Recombinant bacterium for producing nicotinamide mononucleotide as well as preparation method and application of recombinant bacterium

Abstract

The invention discloses a recombinant bacterium for producing nicotinamide mononucleotide as well as a preparation method and application of the recombinant bacterium. According to the invention, a recombinant bacterium capable of producing nicotinamide mononucleotide by using nicotinamide and xylose is obtained by transforming a gene sequence for coding nicotinamide adenylate transferase, constructing recombinant plasmids and transferring the recombinant plasmids into host bacteria. On the basis, coding genes of enzymes related to nicotinamide mononucleotide synthesis are inserted, genes related to a 5-phosphoribose-1-pyrophosphate competition bypass pathway, a carbon flow competition bypass pathway and a nicotinamide mononucleotide downstream metabolic decomposition pathway in host bacteria are knocked out, and catalytic system conditions are optimized; the efficiency of producing nicotinamide mononucleotide by using nicotinamide and xylose through the constructed recombinant bacteria is further improved, and the whole-cell catalysis yield of the recombinant bacteria reaches up to 760 mg / L. The method not only is beneficial to industrial production of nicotinamide mononucleotide, but also has the advantages of simplicity in operation, low cost and the like.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and more specifically, relates to a recombinant bacterium producing nicotinamide mononucleotide, and a preparation method and application thereof. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD + ) is an important regulatory coenzyme in organisms, involved in regulating energy metabolism, cell apoptosis and proliferation. Nicotinamide mononucleotide (NMN) is NAD + Supplementing NMN can effectively increase NAD + level and improve body functions. Existing studies have found that supplementing NMN has a good preventive, therapeutic and repairing effect on the body's heart and brain tissue damage, metabolic diseases, aging and related aging degenerative diseases, and is expected to be used to treat obesity-related liver diseases, Parkinson's and Alzheimer's diseases, and improve tissue function. As people's awareness of the health effects of NMN increases, the market demand for NMN is also increasing day by day.

[0003] At present, there are two main methods for synthesizing NMN: chemical synthesis and biosynthesis. Among them, the chemical synthesis route is complex, the reagents are expensive, and the total yield is relatively low; while the biosynthesis method is green and environmentally friendly, and the raw materials are easily available. Therefore, in recent years, the biosynthesis method for producing NMN has gradually received attention. There are three main metabolic synthesis pathways for NMN; one is NAD + The first is the salvage pathway, which uses 5-phosphoribosyl-1-pyrophosphate (PRPP) and nicotinamide (NAM) as substrates to generate NMN through nicotinamide phosphoribosyltransferase (Nampt, EC2.4.2.12); the second is the NAD+ alternative salvage pathway, which uses nicotinamide ribokinase (EC 2.7.1.173) to catalyze nicotinamide ribose (NR) and ATP to generate NMN; the third is the Preiss-Handler pathway, which uses phosphoribosyltransferase (NaPRT) to catalyze PRPP to synthesize nicotinic acid mononucleotide (NaMN). However, PRPP and NR are both relatively expensive intermediates, and it is too costly to synthesize NMN directly as substrates.

[0004] In addition to the above-mentioned NMN metabolic synthesis pathway, there is also a method for synthesizing NMN using enzymes such as nicotinamide phosphoribosyltransferase and ribose phosphate pyrophosphokinase, using nicotinamide, xylose and ATP as raw materials. Although this method avoids relatively expensive intermediates such as PRPP and NR, it requires the use of ATP, which is also a relatively expensive raw material, and requires the addition of a variety of commercial enzymes. In addition, based on the existing method of biosynthesizing NMN, the enzyme required for synthesizing NMN can be inserted into the engineered bacteria to construct a recombinant bacteria that produces NMN. However, when constructing such recombinant bacteria, it is not possible to significantly increase the NMN production by arbitrarily selecting the required enzyme and inserting it into the engineered bacteria. The enzyme coding gene needs to be selected or modified, and the genetic background of the engineered bacteria needs to be modified, etc., in order to obtain a recombinant bacteria that can efficiently synthesize NMN. However, there is no report on recombinant bacteria that synthesize NMN using nicotinamide and xylose and their preparation methods. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a recombinant bacterium producing nicotinamide mononucleotide and a preparation method thereof.

[0006] The first object of the present invention is to provide a gene encoding nicotinamide adenylate transferase.

[0007] The second object of the present invention is to provide a recombinant plasmid for expressing nicotinamide adenylate transferase.

[0008] The third object of the present invention is to provide a recombinant bacterium that produces nicotinamide mononucleotide.

[0009] The fourth object of the present invention is to provide a method for preparing a recombinant bacterium that produces nicotinamide mononucleotide.

[0010] The fifth object of the present invention is to provide the use of the gene, the recombinant plasmid or the recombinant bacteria in producing nicotinamide mononucleotide or in preparing a preparation for producing nicotinamide mononucleotide.

[0011] The sixth object of the present invention is to provide a method for producing nicotinamide mononucleotide using the recombinant bacteria.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The present invention modifies the coding gene of nicotinamide adenylate transferase from Chitinophaga pinensis (the modified sequence is shown in SEQ ID NO.1) and constructs the corresponding recombinant bacteria. It is found that the obtained recombinant bacteria can not only synthesize nicotinamide mononucleotide using nicotinamide and xylose, but also its yield is significantly improved compared with the recombinant bacteria constructed with the sequence before modification. Therefore, the present invention requests protection of the modified sequence and its application.

[0014] The present invention provides a gene encoding nicotinamide adenylate transferase, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] The present invention also provides a recombinant plasmid for expressing nicotinamide adenylate transferase, wherein the recombinant plasmid contains the gene sequence shown in SEQ ID NO.1.

