Method for producing ethanol-producing yeast with thermotolerance and culture stress tolerance

Abstract

To provide methods for obtaining ethanol-producing yeasts having heat resistance and culture stress tolerance.SOLUTION: Disclosed is a method for obtaining a mutant Kluyveromyces marxianus having heat resistance and tolerance to culture stress, the method comprising the steps of: culturing a strain of Kluyveromyces marxianus under aerobic conditions and temperature conditions of 40°C or higher beyond the logarithmic growth phase, then transferring the cultured cells to a fresh culture medium to culture under the same culture conditions except that the temperature is gradually raised where the latter step is repeated.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing an ethanol-producing yeast having thermotolerance and culture stress tolerance, and also to a mutant of the ethanol-producing yeast obtained by said method. [Background technology]

[0002] As the effects of global warming become apparent, bioethanol has been attracting attention as a renewable and environmentally friendly alternative energy source. However, due to the cost and the shortage of biomass as a raw material, bioethanol production is hardly carried out in Japan. Even overseas, it is considered that ultra-large-scale facilities are necessary to use second-generation biomass as a raw material from the standpoint of profitability. Innovative technology development that can reduce costs is also required to achieve the SDGs. As such a technology, the inventor has developed a high-temperature fermentation system that is expected to have many benefits, such as reducing cooling energy, reducing the amount of hydrolytic enzymes during parallel fermentation, and suppressing the introduction of miscellaneous bacteria. On the other hand, since various stresses in addition to temperature suppress the growth of fermentation yeast during fermentation, the development of more robust yeast is desired, but there was no easy method for breeding such superior strains (stress-resistant strains).

[0003] The inventors have been developing a thermotolerant yeast that is essential for high-temperature fermentation (Non-Patent Document 1). Thermotolerant yeast can efficiently ferment ethanol at 40-43°C, and it has been shown that it can ferment stably at a temperature about 10°C higher than yeast (Saccharomyces cerevisiae) that is used worldwide for ethanol production. In fermentation using S. cerevisiae, the temperature of the fermentation tank rises due to the heat of fermentation, and the growth and fermentation ability of S. cerevisiae are suppressed by the temperature rise, so cooling is essential. On the other hand, such cooling is not necessary for thermotolerant yeast, which leads to a reduction in cooling energy (cost) and therefore a reduction in CO2. However, compared to S. cerevisiae, thermotolerant yeast is vulnerable to stress such as ethanol and acetic acid that are generated in the culture solution during culture for fermentation. As fermentation progresses, in addition to the rise in temperature, the ethanol concentration increases and organic acids such as acetic acid accumulate. Inhibitory substances such as furfural or hydroxymethylfurfural (HMF) also accumulate. Therefore, if a robust strain that can withstand these stresses can be developed, more stable and higher production can be expected. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lertwattanasakul, et al, “Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses” Biotechnology for Biofuels, volume 8, Article number: 47 (2015) Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a method for obtaining an ethanol-producing yeast having thermotolerance and culture stress resistance. [Means for solving the problem]

[0006] In breeding to improve the heat resistance of strains, the temperature is gradually increased while the culture is repeated to isolate strains that are more adapted to high temperatures (e.g., Kosaka et al., PLoS One 2019 doi: 10.1371 / journal.pone.0215614, 2019). In this case, because growth is the most important factor, culture is repeated during the growth period (logarithmic growth phase: about half a day to a day). On the other hand, the present inventors have investigated culturing until the logarithmic growth phase or later in order to separate strains resistant to organic acids such as ethanol and acetic acid that accumulate during culture. In particular, in order to easily separate strains resistant to various stresses from the cell population, it was decided to culture until the death phase or later so that the viable cell count (colony-forming units) reaches the detection limit, so that most strains die and only stress-resistant strains survive. Specifically, it was decided to repeatedly carry out long-term culture (one week) while gradually increasing the temperature. The present invention was completed based on the above findings.

[0007] That is, the present invention is as follows. [1] A method for producing an ethanol-producing yeast having thermotolerance and culture stress resistance, comprising the following steps (a) to (c): (a) aerobically culturing an ethanol-producing yeast under high temperature conditions of 40° C. or higher, the culturing being beyond the logarithmic growth phase of the ethanol-producing yeast; (b) aerobically culturing the cells obtained by the culturing step under high temperature conditions of 40° C. or higher, the high temperature conditions being higher than the temperature conditions immediately before the culturing step, and culturing the cells beyond the logarithmic growth phase of the ethanol-producing yeast; (c) repeating the culture step (b) at least once; (c) a method of production. [2] A method for producing the ethanol-producing yeast according to [1] above, Following step (a), (a') further culturing the bacterial cells obtained in the culturing step in a new medium under the same temperature conditions as in the immediately preceding culturing step under aerobic conditions; and / or Following step (b), (b') further culturing the bacterial cells obtained in the culturing step in a fresh medium under aerobically at the same temperature conditions as in the immediately preceding culturing step. [3] A method for producing the ethanol-producing yeast according to [2] above, The method, wherein the culturing step of step (a') and / or (b') is a step of culturing the ethanol-producing yeast beyond the logarithmic growth phase. [4] A method for producing the ethanol-producing yeast according to any one of [1] to [3] above, The method, wherein the culturing step (a) and / or (b) is a step of continuing the culture until the ethanol-producing yeast reaches its death stage or later. [5] A method for producing the ethanol-producing yeast according to any one of [1] to [4] above, The method, wherein the culturing step of step (a) and / or (b) is for 5 days or more. [6] A method for producing the ethanol-producing yeast according to any one of [1] to [5] above, The method, wherein the temperature condition higher than the immediately preceding culture condition in step (b) is a temperature condition higher by 1°C or more than the immediately preceding culture temperature. [7] A method for producing the ethanol-producing yeast according to any one of [1] to [6] above, The method, wherein said ethanol-producing yeast is Kluyveromyces marxianus. [8] Kluyveromyces marxianus ACT001 strain (received number: NITE AP-03143), Kluyveromyces marxianus ACT002 strain (received number: NITE AP-03144), Kluyveromyces marxianus ACT003 strain (received number: NITE AP-03145), Kluyveromyces marxianus TML001 strain (received number: NITE AP-03146), or These mutants have at least one culture stress tolerance of the group consisting of acetic acid tolerance, ethanol tolerance, low pH tolerance, furfur tolerance, and formic acid tolerance under a temperature condition of 45°C. [9] A method for producing ethanol, comprising a step of culturing the yeast or a mutant strain thereof according to [8] above under aerobic conditions. Effect of the Invention

