JP7675417B2 - Method for producing ethanol-producing yeast with thermotolerance and culture stress tolerance - Google Patents

Description

NPMD NITE P-03143 NPMD NITE P-03144 NPMD NITE P-03145 NPMD NITE P-03146

The present invention relates to a method for producing an ethanol-producing yeast having thermotolerance and culture stress resistance. The present invention also relates to a mutant of the ethanol-producing yeast obtained by the method.

As the effects of global warming become more evident, bioethanol is 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 almost nonexistent 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 unwanted 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 has been no easy method for breeding such superior strains (stress-resistant strains).

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 from ethanol, acetic acid, and the like that occurs 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 is resistant to these stresses can be developed, more stable and higher production can be expected.

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)

The objective of the present invention is to provide a method for obtaining ethanol-producing yeast that is thermotolerant and resistant to culture stress.

In breeding to improve the thermotolerance of strains, the temperature is gradually increased while the strains are repeatedly cultured 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, the 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 considered culturing the cells 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, they decided to culture the cells until the death phase or later, so that the number of live bacteria (colony-forming units) reaches the detection limit, so that most of the strains die and only the stress-resistant strains survive. Specifically, they decided to repeatedly perform long-term culture (one week) while gradually increasing the temperature. The present invention was completed based on the above findings.

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) further culturing the cells obtained by the culturing step under high temperature conditions of 40° C. or higher aerobically, 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 fresh medium under the same temperature conditions as in the immediately preceding culturing step under aerobical 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 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.
[7] A method for producing the ethanol-producing yeast according to any one of [1] to [6] above,
The method, wherein the 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 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.
[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.

The method of the present invention makes it possible to easily isolate strains with multiple stress resistances through long-term culture. More specifically, it is possible to obtain strains that are resistant to culture stresses such as ethanol and acetic acid while maintaining heat resistance, making it possible to provide strains that can overcome the weaknesses of conventional heat-resistant yeast.

1 is a diagram showing 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., or 45° C. WT indicates the DMKU3-1042 strain. 2 is a diagram showing the results of an ethanol resistance test performed on Kluyveromyces marxianus strains ACT001, ACT002, ACT003, and TML001 at temperatures of 30° C., 37° C., 40° C., or 45° C. WT indicates the DMKU3-1042 strain. 3 is a diagram showing the results of a low pH resistance test performed on Kluyveromyces marxianus strains ACT001, ACT002, ACT003, and TML001 under temperature conditions of 30° C., 37° C., 40° C., or 45° C. WT indicates the DMKU3-1042 strain. 4 is a diagram showing 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., or 45° C. WT indicates the DMKU3-1042 strain. 5 is a diagram showing the results of a formic acid resistance test performed on Kluyveromyces marxianus strains ACT001, ACT002, ACT003, and TML001 at temperatures of 30° C., 37° C., 40° C., or 45° C. WT indicates the DMKU3-1042 strain. 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. 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.

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 steps of: (a) preparing a yeast strain having a thermotolerance and a culture stress resistance;
(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) 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 culture step (b) at least once;

In this specification, ethanol-producing yeast means yeast capable of decomposing sugar to produce ethanol. Examples of ethanol-producing yeast include 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, examples of ethanol-producing yeast include 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 glucose-containing YP medium,

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 erythrorhiz ... 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 such yeasts 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.

As used herein, "thermostable" means that the strain can grow well even under high temperature conditions, specifically, that the strain can 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 solution, 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.

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 with 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. An even more preferred embodiment of the ethanol-producing yeast with acetic acid resistance is a yeast that has acetic acid resistance even under high temperature conditions (e.g., 40°C to 45°C).

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 an ethanol-producing yeast with ethanol tolerance is a yeast that can grow even after 48 hours of culture under temperature conditions of 45°C and in the presence of 9% ethanol.

In this specification, "low pH resistance" refers to the ability to grow well even under low pH culture conditions. A preferred embodiment of an ethanol-producing yeast with low pH resistance 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.

In this specification, "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 an ethanol-producing yeast with furfural resistance is a yeast that can grow better than 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 an ethanol-producing yeast with furfural resistance is a yeast that can grow better than Kluyveromyces marxianus DMKU3-1042 strain after 48 hours of culture under temperature conditions of 40°C and in the presence of 15 mM hydroxymethylfurfural.

In this specification, "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 with formic acid resistance is a yeast that can grow well even after 48 hours of culture in the presence of 0.2% formic acid. An even more preferred embodiment of the ethanol-producing yeast with formic acid resistance is a yeast that has formic acid resistance even under high temperature conditions (e.g., 40°C to 45°C).

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 culturing is carried out beyond the logarithmic growth phase of the ethanol-producing yeast.
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. Furthermore, "cultivating aerobically" means culturing in the presence of molecular oxygen.

