Saccharomyces cerevisiae Kwon P1, 2, and 3 producing glutathione and aldehyde dehydrogenase

Abstract

To provide methods for mutating wild type Saccharomyces cerevisiae yeast to enhance the ability to produce both glutathione and aldehyde dehydrogenase.SOLUTION: The invention relates to a yeast strain that produces glutathione (GSH) and an aldehyde dehydrogenase (acetaldehyde dehydrogenase). More specifically, the invention relates to Saccharomyces cerevisiae Kwon P-1 KCTC13925BP, Saccharomyces cerevisiae Kwon P-2 KCTC14122BP and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP that produce both glutathione and an aldehyde dehydrogenase.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to yeast strains that produce glutathione (GSH) and aldehyde dehydrogenase. More specifically, the present invention relates to yeast strains Saccharomyces cerevisiae (Kwon P-1 KCTC13925BP), Saccharomyces cerevisiae Kwon P-2 KCTC14122BP, and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP that simultaneously produce glutathione and aldehyde dehydrogenase. [Background technology]

[0002] Glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH) is a physiologically active substance present in cells. It is a tripeptide composed of three amino acids, glutamate, cysteine, and glycine. It exists in animal, plant, and microbial cells at concentrations of 0.1 to 10 mM, accounting for more than 90% of the total non-protein active components of the cell.

[0003] Glutathione in the body is known to play an important role as an antiviral agent by increasing immune activity through the production of white blood cells. It also plays an important role in detoxification by acting as a substrate for GST (glutathione S-transferase) and binding toxic substances such as xenobiotics that are harmful to the body in the form of conjugation.

[0004] Glutathione also prevents damage to cell membranes, nucleic acids, and cell structures through intracellular oxidation, leading to necrosis, and alleviates the toxicity of reactive oxygen species (ROS), which are the cause of aging. ROS are formed during the metabolic processes of various biological substances and include superoxide, peroxide, and hydroxyl radicals. They can be divided into endogenous ROS, which are produced as metabolic products of biological substances, and exogenous ROS, which are derived from tobacco, radioactivity, etc.

[0005] Oxidative stress caused by reactive oxygen species can damage cognitive function (Liu et al. 2002), damage sperm DNA, causing male infertility (Wright et al. 2014), damage cellular proteins, lipids, and nucleic acids, leading to cancer, and impair physiological functions, acting as a causative factor for various diseases and aging. Therefore, antioxidants that play roles in disease prevention, immune enhancement, and anti-aging are extremely important in our bodies, and the function of glutathione as an antioxidant within cells has attracted attention in many medical fields, including enzymology, pharmacology, therapy, toxicology, endocrinology, and microbiology.

[0006] Glutathione is basically synthesized in the body, but as abnormal conditions such as disease onset, weakened immunity, and aging progress, the absolute amount in the human body decreases, leading to deterioration of health. Therefore, glutathione supplied from the outside can remove reactive oxygen species from cells, maintaining health and delaying aging.

[0007] Due to these physiologically active factors of glutathione in the human body, glutathione is currently used in foods, cosmetics, feeds, and pharmaceuticals, and its usage is tending to increase.

[0008] Meanwhile, glutathione is currently produced using edible microorganisms, but the inherent amount of glutathione that can be produced by microorganisms is very low. Therefore, research is being actively conducted to increase the glutathione content of microorganisms using mutation and recombinant technology, and then mass-produce high-content glutathione-producing strains using fermentation techniques.

[0009] Therefore, the development of a bacterial strain with a high glutathione content will develop a fundamental material that will increase economic value, making glutathione more competitive in the market and allowing it to be used in a wide range of applications, including health foods, pharmaceuticals, and feed. However, the development of bacterial strains using genetic engineering technology is limited in its scope of use due to the current issue of GMOs, whereas the breeding of bacterial strains using mutation technology is not limited in its use and can be used in a wide range of applications. Therefore, the breeding of bacterial strains that produce high amounts of glutathione using mutation technology is a highly necessary technology.

[0010] Meanwhile, South Korea's alcohol consumption has skyrocketed alongside its economic growth, making national health management a key issue, with excessive alcohol consumption becoming a major social problem in terms of both national health and socioeconomic impacts. According to a survey conducted by the Ministry of Health and Welfare and the Korea Centers for Disease Control and Prevention (KCDC), approximately 75% of South Korean adults aged 20 and over reported drinking alcohol at least once a month in 2016, a percentage that is increasing every year. Furthermore, according to a WHO survey on alcohol consumption by country, the average per capita alcohol consumption for South Koreans aged 15 and over in 2016 was 11.9 liters, 4.8 liters higher than the global average, ranking South Korea 17th in alcohol consumption worldwide and first among countries in the Western Pacific region.

[0011] In particular, it has been reported that excessive levels of aldehydes produced by alcohol consumption in the body of Koreans can lead to oxidative diseases such as cardiovascular disease, diabetes, neurodegenerative diseases, upper digestive and respiratory cancers, radiation dermatitis, Fanconi anemia, peripheral nerve damage, inflammation, osteoporosis, and aging (Chen et al. 2014).

[0012] It has also been reported that the socioeconomic losses caused by drinking amount to approximately 0.5-2.7% of GDP in most countries. In South Korea, the socioeconomic costs of drinking in 2000 were estimated at 935.2 billion won, of which the loss of productivity and losses due to illness, accidents, and hangovers were estimated at 6,284.5 billion won (Jung Woo-jin et al. 2006).

