JP7648050B2 - Yeast with cellobiose-decomposing ability - Google Patents

Description

NPMD NITE P-03253 NPMD NITE P-03284 NPMD NITE P-03285 NPMD NITE P-03286 NPMD NITE BP-283

The present invention relates to a yeast having the ability to decompose cellobiose.

As the effects of global warming become more apparent, bioethanol is attracting attention as a renewable and environmentally friendly alternative energy source. However, due to costs and a shortage of biomass as a raw material, bioethanol is hardly produced in Japan. Even overseas, it is thought that ultra-large-scale facilities would be necessary to use second-generation biomass as a raw material in order to be profitable. In order to achieve the SDGs, there is a need to develop innovative technologies that can reduce costs.

When producing ethanol from cellulose, which is second-generation biomass, three types of cellulase enzymes are essential: cellobiohydrolase (CBH), endoglucanase (EG), and β-glucosidase (BGL). CBH is a cellulase that hydrolyzes the cellulose chain from the end, and EG is a cellulase that hydrolyzes the cellulose chain from the inside. Meanwhile, BGL hydrolyzes cellooligosaccharides such as cellobiose (a compound formed by the linkage of two glucose molecules) that are generated by the decomposition of CBH and EG, to generate glucose.
These cellulolytic enzymes are several times more expensive than starch-degrading enzymes for first-generation biomass, and cellulases used in the saccharification reaction are required in larger quantities than amylases used in the enzymatic saccharification reaction of starch-based biomass, making the enzymes expensive and preventing their practical use. Thus, in the production of ethanol from cellulosic biomass, which is second-generation biomass, there is a demand for reducing the cost of hydrolytic enzymes in particular.

If the amount of BGL, a commercially available cellulase, used is reduced to solve the above problems in ethanol production from cellulosic biomass, the product glucose inhibits BGL, resulting in the accumulation of cellobiose. The accumulated cellobiose inhibits CBH and EG, resulting in strong inhibition of the saccharification reaction. Furthermore, when homemade cellulase is used to reduce costs, the cellulase of filamentous fungi (mainly Trichoderma reesei) has low BGL activity, resulting in a low cellulose saccharification rate and a low final ethanol production. The common point between these problems is the accumulation of cellobiose.
Many attempts have been made to produce saccharifying enzymes in Saccharomyces cerevisiae by DNA recombination (e.g., Patent Document 1). However, because this involves DNA recombination, it requires the cost of containment equipment to prevent the recombinant yeast from spreading and the cost of waste disposal after production, and therefore, it has not been used for ethanol fermentation production.

International Publication No. 2013/146540

In view of the above problems, the present invention aims to provide a yeast strain that has been mutated to have a saccharification enzyme that can be used for ethanol production using cellulosic biomass, by a breeding method that does not rely on DNA recombinant technology. In particular, the present invention aims to provide a mutant strain that has the ability to decompose cellobiose.

The present inventors have been developing thermotolerant yeast, which is essential for high-temperature fermentation (Lertwattanasakul, et al, Biotechnol Biofuels, 8:47. doi:10.1186/s13068-015-0227-x, 2015). Thermotolerant yeast can efficiently ferment ethanol at 40 to 43°C, and it has been shown that it can ferment stably at temperatures approximately 10°C higher than yeast (Saccharomyces cerevisiae) that is used worldwide for ethanol production. In fermentation, the temperature of the fermentation tank rises due to the heat of fermentation, and this temperature increase suppresses the growth and fermentation ability of Saccharomyces cerevisiae, making cooling essential. However, when using thermotolerant yeast, such cooling is not necessary, which leads to a reduction in cooling energy (cost) and therefore a reduction in _CO2 emissions. In addition, in parallel multiple fermentation, where saccharification and fermentation are performed simultaneously, the thermotolerant yeast can ferment at a temperature approximately 10°C higher, so the amount of saccharification enzyme can be reduced to half compared to Saccharomyces cerevisiae, thereby reducing costs.
Like Saccharomyces cerevisiae, when thermotolerant yeast uses cellulosic biomass, three types of enzymes for saccharification reaction, namely cellobiohydrolase (CBH), endoglucanase (EG), and β-glucosidase (BGL), are required. If thermotolerant yeast can produce these enzymes, the cost of ethanol production can be further reduced.