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 is obtained. Preferably, a medium having the same composition is used in all steps.

"Cultivating beyond the logarithmic growth phase" refers to culturing for a period of about half a day to a day from the start of culture, which is the logarithmic growth phase of yeast. In a preferred embodiment, "cultivating beyond the logarithmic growth phase" refers to continuing the culture until the death phase or later, so that only strains that are 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). It can be determined that the culture has been continued until the death phase or later 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 1/100,000 or less, 1/1,000,000 or less, or 1/10,000,000 of the maximum number of live bacteria (cfu) of the yeast during culture in the logarithmic growth phase. 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 a mutant strain of ethanol-producing yeast can be obtained, but is, for example, 21 days from the start of culture, and preferably 14 days.

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 new 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, the culture medium of step (a) may be transferred so that the new culture medium becomes a suspension with an OD ₆₆₀ 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, in order to select mutant strains having heat resistance and culture stress resistance.

The method for producing an ethanol-producing yeast having heat tolerance and culture stress resistance 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 culture 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 culture is performed beyond the logarithmic growth phase of the ethanol-producing yeast.
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 a temperature 0.5°C, 1°C, 2°C, or 3°C or 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.

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), a step (b') of further aerobically culturing the bacterial cells obtained in the culture step in a new medium 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 was performed at 42°C, the culture in this step (b') is also performed at 42°C.

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 (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 (b') following the step (b). When the culture step is repeated multiple times, the culture 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 condition higher than the immediately preceding culture condition" 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.

Note that "repeating the culture step (b) at least once" in step (c) means "a step of further culturing the 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, and culturing beyond the logarithmic growth phase of the ethanol-producing yeast." In other words, the cells used in the "step of repeating the culture step (b) at least once" during the repetition in step (c) means the cells obtained in the immediately preceding step (b).

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 at a high temperature 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 temperature condition higher than the immediately preceding culture 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 high temperature conditions 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 are 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-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:

In another aspect, the present invention provides Kluyveromyces marxianus strain ACT001 (Accession No.: NITE AP-03143), Kluyveromyces marxianus strain ACT002 (Accession No.: NITE AP-03144), Kluyveromyces marxianus strain ACT003 (Accession No.: NITE AP-03145), Kluyveromyces marxianus strain TML001 (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 Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on March 2, 2020, respectively. They are stored in the Department of Biological Function Science, Faculty of Agriculture, Yamaguchi University, National University Corporation, and can be sold under certain conditions.
The ACT001, ACT002, and ACT003 strains have acetic acid resistance, low pH resistance, furfural resistance, and formic acid resistance, while the TML001 strain has ethanol resistance and furfural resistance.

In another aspect, the present invention provides a method for producing ethanol, which includes a step of culturing Kluyveromyces marxianus strain 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.

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 media 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% yeast nitrogen base without L-amino acids), 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.

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 are 1 to 10 days, preferably 2 to 7 days, and more preferably 2 to 3 days.

In the ethanol production method of the present invention, any conventionally known method can be used to recover the ethanol produced from the medium. For example, a method can be used in which a liquid layer containing ethanol is separated from a solid layer containing yeast and solid components by solid-liquid separation, and then the ethanol contained in the liquid layer is separated and purified by distillation to recover it.

(Breeding methods for strains tolerant to high temperature stress)
In the breeding method of the stress-resistant strain in this example, a 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 culture medium was placed in 10 test tubes, and a loopful of the heat-resistant yeast Kluyveromyces marxianus DMKU3-1042 strain was inoculated, and long-term culture was started at 40°C, where the high fermentation ability of K. marxianus is maintained (10 test sections). One week after the start of culture, the culture medium was transferred to 3 ml of culture medium in a new test tube so that the suspension (OD ₆₆₀ ) was 0.1, and the medium was replaced for each test section. After the medium replacement, the culture was further performed at 40°C with shaking for one week. This 2-week culture was defined as one period, and the shake culture was continued for one period (i.e., after one week of shake culture, the medium was replaced, and another week of shake culture) every time the culture temperature was increased by 1°C. The culture temperature was raised from 40° C. by 1° C. at a time, 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 groups, 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 ACT001, ACT002, ACT003, and TML001, the resistance to various stresses that may occur during yeast culture was examined in the following tests.

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

(Acetic acid resistance test)
YPD agar medium was prepared, and the stock solution of each strain was diluted 1-fold, 10-fold, 100-fold, 1000-fold, and 10000-fold with YP liquid medium, and then spotted on the YPD agar medium at five locations from the left. Subsequently, 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.

