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

Abstract

The present invention provides a strain of Escherichia coli resistant to nicotinamide stress and its application, which belongs to the field of microbial engineering technology. The strain is Escherichia coli AE74, which has been deposited in the China Center for Type Culture Collection on December 23, 2022, with a deposit number of CCTCC NO: M 20222059. The strain resistant to nicotinamide (NAM) stress has both excellent anti-nicotinamide performance and high nicotinamide mononucleotide (NMN) yield. On the one hand, under non-nicotinamide stress conditions, strain AE74 has a higher NMN yield than the starting strain. On the other hand, strain AE74 overcomes the problem of low NAM tolerance of the starting strain, and can maintain a high survival rate and NMN yield at high NAM concentrations, providing a new strain with high NMN yield suitable for industrial production.

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 stress and its application. Background Art

[0002] Nicotinamide mononucleotide (NMN) is a molecule that synthesizes NAD in the human body. + (nicotinamide adenine dinucleotide), its physiological function is also through the conversion to NAD + To play a role, such as activating NAD + Substrate-dependent enzymes, regulating cell survival and death, maintaining redox status, etc. Due to its potential as an anti-aging drug, nicotinamide mononucleotide has attracted great attention and has a certain social influence, which has laid the foundation for its effective production now and in the future.

[0003] Adaptive laboratory evolution (ALE) is a method that subjects microorganisms to selective pressure in the laboratory and, through long-term domestication, screens for mutant strains with specific phenotypes. Compared to metabolic engineering, ALE does not require consideration of the complex and intersecting metabolic networks within the microorganism. Instead, it simply designs corresponding interference factors based on the target. This method has the advantages of broad microbial applicability and high practicality, making it easy to discover new mechanisms and achieve phenotypic optimization. In recent years, this method has been widely used to screen industrial production strains with excellent characteristics by improving specific evolutionary conditions and screening strategies. For example, specific phenotypic screening is used to select industrial microbial strains for efficient substrate utilization, target product synthesis, and growth characteristic optimization.

[0004] The NMN-producing engineered bacteria NMN02 (recorded in the patent document with application number 202210781815.2: A recombinant Escherichia coli for producing NMN and its application) obtained in the early stage can synthesize 20.3g / L of NMN in a 5L fermenter after induction for 25h, and the OD 600 It can reach more than 30. However, in the actual production process, it was found that the tolerance of the engineered E. coli chassis cells to the substrate nicotinamide (NAM) was low. When the NAM concentration in the fermentation broth reached 5g / L, the bacterial growth was inhibited by 50% compared to the absence of NAM. Since the production of NMN is closely related to bacterial growth, improving the NAM tolerance of NMN-producing engineered bacteria is of great significance for the efficient fermentation production of NMN. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of Escherichia coli (Escherichia coli) resistant to nicotinamide stress, specifically Escherichia coli AE74. In this application, the starting strain is continuously passaged in a culture medium containing nicotinamide (NAM), and the concentration of NAM in the culture medium is gradually increased to force the chassis cells to evolve in a directed manner, thereby obtaining a new strain with both nicotinamide stress resistance and high nicotinamide mononucleotide yield, which can be used in the fermentation production of NMN to improve NMN production efficiency.

[0006] On the one hand, the present application provides a strain of Escherichia coli that is resistant to nicotinamide stress, wherein the Escherichia coli is Escherichia coli AE74, which was deposited in the China Center for Type Culture Collection on December 23, 2022, with a deposit number of CCTCC NO: M20222059.

[0007] Preferably, the nicotinamide concentration in the nicotinamide stress is less than 20 g / L.

[0008] More preferably, the nicotinamide concentration in the nicotinamide stress is less than 15 g / L.

[0009] In a preferred embodiment, Escherichia coli AE74 can still survive in NAM stress culture with a final concentration of 20 g / L, and the survival rate is 31 times that of the starting strain NMN02.

[0010] Preferably, the Escherichia coli is capable of synthesizing nicotinamide mononucleotide, and the nicotinamide mononucleotide production of the Escherichia coli is 15-22.4 g / L. Specifically, the nicotinamide mononucleotide can be 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 22.4 g / L.

[0011] On the other hand, the present application also provides the use of the above-mentioned Escherichia coli in the preparation of nicotinamide mononucleotide.

