A strain of Escherichia coli resistant to nicotinamide mononucleotide stress and its application

Abstract

The present invention provides an Escherichia coli strain resistant to nicotinamide mononucleotide stress and its application, which belongs to the field of microbial engineering technology. The strain is Escherichia coli ME59, which has been deposited in the China Center for Type Culture Collection on December 23, 2022, with a deposit number of CCTCC NO: M 20222060. The strain resistant to nicotinamide mononucleotide (NMN) stress provided by the present invention has both excellent anti-NMN performance and high NMN yield. On the one hand, under non-nicotinamide mononucleotide stress, strain ME59 has a higher NMN yield than the starting strain. On the other hand, strain ME59 overcomes the problem of low NMN tolerance of the starting strain, and can still maintain a high survival rate and NMN yield at high NMN concentrations, providing a high-yield NMN engineering strain.

Description

Technical Field

[0001] The present application relates to the field of microbial engineering technology, and in particular to a strain of Escherichia coli resistant to nicotinamide mononucleotide stress and its application. Background Art

[0002] Nicotinamide mononucleotide (NMN) is a naturally occurring substance in the human body that is used to synthesize NAD. + NMN is the main precursor of NAD, and is also found in some fruits and vegetables. NMN plays an important role in the energy production of human cells and is the main precursor of NAD. + (nicotinamide adenine dinucleotide), its physiological function is also through the conversion into NAD + To play a role, such as activating NAD + Substrate-dependent enzymes, regulating cell survival and death, maintaining redox status, etc. Harvard Medical School has conducted relevant research on NMN and found that NMN can restore the body functions of 60-year-old mice to the state of 20 years old, playing a role in reversing aging and prolonging life.

[0003] Adaptive laboratory evolution (ALE) is a method of artificially simulating the variation and selection process in natural evolution under laboratory conditions, achieving the directed evolution of microorganisms with the help of artificial selection pressure, and selecting individuals with excellent traits from the evolutionary population. Microbial adaptive evolution is a new technology for the study of biological evolution, which is called "directed evolution, laboratory evolution or domestication". Microbial evolution is achieved by imposing artificial interference and controlling the growth environment of microorganisms. The source of evolution is the natural mutation or artificial mutagenesis of the microorganisms themselves, the direction of evolution is the resistance to artificial selection pressure, and the driving force of evolution is the interaction between microorganisms and the environment. The strains are finally screened to obtain the desired target strains through individual competition and survival of the fittest during continuous generation. Compared with metabolic engineering, ALE does not need to consider the intricate and intersecting metabolic networks in the bacteria. It only needs to design the corresponding interference factors according to the target. It has the advantages of wide applicability and strong practicality of microorganisms, and it is easy to discover new mechanisms and achieve phenotypic optimization.

[0004] The NMN-producing engineered bacteria NMN02 obtained in the early stage (recorded in the patent document with patent application number 202210781815.2: A recombinant Escherichia coli producing NMN and its application) can synthesize 20.3g / L of NMN in a 5L fermenter after 25h of induction, and the OD 600It can reach more than 30. However, in the actual production process, it was found that the engineered E. coli chassis cells had a low tolerance to the product NMN. When the NMN concentration in the fermentation broth reached 15g / L, the bacterial growth was inhibited by 50% compared to the absence of NMN. Since the production of NMN is closely related to bacterial growth, in order to obtain an engineered strain with a high yield of more than 15g / L NMN, this inhibitory effect must be alleviated.

[0005] Therefore, improving the NMN tolerance of NMN-producing engineered bacteria is of great significance for the efficient fermentation production of NMN. Summary of the invention

[0006] The purpose of the present invention is to provide an Escherichia coli (Escherichiacoli) resistant to nicotinamide mononucleotide stress, specifically Escherichia coli ME59. In the present application, the starting strain is continuously passaged in a culture medium containing nicotinamide mononucleotide (NMN), and the concentration of NMN is gradually increased in the culture medium to force the chassis cells to evolve in a directed manner, thereby obtaining a new strain with both nicotinamide mononucleotide stress resistance and high nicotinamide mononucleotide yield, which can be used in the process of fermentation production of NMN to improve the production efficiency of NMN.

