CN113755353B - Saccharomyces cerevisiae for preparing NMN - Google Patents
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Abstract
The saccharomyces cerevisiae for catalyzing NR to prepare NMN is obtained by mutagenesis and is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23422. Compared with the original strain, the ability of the saccharomyces cerevisiae for catalyzing NR to synthesize NMN is improved by more than 3 times, and the industrial application feasibility is realized.
Description
Technical Field
The invention belongs to the field of industrial microorganism genetic breeding, and particularly relates to saccharomyces cerevisiae for preparing NMN by catalyzing NR and application thereof.
Background
Nicotinamide mononucleotide (beta-Nicotinamide monoglucoside, short for beta-NMN or NMN) is one endogenous matter in human body, is the product of Nicotinamide phosphoribosyltransferase reaction and participates in intracellular Nicotinamide purine dinucleotide (NAD) + ) Is an important and most direct prerequisite for its synthesis. In humans, NMN is converted to NAD + To exert its physiological function, e.g. to activate NAD + The substrate-dependent enzyme Sirt1 (histone deacetylase, also called sirtuin) regulates cell survival and death, maintains redox status, etc. But it decreases gradually with age. Due to NAD + Too large molecular weight to be absorbed by the body, NAD + The supplement of (A) is realized only by taking precursor substances such as NMN with smaller molecular weight. NMN is also present in many vegetables and fruits on a daily diet, but in very trace amounts, ingested in far insufficient amounts, and therefore requires additional supplementation. In recent years, the research shows that NMN supplementation has the effects of inhibiting cognitive decline caused by aging, protecting cardiovascular and cerebrovascular functions and the like.
Three methods for preparing beta-NMN are reported at present, including chemical synthesis, enzyme catalysis and microbial fermentation. The chemical synthesis method takes nicotinamide and ribose as raw materials, wherein the ribose is subjected to group protection to obtain tetraacetyl ribose, then the tetraacetyl ribose reacts with the nicotinamide, the Nicotinamide Ribose (NR) is obtained through deprotection, and finally the NMN is obtained through phosphorylation. Two schemes of enzyme catalysis are available, namely, NR and ATP (adenosine disodium triphosphate) are used as substrates, and NMN is synthesized under the catalysis of kinase of the substrates; the other one is catalytic synthesis of NMN under a multi-enzyme system by taking nucleoside, nicotinamide and ATP as substrates. For example, CN106755209A discloses the production of nicotinamide mononucleotide by the catalysis of nicotinamide ribokinase using nicotinamide ribose and ATP as substrates; CN108949865A discloses the synthesis of nicotinamide mononucleotide by whole-cell catalysis of immobilized phosphoribosyl pyrophosphate synthetase and nicotinamide phosphoribosyl transferase from D-5-phosphoribosyl, ATP and nicotinamide as raw materials. CN108026130A discloses a method for preparing nicotinamide mononucleotide by reacting nicotinamide, ATP and ribose as raw materials under the catalysis of nicotinamide phosphoribosyltransferase, ribose phosphoribosyl pyrophosphate kinase and ribose kinase; CN108026535A discloses nicotinamide mononucleotide prepared by reacting nicotinamide, ATP and AMP as raw materials with nicotinamide phosphoribosyltransferase, nucleic acid phosphopyrophosphatase kinase and nucleosidase as catalysts. However, NMN is easily degraded in the cell, so a pure enzyme system is mostly used for catalysis, which results in higher production cost. The microbial fermentation method has only a few literature reports, the yield is milligram per liter, and the method has no value of industrial application.
Disclosure of Invention
Considering that ATP is an endogenous substance autonomously produced in cells during the proliferation of microorganisms, the inventors have made efforts to integrate enzyme catalysis with microbial fermentation, and to use an enzyme system existing in a microorganism itself by adding Nicotinamide Riboside (NR) to a medium so that the microorganism can produce NMN by fermentation.