[0013] To solve this social problem, research and experiments are being conducted on many substances that can reduce the toxicity of ethanol or inhibit its manifestation, and the results are being developed into various health supplement products. Alcohol that enters the body is absorbed in the gastrointestinal tract or small intestine, enters the bloodstream, and is transported to the liver where it is broken down and detoxified.

[0014] Alcohol dehydrogenase (ADH) present in liver cells first oxidizes alcohol to acetaldehyde, which is then broken down into acetate by aldehyde dehydrogenase (ALDH) also present in liver cells, and then transported to muscles and fat tissues throughout the body, where it is ultimately broken down into carbon dioxide and water. Acetaldehyde, the first metabolic product of ethanol, is much more reactive and toxic than ethanol, making it the main cause of hangovers and alcoholic liver damage.

[0015] It has been reported that humans have 19 types of aldehyde dehydrogenases (Marchitti et al. 2007, 2008). Among these, acetaldehyde dehydrogenase 2, which is primarily found in mitochondria, was analyzed using enzyme engineering and found to have the lowest Km value (~0.2 μM) when acetaldehyde was used as the enzyme substrate, compared to when other types of aldehydes were used as substrates, and to be the enzyme that best oxidizes and removes alcohol-derived acetaldehyde.

[0016] Acetaldehyde, a substance produced by the metabolism of ethanol in the body that causes hangovers, can be effectively converted to acetate, which is essential for human health (Eriksson et al. 1977, Klyosov et al. 1996-1). Acetaldehyde dehydrogenase is also used in the metabolic processes of not only acetaldehyde but also other aldehydes, such as aliphatic aldehydes, aromatic aldehydes, and polycyclic aldehydes, to remove toxic substances from the body (Klyosov et al. 1996-2).

[0017] Representative examples include the removal of oxidized aldehydes 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) produced during oxidative stress, and the removal of acrolein produced in cigarette smoke and automobile fumes (Chen et al. 2010, Yoval-Sanchez et al. 2012). Individuals with low expression of acetaldehyde dehydrogenase in the human body or with the 487th amino acid residue of this enzyme mutated from glutamic acid to lysine not only show a sensitive reaction to even small amounts of alcohol, such as facial flushing, but also have high blood acetaldehyde levels after drinking because they are unable to convert the enzyme (Yoshida et al. 1984).

[0018] In particular, many studies have shown that people who are homozygous for acetaldehyde dehydrogenase, ALDH2-2, are more susceptible to alcohol. This genetic mutation is rarely found in Westerners, but is found in 50% of the Korean, Chinese, and Japanese populations (Brooks et al. 2009).

[0019] Research and development into aldehyde dehydrogenase has been actively conducted, with the importance of aldehyde dehydrogenase being emphasized through active research into stimulators and inhibitors of aldehyde dehydrogenase in the body for medical purposes (Budas et al. 2009, Chen et al. 2014, M.zel et al. 2018). However, research into breeding microorganisms with high levels of aldehyde dehydrogenase 2 and the development of mass production techniques is still lacking.

[0020] To develop a highly efficient strain for producing aldehyde dehydrogenase 2, a protein expression system using Escherichia coli (E. coli) as a host was used to express human aldehyde dehydrogenase 1 and 2 proteins. It was reported that only 30% of the proteins were expressed as active soluble enzymes, producing only 2-4 mg / L of protein (Zheng et al. 1993). In the case of rat aldehyde dehydrogenase 2, 95% was expressed as active soluble protein, but only 1-2 mg / L of protein was produced (Jeng et al. 1991).

[0021] However, there have been no reported cases of increasing the production of aldehyde dehydrogenase 2 using mutation methods, which are easy to use and have few legal restrictions. Therefore, in order to expand the range of uses of aldehyde dehydrogenase 2, there is an urgent need to develop microorganisms that have highly active aldehyde dehydrogenase 2 using mutation methods.

[0022] Generally, genetic engineering and mutation techniques are widely used to breed strains to increase the productivity of target products from microbial strains. However, genetic engineering techniques for improving strains are subject to many legal restrictions and have limited scope of application, making mutation induction methods highly advantageous.

[0023] Mutagenesis methods for improving strains generally involve inducing mutations in the genes of strains using chemicals such as ethylmethanesulfonate (EMS) or methylnitronitrosoguanidine (NTG) or ultraviolet light (UV) to change their characteristics, and then adding selection factors suitable for the production of the target product to adapt the desired traits, thereby inducing and selecting the desired mutants.

[0024] In biological genes, the genetic bond between guanine and cytosine is a triple hydrogen bond, and adenine and thymine are paired with a double hydrogen bond.The base sequences of genes that store genetic information are always paired with guanine and cytosine, and adenine and thymine, due to triple and double hydrogen bonds.

[0025] When chemicals are used as mutagenizers, primarily the chemical inducers EMS and NTG, guanine is alkylated to form O-6-ethyl guanine, which interrupts the triple bond and forms a double bond with the final thymine, potentially changing the genetic base pair in this region.

[0026] When DNA replication occurs with these altered gene bases, the thymine moiety pairs with adenine, and the guanine and cytosine moieties are replaced by adenine-thymine (Nahafi et al. 2013), resulting in a mutation.