[0008] According to the method of the present invention, it is possible to easily isolate strains having resistance to multiple stresses by long-term culture. More specifically, it is possible to obtain strains that have resistance to culture stresses such as ethanol and acetic acid while maintaining heat tolerance, and it is possible to provide strains that can overcome the weaknesses of conventional heat-resistant yeasts. [Brief description of the drawings]

[0009] [Figure 1] Fig. 1 shows the results of an acetic acid resistance test performed on Kluyveromyces marxianus strains ACT001, ACT002, ACT003, and TML001 at temperatures of 30°C, 37°C, 40°C, and 45°C. WT indicates the DMKU3-1042 strain. [Diagram 2] Figure 2 shows the results of an ethanol resistance test performed on the Kluyveromyces marxianus ACT001, ACT002, ACT003, and TML001 strains at temperatures of 30°C, 37°C, 40°C, and 45°C. WT indicates the DMKU3-1042 strain. [Diagram 3]3 shows the results of a low pH resistance test performed on Kluyveromyces marxianus strains ACT001, ACT002, ACT003, and TML001 at temperatures of 30° C., 37° C., 40° C., and 45° C. WT indicates the DMKU3-1042 strain. [Figure 4] 4 shows the results of a furfural resistance test performed on Kluyveromyces marxianus strains ACT001, ACT002, ACT003, and TML001 at temperatures of 30° C., 37° C., 40° C., and 45° C. WT indicates the DMKU3-1042 strain. [Diagram 5] 5 shows the results of a formic acid resistance test performed on the Kluyveromyces marxianus ACT001, ACT002, ACT003, and TML001 strains at temperatures of 30° C., 37° C., 40° C., and 45° C. WT indicates the DMKU3-1042 strain. [Figure 6] FIG. 1 shows the results of a growth test performed on Kluyveromyces marxianus strains ACT001, ACT002, ACT003, and TML001 by culturing at 45° C. WT indicates the DMKU3-1042 strain. [Figure 7] FIG. 1 shows the results of an ethanol productivity test performed on Kluyveromyces marxianus strains ACT001, ACT002, ACT003, and TML001 when cultured at 45° C. WT indicates the DMKU3-1042 strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] One aspect of the present invention relates to a method for producing an ethanol-producing yeast having thermotolerance and culture stress resistance, the method comprising the following steps (a) to (c): (a) aerobically culturing an ethanol-producing yeast under high temperature conditions of 40° C. or higher, the culturing being carried out beyond the logarithmic growth phase of the ethanol-producing yeast; (b) further aerobically culturing the cells obtained in the culturing step under high temperature conditions of 40°C or higher, the high temperature conditions being higher than the temperature conditions immediately before the culturing step, and culturing the ethanol-producing yeast beyond the logarithmic growth phase; (c) repeating the culturing step (b) at least once;

[0011] As used herein, ethanol-producing yeast refers to yeast capable of decomposing sugar to produce ethanol. An example of the ethanol-producing yeast is a yeast that produces 25% or more, preferably 30% or more, and more preferably 35% or more ethanol relative to the glucose contained in a glucose-containing YP medium when the yeast is cultured for 24 to 40 hours at 30°C. For example, an example of the ethanol-producing yeast is a yeast that produces 4% (W / V) or more, preferably 4.8% (W / V) or more, and more preferably 5.6% (W / V) or more ethanol when the yeast is cultured for 16 to 40 hours at 30°C in a YP medium containing 16% glucose.

