Recombinant bacterium for producing nicotinamide mononucleotide, preparation method therefor, and use thereof

Abstract

Provided are a recombinant bacterium for producing nicotinamide mononucleotide, a preparation method therefor, and a use thereof. By modifying a gene sequence for coding a nicotinamide adenylate transferase, constructing a recombinant plasmid and transferring the recombinant plasmid into a host bacterium, a recombinant bacterium capable of using nicotinamide and xylose to produce nicotinamide mononucleotide is obtained. On this basis, by inserting a coding gene for an enzyme related to nicotinamide mononucleotide synthesis, knocking out genes in the host bacterium related to a 5-phosphoribosyl-1-pyrophosphate competitive bypass pathway, a carbon flux competitive bypass pathway and a nicotinamide mononucleotide downstream metabolic decomposition pathway, and optimizing catalytic system conditions, the efficiency of the constructed recombinant bacterium using nicotinamide and xylose to produce nicotinamide mononucleotide is further improved, and the whole-cell catalytic yield is as high as 760 mg / L. The present invention facilitates industrial production of nicotinamide mononucleotide, and also has the advantages of simple operation, low cost, and the like.

Description

A recombinant bacterium producing nicotinamide mononucleotide and its preparation method and application 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, a preparation method thereof, and an 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 + Current research has found that NMN supplementation has a significant preventive, therapeutic, and repairing effect on cardiovascular and cerebral tissue damage, metabolic diseases, aging, and related degenerative diseases. It is expected to be used to treat obesity-related liver disease, Parkinson's disease, and Alzheimer's disease, and to improve tissue function. As people become more aware of the health benefits of NMN, the market demand for NMN is also growing.

[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, EC 2.4.2.12); 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 the cost of directly using them as substrates to synthesize NMN is too high.

[0004] In addition to the aforementioned NMN metabolic synthesis pathways, there is also a method for synthesizing NMN using nicotinamide, xylose, and ATP as raw materials, utilizing enzymes such as nicotinamide phosphoribosyltransferase and ribose phosphate pyrophosphokinase. While this method avoids relatively expensive intermediates such as PRPP and NR, it requires the equally expensive raw material ATP and the addition of multiple commercial enzymes. Furthermore, while existing methods for biosynthesizing NMN allow the enzymes required for NMN synthesis to be inserted into engineered bacteria to construct recombinant NMN-producing bacteria, constructing such recombinant bacteria does not require simply inserting the desired enzyme into the engineered bacteria to significantly increase NMN production. Enzyme-encoding genes must be selected or modified, and the genetic background of the engineered bacteria must be modified to obtain recombinant bacteria capable of efficiently synthesizing NMN. Currently, there are no reports of recombinant bacteria synthesizing NMN from nicotinamide and xylose, nor of methods for their preparation.

[0005] Summary of the Invention

[0006] In view of the above problems in the prior art, the present invention provides a recombinant bacterium producing nicotinamide mononucleotide and a preparation method thereof.

[0007] The first object of the present invention is to provide a gene encoding nicotinamide adenylate transferase.

[0008] The second object of the present invention is to provide a recombinant plasmid for expressing nicotinamide adenylate transferase.

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

[0010] A fourth object of the present invention is to provide a method for preparing a recombinant bacterium that produces nicotinamide mononucleotide.

[0011] A fifth object of the present invention is to provide use of the gene, the recombinant plasmid or the recombinant bacterium in producing nicotinamide mononucleotide or in preparing a preparation for producing nicotinamide mononucleotide.

[0012] The sixth object of the present invention is to provide a method for producing nicotinamide mononucleotide using the recombinant bacteria.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] The present invention modifies the gene encoding nicotinamide adenylyltransferase from Chitinophaga pinensis (the modified sequence is shown in SEQ ID NO. 1) and constructs the corresponding recombinant bacteria. The resulting recombinant bacteria can not only synthesize nicotinamide mononucleotide from nicotinamide and xylose, but also achieves significantly higher yields than recombinant bacteria constructed using the unmodified sequence. Therefore, the present invention seeks protection for the modified sequence and its applications.