[0007] On the one hand, the present application provides an Escherichia coli resistant to nicotinamide mononucleotide stress, wherein the Escherichia coli is Escherichia coli ME59, which has been deposited in the China Center for Type Culture Collection on December 23, 2022, with the deposit number CCTCCNO:M 20222060.

[0008] The nicotinamide mononucleotide includes α-nicotinamide mononucleotide and β-nicotinamide mononucleotide. Preferably, the concentration of nicotinamide mononucleotide in the nicotinamide mononucleotide stress is less than 40 g / L.

[0009] More preferably, the concentration of β-nicotinamide mononucleotide in the nicotinamide mononucleotide stress is less than 30 g / L.

[0010] In a preferred embodiment, Escherichia coli ME59 can still survive in a β-nicotinamide mononucleotide stress culture with a final concentration of 40 g / L, and the survival rate is 21 times that of the starting strain NMN02.

[0011] Preferably, the Escherichia coli is capable of synthesizing nicotinamide mononucleotide, and the nicotinamide mononucleotide production of the Escherichia coli is 12-23 g / L. Specifically, the nicotinamide mononucleotide may be 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, or 23 g / L.

[0012] On the other hand, the present application also provides the use of the above-mentioned Escherichia coli in the fermentation production of nicotinamide mononucleotide.

[0013] The Escherichia coli ME59 described in the present application is a new strain that has both resistance to nicotinamide mononucleotide stress and high nicotinamide mononucleotide yield. Under non-nicotinamide mononucleotide stress conditions, ME59 has a better growth trend and a higher NMN yield than the starting strain NMN02. Moreover, ME59 overcomes the problem of low NMN tolerance of the starting strain and still maintains a relatively high NMN yield. Therefore, it can be used in the process of fermentation production of NMN to improve the NMN production efficiency.

[0014] On the other hand, the present application also provides a microbial preparation containing the above-mentioned Escherichia coli.

[0015] Preferably, the microbial preparation contains viable bacteria count ≥ 10 6 CFU / g of the above-mentioned Escherichia coli dry cells and / or wet cells.

[0016] On the other hand, the present application also provides the use of the above-mentioned microbial preparation in the preparation of nicotinamide mononucleotide.

[0017] On the other hand, the present application also provides a method for producing nicotinamide mononucleotide by fermentation, which uses the above-mentioned strain or the above-mentioned microbial preparation to ferment and produce nicotinamide mononucleotide.

[0018] Furthermore, the culture method includes: using nicotinamide as a fermentation substrate, inoculating the above strain into a fermentation medium for fermentation culture.

[0019] Furthermore, the fermentation temperature is 30°C-37°C, and the time is 24-36h. Specifically, the fermentation temperature may be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C; the time may be 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h.

[0020] In a preferred embodiment, a method for producing nicotinamide mononucleotide by fermentation comprises the following steps:

[0021] Step 1: Seed culture stage: Inoculate E. coli ME59 into the seed culture medium at a temperature of 25°C-37°C and culture at 200-300r / min until OD 600 2-3, to obtain seed solution; preferably, the temperature is 37 ° C, 220r / min culture until OD 600 is 3;

[0022] Step 2, fermentation stage: using nicotinamide as the fermentation substrate, inoculating the above seed liquid into the fermentation medium at an inoculation rate of 2-8%, and culturing at a temperature of 25°C-37°C and 200-300r / min for 24-36h; preferably, the inoculation rate of the seed liquid is 5%, at a temperature of 37°C and 220r / min.

[0023] Preferably, the method further comprises an inducer, wherein the inducer comprises isopropyl-β-D-thiogalactoside (IPTG), and the concentration of the isopropyl-β-D-thiogalactoside is 0.5 mmol / L; preferably, the OD 600 Add inducer when the pH is 7.0-13.0.

[0024] Preferably, the seed culture medium is LB culture medium; more preferably, the LB culture medium contains 50 μg / μL of kanamycin and 50 μg / μL of streptomycin.

[0025] Preferably, the fermentation medium comprises 10 g / L glucose, 1 g / L MgSO4·7H2O, 6 g / L K2HPO4, 16.4 g / L KH2PO4, 7 g / L ammonium sulfate, 10 g / L yeast powder, 1.1 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 1-10 g / L nicotinamide, wherein the nicotinamide feed medium is 200 g / L and the feed is added at 5 mL / h.

