CN112322511B

CN112322511B - Saccharomyces cerevisiae for producing coenzyme I

Info

Publication number: CN112322511B
Application number: CN202011266455.XA
Authority: CN
Inventors: 陶荣盛; 朱傅赟; 郑云; 沈正权; 原犇犇; 孙梁栋; 潘震华; 沈青; 胡海亮; 刘萍
Original assignee: Huzhou Yisheng Biotechnology Co ltd
Current assignee: Huzhou Yisheng Biotechnology Co ltd
Priority date: 2020-11-13
Filing date: 2020-11-13
Publication date: 2022-11-29
Anticipated expiration: 2040-11-13
Also published as: CN112322511A

Abstract

The saccharomyces cerevisiae for producing coenzyme I is obtained by mutagenesis, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has the preservation number of CGMCC No.20814. The capability of producing coenzyme I by the saccharomyces cerevisiae is improved by more than 4 times compared with that of the original strain, and the saccharomyces cerevisiae has development and application values.

Description

Saccharomyces cerevisiae for producing coenzyme I

Technical Field

The invention belongs to the field of genetic breeding of industrial microorganisms, and particularly relates to a saccharomyces cerevisiae mutant strain with high coenzyme I yield and application thereof.

Background

Coenzyme I, nicotinamide adenine dinucleotide (NAD +), is a coenzyme for many dehydrogenases in the body, and is used to link the tricarboxylic acid cycle and the respiratory chain to realize electron transfer. It is involved in various physiological activities such as cell metabolism, energy synthesis, cell DNA repair, signal transmission and the like, plays an irreplaceable role in glycolysis, gluconeogenesis, tricarboxylic acid cycle and respiratory chain, and has the following structural formula:

in the 40 th 19 th century, NAD + was first discovered, and researchers' exploration of NAD + increasingly focused on aspects of angiogenesis, gene repair, anti-aging, birth defects reduction, and the like, so that NAD + exhibited vitality in the anti-aging and medical fields. Because NAD + has a large molecular weight and is not easy to be absorbed by human bodies, the supplementation of Nicotinamide Mononucleotide (NMN) or Nicotinamide Ribose (NR) which is a precursor of NAD + is probably the most scientific and effective way for supplementing NAD +, but NMN and NR can not pass through a blood brain barrier, and NAD + can be supplemented, so that NAD + has irreplaceable effects in the aspect of treating addiction and other brain imbalance diseases, can be used for treating diseases and symptoms such as chronic diseases, weight control, mood disorders, alcohol addiction and drug addiction, and can also be used for preventing liver injury, multiple sclerosis autoimmune neurodegeneration, heart disease, heart injury caused by stroke, brain injury caused by injury and the like and repairing sequelae thereof.

The preparation method of NAD + comprises the following steps: there are several salvage pathways using nicotinamide mononucleotide, nicotinamide riboside, nicotinamide or nicotinic acid as a starting material in the de novo synthesis pathway, but the yield of NAD + in the de novo synthesis pathway is limited, and thus NAD + is produced by a biological enzyme method or a whole cell transformation method from the salvage pathway of nicotinamide mononucleotide, nicotinamide riboside, nicotinamide or nicotinic acid, which is a NAD + precursor, in many cases. Examples of the biological enzyme method include: starting from nicotinamide, nicotinamide mononucleotide is obtained under the action of nicotinamide phosphoribosyltransferase, and then NAD + is obtained under the action of nicotinamide phosphoribosyl acyltransferase; there are also reactions starting from nicotinamide riboside, which under the action of nicotinamide ribokinase gives nicotinamide mononucleotide, which in turn gives NAD +. However, the biological enzyme method needs to prepare various enzyme solutions, and the process is complicated, so that the industrial cost is relatively high; in addition, NAD + is produced directly from nicotinamide mononucleotide by biological enzyme catalysis, but the method needs chemical synthesis of nicotinamide mononucleotide, so the method has the problems of high cost and chiral compounds. Yeast (Sakai, T.T., et al, accumulation of Nitatinamide Adenosine Dinuclear culture. Agr. Biol. Chem.,1973 (5), 1049-1056.), corynebacterium ammoniagenes (Elhary, H.M., et al, S434F in NrdE genes the thermal sensitivity type of Corynebacterium ammoniagenes CH31and Enhans thermolabile by fermentation of the Surface hydrophilicity of the NrdE (Ts) protein. Applied. Environ. Microbiol.2005,71 (9), 5582-5586.) Nicotinamide, bacillus, and the like have been reported to be used for whole-cell transformation and production of substances such as NAD, but the production cost is low, resulting in high production cost

The production of coenzyme I by either a biological enzymatic process or a whole-cell transformation process requires the culture of a microorganism alone to produce the enzyme or to act as a catalyst to catalyze the enzymatic reaction of a substrate compound. It is clear that it would be a perfect solution if NAD + could be directly produced by a one-step fermentation of a microorganism.