[0027] In addition, for the selection of mutants based on adaptation, methylglyoxal is a toxic substance synthesized in the process and the circulation of aminoacetone, and is converted to lactic acid by glyoxal reductase. Taking advantage of the fact that glutathione is involved as a coenzyme in this system, a medium containing glyoxal is used to induce strains with high glutathione productivity.

[0028] In other words, a lack of glutathione can prevent this system from operating, resulting in increased sensitivity, while overproduction of glutathione can increase resistance. As a result, methylglyoxal can be used as a selection factor to select strains that overproduce glutathione (Ohtake et al. 1990) (Hamad et al. 2018).

[0029] Mutants that produce large amounts of aldehyde dehydrogenase can be selected by adding lycine to the culture medium. Lysine acts on the negatively charged bacterial membrane, disrupting the structure of the cell membrane and inhibiting bacterial growth. It also penetrates into the cell and acts on nucleic acids, inhibiting protein synthesis, thereby inhibiting microbial growth.

[0030] Therefore, resistant strains grown under conditions containing high concentrations of lysine can increase their protein synthesis ability, and as a result, can be used as a method for selecting strains that overproduce aldehyde dehydrogenase.

[0031] However, as seen above, using glutathione and aldehyde dehydrogenase simultaneously is highly efficient in removing various harmful substances that accumulate in the human body, particularly chemicals such as reactive oxygen species and various aldehydes. However, no previous research has reported the development of a strain that simultaneously produces glutathione and aldehyde dehydrogenase.

[0032] In the present invention, a strain capable of simultaneously and highly efficiently producing aldehyde dehydrogenase and glutathione was developed by first creating mutant strains using a chemical mutation method, and then selecting adaptation mutant strains using a secondary selection factor.

[0033] A chemical method using ethyl ethanesulfonate (EMS) or methylnitronitrosoguanidine (NTG) was used as the first selection method, and in the second selection method, two previously untried adaptation tests using methylglyoxal and lysine were used to select strains that simultaneously overproduced glutathione and aldehyde dehydrogenase.

[0034] The mutant strain that overproduces glutathione and aldehyde dehydrogenase simultaneously has been reported as GRAS (Generally Recognized As Safe), meaning that it can be used in foods, health foods, feed, cosmetics, and medicines without any problems. We selected wild-type Saccharomyces cerevisiae, which is known to produce both glutathione and aldehyde dehydrogenase, although the production efficiency is already low, and used it.

[0035] Thus, the present invention was accomplished by obtaining a new improved strain of Saccharomyces cerevisiae sp. that has increased glutathione production capacity through mutation, and at the same time, increased aldehyde dehydrogenase production capacity. [Prior art documents] [Non-patent literature]

[0036] [Non-Patent Document 1] 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.

[0037] [Non-patent document 2] Chen, CH, Ferreira, JCB, Gross, ER, & Mochly-Rosen, D. (2014). Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiological reviews, 94(1), 1-34.

[0038] [Non-patent document 3] Eriksson, CJ (1977). Acetaldehyde metabolism in vivo during ethanol oxidation. Advances in experimental medicine and biology, 85, 319-341.

[0039] [Non-patent document 4] 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.

[0040] [Non-Patent Document 5] Jeng, J., & Weiner, H. (1991). Purification and characterization of catalytically active precursor of rat liver mitochondrial aldehyde dehydrogenase expressed in Escherichia coli. Archives of biochemistry and biophysics, 289(1), 214 - 222.

[0041] [Non - Patent Document 6] Klyosov, A. A., Rashkovetsky, L. G., Tahir, M. K., & Keung, W. M. (1996 - 1). Possible role of liver cytosolic and mitochondrial aldehyde dehydrogenases in acetaldehyde metabolism. Biochemistry, 35(14), 4445 - 4456.

[0042] [Non - Patent Document 7] 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.

[0043] [Non - Patent Document 8] 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.

[0044]

Non-patent Document 9

[0045]

Non-patent Document 10

[0046]

Non-patent Document 11

[0047] [Non-Patent Document 12] 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.

[0048] [Non-Patent Document 13] 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.

[0049] [Non-Patent Document 14] Yoshida, A., Huang, IY, & Ikawa, M. (1984). Molecular abnormality 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 [Problem to be solved by the invention]

[0050] The object of the present invention is to provide a mutation method for mutating wild-type Saccharomyces cerevisiae yeast to increase its ability to simultaneously produce glutathione and aldehyde dehydrogenase.

[0051] Another object of the present invention is to provide a mutant yeast strain of Saccharomyces cerevisiae KwonP-1 (KCTC13925BP) that exhibits the ability to simultaneously produce glutathione and aldehyde dehydrogenase with high efficiency.

[0052] Another object of the present invention is to provide a novel yeast, Saccharomyces cerevisiae KwonP-2 (accession number KCTC14122BP), which is obtained by culturing a mutated wild-type Saccharomyces cerevisiae and has an excellent ability to simultaneously produce glutathione and aldehyde dehydrogenase and is highly resistant to methylglyoxal.

[0053] Another object of the present invention is to provide a novel strain, KCTC14123BP, Saccharomyces cerevisiae KwonP-3, which has strong lysine resistance and is prepared by mutating a wild-type Saccharomyces cerevisiae yeast. [Means for solving the problem]

[0054] In order to achieve the above-mentioned objectives of the present invention, a wild-type Saccharomyces cerevisiae strain capable of producing both glutathione and aldehyde dehydrogenase, albeit in trace amounts, was selected and used to create a mutant microorganism with increased ability to simultaneously produce glutathione and aldehyde dehydrogenase.

