<i>Saccharomyces cerevisiae </i>kwon P-1, 2, 3 which produce aldehyde dehydrogenase and glutathione
11618889 · 2023-04-04
Assignee
Inventors
Cpc classification
C12N15/01
CHEMISTRY; METALLURGY
C12N9/0008
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
International classification
C12N15/01
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
Abstract
A yeast strain producing glutathione (GSH) and aldehyde dehydrogenase, and more specifically, the yeast strains Saccharomyces cerevisiae Kwon P-1 KCTC13925BP, Saccharomyces cerevisiae Kwon P-2 KCTC14122BP, and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP, which produce both glutathione and aldehyde dehydrogenase.
Claims
1. A Saccharomyces cerevisiae yeast having an enhanced ability to produce aldehyde dehydrogenase and glutathione concurrently, the Saccharomyces cerevisiae yeast is 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, wherein the Saccharomyces cerevisiae yeast is obtained by a process comprising: a first step for treating a Saccharomyces cerevisiae yeast with ethylmethanesulfonate or nitrosoguanidine to induce mutation, a second step for treating the induced mutant yeast obtained in the first step with methylglyoxal to select a methylglyoxal-adapted mutant yeast from the induced mutant yeast, and a third step for treating the methylglyoxal-adapted mutant yeast selected in the second step with lysine to select a lysine-adapted mutant yeast from the methylglyoxal-adapted mutant yeast selected in the second step.
2. The Saccharomyces cerevisiae yeast according to claim 1, wherein the yeast is Saccharomyces cerevisiae Kwon P-1 KCTC13925BP.
3. The Saccharomyces cerevisiae yeast according to claim 1, wherein the yeast is Saccharomyces cerevisiae Kwon P-2 KCTC14122BP.
4. The Saccharomyces cerevisiae yeast according to claim 1, wherein the yeast is Saccharomyces cerevisiae Kwon P-3 KCTC14123BP.
5. The Saccharomyces cerevisiae yeast according to claim 1, wherein the induced mutant yeast is treated with 10 mM methylglyoxal in the second step to select the methylglyoxal-adapted mutant yeast, and wherein the methylglyoxal-adapted mutant yeast is treated with lysine at concentrations of 3% to 5% in the third step to select lysine-adapted mutant yeast.
6. A method for producing both glutathione and aldehyde dehydrogenase comprising culturing a Saccharomyces cerevisiae yeast of claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
(9) Hereinafter, constitutions and effects of the present disclosure will be described in more detail with reference to the following examples. However, these examples are used for illustration only, and the scope of the present disclosure is not limited by these examples.
Example 1
(10) Selection of Strains Having Enhanced Ability to Produce Glutathione
(11) In order to select novel mutant strains having an enhanced ability to produce glutathione, the wild Saccharomyces cerevisiae strain was treated with ethyl-methane-sulfonate (EMS) or nitrosoguanidine (NTG) for mutation induction, followed by evaluation for resistance to methylglyoxal, thereby finally selecting strains having an enhanced ability to produce glutathione. Specifically, experiments were carried out as follows.
Example 1-1: Analysis of Survival Rate of Wild Saccharomyces cerevisiae Strains in Methylglyoxal
(12) In order to establish experimental conditions for selecting novel mutant yeast strains having an enhanced ability to produce glutathione, the survival rate was measured to determine the range of the treatment concentration and then the treatment concentration was intended to be selected, before the selection of strains having resistance to methylglyoxal. Saccharomyces cerevisiae selected from the wild was used as a mother strain.