[0026] More preferably, the fermentation medium contains 50 μg / μL of kanamycin and 50 μg / μL of streptomycin.

[0027] On the other hand, the present application also provides nicotinamide mononucleotide produced by fermentation using the above method.

[0028] On the other hand, the present application also provides a composition comprising the above-mentioned nicotinamide mononucleotide.

[0029] On the other hand, the present application also provides the use of the above-mentioned Escherichia coli or the above-mentioned microbial preparation or the above-mentioned nicotinamide mononucleotide or the above-mentioned composition in the chemical field, pharmaceutical field and / or cosmetic field.

[0030] In a preferred embodiment, a method for obtaining a strain resistant to nicotinamide mononucleotide stress comprises the following steps:

[0031] Step 1: taking out the emerging strain and adding it to a medium containing nicotinamide mononucleotide for multiple rounds of transfer culture, and gradually increasing the concentration of nicotinamide mononucleotide in the medium as the number of generations increases to obtain an evolved population;

[0032] Step 2: Use cell sorting technology to perform single cell sorting on the evolved population to ultimately obtain domesticated strains.

[0033] Among them, until the strain OD under the same nicotinamide mononucleotide stress concentration 600 The OD value of the starting strain in the middle and late stages of logarithmic growth under no stress 600 value, then transfer to the next concentration of culture medium.

[0034] The NMN-producing engineered bacteria NMN02 is used as the starting strain. For details about the strain, see the patent document with application number 202210781815.2, a recombinant Escherichia coli for producing NMN and its application.

[0035] The present invention has the following beneficial effects:

[0036] 1. The present application provides an Escherichia coli ME59 strain resistant to NMN stress, which has excellent growth trend, NMN resistance and high NMN yield. On the one hand, under non-nicotinamide mononucleotide stress, the Escherichia coli ME59 described in the present application has a better growth trend and higher NMN yield than the starting strain, wherein the OD of the strain is 0.04477 g / cm3 after fermentation for about 24 hours. 600 Reaching about 110, which is 2.4 times higher than the starting strain, showing excellent growth ability, and the NMN yield is 13% higher than the starting strain. On the other hand, the ME59 strain provided in this application overcomes the problem of low NMN tolerance of the starting strain, and can maintain a high survival rate and NMN yield at high NMN concentrations, with a survival rate of up to 21 times that of the starting strain, providing a new strain with high NMN yield suitable for industrial production;

[0037] 2. This application also provides a method for obtaining a strain resistant to nicotinamide mononucleotide stress, which domesticates NMN engineered bacteria through the method of adaptive laboratory evolution, and has the advantages of wide applicability and strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0039] Figure 1 Growth curve of NMN02 with different concentrations of NMN added;

[0040] Figure 2 This is the growth curve of the NMN-producing engineered strain NMN02;

[0041] Figure 3 This is a graph showing the growth of the NMN domesticated strain population;

[0042] Figure 4is the OD of 93 NMN-acclimated bacteria in deep-well plates 600 Comparison chart with the starting strain, where the left picture is 24h OD 600 , right picture is 48h OD 600 ;

[0043] Figure 5 This is a statistical chart of the top 15 strains in terms of bacterial density growth;

[0044] Figure 6 OD of ten NMN-acclimated bacteria at 24h 600 Comparison chart with the starting strain NMN02;

[0045] Figure 7 This is a comparison chart of the NMN production of ten NMN-acclimated bacteria and the starting strain NMN02 at 24 hours;

[0046] Figure 8 This is a diagram of the fermentation process of ME59 in a 5L tank;

[0047] Fig. 9 The figure shows the survival rate (%) of the domesticated strain and the control strain under 40 g / L NMN stress culture.

[0048] Deposit of biological materials:

[0049] A strain of Escherichia coli ME59 was deposited in the China Center for Type Culture Collection on December 23, 2022, with the deposit number CCTCC NO:M 20222060, and the deposit address is Wuhan University, Wuhan City, Hubei Province, China. DETAILED DESCRIPTION

[0050] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0051] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0052] Among them, the starting strain adopts the strain NMN02 constructed in the patent document with application number 202210781815.2.

[0053] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0054] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0055] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0056] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields conventional in the technical field.