[0055] After artificially chemically mutating wild yeast selected based on these criteria, we selected strains that overproduced glutathione, methylglyoxal-adapted mutants, and aldehyde dehydrogenase-overproducing strains, as a method for selecting lysine-adapted mutants.Finally, we selected Saccharomyces cerevisiae that overproduced both glutathione and aldehyde dehydrogenase. (1) Saccharomyces cerevisiae Kwon P-1 KCTC13925BP. (2) Saccharomyces cerevisiae Kwon P-2 KCTC14122BP. (3) Saccharomyces cerevisiae Kwon P-3 KCTC14123BP. (4) A method for producing mutant yeast with improved glutathione production ability by treating Saccharomyces cerevisiae yeast with ethylmethanesulfonate or nitrosoguanidine to induce mutations, selecting yeast that have adapted to methylglyoxal, and then producing mutant yeast with improved glutathione production ability. (5) A method for producing mutant yeast with improved aldehyde dehydrogenase production ability by treating Saccharomyces cerevisiae yeast with ethyl methanesulfonate or nitrosoguanidine to induce mutations and selecting lysine-adapted yeast. (6) A method for simultaneously producing glutathione and aldehyde dehydrogenase by culturing Saccharomyces cerevisiae yeast. (7) The method for simultaneously producing glutathione and aldehyde dehydrogenase according to (6), wherein the Saccharomyces cerevisiae yeast is any one selected from the group consisting of Saccharomyces cerevisiae Kwon P-1 KCTC13925BP, Saccharomyces cerevisiae Kwon P-2 KCTC14122BP, and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP, or a mixture of these yeasts.

[0056] The mutation induction method of the present invention and the selected mutant yeast will be described in more detail below.

[0057] The mutant strain of Saccharomyces cerevisiae KwonP-1 (KCTC13925BP) was selected from 250 wild strains of Saccharomyces cerevisiae selected from Korean makgeolli, koji, etc., and 20 strains that simultaneously produce glutathione and aldehyde dehydrogenase, albeit in small amounts, were selected. One strain with the highest simultaneous productivity was then finally selected.

[0058] The selected 20 strains were then treated with NTG (Nitrosoguanidine) or EMS (Ethyl-methane-sulfonate) to induce chemical mutations. To increase the glutathione productivity of the mutant microorganisms, yeast microorganisms that are highly adaptable to methylglyoxal concentrations, including but not limited to 5 to 15 mM, preferably 10 mM, were initially selected.

[0059] Then, in the second round of selection, to select a new yeast mutant strain with adaptive tolerance to lysine, a secondary selection was conducted to select yeast microorganisms with tolerance to lysine at a concentration of 3-5%, preferably 3%, without limitation, to select microorganisms with high aldehyde dehydrogenase productivity. Finally, Saccharomyces cerevisiae KwonP-1, which exhibited high simultaneous productivity of glutathione and aldehyde dehydrogenase, was selected.

[0060] The Saccharomyces cerevisiae KwonP-1 strain has been deposited with the International Depository Organization under the accession number KCTC13925BP and is characterized by its excellent glutathione production and aldehyde dehydrogenase production capabilities.

[0061] In the examples of the present invention, the mutant strain (KCTC13925BP) of Saccharomyces cerevisiae KwonP-1 was prepared by inducing mutation in a selected wild-type strain of Saccharomyces cerevisiae that was capable of simultaneously producing glutathione and aldehyde dehydrogenase, albeit in trace amounts, by treating it with NTG (nitrosoguanidine) or EMS (ethyl-methane-sulfonate), and selecting a strain that was resistant to methylglyoxal and lysine from the selected strain. It was confirmed that the mutant strain had superior productivity of both glutathione and aldehyde dehydrogenase compared to the wild-type strain that was initially selected. [Brief explanation of the drawings]

[0062] [Figure 1] 1 is a graph showing a survival curve of a wild-type strain of Saccharomyces cerevisiae depending on the treatment concentration of methylglyoxal according to an embodiment of the present invention. [Figure 2]1 is a graph showing the survival curve of a wild-type strain of Saccharomyces cerevisiae as a function of NTG treatment concentration according to an embodiment of the present invention. The blue line indicates the survival rate of wild-type Saccharomyces cerevisiae cultured for 24 hours, and the orange line indicates wild-type Saccharomyces cerevisiae cultured after reaching an OD value of 0.5. [Figure 3] 1 is a graph showing the survival curve of a wild-type strain of Saccharomyces cerevisiae as a function of the treatment concentration of EMS according to an embodiment of the present invention. The blue line represents the survival rate of wild-type Saccharomyces cerevisiae cultured for 24 hours, and the orange line represents wild-type Saccharomyces cerevisiae cultured until the OD value reached 0.5. [Figure 4] 1 is a graph showing the concentration distribution of glutathione produced from a mutant strain of Saccharomyces cerevisiae according to an embodiment of the present invention, where the horizontal axis represents the glutathione content per cell and the vertical axis represents the number of colonies. [Figure 5] 1 is a graph showing the survival curve of a wild-type strain of Saccharomyces cerevisiae depending on the treatment concentration of lysine according to an embodiment of the present invention. [Figure 6] 1 is a graph showing the results of analyzing the concentration of aldehyde dehydrogenase produced from mutant strains of Saccharomyces cerevisiae according to an embodiment of the present invention, where the numbers on the horizontal axis represent the respective mutant strains of Saccharomyces cerevisiae and the numbers on the vertical axis represent the relative aldehyde dehydrogenase activity of the mutant strains of Saccharomyces cerevisiae compared to the aldehyde dehydrogenase activity of a wild-type strain of Saccharomyces cerevisiae. [Figure 7] FIG. 2 is a diagram showing the cell morphology of the mutant strain KwonP-1 of Saccharomyces cerevisiae according to one embodiment of the present invention, observed under an optical microscope. [Figure 8] FIG. 2 is a diagram showing the cell morphology of a wild-type strain of Saccharomyces cerevisiae according to one embodiment of the present invention, observed with an optical microscope. DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention will be described in more detail with reference to the following examples, which are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0064] [Example 1] Selection of strains with improved glutathione production capacity