The present inventors were able to obtain a mutant yeast strain capable of decomposing cellobiose by subjecting the thermotolerant yeast to ultraviolet irradiation treatment and, in some cases, culturing the yeast in a medium containing a specific drug, and thus succeeded in establishing a method for breeding yeast with cellobiose-decomposing properties. The mutant strain was also capable of assimilating cellobiose into ethanol. The present invention was completed based on the above findings, and includes the following aspects:
That is, one aspect of the present invention is
[1] The present invention relates to a mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having an ability to decompose cellobiose.
In one embodiment, the mutant strain of the present invention comprises:
[2] The mutant strain according to [1] above,
It is characterized by having an increased ability to decompose cellobiose as compared to the wild-type strain.
In one embodiment, the mutant strain of the present invention comprises:
[3] The mutant strain according to [1] or [2] above,
The strain is obtained by mutation using the Kluyveromyces marxianus DMKU3-1042 strain as a parent strain, and is characterized in that it has an increased cellobiose-decomposing ability compared to the Kluyveromyces marxianus DMKU3-1042 strain.
In one embodiment, the mutant strain of the present invention comprises:
[4] The mutant strain according to [3] above, characterized in that it is obtained by subjecting the Kluyveromyces marxianus DMKU3-1042 strain to ultraviolet irradiation treatment and culturing it in a medium containing at least one of an acetaldehyde inhibitor, an erygosterol synthesis inhibitor, and a growth inhibitor.
In one embodiment, the mutant strain of the present invention comprises:
[5] The mutant strain according to any one of [1] to [4] above,
The strain is characterized in that it is SY13-1-4 strain (accession number: P-03284), SY15-2-12 strain (accession number: P-03286), SY15-1-43 strain (accession number: P-03285), SY15-1-19 strain (accession number: P-03253), or a mutant strain thereof.
In another aspect, the present invention comprises:
[6] The present invention relates to a composition for decomposing cellobiose, comprising the mutant strain according to any one of [1] to [5] above or a lysate thereof.
In another aspect, the present invention comprises:
[7] The present invention relates to a method for decomposing cellobiose, which uses the mutant strain according to any one of [1] to [5] above or the composition for decomposing cellobiose according to [6] above.
In another aspect, the present invention comprises:
[8] A method for producing ethanol, comprising:
The present invention relates to a method for producing ethanol, comprising a step of saccharifying cellobiose using the mutant strain according to any one of [1] to [5] above or the composition for decomposing cellobiose according to [6] above.
In one embodiment, the method for producing ethanol of the present invention comprises:
[9] The method for producing ethanol according to [8] above,
The method is characterized in that a glucose-utilizing ethanol-fermenting microorganism is further used in the step of saccharifying cellobiose.
In one embodiment, the method for producing ethanol of the present invention further comprises:
[10] The method for producing ethanol according to [8] or [9] above,
The method is characterized by further comprising a step of saccharifying cellulose into cellobiose using a cellulase-producing bacterium, or cellobiohydrolase or endoglucanase.

The mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having the ability to decompose cellobiose according to the present invention can reduce the cost of hydrolytic enzymes during the saccharification reaction of cellulosic biomass and can solve the problem of BGL inhibition due to cellobiose accumulation.