[0016] As an optional embodiment, the plasmid used to construct the recombinant plasmid of the present invention is pRSFDuet-1.

[0017] The present invention also provides a recombinant bacterium producing nicotinamide mononucleotide, wherein the recombinant bacterium expresses the gene shown in SEQ ID NO.1.

[0018] Optionally, the method for expressing the gene shown in SEQ ID NO.1 is: constructing a recombinant plasmid containing the gene sequence shown in SEQ ID NO.1 and transferring it into a host bacterium (starting strain).

[0019] The present invention also provides a recombinant bacterium producing nicotinamide mononucleotide, which, in addition to expressing the gene shown in SEQ ID NO.1, also expresses genes encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosylpyrophosphate synthetase.

[0020] Specifically, the ribose-5-phosphate isomerase of the present invention includes RipA and RipB.

[0021] Specifically, the amino acid sequence of the xylose isomerase of the present invention is shown in SEQ ID NO.8, the amino acid sequence of the xylulokinase is shown in SEQ ID NO.9, the amino acid sequence of the ribulose-3-phosphate isomerase is shown in SEQ ID NO.10, the amino acid sequence of the ribose-5-phosphate isomerase RipA is shown in SEQ ID NO.11, the amino acid sequence of the ribose-5-phosphate isomerase RipB is shown in SEQ ID NO.12, and the amino acid sequence of the phosphoribosyl pyrophosphate synthetase is shown in SEQ ID NO.13.

[0022] If the recombinant bacteria, in addition to expressing the gene shown in SEQ ID NO.1, also express genes encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosyl pyrophosphate synthetase, the efficiency and yield of the recombinant bacteria in producing nicotinamide mononucleotide using nicotinamide and xylose will be further improved.

[0023] Optionally, the gene sequence encoding xylose isomerase is shown as SEQ ID NO.2; the gene sequence encoding xylulokinase is shown as SEQ ID NO.3; the gene sequence encoding ribulose-3-phosphate isomerase is shown as SEQ ID NO.4; the gene sequence encoding ribose-5-phosphate isomerase RipA is shown as SEQ ID NO.5; the gene sequence encoding ribose-5-phosphate isomerase RipB is shown as SEQ ID NO.6; the gene sequence encoding ribose pyrophosphate synthetase is shown as SEQ ID NO.7.

[0024] The above gene sequence is only one of the optional embodiments provided by the present invention. On the basis of the amino acid and gene sequences provided by the present invention, other gene sequences similar to the gene sequences described in the present invention (SEQ ID NO. 2 to 7) can be derived. By constructing a recombinant plasmid and transferring it into a host bacterium, the expression of genes encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosyl pyrophosphate synthetase can be achieved, thereby achieving the same technical effect as the present invention.

[0025] The present invention also provides a recombinant plasmid for expressing nicotinamide adenylate transferase, xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosyl pyrophosphate synthetase, wherein the plasmid contains gene sequences encoding nicotinamide adenylate transferase, xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosyl pyrophosphate synthetase.

[0026] Specifically, the ribose-5-phosphate isomerase includes RipA and RipB.

[0027] Optionally, the gene sequence encoding xylose isomerase is shown as SEQ ID NO.2; the gene sequence encoding xylulokinase is shown as SEQ ID NO.3; the gene sequence encoding ribulose-3-phosphate isomerase is shown as SEQ ID NO.4; the gene sequence encoding ribose-5-phosphate isomerase RipA is shown as SEQ ID NO.5; the gene sequence encoding ribose-5-phosphate isomerase RipB is shown as SEQ ID NO.6; the gene sequence encoding ribose pyrophosphate synthetase is shown as SEQ ID NO.7.

[0028] The present invention also provides a recombinant bacterium producing nicotinamide mononucleotide, wherein the recombinant bacterium, on the basis of expressing the gene shown in SEQ ID NO.1, has any one or several of the genes related to the PRPP competition bypass pathway, the carbon flow competition bypass pathway and the NMN downstream metabolic decomposition pathway of the recombinant bacterium knocked out; or on the basis of expressing the gene shown in SEQ ID NO.1 and the gene encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosyl pyrophosphate synthetase, has any one or several of the genes related to the PRPP competition bypass pathway, the carbon flow competition bypass pathway and the NMN downstream metabolic decomposition pathway of the recombinant bacterium knocked out, thereby transforming the chassis cells. The efficiency and yield of the recombinant bacterium producing nicotinamide mononucleotide using nicotinamide and xylose are further improved.

[0029] Specifically, the PRPP competitive bypass pathway includes the ATP phosphoribosyltransferase encoding gene hisG, the adenine phosphoribosyltransferase encoding gene apt and the hypoxanthine guanine phosphoribosyltransferase encoding gene hpt; the carbon flow competitive bypass pathway includes the transketolase 2 encoding gene tktB and the transaldolase encoding genes talA and talB; the NMN (nicotinamide mononucleotide) downstream metabolic decomposition pathway includes the NMN aminohydrolase encoding gene pncC, the regulation of NAD + (nicotinamide adenine dinucleotide) derivative gene nadR.

[0030] Optionally, the accession number of the gene hisG is WP_005128078.1; the accession number of the gene apt is WP_005125198.1; the accession number of the gene hpt is WP_004204384.1; the accession number of the gene tktB is NP_416960.1; the accession number of the gene talA is NP_311353.1; the accession number of the gene talB is NP_308035.1; the accession number of the gene pncC is NP_417180.1; and the accession number of the gene nadR is NP_418807.4.

[0031] Optionally, the recombinant bacteria are based on Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Pichia pastoris as starting strains. In the embodiment of the present invention, Escherichia coli is used as the starting strain.

[0032] Specifically, the Escherichia coli is a BL21 (DE3) strain.