Disclosure of Invention

In order to search and develop coenzyme I producing bacteria with industrial application prospect, the microorganisms in the prior art capable of producing NAD + are screened widely, the fermented NAD + yield of the microorganisms is found to be generally too low, and the microorganisms need to be subjected to genetic engineering modification or mutation breeding to greatly improve the NAD + production capacity of the strains, so that the industrial application and development are possible. In both research directions, we have made some progress, for example, by mutagenizing Saccharomyces cerevisiae to obtain a mutant strain with vigorous growth and significantly improved NAD + production capacity. Specifically, the present invention includes the following technical solutions.

A saccharomyces cerevisiae for producing coenzyme I 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.20814.

According to a second aspect of the present invention, there is provided the use of a Saccharomyces cerevisiae strain as described above for the production of coenzyme I.

Preferably, coenzyme I is produced by fermentation of Saccharomyces cerevisiae as described above.

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 ℃.

In one embodiment, inosine and nicotinamide may also be added to the fermentation medium as substrates.

The saccharomyces cerevisiae CGMCC No.20814 can realize the effective accumulation of the coenzyme I in the fermentation liquor through fermentation, improves the coenzyme I yield by at least 4 times compared with the coenzyme I yield of the original strain, and has industrial application prospect.

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 09 and 24 days in 2020, and the preservation address is microbial research institute of China academy of sciences No. 3 of West Lu 1 of Beijing Korean district, and the preservation number is CGMCC No.20814.

The mutant strain CGMCC No.20814 has the morphological and physiochemical characteristics that: colony color: milky white; growth temperature: 30, of a nitrogen-containing gas; optimum pH at C: 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.

Drawings

FIG. 1 is a photograph showing the state of plate culture of the starting strain SC001 and the mutant strain SC0472 cultured for 48 hours.

Detailed Description

An Angel low-sugar Saccharomyces cerevisiae producing NAD + intracellularly is screened from a plurality of microorganisms, and is numbered SC001 (provided by Huzhou Industrial biotechnology center of Shanghai Life sciences research institute of Chinese academy of sciences). However, the capability of expressing coenzyme I is too low, and strain improvement is required to greatly improve the expression level of the coenzyme I for industrial application and development. We have carried out genome transformation and mutant screening on the yeast through genetic engineering and mutation breeding respectively.

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 inventor adopts an ultraviolet mutagenesis form to carry out multiple rounds of mutagenesis treatment on the starting strain SC001 to obtain a variant strain which has obviously improved coenzyme I yield, vigorous growth (the multiplication speed is faster than that of the original strain SC 001) and stable hereditary characters, and is named as SC0472. The mutant strain has the potential of industrial development and can be used as an object for genetic engineering modification. Therefore, the strain is submitted for preservation with the preservation number of CGMCC No.20814.

In this context, the terms "coenzyme I" and "NAD +" mean the same meaning and are used interchangeably as is well known in the art. Similarly, the terms "ultraviolet" or "ultraviolet" and "UV" are used synonymously.

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

The product NAD + Saccharomyces cerevisiae (number SC 001) is a gift from Huzhou industrial biotechnology center of Shanghai Life sciences research institute of Chinese academy of sciences.

YPD solid Medium: 10g/L of yeast extract, 20g/L of peptone, 20g/L of glucose, 20g/L of agar and pH of 5.8-6.0;

YPD liquid seed Medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, pH 5.8-6.0;

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 appearance: beijing Saiboyao technologies, inc., type CB10-UV 8A.

Example 1: UV mutagenesis and screening

Yeast cell suspension preparation:

inoculating 1-ring Saccharomyces cerevisiae SC001 activated strain into YPD liquid seed culture medium test tube, culturing at 30 deg.C and 200rpm for 24 hr, inoculating 5ml into 250ml triangular shake flask containing 50ml seed culture medium, and culturing at 30 deg.C and 200rpm for 12 hr to make the cell in middle logarithmic growth phase. Diluting with sterile water to obtain a solution with a concentration of 10 ⁶ CFU/mL of bacterial suspension.

