Kwon P-1,2,3, a brewing yeast that produces glutathione and aldehyde dehydrogenase

Abstract

The present invention relates to a yeast strain producing glutathione (GSH) and acetaldehyde dehydrogenase, and more particularly, to a Saccharomyces cerevisiae Kwon P-1 KCTC 13925BP, a Saccharomyces cerevisiae Kwon P-2 KCTC 14122BP, and a Saccharomyces cerevisiae Kwon P-3 KCTC 14123BP yeast strain for simultaneously producing glutathione and acetaldehyde dehydrogenase.

Description

Technical Field

[0001] This invention relates to a yeast strain that produces glutathione (GSH) and acetaldehyde dehydrogenase. More specifically, this invention relates to three yeast strains, *Saccharomyces cerevisiae* Kwon P-1KCTC 13925BP, Kwon P-2KCTC14122BP, and Kwon P-3KCTC14123BP, that simultaneously produce glutathione and acetaldehyde dehydrogenase. Background Technology

[0002] Glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH), a bioactive substance found in cells, is a tripeptide composed of three amino acids: glutamate, cysteine, and glycine. It exists in the cells of animals, plants, and microorganisms at a concentration of 0.1–10 mM and accounts for more than 90% of the total non-protein active components of cells.

[0003] Biological glutathione enhances immune activity through leukocyte production, playing an important role as an antiviral agent. Furthermore, as the matrix of GST (glutathione S-transferase), it binds harmful toxic substances into a conjugated form, playing a crucial role in detoxification, such as xenobiotics.

[0004] Furthermore, glutathione plays an oxidative role within cells, preventing damage and necrosis of cell membranes, nucleic acids, and cell structures, and alleviating the toxicity of reactive oxygen species (ROS), which contribute to aging. ROS are formed through the metabolic processes of various biological substances, including superoxide, peroxide, and hydroxyl radicals. They can be categorized into endogenous ROS generated from biological metabolic products and exogenous ROS generated from sources such as cigarettes and radiation.

[0005] Oxidative stress caused by reactive oxygen species can impair cognitive function (Liu et al. 2002), damage sperm DNA, becoming a cause of male infertility (Wright et al. 2014), and also damage cellular proteins, lipids, and nucleic acids, potentially leading to cancer, reducing physiological function, and contributing to various diseases and aging. Therefore, antioxidants, which play a crucial role in preventing disease, enhancing immunity, and preventing aging, are extremely important for the human body. The function of glutathione, which acts as an antioxidant within cells, is attracting attention from numerous medical fields, including enzymology, pharmacology, medicine, toxicology, endocrinology, and microbiology.

[0006] Glutathione is primarily synthesized within the body, but its absolute content decreases due to disease, weakened immunity, aging, and other abnormal conditions, leading to a decline in health. Therefore, exogenous glutathione can scavenge intracellular reactive oxygen species, maintaining health and slowing aging.

[0007] Due to its physiological activity in vivo, glutathione is currently being used in food, cosmetics, feed, and pharmaceuticals, and its usage is gradually increasing.

[0008] Furthermore, although glutathione can be produced using edible microorganisms, the inherent glutathione content produced by these microorganisms is extremely low. Therefore, research is underway to increase the glutathione content of microorganisms through mutation and recombination technologies, and to utilize their fermentation processes to mass-produce high-content glutathione-producing strains.

[0009] Therefore, the development of high-glutathione-content strains is a key source of material for enhancing economic value, enabling glutathione to be widely used in health foods, pharmaceuticals, and animal feed, thus gaining market competitiveness. When developing strains using genetic recombination technology, the current hot topic of GMOs cannot be ignored, as its application is limited. However, when using mutation technology to breed strains, its application is unrestricted, allowing for a wide range of uses. Therefore, the breeding of high-glutathione-content production strains using mutation technology is a highly needed technology.

[0010] Furthermore, with South Korea's economic growth, alcohol consumption has surged, making public health a critical issue. Excessive alcohol consumption has become a significant social problem, impacting both public health and the socio-economic landscape. According to surveys by the Ministry of Health and Welfare and the Korea Centers for Disease Control and Prevention, as of 2016, approximately 75% of South Korean adults aged 20 and over consumed alcohol more than once a month, a rate that increases annually. Meanwhile, according to a WHO survey of alcohol consumption across countries, as of 2016, the average alcohol consumption per capita for citizens aged 15 and over was 11.9 liters, exceeding the global average by 4.8 liters, ranking 17th globally and 1st among countries in the Western Pacific region.

[0011] In particular, reports indicate that excessive aldehydes produced from alcohol consumption in South Koreans can lead to oxidative illnesses such as cardiovascular disease, diabetes, neurodegenerative diseases, cancers of the upper digestive and respiratory organs, radiation dermatitis, Fanconi anemia, peripheral nerve damage, inflammation, osteoporosis, and aging (Chen et al. 2014).

[0012] Furthermore, according to reports, in most countries, the socioeconomic losses caused by alcohol consumption account for approximately 0.5-2.7% of GDP. It is estimated that in South Korea alone, the socioeconomic costs incurred due to alcohol consumption in 2000 reached 14.9352 trillion won, of which 6.2845 trillion won was due to reduced productivity and losses caused by illness, accidents, and hangovers (Jung Woo-jin et al. 2006).

[0013] To address these social problems, research and testing are underway on numerous substances that reduce or prevent the expression of ethanol toxicity, and the results are being developed into various health food products. Alcohol entering the body is absorbed by the gastrointestinal tract or small intestine and enters the bloodstream, then travels to the liver where it is broken down and detoxified.

