CN113755353A

CN113755353A - Saccharomyces cerevisiae for preparing NMN

Info

Publication number: CN113755353A
Application number: CN202111214400.9A
Authority: CN
Inventors: 葛永超
Original assignee: Fenghuolun Shanghai Biotechnology Co ltd
Current assignee: Fenghuolun Shanghai Biotechnology Co ltd
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2021-12-07
Anticipated expiration: 2041-10-19
Also published as: CN113755353B

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

Saccharomyces cerevisiae for preparing NMN

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 the synthesis of (a). In humans, NMN is converted to NAD⁺To exert its physiological function, e.g. to activate NAD⁺Substrate-dependent enzyme Sirt1 (histone deacetylase, also known as sirtuin), regulates cell survival and death, maintains redox status, and the like. But it decreases gradually with age. Due to NAD⁺Too high 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. Recent studies have found that supplementary NMN hasInhibiting cognitive decline caused by aging, and protecting cardiovascular and cerebrovascular functions.

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. The enzyme catalysis has two schemes, one is that NR and ATP (adenosine triphosphate disodium) are taken as substrates, and NMN is synthesized under the catalysis of kinase thereof; 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 that nicotinamide riboside and ATP are used as substrates, and nicotinamide mononucleotide is generated under catalysis of nicotinamide ribokinase; CN108949865A discloses that nicotinamide mononucleotide is synthesized by using D-5-phosphoribosyl, ATP and nicotinamide as raw materials through whole-cell catalysis of immobilized phosphoribosyl pyrophosphate synthetase and nicotinamide phosphoribosyl transferase. CN108026130A discloses that nicotinamide mononucleotide is prepared by reacting nicotinamide, ATP and ribose as raw materials under the catalytic action of nicotinamide phosphoribosyltransferase, ribose phosphopyrophosphate kinase and ribose kinase; CN108026535A discloses that nicotinamide mononucleotide is prepared by reacting nicotinamide, ATP and AMP as raw materials under the catalytic action of nicotinamide phosphoribosyltransferase, nucleic acid phosphopyrophosphorykinase and nucleosidase. However, NMN is easily degraded in the cell, so a pure enzyme system is often 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 focused on integrating enzyme catalysis with microbial fermentation, by adding Nicotinamide Riboside (NR) to a medium, using an enzyme system present in the microorganism itself, 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 (high-activity dry yeast (low-sugar type) produced by Hippocampus 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.

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.

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 10-40 g/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 and 15g/L, NaCl 5g/L, KH yeast extract₂PO₄ 1g/L、K₂HPO₄ 1g/L、MgSO₄·7H₂O 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 (high-activity dry yeast (low-sugar type) immediately after the fermentation in Okinawa mountain and the mutant strain FHR2021032 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 Ribose (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., Yinjianmari Yanshan instant high-activity dry yeast (low-sugar type)) 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 repeated multiple rounds of mutagenesis on the starting strain, i.e., high activity dry yeast (low sugar type) in Olympic Marie Yanshan, using a combined mutagenesis format of Ultraviolet (UV) mutagenesis + 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 FHR 2021032. 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 mutant strain CGMCC No.23422 has the following morphological and physiochemical characteristics: 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 should be understood that the following examples are illustrative 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 (high activity dry yeast (low sugar type) from Hibei Marie food Co., Ltd.) was purchased.

YPD medium: 10g/L yeast extract, 20g/L peptone, 20g/L glucose, pH 5.8-6.0. (solid medium additionally agar 20g/L)

Fermentation medium: 50g/L glucose, 15g/L peptone and 15g/L, NaCl 5g/L, KH yeast extract₂PO₄1g/L、K₂HPO₄ 1g/L、MgSO₄·7H₂O 0.3g/L，pH 5.4。

Ultraviolet mutagenesis instrument: beijing Saiboyao technologies, Inc., model 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(4.6 × 150mm, 5 μm), and the wavelength is 254nm when detected by an ultraviolet detector; the flow rate is 1.0mL/min, the sample amount is 5. mu.L, the column temperature is 35 ℃, the mobile phase A is 50mM potassium dihydrogen phosphate + 10% methanol, and the pH is adjusted to 6.5 by TBAH; the mobile phase B is methanol, A: b ═ 97: 3, running time is 2.5 min; 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, taking 5ml, inoculating in 50ml YPD liquid culture medium triangular shake flask, and culturing at 30 deg.C and 200rpm for 16 hr. At this point, the cells are in logarithmic growth phase. Diluting the fermentation broth with sterile water to a concentration of 10⁵CFU/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) carrying out gradient dilution on the mutagenized bacteria liquid and the non-mutagenized bacteria liquid by 10 times, coating a flat plate, carrying out parallel sampling on 3 samples, culturing for 2 days in a 30 ℃ biochemical incubator, counting and observing results, and calculating the lethality.

Lethality (%) - (control colony count-treated colony count)/control colony count × 100%.

Selecting mutagenesis measurement (4min) 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 (40g/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 by a liquid phase, and 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 mutant strain is numbered FHR 2021032.

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 220rpm) for 48 h. 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

Bacterial strains	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

The mutant FHR2021032 is subjected to chemical mutagenesis, and Nitrosoguanidine (NTG) is selected 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. Centrifuging at 4000g for 3 minutes, collecting yeast cells, resuspending with 0.1M potassium phosphate buffer (pH6.0), diluting with sterile water to give a concentration of 10⁶CFU/mL of bacterial suspension.

Chemical mutagenesis of Nitrosoguanidine (NTG):

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^-5And 10^-4And (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 adjustment and further attempts.

Example 4: investigation of genetic stability of strains

Coating the mutant FHR2021032 on a YPD plate, culturing for 48h 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 the 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 between generations 1 and 5 in 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 10 v/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 checked to be 9.1 g/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

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 10-60 g/L.

5. Use according to claim 3, wherein the fermentation medium consists of: 50g/L glucose, 15g/L peptone and 15g/L, NaCl 5g/L, KH yeast extract₂PO₄ 1g/L、K₂HPO₄ 1g/L、MgSO₄·7H₂O0.3g/L，pH 5.4。

6. Use according to claim 3, wherein the fermentation temperature is 30 ± 2 ℃.