(13) Specifically, in order to select resistant strains of mutant strains, the survival rate of the prepared yeast strain was checked for different methylglyoxal treatment concentrations. The strain was seeded in YPD media (2% peptone, 1% yeast extract, 2% glucose) and grown at 30° C. until the OD.sub.600 nm (optical density at 600 nm) value reached 0.5, and then the cells were recovered. The recovered cells were seeded by plating on YPD agar media (with 1.5% agar added) supplemented with methylglyoxal at concentrations of 0 mM, 5 mM, 10 mM and 15 mM, separately, and then cultured. During this procedure, the recovered cells were washed with 0.1 M citric buffer (pH 5.5), and then, the cells were plated on YPD agar media supplemented with methylglyoxal after the OD.sub.600 nm value was adjusted to 1.0. After plating, the cells were cultured at 30° C. for 48 hours, and the survival curve of the strain was plotted on the graph.
(14) As shown in the results in
(15) Therefore, in order to select novel mutant strains having an enhanced ability to produce glutathione, YPD agar media supplemented with 10 mM methylglyoxal were used on the basis of the result, so as to select methylglyoxal-resistant strains of the wild strain.
Example 1-2: Establishment of Mutation Conditions for Selecting Candidate Mutant Strains Having Enhanced Ability to Produce Glutathione
(16) Experimental conditions for selecting novel mutant strains having an enhanced ability to produce glutathione were established in Example 1-1. Specifically, in order to select methylglyoxal-resistant strains of the wild strain, 10 mM methylglyoxal was added to YPD agar media.
(17) Therefore, in order to select mutant strains having an enhanced ability to produce glutathione from the wild strain, a curve of survival rate versus treatment concentration of ethyl-methane-sulfonate (EMS) or nitrosoguanidine (NTG) was created.
(18) Specifically, the wild strain was seeded in YPD media, and grown until the OD.sub.600 nm value reached 0.5 or for 24 hours. Then, the two types of cultures were centrifuged at 4,000 rpm for 10 minutes to recover precipitated cells. The recovered cells were washed two times with 0.1 M citric buffer (pH 5.5), followed by centrifugation, and finally, the cells were diluted with 0.1 M citric buffer (pH 5.5) until the OD.sub.600 nm value reached 1.0, and then used. Thereafter, for induction of mutation, 0.1 M citric buffers (pH 5.5) containing 1%, 2%, 3%, and 4% NTG were added to the cells recovered after centrifugation, wherein the cells were treated with NTG in a temperature condition of 30° C. for 30 minutes. The NTG-treated mutant strains were centrifuged at 4,000 rpm for 10 minutes to recover the cells, and then the cells were washed two times with 0.1 M citric buffer (pH 5.5). After 1 mL of 0.1 M citric buffer (pH 5.5) was added to and mixed with the washed cells, the mixture was plated on YPD agar media.
(19) Meanwhile, in order to select methylglyoxal-resistant mutant strains by the treatment with EMS, which is a substance for mutation induction, 1 mL of the wild yeast strain grown in the same conditions as in the case of NTG treatment was taken and centrifuged. 0.1 M citric buffers (pH 5.5) containing 1%, 2%, 3%, and 4% EMS were added to the cells recovered after centrifugation, wherein the microorganisms were treated in a temperature condition of 30° C. for 60 minutes. The EMS-treated mutant strains were centrifuged at 4,000 rpm for 10 minutes to recover the cells, and then the cells were washed two times with 0.1 M citric buffer (pH 5.5). After 1 mL of 0.1 M citric buffer (pH 5.5) was added to and mixed with the washed cells, the mixture was plated on YPD agar media.
(20) As shown in the results of
(21) Therefore, in order to select novel mutant yeast strains having an enhanced ability to produce glutathione, the optimal treatment concentrations of NTG and EMS for selecting glutathione-resistant strains of the wild strain were selected to be 1% and 3%, respectively, on the basis of the above results.
Example 1-3: Selection of Candidate Mutant Strains Having Enhanced Ability to Produce Glutathione
(22) Experimental conditions for selecting novel mutant yeast strains having an enhanced ability to produce glutathione were established in Example 1-2. Specifically, for the selection of methylglyoxal-resistant strains of the wild yeast strain, methylglyoxal was intended to be added at a concentration of 10 mM to YNB agar media, and for mutation, the conditions of 1% NTG and 3% EMS were established.
