE_Ba-R, amplifying the target fragment obtained by amplifying pET28a-BaPRS using primers BaPRS-F and primer dE_BaPRS-R, and linearizing pRSFDuet using primers RSF-fan1-F and primer RSF-fan1-R to obtain a linearized fragment, thereby obtaining pRSF-FtnadE-BaPRS. Subsequently, the target fragment obtained by amplifying the Escherichia coli K-12MG1655 genome using primers nadC-F and primer nadC-R, and the linearized fragment obtained by linearizing the pRSF-FtnadE-BaPRS vector using primers RSF-fan2-F and primer RSF-fan2-R were amplified by a one-step cloning method to obtain a recombinant vector. The plasmid map is shown in FIG. Figure 7 shown.

[0107] pET-NadA-NadB_BmpnuC was constructed by first amplifying the Escherichia coli K-12MG1655 genome using primers nadA-F and nadA_dB-R to obtain the target fragment, linearizing the pETDuet vector using primers ET-fan1-F and ET-fan1-R to obtain the linearized fragment, and then using a one-step cloning method to obtain pET-NadA-NadB. Subsequently, the target fragment amplified from pET28a-BmpnuC using primers BmpnuC-F and BmpnuC-R, and the linearized fragment linearized from the pET-NadA-NadB vector using primers ET-fan2-F and ET-fan2-R were used to construct a recombinant vector. The plasmid map is shown in FIG. Figure 8 shown.

[0108] pET-NadA_BmpnuC-NadB was first constructed by amplifying the genome of Escherichia coli K-12MG1655 using primers nadA-F and nadA-R to obtain the target fragment, and linearizing the pETDuet vector using primers ET-fan1-F and ET-fan1-R to obtain the linearized fragment, thereby obtaining pET-NadA. Subsequently, a recombinant vector was constructed by amplifying the target fragment of pET28a-BmpnuC using primers BmpnuC-F and BmpnuC_dB-R, amplifying the genome of Escherichia coli K-12MG1655 using primers nadB-F and bm_nadB-R to obtain the target fragment, and linearizing the pET-NadA vector using primers ET-fan2-F and ET-fan2-R to obtain the linearized fragment, thereby obtaining the plasmid map. Figure 9 shown.

[0109] pRSF-FtnadE_NadC-BaPRS, pRSF-FtnadE-BaPRS_NadC, pET-NadA-NadB_BmpnuC, and pET-NadA_BmpnuC-NadB were electroporated into the chassis cell NT04 constructed by patent CN 118222525 A according to the combination of Tables 1-6 to obtain strains NT04PDN01, NT04PDN02, NT04PDN03, and NT04PDN04.

[0110] Table 1-5 One-step cloning system

[0111] Components Volume (μL) Target segment 1 0.5 Target segment 2 0.5 Linearized vector 4 2×CEMix 5

[0112] Table 1-6 Recombinant plasmid and recombinant bacteria information

[0113]

[0114]

[0115] 2. Shake flask fermentation of recombinant bacteria

[0116] Pick fresh NT04PDN01, NT04PDN02, NT04PDN03, and NT04PDN04 into 4 mL of LB liquid medium containing kanamycin (50 μg / mL) and ampicillin (100 μg / mL) and culture at 37°C, 220 rpm for 10 h. Transfer the bacterial solution into fresh 50 mL of LB liquid medium containing kanamycin (50 μg / mL) and ampicillin (100 μg / mL) at a 1% (v / v) inoculum volume and culture at 37°C, 220 rpm until OD 600 =1.0, IPTG was added to a final concentration of 0.2 mM, and the culture was continued at 28°C, 220 rpm for 24 h. After fermentation, 1 mL of the fermentation broth was removed and centrifuged at 14,000 rpm for 10 min. The supernatant was diluted 5-fold with ultrapure water and the NMN content was determined by HPLC.

[0117] We successfully constructed four different genetically engineered bacteria, NT04PDN01, NT04PDN02, NT04PDN03, and NT04PDN04, and investigated their ability to synthesize NMN from scratch. Figure 2 As shown in the figure, it can be seen that the NMN production of NT04PDN01 engineered bacteria is the highest, reaching 51.1 mg / L.

[0118] Example 2, Optimization of fermentation conditions

[0119] Four culture media, LB, SB, 2YT, and TB, were prepared. LB included 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 20 g / L glucose; SB included 20 g / L yeast extract, 32 g / L tryptone, 5 g / L NaCl, and 20 g / L glucose; 2YT included 10 g / L yeast extract, 16 g / L tryptone, 5 g / L NaCl, and 20 g / L glucose; TB included 24 g / L yeast extract, 12 g / L tryptone, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, and 20 g / L glucose.