[0012] The Escherichia coli AE74 described in this application is a new strain that has both nicotinamide stress resistance and high nicotinamide mononucleotide yield. Under non-nicotinamide stress conditions, strain AE74 has a better growth trend and higher NMN yield than the starting strain NMN02. In addition, AE74 overcomes the low NAM tolerance problem of the starting strain and still maintains a high NMN yield. Therefore, it can be used in the fermentation production of NMN to improve NMN production efficiency.

[0013] On the other hand, the present application also provides a microbial preparation containing the above-mentioned Escherichia coli.

[0014] Preferably, the microbial preparation contains viable bacteria ≥ 10 6CFU / g of the above-mentioned Escherichia coli dry cells and / or wet cells.

[0015] On the other hand, the present application also provides the use of the above-mentioned microbial preparation in the preparation of nicotinamide mononucleotide.

[0016] On the other hand, the present application also provides a method for producing nicotinamide mononucleotide by fermentation, which utilizes the above-mentioned Escherichia coli or the above-mentioned microbial preparation to ferment and produce nicotinamide mononucleotide.

[0017] Furthermore, the culture method includes: using nicotinamide as a fermentation substrate, inoculating the above-mentioned Escherichia coli into a fermentation medium for fermentation culture.

[0018] Furthermore, the fermentation temperature is 30°C-37°C, and the culture time is 24-36h. Specifically, the fermentation temperature can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C; the time can be 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h.

[0019] In a preferred embodiment, a method for producing nicotinamide mononucleotide by fermentation comprises the following steps:

[0020] Step 1: Seed culture stage: Escherichia coli AE74 was inoculated into the seed culture medium at a temperature of 25℃-37℃ and cultured at 200-300r / min until OD 600 More preferably, the temperature is 37 ° C, 220 r / min and the OD is raised to 0. 600 is 3;

[0021] Step 2, fermentation stage: using nicotinamide as the fermentation substrate, inoculating the above seed liquid into the fermentation medium at an inoculum size of 2-8%, and culturing at a temperature of 25°C-37°C and 200-300 rpm for 24-36 hours; more preferably, the inoculum size of the seed liquid is 5%, and culturing is carried out at a temperature of 37°C and 220 rpm for 24-36 hours.

[0022] Preferably, the method further comprises an inducer, wherein the inducer comprises isopropyl-β-D-thiogalactopyranoside (IPTG), and the concentration of the isopropyl-β-D-thiogalactopyranoside is 0.5 mmol / L; preferably, the OD 600 Add inducer when the pH is 1.0-3.0.

[0023] 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.

[0024] 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 extract, 1.1 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 1-10 g / L nicotinamide, wherein 200 g / L nicotinamide is added at a rate of 5 mL / h.

[0025] More preferably, the fermentation medium contains 50 μg / μL of kanamycin and 50 μg / μL of streptomycin.

[0026] On the other hand, the present application also provides nicotinamide mononucleotide produced by fermentation using the above method.

[0027] On the other hand, the present application also provides a composition comprising the above-mentioned nicotinamide mononucleotide.

[0028] On the other hand, the present application also provides the use of the aforementioned Escherichia coli or the aforementioned microbial preparation or the aforementioned nicotinamide mononucleotide or the aforementioned composition in the chemical field, pharmaceutical field and / or cosmetic field.

[0029] In a preferred embodiment, a method for obtaining a nicotinamide stress-resistant strain comprises the following steps:

[0030] Step 1: taking out the emerging strain and adding it to a medium containing nicotinamide for multiple rounds of transfer culture, while gradually increasing the concentration of nicotinamide in the medium as the number of passages increases to obtain an evolved population;

[0031] Step 2: Use cell sorting technology to perform single cell sorting on the evolved population to finally obtain the domesticated strain.

[0032] Among them, until the strain OD 600 Reach the OD of the middle and late stages of logarithmic growth of the starting strain under no stress 600 value, then transfer to the next concentration medium.

[0033] The NMN-producing engineered bacteria NMN02 was used as the starting strain. For details about this strain, please refer to the patent document with application number 202210781815.2, "A recombinant Escherichia coli for producing NMN and its application."