Since fermentation products of generally recognized safe microorganisms (GRAS) such as baker's yeast, saccharomyces cerevisiae, lactic acid bacteria, bacillus subtilis, etc. do not contain endotoxin and are easily accepted by a wide range of food and drug vendors and consumers, emphasis on strain screening is focused on these microorganism species. Then, a strain which can convert exogenous NR into NMN through fermentation is screened out by taking various microorganisms as starting bacteria and is low-sugar saccharomyces cerevisiae (instant high-activity dry yeast (low-sugar type)) provided by Hebei Mali food Co., ltd; and then, the method is subjected to multiple rounds of mutagenesis, so that the capacity of catalyzing NR to synthesize NMN is obviously improved by more than 3 times, the requirement of industrial application is met, the method is named as FHR2021032, and strain preservation is submitted. Therefore, the present invention includes the following technical solutions.
A Saccharomyces cerevisiae (Saccharomyces cerevisiae) for preparing NMN is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23422.
According to a second aspect of the present invention, there is provided the use of the above mentioned saccharomyces cerevisiae for the preparation of NMN.
Specifically, the NMN is prepared by fermentation of the saccharomyces cerevisiae.
Preferably, nicotinamide Riboside (NR) is added to the fermentation medium during the fermentative conversion, and the amount of nicotinamide riboside added may be 10-60g/L, for example 10-40g/L.
The medium used in the fermentation can be any medium suitable for growth and fermentation of saccharomyces cerevisiae.
Preferably, the fermentation medium consists of: 50g/L glucose, 15g/L peptone, 15g/L yeast extract, 5g/L NaCl, KH 2 PO 4 1g/L、K 2 HPO 4 1g/L、MgSO 4 ·7H 2 O 0.3g/L，pH 5.4。
Preferably, the fermentation temperature is 30. + -. 3 ℃ such as 30. + -. 2 ℃ or 30. + -. 1 ℃.
The Latin chemical name of the Saccharomyces cerevisiae mutant screened by mutagenesis is Saccharomyces cerevisiae, the Chinese name is Saccharomyces cerevisiae, the Saccharomyces cerevisiae mutant is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 16 days at 09 months at 2021, the preservation address is microbial research institute of China academy of sciences No. 3 of West Lu 1 of North Chen of the Korean area in Beijing, and the preservation number is CGMCC No.23422.
Drawings
FIG. 1 is a photograph showing the state of plate culture of the starting strain (highly active dry yeast (low sugar type)) and the mutant strain FHR2021032 cultured for 48 hours.
Detailed Description
Many microorganisms contain enzyme systems composed of enzymes such as purine nucleoside phosphorylase, nicotinamide ribokinase, and uridine phosphatase, and can catalyze the reaction of NAD + precursor supplements such as Nicotinamide (NAM), nicotinic Acid (NA), and Nicotinamide Riboside (NR) with ATP to produce Nicotinamide Mononucleotide (NMN). Therefore, microorganisms can theoretically convert NR added in vitro into NMN by fermentation, so that enzyme catalysis and microbial fermentation methods are combined to synthesize NMN in one pot.
Through experimental screening of nearly hundreds of microorganisms of different types or subspecies, the low-sugar saccharomyces cerevisiae (purchased from north Hebei Mali food Co., ltd., high-activity dry yeast (low-sugar type) instantly produced by Onjianmari Yanshan) can realize the conversion of NR to NMN. However, the ability of the compound to catalyze NR reaction is too low, and strain improvement is required for industrial application and development.
Mutation breeding is a common and effective means for microbial improvement, a method for artificially mutating and improving strains is still an effective means for breeding high-yield strains, and currently used mutagens can be basically divided into four categories, namely physical mutagens (such as ultraviolet rays, X rays, fast neutrons, normal pressure room temperature plasma (ARTP) and the like), chemical mutagens (such as nitrogen mustard, diethyl sulfate, nitrosoguanidine and the like), biological mutagens (such as bacteriophage) and compound mutagens, which can improve the mutation frequency of organisms and cause mass death of the organisms.
The inventors repeatedly performed multiple rounds of mutagenesis treatment on the developed strain (high-activity dry yeast (low-sugar type) by using a combined mutagenesis form of Ultraviolet (UV) mutagenesis and Nitrosoguanidine (NTG) chemical mutagenesis. However, the mutation strain with obviously improved catalytic activity and stable genetic character is obtained by Ultraviolet (UV) mutagenesis which is carried out firstly, and is named as FHR2021032. The mutant strain FHR2021032 is then subjected to chemical mutagenesis by using nitrosoguanidine, but no further great improvement is achieved, the effect of forward mutation is not obvious and the expected effect is not achieved, and new physical mutagenesis and/or chemical mutagenesis is awaited to be further tried so as to obtain a new unexpected mutant strain.