[0012] Examples of such ethanol-producing yeast include yeasts belonging to the genera Kluyveromyces, Saccharomyces, Candida, Pichia, and Mellozyma, and preferably yeasts belonging to the genus Kluyveromyces. Yeasts belonging to the genus Kluyveromyces include Kluyveromyces marxianus, Kluyveromyces aestuarii, Kluyveromyces africanus, Kluyveromyces bacillisporus, Kluyveromyces blattae, Kluyveromyces bulgaricus, Kluyveromyces ul ... Kluyveromyces delphensis, Kluyveromyces dobzhanskii, Kluyveromyces drosophilarum, Kluyveromyces hubeiensis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces nonfermentus entans, Kluyveromyces piceae, Kluyveromyces phaffii, Kluyveromyces phaseolosporus, Kluyveromyces polysporus, Kluyveromyces sinensis, Kluyveromyces thermotolerans, Kluyveromyces vanudenii Examples of yeast include Kluyveromyces vanudenii, Kluyveromyces veronae, Kluyveromyces wikenii, Kluyveromyces waltii, Kluyveromyces wickerhamii, and Kluyveromyces yarrowii, with Kluyveromyces marxianus, which is known as a thermotolerant yeast, being a preferred example.Examples of yeasts belonging to the genus Candida include Candida utilis, Candida tropicalis, Candida glabrata, Candida maltosa, Candida manshurica, Candida nivariensis, and Candida stellimalicola, with Candida tropicalis being a preferred example. Examples of yeasts belonging to the genus Pichia include Pichia kudriavzevii and Pichia manshurica. Examples of yeasts belonging to the genus Meerozyma include Meerozyma caribbica.

[0013] As used herein, "thermostable" means the ability to grow well even under high temperature conditions, specifically the ability to grow under conditions of 40°C, preferably under conditions of 43°C, and more preferably under conditions of 45°C. In this specification, "culture stress resistance" refers to resistance to stresses on yeast caused by culture. Examples of stresses on yeast caused by culture include stresses due to accumulation of acetic acid, ethanol, furfural, formic acid, etc. in the culture medium, and stresses due to a decrease in pH. "Having culture stress resistance" refers to having at least one resistance to the stresses listed above. More specifically, it refers to having at least one resistance selected from the group consisting of acetic acid resistance, ethanol resistance, low pH resistance, furfural resistance, and formic acid resistance. In a preferred embodiment, it refers to having two, three, or four resistances selected from the group consisting of acetic acid resistance, ethanol resistance, low pH resistance, furfural resistance, and formic acid resistance.

[0014] As used herein, "acetic acid resistance" refers to the ability to grow well even in the presence of acetic acid. A preferred embodiment of the ethanol-producing yeast having acetic acid resistance is a yeast that can grow well even after 48 hours of culture in the presence of 0.2% acetic acid, and more preferably a yeast that can grow well even after 48 hours of culture in the presence of 0.3% acetic acid. A further preferred embodiment of the ethanol-producing yeast having acetic acid resistance is a yeast that has acetic acid resistance even under high temperature conditions (e.g., 40°C to 45°C).

[0015] As used herein, "ethanol tolerance" refers to the ability to grow well under high temperature conditions and in the presence of ethanol. A preferred embodiment of the ethanol-tolerant ethanol-producing yeast is a yeast that can grow even after 48 hours of culture at 45°C in the presence of 9% ethanol.

[0016] As used herein, "low pH tolerance" refers to the ability to grow well even under low pH culture conditions. A preferred embodiment of the ethanol-producing yeast having low pH tolerance is a yeast that can grow well even after 48 hours of culture under pH 3.5 conditions, and more preferably a yeast that can grow well even after 48 hours of culture under pH 3 conditions.

[0017] As used herein, "furfural resistance" refers to the ability to grow well even under high temperature conditions and in the presence of furfural (or hydroxymethylfurfural). A preferred embodiment of the ethanol-producing yeast having furfural resistance is a yeast that can grow better than the Kluyveromyces marxianus DMKU3-1042 strain after 48 hours of culture under temperature conditions of 45°C and in the presence of 15 mM furfural. Alternatively, a preferred embodiment of the ethanol-producing yeast having furfural resistance is a yeast that can grow better than the Kluyveromyces marxianus DMKU3-1042 strain after 48 hours of culture under temperature conditions of 40°C and in the presence of 15 mM hydroxymethylfurfural.

[0018] As used herein, "formic acid resistance" refers to the ability to grow well even in the presence of formic acid. A preferred embodiment of the ethanol-producing yeast having formic acid resistance is a yeast that can grow well even after 48 hours of culture in the presence of 0.2% formic acid. A more preferred embodiment of the ethanol-producing yeast having formic acid resistance is a yeast that has formic acid resistance even under high temperature conditions (e.g., 40°C to 45°C).

[0019] The method of the present invention for producing an ethanol-producing yeast having heat tolerance and culture stress resistance includes: (a) aerobically culturing the ethanol-producing yeast under high-temperature conditions of 40°C or higher, wherein the ethanol-producing yeast is cultured beyond its logarithmic growth phase. The term "high temperature conditions of 40°C or higher" is not limited as long as it allows the production of a mutant strain of ethanol-producing yeast to be used for the culture. Examples of high temperature conditions include, but are not limited to, 40°C, 41°C, 42°C, 43°C, 44°C, and 45°C. In addition, "cultivating aerobically" means culturing in the presence of molecular oxygen.

[0020] In the present invention, for culturing the ethanol-producing yeast, YPD medium (1% yeast extract, 2% peptone, 2% glucose), YPAD medium (2% bactopeptone, 1% yeast extract, 2% glucose, 40 μl / ml adenine sulfate), SD medium (2% glucose, 0.67% L-amino acid-free yeast nitrogen base), YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose), etc., which are generally used for culturing yeast, can be used. The pH of the medium can be, for example, pH 4 to 8, preferably pH 5 to 7. In addition, the same medium can be used in the following steps (a'), (b), (b'), and (c) unless otherwise specified. The medium used in each step may have the same composition as that in each step or a different composition, as long as an ethanol-producing yeast having heat resistance and culture stress resistance can be obtained. Preferably, a medium with the same composition is used in all steps.

