Fermentation control process method for improving synthesis of nicotinamide riboside

Abstract

The invention relates to a fermentation control process method for improving synthesis of nicotinamide riboside, which is characterized in that the final yield is greatly improved by controlling the supplementing time of a nicotinamide solution substrate, adding supplementary materials after induction, induction temperature and the amount of grafting, so that the target of industrial production is achieved. According to the optimized conditions, the content of 45H and NR is 56.29 g / L after fermentation, the conversion efficiency of a nicotinamide substrate reaches 99.3%, the metabolic byproducts of NA and NAM are few, and the separation and purification burden is relieved. And the highest NR production rate in unit time is 1.5 g / (L.H).

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a fermentation control process method for improving the synthesis of nicotinamide riboside (NR). Background Art

[0002] Nicotinamide riboside (NR) is an important compound, a derivative of vitamin B3, and a precursor of coenzymes NAD+ and NADP+. NAD+ plays a key role in cell metabolism and participates in a variety of biochemical reactions. As research deepens, people have found that supplementing NR can increase NAD+ levels in cells, thereby fighting aging and some chronic diseases.

[0003] At present, the main method is chemical synthesis. Nicotinamide and 1,2,3,5-tetraacetyl-β-D-furanose are used as substrates, and the finished product NR is obtained through condensation reaction, acetyl deprotection reaction, column chromatography, distillation drying, and recrystallization. NR is synthesized by adding trimethylsilyl trifluoromethanesulfonate as a catalyst with furanose, methyl nicotinate, and chloroform. The conversion efficiency of chemical synthesis is relatively high, but the addition of catalysts and toxic reagents limits the application of the generated NR in the food field.

[0004] At the same time, attempts have also been made to produce nicotinamide riboside through genetic engineering in the field. For example, in the Chinese invention patent application with publication number CN114854656 A, the inventors used recombinant Escherichia coli modified by genetic engineering and used low-cost nicotinamide as a substrate to efficiently convert and produce nicotinamide riboside through whole-cell catalysis. At the level of a 5L fermentation tank, a maximum NR content of 20g / L was obtained, but this output is still a long way from industrial production.

[0005] In order to overcome the shortcomings of the above methods, the present invention provides a method for efficiently converting nicotinamide riboside (NR) using a strain shown in publication number CN 114854656 A by using a 10% tricyanopyridine solution supplementation strategy with a cheap carbon source such as glucose as a substrate, with high conversion rate and stable production performance. The NR content can reach 55 g / L in 45 hours, and the specific production rate can reach 1.5 g / (L·h). Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a fermentation control process method for improving the synthesis of nicotinamide riboside (NR). The present invention creatively achieves a significant increase in the unit yield of nicotinamide riboside (NR), shortens the fermentation cycle, and reduces production costs.

[0007] The nicotinamide riboside-producing strain used in the present invention was purchased from the laboratory of Jiangnan University and has been disclosed in the publication number CN114854656A, specifically the recombinant strain numbered NR011 that can synthesize nicotinamide riboside using nicotinamide.

[0008] The technical solution of the present invention is as follows:

[0009] A nicotinamide riboside synthesis and fermentation method, the method comprising the following steps:

[0010] S1. Seed solution preparation

[0011] S1-1. Prepare LB solid medium and LB liquid medium for standby use;

[0012] S1-2 pour plate: add carnamycin sulfate and streptomycin sulfate antibiotics to LB solid medium to obtain a double-antibody plate;

[0013] S1-3 plate coating: take a strain capable of synthesizing nicotinamide riboside, dilute, apply to the double-antibody plate prepared in S1-2, and culture;

[0014] S1-4. Second streaking: Pick the single bacteria obtained by culture in S1-3, streak them on the double-antibody plate prepared in S1-2, and culture them;

[0015] S1-5. Take the bacterial cells obtained in S1-4, take a single colony and inoculate it into 50 mL LB liquid culture medium, add carnamycin sulfate and streptomycin sulfate antibiotics, and culture at 37°C with a shaker at 250 rpm. OD 600 =3-4, obtain seed solution;

[0016] S2. Fermentation in fermenter

[0017] S2-1. The seed solution obtained from S1-5 was inoculated into the fermenter at a volume percentage of 6% to 12% with the fermentation medium, and the pH was controlled at 6.7-7.2 during the fermentation process, the temperature was maintained at 28°C-37°C, and the dissolved oxygen was between 10-30%;