[0034] The present invention has the following beneficial effects:

[0035] 1. The present application provides a strain of Escherichia coli AE74 that is resistant to NAM stress, which has both excellent anti-NAM performance and high NMN yield. On the one hand, under non-nicotinamide stress conditions, the Escherichia coli AE74 described in the present application has a better growth trend and higher NMN yield than the starting strain. After 24 hours of fermentation, the OD of the strain is 600 The NMN yield of E. coli AE74 can reach 68, which is 1.5 times that of the starting strain, and the NMN yield is increased by 10% compared with the starting strain. On the other hand, the E. coli AE74 provided in this application overcomes the problem of low NAM tolerance of the starting strain and can maintain a high survival rate and NMN yield at high NAM concentrations. The survival rate can reach 31 times that of the starting strain, providing a new strain with high NMN yield suitable for industrial production.

[0036] 2. This application also provides a method for obtaining a nicotinamide stress-resistant strain, which domesticates NMN engineered bacteria through adaptive laboratory evolution, and has the advantages of wide applicability and strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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:

[0038] Figure 1 Growth curve of NMN02 with different concentrations of NAM added;

[0039] Figure 2 This is the growth curve of the NMN-producing engineered strain NMN02;

[0040] Figure 3 This is the growth curve of the NAM domesticated strain population;

[0041] Figure 4 The OD600 of 93 NAM-acclimated bacteria in deep-well plates is compared with that of the starting strains. The left picture shows the OD600 of 93 NAM-acclimated bacteria in deep-well plates. 600 , the right picture is 48h OD 600 ;

[0042] Figure 5 This is the statistical chart of the top 15 NAM domesticated bacteria in terms of bacterial density growth;

[0043] Figure 6 OD of ten NAM acclimated bacteria after 24h fermentation 600 Comparison chart with the starting strain NMN02;

[0044] Figure 7 The figure shows the comparison of NMN production of ten NAM-acclimated strains and the starting strain NMN02 after 24 hours of fermentation.

[0045] Figure 8 This is the fermentation process of AE74 in a 5L tank;

[0046] Figure 9 The figure shows the survival rate (%) of the acclimated strain and the control strain under 20 g / L NAM stress culture.

[0047] Deposit of biological materials:

[0048] A strain of Escherichia coli AE74 was deposited in the China Center for Type Culture Collection on December 23, 2022, with the deposit number CCTCC NO: M 20222059, and the deposit address is Wuhan University, Wuhan City, Hubei Province, China. DETAILED DESCRIPTION

[0049] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. 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.

[0050] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0051] Among them, the starting strain adopts the strain NMN02 constructed in the patent document with application number 202210781815.2.

[0052] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0053] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the specific embodiments described below. It should also be understood that the terminology used in the examples is intended to describe specific embodiments and is not intended to limit the scope of the present invention. The experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0054] When the embodiments provide 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 may 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 understanding of the prior art by those skilled in the art and the description 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 may also be used to implement the present invention.

[0055] 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 art.

[0056] 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 substrate NAM. When the NAM concentration in the fermentation broth reaches 5g / L, the bacterial growth is inhibited by 50% compared to the case where there is no NAM. The growth curve of strain NMN02 was measured under the addition of different concentrations of NAM, and the results are as follows. Figure 1 As shown, the growth of strain NMN02 was significantly inhibited in a high NAM environment.

[0057] In this application, the starting strain is continuously passaged in a culture medium containing NAM, and the concentration of NAM in the culture medium is gradually increased to force the chassis cells to evolve in a directed manner, thereby obtaining a new engineered bacterium with excellent anti-NAM performance and high NMN yield.

[0058] In the previous fermentation process of the NMN-producing strain NMN02, the maximum concentration of NAM feed was 8 g / L at one time, and the maximum NMN production could reach 20 g / L. Therefore, it can be considered that the growth of the cells transferred to the chassis under the condition of 15 g / L NAM culture medium was basically not inhibited, which was the evolutionary end point.

[0059] In this application, single cells were sorted from the evolved population using cell sorting technology. Initial screening was performed on 96-well plates. The 10 best-growing strains were selected by measuring the growth of each single cell in a culture containing 15 g / L NAM. Rescreening was performed in shake flasks 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.

[0060] The culture medium components in this application are as follows:

[0061] LB plate: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar powder 15 g / L;

[0062] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L;

[0063] 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 extract 10 g / L, citric acid monohydrate 1.1 g / L, vitamin B1 0.1 g / L, nicotinamide 1-10 g / L, nicotinamide 200 g / L, fed at 5 mL / h.