(23) Therefore, in order to select mutant strains having an enhanced ability to produce glutathione from the wild strain, methylglyoxal-resistant mutant strains were selected and the cells were grown in YPD media. Then, the concentration of glutathione was measured and expressed as the content of glutathione (%, g/g-cell) in cells.
(24) Specifically, the wild strain was seeded in YPD media and grown for 24 hours, and then the culture was obtained and centrifuged at 4,000 rpm for 10 minutes, thereby recovering cells. Thereafter, the recovered cells were washed two times with 0.1 M citric buffer (pH 5.5), and finally diluted with 0.1 M citric buffer (pH 5.5) until the OD.sub.600 nm value reached 1.0, and then used. For mutation induction, the cells were separately treated with 0.1 M citric buffer (pH 5.5) containing 3% EMS for 10 minutes and 0.1 M citric buffer (pH 5.5) containing 1% NTG for 30 minutes, the mutated microorganisms were centrifuged at 4,000 rpm for 10 minutes to recover cells, 0.1 M citric buffer (pH 5.5) was added and mixed with the cells, and then the mixtures were plated on YPD agar media supplemented with 10 mM methylglyoxal. After plating and culture, the surviving strains were recovered, and cultured in YPD media for 48 hours. As for culture conditions, the culture temperature was 30° C. and the stirring rate was 160 rpm. After the cells were cultured for 48 hours, the concentration of glutathione was measured and expressed as the content of glutathione (%, g/g-cell).
(25) For the analysis of glutathione concentration, the cultured cells were centrifuged, and 1 mL of water was added to the precipitated cells, followed by stirring at 1,000 rpm for 2 hours at 85° C., and then extraction was conducted. After extraction, the cells were removed using a centrifuge, and the supernatant was recovered by filtration through a 0.22 μm-filter. Through HPLC (Shimazu LC-20AD) analysis, the concentration of glutathione contained in the recovered filtrate was measured. The glutathione concentration was analyzed using a standard curve of glutathione, and as for HPLC analysis conditions, a C18 column was used for analysis. A mobile-phase solvent (a mixture of 2.02 g/L sodium 1-heptanesulfonate monohydrate, 6.8 g/L potassium dihydrogen phosphate, and methanol of pH 3.0) was allowed to flow at a flow rate of 1 ml/min, and the concentration of glutathione was determined by detection at a wavelength of 210 nm in an ultraviolet detector.
(26) A total of 130 mutant strains were generated, and as a result of analyzing the glutathione concentration of each mutant strain, as shown in
(27) Based on the above results, Saccharomyces cerevisiae ems c7, which had the best glutathione-producing ability among methylglyoxal-resistant mutant strains by EMS treatment, was selected as the final candidate mutant strain having excellent glutathione-producing ability.
(28) Hereinafter, in Example 2 below, the aldehyde dehydrogenase-producing ability of the candidate strains selected using mutation through NTG treatment was enhanced.
(29) TABLE-US-00001 TABLE 1 Culture results of methylglyoxal-resistant mutant strains of yeast by EMS or NTG treatment Glutathione Mutation treatment content No. Strain name method (%) Control Saccharomyces — 0.42 cerevisiae WT Test group 1 Saccharomyces EMS/methylglyoxal 0.95 cerevisiae ems c7 resistance Test group 2 Saccharomyces EMS/methylglyoxal 0.88 cerevisiae ems d5 resistance Test group 3 Saccharomyces EMS/methylglyoxal 0.92 cerevisiae ems d6 resistance Test group 4 Saccharomyces EMS/methylglyoxal 0.93 cerevisiae ems e1 resistance Test group 5 Saccharomyces EMS/methylglyoxal 0.85 cerevisiae ems e3 resistance Test group 6 Saccharomyces NTG/methylglyoxal 0.86 cerevisiae ntg g6 resistance Test group 7 Saccharomyces NTG/methylglyoxal 0.88 cerevisiae ntg h1 resistance
Example 2
(30) Selection of Strains Having Enhanced Ability to Produce Aldehyde Dehydrogenase (ALDH)
(31) In Order to Select Novel Mutant Strains Having an Enhanced Ability to Produce Aldehyde dehydrogenase, Saccharomyces cerevisiae ems c7 showing the highest glutathione production, which was selected in Example 1, was evaluated for resistance to lysine, and finally, strains having an enhanced ability to produce aldehyde dehydrogenase were selected. Specifically, the following experiments were conducted.