[0021] "Culturing beyond the logarithmic growth phase" refers to culturing for a period of about half a day to one day from the start of culture, which is the logarithmic growth phase of yeast. In a preferred embodiment, "culturing beyond the logarithmic growth phase" refers to a form in which the culture is continued until the death phase or later, so that only strains resistant to stress survive. Whether the culture has been continued until the death phase or later can be evaluated, for example, by the number of live bacteria (colony-forming units: cfu). When the number of live bacteria (cfu) of the yeast during culture is below the detection limit, or when the number of live bacteria (cfu) of the yeast during culture is 1 / 100000 or less, 1 / 1000000 or less, or 1 / 10000000 of the maximum number of live bacteria (cfu) of the yeast during culture in the logarithmic growth phase, it can be determined that the yeast has been cultured until the death phase or later. The culture period beyond the logarithmic growth phase can be, for example, 5 days or more from the start of culture. The culture period is preferably 6 days or more from the start of culture, and more preferably 7 days or more. The upper limit of the culture period is not limited as long as an ethanol-producing yeast mutant can be obtained, but is, for example, 21 days, preferably 14 days, from the start of culture.

[0022] The method for producing an ethanol-producing yeast having heat tolerance and culture stress resistance according to the present invention may further include, after the above-mentioned step (a), (a') a step of further culturing the bacterial cells obtained in the culture step in a fresh medium under aerobically under the same temperature conditions as in the immediately preceding culture step. The mutant strain (adapted strain) obtained in step (a) is grown by the culture in step (a'). The method of replacing the medium is not limited, and can be carried out according to a known method. For example, when a new culture medium is added at OD 660 The culture medium from step (a) should be transferred so that the suspension has a pH of 0.1. The temperature conditions in this step (a') are preferably the same as those in step (a). The culture period in this step is not limited as long as the mutant strain (adapted strain) obtained in step (a) can grow, and can be, for example, half a day to 21 days. In one embodiment, the culture period in this step (a') may be beyond the logarithmic growth phase as in step (a), or may be cultured until the death phase or later. In this step, it is preferable to culture beyond the logarithmic growth phase or until the death phase or later, since this allows the selection of mutant strains having heat resistance and culture stress resistance.

[0023] The method for producing an ethanol-producing yeast having heat tolerance and resistance to culture stress according to the present invention includes, after the above-mentioned step (a) or step (a'), (b) a step of further aerobically culturing the bacterial cells obtained in the culturing step under high-temperature conditions of 40°C or higher, wherein the high-temperature conditions are temperature conditions higher than the immediately preceding culture conditions, and the ethanol-producing yeast is cultured beyond its logarithmic growth phase. The term "high temperature conditions higher than the immediately preceding culture conditions" is not limited as long as it is a temperature condition higher than the temperature condition in the immediately preceding culture. For example, the culture conditions may be at least 0.5°C, 1°C, 2°C, or 3°C higher than the immediately preceding temperature, without being limited thereto. In a preferred embodiment, the "high temperature condition is a temperature condition higher than the immediately preceding culture condition" refers to a temperature condition that is 1° C. or more higher than the immediately preceding temperature. In this embodiment, for example, if the immediately preceding culture temperature was 40° C., a temperature 1° C. higher means 41° C.

[0024] The method for producing an ethanol-producing yeast having heat tolerance and culture stress resistance according to the present invention may further include, after the above-mentioned step (b), (b') a step of further culturing the bacterial cells obtained in the culture step in a new medium under aerobically under the same temperature conditions as in the immediately preceding culture step. This step is a culture performed to grow the adapted strain obtained in the culture step of step (b). The culture in step (b') is preferably performed under the same temperature conditions as the immediately preceding culture step, and can be performed in the same manner as in step (a') above. The same temperature conditions as the immediately preceding culture step means, for example, that if the immediately preceding culture step was performed at 42°C, the culture in this step (b') is also performed at 42°C.

[0025] The method for producing an ethanol-producing yeast having thermotolerance and culture stress resistance according to the present invention includes, after the above-mentioned step (b), (c) a step of repeating the culture step of the step (b) at least once or more. When the step (b) is followed by the step (b'), the culture may be repeatedly performed including the culture step of the step (b') following the step (b). When the culture step is repeated multiple times, the culture step of the step (b') may be included in only some of the steps. By gradually increasing the culture temperature in this manner, it is possible to obtain mutant strains that are heat-resistant and resistant to culture stress. When step (c) is repeated, the "high-temperature conditions higher than the immediately preceding culture conditions" in step (b) may be increased by a constant amount, or may be increased by a different amount. For example, the temperature increase in the repeated culture in step (b) may be increased by 1°C each time, or the temperature increase may be changed, such as increasing the temperature by 1°C and then increasing the temperature by 0.5°C. In a preferred embodiment, the temperature is increased by 1° C. each time in the repeated culture in step (c). In addition, when step (c) is repeated, the culture period may be kept constant as long as it is beyond the logarithmic growth phase, or a different period may be adopted. For example, the culture period in the repeated culture of step (b) may be 7 days each time, or a combination of 7 days and 8 days may be used.