[0018] S2-2. After DO rises and glucose in the fermentation medium is consumed, add a glucose solution containing 60% reducing sugar and maintain the glucose concentration in the fermentation medium <0.1 g / L. 600 ≥25, start cooling to 25℃~28℃, add 0.1mM-0.5mM / L IPTG inducer, and maintain the induction temperature at 25℃~28℃;

[0019] S2-3. After the addition of the inducer, yeast powder and peptone were added once so that the concentration of yeast powder and peptone in the fermentation system was 5g-10g / L;

[0020] S2-4. 1.5 to 2 hours after the addition of the inducer, according to the volume of the fermentation system, a 12% mass percentage of nicotinamide solution was added, the flow acceleration was 40ml-60ml / H, and the nicotinamide content in the fermentation system was detected. The amount of nicotinamide solution added was adjusted to control the nicotinamide content within 100mg-500mg / L, and the fermentation cycle was 40-45 hours.

[0021] Furthermore, the LB solid culture medium formula of S1-1 is: 5 g / L yeast extract powder, 10 g / L peptone, 10 g / L sodium chloride, and 15 g / L agar strips.

[0022] Furthermore, the LB liquid culture medium formula of S1-1 is 5 g / L yeast extract powder, 10 g / L peptone, and 10 g / L sodium chloride.

[0023] Furthermore, the concentrations of the kanamycin sulfate and streptomycin sulfate antibiotics described in S1-2 in the LB solid culture medium are both 0.8‰.

[0024] In a specific embodiment, S1-2 is to add 40 μL of kanamycin sulfate and streptomycin sulfate antibiotics to 50 ml of LB solid culture medium, so that the concentration of both is 0.8‰.

[0025] In a specific embodiment, the culture medium prepared by S1-2 is sterilized by filtration using a 0.2 μm filter membrane.

[0026] Furthermore, the culture conditions of S1-3 and S1-4 are culturing at 37° C. for 12 hours.

[0027] Furthermore, in S1-5, the final concentration of carnamycin sulfate and streptomycin sulfate antibiotics in the LB liquid culture medium is 50 mg / L.

[0028] Further, the fermentation medium of S2-1: KH2PO4 5g-15g / L, K2 HPO4 10g-20g / L, citric acid 1g-3g / L, ammonium sulfate 2g-5g / L, metal ion solution 2mL-10ml, glucose 10g-20g / L, MgSO4.7H2O 1.5g-5g / L, yeast powder 5g-15g / L, defoamer 0.01%, carnamycin sulfate 0.02g / L, streptomycin sulfate 0.02g / L; the metal ion solution is: FeSO4·7H2O 10g / L, CaCl2 1.53g / L, ZnSO4·7H2O 2.2g / L, MnSO4·4H2O 1.0g / L, CuSO4·5H2O 1g / L, (NH4)6Mo7O 24·4H2O 0.1g / L, Na2B4O7·10H2O 0.2g / L, NiCl2 1.0g / L, H3BO31.0g / L.

[0029] In a particular embodiment, the carnamycin sulfate and streptomycin sulfate in the fermentation medium are sterilized by filtration with a 0.2 μm filter membrane, and then added after the remaining components of the fermentation medium are sterilized and cooled.

[0030] Furthermore, the inoculation amount in S2-1 is 10%.

[0031] Furthermore, the concentration of the IPTG inducer in S2-2 in the fermentation medium is 0.1 mM-0.5 mM / L.

[0032] Furthermore, the induction temperature in S2-2 is 28°C.

[0033] Furthermore, the time for adding the 12% by mass nicotinamide solution in S2-4 is 1.5 hours after the addition of the inducer is completed.

[0034] The beneficial effects of the present invention are:

[0035] The method provides a fermentation control process for improving the synthesis of nicotinamide riboside (NR).

[0036] 1. The present invention creatively achieves a NR synthesis rate of 1.5 g / (L·h), relieves bacterial growth inhibition, and improves sugar metabolism pathways.

[0037] 2. The method of the present invention can shorten the fermentation period to 40-45 hours, and the nicotinamide substrate conversion efficiency reaches 99.3%, thereby reducing production costs. DETAILED DESCRIPTION

[0038] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0039] Example 1

[0040] The strain used in the experiment of the present invention was purchased from the laboratory of Jiangnan University, and the publication number is the strain shown in CN 114854656 A.