[0065] To screen for novel mutant strains with improved glutathione production, wild-type strains of Saccharomyces cerevisiae were treated with EMS (Ethyl-methane-sulfonate) or NTG (Nitrosoguanidine) to induce mutations, and their resistance to methylglyoxal was evaluated. Finally, strains with improved glutathione production were selected. Specifically, the experiment was carried out as follows.

[0066] Example 1-1: Survival rate analysis of wild-type strain of Saccharomyces cerevisiae in methylglyoxal

[0067] To establish experimental conditions for screening novel yeast mutant strains with improved glutathione production, we first measured survival rates and determined the range of treatment concentrations before selecting methylglyoxal-resistant strains. Wild-selected Saccharomyces cerevisiae was used as the parent strain.

[0068] Specifically, to select resistant mutant strains, the survival rate of the prepared yeast strains was investigated by varying the concentration of methylglyoxal. To this end, the strains were inoculated into YPD medium (2% peptone, 1% yeast extract, 2% glucose) and incubated at 30°C for 1 hour at OD 20°C. 600nmAfter growth until the optical density (Optical Density at 600 nm) value reached 0.5, the cells were harvested. The harvested cells were inoculated onto YPD agar medium (containing 1.5% agar) supplemented with methylglyoxal at concentrations of 0 mM, 5 mM, 10 mM, and 15 mM, and then cultured. The harvested cells were washed with 0.1 M citrate buffer (pH 5.5) and then incubated at OD . 600nm The value was adjusted to 1.0 and the resultant was spread on a YPD agar medium supplemented with methylglyoxal. After spreading, the resultant was cultured at 30°C for 48 hours, and a survival rate graph of the strain was created.

[0069] As a result, as shown in Figure 1 below, when methylglyoxal was applied at a concentration of 10 mM, a mortality rate of 99.995% was confirmed, and when methylglyoxal was applied at a concentration of 15 mM, 100% of the test microorganisms were killed.

[0070] Based on the above results, in order to select novel mutant strains with improved glutathione production ability, YPD agar medium supplemented with 10 mM methylglyoxal was used to select methylglyoxal-resistant strains of wild-type strains.

[0071] Example 1-2: Establishment of mutation conditions for selecting mutant candidate strains with superior glutathione production ability

[0072] In Example 1-1, experimental conditions were established to screen for novel mutant strains with improved glutathione production. Specifically, to screen for methylglyoxal-resistant strains of wild-type strains, methylglyoxal was added to YPD agar medium at a concentration of 10 mM.

[0073] Therefore, in order to select mutant strains with superior glutathione production from the wild-type strain, survival curves were created for each treatment concentration of EMS (Ethyl-methane-sulfonate) or NTG (Nitrosoguanidine).

[0074] Specifically, the wild-type strain was inoculated into YPD medium, and the OD 600nm After 24 hours of cultivation, the two cultures were centrifuged at 4,000 rpm for 10 minutes to recover the precipitated bacterial cells. The recovered bacterial cells were washed twice with 0.1 M citrate buffer (pH 5.5), centrifuged, and finally measured at OD 0.5. 600nm The strain was diluted with 0.1 M citrate buffer (pH 5.5) to a value of 1.0. To induce mutations, the cells were collected after centrifugation and treated with NTG at 30°C for 30 minutes in 0.1 M citrate buffer (pH 5.5) containing 1%, 2%, 3%, or 4% NTG. The NTG-treated mutant strain was centrifuged at 4,000 rpm for 10 minutes to collect the cells, which were then washed twice with 0.1 M citrate buffer (pH 5.5). The washed cells were mixed with 1 ml of 0.1 M citrate buffer (pH 5.5), and then plated on YPD agar medium.

[0075] To screen for methylglyoxal-resistant mutant strains using EMS, a mutation-inducing agent, 1 mL of wild-type yeast strain grown under the same conditions as the NTG treatment was centrifuged. After centrifugation, the collected cells were treated with 0.1 M citrate buffer (pH 5.5) containing 1%, 2%, 3%, and 4% EMS at 30°C for 60 minutes. The EMS-treated mutant strains were centrifuged at 4,000 rpm for 10 minutes to collect the cells, which were then washed twice with 0.1 M citrate buffer (pH 5.5). The washed cells were mixed with 1 mL of 0.1 M citrate buffer (pH 5.5) and plated on YPD agar medium.