[0014] Alcohol dehydrogenase (ADH), present in stem cells, first oxidizes alcohol to acetaldehyde. Acetaldehyde is then further broken down into acetic acid by aldehyde dehydrogenase (ALDH) located in liver cells. Acetaldehyde is then transported to muscle and adipose tissue throughout the body, ultimately decomposed into carbon dioxide and water. As the initial metabolite of ethanol, acetaldehyde is far more reactive and toxic than ethanol, making it a major contributing factor to hangovers and alcoholic liver dysfunction.

[0015] According to the report, there are 19 types of aldehyde dehydrogenases in the human body (Marchitti et al. 2007, 2008). Among them, according to the analysis results of enzyme engineering, the enzyme mainly exists in mitochondria, acetaldehyde dehydrogenase 2. When acetaldehyde is used as the substrate for analysis, it has a lower Km value (~0.2 μM) compared with other types of aldehydes, and it can better oxidize and remove acetaldehyde derived from alcohol.

[0016] The efficient conversion of acetaldehyde, a hangover-inducing substance generated during the metabolism of ethanol in the body, into acetic acid and the removal of aldehydes are extremely important for human health (Eriksson et al. 1977, Klyosov et al. 1996-1). Furthermore, acetaldehyde dehydrogenase 2 is also involved in the metabolism of acetaldehyde, as well as aliphatic aldehydes, aromatic aldehydes, and polycyclic aldehydes, clearing toxic substances from the body (Klyosov et al. 1996-2).

[0017] As a representative example, it plays the following roles: clearing oxidized aldehydes 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) produced during oxidative stress, and clearing acrolein produced in smoke and automobile exhaust (Chen et al. 2010, Yoval-Sanchez et al. 2012). Individuals with low expression of acetaldehyde dehydrogenase 2 (ALD2) or whose 487th amino acid residue has been mutated from glutamate to lysine exhibit facial flushing, and even small amounts of alcohol consumption will elicit a sensitive reaction. Furthermore, due to the inability to convert ALD2, the concentration of acetaldehyde in the blood is high during alcohol consumption (Yoshida et al. 1984).

[0018] In particular, research has shown that aldehyde dehydrogenase 2 (ALDH2)-2 is an isoform of aldehyde dehydrogenase 2, and when humans possess it, they are more vulnerable to alcohol consumption. This genetic variant is almost non-existent in Western populations, but it is present in 50% of the populations of Koreans, Chinese, and Japanese (Brooks et al. 2009).

[0019] Regarding the research and development of aldehyde dehydrogenase 2, there is active research on in vivo aldehyde dehydrogenase 2 promoters and inhibitors for medical use, emphasizing the importance of aldehyde dehydrogenase 2 (Budas et al. 2009, Chen et al. 2014, M. zeletal. 2018). However, in reality, the development of high-content microbial breeding or mass production technologies for aldehyde dehydrogenase 2 is still insufficient.

[0020] According to reports, when developing strains that produce aldehyde dehydrogenase 2 efficiently, human aldehyde dehydrogenase 1 and 2 proteins were expressed using a protein expression system with E. coli as the host. However, only 30% of these proteins were expressed as active soluble enzymes, producing only 2-4 mg / L of protein (Zheng et al. 1993). In contrast, 95% of mouse aldehyde dehydrogenase 2 was expressed as active soluble protein, producing only a very small amount of protein, 1-2 mg / L (Jeng et al. 1991).

[0021] However, there have been no reported instances of increased aldehyde dehydrogenase 2 production using mutation methods that are less restricted by law and easier to apply. Therefore, in order to expand the use of aldehyde dehydrogenase 2, there is an urgent need to develop microorganisms with high aldehyde dehydrogenase 2 activity using mutation methods.

[0022] Typically, the purpose of strain propagation methods is to enhance the efficiency of producing target products from microbial strains. Genetic recombination or mutation techniques are often used. However, when using genetic recombination to improve strains, there are many legal restrictions and the scope of application is limited. Therefore, mutation-induced methods are extremely advantageous.

[0023] Mutation induction methods for improved strains typically utilize chemicals such as ethylmethanesulfonate (EMS) or methylnitronitrosoguanidine (NTG) or ultraviolet light (UV) to induce mutations in the strain's genetic material. After altering its characteristics, a selection factor suitable for the production of the target product is added to adapt the strain to the desired form, induce the desired mutant, and then screen for it.

[0024] In biological genetic material, the genetic bonds between guanine and cytosine are triple hydrogen bonds, while those between adenine and thymine are double hydrogen bonds. Because the base sequence of genetic material storing gene information consists of triple and double hydrogen bonds, guanine must form bonds with cytosine, adenine, and thymine.

[0025] When chemical substances are used as mutation inducers, such as ethyl methanesulfonate (EMS) or nitrosoguanidine (NTG), guanine is alkylated to form O-6-ethylguanine. As a result, the triple bond is blocked, and the guanine is eventually formed with thymine to form a double bond, thereby altering the genetic base bonds of that part.

[0026] In this state, if DNA replication occurs while the genetic bases are altered, the thymine portion bonds with adenine, while the guanine and cytosine portions are replaced with adenine and thymine (Nahafi et al. 2013), thus causing a mutation.

[0027] Furthermore, based on the adaptation screening of mutants, methylglyoxal is a toxic substance synthesized in this process and the aminoacetone cycle. It is converted into lactic acid by glyoxal reductase. The properties of this system are intervened by glutathione as a coenzyme. Using a medium supplemented with glyoxal, strains with high glutathione production efficiency are induced.

[0028] That is, when glutathione is insufficient, the system does not activate, thus enhancing sensitivity, while when glutathione is overproduced, resistance is enhanced. As a result, acetone aldehyde has been used as a screening factor for strains that overproduce glutathione (Ohtake et al. 1990) (Hamad et al. 2018).

[0029] Lycine can be added to the culture medium to screen for mutant inducers that produce more acetaldehyde dehydrogenase 2. Lycine acts on the negatively charged bacterial membrane, destroying its structure and inhibiting bacterial growth. It also penetrates the cell interior and acts on nucleic acids, hindering protein synthesis and thus inhibiting microbial growth.