Ultraviolet mutagenesis:

preheating for 30min with ultraviolet lamp, placing 4ml yeast cell suspension in a culture dish with diameter of 9cm and liquid thickness of about 2mm, irradiating the culture dish at 30cm position of lamp tube for 1, 2, 3, 4min, and taking out. And diluting the mutagenized liquid and the non-mutagenized bacterial liquid by a 10-fold dilution method, coating the liquid on a flat plate, performing parallel sampling on 3 samples, culturing the samples in a 30-DEG C biochemical incubator for 2 days, counting the observation results, and calculating the lethality.

Lethality (%) = (number of control colonies-number of treated colonies)/number of control colonies X100%.

And selecting mutagenesis measurement (2 min) with 85% of lethality as mutagenesis irradiation time of the experiment, carrying out UV irradiation mutagenesis on the saccharomyces cerevisiae SC001 in the logarithmic growth phase, coating the saccharomyces cerevisiae SC001 on a YPD solid culture medium, and culturing for 2d in a biochemical incubator at 30 ℃ to enable a single colony to grow on a flat plate, thereby obtaining an ultraviolet mutagenesis mutant library.

Example 2: high throughput screening of mutant strains

A single colony in the ultraviolet mutation mutant library is inoculated into a 96-hole deep-hole culture plate containing 200 mu L of YPD medium by using a toothpick, and cultured for 24 hours at 30 ℃ and 220 rpm.

Extraction of coenzyme NAD +:

vibrating 96-well plate, beating the bacteria evenly, transferring 50 μ L of bacteria liquid to another 96-well plate, adding equal volume of HClO ₄ The solution (3.5 g/L, -20 ℃) stopped the cell metabolism. Freeze thawing at 30 deg.C and-70 deg.C for 2 times repeatedly, destroying cell structure, extracting NAD + and destroying NADH. After the completion of freeze thawing, the pH of the extract was adjusted to 7.0 by titration with 2M KOH under ice bath conditions, centrifuged (12000rpm 10min,4 ℃ C.), 50ul of the supernatant was transferred to another 96-well plate by 8-channel pipette for the enzymatic reaction for L-2-aminobutyric acid production.

Specifically, substrates tetronic acid and ammonia are reduced into L-2-aminobutyric acid by taking NADH as coenzyme under the action of L-amino acid dehydrogenase, NAD + is reduced into NADH by coupling a glucose dehydrogenase coenzyme regeneration system, and the pH of the reaction system is reduced by generated gluconic acid. When the concentration of NAD + added into the reaction system is higher, the reaction is quicker, the pH is reduced more quickly, an acid-base indicator is added to observe the color, and the mutant corresponding to the hole with low pH represents that the yield of NAD + is higher.

mu.L of a reaction mixture (containing 0.5M tetronic acid, 1.0M glucose, 0.05M ammonium sulfate in solution at 30 ℃ in a warm bath, pH7.6 adjusted with ammonia water, glucose dehydrogenase (20000U/L) and valine dehydrogenase (6000U/L), see example 3 of CN 102212567A) was added to each well of a 96-well plate containing 50ul of the supernatant, and reacted at 30 ℃ and 150rpm for 1.5 hours, 50. Mu.L of bromothymol blue was added to each well to observe color change, and mutants with yellow color were detected from the corresponding mother plate and rescreened and shaken for fermentation.

Preparing bromothymol blue: 0.05g of indicator bromothymol blue is dissolved in 100mL of 20% ethanol.

Example 3: investigation of genetic stability of strains

And respectively inoculating the mutant strains with the yield of the NAD + increased by more than two times to YPD plates, culturing for 2d in a biochemical incubator at 30 ℃, continuously passaging for 6 times, measuring the content of the NAD + in strain fermentation liquor according to the method in the example 2, and determining the genetic stability of the mutant strains. Screening out mutant strains with the NAD + yield still keeping more than 80% before passage after 6 times of continuous passage to obtain mutant strains with stable heredity.

Finally, 10 mutant strains with high genetic stability and the yield of NAD + improved by more than 2 times than that of the saccharomyces cerevisiae SC001 are screened. One mutant strain numbered as SC0472 grows faster than SC001, and the fact that the mutant strain is vigorous in vitality and excellent in biological properties is proved, and the reference is made in figure 1.

The mutant strain is comprehensively judged to have development potential, including genetic engineering modification and further mutagenesis, and the important research is carried out below.