[0033] The present invention also provides a method for preparing a recombinant bacterium producing nicotinamide mononucleotide, comprising scheme 1: constructing a recombinant plasmid containing the sequence shown in SEQ ID NO.1 and transferring it into a starting strain to obtain a recombinant bacterium;

[0034] Or scheme 2: construct a recombinant plasmid containing the sequence shown in SEQ ID NO.1 and the gene sequences encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosyl pyrophosphate synthetase and transfer it into the starting strain to obtain a recombinant bacterium;

[0035] Or scheme three: based on the recombinant bacteria obtained in scheme one or scheme two, knock out any one or any several of the recombinant bacteria's ATP phosphoribosyltransferase encoding gene hisG, adenine phosphoribosyltransferase encoding gene apt, hypoxanthine guanine phosphoribosyltransferase encoding gene hpt, carbon flow competition bypass pathway including transketolase 2 encoding gene tktB, transaldolase encoding genes talA, talB, nicotinamide adenylate aminohydrolase encoding gene pncC, and nicotinamide adenine dinucleotide derivative encoding gene nadR.

[0036] The present invention also claims to protect the use of the gene, the recombinant plasmid or the recombinant bacteria in producing nicotinamide mononucleotide or in preparing a preparation for producing nicotinamide mononucleotide.

[0037] The present invention also claims a method for producing nicotinamide mononucleotide using the recombinant bacteria, characterized in that it comprises the following steps:

[0038] S1. Preparation of whole-cell catalyst: First, culture the recombinant bacteria to OD 600 is 0.6 to 0.8, then adding isopropyl-β-D-thiogalactopyranoside (IPTG) for induction culture, collecting the cells after induction culture, resuspending and washing the cells with phosphate buffer to obtain whole-cell catalyst;

[0039] S2. Add the whole cell catalyst obtained in step S1, nicotinamide and xylose into the catalytic system for reaction.

[0040] The recombinant bacteria of the present invention need to be induced by IPTG to produce enzymes.

[0041] Specifically, the final concentration of IPTG is 0.1-1.5 mmol / L.

[0042] More specifically, the final concentration of IPTG is 1 mmol / L.

[0043] Specifically, the culture temperature of the recombinant bacteria is 16-37°C; the induction culture temperature is 10-30°C.

[0044] More specifically, the induction culture temperature of the recombinant bacteria is 25°C.

[0045] Specifically, the induction culture time of the recombinant bacteria is 5 to 20 hours.

[0046] More specifically, the induction culture time of the recombinant bacteria is 8 to 12 hours.

[0047] Specifically, the pH of the phosphate buffer is 7.2.

[0048] Specifically, the method for resuspending and washing the bacteria is as follows: centrifuging the bacterial solution after induction culture at 4°C and 8000rpm for 10 minutes to collect the bacteria; resuspending the bacteria with pH 7.2 phosphate buffer, centrifuging at 4°C and 8000rpm for 10 minutes to collect the bacteria; repeating this step.

[0049] Specifically, the catalytic system is LB culture medium or phosphate buffer.

[0050] Specifically, the reaction temperature of the catalytic system is 16-37°C.

[0051] Specifically, in the catalytic system, the final concentration of xylose is in the range of 0.5 to 20 g / L; the final concentration of nicotinamide is in the range of 0.5 to 20 g / L; the OD of the whole cell catalyst is 600 It ranges from 1 to 30.

[0052] The present invention also provides a method for producing nicotinamide mononucleotide using whole-cell catalysis on a tank, the method comprising: transferring the secondary seed solution of the recombinant bacteria to a 5L fermentation tank at a 10% (V / V) inoculation rate, the initial culture temperature being 37°C, the pH being controlled at 7.0±0.2, the ventilation being set to 1vvm, the rotation speed being automatically adjusted in association with the dissolved oxygen content, the dissolved oxygen content being maintained at 10-30%, the liquid volume being 3L, and the bacteria being cultured to an OD of 600 When the pH value was 5, IPTG with a final concentration of 1 mmol / L was added for induction. At the same time, the fermentation tank was slowly lowered to the induction temperature (25°C), the feeding rate was 20 mL / h, and the tank was fed with 3-fold concentrated fermentation medium.

[0053] Specifically, the preparation method of the secondary seed solution is: transfer the primary seed solution to 50 mL of liquid LB culture medium at a 2% inoculation rate, and culture in a shaking incubator at 37° C. and 220 rpm for 8 to 10 hours.

[0054] Specifically, the preparation method of the first-level seed liquid is as follows: aspirate an appropriate amount of bacterial liquid, streak on a solid LB plate to ensure that a single colony can be isolated, invert the solid LB plate in a 37°C incubator and culture for 12 hours, pick a single colony in 10 mL of liquid LB culture medium, and culture it in a shaker at 37°C and 220 rpm for 12 hours.

[0055] The present invention has the following beneficial effects:

[0056] The present invention obtains a recombinant bacterium that can produce nicotinamide mononucleotide using nicotinamide and xylose by modifying the gene sequence encoding nicotinamide adenylate transferase, constructing a recombinant plasmid and transferring it into a host bacterium. On this basis, the present invention further improves the efficiency of the constructed recombinant bacteria in producing nicotinamide mononucleotide using nicotinamide and xylose by inserting the coding gene of the enzyme related to nicotinamide mononucleotide synthesis, knocking out the genes related to the 5-phosphoribosyl-1-pyrophosphate competition bypass pathway, the carbon flow competition bypass pathway, and the downstream metabolic decomposition pathway of nicotinamide mononucleotide in the host bacteria, and optimizing the catalytic system conditions, so that the whole cell catalytic yield is as high as 760 mg / L. The present invention not only contributes to the industrial production of nicotinamide mononucleotide, but also has the advantages of simple operation and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the plasmid map of the recombinant plasmid pRSFDuet-nampt.