[0030] Therefore, tolerant strains that can grow under conditions containing high concentrations of lysine can enhance the protein synthesis capacity of the strain, which can be used as a screening method for strains that overproduce acetaldehyde dehydrogenase 2.

[0031] However, as mentioned above, while using glutathione and aldehyde dehydrogenase is highly efficient in removing various harmful substances accumulated in the human body, especially reactive oxygen species or various acetaldehyde chemicals, no strains that simultaneously produce glutathione and aldehyde dehydrogenase have been reported to date.

[0032] In this invention, a single chemical mutagenesis method was used to prepare mutant strains from strains that simultaneously and efficiently produce aldehyde dehydrogenase 2 and glutathione. Mutant strains adapted to secondary selection factors were screened and developed.

[0033] A chemical method based on ethylmethanesulfonate (EMS) or ethylnitronitrosoguanidine (NTG) was used in the first screening. In the second screening, two adaptation tests, which had not been implemented to date, were conducted using methylglyoxal and lysine. Finally, strains that simultaneously overproduce glutathione and acetaldehyde dehydrogenase 2 were screened out.

[0034] Reports have been submitted regarding mutant strains that simultaneously overproduce glutathione and aldehyde dehydrogenase 2: GRAS (Generally Recognized As Safe) strains that can be safely used in food, health foods, feed, cosmetics, and pharmaceuticals. These strains were screened and used from wild-type Saccharomyces cerevisiae, which, although having low production efficiency, can still produce both glutathione and aldehyde dehydrogenase.

[0035] Thus, a new improved strain of Saccharomyces cerevisiae sp. was obtained, which, through mutation, enhanced the production capacity of glutathione and acetaldehyde dehydrogenase 2, thereby completing the present invention.

[0036] [Preliminary Technology Documents]

[0037] (Non-patent literature 0001) Budas, GR, Disatnik, MH, & Mochly-Rosen, D. (2009). Aldehyde dehydrogenase 2 in cardiac protection: a new therapeutic target? Trends in cardiovascular medicine, 19(5), 158-164.

[0038] (Non-patent document 0002) Chen, CH, Ferreira, JCB, Gross, ER, & Mochly-Rosen, D. (2014). Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiological reviews, 94 (1), 1-34.

[0039] (Non-Patent Literature 0003) Eriksson, CJ (1977). Acetaldehyde metabolism in vivo during ethanol oxidation. Advances in experimental medicine and biology, 85, 319-341.

[0040] (Non-patent document 0004) Hamad, GM, Taha, TH, Alshehri, AM & Hafez, EE, (2018). Enhancement of the Glutathione Production by Mutated Yeast Strains and its Potential as Food Supplement and Preservative. Res. J. Microbiol., 13: 28-36.

[0041] (Non-patent document 0005) Jeng, J., & Weiner, H. (1991). Purification and characterization of catalytically active precursor of rat liver mitochondrialaldehyde dehydrogenase expressed in Escherichia coli. Archives of biochemistry and biophysics, 289 (1), 214-222.

[0042] (Non-Patent Literature 0006) Klyosov, AA, Rashkovetsky, LG, Tahir, MK, & Keung, WM (1996-1). Possible role of liver cytosolic and mitochondrial aldehyde dehydrogenases in acetaldehyde metabolism. Biochemistry, 35(14), 4445-4456.

[0043] (Non-Patent Document 0007) Klyosov, A.A. (1996-2). Kinetics and specificity of human liver aldehyde dehydrogenases toward aliphatic, aromatic, and fused polycyclic aldehydes. Biochemistry, 35(14), 4457-4467.

[0044] (Non-Patent Document 0008) Liu, J., Head, E., Gharib, A.M., Yuan, W., Ingersoll, R.T., Hagen, T.M., & Ames, B.N. (2002). Memory loss in old rats is associated with brain mitochondrial decay and RNA / DNA oxidation: Partial reversal by feeding acetyl-L-carnitine and / or R-α-lipoic acid. Proc. Natl. Acad. Sci., 99(4), 2356-2361.

[0045] (Non-Patent Document 0009) Marchitti, S.A., Deitrich, R.A., & Vasiliou, V. (2007). Neurotoxicity and metabolism of the atecholamine-derived 3,4-dihydroxyphenyla cetaldehyde and 3,4-dihydroxyphenylglycolaldehyde: the role of aldehyde dehydrogenase. Pharmacological reviews, 59(2), 125-150.

[0046] (Non-Patent Document 0010) Marchitti, S.A., Brocker, C., Stagos, D., & Vasiliou, V. (2008). Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily. Expert opinion on drug metabolism&toxicology, 4(6), 697-720.

[0047] (Non-patent literature 0011) Najafi, MBH, & Pezechki, P. (2013) Bacterial mutation; types, mechanisms and mutant detection methods: a review. European Scientific Journal, 4

[0048] (Non-patent document 0012) Ohtake, Y., Satou, A., & Yabuuchi, S. (1990). Isolation and Characterization of Glutathione Biosynthesis-deficient Mutants in Saccharomyces cerevisiae. Agric. Biol. Chem., 54(12), 3145-3150.

[0049] (Non-patent document 0013) Wright, C., Milne, S., & Leeson, H. (2014). Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod. BioMed. Online, 28 (6), 684-703.

[0050] (Non-patent document 0014) Yoshida, A., Huang, IY, & Ikawa, M. (1984). Molecularabnormality of an inactive aldehyde dehydrogenase variant commonly found in Orientals. Proceedings of the National Academy of Sciences, 81(1), 258-261. Summary of the Invention

[0051] The problem to be solved

[0052] The purpose of this invention is to provide a mutation method for inducing mutations in wild brewer's yeast to enhance the simultaneous production of glutathione and aldehyde dehydrogenase 2.