[0026] In step (c), "repeating the culture step (b) at least once" means "a step of further culturing the bacterial cells obtained in the culture step (b) aerobically under high-temperature conditions of 40° C. or higher, the high-temperature conditions being higher than the temperature conditions immediately before the culture, and culturing the ethanol-producing yeast beyond the logarithmic growth phase." In other words, the bacterial cells used in "the step of repeating the culture step (b) at least once" during the repetition in step (c) means the immediately preceding "microbial cells obtained by step (b)."

[0027] Specifically, when the step (c) is repeated four times and the method of the present invention includes steps (a') and (b'), (a) aerobically culturing an ethanol-producing yeast under a high temperature condition of 40° C., the culturing being beyond the logarithmic growth phase of the ethanol-producing yeast; (a') further culturing the bacterial cells obtained in the culturing step (a) in a new medium under aerobic conditions at the same temperature as in the immediately preceding culturing step, i.e., 40°C; (b) further culturing the cells obtained in the culturing step (a') under aerobically controlled conditions at 41°C, which is a higher temperature than the previous culturing conditions, and culturing the cells beyond the logarithmic growth phase of the ethanol-producing yeast; (b') further culturing the bacterial cells obtained in the culturing step (b) in a new medium under aerobic conditions at 41°C, which is the same temperature as in the immediately preceding culturing step; (c-1) a step of further aerobically culturing the cells obtained in the culturing step (b') under a high temperature condition of 42°C, which is a temperature condition higher than the immediately preceding culture condition, and culturing the ethanol-producing yeast beyond the logarithmic growth phase; (c-1') further culturing the bacterial cells obtained in the culturing step (c-1) in a new medium under aerobic conditions at 42°C, which is the same temperature as in the immediately preceding culturing step; (c-2) a step of further aerobically culturing the cells obtained in the culturing step (c-1') under high temperature conditions of 43°C, which is a temperature condition higher than the immediately preceding culture condition, and culturing the ethanol-producing yeast beyond the logarithmic growth phase; (c-2') further culturing the bacterial cells obtained in the culturing step (c-2) in a new medium under aerobic conditions at 43°C, which is the same temperature as in the immediately preceding culturing step; (c-3) a step of further aerobically culturing the cells obtained in the culturing step (c-2') under high temperature conditions of 44°C, which is a temperature condition higher than the immediately preceding culture condition, and culturing the ethanol-producing yeast beyond the logarithmic growth phase; (c-3') further culturing the bacterial cells obtained in the culturing step (c-3) in a new medium under aerobic conditions at 44°C, which is the same temperature as in the immediately preceding culturing step; (c-4) a step of further aerobically culturing the cells obtained in the culturing step (c-3') under high temperature conditions of 45°C, which is a temperature condition higher than the immediately preceding culture condition, and culturing the ethanol-producing yeast beyond the logarithmic growth phase; (c-4') further culturing the bacterial cells obtained in the culturing step (c-4) in a new medium under aerobic conditions at 45°C, which is the same temperature as in the immediately preceding culturing step; An example of the method includes a method for producing an ethanol-producing yeast having thermotolerance and culture stress tolerance, comprising the steps of:

[0028] In another aspect, the present invention provides Kluyveromyces marxianus ACT001 strain (Accession No.: NITE AP-03143), Kluyveromyces marxianus ACT002 strain (Accession No.: NITE AP-03144), Kluyveromyces marxianus ACT003 strain (Accession No.: NITE AP-03145), Kluyveromyces marxianus TML001 strain (Accession No.: NITE AP-03146), or a mutant strain thereof having at least one culture stress resistance selected from the group consisting of acetic acid resistance, ethanol resistance, low pH resistance, furfur resistance, and formic acid resistance under a temperature condition of 45°C. The Kluyveromyces marxianus ACT001 strain, Kluyveromyces marxianus ACT002 strain, Kluyveromyces marxianus ACT003 strain, and Kluyveromyces marxianus TML001 strain were received by the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on March 2, 2020, and are stored in the Department of Biofunctional Science, Faculty of Agriculture, Yamaguchi University, National University Corporation, and can be distributed under certain conditions. The ACT001, ACT002, and ACT003 strains have acetic acid tolerance, low pH tolerance, furfural tolerance, and formic acid tolerance, while the TML001 strain has ethanol tolerance and furfural tolerance.

[0029] In another aspect, the present invention provides a method for producing ethanol, which includes a step of culturing Kluyveromyces marxianus ACT001, ACT002, ACT003, or TML001, or a mutant strain thereof, under aerobic conditions. The method for producing ethanol in the present invention is not particularly limited as long as it is a method for producing ethanol in which the ethanol-producing yeast having the above-mentioned heat tolerance and culture stress resistance is cultured in an appropriate medium at a temperature condition, preferably at 30°C or higher.

[0030] The medium that can be used in the ethanol production method of the present invention means a medium containing sugar components (e.g., glucose and xylose) that are substrates for ethanol synthesis. Examples of such a medium include YPD medium (1% yeast extract, 2% peptone, 2% glucose), YPAD medium (2% bactopeptone, 1% yeast extract, 2% glucose, 40 μl / ml adenine sulfate), SD medium (2% glucose, 0.67% L-amino acid-free yeast nitrogen base), and YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose). Pure sugars such as xylose, glucose, sucrose, or fructose, or mixtures thereof may be added depending on the assimilation ability of the yeast used. Trace metals such as a nitrogen source, a potassium source, or a magnesium source may also be added as auxiliary components.