[0058] Figure 2 The recombinant plasmid pRSFDuet-xylA-xylB-rpe-rpiA-rpiB-prs-P T7 -nampt (pRSFDuet-xylA→nampt) plasmid map.

[0059] Figure 3 Figure 1 is the SDS-PAGE profile of nicotinamide phosphoribosyltransferase; M in the figure is a protein standard marker; 1 is the supernatant of the E. coli BL21 (DE3) strain transformed with the pRSFDuet-1 plasmid; 2 is the supernatant of the E. coli BL21 (DE3) strain transformed with the pET-28a plasmid; 3 is the supernatant of the E. coli BL21 (DE3) strain transformed with the pRSFDuet-nampt plasmid.

[0060] Figure 4 : is the SDS-PAGE map of the xylose pathway; in the figure, M is a protein standard marker; 1 is the supernatant of the E. coli BL21 (DE3) strain transformed with the pRSFDuet-1 plasmid; 2 is the supernatant of the E. coli BL21 (DE3) strain transformed with the pRSFDuet-xylA→nampt plasmid.

[0061] Figure 5The figures are the results of NMN production determination of different recombinant Escherichia coli strains constructed; Figure 1 is the result of NMN production determination of BL21(DE3) / pRSFDuet-nampt; Figure 2 is the result of NMN production determination of BL21(DE3) / pRSFDuet-xylA→nampt; Figure 3 is the result of NMN production determination of BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt; Figure 4 is the result of NMN production determination on BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt5 L tank after optimized conditions.

[0062] Figure 6 This is a time curve of NMN production in the fermentation tank described in Example 4. DETAILED DESCRIPTION

[0063] The present invention is further described below in conjunction with specific examples and accompanying drawings, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0064] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0065] Example 1

[0066] Nicotinamide adenylate transferase (Nampt) is a key enzyme for NMN synthesis. The present invention transforms the coding gene of Nampt from Chitinophaga pinensis (gene accession number CP001699.1) and constructs the corresponding recombinant bacteria, so that the yield of NMN synthesized by nicotinamide and xylose is significantly improved compared with the recombinant bacteria constructed by using the gene sequence before the transformation. The coding gene of Nampt after the transformation of the present invention is shown in SEQ ID NO.1.

[0067] The process of constructing a recombinant bacterium using the modified Nampt coding gene and performing whole-cell catalytic synthesis of NMN is as follows:

[0068] (1) Amplification of the Nampt encoding gene

[0069] The coding sequence of Nampt from Chitinophaga spp. (shown in SEQ ID NO.1) was used as a template for PCR amplification. The primers (P1 / P2) used for amplification are as follows:

[0070] Primer P1: 5′-TAAGAAGGAGATATACATATGATGACTAAGGAGAATCTGA TTTTGCTG-3′

[0071] Primer P2: 5'-TTTACCAGACTCGAGGGTACCTTAGATTGTGGCATTCTTG CGA-3'

[0072] PCR amplification reaction system (40 μL): 20 μL Max, 17 μL ddH2O, 1 μL template DNA, 1 μL upstream and downstream primers (10 μmol / L) each. The reaction conditions were: 95°C pre-denaturation for 3 min, then cycle: 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles in total; 72°C final extension for 5 min. The PCR product was identified by nucleic acid electrophoresis and purified by gel cutting.

[0073] (2) Construction of recombinant plasmid pRSFDuet-nampt

[0074] The pRSFDuet-1 plasmid and the PCR product amplified in (1) were double-digested with Nde I and Kpn I, respectively. After digestion at 37°C for 1 hour, nucleic acid electrophoresis was performed for verification and gel-cutting recovery and purification were performed. The linearized plasmid and the recovered fragment after digestion were connected with the homologous recombinase ClonExpress MultiS at 37°C for 30 minutes. The connection product was transformed into E. coli JM 109 competent cells and spread on solid LB medium (containing 50 mg / L kanamycin sulfate (Kanr)), cultured overnight, and multiple transformants were picked to liquid LB medium (50 mg / L Kanr), cultured at 37°C and 220 rpm for 10 to 12 hours, and PCR verification was performed after plasmid extraction.

[0075] (3) Transformation of recombinant plasmid into E. coli BL21(DE3)

[0076] The plasmid extracted in step (2) was sent to a sequencing company for sequence determination. The recombinant plasmid with the correct sequence was named pRSFDuet-nampt. The plasmid map of pRSFDuet-nampt is shown in Figure 1 As shown; the recombinant plasmid pRSFDuet-nampt was transformed into E.coli BL21(DE3), and the recombinant bacteria E.coli BL21(DE3) / pRSFDuet-nampt were obtained by PCR identification.

[0077] The present invention also used SDS-PAGE to detect the supernatant of E. coli BL21 (DE3) transformed with pRSFDuet-1 plasmid, pET-28a plasmid and pRSFDuet-nampt recombinant plasmid. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that the present invention successfully expressed the modified Nampt in E. coli BL21 (DE3).