[0076] As a result, as shown in Figure 3 below, EMS killed 99.4% of the bacteria when treated at a concentration of 3% after 24 hours of culture. Also, as shown in Figure 2, NTG killed 99.7% of the bacteria when treated at a concentration of 1% after 24 hours of culture.

[0077] Based on the above results, the optimal NTG and EMS treatment concentrations for selecting glutathione-resistant strains from wild-type strains were selected to be 1% and 3%, respectively, in order to select novel mutant yeast strains with improved glutathione production ability.

[0078] Example 1-3: Selection of mutant yeast strains with superior glutathione production ability

[0079] In Examples 1-2 and 1-3, experimental conditions were established to screen for novel mutant yeast strains with improved glutathione production. Specifically, to screen for methylglyoxal-resistant wild-type yeast strains, methylglyoxal was added to YNB agar medium at a concentration of 10 mM, and conditions of 1% NTG and 3% EMS were established for mutation.

[0080] To select mutant strains with superior glutathione production from wild-type strains, methylglyoxal-resistant mutant strains were selected, and the cells were grown in YPD medium to measure the glutathione concentration, which was expressed as the intracellular glutathione content (%, g / g-cell).

[0081] Specifically, the wild-type strain was inoculated into YPD medium and cultured for 24 hours, and then the culture medium was harvested and centrifuged at 4,000 rpm for 10 minutes to recover the bacterial cells. The recovered bacterial cells were then washed twice with 0.1 M citrate buffer (pH 5.5) and finally OD 600nmThe strain was diluted with 0.1 M citrate buffer (pH 5.5) to a value of 1.0. To induce mutations, the strain was treated with 0.1 M citrate buffer (pH 5.5) containing 3% EMS for 10 minutes and with 0.1 M citrate buffer (pH 5.5) containing 1% NTG for 30 minutes. The mutated microorganisms were then centrifuged at 4,000 rpm for 10 minutes to recover the cells, which were then mixed with 0.1 M citrate buffer (pH 5.5) and plated onto YPD agar medium supplemented with 10 mM methylglyoxal. After culturing, the surviving strains were collected and cultured in YPD medium for 48 hours. The incubation conditions were a temperature of 30°C and a stirring speed of 160 rpm. After 48 hours of culture, the glutathione concentration was measured and expressed as the intracellular glutathione content (%, g / g-cell).

[0082] To analyze glutathione concentration, the cultured cells were centrifuged, and 1 mL of water was added to the precipitated cells. The cells were then extracted by stirring at 85°C and 1,000 rpm for 2 hours. After extraction, the cells were removed using a centrifuge, and the supernatant was collected by filtration through a 0.22 μm filter. The glutathione concentration in the collected filtrate was measured using HPLC (Shimazu LC-20AD). The glutathione concentration was analyzed using a glutathione standard curve. The HPLC analysis conditions were a C18 column. The mobile phase (2.02 g / L sodium 1-heptanesulfonate monohydrate, 6.8 g / L potassium dihydrogen phosphate, pH 3.0, methanol mixture) was run at a flow rate of 1 mL / min, and the glutathione concentration was measured using a UV detector at a wavelength of 210 nm.

[0083] A total of 130 mutant strains were generated, and the glutathione concentrations of each mutant strain were analyzed. As shown in Figure 4, 70 mutant strains had intracellular glutathione contents of 0.3% to <0.5%, 30 mutant strains had intracellular glutathione contents of 0.5% to <0.7%, 23 mutant strains had intracellular glutathione contents of 0.7% to <0.85%, and 7 mutant strains had intracellular glutathione contents of 0.85% to <1.1%. That is, the glutathione production capacity of the methylglyoxal-resistant strains treated with EMS or NTG was 48.6%, which was superior to the intracellular glutathione content of the wild-type strain (0.42%). Of these, seven mutant strains showed glutathione production capacity that was more than twice that of the parent strain, as shown in Table 1 below.

[0084] Based on this, Saccharomyces cerevisiae ems c7, which had the highest glutathione production ability among the methylglyoxal-resistant mutant strains treated with EMS, was selected as the final candidate mutant strain with excellent glutathione production ability.

[0085] In Example 2 below, mutation using NTG treatment was used to improve the aldehyde dehydrogenase-producing ability of the selected candidate strain.

[0086] Cultivation results of methylglyoxal-resistant mutant strains of yeast treated with EMS or NTG. [Table 1]

[0087] [Example 2] Selection of strains with improved aldehyde dehydrogenase (ALDH) production

[0088] To screen for novel mutant strains with improved aldehyde dehydrogenase production, the lysine resistance of Saccharomyces cerevisiae ems c7, which was selected in Example 1 to have the highest glutathione production, was evaluated, and a strain with improved aldehyde dehydrogenase production was finally selected. Specifically, the experiment was carried out as follows.

[0089] Example 2-1: Viability analysis of Saccharomyces cerevisiae EMS C7 strain in lysine

[0090] To establish experimental conditions for screening novel mutant strains with improved aldehyde dehydrogenase production, we sought to determine the optimal lysine concentration for screening lysine-resistant strains of the glutathione mutant Saccharomyces cerevisiae ems c7. For this study, we prepared a Saccharomyces cerevisiae ems c7 strain with improved glutathione production and used it as the parent strain.