Example 2-1: Analysis of Survival Rate of Saccharomyces cerevisiae Ems c7 Strain in Lysine
(32) To establish experimental conditions for selecting novel mutant strains having an enhanced ability to produce aldehyde dehydrogenase, the optimal lysine treatment concentration for selecting lysine-resistant strains of the glutathione mutant strain Saccharomyces cerevisiae ems c7 was intended to be selected. Saccharomyces cerevisiae ems c7 having an enhanced ability to produce glutathione was prepared, and this strain was used as a mother strain.
(33) Specifically, in order to select lysine-resistant strains of the Saccharomyces cerevisiae mutant strain, the survival rate of the prepared Saccharomyces cerevisiae ems c7 strain versus the lysine treatment concentration was checked. To this end, the strain was seeded in YPD media (2% peptone, 1% yeast extract, 2% glucose) and grown at 30° C. until the OD.sub.600 nm value reached 0.5, and then the cells were recovered. The recovered cells were plated on YPD agar media (with 1.5% agar added) supplemented with lysine at concentrations of 0%, 3%, 4%, 5%, 6%, and 7%, separately. The recovered cells were washed with 0.1 M citric buffer (pH 5.5), and then, the cells were plated on YPD agar media supplemented with lysine after the OD.sub.600 nm value was adjusted to 1.0. After plating, the cells were cultured at 30° C. for 48 hours, and a curve of survival rate of the strain was created.
(34) As shown in the results in
(35) Therefore, in order to select novel mutant strains having an enhanced ability to produce aldehyde dehydrogenase, the optimal lysine treatment concentration for selecting lysine-resistant strains of the Saccharomyces cerevisiae ems c7 strain was selected to be 3% on the basis of the above results, and strain selection was carried out by using YPD agar media supplemented with 3% lysine.
Example 2-2: Selection of Candidate Mutant Strains Having Excellent Ability to Produce Aldehyde Dehydrogenase
(36) Experimental conditions for selecting novel yeast mutant strains having enhanced aldehyde dehydrogenase activity in Examples 1-2 and 2-1 were established. Specifically, for the selection of lysine-resistant strains of the mutant Saccharomyces cerevisiae ems c7 strain, 3% lysine was added to YPD agar media, and for mutation, 1% NTG conditions were established.
(37) Therefore, in order to select mutant strains having an excellent ability to produce glutathione from the Saccharomyces cerevisiae ems c7 strain, the cells were grown in YPD media containing 3% lysine and the concentration of aldehyde dehydrogenase was measured.
(38) Specifically, the wild yeast strain was seeded in YPD media and grown for 24 hours, and then the culture was centrifuged at 4,000 rpm for 10 minutes to recover cells. The recovered cells were washed two times with 0.1 M citric buffer (pH 5.5) and diluted by addition of 0.1 M citric buffer (pH 5.5) until the OD.sub.600 nm value reached 1.0. For mutation induction, the cells were treated with 0.1 M citric buffer (pH 5.5) containing 1% NTG for 10 min, and then the mutant strains were centrifuged at 4,000 rpm for 10 minutes to recover cells. Thereafter, 0.1 M citric buffer (pH 5.5) was added and mixed with the cells, and then the mixture was plated on YPD agar media supplemented with 3% lysine. After plating and culture, the surviving strains were recovered, and cultured in YPD media for 48 hours. As for culture conditions, the culture temperature was 30° C. and the stirring rate was 160 rpm. After the cells were cultured for 48 hours, the aldehyde dehydrogenase activity was measured.