[0041] (1) Seed solution preparation

[0042] S1-1. Prepare LB solid culture medium and LB liquid culture medium, sterilize them at 121°C for 20 min, and set aside.

[0043] S1-2. Pour plates: Add 40 μL of kanamycin sulfate and streptomycin sulfate antibiotics to 50 ml of LB solid culture medium to make the concentration of both 0.8‰ and store at 4°C for later use.

[0044] S1-3. Plate coating: dilute the strain stored in a -80℃ glycerol tube, spread it on a double-antibody plate, and culture at 37℃ for 12h.

[0045] S1-4. Second streaking: Pick about 30 single bacteria from the double-resistance plate, streak them on a new double-resistance plate, and culture at 37°C for 12 hours.

[0046] S1-5. Take a single colony and inoculate it into a 50mL LB liquid medium shake flask, add Kan and Sm with a final concentration of 50mg / L (50mL LB liquid medium plus 40μL antibiotics), culture at 37℃250rpm for 10-11h, and measure OD after the culture. 600 About 3-4.

[0047] (2) Fermentation tank control parameters

[0048] S2-1. The seed solution was inoculated with a volume percentage of 10% of the fermentation medium into a 10L fermenter. During the fermentation process, the pH was controlled at 6.7-7.2 (ammonia to adjust the pH), the temperature was maintained at 37 ° C, and the dissolved oxygen was between 10-30%;

[0049] S2-2. After DO rises and glucose in the fermentation medium is consumed, add (% 60 reducing sugar) glucose solution and maintain the glucose concentration in the fermentation medium <0.1g / L, OD 600 ≥25, start cooling to 28℃, add 0.1mM-0.5mM / L IPTG for induction, and keep the induction temperature at 28℃

[0050] 0H after the addition of the inducer, add 5g-10g / L of yeast powder and peptone (1:1) at one time according to the volume of the fermentation liquid.

[0051] 1.5 to 2 hours after the addition of the inducer, add 12% nicotinamide 40 ml-60 ml / (L·H) according to the volume of the fermentation liquid. According to the nicotinamide content in the fermentation liquid, adjust the amount of nicotinamide added to control the nicotinamide content within 100 mg-500 mg / L. The fermentation cycle is 40-45 hours.

[0052] (3) HPLC detection of nicotinamide, NAD derivatives and NR

[0053] A chromatographic column (250×4.6 mm, 5 μm, Thermo-Fisher, MA, USA) was used and the detection conditions were 30°C with a Shimadzu SPD-20A detector. The mobile phase was 20 mM ammonium acetate containing 5% acetonitrile; flow rate: 1.0 mL / min; detection wavelength: 259 nm; column oven temperature: 30°C.

[0054] Test content: the content of tricyanopyridine, nicotinamide, NAD derivatives and NR.

[0055] (4) Culture medium configuration

[0056] LB medium:

[0057] The formula of LB solid culture medium is: yeast extract powder 5g / L, peptone 10g / L, sodium chloride 10g / L, and 15g / L agar strip.

[0058] The formula of LB liquid culture medium is 5 g / L yeast extract powder, 10 g / L peptone, and 10 g / L sodium chloride.

[0059] 5g / L yeast extract powder, 10g / L peptone, 10g / L sodium chloride. Add 15g / L agar strip to prepare LB solid medium.

[0060] Fermentation medium: KH2PO4 5g-15g / L, K2 HPO4 10g-20g / L, citric acid 1g-3g / L, ammonium sulfate 2g-5g / L, metal ion solution 2mL-10ml, glucose 10g-20g / L, MgSO4.7H2O 1.5g-5g / L, yeast powder 5g-15g / L, defoamer 0.01%, streptomycin sulfate: 0.02g / L, streptomycin sulfate: 0.02g / L. Feed medium: yeast powder, peptone (1:1).

[0061] (5) Solution preparation

[0062] 10% tricyanopyridine solution: Dissolve 100g tricyanopyridine in 1000mL pure water and sterilize in an autoclave at 121℃ for 20min.

[0063] The metal ion solution is: FeSO4·7H2O 10g / L, CaCl2 1.53g / L, ZnSO4·7H2O 2.2g / L, MnSO4·4H2O1.0g / L, CuSO4·5H2O 1g / L, (NH4)6Mo7O 24 ·4H2O 0.1g / L, Na2B4O7·10H2O 0.2g / L, NiCl2 1.0g / L, H3BO3 1.0g / L, add concentrated hydrochloric acid to dissolve, the ratio of metal ions to concentrated hydrochloric acid is 10mL / L.