[0053] Another objective of this invention is to provide a mutant yeast strain of Saccharomyces cerevisiae KwonP-1 (KCTC13925BP) that has the ability to simultaneously and efficiently produce glutathione and aldehyde dehydrogenase 2.

[0054] Another objective of this invention is to develop a new yeast strain, Saccharomyces cerevisiae KwonP-2, cultured by mutating wild Saccharomyces cerevisiae. This new yeast strain exhibits excellent ability to produce both glutathione and aldehyde dehydrogenase 2, and strong tolerance to methylglyoxal.

[0055] Another objective of this invention is to produce a new strain of Saccharomyces cerevisiae, KCTC14123BP, prepared by mutating wild Saccharomyces cerevisiae, which exhibits strong lysine tolerance.

[0056] Problem Solution

[0057] In order to achieve the above-mentioned objectives of the present invention, in order to prepare a variant microorganism with enhanced production capacity of both glutamate and aldehyde dehydrogenase 2, the present invention screened wild Saccharomyces cerevisiae and used trace strains capable of producing glutathione and aldehyde dehydrogenase 2.

[0058] For strains of wild yeast that were artificially mutated based on this standard and then overproduced glutathione again, methylglyoxal-adapted mutant strains were screened, while lysine-adapted mutant strains were screened when screening for strains that overproduced aldehyde dehydrogenase 2. Finally, brewer's yeast that simultaneously overproduced glutathione and aldehyde dehydrogenase 2 was screened out.

[0059] The mutation induction method and the selected mutant yeast of the present invention will be further described in detail below.

[0060] Regarding the KwonP-1 mutant strain of Saccharomyces cerevisiae (KCTC13925BP), 20 strains were screened from 250 wild Saccharomyces cerevisiae strains selected from domestic rice wine, yeast, etc. Although they produced trace amounts of these strains, they simultaneously produced glutathione and acetaldehyde dehydrogenase 2. Finally, a strain with extremely high simultaneous production efficiency was selected.

[0061] Then, in order to treat the 20 selected strains with nitrosoguanidine (NTG) or ethyl methane-sulfonate (EMS) to improve the production efficiency of glutathione by the mutant microorganisms that induce chemical mutations, a highly adaptable and resistant yeast microorganism was screened in one step, with a methylglyoxal concentration of 5 to 15 mM, preferably 10 mM, but not limited thereto.

[0062] Then, in a second screening, to identify microorganisms with high aldehyde dehydrogenase 2 production efficiency and new yeast mutant strains adapted to lysine, yeast microorganisms tolerant to lysine were screened at a concentration of 3 to 5%, preferably 3%, but not limited to this. Finally, the Saccharomyces cerevisiae KwonP-1, which has high simultaneous production efficiency of glutathione and aldehyde dehydrogenase 2, was selected.

[0063] The brewing yeast KwonP-1 is deposited in the International Collections of Cultural Relics (ICC) with accession number KCTC13925BP, brewing yeast Kwon P-2 is deposited in the ICC with accession number KCTC14122BP, and brewing yeast Kwon P-3 is deposited in the ICC with accession number KCTC14123BP. Its characteristic feature is that it possesses excellent glutathione and acetaldehyde dehydrogenase 2 production capabilities.

[0064] In this embodiment of the invention, the KwonP-1 mutant strain of Saccharomyces cerevisiae (KCTC13925BP), although present in trace amounts compared to wild-type Saccharomyces cerevisiae, was induced to mutate by treatment with nitrosoguanidine (NTG) or ethylmethane-sulfonate (EMS) on selected strains capable of simultaneously producing glutathione and aldehyde dehydrogenase 2. Strains resistant to methylglyoxal and lysine were then selected from these strains. It was confirmed that this strain exhibited superior glutathione and aldehyde dehydrogenase 2 production efficiency compared to the initially selected wild-type strain. Attached Figure Description

[0065] Figure 1 This is a graph showing the survival rate curve of wild-type Saccharomyces cerevisiae based on the acetone aldehyde treatment concentration according to an embodiment of the present invention.

[0066] Figure 2This is a graph showing the survival rate curve of wild-type Saccharomyces cerevisiae strains based on the concentration of nitrosoguanidine (NTG) treatment according to an embodiment of the present invention. Blue represents the survival rate of wild-type Saccharomyces cerevisiae after 24 hours of culture, and red represents the effect after culturing wild-type Saccharomyces cerevisiae and achieving an OD value of 0.5.

[0067] Figure 3 This is a graph showing the survival rate curve of wild-type Saccharomyces cerevisiae strains based on the concentration of ethyl methanesulfonate (EMS) treatment according to an embodiment of the present invention. Blue represents the survival rate of wild-type Saccharomyces cerevisiae after 24 hours of culture, and red represents the effect after culturing wild-type Saccharomyces cerevisiae and achieving an OD value of 0.5.

[0068] Figure 4 This is a graph showing the concentration distribution of glutathione generated from the mutant strain of Saccharomyces cerevisiae in an embodiment of the present invention. In this case, the horizontal axis shows the glutathione content of each cell, and the vertical axis shows the number of colonies.

[0069] Figure 5 This is a graph showing the survival rate curve of wild-type Saccharomyces cerevisiae strains based on lysine treatment concentration according to an embodiment of the present invention.

[0070] Figure 6 This is a graph showing the concentration analysis results of aldehyde dehydrogenase 2 generated from mutant strains of Saccharomyces cerevisiae according to an embodiment of the present invention. In this case, the numbers on the horizontal axis indicate the mutant strains of Saccharomyces cerevisiae, and the vertical axis compares the activity of aldehyde dehydrogenase 2 of mutant strains of Saccharomyces cerevisiae with the activity of aldehyde dehydrogenase 2 of wild strains of Saccharomyces cerevisiae, showing the relative activities of aldehyde dehydrogenase 2.