[0031] In the ethanol production method of the present invention, the culture temperature is preferably 30° C. or higher, and more preferably 30° C. to 45° C. Examples of the culture method include shaking culture, stirring culture, shaking and stirring culture, continuous culture stationary culture, and combinations thereof, and shaking culture or stirring culture is preferable. Examples of the culture time include 1 to 10 days, preferably 2 to 7 days, and more preferably 2 to 3 days.

[0032] In the method for producing ethanol according to the present invention, any conventionally known method can be used as a method for recovering ethanol produced from the medium. For example, there can be mentioned a method in which a liquid layer containing ethanol is separated from a solid layer containing yeast and solid components by a solid-liquid separation operation, and then the ethanol contained in the liquid layer is separated and purified by a distillation method to recover it. EXAMPLES

[0033] (Breeding methods for strains tolerant to high temperature stress) In the breeding method of stress-tolerant strains in this example, yeast extract peptone (YP) medium containing 2% glucose was used as the culture medium, and the culture was performed by shaking (160 rpm). 3 ml of the culture medium was placed in 10 test tubes, and a loopful of the heat-resistant yeast Kluyveromyces marxianus DMKU3-1042 strain was inoculated into each test tube, and long-term culture was started at 40°C, where the high fermentation ability of K. marxianus is maintained (10 test groups). One week after the start of culture, the suspension (OD 660 The culture medium was replaced for each test group by transferring the culture medium so that the ratio of the culture medium to the culture medium was 0.1. After the culture medium was replaced, shaking culture was continued for another week at 40°C. This 2-week culture was counted as one period, and shaking culture was continued for that period every time the culture temperature was raised by 1°C (i.e., shaking culture was continued for one week, followed by changing the medium and shaking culture for another week). The culture temperature was raised by 1°C from 40°C, and long-term culture was repeated up to 45°C (a total culture period of 12 weeks). As a result, growth was observed in only four test tubes out of the ten test plots, and four adapted strains were obtained from each test tube. The adapted strains were named ACT001, ACT002, ACT003, and TML001, respectively. To confirm the industrial usefulness of the ACT001, ACT002, ACT003, and TML001 strains, the resistance to various stresses that may occur during yeast culture was examined in the following tests.

[0034] (Preparation of original solution) A loopful of ACT001, ACT002, ACT003, and TML001 strains, as well as the control strain DMKU3-1042, were inoculated into YPD liquid medium (pH 7.0) and cultured at 30°C for 18 hours under aerobic conditions to prepare stock solutions. The stock solutions were used in the following resistance tests (acetic acid, ethanol, low pH, furfural, HMF, or formic acid resistance tests).

[0035] (Acetic acid resistance test) YPD agar medium was prepared, and the stock solution of each strain was diluted 1x, 10x, 100x, 1000x, and 10000x with YP liquid medium, and then spotted on the YPD agar medium at 5 points from the left. Next, acetic acid was added to the YPD agar medium to a concentration of 0.2% or 0.3%, and each strain was cultured for 48 hours under four temperature conditions of 30°C, 37°C, 40°C, and 45°C to examine its growth.

[0036] The results of the acetic acid resistance test are shown in Figure 1. The figure is in the form of a matrix, with the columns representing temperature conditions (30°C, 37°C, 40°C, 45°C) and the rows representing acetic acid stress (upper row: YPD agar medium without acetic acid, middle row: YPD agar medium containing 0.2% acetic acid, lower row: YPD agar medium containing 0.3% acetic acid). The ACT001, ACT002, ACT003, TML001, and DMKU3-1042 strains were spotted side by side on one YPD agar plate and photographed.

[0037] In response to acetic acid stress in a 0.2% acetic acid-containing medium, the ACT001 strain, the ACT002 strain, and the ACT003 strain grew well at any of the temperatures of 30° C., 37° C., 40° C., and 45° C. In response to acetic acid stress in a 0.3% acetic acid-containing medium, the ACT001 strain, the ACT002 strain, and the ACT003 strain grew well at 30° C., 37° C., or 40° C. This demonstrated that the ACT001 strain, the ACT002 strain, and the ACT003 strain can be used under high temperature conditions and under acetic acid stress conditions.

[0038] (Ethanol tolerance test) YPD agar medium was prepared, and the stock solution of each strain was diluted 1x, 10x, 100x, 1000x, and 10000x with YP liquid medium, and then spotted on the YPD agar medium at 5 points from the left. Ethanol was then added to the YPD agar medium to a concentration of 9%, and each strain was cultured for 48 hours under four temperature conditions of 30°C, 37°C, 40°C, and 45°C to examine its growth.

[0039] The results of the ethanol resistance test are shown in Figure 2. The figure is in the form of a matrix, with the columns representing temperature conditions (30°C, 37°C, 40°C, 45°C) and the rows representing ethanol stress (upper row: YPD agar medium without ethanol, lower row: YPD agar medium containing 9% ethanol). The ACT001, ACT002, ACT003, TML001, and DMKU3-1042 strains were spotted side by side on one YPD agar medium plate and photographed.

[0040] In response to ethanol stress in a 9% ethanol-containing medium, the TML001 strain grew well under the following temperature conditions: 30°C, 37°C, 40°C, and 45°C. This demonstrated that the TML001 strain can be used under high temperature and ethanol stress conditions.