[0015] The present invention provides a gene encoding nicotinamide adenylate transferase, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0016] 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.

[0017] As an optional embodiment, the plasmid used to construct the recombinant plasmid of the present invention is pRSFDuet-1.

[0018] The present invention also provides a recombinant bacterium that produces nicotinamide mononucleotide, wherein the recombinant bacterium expresses the gene shown in SEQ ID NO.1.

[0019] 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).

[0020] The present invention also provides a recombinant bacterium that produces nicotinamide mononucleotide. In addition to expressing the gene shown in SEQ ID NO. 1, the recombinant bacterium also expresses genes encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase, and phosphoribosylpyrophosphate synthetase.

[0021] Specifically, the ribose-5-phosphate isomerase of the present invention includes RipA and RipB.

[0022] 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.

[0023] 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 phosphoribosylpyrophosphate synthetase, the efficiency and yield of nicotinamide mononucleotide production by the recombinant bacteria using nicotinamide and xylose will be further improved.

[0024] 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 phosphoribosylpyrophosphate synthetase is shown as SEQ ID NO.7.

[0025] The above gene sequence is only one of the optional embodiments provided by the present invention. Based on 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 NOs. 2 to 7) can be derived. By constructing recombinant plasmids and transferring them into host bacteria, the expression of genes encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosylpyrophosphate synthetase can be achieved, thereby achieving the same technical effects as the present invention.

[0026] 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 phosphoribosylpyrophosphate synthetase, wherein the plasmid contains gene sequences encoding nicotinamide adenylate transferase, xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase and phosphoribosylpyrophosphate synthetase.

[0027] Specifically, the ribose-5-phosphate isomerase includes RipA and RipB.

[0028] 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 phosphoribosylpyrophosphate synthetase is shown as SEQ ID NO.7.

[0029] The present invention also provides a recombinant bacterium for producing nicotinamide mononucleotide. The recombinant bacterium, while expressing the gene shown in SEQ ID NO. 1, has any one or more genes associated with the PRPP competitive bypass pathway, the carbon flux competitive bypass pathway, and the NMN downstream metabolic decomposition pathway knocked out. Alternatively, the recombinant bacterium, while expressing the gene shown in SEQ ID NO. 1 and genes encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase, and phosphoribosylpyrophosphate synthetase, has any one or more genes associated with the PRPP competitive bypass pathway, the carbon flux competitive bypass pathway, and the NMN downstream metabolic decomposition pathway knocked out, thereby transforming the chassis cells. The efficiency and yield of nicotinamide mononucleotide production from nicotinamide and xylose are further improved in this recombinant bacterium.

[0030] 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.

[0031] 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.

[0032] Optionally, the recombinant bacteria are Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or Pichia pastoris as starting strains. In the embodiment of the present invention, Escherichia coli is used as the starting strain.

[0033] Specifically, the Escherichia coli is a BL21 (DE3) strain.

[0034] The present invention also provides a method for preparing a recombinant bacterium producing nicotinamide mononucleotide, comprising the following steps: constructing a recombinant plasmid containing the sequence shown in SEQ ID NO. 1 and transferring the plasmid into a starting strain to obtain a recombinant bacterium;

[0035] Alternatively, a recombinant plasmid containing the sequence shown in SEQ ID NO. 1 and gene sequences encoding xylose isomerase, xylulokinase, ribulose-3-phosphate isomerase, ribose-5-phosphate isomerase, and phosphoribosylpyrophosphate synthetase is constructed and transformed into the starting strain to obtain a recombinant bacterium.

[0036] Or scheme three: based on the recombinant bacteria obtained in scheme one or scheme two, 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.

[0037] The present invention also seeks 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.

[0038] The present invention also seeks to protect a method for producing nicotinamide mononucleotide using the recombinant bacteria, characterized in that it comprises the following steps:

[0039] 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 induced cultured cells, resuspending and washing the cells with phosphate buffer to obtain whole-cell catalysts;

[0040] S2. Add the whole-cell catalyst obtained in step S1, nicotinamide, and xylose to the catalytic system for reaction.