1 shows a schematic diagram of one embodiment of ethanol production from cellulosic biomass using thermotolerant fermentation yeast. Cellulose is decomposed into cellobiose by the CBH enzyme and EG enzyme of cellulase-producing bacteria, for example, filamentous fungi. Cellobiose is decomposed into glucose by the BGL enzyme and used as a resource for ethanol production by thermotolerant fermentation yeast. On the other hand, when a mutant strain of thermotolerant fermentation yeast having the ability to decompose cellobiose provided by the present invention is used, ethanol production is possible using cellobiose as a direct resource. FIG. 2 is a flow chart showing the process of isolating a thermotolerant mutant yeast having cellobiose-decomposing ability by a breeding method using Kluyveromyces marxianus DMKU3-1042 strain as a parent strain and culturing it in a drug-containing medium in the following Examples. 3 is a graph showing the results of a cell growth test, a cellobiose decomposition test, and an ethanol production test for the SY13-1-4 strain, which is a mutant strain of the Kluyveromyces marxianus DMKU3-1042 strain. The parent strain DMKU3-1042 was used as a control. 4 is a graph showing the results of a cell growth test, a cellobiose decomposition test, and an ethanol production test for the SY14-3-7 strain, which is a mutant strain of the Kluyveromyces marxianus DMKU3-1042 strain. The parent strain DMKU3-1042 was used as a control. 5 is a graph showing the results of a cell growth test, a cellobiose decomposition test, and an ethanol production test for the SY15-1-19 strain, which is a mutant strain of the Kluyveromyces marxianus DMKU3-1042 strain. The parent strain, DMKU3-1042, was used as a control. 6 is a graph showing the results of a cell growth test, a cellobiose decomposition test, and an ethanol production test for the SY15-1-43 strain, which is a mutant strain of the Kluyveromyces marxianus DMKU3-1042 strain. The parent strain DMKU3-1042 was used as a control. 7 is a graph showing the results of a cell growth test, a cellobiose decomposition test, and an ethanol production test for the SY15-2-12 strain, which is a mutant strain of the Kluyveromyces marxianus DMKU3-1042 strain. The parent strain DMKU3-1042 was used as a control.

One aspect of the present invention provides a mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having cellobiose decomposition ability.
The mutant strain according to the present invention has the ability to decompose cellobiose, and therefore enables the saccharification of cellobiose. Furthermore, the mutant strain according to the present invention enables cellobiose assimilation and ethanol fermentation in ethanol production using cellulosic biomass.

"Cellobiose" refers to a disaccharide in which two glucose molecules are linked by a β1,4 bond. In ethanol production using cellulosic biomass, cellobiose is produced as a decomposition product of cellulose by the action of cellobiohydrolase (CBH) and endoglucanase (EG). In this specification, "a mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having the ability to decompose cellobiose contained in a culture solution into glucose" refers to a mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having the ability to decompose cellobiose contained in a culture solution into glucose. Bacteria belonging to the genus Kluyveromyces marxianus or Kluyveromyces lactis do not substantially have the ability to decompose cellobiose. Therefore, the mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having the ability to decompose cellobiose according to the present invention is a mutant with an increased ability to decompose cellobiose. In one embodiment, the mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having cellobiose-decomposing ability has increased cellobiose-decomposing ability compared to a wild-type strain. An example of a wild-type strain of Kluyveromyces marxianus is the DMKU3-1042 strain, and an example of a wild-type strain of Kluyveromyces lactis is the NBRC 0433 strain.
Furthermore, one embodiment of the "Kluyveromyces marxianus or Kluyveromyces lactis mutant strain having cellobiose-decomposing ability" is a strain obtained by mutation using the Kluyveromyces marxianus DMKU3-1042 strain as a parent strain, and has increased cellobiose-decomposing ability compared to the Kluyveromyces marxianus DMKU3-1042 strain. Further, a more specific example of a "mutant having the ability to decompose cellobiose" is, but is not limited to, a mutant that decomposes 30% or more, preferably 40% or more, 50% or more, 60% or more, and more preferably 90% or more of the cellobiose contained in a cellobiose-containing medium (e.g., a mixed medium of _0.17 % yeast nitrogen base (containing no amino acids), 0.08% ammonium sulfate, 0.5% yeast extract, and 3% cellobiose) when a culture solution containing the mutant is transferred to 100 ml of the medium so as to have a turbidity (OD 660 ) of 0.1 and the culture is subjected to shaking culture (160 rpm) for a certain period of time (e.g., 48 to 60 hours) at a temperature of 40°C. Furthermore, for example, when a bacterial cell culture solution is transferred to 100 ml of the above cellobiose-containing medium containing 3% cellobiose so that the degree of suspension (OD ₆₆₀ ) is 0.1, and the culture is subjected to shaking culture (160 rpm) at a temperature of 40° C. for a certain period of time (e.g., 20 to 40 hours), a mutant strain capable of decomposing cellobiose at 1% (W/V) or more, preferably 2% (W/V) or more, and more preferably 2.5% (W/V) or more can be exemplified.
As described above, the mutant strain is cultured in a cellobiose-containing medium for a certain period of time, and then the concentration of cellobiose remaining in the medium after the culture is measured to evaluate the cellobiose decomposition ability. The cellobiose concentration in the medium can be measured by a known method, for example, the dinitrosalicylic acid method (DNSA method) or high performance liquid chromatography (HPLC method) shown in the following Examples.