The morphological and physiochemical characteristics of the mutant strain CGMCC No.23422 are as follows: colony color: milky white; growth temperature: 30 ℃; optimum pH:5.0-6.0; colony morphology: the surface is smooth, moist and sticky, and is easy to pick up and uniform in texture; the reproduction mode is as follows: budding, see figure 1.
The present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.
In the examples, the addition, content and concentration of various substances are mentioned, wherein the percentages refer to mass percentages unless otherwise indicated.
Examples
Materials and methods
Saccharomyces cerevisiae (instant high-activity dry yeast (low-sugar type)) is purchased from Hebei Mali food Co., ltd.
YPD medium: 10g/L yeast extract, 20g/L peptone, 20g/L glucose, pH 5.8-6.0. (solid medium additionally agar 20 g/L)
Fermentation medium: 50g/L glucose, 15g/L peptone, 15g/L yeast extract, 5g/L NaCl, KH 2 PO 4 1g/L、K 2 HPO 4 1g/L、MgSO 4 ·7H 2 O 0.3g/L，pH 5.4。
Ultraviolet mutagenesis instrument: beijing Saiboyao technologies, inc., type CB10-UV 8A.
Nicotinamide ribose, medium ingredients, nitrosoguanidine, and other reagents were purchased from Shanghai chemical Co., ltd., china pharmaceutical Co., ltd.).
HPLC detection method of substrate NR and product NMN content:
agilent 1260 liquid chromatograph. The chromatographic column is XDB-C18 (4.6X 150mm,5 μm), and the wavelength is =254nm when detected by an ultraviolet detector; flow =1.0mL/min, sample size =5 μ L, column temperature =35 ℃, mobile phase a is 50mM potassium dihydrogen phosphate +10% methanol, PH =6.5 with TBAH; the mobile phase B is methanol, A: b =97:3, running time is 2.5min; retention time (min): NR: about 1.4; NMN: about 1.9.
Example 1: UV mutagenesis and screening
Yeast cell suspension preparation:
inoculating Saccharomyces cerevisiae (instant high-activity dry yeast (low-sugar type)) in YPD liquid culture medium test tube, culturing at 30 deg.C and 220rpm for 24 hr, collecting 5ml, and inoculating to 500 containing 50ml YPD liquid culture mediumThe cells were incubated in a ml Erlenmeyer flask at 30 ℃ and 200rpm for 16h. At this point, the cells are in logarithmic growth phase. Diluting the fermentation broth with sterile water to a concentration of 10 5 CFU/mL of starting bacterial suspension.
Ultraviolet (UV) physical mutagenesis:
starting an ultraviolet lamp of an ultraviolet mutagenic instrument, preheating for 30min, putting 3ml of yeast cell suspension into a culture dish with the diameter of 9cm and the liquid thickness of about 1.5mm, placing the culture dish at a position which is about 30cm away from a lamp tube, respectively irradiating for 1, 2, 3, 4 and 5min, and taking out. And (3) performing 10-fold gradient dilution on the mutagenized liquid and the non-mutagenized bacterial liquid, coating a flat plate, performing parallel sampling, culturing for 2 days in a 30 ℃ biochemical incubator, counting and observing results, and calculating the lethality.
Lethality (%) = (number of control colonies-number of treated colonies)/number of control colonies × 100%.
Selecting mutagenesis measurement (4 min) with 95% lethality as mutagenesis irradiation time of the test, coating starting bacteria in logarithmic growth phase after UV irradiation mutagenesis on YPD solid culture medium, culturing in 30 ℃ biochemical incubator for 48h to grow single colony on the plate, obtaining ultraviolet mutagenesis mutant library, and collecting 1 ten thousand strains in total.
Example 2: high throughput screening of mutant strains
Single colonies were picked from YPD plates of the UV-mutagenized mutant pool, inoculated into 96-well deep-well plates containing 200. Mu.L of YPD medium, and cultured at 30 ℃ and 220rpm for 48 hours. Then, 200. Mu.L of prepared NR mother liquor (40 g/L) was added thereto, and after further culturing at 30 ℃ and 220rpm for 24 hours, the fermentation broth was transferred to a 1ml EP tube, centrifuged at 12000g for 1 minute, and subjected to HPLC analysis.