[0041] The recombinant bacteria of the present invention need to be induced by IPTG to produce enzymes.

[0042] Specifically, the final concentration of IPTG is 0.1-1.5 mmol / L.

[0043] More specifically, the final concentration of IPTG is 1 mmol / L.

[0044] Specifically, the culture temperature of the recombinant bacteria is 16-37°C; the induction culture temperature is 10-30°C.

[0045] More specifically, the induction culture temperature of the recombinant bacteria is 25°C.

[0046] Specifically, the induction culture time of the recombinant bacteria is 5 to 20 hours.

[0047] More specifically, the induction culture time of the recombinant bacteria is 8 to 12 hours.

[0048] Specifically, the pH of the phosphate buffer is 7.2.

[0049] Specifically, the method for resuspending and washing the bacteria is as follows: centrifuging the bacterial solution after induction culture at 4°C and 8000 rpm for 10 minutes to collect the bacteria; resuspending the bacteria with pH 7.2 phosphate buffer, centrifuging at 4°C and 8000 rpm for 10 minutes to collect the bacteria; and repeating this step.

[0050] Specifically, the catalytic system is LB culture medium or phosphate buffer.

[0051] Specifically, the reaction temperature of the catalytic system is 16-37°C.

[0052] 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.

[0053] The present invention also provides a method for producing nicotinamide mononucleotide using whole-cell catalysis on a tank, wherein the method comprises: 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 is 37°C, the pH is controlled at 7.0±0.2, the ventilation volume is set to 1vvm, the speed is automatically adjusted in conjunction with the dissolved oxygen content, and the dissolved oxygen content is maintained at 10-30%, the liquid volume is 3L, and the bacteria are 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 a 3-fold concentrated fermentation medium.

[0054] Specifically, the preparation method of the secondary seed liquid is as follows: the primary seed liquid is transferred to 50 mL of liquid LB culture medium at a 2% inoculation rate, and cultured in a shaking incubator at 37° C. and 220 rpm for 8 to 10 hours.

[0055] 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, place the solid LB plate upside down in a 37°C incubator and culture for 12 hours, pick a single colony and place it in 10 mL of liquid LB medium, and culture it in a shaker at 37°C and 220 rpm for 12 hours.

[0056] The present invention has the following beneficial effects:

[0057] The present invention modifies the gene sequence encoding nicotinamide adenylyltransferase, constructs a recombinant plasmid, and transfers it into a host bacterium to obtain a recombinant bacterium capable of producing nicotinamide mononucleotide from nicotinamide and xylose. Furthermore, by inserting genes encoding enzymes related to nicotinamide mononucleotide synthesis, knocking out genes related to the 5-phosphoribosyl-1-pyrophosphate competitive bypass pathway, the carbon flow competitive bypass pathway, and the downstream metabolic decomposition pathway of nicotinamide mononucleotide in the host bacterium, and optimizing the catalytic system conditions, the efficiency of the constructed recombinant bacterium in producing nicotinamide mononucleotide from nicotinamide and xylose is further improved, achieving a whole-cell catalytic yield of up to 760 mg / L. This invention not only facilitates the industrial production of nicotinamide mononucleotide but also has the advantages of simple operation and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is the plasmid map of the recombinant plasmid pRSFDuet-nampt.

[0059] Figure 2 is the recombinant plasmid pRSFDuet-xylA-xylB-rpe-rpiA-rpiB-prs-P T7 Plasmid map of -nampt (pRSFDuet-xylA→nampt).

[0060] Figure 3 is an SDS-PAGE profile of nicotinamide phosphoribosyltransferase; M in the figure is a protein marker; 1 is the supernatant of E. coli BL21 (DE3) strain transformed with the pRSFDuet-1 plasmid; 2 is the supernatant of E. coli BL21 (DE3) strain transformed with the pET-28a plasmid; 3 is the supernatant of E. coli BL21 (DE3) strain transformed with the pRSFDuet-nampt plasmid.