[0078] (4) Whole-cell catalytic synthesis of NMN

[0079] The primary seed solution of E. coli BL21(DE3) / pRSFDuet-nampt recombinant bacteria was transferred to 50 mL fermentation medium (LB liquid medium containing 10.0 g / L peptone, 5.0 g / L yeast powder and 5.0 g / L NaCl) at a 2% (v / v) inoculation volume and cultured at 37°C until OD 600 When the concentration of IPTG was 0.6 to 0.8, IPTG was added at a final concentration of 1 mmol / L, and the cells were induced at 25°C for 8 to 12 hours. After the induction culture, the bacterial solution was transferred to a 50 mL centrifuge tube, centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the cells were collected. 10 mL of phosphate buffer with a pH of 7.2 was added to resuspend the cells, and the cells were centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was discarded. This step is to wash the cells. The washing was repeated once, and an appropriate amount of phosphate buffer was added to resuspend the cells to obtain a whole-cell catalyst. The whole-cell catalyst was added to the catalytic system (LB medium) (the amount added was: the OD of the system). 600 15), and nicotinamide and xylose were added at a final concentration of 5 g / L, respectively. The NMN production was measured after reacting at 30°C and 220 rpm for 5 hours. The NMN production was measured to be 21 mg / L, which was 71.3% higher than the production of the recombinant bacteria constructed using the Nampt coding gene (CP001699.1) before the transformation.

[0080] The preparation method of the primary seed liquid is as follows: aspirate an appropriate amount of bacterial liquid, draw a line on a solid LB plate to ensure that a single colony can be isolated, invert the solid LB plate in a 37°C incubator for 12 hours, pick a single colony in a 50mL small shake flask (containing 10mL of liquid LB culture medium), and culture it in a shaker at 37°C and 220rpm for 12 hours.

[0081] The method for determining NMN production is as follows: take 1 mL of the reaction solution, centrifuge and take the supernatant as the reaction supernatant; wash the bacteria with PBS and then ultrasonically disrupt them, and take the supernatant after centrifugation as the intracellular supernatant; filter the sample through a filter membrane with a pore size of 0.22 μm before measurement, and then detect it with high performance liquid chromatography.

[0082] The liquid chromatography column was Diamonsil 5μL C18250x4.6 mm; mobile phase A: 100% methanol, mobile phase B: 5 mmol / L tetrabutyl sodium hydroxide in 5% methanol aqueous solution; the flow rate was 0.7 mL / min; the detection wavelength was 254 nm, the injection volume was 10 μL, and the column temperature was 30°C.

[0083] Elution scheme: 0-20 min, 100% mobile phase A; 20-25 min, 40% mobile phase A and 60% mobile phase B; 25-35 min, 20% mobile phase A and 80% mobile phase B; 35-40 min, 100% mobile phase A.

[0084] Example 2

[0085] In order to further improve the efficiency of the recombinant bacteria obtained in Example 1 in synthesizing NMN using nicotinamide and xylose, the present invention, based on the recombinant plasmid pRSFDuet-nampt constructed in Example 1, sequentially inserted gene sequences encoding xylose isomerase (XylA), xylulokinase (XylB), ribulose-3-phosphate isomerase (Rpe), ribose-5-phosphate isomerase (RipA, RipB) and phosphoribosyl pyrophosphate synthetase (PRPP synthase, Prs) and a T7 promoter before the gene sequence encoding Nampt. The amino acid sequence of xylose isomerase is shown in SEQ ID NO.8, the amino acid sequence of xylulokinase is shown in SEQ ID NO.9, the amino acid sequence of ribulose-3-phosphate isomerase is shown in SEQ ID NO.10, the amino acid sequence of ribose-5-phosphate isomerase RipA is shown in SEQ ID NO.11, the amino acid sequence of ribose-5-phosphate isomerase RipB is shown in SEQ ID NO.12, and the amino acid sequence of phosphoribosyl pyrophosphate synthetase is shown in SEQ ID NO.13.

[0086] The gene sequence encoding the XylA used in this example is shown in SEQ ID NO.2; the gene sequence encoding the XylB is shown in SEQ ID NO.3; the gene sequence encoding the Rpe is shown in SEQ ID NO.4; the gene sequence encoding the RipA is shown in SEQ ID NO.5; the gene sequence encoding the RipB is shown in SEQ ID NO.6; and the gene sequence encoding the Prs is shown in SEQ ID NO.7.

[0087] The process of using the above enzyme to construct a recombinant bacterium and perform whole-cell catalytic synthesis of NMN is as follows:

[0088] (1) Amplification of the gene sequences encoding each enzyme

[0089] Using the genomic DNA of E. coli BL21 (DE3) as a template, primers were used to amplify the gene sequences encoding xylose isomerase (P3 / P4), xylulose kinase (P5 / P6), ribulose-3-phosphate isomerase (P7 / P8), ribose-5-phosphate isomerase (RpiA: P9 / P10; RpiB: P11 / P12), and PRPP synthase (P13 / P14). The sequences of primers P3 to P14 are shown below:

[0090] Primer P3: 5'-TCATCACCACAGCCAGGATCCATGCAAGCCTATTTTGAC CAGC-3'

[0091] Primer P4: 5'-cccgatatacatggtatatctccttttatttgtcgaacagataatg-3'

[0092] Primer P5: 5′-TTCGACAAATAAAAGGAGATATACCATGTATATCGGGATAG ATCTT-3′

[0093] Primer P6: 5'-ATACTGTTTCATGGTATATCTCCTTTTACGCCATTAATGGCA GAAG-3'

[0094] Primer P7: 5′-TTAATGGCGTAAAAGGAGATATACCATGAAACAGTATTTGA TTGCCCC-3′

[0095] Primer P8: 5′-CATCCTGCGTCATGGTATATCTCCTTGAATTCTTATTCATGA CTTACCTTTGCCA-3′

[0096] Primer P9: 5′-AGTCATGAATAAGAATTCAAGGAGATATACCATGACGCAG GATGAATTGAAAA-3′

[0097] Primer P10: 5′-CAATCTTTTTCATGGTATATCTCCTTTCATTTCACAATGGTT TTGACACC-3′

[0098] Primer P11: 5′-ATTGTGAAATGAAAGGAGATATACCATGAAAAAGATTGCA TTTGGCTG-3′

[0099] Primer P12: 5′-TTCATATCAGGCACGGTATATCTCCTTTCAATTTCTCCGCT GCTCTATTG-3′

[0100] Primer P13: 5′-GCGGAGAAATTGAAAGGAGATATACCGTGCCTGATATGAAGCTTTTTGC-3′

[0101] Primer P14: 5′-TGCAGGCGCGCCGAGCTCTTAGTGTTCGAACATGGCAGA GA-3′

[0102] PCR amplification reaction system (40 μL): 20 μL Max, 17 μL ddH2O, 1 μL template DNA, 1 μL upstream and downstream primers (10 μmol / L) each. The reaction conditions were: 95°C pre-denaturation for 3 min, then cycle: 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles in total; 72°C final extension for 5 min. The PCR product was identified by nucleic acid electrophoresis and purified by gel cutting.