(39) However, aldehydes are volatile and are produced in trace amounts, and thus the measurement by an existing method causes a large deviation for the same sample, so that a stable aldehyde measurement method needs to be established. Therefore, the development of new accurate enzyme activity measurement methods based on the above results is absolutely needed in the quality control (QC) of the produced aldehyde dehydrogenase, and thus, a new method was developed in Example 3.
Example 3
(40) Establishment of New Aldehyde Dehydrogenase Activity Measurement Method and Measurement of Enzyme Activity of Mutant Strains
(41) As for existing methods for measuring aldehyde dehydrogenase activity, the measurement of NAD(P)+ absorbance at a wavelength of 340 nm was widely used, but this method could not be favorably used since this method checked a change in coenzyme amount and thus corresponds to an indirect method. In order to directly measure the Km value of an enzyme to a substrate of the enzyme, the present inventors employed HPLC analysis for aldehyde quantification. However, the amount of aldehyde consumed by the enzyme ALDH is very small and aldehyde is highly volatile even at room temperature, and thus the direct quantification of aldehyde in the reaction product is technically difficult.
(42) To solve the problem, the present inventors analyzed the amount of aldehyde reduced by the reaction of aldehyde dehydrogenase, on the basis of the reference document (Guan. et. al, 2012) describing that when dinitrophenylhydrazine (DNPH) is added to aldehyde at a certain ratio and reacted at certain concentrations, aldehyde-hydrazone (AcH-DNPH) is formed, and this compound is developed on C18 column for HPLC using acetonitrile as a mobile phase and water as a solvent and thus can be detected and quantified at 360 nm. As for an enzyme reaction solution, 1 mM NADP+ as an enzyme cofactor was added to 50 mM potassium phosphate buffer (pH 8.0), 1 mM acetaldehyde, and 10 μL of a lysate of microorganisms to be measured, followed by culture at 30° C. Then, 50 μL of 10 mM DNPH was added thereto, followed by acetaldehyde-hydrazone (AcH-DNPH) labeling at 22° C. for 1 hour. The labeling reaction was stopped by addition of 3 M sodium acetate (pH 9), and a 2-fold volume of acetonitrile was added to separate a layer containing the acetaldehyde-DNPH compound dissolved therein, and then the layer was injected into HPLC and analyzed. The concentration of the labeled aldehyde was analyzed using a substance standard curve of aldehyde-DNPH (Sigma-Aldrich). As for HPLC analysis conditions, C18 column was used, and a solvent (acetonitrile and water) was allowed to flow at a flow rate of 1 ml/min, and the concentration of aldehyde was detected at a wavelength of 360 nm in an ultraviolet detector. Meanwhile, 1 unit of aldehyde dehydrogenase is designated as the concentration of acetaldehyde-DNPH reduced per minute, 1 mM, and the aldehyde dehydrogenase activity was expressed as the unit per mg of protein.
(43) As a result of analyzing the concentration of aldehyde dehydrogenase, as shown in
(44) Table 2 shows the results of the four species of mutant strains, of which the aldehyde dehydrogenase activity was enhanced by 1.4 times or more compared with the mother strain. Based on these results, the lysine-resistant mutant Saccharomyces cerevisiae #8 by NTG treatment was selected as a mutant strain having an excellent ability to produce aldehyde dehydrogenase and named Saccharomyces cerevisiae Kwon P-1, the lysine-resistant mutant Saccharomyces cerevisiae #16 by NTG treatment was selected as a mutant strain having an excellent ability to produce aldehyde dehydrogenase and named Saccharomyces cerevisiae Kwon P-2, and the lysine-resistant mutant Saccharomyces cerevisiae #21 by NTG treatment was selected as a mutant strain having an excellent ability to produce aldehyde dehydrogenase and named Saccharomyces cerevisiae Kwon P-3.