Then, single colonies with high NMN content are respectively inoculated into 3 holes, re-cultured and transformed according to the method, and the content is determined through a liquid phase, so that the single colonies with high NMN content are taken as high-efficiency bacteria. And then the UV mutation breeding is carried out repeatedly by taking the strain as a starting strain. After 5 rounds of ultraviolet mutagenesis, a mutant strain with obviously improved activity is finally obtained, and the serial number of the mutant strain is FHR2021032.
Verification and comparison of mutant FHR2021032 and starting bacterium:
fermentation preparation of yeast: single colonies of the initial strain and the mutant strain FHR2021032 are respectively picked out, and are fermented in a 250mL shaking flask filled with 30mL of fermentation medium by YPD liquid medium (the liquid loading is 30mL, the temperature is 28 ℃, and the rotating speed is 220 rpm) for 48h. Then adding NR with the final concentration of 10g/L and glucose with the final concentration of 10g/L into the fermentation liquor, continuing to ferment for 24h, centrifuging the fermentation liquor at 12000g for 1 min, taking the supernatant, and detecting the content of NMN. The results are shown in Table 1.
Table 1 comparison of the conversion Effect of mutant bacterium FHR2021032 and the developing bacterium on NR
| Strain of bacillus | NMN content (g/L) of fermentation broth |
| Growth promoting bacteria | 0.7 |
| FHR2021032 | 2.9 |
The shake flask experiment result shows that the ability of mutant bacterium FHR2021032 to catalyze NR to be converted into NMN is improved by more than 3 times than that of the developed bacterium.
Example 3: chemical mutagenesis of mutant bacterium FHR2021032
And continuously carrying out chemical mutagenesis on the mutant bacterium FHR2021032, and selecting Nitrosoguanidine (NTG) as a mutagen.
Yeast cell suspension preparation:
the mutant FHR2021032 is inoculated into a test tube containing YPD liquid culture medium, cultured for 24h at 30 ℃ and 220rpm, then 5ml of the mutant is inoculated into a 500ml triangular shaking flask containing 50ml of YPD liquid culture medium, and cultured for 12h at 30 ℃ and 200 rpm. At this point, the cells are already in logarithmic growth phase. 4000g of centrifugation for 3 minutes, yeast cells were collected, resuspended in 0.1M potassium phosphate buffer, pH6.0, diluted with sterile water,is prepared to have a concentration of 10 6 CFU/mL of bacterial suspension.
Nitrosoguanidine (NTG) chemical mutagenesis:
10-20mg of nitrosoguanidine is respectively weighed and dissolved in yeast suspension (when the concentration of the nitrosoguanidine is high, a small amount of acetone is added for assisting dissolution) to obtain 3 nitrosoguanidine treatment solutions with concentration of 1mg/ml, 1.5mg/ml and 2mg/ml respectively. Mutagenizing at 28 deg.C and 250r/min with shaking for 60min, 90min and 120min, and terminating mutagenesis with sterile water. Post mutagenesis dilution 10 -5 And 10 -4 And (4) coating. And (3) placing the coated plate upside down into an incubator at 28 ℃ for 3 days, counting the plate, and selecting the colony number within 50 as a standard for counting.
YPD plates treated with nitrosoguanidine with a lethality of 95%, i.e., 1.5mg/ml, for 120min were selected, single colonies were picked from the plates, and the ability of the strain to catalyze the synthesis of NMN by NR was determined as in example 2.
After 3 rounds of chemical mutagenesis, most of the mutagenized strains have negative mutation relative to FHR2021032, namely the capacity of catalyzing NR reaction is reduced. The catalytic activity of only a very individual mutant strain is improved by about 10-20% compared with FHR2021032, and unfortunately, negative mutation occurs after continuous mutagenesis.
The strategy of chemical mutagenesis requires adaptation and further attempts.
Example 4: investigation of genetic stability of strains
Coating the mutant bacterium FHR2021032 on a YPD plate, culturing for 48 hours in a biochemical incubator at 30 ℃, growing a single colony on the plate, and selecting the single colony as a generation 1 bacterium; coating the 1 generation bacteria on a YPD solid culture medium, culturing for 2 days according to the same culture conditions, and selecting a single colony as 2 generation bacteria; the passage is repeated until 5 generations of bacteria are obtained.