[0103] (2) Multiple fragment overlap extension fusion

[0104] The fragments xylA, xylB, and rpe purified in (1) were fused by overlapping extension PCR to obtain xylA-xylB-rpe. The reaction system (40 μL) for PCR fusion was: 20 μL Max, 17 μL ddH2O, 1 μL each of xylA, xylB and rpe; reaction conditions: pre-denaturation at 95°C for 3 min and then cycling: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for a total of 10 cycles; final extension at 72°C for 5 min to fuse the three fragments into one fragment; referring to the amplification procedure in (1), adding upstream and downstream primers (P3 / P8) for amplification, changing the extension time to 2 min, and keeping other conditions unchanged, to obtain the multi-fragment xylA-xylB-rpe; using a method similar to the above, to obtain the multi-fragment rpiA-rpiB-prs.

[0105] (3) Recombinant plasmid pRSFDuet-xylA-xylB-rpe-rpiA-rpiB-prs-P T7 -nampt construction

[0106] The pRSFDuet-nampt plasmid and the fusion fragment obtained in (2) were double-digested with BamH I and Sac I, respectively. After digestion at 37°C for 1 hour, nucleic acid electrophoresis was performed for verification and gel purification. The linearized plasmid and the fusion fragment were connected with multi-fragment homologous recombinase (ClonExpress MultiS) at 50°C for 30 minutes. The connection product was transformed into E. coli JM 109 competent cells and spread on solid LB medium (containing kanamycin sulfate (Kanr) 50 mg / L), cultured overnight, and multiple transformants were picked to liquid LB medium (50 mg / L Kanr), cultured at 37°C, 220 rpm for 10-12 hours, and PCR verification was performed after plasmid extraction.

[0107] (4) Transformation of recombinant plasmid into E. coli BL21 (DE3)

[0108] The recombinant plasmid in step (3) was sent to a sequencing company for sequence determination, and the recombinant plasmid with the correct sequence was named pRSFDuet-xylA-xylB-rpe-rpiA-rpiB-prs-P T7 -nampt, referred to as pRSFDuet-xylA→nampt, the plasmid map of pRSFDuet-xylA→nampt is as follows Figure 2 As shown; the recombinant plasmid pRSFDuet-xylA→nampt with the correct sequence was transformed into E.coli BL21(DE3), and the recombinant bacteria E.coli BL21(DE3) / pRSFDuet-xylA→nampt were obtained by PCR identification.

[0109] The present invention also used SDS-PAGE to detect the supernatant of E. coli BL21 (DE3) transformed with pRSFDuet-1 plasmid and pRSFDuet-xylA→nampt recombinant plasmid. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that the present invention successfully expressed the modified Nampt as well as xylA, xylB, rpe, rpiA, rpiB and prs in E. coli BL21 (DE3).

[0110] (5) Whole-cell catalytic synthesis of NMN

[0111] The first-stage seed solution of the recombinant E. coli BL21(DE3) / pRSFDuet-xylA→nampt was transferred to 50 mL of fermentation medium at a 2% inoculum volume and cultured at 37°C until OD 600When the concentration of IPTG was 0.6 to 0.8, IPTG was added at a final concentration of 1 mmol / L and induced at 25°C for 8 to 12 hours. After the induction expression was completed, the bacterial solution was transferred to a 50 mL centrifuge tube, centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the cells were collected. 10 mL of phosphate buffer with a pH of 7.2 was added to resuspend the cells, and the cells were centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was discarded. This step is to wash the cells. Repeat the washing once, add an appropriate amount of buffer to resuspend the cells, and the whole-cell catalyst is obtained. Add the whole-cell catalyst (OD 600 15), and nicotinamide and xylose were added at a final concentration of 5 g / L, respectively. The NMN yield was measured after reacting at 30° C. and 220 rpm for 5 h. The NMN yield was measured to be 190 mg / L. The method for determining the NMN yield was the same as in Example 1.

[0112] Example 3

[0113] In order to further improve the efficiency of the recombinant bacteria obtained in Example 2 in synthesizing NMN using nicotinamide and xylose, the present invention uses the CRISPR / Cas9 dual-plasmid system to knock out genes related to the PRPP competition bypass pathway, carbon flow competition bypass pathway and NMN downstream metabolic decomposition pathway of E. coli BL21 (DE3), thereby transforming the chassis cells. The PRPP competition bypass pathway includes the ATP phosphoribosyltransferase encoding gene hisG (gene accession number WP_005128078.1), the adenine phosphoribosyltransferase encoding gene apt (gene accession number WP_005125198.1) and the hypoxanthine guanine phosphoribosyltransferase encoding gene hpt (gene accession number WP_004204384.1); the carbon flow competition bypass pathway includes the transketolase 2 encoding gene tktB (gene accession number NP_416960.1) and the transaldolase encoding genes talA (gene accession number NP_311353.1) and talB (gene accession number NP_308035.1); the NMN (nicotinamide mononucleotide) downstream metabolic decomposition pathway includes the NMN aminohydrolase encoding gene pncC (gene accession number NP_417180.1), the NAD regulating gene pncC (gene accession number NP_417180.1), the NAD regulating gene pncC (gene accession number NP_417180.1), the NAD regulating gene pncC (gene accession number NP_417180.1), the NAD regulating gene pncC (gene accession number NP_417180.1), the NAD regulating gene pncC (gene accession number NP_417180.1), the NMN regulating gene pncC (gene accession number NP_417180.1), the NAD regulating gene pncC (gene accession number NP_417180.1). + (nicotinamide adenine dinucleotide) derivative gene nadR (gene accession number is NP_418807.4).