Furthermore, the mutant strain according to the present invention is capable of cellobiose assimilation and ethanol fermentation. The mutant strain according to the present invention can decompose cellobiose into glucose by saccharification reaction, and the obtained glucose can be used for ethanol fermentation. In one embodiment, the mutant strain according to the present invention can be said to have increased cellobiose assimilation and ethanol fermentation compared to the Kluyveromyces marxianus DMKU3-1042 strain. More specifically, a "mutant capable of cellobiose assimilation and ethanol fermentation" is, but is not limited to, a mutant capable of producing 1% (W/V) or more, preferably 2% (W/V) or more, and more preferably 2.5% (W _/V ) or more of ethanol when a mutant-containing culture solution is transferred to 100 ml of a cellobiose-containing medium (e.g., a mixed medium of 0.17% yeast nitrogen base (containing no amino acids), 0.08% ammonium sulfate, 0.5% yeast extract, and 3% cellobiose) so that the suspension degree (OD 660 ) becomes 0.1 and the culture is subjected to shaking culture (160 rpm) at a temperature of 40° C. for a certain period of time (e.g., 48 to 60 hours).
As described above, the mutant can be cultured in a cellobiose-containing medium for a certain period of time, and then the concentration of ethanol produced in the medium after the culture can be measured to evaluate the ethanol production ability in cellobiose-utilizing ethanol fermentation. The ethanol concentration can be measured by a known method, for example, high performance liquid chromatography as shown in the following examples.

In this specification, the term "mutant" refers to a strain in which a given trait has been changed from a parent strain, and includes, but is not limited to, a mutant that has newly acquired a trait not possessed by the parent strain, or a mutant in which the function of a trait possessed by the parent strain has been improved or changed. In addition, "mutants" include natural mutants, mutants induced by UV irradiation, X-ray irradiation, mutagenized with a mutagen (e.g., N-methyl-N-nitro-N-nitrosoguanidine, etc.), mutants obtained by screening using a drug-containing medium , and polyploidized mutants. In a preferred embodiment, the mutant is a mutant that can be selected by UV irradiation and/or culture using a drug-containing medium. Unlike mutants produced using DNA recombination technology, mutants selected by UV irradiation or culture using a drug-containing medium are preferable in that they do not require the cost of containment equipment to prevent yeast from spreading and the disposal process after production.

The above-listed mutant strains can be produced according to known methods. For example, ultraviolet treatment methods for producing yeast mutant strains are known, and ultraviolet rays can be irradiated onto the cells using an ultraviolet irradiation device for microbial breeding. The ultraviolet irradiation dose is preferably set to a level that allows about 10% of the target strain to survive. Specifically, cells are added to a glass petri dish with an outer diameter of 10 cm in a clean bench so that the cell turbidity is OD ₆₆₀ = 1.0, and ultraviolet irradiation can be performed by irradiating the cells with a germicidal lamp (GL-15, manufactured by Hotalux Co., Ltd., wavelength = 250 nm) from a distance of 20 cm for 5 minutes.