[0091] Specifically, to select a lysine-resistant mutant strain of Saccharomyces cerevisiae, the survival rate of the prepared Saccharomyces cerevisiae ems c7 strain was investigated depending on the concentration of lysine treatment. To this end, the strain was inoculated into YPD medium (2% peptone, 1% yeast extract, 2% glucose) and incubated at 30°C until the OD 600nm The cells were grown until the OD value reached 0.5, and then harvested. The harvested cells were smeared on YPD agar medium (containing 1.5% agar) supplemented with lysine at concentrations of 0%, 3%, 4%, 5%, 6%, and 7%. The harvested cells were washed with 0.1 M citrate buffer (pH 5.5) and then subjected to OD 600nm The value was adjusted to 1.0 and the resultant was spread on a YPD agar medium supplemented with lysine. After spreading, the strain was cultured at 30°C for 48 hours, and a survival curve of the strain was created.

[0092] As a result, as shown in Figure 5 below, 87.5% of the bacteria were killed when treated with ricin at a concentration of 3%.

[0093] Therefore, to select novel mutant strains with improved aldehyde dehydrogenase production, the optimal lysine treatment concentration for selecting lysine-resistant strains of Saccharomyces cerevisiae ems c7 strain was selected as 3% based on the above results, and selection was performed using YPD agar medium supplemented with lysine at a concentration of 3%.

[0094] Example 2-2: Selection of mutant candidate strains with superior aldehyde dehydrogenase production ability

[0095] In Examples 1-2 and 2-1, experimental conditions were established to screen for novel yeast mutant strains with improved aldehyde dehydrogenase activity. Specifically, to screen for lysine-resistant mutant Saccharomyces cerevisiae ems c7 strains, lysine was added to YPD agar medium at a concentration of 3%, and NTG was added at 1% for mutation.

[0096] To select mutant strains with superior glutathione production from the Saccharomyces cerevisiae ems c7 strain, the cells were grown in YPD medium containing 3% lysine and the aldehyde dehydrogenase concentration was measured.

[0097] Specifically, the wild-type yeast strain was inoculated into YPD medium and cultured for 24 hours. The culture was then centrifuged at 4,000 rpm for 10 minutes to collect the cells. The collected cells were washed twice with 0.1 M citrate buffer (pH 5.5) and finally measured at OD . 600nm The strains were diluted with 0.1 M citrate buffer (pH 5.5) to a value of 1.0. To induce mutations, each strain was treated with 0.1 M citrate buffer (pH 5.5) containing 1% NTG for 10 minutes. The mutants were then centrifuged at 4,000 rpm for 10 minutes to collect the cells, which were then mixed with 0.1 M citrate buffer (pH 5.5) and plated onto YPD agar medium supplemented with 3% lysine. After plating and incubation, surviving strains were collected and cultured in YPD medium for 48 hours. The incubation conditions were a temperature of 30°C and an agitation speed of 160 rpm. After 48 hours of incubation, aldehyde dehydrogenase activity was measured.

[0098] However, because aldehydes are volatile and produced in trace amounts, existing methods have significant sample-to-sample variability. Therefore, it is necessary to establish a stable method for measuring aldehydes. Based on this, the development of a new, accurate method for measuring enzyme activity is absolutely necessary for quality control (QC) of the produced aldehyde dehydrogenase. A new method was developed in Example 3.

[0099] [Example 3] Establishment of a new method for measuring aldehyde dehydrogenase activity and measurement of enzyme activity in mutant strains

[0100] A commonly used method for measuring aldehyde dehydrogenase activity is measuring NAD(P) absorbance at a wavelength of 340 nm. However, this method is indirect and cannot be used because it measures the change in coenzyme. Therefore, to directly measure the Km value of the enzyme for the substrate, the inventors used HPLC analysis to quantify aldehydes. However, the amount of aldehyde consumed by the enzyme ALDH is very small, and acetaldehyde is highly volatile even at room temperature, making it technically difficult to directly quantify the reaction product.

[0101] Therefore, in order to solve this problem, the inventors analyzed the amount of aldehyde reduced by the reaction of aldehyde dehydrogenase based on the reference (Guan et al., 2012) that when a certain amount of dinitrophenylhydrazine (DNPH) is added to acetaldehyde and reacted at a certain concentration, an acetaldehyde-hydrazone (AcH-DNPH) compound is formed, which can be quantified by developing it on an HPLC C18 column with a mobile phase of acetonitrile and water, and detecting it at 360 nm. The enzyme reaction mixture consisted of 50 mM potassium phosphate buffer (pH 8.0), 1 mM acetaldehyde, and 10 μL of the microbial lysate to be tested, plus the enzyme cofactor 1 mM NADP+. The reaction was then incubated at 30°C, followed by the addition of 50 μL of 10 mM DNPH, followed by labeling with acetaldehyde-hydrazone (AcH-DNPH) for 1 hour at 22°C. The reaction was terminated by the addition of 3 M sodium acetate (pH 9), and the layer containing the acetaldehyde-DNPH compound was separated by the addition of a two-fold volume of acetonitrile, after which it was injected into an HPLC for analysis. The concentration of labeled aldehyde was analyzed using a standard curve of Aldehyde-DNPH (Sigma-Aldrich). The HPLC analysis conditions were a C18 column with a solvent (acetonitrile, water) at a flow rate of 1 ml / min and a UV detector at 360 nm. One unit of aldehyde dehydrogenase was defined as the reduction of acetaldehyde-DNPH at a concentration of 1 mM per minute, and aldehyde dehydrogenase activity was expressed as units per mg of protein.