[0071] Figure 7 The illustration shows the cell morphology of a mutant strain of Saccharomyces cerevisiae, KwonP-1, as observed using an optical microscope in one embodiment of the present invention.

[0072] Figure 8 The illustration shows the cell morphology of a wild-type strain of Saccharomyces cerevisiae as observed using an optical microscope in one embodiment of the present invention. Detailed Implementation

[0073] The structure and effects of the present invention will be further described in detail below through the following embodiments. These embodiments are only for illustrating the present invention, and the scope of the present invention is not limited by these embodiments.

[0074]

Example 1

[0075] Screening strains with enhanced glutathione production capacity

[0076] To screen for novel mutant strains with enhanced glutathione production capacity, *Saccharomyces cerevisiae* wild-type strains were treated with ethyl-methane-sulfonate (EMS) or nitrosoguanidine (NTG) to induce mutations. Tolerance to methylglyoxal was assessed, and strains exhibiting enhanced glutathione production were ultimately selected. The specific experiments were conducted as follows.

[0077] Example 1-1: Survival analysis of wild-type Saccharomyces cerevisiae strains in methylglyoxal.

[0078] To create screening conditions for a new yeast mutant strain with enhanced glutathione production performance, the treatment concentration range was determined by detecting the survival rate before selecting a treatment concentration, prior to screening for strains resistant to acetone aldehyde. At this point, wild-type Saccharomyces cerevisiae was used as the parent strain.

[0079] Specifically, firstly, to screen for resistant mutant strains, the acetone aldehyde treatment concentration of the prepared yeast strains was varied, and the survival rate was investigated. For this purpose, the strains were inoculated into YPD medium (2% peptone, 1% yeast extract, 2% glucose) and grown at 30°C until the OD600nm (Optical Density at 600nm) value reached 0.5. The cells were then recovered. The recovered cells were spread onto YPD agar medium (with 1.5% agar added) containing 0mM, 5mM, 10mM, and 15mM methylglyoxal, respectively, and cultured. Then, the recovered cells were washed with 0.1M citric buffer (pH 5.5), and the OD600nm value was adjusted to 1.0 before being spread onto YPD agar medium containing acetone aldehyde. After spreading, the cultures were incubated at 30°C for 48 hours, and a survival rate curve was plotted.

[0080] The result is as follows: Figure 1 As shown, when acetone aldehyde was treated at a concentration of 10 mM, the mortality rate was confirmed to be 99.995%, and when acetone aldehyde was treated at a concentration of 15 mM, the mortality rate of the test microorganisms reached 100%.

[0081] In view of this, in order to screen out new mutant strains with enhanced glutathione production performance, YPD agar medium supplemented with 10 mM acetone aldehyde was used based on the above results to screen out acetone aldehyde-resistant strains of wild-type strains.

[0082] Examples 1-2: Creating mutation conditions to screen mutant candidate strains with excellent glutathione production performance.

[0083] In Example 1-1, screening conditions were created for a new mutant strain with enhanced glutathione production performance. Specifically, to screen for methylglyoxal-resistant strains of wild-type strains, 10 mM acetone aldehyde was added to YPD agar medium.

[0084] Therefore, in order to screen out mutant strains with excellent glutathione production performance from wild strains, survival curves of various treatment concentrations of ethyl-methane-sulfonate (EMS) or nitrosoguanidine (NTG) were constructed.

[0085] Specifically, the wild-type strain was inoculated into YPD medium and cultured for 24 hours until the OD600nm value reached 0.5. Then, for both cultures, the precipitated bacterial cells were recovered by centrifugation at 4,000 rpm for 10 minutes. The recovered bacterial cells were washed twice with 0.1M citric buffer (pH 5.5) and centrifuged again. Finally, the cells were diluted with 0.1M citric buffer (pH 5.5) to an OD600nm value of 1.0 before use. Then, to induce mutagenesis, after centrifugation, 0.1M citric buffer (pH 5.5) containing 1%, 2%, 3%, and 4% NTG was added to the recovered bacterial cells, and the cells were treated with NTG at 30°C for 30 minutes. After centrifuging at 4,000 rpm for 10 minutes to separate the NTG-treated mutant strain and recovering the bacterial cells, the cells were washed twice with 0.1 M citrate buffer (pH 5.5). The washed bacterial cells were then treated with... After mixing with 0.1M citric buffer (pH 5.5), the mixture was spread onto YPD agar medium.

[0086] In addition, to screen for the substance that induces mutations, the substance was an acetone aldehyde-resistant mutant strain treated with ethyl methane sulfonate (EMS), which was extracted under the same conditions as the nitrosoguanidine (NTG) treatment. Wild-type yeast strains were grown and centrifuged. The recovered cells after centrifugation were treated with 0.1M citrate buffer (pH 5.5) containing 1%, 2%, 3%, and 4% ethyl methanesulfonate (EMS) at 30°C for 60 minutes. After centrifugation at 4,000 rpm for 10 minutes to separate the EMS-treated mutant strains and recovering the cells, they were washed twice with 0.1M citrate buffer (pH 5.5). The washed cells were then treated with... After mixing with 0.1M citric buffer (pH 5.5), the mixture was spread onto YPD agar medium.

[0087] The result is as follows: Figure 3 As shown, for ethyl methanesulfonate (EMS), after 24 hours of culture, treatment at a 3% concentration resulted in a confirmed mortality rate of 99.4%. Furthermore, as... Figure 2 As shown, for nitrosoguanidine (NTG), after 24 hours of culture, the mortality rate was 99.7% when treated with a 1% concentration.

[0088] In view of this, in order to screen out new yeast mutant strains with improved glutathione production performance, based on the above results, the optimal treatment concentrations of nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS) for screening wild-type glutathione resistant strains were selected as 1% and 3%, respectively.