Mutant FHR2021032 and 1 to 5 generations of the bacteria were cultured and NR-transformed in the same manner as in example 2, and the final NMN content of each bacteria was as shown in Table 2 below.
Table 2, results of examining genetic stability of NR transformation at passages 1 to 5 of mutant bacterium FHR2021032
| Bacterial strains | NMN content (g/L) of fermentation broth |
| Mutant bacterium FHR2021032 | 2.7 |
| Generation 1 | 2.5 |
| Generation 2 | 2.5 |
| Generation 3 | 2.4 |
| Generation 4 | 2.7 |
| 5 generation | 2.9 |
Passage experiments show that the ability of the mutant bacterium FHR2021032 to catalyze NR to NMN is not attenuated due to increase of passage times, and the mutant bacterium FHR has high genetic stability.
Example 5: fermentation amplification experiment of mutant bacterium FHR2021032
The mutant FHR2021032 is inoculated into a plurality of 500ml triangular shaking flasks containing 50ml YPD liquid culture medium, cultured for 12h at 30 ℃ and 200rpm, and then transferred into 2.5L shaking flasks containing 500ml YPD liquid culture medium according to the proportion of 10v/v%, and cultured for 48h at 28 ℃ and 220 rpm. Then, the yeast cells were collected by centrifugation, and the obtained fresh yeast cells were resuspended in 0.1M potassium phosphate buffer solution, pH6.0, and then transferred to a 250ml shake flask containing 30ml YPD liquid medium, after addition of NR at a final concentration of 10g/L and glucose at a final concentration of 10g/L, fermentation was continued at 220rpm at 28 ℃ for 24 hours, and the NMN content in the fermentation broth was detected to be 9.1g/L.
The experiment shows that the mutant strain FHR2021032 has stable genetic character, can catalyze NR to be converted into NMN efficiently, and has the possibility of industrial application.
Claims (6)
1. The Saccharomyces cerevisiae (Saccharomyces cerevisiae) for preparing NMN is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23422.
2. Use of the saccharomyces cerevisiae yeast according to claim 1 for the preparation of NMN.
3. The use according to claim 2, wherein NMN is produced by fermentation of saccharomyces cerevisiae according to claim 1.
4. The use of claim 3, wherein nicotinamide riboside is added to the fermentation medium during fermentative conversion, wherein the amount of nicotinamide riboside added is 10-60g/L.
5. Use according to claim 3, wherein the fermentation medium consists of: 50g/L glucose, 15g/L peptone, 15g/L yeast extract, 5g/L NaCl, KH 2 PO 4 1g/L、K 2 HPO 4 1g/L、MgSO 4 ·7H 2 O0.3g/L，pH 5.4。
6. Use according to claim 3, wherein the fermentation temperature is 30 ± 2 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018120069A1 (en) * | 2016-12-29 | 2018-07-05 | 苏州汉酶生物技术有限公司 | METHOD FOR ENZYMATICALLY PREPARING β-NICOTINAMIDE MONONUCLEOTIDE |
| CN112322511A (en) * | 2020-11-13 | 2021-02-05 | 湖州颐盛生物科技有限公司 | Saccharomyces cerevisiae for producing coenzyme I |
| CN112662699A (en) * | 2021-01-07 | 2021-04-16 | 钇澜杉生物科技(北京)有限公司 | Nicotinamide riboside kinase whole yeast cell and process for synthesizing NMN by biocatalysis thereof |
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| WO2018120069A1 (en) * | 2016-12-29 | 2018-07-05 | 苏州汉酶生物技术有限公司 | METHOD FOR ENZYMATICALLY PREPARING β-NICOTINAMIDE MONONUCLEOTIDE |
| CN112322511A (en) * | 2020-11-13 | 2021-02-05 | 湖州颐盛生物科技有限公司 | Saccharomyces cerevisiae for producing coenzyme I |
| CN112662699A (en) * | 2021-01-07 | 2021-04-16 | 钇澜杉生物科技(北京)有限公司 | Nicotinamide riboside kinase whole yeast cell and process for synthesizing NMN by biocatalysis thereof |
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| 酿酒酵母发酵制备RNA的研究;杨依顺等;《药物生物技术》;20130815(第04期);310-313 * |
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