[0057] In this application, the NMN-producing engineered bacteria NMN02 is used as the starting strain (recorded in the patent document with application number 202210781815.2). In this application, it is found that if the starting strain is used to directly produce NMN, the strain will have a low tolerance to the product NMN. When the NMN concentration in the fermentation broth reaches 15g / L, the bacterial growth is inhibited by 50% compared to the absence of NMN. The growth curve of strain NMN02 was measured under the addition of different concentrations of NMN, and the results are as follows: Figure 1 As shown, the growth of strain NMN02 was significantly inhibited in a high NMN environment.

[0058] In the present application, the starting strain is continuously subcultured in a culture medium containing NMN, while gradually increasing the concentration of NMN in the culture medium, forcing the chassis cells to evolve in a directed manner, thereby obtaining a new engineered bacterium with both excellent anti-NMN performance and high NMN yield.

[0059] In the previous fermentation process of the NMN-producing strain NMN02, the maximum concentration of NAM feed was 8g / L at one time, and the maximum NMN production could reach 20g / L. Therefore, it can be considered that the growth of the cells transferred to the chassis under the condition of 30g / L NMN culture medium was basically not inhibited, which was the evolutionary end point.

[0060] In this application, single cells of the evolved population were sorted by cell sorting technology. The initial screening was performed on a 96-well plate, and the growth of each single cell under the culture condition containing 30g / L NMN was detected. The 10 strains with the best growth were selected, and rescreened in a shake bottle under the same culture conditions. Finally, the strain with the best growth and a yield not lower than that of the starting strain was selected for subsequent experiments and production.

[0061] The components of the culture medium in this application are as follows:

[0062] LB plate: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar powder 15 g / L;

[0063] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L;

[0064] Fermentation medium: glucose 10 g / L, MgSO4·7H2O 1 g / L, K2HPO4 6 g / L, KH2PO4 16.4 g / L, ammonium sulfate 7 g / L, yeast powder 10 g / L, citric acid monohydrate 1.1 g / L, vitamin B1 0.1 g / L, nicotinamide 1-10 g / L, of which nicotinamide feed medium was 200 g / L, and feed was added at 5 mL / h.

[0065] Example 1 NMN adaptive evolution process

[0066] Take the original strain of NMN02 and streak it on the LB plate, pick a single colony and activate it in LB liquid medium, and take samples in real time to measure OD 600 , draw a growth curve (such as Figure 2 Generally speaking, strains have higher metabolic activity and are prone to mutation in the middle and late stages of logarithmic growth. According to the growth curve determined in previous experiments, NMN02 can grow to OD 600 The value is around 4, OD 600 When the value is 2-3, it is in the middle and late stages of logarithmic growth, so it is cultured until the OD 600 2-3 are suitable as transfer points for multiple rounds of adaptive evolution.

[0067] The specific culture process includes: using NMN02 as the starting strain, streaking culture of NMN02, selecting 4-5 single colonies and inoculating them into LB liquid culture medium, culturing for about 12 hours, inoculating them into LB supplemented with NMN at a 2% inoculum, and performing multiple rounds of transfer culture until the OD at the same concentration is 600 When the concentration reaches 2-3 within 12 hours, it is transferred to the next concentration medium and finally evolves to the end point to obtain the domesticated strain population (which can be considered as the evolutionary population). The specific concentration of NMN and the passage process are shown in Table 1.

[0068] Table 1

[0069]

[0070]

[0071] Note: The passage number is counted from 1 after transferring to the next concentration of culture medium.

[0072] After multiple rounds of product stress cultivation as mentioned above, the growth curves of the domesticated strain population and the starting strain are measured until the growth trend of the domesticated strain population is close to that of NMN02 or the inhibitory effect is significantly improved, reaching the end point of adaptive evolution.

[0073] The growth curves of the domesticated strain population and the starting strain are as follows: Figure 3 As shown in the figure, it can be seen that after multiple rounds of subculture and domestication, the growth of the NMN domesticated strain group in ordinary LB medium is significantly faster than that of the starting strain NMN02, and the growth in LB medium supplemented with 30g / LNMN is also better than that of NMN02. That is, it can be considered that the NMN domesticated strain group can tolerate 30g / L NMN and reach the domestication endpoint.

[0074] Example 2 Screening of NMN domesticated strains

[0075] In order to further screen out several single colonies with good growth trends, the domesticated strain population in Example 1 was cultured overnight on a plate supplemented with 30 g / L NMN using the gradient dilution coating method, and the larger single colonies were screened and marked, inoculated into a 96-deep-well plate for culture, and the OD 600 To screen out fast-growing domesticated strains.