[0089] Examples 1-3: Screening for mutant candidate strains with excellent glutathione production performance

[0090] In Examples 1-2 and 1-3, screening conditions were created for novel yeast mutant strains with enhanced glutathione production performance. Specifically, to screen for acetone-resistant strains of wild-type yeast, 10 mM acetone was added to YNB agar medium, and conditions of 1% nitrosoguanidine (NTG) and 3% ethyl methanesulfonate (EMS) were created for mutation.

[0091] Therefore, in order to screen out mutant strains with excellent glutathione production performance from wild strains, acetone aldehyde-resistant mutant strains were screened out. The strains were grown in YPD medium, and the glutathione concentration was measured and labeled as the glutathione content in the cells (%), g / g-cell.

[0092] Specifically, the wild-type strain was inoculated into YPD medium and cultured for 24 hours. The resulting culture was centrifuged at 4,000 rpm for 10 minutes to recover the bacterial cells. The recovered cells were then washed twice with 0.1 M citric buffer (pH 5.5) and finally diluted with 0.1 M citric buffer (pH 5.5) to an OD600 nm value of 1.0 before use. To induce mutations, the microorganisms were treated with 0.1 M citric buffer (pH 5.5) containing 3% ethyl methanesulfonate (EMS) for 10 minutes and with 0.1 M citric buffer (pH 5.5) containing 1% nitrosoguanidine (NTG) for 30 minutes. The mutated microorganisms were then centrifuged at 4,000 rpm for 10 minutes and the cells recovered. After mixing with 0.1 M citric buffer (pH 5.5), the mixture was spread onto YPD agar medium supplemented with 10 mM acetone aldehyde. After spreading and culturing, the surviving strains were recovered and cultured in YPD medium for 48 hours. The culture conditions were 30°C and 160 rpm. After 48 hours of culture, the glutathione concentration was measured and labeled as the glutathione content per cell (%, g / g-cell).

[0093] To analyze the glutathione concentration, the cultured bacterial cells were centrifuged, and 1 mL of water was added to the precipitated cells. The mixture was stirred at 1,000 rpm for two hours at 85°C and extracted. After extraction, the bacterial cells were removed using a centrifuge, filtered through a 0.22 μm filter, and the supernatant was recovered. The concentration of glutathione in the recovered filtrate was determined by high-performance liquid chromatography (HPLC) (Shimazu LC-20AD). Furthermore, the glutathione concentration was analyzed using a standard curve, and the HPLC analytical conditions were analyzed using a C18 column. The mobile phase (a mixture of 2.02 g / L Sodium 1-heptanesulfonate monohydrate, 6.8 g / L Lotussium dihydrogenphosphate, pH 3.0m, and methanol) was used... The flow rate was measured, and the concentration of glutathione was detected by a 210 nm wavelength ultraviolet detector.

[0094] 130 mutant strains were generated. Based on the glutathione concentration analysis results of each mutant strain, such as... Figure 4As shown, there were 70 mutant strains with glutathione content of 0.3% to 0.5%, 30 mutant strains with 0.5% to 0.7%, 23 mutant strains with 0.7% to 0.85%, and 7 mutant strains with 0.85% to 1.1%. That is, the glutathione production capacity of acetone aldehyde-resistant strains treated with ethyl methanesulfonate (EMS) or nitrosoguanidine (NTG) was superior to that of the wild-type strain at a glutathione content of 48.6% (0.42%). Among them, as recorded in Table 1 below, the 7 mutant strains showed more than twice the glutathione production capacity compared to the parent strain.

[0095] Based on this, among the acetone aldehyde-resistant mutant strains treated with ethyl methane sulfonate (EMS), the Saccharomyces cerevisiae EMS C7 with the best glutathione production performance was selected as the final candidate mutant strain with excellent glutathione production performance.

[0096] In Example 2, a mutation treated with nitrosoguanidine (NTG) was used to improve the aldehyde dehydrogenase 2 production performance of the selected candidate strain.

[0097] Table 1

[0098] Culture results of methylglyoxal-resistant mutant strains of yeast treated with ethyl methanesulfonate (EMS) or nitrosoguanidine (NTG).

[0099]

[0100]

Example 2

[0101] Screening for strains with enhanced aldehyde dehydrogenase 2 (ALDH2) production performance

[0102] To screen for novel mutant strains with enhanced aldehyde dehydrogenase 2 production performance, the lysine tolerance of *Saccharomyces cerevisiae* ems c7, which had the highest glutathione production among the strains screened in Example 1, was evaluated, and strains with enhanced aldehyde dehydrogenase 2 production performance were ultimately selected. The following experiments were specifically conducted.

[0103] Example 2-1: Analysis of the survival rate of Saccharomyces cerevisiae EMS C7 strain in lysine.

[0104] To create screening conditions for a new mutant strain with enhanced aldehyde dehydrogenase 2 production performance, the optimal lysine treatment concentration for screening lysine-tolerant strains of the glutathione mutant *Saccharomyces cerevisiae* EMS C7 was selected. *Saccharomyces cerevisiae* EMS C7 with enhanced glutathione production performance was prepared and used as the parent strain.

[0105] Specifically, to screen for lysine-tolerant mutant strains of *Saccharomyces cerevisiae*, the survival rate of the prepared *Saccharomyces cerevisiae* EMS C7 strain was investigated based on the lysine treatment concentration. For this purpose, the strain was inoculated into YPD medium (2% peptone, 1% yeast extract, 2% glucose) and grown at 30°C until the OD600nm value reached 0.5, and the cells were recovered. The recovered cells were spread onto YPD agar medium (with 1.5% agar added) supplemented with 0%, 3%, 4%, 5%, 6%, and 7% lysine, respectively. After washing the recovered cells with 0.1M citric buffer (pH 5.5), the OD600nm value was adjusted to 1.0, and the cells were spread onto YPD agar medium supplemented with lysine. After spreading, the cells were incubated at 30°C for 48 hours, and a survival curve was constructed.