The method for producing a mutant strain using a drug-containing medium can be carried out, for example, as follows. Examples of drugs that can be used in the drug-containing medium include acetaldehyde inhibitors, erygosterol synthesis inhibitors, and growth suppressants. These drugs may be used alone or in combination of two or more.
An example of an acetaldehyde inhibitor is, but is not limited to, Disulfiram (DSF). The concentration of the acetaldehyde inhibitor in the medium can be appropriately set depending on the conditions such as the drug used. When DSF is used as the acetaldehyde inhibitor, the concentration can be, but is not limited to, in the range of, for example, 0.1 μM to 10 μM.
Examples of erigosterol synthesis inhibitors include, but are not limited to, clotrimazole (CTZ) and miconazole (MCZ). The concentration of the erigosterol synthesis inhibitor in the medium can be appropriately set depending on conditions such as the drug used. When CTZ or MCZ is used as the erigosterol synthesis inhibitor, the concentration can be, but is not limited to, in the range of 0.4 μg/mL to 4 μg/mL for CTZ and in the range of 5 μM to 50 μM for MCZ.
The growth inhibitor may be, but is not limited to, 2-deoxyglucose (2-DG), a glucose analog. The concentration of the growth inhibitor in the medium may be appropriately set depending on the conditions of the drug used. When 2-DG is used as the growth inhibitor, the concentration may be, but is not limited to, in the range of 0.01% (w/v) to 0.5% (w/v).
In addition to the drugs listed above, Amphotericin B (AMB) and other drugs that increase the permeability of cell membranes may also be used.
The medium to which the drug is added may be any known medium suitable for culturing yeast, and preferably contains cellobiose. Examples of the medium include, but are not limited to, a mixed medium of 0.17% yeast nitrogen base (containing no amino acids), 0.08% ammonium sulfate, 0.5% yeast extract, and 3% cellobiose, and the desired drug can be added to the mixed medium to a preferred concentration.

The method of selecting mutant strains using a drug-containing medium involves culturing the bacteria for a certain period of time in a drug-containing medium. The culture conditions are not limited to the following, but can be 30 to 41°C for 48 to 60 hours.

It is also preferable to carry out long-term subculture using a cellobiose-containing medium after ultraviolet irradiation treatment or cultivation using a drug-containing medium. By carrying out long-term subculture, mutations adapted to cellobiose decomposition ability or cellobiose assimilation can be established. Examples of subculture are not limited as long as they can establish mutations, but for example, the cultured mutant strain can be plated on a plate medium using a cellobiose-containing medium, and subculture can be carried out at 24-hour, 48-hour, 74-hour, or 96-hour intervals at 37°C for 240 to 432 hours.

Mutants that have been subjected to mutation treatments such as UV irradiation or cultivation in a drug-containing medium, or that have been cultivated for a long period of time after mutation treatment, can be used to select preferred mutants by evaluating the size of colonies formed by plate cultivation, growth ability, cellobiose decomposition ability, or ethanol production ability.

The process of isolating mutant strains by ultraviolet irradiation or the process of culturing the strain in a drug-containing medium and isolating mutant strains may be repeated multiple times until a mutant strain having the desired properties is obtained.

In one embodiment, the mutant strain of the present invention is a mutant strain having heat resistance. As used herein, having heat resistance means being able to grow under conditions of at least 40°C. "Able to grow under conditions of at least 40°C" means that the mutant strain can grow well even under high temperature conditions of 40°C or higher. More specifically, it means that the mutant strain can grow under conditions of 40°C, preferably under conditions of 43°C, and more preferably under conditions of 45°C.
In a preferred embodiment, the mutant strain of the present invention has the ability to decompose cellobiose or the ability to utilize cellobiose for ethanol fermentation under conditions of at least 40°C.

To obtain a mutant strain that has the ability to decompose cellobiose and can grow at a temperature of at least 40°C, a thermotolerant strain capable of growing at a temperature of at least 40°C may be used as a parent strain for breeding to obtain a mutant strain having the ability to decompose cellobiose. A known strain may be used as the thermotolerant strain capable of growing at a temperature of at least 40°C, for example, the Kluyveromyces marxianus DMKU3-1042 strain.

In one embodiment, the mutant strain of the present invention is a strain obtained by mutation using Kluyveromyces marxianus DMKU3-1042 strain as a parent strain. More specifically, the mutant strain is obtained by subjecting Kluyveromyces marxianus DMKU3-1042 strain to ultraviolet irradiation treatment and culturing in a drug-containing medium containing at least one of an acetaldehyde inhibitor, an erygosterol synthesis inhibitor, or a growth inhibitor. The Kluyveromyces marxianus DMKU3-1042 strain is a strain disclosed in Japanese Patent No. 5051727 as a thermotolerant yeast strain (Accession No.: NITE BP- 283 ).
In one embodiment, the mutant strain of the present invention is SY13-1-4 strain, SY15-2-12 strain, SY15-1-43 strain, or SY15-1-19 strain, or a mutant strain derived therefrom. The above SY13-1-4 strain, SY15-2-12 strain, SY15-1-43 strain, and SY15-1-19 strain are deposited on August 5, 2020 or September 15, 2020 at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, under the accession numbers NITE P-03284, NITE P-03286, NITE P-03285, and NITE P-03253, respectively, and can be distributed under certain conditions.