[0061] Figure 4 is an SDS-PAGE profile of the xylose pathway; M in the figure is a protein 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.

[0062] Figure 5 shows the NMN production determination results of different recombinant Escherichia coli strains constructed; Figure 1 shows the NMN production determination results of BL21(DE3) / pRSFDuet-nampt; Figure 2 shows the NMN production determination results of BL21(DE3) / pRSFDuet-xylA→nampt; Figure 3 shows the NMN production determination results of BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt; Figure 4 shows the NMN production determination results of BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt5L tank after optimized conditions.

[0063] FIG6 is a time curve diagram of the NMN production in the fermentation tank described in Example 4. DETAILED DESCRIPTION

[0064] The present invention will be further described below with reference to specific examples and accompanying drawings, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0065] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0066] Example 1

[0067] Nicotinamide adenylyltransferase (Nampt) is a key enzyme in the synthesis of NMN. The present invention modifies the Nampt encoding gene (gene accession number CP001699.1) from Chitinophaga pinensis and constructs corresponding recombinant bacteria, thereby significantly improving the yield of NMN synthesized from nicotinamide and xylose compared to the recombinant bacteria constructed using the gene sequence before the modification. The encoding gene of Nampt after the modification of the present invention is shown in SEQ ID NO.1.

[0068] The present invention utilizes the modified Nampt coding gene to construct a recombinant bacterium and perform whole-cell catalytic synthesis of NMN as follows:

[0069] (1) Amplification of the Nampt encoding gene

[0070] PCR amplification was performed using the coding sequence of Nampt (shown in SEQ ID NO. 1) synthesized from Chitinophaga piniporus as a template. The primers (P1 / P2) used for amplification are as follows:

[0071] Primer P1: 5′-TAAGAAGGAGATATACATATGATGACTAAGGAGAATCTGATTTTGCTG-3′

[0072] Primer P2: 5′-TTTACCAGACTCGAGGGTACCTTAGATTGTGGCATTCTTGCGA-3′

[0073] PCR amplification reaction system (40 μL): 20 μL 17 μL ddH2O, 1 μL template DNA, and 1 μL each of upstream and downstream primers (10 μmol / L) were used. Reaction conditions were: 95°C denaturation for 3 minutes followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds; a final extension at 72°C for 5 minutes. PCR products were identified by electrophoresis and purified by gel extraction.

[0074] (2) Construction of recombinant plasmid pRSFDuet-nampt

[0075] The pRSFDuet-1 plasmid and the PCR product amplified in (1) were double-digested with Nde I and Kpn I, respectively. After enzyme digestion at 37°C for 1 hour, nucleic acid electrophoresis verification was performed and the recovered fragments were purified by gel tapping. The linearized plasmid and the recovered fragments after enzyme digestion were ligated with the homologous recombinase ClonExpress MultiS at 37°C for 30 minutes. The ligation products were transformed into E. coli JM 109 competent cells and spread on solid LB medium (containing 50 mg / L kanamycin sulfate (Kanr)) and cultured overnight. Several transformants were picked and transferred to liquid LB medium (50 mg / L Kanr) and cultured at 37°C and 220 rpm for 10-12 hours. The plasmids were extracted and verified by PCR.

[0076] (3) Transformation of the recombinant plasmid into E. coli BL21 (DE3)

[0077] The plasmid extracted in step (2) was sent to a sequencing company for sequence determination, and the recombinant plasmid with the correct sequence was named pRSFDuet-nampt. The plasmid map of pRSFDuet-nampt is shown in Figure 1; 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.

[0078] 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, and the results are shown in Figure 3. As shown in Figure 3, the present invention successfully expressed the modified Nampt in E. coli BL21 (DE3).