[0064] Example 1 NAM adaptive evolution passage process

[0065] Take the original strain of NMN02 and streak it on the LB plate. Pick a single colony and activate it in LB liquid medium. Take samples in real time to measure OD. 600 , draw a growth curve (such as Figure 2 Generally speaking, the strain has higher metabolic activity and is prone to mutation in the middle and late stages of logarithmic growth. According to the growth curve determined in the previous experiment, 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 to OD 600 2-3 are suitable as transfer points for multiple rounds of adaptive evolution.

[0066] NAM has a strong inhibitory effect on the bacteria, so stress culture was carried out with a concentration gradient of 2 g / L.

[0067] Streak culture of NMN02, select 4-5 single colonies and inoculate them into LB liquid medium, culture for about 12 hours, inoculate 2% of the inoculum into LB supplemented with NAM, and perform multiple rounds of transfer culture until the OD value reaches 0. 600 When the concentration reaches 2-3 within 12 hours, it is transferred to the next concentration culture medium and eventually evolves to the end point to obtain an acclimated strain population (which can be considered an evolutionary population). The specific NAM concentration and passage process are shown in Table 1.

[0068] Table 1

[0069] NAM concentration Algebra 2g / L 1、2、3、4、5 4g / L 1、2、3、4、5、6、7 6g / L 1、2、3、···6、7、8 8g / L 1、2、3、···12、13、14 10g / L 1、2、3、···17、18、19、20 12g / L 1、2、3、···21、22、23、24、25 14g / L 1、2、3、···27、28、29、30、31 15g / L 1、2、3、…12、13、14、15、16、17

[0070] Note: The passage number is counted from 1 after transferring to the next concentration of culture medium.

[0071] After multiple rounds of substrate stress cultivation, the growth curves of the domesticated strain population and the starting strain were measured until the growth trend of the domesticated strain population approached that of NMN02 or the inhibitory effect was significantly improved, reaching the end point of adaptive evolution.

[0072] The growth curves of the domesticated strain population and the starting strain are as follows Figure 3 As shown in the figure, after multiple rounds of passage and domestication, the growth of the NAM domesticated strain group in ordinary LB medium is significantly faster than that of the starting strain NMN02, and in the LB medium supplemented with 15g / LNAM, the growth of the domesticated strain group is also better than that of NMN02. It is believed that the NAM domesticated strain group has been greatly improved by the inhibition of 15g / L NAM and has reached the end point of domestication.

[0073] Example 2 Screening of NAM Acclimated Bacteria

[0074] 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 15 g / L NAM using a gradient dilution spreading method. The larger single colonies were screened and marked, inoculated into 96 deep-well plates, and the OD values ​​were measured. 600 To screen out fast-growing domesticated strains.

[0075] The specific operation method includes: streaking the acclimated strain population that tolerates 15g / L NAM on an LB plate supplemented with 15g / L NAM overnight, then scraping a section of the bacterial line and inoculating it into LB liquid culture medium supplemented with 15g / L NAM, culturing for about 12 hours, gradient dilution, controlling the bacterial solution concentration to 100 cfu / plate, and spreading it on an LB plate supplemented with 15g / L NAM, with 10 plates spread for each gradient, culturing overnight, and selecting 8-9 single colonies with the largest growth on each plate.

[0076] Sterilize a 96-deep-well plate and dry it overnight. Add 1 mL of LB liquid culture medium supplemented with 15 g / L NAM to each well. Use a pipette tip to pick up the above single colonies to the corresponding wells and mark them. A total of 93 single colonies of the domesticated strain were added. The remaining 3 wells were inoculated with the starting strain NMN02 as a control and cultured in a 37°C shaker overnight.

[0077] Pipette a certain amount of bacterial solution into the ELISA plate, dilute to an appropriate concentration, and measure the OD using 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.

[0078] The bacterial density in each well was measured at 24h and 48h. At 24h, the bacterial density of 92 strains of domesticated bacteria was not lower than that of the starting strain, accounting for 98.9%; at 48h, the bacterial density of 80 strains of domesticated bacteria was not lower than that of the starting strain, accounting for 86%. Figure 4 shown.

[0079] The top 20 strains were taken 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 were 600 The growth is greater than 20%, such as Figure 5 As shown, the top ten domesticated strains were determined.