[0041] (Low pH resistance test) YPD agar medium was prepared, and the stock solution of each strain was diluted 1x, 10x, 100x, 1000x, and 10000x with YP liquid medium, and then spotted on the YPD agar medium at 5 points from the left. Next, hydrochloric acid was added to the YPD agar medium so that the pH of the medium became 3.5 or 3.0, and each strain was cultured for 48 hours under four temperature conditions of 30°C, 37°C, 40°C, and 45°C to examine its growth.

[0042] The results of the low pH resistance test are shown in Figure 3. The figure is in the form of a matrix, with the columns representing temperature conditions (30°C, 37°C, 40°C, 45°C) and the rows representing low pH stress (upper row: pH 7.0, middle row: pH 3.5, lower row: pH 3). The ACT001, ACT002, ACT003, TML001, and DMKU3-1042 strains were spotted side by side on a single YPD agar medium plate and photographed.

[0043] In response to low pH stress at both pH 3.5 and pH 3.0, the ACT001, ACT002, and ACT003 strains grew well under the temperature conditions of 30°C, 37°C, 40°C, and 45°C. This demonstrated that the ACT001, ACT002, and ACT003 strains can be used under high temperature and low pH stress conditions.

[0044] (Furfural resistance test) YPD agar medium was prepared, and the stock solution of each strain was diluted 1x, 10x, 100x, 1000x, and 10000x with YP liquid medium, and then spotted on five spots on the YPD agar medium starting from the left. Next, furfural or hydroxymethylfurfural (HMF) was added to the YPD agar medium so that it contained 15 mM, and each strain was cultured for 48 hours under four temperature conditions of 30°C, 37°C, 40°C, and 45°C to examine its growth.

[0045] The results of the furfural resistance test are shown in Figure 4. The figure is in the form of a matrix, with the columns representing temperature conditions (30°C, 37°C, 40°C, 45°C) and the rows representing furfural stress (upper row: YPD agar medium without furfural, middle row: YPD agar medium containing 15 mM furfural, lower row: YPD agar medium containing 15 mM HMF). The ACT001, ACT002, ACT003, TML001, and DMKU3-1042 strains were spotted side by side on one YPD agar plate and photographed.

[0046] In response to furfural stress in a 15 mM furfural-containing medium, the ACT001, ACT002, ACT003, and TML001 strains grew well at temperatures of 37° C. and 40° C., and also at temperatures of 30° C. and 45° C. At a temperature of 45° C., the ACT001, ACT002, ACT003, and TML001 strains showed better growth than the DMKU3-1042 strain, and the TML001 strain in particular showed the best growth. In response to furfural stress in a 15 mM HMF-containing medium, the ACT001, ACT002, ACT003, and TML001 strains grew well at 37°C. The ACT001, ACT002, ACT003, and TML001 strains also showed better growth than the DMKU3-1042 strain at 40°C. Furthermore, the TML001 strain showed HMF resistance compared to the other strains even at 45°C.

[0047] (Formic acid resistance test) YPD agar medium was prepared, and the stock solution of each strain was diluted 1x, 10x, 100x, 1000x, and 10000x with YP liquid medium, and then spotted on the YPD agar medium at 5 points from the left. Next, formic acid was added to the YPD agar medium to a concentration of 0.1%, and each strain was cultured for 48 hours under four temperature conditions of 30°C, 37°C, 40°C, and 45°C to examine its growth.

[0048] The results of the formic acid resistance test are shown in Figure 5. The figure is in the form of a matrix, with the columns representing temperature conditions (30°C, 37°C, 40°C, 45°C) and the rows representing formic acid stress (upper row: YPD agar medium without formic acid, lower row: YPD agar medium containing 0.1% formic acid). The ACT001, ACT002, ACT003, TML001, and DMKU3-1042 strains were spotted side by side on a single YPD agar plate and photographed.

[0049] In response to formic acid stress in a medium containing 0.1% formic acid, the ACT001, ACT002, and ACT003 strains grew well under any of the temperature conditions of 30° C., 37° C., 40° C., and 45° C. This demonstrated that the ACT001, ACT002, and ACT003 strains can be used under high temperature conditions and under formic acid stress conditions.

[0050] (Growth of each adapted strain and ethanol concentration in the medium) The adapted strains (ACT001, ACT002, ACT003, TML001) and Kluyveromyces marxianus DMKU3-1042 (parent strain) obtained above were inoculated into 5 ml of YP medium (pH 7) in a test tube with a platinum loopful, and cultured at 30°C for 18 hours with shaking (160 rpm) to obtain a preculture solution. Next, the culture solution was inoculated into 100 ml of YP medium containing 16% glucose in an Erlenmeyer flask until the OD 660 The preculture medium was transferred so that the concentration of the culture medium became 0.1, and the preculture was carried out at 45° C. with shaking (160 rpm).

[0051] The growth of each adapted strain and the DMKU3-1042 strain and the ethanol concentration in the medium were measured 8, 16, 24, 32, 40, and 48 hours after the start of cultivation. The growth of the adapted strain and the DMKU3-1042 strain was measured based on the turbidity of the culture solution (OD 660 ) was measured with a spectrophotometer. The ethanol concentration in the medium was determined by centrifuging the culture solution (1,400 rpm, 1 minute), filtering the supernatant with a membrane filter (manufactured by Nippon Pall Corporation) to obtain a test liquid, and measuring it with high performance liquid chromatography (manufactured by Hitachi High-Technologies Corporation). The analytical conditions for high performance liquid chromatography in measuring the ethanol concentration in the medium were as follows: a Gelpack (registered trademark) GL-C610-S (manufactured by Hitachi High-Technologies Corporation) was used as the column, the oven temperature during measurement was 60°C, the flow rate in the column was 0.3 ml / min, and ion-exchanged water was used as the mobile phase.