[0079] (4) Whole-cell catalytic synthesis of NMN

[0080] 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) inoculum and cultured at 37°C until the OD 600 When the pH value is 0.6 to 0.8, add IPTG with a final concentration of 1 mmol / L, and induce culture at 25°C for 8 to 12 hours; after the induction culture is completed, transfer the bacterial solution to a 50 mL centrifuge tube, centrifuge at 4°C and 8000 rpm for 10 minutes, pour out the supernatant, and collect the bacteria; add 10 mL of phosphate buffer with pH 7.2 to resuspend the bacteria, centrifuge at 4°C and 8000 rpm for 10 minutes, pour out the supernatant, this step is to wash the cells; repeat the washing once, add an appropriate amount of phosphate buffer to resuspend the bacteria, and obtain the whole-cell catalyst; add the whole-cell catalyst to the catalytic system (LB medium) (the amount added is: the OD value 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 the reaction at 30 ° C and 220 rpm for 5 hours. The NMN production was determined 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.

[0081] The preparation method of the primary 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, place the solid LB plate upside down in a 37°C incubator and culture for 12 hours, pick a single colony in a 50 mL small shake flask (containing 10 mL of liquid LB medium), and culture it in a shaker at 37°C and 220 rpm for 12 hours.

[0082] The method for determining NMN production is as follows: take 1 mL of the reaction solution, centrifuge it, and take the supernatant as the reaction supernatant; the bacteria are washed with PBS and then ultrasonically disrupted, and the supernatant after centrifugation is the intracellular supernatant; before the sample is measured, it is filtered through a filter membrane with a pore size of 0.22 μm, and then detected by high-performance liquid chromatography.

[0083] The liquid chromatography column was a Diamonsil 5 μL C18 250x4.6 mm; mobile phase A was 100% methanol, and mobile phase B was 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.

[0084] 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.

[0085] Example 2

[0086] 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 the gene sequences encoding xylose isomerase (XylA), xylulokinase (XylB), ribulose-3-phosphate isomerase (Rpe), ribose-5-phosphate isomerase (RipA, RipB) and phosphoribosylpyrophosphate synthetase (PRPP synthase, Prs) and the 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 phosphoribosylpyrophosphate synthetase is shown in SEQ ID NO.13.

[0087] 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.

[0088] The process of the present invention using the above enzyme to construct a recombinant bacterium and perform whole-cell catalytic synthesis of NMN is as follows:

[0089] (1) Amplification of the gene sequences encoding each enzyme

[0090] Using E. coli BL21 (DE3) genomic DNA as a template, primers were used to amplify the gene sequences encoding xylose isomerase (P3 / P4), xylulokinase (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:

[0091] Primer P3: 5'-TCATCACCACAGCCAGGATCCATGCAAGCCTATTTTGAC CAGC-3'

[0092] Primer P4: 5'-cccgatatacatggtatatctccttttatttgtcgaacagataatg-3'

[0093] Primer P5: 5′-TTCGACAAATAAAAGGAGATATACCATGTATATCGGGATAGATCTT-3′

[0094] Primer P6: 5′-ATACTGTTTCATGGTATATCTCCTTTTACGCCATTAATGGCAGAAG-3′

[0095] Primer P7: 5′-TTAATGGCGTAAAAGGAGATATACCATGAAACAGTATTTGATTGCCCC-3′

[0096] Primer P8: 5′-CATCCTGCGTCATGGTATATCTCCTTGAATTCTTATTCATGACTTACCTTTGCCA-3′

[0097] Primer P9: 5′-AGTCATGAATAAGAATTCAAGGAGATATACCATGACGCAGGATGAATTGAAAA-3′

[0098] Primer P10: 5′-CAATCTTTTTCATGGTATATCTCCTTTCATTTCACAATGGTTTTGACACC-3′

[0099] Primer P11: 5′-ATTGTGAAATGAAAGGAGATATACCATGAAAAAGATTGCATTTGGCTG-3′

[0100] Primer P12: 5′-TTCATATCAGGCACGGTATATCTCCTTTCAATTTCTCCGCTGCTCTATTG-3′

[0101] Primer P13: 5′-GCGGAGAAATTGAAAGGAGATATACCGTGCCTGATATGAAGCTTTTTGC-3′

[0102] Primer P14: 5′-TGCAGGCGCGCCGAGCTCTTAGTGTTCGAACATGGCAGAGA-3′

[0103] PCR amplification reaction system (40 μL): 20 μL 17 μL ddH2O, 1 μL template DNA, and 1 μL each of upstream and downstream primers (10 μmol / L) were used. Reaction conditions were: 95°C denaturation for 3 minutes followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds; a final extension at 72°C for 5 minutes. PCR products were identified by electrophoresis and purified by gel extraction.