In another aspect, the present invention provides a composition for decomposing cellobiose, comprising the mutant strain of the present invention or a lysate thereof.
The cellobiose decomposition composition of the present invention can be used for decomposing cellobiose. In one embodiment, the cellobiose decomposition composition of the present invention can contain a lysate of the mutant strain of the present invention, and can thus contain a cellobiose-decomposing enzyme derived from the mutant strain of the present invention. The dissolution method for preparing the lysate is not particularly limited, and examples thereof include an enzymatic decomposition method in which an enzyme preparation obtained from a microorganism or a plant is added to solubilize the enzyme, a hydrolysis method in which an acid or an alkali is used to solubilize the enzyme, and a method in which the cell wall is physically broken by ultrasonication or the like to elute the cellobiose-decomposing enzyme out of the bacterial cells.
The composition for decomposing cellobiose of the present invention may further contain additives such as a culture medium for maintaining the mutant strain and the enzyme, and a stabilizer.

Another aspect of the present invention provides a method for decomposing cellobiose, which uses the above-mentioned mutant strain of the present invention or the above-mentioned composition for decomposing cellobiose of the present invention.
The method for decomposing cellobiose is not limited as long as the mutant strain having the cellobiose decomposition ability of the present invention or the composition for decomposing cellobiose of the present invention is used. The method for decomposing cellobiose using the mutant strain or the composition for decomposing cellobiose of the present invention also includes a method for producing glucose from cellobiose.
For example, the mutant strain having cellobiose decomposition ability of the present invention or the composition for cellobiose decomposition of the present invention may be added to a solvent containing cellobiose, and cultured or reacted for a certain period of time. The solvent is not limited as long as it allows the mutant strain or the composition for cellobiose decomposition to decompose cellobiose. When cellobiose is decomposed using the mutant strain, for example, a yeast culture liquid such as 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), or YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose) may be used.
When cellobiose is decomposed using the mutant strain, 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 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.

The glucose derived from cellobiose obtained in the above embodiment may be used as a raw material for combination with another fermentation process. Furthermore, the mutant strain of the present invention is also capable of cellobiose assimilation and ethanol fermentation.
Thus, another aspect of the present invention provides a method for producing ethanol, comprising a step of saccharifying cellobiose using the mutant strain of the present invention or the composition for decomposing cellobiose of the present invention. The method for producing ethanol is not limited as long as it includes a step of saccharifying glucose from cellobiose using the mutant strain having the cellobiose-decomposing ability of the present invention or the composition for decomposing cellobiose of the present invention.
In this embodiment, the mutant strain having cellobiose decomposition ability of the present invention can perform ethanol fermentation using glucose obtained by saccharification of cellobiose, and thus it is possible to produce ethanol by culturing the mutant strain in a medium containing cellobiose. In one embodiment, a microorganism capable of glucose assimilation ethanol fermentation may be further used to promote alcoholic fermentation to produce ethanol from glucose. Such a microorganism capable of glucose assimilation ethanol fermentation can be a known one, and examples of the microorganism include yeasts such as Saccharomyces yeast and Kluyveromyces yeast, and bacteria such as Zymomonas and Zymobacter, and preferably includes thermotolerant yeast (e.g., Kluyveromyces marxianus DMKU3-1042 strain), thermotolerant Zymomonas mobilis, and the like.