[0064] Kan, ST antibiotics: 50g / L stock solution, 0.2μm filter membrane, filter sterilize, store at -20℃, working concentration 1.0 ‰.

[0065] IPTG: 0.5M stock solution, 0.2μm filter membrane, filter sterilize, store at -20℃, working concentration: 0.1mM-0.5mM.

[0066] Preparation of glucose solution containing 60% reducing sugar: 660 g of glucose monohydrate, add water to make up to 1 liter, sterilize in an autoclave at 115°C for 20 minutes.

[0067] Example 2 Seed solution inoculation amount

[0068] Table 1

[0069] Inoculation volume Fermentation tank volume <![CDATA[OD 600 ≥25 hours (H)]]> Control group 4％ 4L 5.3 Experimental Group 1 6％ 4L 4.8 Experimental Group 2 8％ 4L 3.5 Experimental Group 3 10％ 4L 2.5 Experimental Group 4 12％ 4L 2.5 Experimental Group 5 15％ 4L 2.4

[0070] The experimental groups were set up according to the experimental conditions shown in Table 1. Except for the experimental conditions shown in Table 1, the rest were consistent with Example 1. The control group had an inoculum size of 4%, the fermenter was set to 4L, the inoculum size was gradually increased, and the time to reach OD=25 gradually increased. The inoculum size was increased to 12%, and 15% could not continue to increase the bacterial growth rate. Considering that the inoculum size was too large, the demand for fermentation equipment conditions, and the excessive consumption of nutrients was not conducive to late fermentation, the inoculum size was selected to be 10%.

[0071] Example 3 Induction Temperature

[0072] Table 2

[0073]

[0074]

[0075] The experimental groups were set up according to the experimental conditions shown in Table 2. Except for the experimental conditions shown in Table 2, the rest were consistent with Example 1. The induction temperature of the control group was 37°C, and the final yield was 5.3 g / L, which showed rapid bacterial growth, sugar consumption, and large oxygen demand. The highest OD value was 177. In the protein gel experiment, the intracellular protein was insoluble protein. The year-on-year verification temperature was 31°C, 28°C, 25°C, and 22°C for induction. The temperature was 28°C, and the final yield was 27.9 g / L, which was the highest. Continuing to lower the temperature, the NR yield was significantly reduced, the bacteria consumed sugar, the oxygen consumption was reduced, and the growth of the bacteria was inhibited.

[0076] Example 4 12% Nicotinamide Solution Start Addition Time

[0077] Table 3

[0078] Post-induction timing Nicotinamide solution addition rate NR content Control group 0H 60ml / (L·H) 22g / L Experimental group 0.5H 60ml / (L·H) 25.3g / L Experimental group 1H 60ml / (L·H) 33.4g / L Experimental group 1.5H 60ml / (L·H) 43.3g / L Experimental group 2H 60ml / (L·H) 41.0g / L Experimental group 3H 60ml / (L·H) 36.3g / L

[0079] The experimental groups were set up according to the experimental conditions shown in Table 3. Except for the experimental conditions shown in Table 3, the rest were consistent with Example 1. When 12% nicotinamide solution was added directly after induction, bacterial growth was inhibited. In the protein gel experiment, the protein expression was low. In the comparative experimental group, 12% nicotinamide solution was added 1.5 hours after the addition of the inducer, and the final yield was 43.3 g / L. The nicotinamide solution was added within 1.5 to 2 hours after the addition of the inducer, and a higher yield could be obtained. The addition of nicotinamide solution at other times inhibited bacterial growth due to other reasons such as the incomplete expression of proteins in the bacteria after induction, and the yield was significantly different from that added within 1.5 to 2 hours after the addition of the inducer.

[0080] Example 5 Post-induction feeding

[0081] After the addition of the inducer, yeast powder and peptone (1:1) 5g-10g / L were added at one time according to the volume of the fermentation liquid. After 33H-45H of fermentation, the trend of slowing down the accumulation rate of NR was lifted, the activity of the bacteria was improved in the later stage, and the final yield was 56.29g / L.