[0102] Analysis of aldehyde dehydrogenase levels revealed that the lysine-resistant mutants of Saccharomyces cerevisiae ems c7 strains treated with NTG exhibited aldehyde dehydrogenase production abilities of approximately 42% of the lysine-resistant mutants (#4, #8, #16, and #21) superior to the parent strain and Saccharomyces cerevisiae ems c7 (110% superior to the parent strain). Four strains (#4, #8, #16, and #21) exhibited over 140% improved production. The glutathione content of the four strains with superior aldehyde dehydrogenase production abilities was 0.9% for mutant #4, 0.96% for mutant #8, 0.93% for mutant #16, and 0.92% for mutant #21. There was no change in glutathione production ability after NTG treatment.

[0103] The results for the four mutant strains, whose aldehyde dehydrogenase activity was increased by 1.4 times or more compared to the parent strain, are shown in Table 2. Based on this, the lysine-resistant mutant Saccharomyces cerevisiae #8, which was treated with NTG, was selected as a mutant strain with excellent aldehyde dehydrogenase production ability and named Saccharomyces cerevisiae Kwon P-1.

[0104] Aldehyde dehydrogenase activity in lysine-resistant mutants of yeast treated with NTG. [Table 2]

[0105] [Example 4] Morphological changes in mutants with superior glutathione and aldehyde dehydrogenase production

[0106] In Example 2-2, novel yeast mutant strains with improved glutathione and aldehyde dehydrogenase production were selected, and the mutant strains were observed using an optical microscope to observe morphological changes. The cell morphology of the mutant strain Saccharomyces cerevisiae Kwon P-1 is shown in Figure 7, and the wild-type morphology of the wild-type yeast strain is shown in Figure 8. Optical microscope observation confirmed that the mutant strain Saccharomyces cerevisiae Kwon P-1 had a cell diameter that increased by more than 60%, and that the size of the intracellular organelle, the vacuole, was enlarged. In the above example, the unique morphological characteristics of the Saccharomyces cerevisiae Kwon P-1 strain of this patent, with a 60% increase in cell size and large vacuoles, are specialized morphologies that can be used as an important means to prevent patent infringement.

[0107] [Example 5] Simultaneous production of glutathione and aldehyde dehydrogenase using Saccharomyces cerevisiae Kwon P-1

[0108] Saccharomyces cerevisiae Kwon P-1 was inoculated into sterilized YPD liquid medium (2% peptone, 1% yeast extract, 2% glucose) and incubated for 16 hours at 30°C with an agitation speed of 160 rpm. For the main culture, sterilized YPD liquid medium was inoculated at a 1% level and incubated for 48 hours under the same conditions as for the seed culture. The glutathione concentration and aldehyde dehydrogenase activity of the culture medium were then measured. Using 20 flasks and 20 replicate culture experiments under the same conditions, the intracellular glutathione content and aldehyde dehydrogenase activity results were obtained, as shown in Table 3. The patented strain, Saccharomyces cerevisiae Kwon P-1, can simultaneously produce glutathione and aldehyde dehydrogenase, and the average production values ​​of 20 repeated experiments were confirmed to be 0.97% intracellular glutathione content and 0.173 Unit / mg-protein activity. This indicates that the mutant strain of the patented strain, Saccharomyces cerevisiae Kwon P-1, has a 2.3-fold increase in glutathione content and a 1.7-fold increase in aldehyde dehydrogenase production capacity compared to the parent strain.

[0109] Simultaneous production of glutathione and aldehyde dehydrogenase by Saccharomyces cerevisiae Kwon P-1 in flask liquid culture [Table 3] [Accession number]

[0110] Depository institution name: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13925BP Date of acceptance: 20190822 Depository institution name: Korea Institute of Bioscience and Biotechnology Accession number: KCTC14122BP Date of acceptance: 20200130 Depository institution name: Korea Institute of Bioscience and Biotechnology Accession number: KCTC14123BP Date of acceptance: 20200130

Claims

1. Saccharomyces cerevisiae Kwon P-1 KCTC13925BP.

2. Saccharomyces cerevisiae Kwon P-2 KCTC14122BP.

3. Saccharomyces cerevisiae Kwon P-3 KCTC14123BP.

4. A method for producing a mutant yeast having an improved ability to simultaneously produce glutathione and aldehyde dehydrogenase, comprising: a primary selection step of treating Saccharomyces cerevisiae yeast with ethyl methanesulfonate or nitrosoguanidine to induce mutations, and then treating the resulting mutant yeast with 5 mM to 15 mM methylglyoxal to select methylglyoxal-adapted yeast from the induced mutant yeast; and a second selection step of treating the methylglyoxal-adapted yeast selected in the first selection with lysine at a concentration of 3% to 5% in order to select a lysine-adapted mutant yeast from the methylglyoxal-adapted mutant yeast selected in the first selection.

5. A method for simultaneously producing glutathione and aldehyde dehydrogenase, comprising: a first selection step in which Saccharomyces cerevisiae yeast is treated with ethyl methanesulfonate or nitrosoguanidine to induce mutations, and the resulting mutant yeast is treated with 10 mM methylglyoxal to select methylglyoxal-adapted yeast from the induced mutant yeast; and (ii) a second selection step in which the methylglyoxal-adapted yeast selected in the first selection step is treated with 3% to 5% lysine to select a lysine-adapted mutant yeast from the methylglyoxal-adapted mutant yeast selected in the first selection step.

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