Example 4: investigation of the capacity of SC0472 to produce NAD + without addition of exogenous substrate

Shake flask fermentations without addition of exogenous substrate investigate the ability of the mutagenized strain to produce NAD +:

a method for producing NAD + by flask fermentation comprises the following steps: selecting a ring bacterial liquid from a glycerol cryopreservation tube of the strain SC0472 obtained in the embodiment 3, marking a YPD plate, placing the YPD plate in an incubator at 30 ℃, and culturing for 2-3 days; respectively picking activated saccharomyces cerevisiae SC001 and SC0472 by using inoculating loops to be monoclonal into a 500mL shaking flask containing 50mL fermentation medium, placing the shaking flask at 30 ℃ and 220rpm, and stopping fermentation after culturing for 6d to obtain fermentation liquor;

fermentation extracting solution: and (2) centrifuging 20mL of the fermentation liquid in a 50mL centrifuge tube, collecting thalli, adding 5mL of 0.2% formic acid water into the thalli, mixing uniformly in a vortex manner, stirring for 5min at 1000rpm in a water bath at 95 ℃, quickly transferring to an ice bath, stirring for 5-10min at 1000rpm, centrifuging for 5min at 7500rpm at 4 ℃, and taking supernatant to obtain the fermentation extracting solution.

And (3) detecting the content of NAD +: detecting the content of NAD + in the fermentation extract by using a High Performance Liquid Chromatography (HPLC) method, wherein the detection method comprises the following steps: absorbing 1mL of NAD + extracting solution, filtering by using a 0.22 mu m filter membrane, and then carrying out sample detection;

detection conditions for HPLC: the chromatographic column is XDB-C18 (4.6X 150mm,5 μm), and the wavelength is =254nm when detected by an ultraviolet detector; flow rate =1.0mL/min, sample size =5 μ L, column temperature =35 ℃, mobile phase a 50mM potassium dihydrogen phosphate +10% methanol, pH =6.5 with TBAH, mobile phase B methanol, gradient elution, elution program as follows:

wherein the retention time of the NAD + is 3.07min; the retention time of NADP is 11.93min; the ADP retention time is 14.5min; the ATP retention time was 17.63min.

Through detection: the NAD + production of the strains is shown in Table 1.

TABLE 1 NAD + production of strains without addition of exogenous substrate

Bacterial strains	NAD+(g/kg DCW)
		SC001	2.07
SC0472	10.88

The results show that the mutagenized strain SC0472 produced approximately 5.3 times more NAD + without addition of exogenous substrate than the starting strain SC 001.

Example 5: investigation of the capacity of SC0472 to produce NAD + when exogenous substrate was added

Shake flask fermentations with exogenous substrates (inosine and nicotinamide) added to verify the ability of the mutagenized strain to produce NAD +:

method for producing NAD + by shake flask fermentation: selecting a ring bacterial liquid from the strain SC0472 glycerol frozen tube obtained in the embodiment 3, marking a YPD plate, placing the plate in an incubator at 30 ℃, and culturing for 2-3 days; respectively picking activated saccharomyces cerevisiae SC001 and SC0472 by using inoculating loops to be monoclonal into a 500mL shake flask containing 50mL fermentation medium, placing the shake flask in a shaking table at 30 ℃ and 220rpm, adding exogenous substrates inosine and nicotinamide after culturing for 2 days, wherein the final concentration of the inosine is 3g/L and the final concentration of the nicotinamide is 6g/L, and stopping fermentation after continuously culturing for 3 days at 30 ℃ and 220rpm to obtain fermentation liquor;

the NAD + extraction from the fermentation liquor and the HPLC detection method are the same as those described above; the NAD + production of the strain is shown in table 2.

TABLE 3 NAD + production by different strains with exogenous substrate addition

Bacterial strains	NAD+(g/kg DCW)
		SC001	7.26
SC0472	40.65

The results showed that the mutant strain SC0472 produced approximately 5.6 times more NAD + when exogenous substrates (inosine and nicotinamide) were added than the starting strain SC 001.

As can be seen from tables 1and 2, the NAD + production capacity of the mutant strain SC0472 is improved by more than 4 times than that of the original strain SC001, and the experiment also finds that the proliferation speed is faster than that of the original strain SC001, which shows that the mutant strain has excellent growth traits and is worthy of further development and utilization.

Claims

1. Saccharomyces cerevisiae capable of producing coenzyme I by using inosine as substrateSaccharomyces cerevisiae) The strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.20814.

2. Use of the Saccharomyces cerevisiae according to claim 1 for the production of coenzyme I.

3. Use according to claim 2, wherein coenzyme I is produced by fermentation of the Saccharomyces cerevisiae according to claim 1.

4. 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 O 0.3g/L，pH 5.4。

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

6. Use according to claim 3, wherein inosine and nicotinamide are added to the fermentation medium.