Figure 1 shows the results of the acetic acid resistance test. 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 (top row: YPD agar medium without acetic acid, middle row: YPD agar medium containing 0.2% acetic acid, bottom row: YPD agar medium containing 0.3% acetic acid). The ACT001, ACT002, ACT003, TML001, and DMKU3-1042 strains were spotted side by side on a single YPD agar medium plate and photographed.

In response to acetic acid stress in a 0.2% acetic acid-containing medium, the ACT001, ACT002, and ACT003 strains grew well at 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, ACT002, and ACT003 strains grew well at 30°C, 37°C, and 40°C. This demonstrated that the ACT001, ACT002, and ACT003 strains can be used under high temperature conditions and under acetic acid stress conditions.

(Ethanol tolerance test)
YPD agar medium was prepared, and the stock solution of each strain was diluted 1-fold, 10-fold, 100-fold, 1000-fold, and 10000-fold with YP liquid medium, and then spotted on the YPD agar medium at five locations 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.

Figure 2 shows the results of the ethanol resistance test. 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 a single YPD agar medium plate and photographed.

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 conditions and under ethanol stress conditions.

(Low pH resistance test)
YPD agar medium was prepared, and the stock solution of each strain was diluted 1-fold, 10-fold, 100-fold, 1000-fold, and 10000-fold with YP liquid medium, and then spotted on the YPD agar medium at five locations from the left. Subsequently, 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.

Figure 3 shows the results of the low pH resistance test. 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.

Despite the low pH stress of 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.

(Furfural Resistance Test)
YPD agar medium was prepared, and the stock solution of each strain was diluted 1-fold, 10-fold, 100-fold, 1000-fold, and 10000-fold with YP liquid medium, and then spotted on the YPD agar medium at five locations 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.

Figure 4 shows the results of the furfural resistance test. 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 (top row: YPD agar medium without furfural, middle row: YPD agar medium containing 15 mM furfural, bottom row: YPD agar medium containing 15 mM HMF). The ACT001, ACT002, ACT003, TML001, and DMKU3-1042 strains were spotted side by side on a single YPD agar medium plate and photographed.

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

(Formic acid resistance test)
YPD agar medium was prepared, and the stock solution of each strain was diluted 1-fold, 10-fold, 100-fold, 1000-fold, and 10000-fold with YP liquid medium, and then spotted on the YPD agar medium at five 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.

Figure 5 shows the results of the formic acid resistance test. The figure is in 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 medium plate and photographed.

In response to formic acid stress in a medium containing 0.1% formic acid, the ACT001, ACT002, and ACT003 strains grew well under the following temperature conditions: 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.

(Growth of each adapted strain and ethanol concentration in the medium)
A loopful of the adaptive strains (ACT001, ACT002, ACT003, TML001) and Kluyveromyces marxianus DMKU3-1042 (parent strain) obtained above was inoculated into 5 ml of YP medium (pH 7) in a test tube, and a preculture solution was obtained by shaking culture (160 rpm) at 30° C. for 18 hours. The preculture solution was then transferred to 100 ml of YP medium containing 16% glucose in an Erlenmeyer flask to a turbidity (OD ₆₆₀ ) of 0.1, and shake culture (160 rpm) was performed at 45° C.

The growth of each of the adapted strains and the DMKU3-1042 strain and the ethanol concentration in the medium were measured 8 hours, 16 hours, 24 hours, 32 hours, 40 hours, and 48 hours after the start of the culture. The growth of the adapted strains and the DMKU3-1042 strain was determined by measuring the turbidity (OD ₆₆₀ ) of the culture solution 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 Co., Ltd.) to obtain a test solution, and measuring it with high performance liquid chromatography (manufactured by Hitachi High-Technologies Corporation). The analytical conditions for the 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.

The measurement results of the turbidity (OD ₆₆₀ ) of the culture liquid are shown in FIG. 6. The vertical axis indicates the OD ₆₆₀ value, and the horizontal axis indicates the time (hours) from the start of culture. As shown in FIG. 6, the OD ₆₆₀ value of each of the adapted strains and Kluyveromyces marxianus DMKU3-1042 increased until 16 hours, and then remained almost constant until 48 hours. The TML001 strain grew faster than the other adapted strains and Kluyveromyces marxianus DMKU3-1042, and showed good growth results. The OD ₆₆₀ values of the ACT001 strain, ACT002 strain, and ACT003 strain were almost similar to the OD ₆₆₀ value of Kluyveromyces marxianus DMKU3-1042.

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) in percent, and the horizontal axis represents the time (hours) from the start of culture. As shown in Figure 7, ACT001, ACT002, ACT003, and ACT003 all had higher ethanol concentrations than 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, and 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.

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

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 (8)

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. 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 fresh 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 controlled temperature conditions similar to those in the immediately preceding culturing step. 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. 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. 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. 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. 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). A method for producing ethanol, comprising a step of culturing the yeast according to claim 7 under aerobic conditions.