[0104] (2) Multi-fragment overlapping extension fusion

[0105] The fragments xylA, xylB, and rpe purified in (1) were fused by overlapping extension PCR to obtain xylA-xylB-rpe. The reaction system for PCR fusion (40 μL) was: 20 μL 17 μL ddH2O, 1 μL each of xylA, xylB, and rpe; reaction conditions: pre-denaturation at 95°C for 3 min followed by cycling: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and 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), upstream and downstream primers (P3 / P8) were added for amplification, the extension time was changed to 2 min, and other conditions remained unchanged to obtain the multi-fragment xylA-xylB-rpe; a similar method to the above was used to obtain the multi-fragment rpiA-rpiB-prs.

[0106] (3) Recombinant plasmid pRSFDuet-xylA-xylB-rpe-rpiA-rpiB-prs-P T7 -nampt construction

[0107] The pRSFDuet-nampt plasmid and the fusion fragment obtained in (2) were double-digested with BamH I and Sac I, respectively. After enzyme digestion at 37°C for 1 hour, nucleic acid electrophoresis was performed and the gel was cut and purified. The linearized plasmid and the fusion fragment were ligated with multi-fragment homologous recombinase (ClonExpress MultiS) at 50°C for 30 minutes. The ligation 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, several transformants were picked and transferred 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.

[0108] (4) Transformation of the recombinant plasmid into E. coli BL21 (DE3)

[0109] 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, abbreviated as pRSFDuet-xylA→nampt, the plasmid map of pRSFDuet-xylA→nampt is shown in Figure 2; 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.

[0110] The present invention also used SDS-PAGE to detect the supernatants of E. coli BL21 (DE3) transformed with the pRSFDuet-1 plasmid and the pRSFDuet-xylA→nampt recombinant plasmid, respectively. The results are shown in Figure 4. As can be seen from Figure 4, the present invention successfully expressed the modified Nampt as well as xylA, xylB, rpe, rpiA, rpiB and prs in E. coli BL21 (DE3).

[0111] (5) Whole-cell catalytic synthesis of NMN

[0112] The first-stage seed solution of recombinant E. coli BL21 (DE3) / pRSFDuet-xylA→nampt was transferred to 50 mL fermentation medium at a 2% inoculum volume and cultured at 37°C until the 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 minutes, 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 minutes. 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 reaction was carried out at 30°C and 220 rpm for 5 h, and the NMN yield was measured. The NMN yield was measured to be 190 mg / L. The method for determining the NMN yield was the same as that in Example 1.

[0113] Example 3

[0114] 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 competitive 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 competitive 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 NMN ... + (nicotinamide adenine dinucleotide) derivative gene nadR (gene accession number is NP_418807.4).

[0115] Taking the knockout of the ATP phosphoribosyltransferase encoding gene hisG as an example, the specific steps of the knockout are as follows:

[0116] (1) Construction of homology arm repair template

[0117] PCR amplification primers for the upstream and downstream homology arms of hisG were designed, and then the upstream and downstream fragments were 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 in the present invention are:

[0118] Primer P15: 5′-TGCCTGATGTGCTCCGCTTAT-3′

[0119] Primer P16: 5′-AAAGCTCATGGCGATTTCTTATTCCTCTTTAAACCTGTCTGAA-3′

[0120] The PCR amplification primers for the hisG downstream homology arm designed by the present invention are:

[0121] Primer P17: 5'-AGAGGAATAAGAAATCGCCATGAGCTTTAACACAATCATTGAC-3'

[0122] Primer P18: 5′-GCGCCGCATAAAGGATCTCATCG-3′

[0123] The PCR reaction system and conditions were the same as in Example 1.