[0114] Taking the knockout of the ATP phosphoribosyltransferase encoding gene hisG as an example, the specific steps of the knockout are as follows:

[0115] (1) Construction of homology arm repair template

[0116] PCR amplification primers for the upstream homology arm and the downstream homology arm are designed for hisG, and then the upstream and downstream fragments are fused according to the overlap extension PCR technique to obtain a homology arm repair template of about 1000 bp. Specifically, the PCR amplification primers for the upstream homology arm of hisG designed by the present invention are:

[0117] Primer P15: 5′-TGCCTGATGTGCTCCGCTTAT-3′

[0118] Primer P16: 5′-AAAGCTCATGGCGATTTCTTATTCCTCTTTAAACCTGTCTG AA-3′

[0119] The PCR amplification primers for the hisG downstream homology arm designed by the present invention are:

[0120] Primer P17: 5'-AGAGGAATAAGAAATCGCCATGAGCTTTAACACAATCATT GAC-3'

[0121] Primer P18: 5′-GCGCCGCATAAAGGATCTCATCG-3′

[0122] The PCR reaction system and conditions were the same as in Example 1.

[0123] (2) Preparation of electrocompetent cells

[0124] The pCas9 plasmid containing the Cas9 coding gene was transferred into the expression host (E. coli BL21 (DE3)), and a single colony was picked and cultured at 30°C for more than 18 hours; it was transferred to 50 mL of LB medium at a 2% inoculum, and cultured at 30°C on a shaker for 1 hour, and then arabinose solution was added and cultured until the OD 600 The pH value was 0.6-0.8; after a 10-min ice bath, the cells were centrifuged at 4°C and 4000 rpm for 10 min, the supernatant was discarded, and the cells were collected; the cells were resuspended in 10 mL of a 10% glycerol aqueous solution, and the cells were centrifuged at 4°C and 4000 rpm for 5 min, the supernatant was discarded, and this process was repeated twice; the cells were resuspended in 500 μL of a 10% glycerol aqueous solution, and then dispensed into 100 μL tubes and stored in a -80°C ultra-low temperature refrigerator.

[0125] (3) Construction of targeting plasmid

[0126] The sgRNA sequence was designed through the website CHOPCHOP, and the targeting plasmid pTarget was reversely amplified by PCR using primers. It was then digested with QuickCut Dpn I in a 37°C water bath for 1 hour, recovered and transformed, spread on a streptomycin plate, and placed in a 37°C incubator for growth; a single colony was picked into a small shake flask, cultured in a 37°C shaker, and then the plasmid was extracted and sent to the company for sequencing. After correct sequencing, the hisG gene knockout targeting plasmid was obtained.

[0127] (4) Electroporation

[0128] Add 800 ng of homology arm repair template and 400 ng of pTargetF-hisG plasmid to the electroporation competent medium and mix evenly. After an ice bath for 10 minutes, transfer to an electroporation cup and place on ice. Set the electroporator parameters to 1800 V and 5 ms for electroporation. The electroporator can transform the homology arm repair template and pTargetF-hisG plasmid into E. coli BL21 (DE3) / pCas9 through perforation.

[0129] (5) Induced recombination culture

[0130] After the electroporation experiment, immediately add 1 mL of LB medium mixed with arabinose (final concentration 10 mmol / L), mix well and transfer to a 2 mL EP tube, then place it in a shaker at 30°C and 120 rpm for 3 h. After the culture is completed, spread it on a solid plate containing kanamycin and streptomycin double antibodies, place it in a 30°C incubator for 18 to 24 h, and observe the growth of the colonies on the plate until they grow to a size that allows single colonies to be picked.

[0131] (6) Screening of positive clones

[0132] Pick a single colony and place it in a small shake flask LB containing kanamycin and streptomycin double antibodies, and culture it at 30℃ and 150rpm for at least 16 hours. Extract genomic DNA, amplify it by PCR using sequencing primers, and then perform nucleic acid electrophoresis. The size of the hisG gene is about 900bp. If it is successfully knocked out, the band size is about 1200bp. Send the band with the correct size to the company for sequencing.

[0133] (7) Elimination of targeting plasmid

[0134] After successful knockout, 10 μL of bacterial solution was transferred into a small shake flask containing kanamycin, and IPTG with a final concentration of 0.5 mmol / L was added. The plate was cultured at 30°C and 220 rpm for more than 12 h. An appropriate amount of bacterial solution was spread on a kanamycin LB plate and placed in a 30°C incubator for growth until a single colony could be isolated. Half of the single colonies were picked and cultured in a double-antibody small shake flask. If the culture medium remained clear, it indicated that pTargetF had been eliminated in this single colony.

[0135] (8) Elimination of pCas9 plasmid

[0136] Use a pipette to transfer 10 μL of bacterial solution into a small shake flask LB without resistance, and culture it on a shaker at 42°C and 220 rpm for at least 12 hours. Streak it on a plate without antibiotics and culture it. After a single colony grows, pick it into LB medium containing kanamycin resistance. If the culture medium remains clear, it means that the pCas9 plasmid has been eliminated from this single colony, and E. coli BL21 (DE3) ΔhisG is obtained.