[0106] The result is as follows: Figure 5 As shown, when lysine was treated at a concentration of 3%, the mortality rate was confirmed to be 87.5%.

[0107] In view of this, in order to screen out new mutant strains with improved aldehyde dehydrogenase 2 production performance, based on the above results, the optimal lysine treatment concentration for screening lysine-tolerant strains of Saccharomyces cerevisiae EMS C7 strain was selected as 3%, and screening was carried out using YPD agar medium supplemented with 3% lysine.

[0108] Example 2-2: Screening for mutant candidate strains with excellent aldehyde dehydrogenase 2 production performance

[0109] In Examples 1-2 and 2-1, screening conditions were created for novel yeast mutant strains with enhanced aldehyde dehydrogenase 2 activity. Specifically, to screen for lysine-tolerant mutant Saccharomyces cerevisiae EMS C7 strains, 3% lysine was added to YPD agar medium, and 1% nitrosoguanidine (NTG) was added to induce mutation.

[0110] Therefore, in order to screen out mutant strains with excellent glutathione production performance from Saccharomyces cerevisiae EMS C7, cells were grown in YPD medium containing 3% lysine, and the concentration of aldehyde dehydrogenase 2 was detected.

[0111] Specifically, wild-type yeast strains were inoculated onto YPD medium and cultured for 24 hours. The medium was then centrifuged at 4,000 rpm for 10 minutes to separate the culture, and the cells were recovered. The recovered cells were washed twice with 0.1 M citric buffer (pH 5.5), and finally diluted with 0.1 M citric buffer (pH 5.5) until the OD600nm value reached 1.0. To induce mutations, mutant strains were treated with 0.1 M citric buffer (pH 5.5) containing 1% nitrosoguanidine (NTG) for 10 minutes, centrifuged at 4,000 rpm for 10 minutes to separate the mutant strains, and the cells were recovered. Then, 0.1 M citric buffer (pH 5.5) was added and mixed, and the mixture was spread onto YPD agar medium supplemented with 3% lysine. After spreading and culturing, the surviving strains were recovered and cultured on YPD medium for 48 hours. The culture conditions were 30°C and 160 rpm. After 48 hours of culture, the activity of aldehyde dehydrogenase 2 was measured.

[0112] However, aldehydes are volatile and produced in trace quantities, leading to significant deviations even with the same sample when using traditional detection methods. Therefore, a stable acetaldehyde detection method is needed to develop a precise new enzyme titer detection method. This is absolutely essential for the quality control (QC) of the already prepared acetaldehyde dehydrogenase. In view of this, Example 3 describes the development of a new method.

[0113]

Example 3

[0114] Establish a novel method for detecting the titer of acetaldehyde dehydrogenase and for detecting the enzyme titer of mutant strains.

[0115] Traditional methods for detecting aldehyde dehydrogenase activity, such as measuring NAD(P)+ absorbance at 340 nm, are widely used. However, this method only examines changes in the coenzyme, resulting in an indirect outcome and is therefore unsuitable. To detect the Km value of the enzyme directly targeting the enzyme matrix, the inventors employed high-performance liquid chromatography (HPLC). However, because acetaldehyde is consumed in very small quantities by aldehyde dehydrogenase 2 (ALDH2) and is highly volatile at room temperature, direct quantification of the reaction product presents technical challenges.

[0116] In view of this, in order to solve the above problems, many inventors have added dinitrophenylhydrazine (DNPH) to acetaldehyde in a quantitative ratio, causing it to react at a certain concentration to form an acetaldehyde-hydrazone (AcH-DNPH) compound. Following the reference (Guan et al., 2012), a C18 column was used for high-performance liquid chromatography (HPLC), with acetonitrile and water as the mobile phase. The mixture was detected and quantified at 360 nm, and the amount of aldehyde reduced was analyzed by the reaction of aldehyde dehydrogenase. The enzyme reaction solution consisted of 50 mM potassium phosphate buffer (pH 8.0) and 1 mM acetaldehyde. 10 μL of the lysate from the microbial lysate was added with 1 mM NADP+ cofactor, and the reaction was incubated at 30 °C. Then, 50 μL of 10 mM dinitrophenylhydrazone (DNPH) was added, and the reaction was calibrated at 22 °C for 1 hour to form acetaldehyde-hydrazone (AcH-DNPH). During calibration, the reaction was terminated by adding 3 M sodium acetate (pH 9), and 2 times the volume of acetonitrile was added to separate the layer containing the acetaldehyde-hydrazone (AcH-DNPH) compound. This layer was then injected into a high-performance liquid chromatograph (HPLC) for analysis. The concentration of the calibrated aldehyde was analyzed using an acetaldehyde-hydrazone (Aldehyde-DNPH) (Sigma-Aldrich) standard curve. High-performance liquid chromatography (HPLC) was performed using a C18 column, with the solvent (acetonitrile and water) at a specific concentration. The flow rate was measured and analyzed using a 360 nm ultraviolet detector. At this point, one unit of aldehyde dehydrogenase 2 activity was defined as the decrease in acetaldehyde-hydrazone (AcH-DNPH) concentration by 1 mM per minute. The activity of aldehyde dehydrogenase 2 was expressed as units of enzyme activity per mg of protein.