[0082] Example 6 Substrate conversion rate and production rate

[0083] Table 4

[0084]

[0085]

[0086] According to the above optimization conditions, the substrate conversion rate and production rate under the optimal conditions were tested. The results showed that after 45 hours of fermentation, the NR content was 56.29g / L, the nicotinamide substrate conversion efficiency reached 99.3%, and the NA and NAM metabolic byproducts were small, which reduced the burden of separation and purification. The NR production rate per unit time was at a maximum of 1.5g / (L·H).

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for synthesizing and fermenting nicotinamide riboside, characterized in that: The method comprises the following steps: S1. Seed solution preparation S1-1. Prepare LB solid medium and LB liquid medium for standby use; S1-2. Pour plate: Add carnamycin sulfate and streptomycin sulfate antibiotics to LB solid medium to obtain a double-antibody plate; S1-3 plate coating: take a strain capable of synthesizing nicotinamide riboside, dilute it, and culture it on the double-antibody plate prepared in S1-2; S1-4. Second streaking: Pick the single bacteria obtained by culture in S1-3, streak them on the double-antibody plate prepared in S1-2, and culture them; S1-5. Select the bacteria obtained in step S1-4, take a single colony and inoculate it into 50 mL LB liquid medium, add carnamycin sulfate and streptomycin sulfate antibiotics, and culture at 37°C at a shaking speed of 250 rpm. OD 600 =3-4, stop culturing and obtain seed solution; S2. Fermentation in fermenter S2-1. The seed solution obtained from S1-5 was inoculated into the fermenter at a volume percentage of 6% to 12% with the fermentation medium, and the pH was controlled at 6.7-7.2 during the fermentation process, the temperature was maintained at 28°C-37°C, and the dissolved oxygen was between 10-30%; S2-2. After DO rises and glucose in the fermentation medium is consumed, add a glucose solution containing 60% reducing sugar and maintain the glucose concentration in the fermentation medium <0.1 g / L. 600 ≥25, start cooling to 25℃~28℃, add 0.1mM-0.5mM / L IPTG inducer, and maintain the induction temperature at 25℃~28℃ S2-3. After the addition of the inducer, yeast powder and peptone were added once so that the concentration of yeast powder and peptone in the fermentation system was 5g-10g / L; S2-4. 1.5 to 2 hours after the addition of the inducer, according to the volume of the fermentation system, a 12% mass percentage of nicotinamide solution was added, the flow acceleration was 40ml-60ml / H, and the nicotinamide content in the fermentation system was detected. The amount of nicotinamide solution added was adjusted to control the nicotinamide content within 100mg-500mg / L, and the fermentation cycle was 40-45 hours.

2. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: The formula of LB solid culture medium described in S1-1 is: 5g / L yeast extract powder, 10g / L peptone, 10g / L sodium chloride, and 15g / L agar strips.

3. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: The LB liquid culture medium formula of S1-1 is 5 g / L yeast extract powder, 10 g / L peptone, and 10 g / L sodium chloride.

4. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: The working concentrations of carnamycin sulfate and streptomycin sulfate antibiotics described in S1-2 in LB solid culture medium are both 0.8‰.

5. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: The culture conditions of S1-3 and S1-4 are 37° C. for 12 h.

6. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: S2-1 Fermentation medium: KH2PO4 5g-15g / L, K2 HPO410g-20g / L, citric acid 1g-3g / L, ammonium sulfate 2g-5g / L, metal ion solution 2mL-10ml, glucose 10g-20g / L, MgSO4.7H2O 1.5g-5g / L, yeast powder 5g-15g / L, defoamer 0.01%, carnamycin sulfate 0.02g / L, streptomycin sulfate 0.02g / L; the metal ion solution is: FeSO4·7H2O 10g / L, CaCl21.53g / L, ZnSO4·7H2O 2.2g / L, MnSO4·4H2O 1.0g / L, CuSO4·5H2O 1g / L, (NH4)6Mo7O 24 ·4H2O 0.1g / L,Na2B4O7·10H2O 0.2g / L,NiCl2 1.0g / L,H3BO3 1.0g / L。 7. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: The inoculation amount of S2-1 is 10%.

8. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: S2-2 The concentration of the IPTG inducer in the fermentation medium is 0.1mM-0.5mM / L.

9. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: The induction temperature in S2-2 is 28°C.

10. The method for synthesizing and fermenting nicotinamide riboside according to claim 1, characterized in that: S2-4 The time for adding 12% by weight nicotinamide solution is 1.5 hours after the addition of the inducer is completed.

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