[0080] After deep-well plate screening and sorting by the average growth rate of their bacterial density, the top ten fastest-growing NAM-acclimated bacteria were obtained, namely AE74, AE08, AE96, AE50, AE75, AE89, AE84, AE03, AE90, and AE02.

[0081] Example 3 Shake flask level verification of NAM acclimated bacteria

[0082] The strains screened in Example 2 were inoculated into liquid shake flasks, cultured and rescreened using NMN-producing fermentation medium, samples were taken to draw growth curves, and NMN production was determined by liquid phase analysis. The domesticated strains with fast growth and high NMN production were retained.

[0083] Specifically, the single colony obtained by the above screening was streaked on an LB plate (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 the 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 5% transfer volume, culture at 37°C, 220 rpm, and add IPTG at a final concentration of 0.5 mmol / L to induce when OD600 = 1.0-3.0. Add the substrate nicotinamide at a final concentration of 1-3 g / L. Samples were taken after 24 hours to measure the OD 600 and NMN production, such as Figure 6 、 Figure 7 shown.

[0084] It can be seen that the cell density of the ten NAM domesticated bacteria was not lower than that of the starting strains through shake flask fermentation verification, and the growth was good, among which the OD of AE74 and AE08 was 600The highest, an increase of 15-20% relative to the starting strain. In terms of NMN production, most strains had better NMN production than the starting strain, with AE08, AE62, AE74, AE89, and AE93 having NMN production increased by 2-12.6%, while the remaining strains were equal to or lower than the starting strain.

[0085] AE74 showed significant improvement in bacterial growth and NMN production, so AE74 was selected for fermentation verification at the 5L fermentation tank level.

[0086] Example 4: Fermentation Tank Level Verification of NAM Acclimated Bacteria

[0087] The specific experimental method is as follows: streak the domesticated strain AE74 on an LB plate (kanamycin 50 μg / μL + streptomycin 50 μg / μL), culture at 37°C for 10 h, inoculate the bacteria on one 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 rpm 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%. The culture was carried out at 37°C and 220r / min. The OD was measured. 600 =7.0-13.0, IPTG was added to induce the cells at a final concentration of 0.5 mmol / L, and the substrate nicotinamide was added at a constant rate, with a nicotinamide feed concentration of 200 g / L.

[0088] Strain AE74 grew rapidly in a 5 L jar. Figure 8 As shown, the OD 600 Reached about 68, while the starting strain OD 600 At around 45, it is 1.5 times that of the starting strain, and its growth has been greatly improved.

[0089] The starting strain NMN02 reached its maximum NMN production after 25 hours of induction in a 5L fermenter, producing up to 20.3g / L of NMN. In this application, NMN production was 21.3g / L and 22.4g / L at 24h and 31h of fermentation, respectively.

[0090] Example 5 Determination of the survival rate of NAM acclimated strain AE74

[0091] In order to verify the survival rate of the domesticated strain AE74 under 20g / L NAM stress, a survival rate experiment was conducted under 20g / L NAM stress. The specific experimental steps are as follows:

[0092] 1. Streak the strains NMN02 and AE74 on LB plates respectively;

[0093] 2. Select a single colony and inoculate it into LB medium for activation. Culture at 37°C and 220 rpm for 12-16 hours.

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

[0095] 4. Collect NMN02 and AE74 cells cultured to the middle and late logarithmic growth stages, 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 NAM at a final concentration of 20 g / L and culture for different time periods (0, 1, 2, 4, and 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 viable bacteria was counted and the survival rate was calculated.

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

[0098] The experimental results are shown in Table 2 and Figure 9 As shown in the figure, after stress culture tolerance experiment analysis, the survival rate of NAM-acclimated bacteria AE74 after 6 hours of stress was 31 times that of the control.

[0099] Table 2 Survival rate (%) of acclimated strains and control strains under 20 g / L NAM stress culture

[0100]

[0101] The foregoing is merely 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 modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A strain of Escherichia coli resistant to nicotinamide stress ( Escherichia coli ), characterized in that, The Escherichia coli is Escherichia coli AE74, which was deposited in the China Center for Type Culture Collection on December 23, 2022, with the deposit number CCTCCNO: M 20222059.

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

3. A microbial preparation containing the Escherichia coli according to 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 according to claim 1 or the microbial preparation according to 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 according to 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 culture time is 24-36 h.

Images