[0076] The specific operation method includes: streaking the domesticated strain population tolerant to NMN 30g / L on an LB plate supplemented with 30g / L NMN overnight, then scraping a section of the bacterial line and inoculating it into LB liquid culture medium supplemented with 30g / L NMN, culturing for about 12 hours, gradient dilution, controlling the bacterial solution concentration to 100cfu / plate, and spreading it on an LB plate supplemented with 30g / L NMN, 10 plates for each gradient, culturing overnight, and selecting 8-9 single colonies with the largest growth on each plate.

[0077] Sterilize the 96-deep-well plate and dry it overnight. Add 1 mL of LB liquid culture medium supplemented with 30 g / L NMN to each well. Use a pipette tip to pick up the above single colonies to the corresponding wells and mark them. There are a total of 93 single colonies of domesticated strains. Inoculate the remaining 3 wells with the starting strain NMN02 as a control and culture them in a 37°C shaker overnight.

[0078] Pipette a certain amount of bacterial solution into the ELISA plate, dilute to an appropriate concentration, and measure OD with an ELISA reader. 600 , record the well with the highest bacterial density, find the corresponding single colony on the plate, retain the top ten fastest growing bacterial strains, pick them into test tubes for overnight culture, and preserve the bacteria.

[0079] After measuring the OD of a single colony in a deep-well plate 600 , the growth of 93 domesticated strains was compared with that of 3 starting strains at 24h and 48h. At 24h, 87 strains grew faster than the starting strains, accounting for 93.5%; at 48h, 80 strains grew faster than the starting strains, accounting for 86%. The results are as follows Figure 4 shown.

[0080] The top 20 strains were selected at 24h and 48h for comparison, and the OD values ​​of the two sampling points were analyzed. 600 The growth and average growth of 15 strains of bacteria 600 The growth is greater than 20%, such as Figure 5 As shown, the top ten domesticated strains were determined.

[0081] After the above-mentioned deep-well plate screening, the top ten strains with faster growth were obtained by sorting them according to the average growth rate of their bacterial density, namely ME59, ME52, ME01, ME53, ME56, ME61, ME51, ME72, ME71, and ME33.

[0082] Example 3 Shake flask horizontal fermentation verification of NMN domestication strain

[0083] The strains screened in Example 2 were inoculated into liquid shake flasks, cultured and screened using NMN-producing fermentation medium, samples were taken to draw growth curves, and the NMN production was determined by liquid phase, and the domesticated strains with fast growth and high NMN production were retained.

[0084] Specifically, the single colonies of the ten strains screened above were streaked on LB plates (kanamycin 50 μg / μL + streptomycin 50 μg / μL), cultured at 37°C for 10 h, and the bacteria on one line were inoculated into 20 mL of liquid LB (kanamycin 50 μg / μL + streptomycin 50 μg / μL) in a 100 mL shake flask, and cultured at 37°C, 220 r / min for 10-12 h until OD 600 =3.0. Transfer the cultured seed solution to 100 mL of NMN-producing fermentation medium (kanamycin 50 μg / μL + streptomycin 50 μg / μL) at a transfer volume of 5%, culture at 37°C, 220 r / min, and measure OD 600 =1.0-3.0, add IPTG with a final concentration of 0.5mmol / L for induction, and add substrate nicotinamide with a final concentration of 1-3g / L. Take samples at 24h to measure the OD600 and NMN production, such as Figure 6 , Figure 7 shown.

[0085] It can be seen that through shake flask fermentation, the ten strains of NMN domesticated bacteria grew well, with an increase of 10%-40% relative to the starting strain. In terms of NMN yield, the yield of the starting strain NMN02 was about 2.5g / L after 24h of fermentation, and the yield of NMN domesticated bacteria varied greatly, among which the yields of ME59, ME52, and ME01 were greatly improved, with the highest being 3.3g / L; ME33, ME51, ME53, ME56, ME71, and ME72 were not much different from the starting strain; the yield of ME61 was lower than that of the starting strain, so ME59 was selected as the optimal strain for subsequent fermentation verification at the 5L fermentation tank level.