[0137] Based on E. coli BL21(DE3)ΔhisG, the above method was used to knock out genes apt, hpt, tktB, talA, talB, pncC and nadR in sequence to obtain BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt.

[0138] The present invention adopts the same conditions and methods to measure the NMN production of different NMN-producing recombinant bacteria constructed in Examples 1 to 3, and also measures the NMN production on the BL21 (DE3) / ΔhisG Δhpt Δapt ΔtktB / talA ΔtalB ΔnadR ΔpncC / pRSFDuet-xylA → nampt 5L tank after optimizing the conditions. The results are as follows: Figure 5 As shown in the figure; Figure 1 is the NMN production determination result of BL21(DE3) / pRSFDuet-nampt; Figure 2 is the NMN production determination result of BL21(DE3) / pRSFDuet-xylA→nampt; Figure 3 is the NMN production determination result of BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt; Figure 4 is the NMN production determination result of BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt on a 5L tank after optimization. Figure 5 It can be seen that after the relevant gene was knocked out, the NMN production of the obtained recombinant bacteria reached 373 mg / L.

[0139] Example 4

[0140] In this example, the whole-cell catalytic conditions of the recombinant bacteria BL21 (DE3) / ΔhisG Δhpt Δapt ΔtktB / talA ΔtalB ΔnadR ΔpncC / pRSFDuet-xylA → namp in a 5L tank were optimized as follows:

[0141] The secondary seed solution of the recombinant bacteria was transferred to a 5L fermenter at a 10% (V / V) inoculation rate. The initial culture temperature was 37°C, the pH was controlled at 7.0±0.2, the ventilation was set to 1vvm, the speed was automatically adjusted in conjunction with the dissolved oxygen content, and the dissolved oxygen content was maintained at 10-30%. The liquid volume was 3L, and the bacteria were cultured until OD 600 When the temperature was 5, IPTG with a final concentration of 1 mmol / L was added for induction, and the fermenter was slowly lowered to the induction temperature (25°C). Xylose and nicotinamide were added when the growth reached the steady state, and the feed rate was 20 mL / h. Samples were taken regularly to determine the biomass.

[0142] The time curve of NMN production in the fermentation tank is as follows Figure 6 As shown by Figure 6 It can be seen that under the above catalytic conditions, the maximum NMN production reached 760 mg / L.

[0143] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A gene encoding nicotinamide adenylate transferase, characterized in that: The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. A recombinant plasmid for expressing nicotinamide adenylate transferase, characterized in that: The recombinant plasmid contains the gene according to claim 1.

3. A recombinant bacterium producing nicotinamide mononucleotide, characterized in that: The recombinant bacteria express the gene according to claim 1.

4. The recombinant bacterium according to claim 3, characterized in that The recombinant bacteria also express genes encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosylpyrophosphate synthetase.

5. The recombinant bacterium according to claim 4, characterized in that: The recombinant bacteria knock out any one or more of the ATP phosphoribosyltransferase encoding gene hisG, the adenine phosphoribosyltransferase encoding gene apt, the hypoxanthine guanine phosphoribosyltransferase encoding gene hpt, the carbon flow competition bypass pathway including the transketolase 2 encoding gene tktB, the transaldolase encoding genes talA and talB, the nicotinamide adenylate aminohydrolase encoding gene pncC, and the nicotinamide adenine dinucleotide derivative encoding gene nadR.

6. The recombinant bacterium according to any one of claims 3 to 5, characterized in that: The recombinant bacteria use Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or Pichia pastoris as starting strains.

7. A method for preparing a recombinant bacterium producing nicotinamide mononucleotide, characterized in that: The method comprises the following steps: constructing a recombinant plasmid containing the sequence shown in SEQ ID NO.1 and transferring the plasmid into the starting strain of the engineering bacteria to obtain a recombinant bacterium; Or scheme 2: construct a recombinant plasmid containing the sequence shown in SEQ ID NO.1 and the gene sequences encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosyl pyrophosphate synthetase and transfer it into the starting strain of the engineered bacteria to obtain a recombinant bacterium; Or scheme three: based on the recombinant bacteria constructed by scheme one or scheme two, knock out any one or more of the recombinant bacteria's ATP phosphoribosyltransferase encoding gene hisG, adenine phosphoribosyltransferase encoding gene apt, hypoxanthine guanine phosphoribosyltransferase encoding gene hpt, carbon flow competition bypass pathway including transketolase 2 encoding gene tktB, transaldolase encoding genes talA, talB, nicotinamide adenylate aminohydrolase encoding gene pncC, and nicotinamide adenine dinucleotide derivative encoding gene nadR.

8. Use of the gene according to claim 1, the recombinant plasmid according to claim 2, or the recombinant bacterium according to any one of claims 3 to 5 in producing nicotinamide mononucleotide or in preparing a preparation for producing nicotinamide mononucleotide.

9. A method for producing nicotinamide mononucleotide using the recombinant bacteria according to any one of claims 3 to 5, characterized in that: The following steps are involved: S1. Preparation of whole-cell catalyst: First, culture the recombinant bacteria to OD 600 is 0.6 to 0.8, then adding isopropyl-β-D-thiogalactopyranoside for induction culture, collecting the cells after induction culture and resuspending them in phosphate buffer to obtain whole-cell catalyst; S2. Add the whole cell catalyst obtained in step S1, nicotinamide and xylose into the catalytic system for reaction.

10. The method according to claim 9, characterized in that: The final concentration of the isopropyl-β-D-thiogalactopyranoside is 0.1-1.5 mmol / L.

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