(45) TABLE-US-00002 TABLE 2 Aldehyde dehydrogenase activity of lysine-resistant mutant strains of yeas by NTG treatment ALDH activity Mutation treatment (Unit/mg- No. Strain name method protein) Control 1 Saccharomyces — 0.10 cerevisiae WT Control 2 Saccharomyces EMS/methylglyoxal 0.11 cerevisiae ems c7 resistance Test group 4: Saccharomyces NTG/methylglyoxal and 0.15 cerevisiae #4 lysine resistance Test group 8: Saccharomyces NTG/methylglyoxal and 0.16 cerevisiae #8 lysine resistance Test group 16: Saccharomyces NTG/methylglyoxal and 0.14 cerevisiae #16 lysine resistance Test group 21: Saccharomyces NTG/methylglyoxal and 0.14 cerevisiae #21 lysine resistance
Example 4
Morphological Change of Mutants Having Excellent Ability to Produce Glutathione and Aldehyde Dehydrogenase
(46) The novel mutant yeast strain having an enhanced ability to produce glutathione and aldehyde dehydrogenase were selected in Example 2-2, and for the observation of morphological changes, the mutant strain was observed using an optical microscope. The cell morphology of the mutant strain Saccharomyces cerevisiae KownP-1 is shown in
Example 5
(47) Production of Both Glutathione and Aldehyde Dehydrogenase Using Saccharomyces cerevisiae KownP-1
(48) Saccharomyces cerevisiae Kwon P-1 was seeded in sterilized YPD liquid media (2% peptone, 1% yeast extract, 2% glucose), and seed-cultured for 16 hours wherein the culture temperature was 30° C. and the stirring rate was 160 rpm. As for main culture, the strain was seeded at a level of 1% in sterilized YPD liquid media and cultured for 48 hours under the same conditions as in the seed culture. Thereafter, the glutathione concentration and the aldehyde dehydrogenase activity of the culture were measured. As a result of 20 times of repeated culture experiments using 20 flasks under the same conditions, the results of glutathione content and aldehyde dehydrogenase activity in the cells were obtained as shown in Table 3. The strain Saccharomyces cerevisiae Kwon P-1 can produce both glutathione and aldehyde dehydrogenase and showed 0.97% in glutathione content in cells and 0.173 unit/mg-protein in aldehyde dehydrogenase activity, referring to the average production values from 20 times of repeated experiments. These results indicate that the mutant strain Saccharomyces cerevisiae Kwon P-1 showed an increase in glutathione content by 2.3 times and an increase in aldehyde dehydrogenase activity by 1.7 times compared with the mother strain, indicating enhanced producing ability.
(49) TABLE-US-00003 TABLE 3 Verification of production of both glutathione and aldehyde dehydrogenase by Saccharomyces cerevisiae Kwon P-1 through flask liquid culture Produced amount after flask culture Flask Glutathione ALDH activity No. content (%) (Unit/mg-protein) 1 0.95 0.16 2 0.98 0.18 3 0.93 0.15 4 1.00 0.19 5 0.98 0.17 6 0.97 0.19 7 0.95 0.21 8 0.96 0.16 9 0.94 0.17 10 0.98 0.18 11 0.99 0.19 12 1.00 0.16 13 0.98 0.15 14 0.94 0.18 15 0.96 0.17 16 0.96 0.18 17 0.98 0.16 18 0.99 0.15 19 0.97 0.21 20 1.02 0.15 Average 0.972 0.173
Accession Numbers
Depository Authority: Korea Research Institute of Bioscience and Biotechnology
Accession number: KCTC13925BP
Date of deposit: 20190822
Depository institution name: Korea Research Institute of Bioscience and Biotechnology
Accession number: KCTC14122BP
Date of deposit: 20200130
Depository Authority: Korea Research Institute of Bioscience and Biotechnology
Accession number: KCTC14123BP
Date of deposit: 20200130