[0086] Example 4 5L fermentation tank level verification of NMN domestication bacteria

[0087] The specific experimental method is as follows: streak the domesticated strain ME59 on an LB plate (kanamycin 50 μg / μL + streptomycin 50 μg / μL), culture at 37°C for 10 h, inoculate the bacteria on a section of the line into 100 mL of liquid LB (kanamycin 50 μg / μL + streptomycin 50 μg / μL) in a 500 mL shake flask, and culture at 37°C, 220 r / min for 10-12 h until the OD 600 =3.0. The cultured seed solution was transferred to a 5L fermenter containing 2.5L NMN-producing fermentation medium (kanamycin 50μg / μL + streptomycin 50μg / μL) at a transfer volume of 5%, and cultured at 37°C and 220r / min. OD was measured. 600 =7.0-13.0, IPTG was added to induce at a final concentration of 0.5 mmol / L, and the substrate nicotinamide was added at a constant rate, and the nicotinamide feed concentration was 200 g / L.

[0088] ME59 grows fast in 5L pots. Figure 8 As shown, the OD 600 Reached about 110, while the starting strain OD 600 At around 45, ME59 was higher than the starting strain OD 600 It increased by 2.4 times, showing greater growth potential.

[0089] The starting strain NMN02 reached its maximum value after 25h of induction in a 5L fermenter, and could synthesize up to 20.3g / L of NMN. In this application, the NMN production of ME59 was 20.8g / L and 23g / L at 24h and 30h of fermentation, respectively.

[0090] Example 5 Determination of the survival rate of NMN domesticated strain ME59

[0091] In order to verify that the domesticated strain ME59 can have a better survival rate, it is planned to select the survival rate under 40g / L NMN stress and conduct a survival rate experiment. The specific experimental steps are as follows:

[0092] 1. Streak and culture strains NMN02 and ME59 on LB plates respectively;

[0093] 2. Select a single colony and inoculate it into LB medium for activation, and culture it at 37℃220rpm for 12-16h;

[0094] 3. Transfer the above seed solution to 20 mL of fresh LB liquid medium at an inoculum volume of 2% (V / V) and culture at 37°C in a shaker at 220 rpm;

[0095] 4. Collect the NMN02 and ME59 cells cultured to the middle and late stages of logarithmic growth, centrifuge at 8000 rpm for 5 min, wash twice with 1X PBS buffer, and then resuspend in an equal volume of fresh LB liquid medium, add NMN with a final concentration of 40 g / L and stress culture for different times (stress culture 0, 1, 2, 4, 6 h);

[0096] 5. The bacterial suspension after stress was washed twice and resuspended in an equal volume of physiological saline. Different dilution gradients were spread on LB plates for culture. The number of live bacteria was counted and the survival rate was calculated.

[0097] Bacterial survival rate = (number of living cells after stress ÷ total number of living cells at the beginning) × 100%

[0098] The results are shown in Table 2 and Fig. 9 As shown, after stress culture tolerance experiment analysis, the survival rate of NMN-acclimated bacteria ME59 after 6 hours of stress was 21 times that of the control.

[0099] Table 2 Survival rate (%) of domesticated strains and control strains under 40 g / L NMN stress culture

[0100]

[0101] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A strain of Escherichia coli resistant to nicotinamide mononucleotide stress ( Escherichia coli ), characterized in that, The Escherichia coli is Escherichia coli ME59, which was deposited in the China Center for Type Culture Collection on December 23, 2022, with the deposit number CCTCC NO: M 20222060.

2. Use of the Escherichia coli according to claim 1 in fermentation production of nicotinamide mononucleotide.

3. A microbial preparation containing the Escherichia coli as claimed in claim 1.

4. Use of the microbial preparation according to claim 3 in the preparation of nicotinamide mononucleotide.

5. A method for producing nicotinamide mononucleotide by fermentation, characterized in that: Nicotinamide mononucleotide is produced by fermentation using the Escherichia coli as claimed in claim 1 or the microbial preparation as claimed in claim 3.

6. The method according to claim 5, characterized in that The culture method comprises: using nicotinamide as a fermentation substrate, inoculating the Escherichia coli of claim 1 into a fermentation medium for fermentation culture.

7. The method according to claim 5, characterized in that The fermentation temperature is 30°C-37°C and the fermentation time is 24-36 hours.

Images