[0052] The turbidity of the culture (OD 660 The results of the measurements are shown in Figure 6. The vertical axis is OD 660 The horizontal axis represents the OD value, and the horizontal axis represents the time (hours) from the start of culture. As shown in Figure 6, both the adapted strains and Kluyveromyces marxianus DMKU3-1042 showed high OD values ​​up to 16 hours. 660 The OD value increased and then remained almost constant for 48 hours. The TML001 strain grew faster than the other adapted strains and Kluyveromyces marxianus DMKU3-1042, and showed good growth results. The OD values ​​of the ACT001, ACT002, and ACT003 strains were 660The value of OD in Kluyveromyces marxianus DMKU3-1042 660 The values ​​were almost the same as those of the control.

[0053] Next, the measurement results of the ethanol concentration in the medium are shown in Figure 7. The vertical axis represents the ethanol concentration (w / v) and the horizontal axis represents the time (hours) from the start of culture. As shown in Figure 7, the ethanol concentrations of the ACT001, ACT002, ACT003, and ACT003 strains were higher than that of the parent strain 24 hours after the start of culture, and the ethanol concentrations of all strains decreased after 40 hours from the start of culture. In addition, from 16 to 24 hours after the start of culture, the ethanol concentrations of all the adapted strains were higher than that of Kluyveromyces marxianus DMKU3-1042. Specifically, after 16 hours, the ratios were 1.17 times for the ACT001 strain, 1.15 times for the ACT002 strain, 1.24 times for the ACT003 strain, and 1.25 times for the TML001 strain, while after 24 hours, the ratios were 1.15 times for the ACT001 strain, 1.24 times for the ACT002 strain, 1.22 times for the ACT003 strain, and 1.11 times for the TML001 strain.

[0054] The above results show that each of the adapted strains obtained from the above test has various stress resistances under high temperature conditions of 45°C. When using yeast to industrially produce ethanol, the general culture time can be 16 to 24 hours. It was revealed that the adapted strains obtained from the above test can produce ethanol more efficiently than the parent strain Kluyveromyces marxianus DMKU3-1042 during such a culture period. [Industrial Applicability]

[0055] INDUSTRIAL APPLICABILITY According to the present invention, a yeast capable of efficiently producing ethanol even under high temperature conditions and various stress conditions can be produced, which is useful in the field of ethanol production.

Claims

1. A method for producing an ethanol-producing yeast having culture stress resistance, comprising the steps of: (a) aerobically culturing an ethanol-producing yeast at a high temperature of 40° C. or higher for 5 days or more, the culturing being beyond the logarithmic growth phase of the ethanol-producing yeast; (b) a step of aerobically culturing the bacterial cells obtained in the culturing step under high-temperature conditions of 40° C. or higher for 5 days or more, the high-temperature conditions being higher than the temperature conditions immediately before the culturing step; and culturing the ethanol-producing yeast beyond the logarithmic growth phase; (c) repeating the culture step (b) at least four times; Including, The method for producing said culture stress tolerance is at least two of the culture stress tolerances selected from the group consisting of acetic acid tolerance, ethanol tolerance, low pH tolerance, fullerene tolerance, and formic acid tolerance.

2. 2. A method for producing the ethanol-producing yeast of claim 1, comprising: Following step (a), (a') further culturing the bacterial cells obtained in the culturing step in a new medium under aerobically at the same temperature conditions as in the immediately preceding culturing step; and / or Following step (b), (b') further culturing the bacterial cells obtained in the culturing step in a fresh medium under aerobically at the same temperature conditions as in the immediately preceding culturing step.

3. 3. A method for producing the ethanol-producing yeast of claim 2, comprising: The method, wherein the culturing step of step (a') and / or (b') is a step of culturing the ethanol-producing yeast beyond the logarithmic growth phase.

4. A method for producing the ethanol-producing yeast according to any one of claims 1 to 3, comprising: The method, wherein the culturing step (a) and / or (b) is a step of continuing the culture until the viable cell count (cfu) of the ethanol-producing yeast during the culture becomes 1 / 100,000 or less of the maximum viable cell count (cfu) of the yeast during the culture in the logarithmic growth phase.

5. A method for producing the ethanol-producing yeast according to any one of claims 1 to 4, comprising: The method, wherein the temperature condition in step (b) which is higher than the immediately preceding culture condition is a temperature condition which is 1°C or more higher than the immediately preceding culture temperature.

6. A method for producing the ethanol-producing yeast according to any one of claims 1 to 5, The method, wherein the ethanol-producing yeast is Kluyveromyces marxianus.

7. Kluyveromyces marxianus ACT001 strain (accession number: NITE P-03143), Kluyveromyces marxianus ACT002 strain (accession number: NITE P-03144), Kluyveromyces marxianus ACT003 strain (accession number: NITE P-03145), or Kluyveromyces marxianus TML001 strain (accession number: NITE P-03146).

8. A method for producing ethanol, comprising a step of culturing the yeast according to claim 7 under aerobic conditions.

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