In one embodiment, the method for producing ethanol of the present invention further comprises a step of saccharifying cellulose into cellobiose using a cellulase-producing bacterium, or cellobiohydrolase or endoglucanase.
In the method for producing ethanol of the present invention, the step of decomposing the cellulose into cellobiose may be carried out before or simultaneously with the step of saccharifying the cellobiose.
The cellulase-producing bacteria that can be used in the present invention are not limited as long as they are capable of saccharifying cellulose into cellobiose, and for example, filamentous fungi and bacteria of the genus Bacillus such as Bacillus licheniformis can be used.
When cellulose is saccharified into cellobiose using cellobiohydrolase or endoglucanase, a preferred embodiment is an embodiment in which cellulose is saccharified into cellobiose using cellobiohydrolase and endoglucanase.

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.

(Obtaining a thermotolerant mutant yeast with cellobiose-decomposing ability)
In this example, breeding was carried out using the thermotolerant yeast Kluyveromyces marxianus DMKU3-1042 strain as a parent strain.
The DMKU3-1042 strain was irradiated with ultraviolet light (mutation treatment) for 5 minutes using a germicidal lamp (GL-15, Hotalux Co., Ltd., wavelength = ₂₅₀ nm) from a distance of 20 cm under conditions in which 10% of the heat-resistant yeast cells survive, by adding cells to a glass petri dish with an outer diameter of 10 cm in a clean bench so that the cell turbidity reached OD 660 = 1.0, and the mutated yeast was then inoculated into 10 mL of cellobiose-containing medium (a mixed medium of 0.17% yeast nitrogen base (amino acid-free), 0.08% ammonium sulfate, 0.5% yeast extract, and 3% cellobiose) and cultured at 40°C, 160 rpm, and shaking for 24 hours. The cultured mutant strain was then plated on the cellobiose-containing medium and cultured for 48 to 72 hours at 37° C. Large colonies (96 to 144 colonies) were selected from the colonies that emerged, and a strain with high cellobiose consumption and ethanol production was further selected (SY5-1-17).
The cellobiose concentration and ethanol concentration in the medium after culturing the mutant strain were measured using HPLC. Specifically, the culture solution was centrifuged (1,400 rpm, 1 minute), the supernatant was filtered through a membrane filter (manufactured by Nippon Pall Co., Ltd.) to obtain a test liquid, and the concentration was measured by 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: the column was Gelpack (registered trademark) GL-C610-S (manufactured by Hitachi High-Technologies Corporation), 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.

Next, the SY5-1-17 strain was subjected to the same mutation treatment by UV irradiation as above, followed by shaking culture using the cellobiose-containing medium and plate culture, and a strain with high cellobiose consumption and ethanol production was selected (SY5-1-17-3-40 strain). The obtained SY5-1-17-3-40 strain was further subjected to the same mutation treatment by UV irradiation as above, followed by long-term subculture (subculture for 384 to 432 hours at 48-hour intervals) using the cellobiose-containing medium multiple times, and a strain with high cellobiose consumption and ethanol production was selected (SY12-2-35 strain). The SY12-2-35 strain was further subjected to the above mutation treatment by UV irradiation, followed by shaking culture using a cellobiose-containing medium further containing 1 μM DSF (disulfiram), and plate culture. By selecting the larger colonies from those that emerged, and then selecting strains with high cellobiose consumption and ethanol production, we were finally able to isolate a strain (SY13-1-4) that had the ability to not only degrade cellobiose but also utilize cellobiose to produce ethanol (Figure 2).

In order to obtain a strain having improved cellobiose-decomposing ability or cellobiose-utilizing ethanol-producing ability from the above-obtained SY13-1-4 strain, the following operations were carried out.
The SY13-1-4 strain was further subjected to the above-mentioned UV irradiation mutation treatment, and then shake culture and plate culture were performed using a cellobiose-containing medium further containing 1 μM DSF and 0.1% 2DG (2-deoxyglucose). By selecting larger colonies from the colonies that emerged, and then selecting strains with high cellobiose consumption and ethanol production, we succeeded in isolating a strain (SY14-3-7 strain) with improved cellobiose decomposition ability and cellobiose assimilation ethanol production ability (FIG. 2).
Furthermore, the SY14-3-7 strain was further subjected to the above-mentioned mutation treatment by ultraviolet irradiation. The shaking culture and plate culture after the mutation treatment were carried out using three media ((i) a cellobiose-containing medium further containing 1 μM DSF and 0.5% 2DG, (ii) a cellobiose-containing medium further containing 1 μM DSF, 0.5% 2DG, and 4 μg CTZ, and (iii) a cellobiose-containing medium further containing 10 μM DSF and 0.1% 2DG). By selecting larger colonies from the colonies that appeared and further selecting strains with high cellobiose consumption and ethanol production, strains (SY15-2-12 strain, SY15-1-43 strain, and SY15-1-19 strain) with improved cellobiose decomposition ability and cellobiose assimilation ethanol production ability were successfully isolated (FIG. 2).