[0117] The results of the analysis of aldehyde dehydrogenase 2 concentration are as follows: Figure 6As shown, among the lysine-tolerant strains of *Saccharomyces cerevisiae* EMS C7 treated with nitrosoguanidine (NTG), approximately 42% of the lysine-tolerant mutant strains showed superior aldehyde dehydrogenase 2 production capacity compared to the initial parent strain and *Saccharomyces cerevisiae* EMS C7 (110% superior to the parent strain). The production performance of four mutant strains (#4, #8, #16, and #21) was improved by more than 140%. At this point, the glutathione content of the four mutant strains with excellent aldehyde dehydrogenase 2 production capacity was 0.9% for mutant strain #4, 0.96% for mutant strain #8, 0.93% for mutant strain #16, and 0.92% for mutant strain #21. Furthermore, the glutathione production capacity remained unchanged after lysine treatment.

[0118] Table 2 records the results of four mutant strains with aldehyde dehydrogenase activity increased by more than 1.4 times compared to the parent strain. Based on this, the lysine-tolerant mutant *Saccharomyces cerevisiae* #8 treated with nitrosoguanidine (NTG) was selected as a mutant strain with excellent aldehyde dehydrogenase production performance and named *Saccharomyces cerevisiae* Kwon P-1; the lysine-tolerant mutant *Saccharomyces cerevisiae* #16 treated with nitrosoguanidine (NTG) was selected as a mutant strain with excellent aldehyde dehydrogenase production performance and named *Saccharomyces cerevisiae* Kwon P-2; and the lysine-tolerant mutant *Saccharomyces cerevisiae* #21 treated with nitrosoguanidine (NTG) was selected as a mutant strain with excellent aldehyde dehydrogenase production performance and named *Saccharomyces cerevisiae* Kwon P-3.

[0119] Table 2

[0120] Aldehyde dehydrogenase 2 activity of lysine-tolerant mutant yeast strains treated with nitrosoguanidine (NTG)

[0121]

[0122]

Example 4

[0123] Mutant morphological changes with excellent glutathione and aldehyde dehydrogenase 2 production performance

[0124] In Example 2-2, a novel yeast mutant strain with enhanced glutathione and aldehyde dehydrogenase 2 production performance was screened. Furthermore, the mutant strain was observed using an optical microscope to examine morphological changes. The cell morphology of the mutant strain *Saccharomyces cerevisiae* KongP-1 is as follows: Figure 7 As shown, the wild morphology of wild-type yeast strains is as follows: Figure 8As shown, observations using an optical microscope revealed that over 60% of the mutant strain of *Saccharomyces cerevisiae* Kwon P-1 showed increased cell diameter, and the vacuoles, as small intracellular organelles, became enlarged. The unique morphological characteristics of the *Saccharomyces cerevisiae* Kwon P-1 in the above embodiments, with 60% of cells showing increased size and enlarged vacuoles, represent a specific morphology and can be used as an important means to prevent unauthorized use and infringement of patent rights.

[0125]

Example 5

[0126] Simultaneous production of glutathione and aldehyde dehydrogenase 2 using brewer's yeast Kwon P-1

[0127] Saccharomyces cerevisiae Kwon P-1 was inoculated into sterile YPD liquid medium (2% peptone, 1% yeast extract, 2% glucose) at 30°C and 160 rpm for 16 hours as seed culture. This culture was then inoculated at 1% in sterile YPD liquid medium and cultured under the same conditions for 48 hours. The glutathione concentration and aldehyde dehydrogenase 2 activity in the culture were then measured. The culture was repeated 20 times in 20 flasks under the same conditions. The results, as shown in Table 3, are the glutathione content and aldehyde dehydrogenase 2 activity in the cells. As a patented strain, Saccharomyces cerevisiae Kwon P-1 can simultaneously produce glutathione and aldehyde dehydrogenase 2. After 20 repeated experiments, the average production values ​​were 0.97% glutathione and 0.173 Unit / mg-protein. These results indicate that the patented strain of Saccharomyces cerevisiae Kwon P-1 mutant has 2.3 times higher glutathione content and 1.7 times greater aldehyde dehydrogenase 2 production capacity compared to the parent strain.

[0128] Table 3

[0129] Liquid culture in flasks was used to confirm that the brewer's yeast Kwon P-1 simultaneously produces glutathione and aldehyde dehydrogenase 2.

[0130]

[0131]

[0132] [Collection Number]

[0133] Name of the depository: Korea Institute of Life Sciences

[0134] Collection Number: KCTC13925BP

[0135] Date of preservation: August 22, 2019

[0136]

[0137]

[0138]

Claims

1. A strain of Saccharomyces cerevisiae, Kwon P-1, with accession number KCTC13925BP.

2. A strain of Saccharomyces cerevisiae, Kwon P-2, with accession number KCTC14122BP.

3. A strain of Saccharomyces cerevisiae, Kwon P-3, with accession number KCTC14123BP.

4. A method for preparing a mutant yeast with simultaneously enhanced production capacity of glutathione and aldehyde dehydrogenase, comprising the following steps: The first step involves treating the *Saccharomyces cerevisiae* yeast with ethylmethanesulfonate or nitrosoguanidine to induce mutations. The second step involves treating the induced mutant yeast obtained in the first step with 5 mM to 15 mM methylglyoxal, thereby selecting methylglyoxal-adapted yeast from the induced mutant yeast. The third step involves treating the methylglyoxal-adapted yeast selected in the second step with 3% to 5% lysine to select lysine-adapted mutant yeast from the methylglyoxal-adapted mutant yeast selected in the second step.

5. A method for culturing *Saccharomyces cerevisiae* while simultaneously producing glutathione and aldehyde dehydrogenase, said yeast being prepared by a method comprising the following steps: The first step involves treating the *Saccharomyces cerevisiae* yeast with ethyl methanesulfonate or nitrosoguanidine to induce mutations. The second step involves treating the induced mutant yeast obtained in the first step with 10 mM methylglyoxal to select methylglyoxal-adapted yeast. The third step involves treating the methylglyoxal-adapted yeast selected in the second step with 3% to 5% lysine to select lysine-adapted mutant yeast from the methylglyoxal-adapted mutant yeast selected in the second step.

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