[0120] The engineered bacteria NT04PD01 was inoculated into the above four culture media at the same inoculum volume, and fermented under the same conditions. After the culture was completed, the NMN production was detected. The test results are as follows: Figure 3 As shown, it can be seen that the engineered bacteria has the highest NMN production in nutrient-rich TB medium, which can reach 105.1 mg / L.

[0121] The engineered bacteria NT04PD01 was inoculated into TB culture medium, and the effects of two induction temperatures (28°C and 37°C) and three inducer concentrations (0.2mM, 0.4mM, and 0.6mM) on NMN production were tested. Figure 4 As shown in the figure, when the induction temperature is 37°C and the inducer concentration is 0.6mM, the NMN production of the engineered bacteria is the highest, reaching 231.5mg / L.

[0122] Example 3, 5L fermenter scale-up

[0123] The fermentation process was scaled up in a 5L fermenter to test the production capacity of the strain.

[0124] Pick a single colony of freshly activated NT04PDN01 engineered bacteria from the plate and place it in 200mL of LB liquid culture medium containing kanamycin (50μg / mL) and ampicillin (100μg / mL), and culture it at 30°C and 220rpm for 16h. Under flame protection, inoculate the seed liquid into a fermenter containing 2L of culture medium at a 10% (v / v) inoculation rate. The initial temperature is set to 37°C, the stirring speed is 200rpm, and the ventilation volume is 1vvm (2.2L / min). As the bacteria grow, the dissolved oxygen (DO) gradually decreases. Link DO with the stirring speed to maintain DO at around 10%. After the stirring speed is increased to 700rpm, maintain the speed unchanged. When OD 600 When the OD value reaches about 20, 0.5mM IPTG is added for induction. After induction, the glucose concentration is controlled below 2g / L, the pH is 7.0, and the DO is 10%-20%. Samples are taken every two hours to detect the OD value of the bacteria. 600 and NMN concentration.

[0125] We scaled up the fermentation process in a 5L fermenter to test the production capacity of the strain. The results are as follows: Figure 5 As shown in the figure, within 30 hours after induction, the production of NMN basically increased at a constant rate, but then the bacterial concentration began to drop rapidly, and the NMN synthesis rate slowed down. Finally, after 48 hours of fermentation, the production of NMN reached 1.87 g / L, which was 8.1 times the production in the shake flask.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant Escherichia coli for producing nicotinamide mononucleotide, characterized in that: The recombinant Escherichia coli simultaneously expresses NMN synthase, ribose phosphate pyrophosphokinase, nicotinamide riboside transporter, L-aspartate oxidase, quinolinate synthase and quinolinate phosphoribosyltransferase.

2. The recombinant Escherichia coli according to claim 1, characterized in that The NMN synthase is derived from Francisella tularensisa; and / or, the ribose phosphate pyrophosphokinase is derived from Bacillus myloliquefaciens; and / or, the nicotinamide riboside transporter is derived from Bacillus mycoides; and / or, the L-aspartate oxidase, quinolinate synthase and quinolinate phosphoribosyltransferase are all derived from Escherichiacoli.

3. The recombinant Escherichia coli according to claim 1, characterized in that The pncC, ushA, nadR and pncA genes in the recombinant Escherichia coli genome are knocked out.

4. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli was constructed using E. coli BL21 (DE3) as the starting strain.

5. A recombinant expression vector, characterized in that: Including at least two of the genes encoding NMN synthase, ribose phosphate pyrophosphokinase, nicotinamide riboside transporter, L-aspartate oxidase, quinolinate synthase and quinolinate phosphoribosyltransferase.

6. A method for constructing the recombinant Escherichia coli according to any one of claims 1 to 4, characterized in that: The coding genes corresponding to NMN synthase, ribose phosphate pyrophosphokinase, nicotinamide riboside transporter, L-aspartate oxidase, quinolinate synthase and quinolinate phosphoribosyltransferase are introduced into Escherichia coli to obtain the recombinant Escherichia coli.

7. A product for producing nicotinamide mononucleotide, characterized in that: The invention comprises the recombinant Escherichia coli according to any one of claims 1 to 4.

8. Use of the recombinant Escherichia coli according to any one of claims 1 to 4 or the product according to claim 7 in the production of nicotinamide mononucleotide or its derivatives.

9. A method for producing nicotinamide mononucleotide, characterized in that: include: A culture medium containing glucose is used as a fermentation medium, the recombinant Escherichia coli according to any one of claims 1 to 4 is inoculated into the fermentation medium for fermentation, and nicotinamide mononucleotide is obtained from the fermentation product.

10. The method according to claim 9, characterized in that The fermentation medium at least comprises yeast extract, tryptone, K2HPO4, KH2PO4 and glucose.

11. The method according to claim 9, characterized in that During the fermentation process, an inducer is added to induce gene expression. The induction temperature is 28-37° C. and the concentration of the inducer is 0.2-0.6 mM.

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