[0124] (2) Preparation of electroporation competent cells

[0125] The pCas9 plasmid containing the Cas9 encoding gene was transformed into the expression host (E. coli BL21 (DE3)), and a single colony was picked and cultured at 30°C for more than 18 hours; the inoculum was transferred to 50 mL of LB medium at a 2% inoculum, and cultured at 30°C on a shaker for 1 hour. After that, 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, 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 aliquoted into 100 μL tubes and stored in a -80°C ultra-low temperature freezer.

[0126] (3) Construction of targeting plasmid

[0127] 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 and placed in a small shake flask, cultured in a shaker at 37°C, and then the plasmid was extracted and sent to the company for sequencing. After correct sequencing, the hisG gene knockout targeting plasmid was obtained.

[0128] (4) Electroporation

[0129] Add 800 ng of homology arm repair template and 400 ng of pTargetF-hisG plasmid to the electroporation competent medium and mix evenly. After ice bath for 10 minutes, transfer to an electroporation cup and place on ice. Set the electroporator parameters to 1800 V voltage 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.

[0130] (5) Induced recombination culture

[0131] 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 hours. After the incubation, spread it on a solid plate containing kanamycin and streptomycin double antibodies and place it in a 30°C incubator for 18 to 24 hours. Observe the growth of the colonies on the plate until they grow to a size that can be picked as a single colony.

[0132] (6) Screening of positive clones

[0133] Pick a single colony and place it in a small shake flask containing kanamycin and streptomycin double antibodies LB, place it at 30℃ and 150rpm for at least 16 hours, extract genomic DNA, use sequencing primers to amplify by PCR 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.

[0134] (7) Elimination of targeting plasmids

[0135] After successful knockout, 10 μL of the bacterial solution was transferred to a small shake flask containing kanamycin and IPTG was added at a final concentration of 0.5 mmol / L. The cells were cultured at 30°C and 220 rpm for more than 12 h. An appropriate amount of the bacterial solution was spread on a kanamycin LB plate and placed in a 30°C incubator for growth until single colonies could be isolated. Half of the single colonies were picked and cultured in a small shake flask containing double-antibody culture. If the culture medium remained clear, it indicated that the single colony had eliminated pTargetF.

[0136] (8) Elimination of pCas9 plasmid

[0137] Use a pipette to transfer 10 μL of bacterial solution into a small shake flask containing LB without resistance, and culture it on a shaker at 42°C and 220 rpm for at least 12 hours. Streak the plate 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.

[0138] 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.

[0139] The present invention uses 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 of BL21 (DE3) / ΔhisG Δhpt Δapt ΔtktB / talA ΔtalB ΔnadR ΔpncC / pRSFDuet-xylA → nampt after optimization. The NMN production in the 5L tank is shown in Figure 5. Figure 1 shows the NMN production measurement results of BL21(DE3) / pRSFDuet-nampt; Figure 2 shows the NMN production measurement results of BL21(DE3) / pRSFDuet-xylA→nampt; Figure 3 shows the NMN production measurement results of BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt; and Figure 4 shows the NMN production measurement results of BL21(DE3) / ΔhisGΔhptΔaptΔtktB / talAΔtalBΔnadRΔpncC / pRSFDuet-xylA→nampt 5L tank after optimization. As shown in Figure 5, after the relevant genes were knocked out, the NMN production of the resulting recombinant bacteria reached 373 mg / L.

[0140] Example 4

[0141] 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 5 L tank were optimized as follows:

[0142] 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 volume 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 the OD 600 When the growth temperature was 5, IPTG with a final concentration of 1 mmol / L was added for induction, and the fermentation tank was slowly lowered to the induction temperature (25°C). Xylose and nicotinamide were added when the growth reached the stationary phase. The feeding rate was 20 mL / h, and samples were taken regularly to determine the biomass.

[0143] The time curve of NMN production in the fermentation tank is shown in Figure 6. As shown in Figure 6, under the above catalytic conditions, the maximum NMN production reached 760 mg / L.

[0144] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A gene encoding nicotinamide adenylate transferase, characterized in that: The nucleotide sequence of the gene is shown in 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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