(Growth test, cellobiose decomposition test, and ethanol production test)
The following culture and measurements were carried out to measure the growth ability, cellobiose decomposition ability, and ethanol production ability of the thermotolerant mutant yeasts having cellobiose decomposition ability obtained as described above (SY13-1-4 strain, SY14-3-7 strain, SY15-2-12 strain, SY15-1-43 strain, SY15-1-19 strain). Kluyveromyces marxianus DMKU3-1042 strain (parent strain) was used as a control for the mutant strains. The culture and measurements for the growth test and the culture and measurements for the cellobiose differentiation and ethanol production tests were carried out independently.
Each of the SY13-1-4, SY14-3-7, SY15-2-12, SY15-1-43, SY15-1-19, and DMKU3-1042 strains was inoculated into 10 ml of cellobiose-containing medium in an Erlenmeyer flask with a loopful of inoculation, and the preculture was obtained by shaking (160 rpm) for 18 hours at a temperature of 37 ° C. Next, the preculture was transferred to 30 ml of cellobiose-containing medium (cellobiose content is about 3 to 4% (w / v)) in an Erlenmeyer flask so that the degree of suspension (OD ₆₆₀ ) was 0.1, and the preculture was performed at a temperature of 40 ° C. with shaking (160 rpm).

The growth of the thermotolerant mutant yeast having cellobiose differentiation ability and Kluyveromyces marxianus DMKU3-1042 strain, as well as the cellobiose and ethanol concentrations in the medium were measured 12 hours, 24 hours, 36 hours, and 48 hours after the start of culture. The growth of the thermotolerant mutant yeast having cellobiose differentiation ability and Kluyveromyces marxianus DMKU3-1042 was determined by measuring the turbidity (OD ₆₆₀ ) of the culture solution with a spectrophotometer. The cellobiose concentration in the medium was measured simultaneously with the ethanol concentration as described below. 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 high performance liquid chromatography in measuring the ethanol concentration in the medium were as follows: a Gelpack (registered trademark) GL-C610-S (Hitachi High-Technologies Corporation) column was used, 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 ), cellobiose concentration, and ethanol concentration of the culture solution of the SY13-1-4 strain are shown in FIG.
The SY13-1-4 strain showed better growth after about 10 hours of culture compared to the DMKU3-1042 strain. In addition, the SY13-1-4 strain showed cellobiose differentiation ability. On the other hand, the DMKU3-1042 strain did not substantially decompose cellobiose. Furthermore, the SY13-1-4 strain also showed ethanol production. On the other hand, the DMKU3-1042 strain did not show ethanol production. These results indicate that the SY13-1-4 strain has superior growth ability compared to the DMKU3-1042 strain, and further has the ability to decompose cellobiose and to utilize cellobiose to produce ethanol.

The measurement results of the turbidity (OD ₆₆₀ ), cellobiose concentration, and ethanol concentration of the culture solution of the SY14-3-7 strain are shown in Figure 4. In the graph of cell growth, the vertical axis indicates OD ₆₆₀ , and the horizontal axis indicates time (hours). In the graph of cellobiose concentration in Figure 4, the vertical axis indicates the cellobiose concentration in the medium (% w/v), and the horizontal axis indicates time. In the graph of ethanol concentration, the vertical axis indicates the ethanol concentration in the medium (% w/v), and the horizontal axis indicates time. The vertical and horizontal axes of Figures 5 to 7 also show the same units.
The SY14-3-7 strain showed better growth after about 10 hours of culture than the DMKU3-1042 strain. The SY13-3-7 strain also showed cellobiose differentiation ability. The cellobiose decomposition ability was improved compared to the SY13-1-4 strain. Furthermore, the SY14-3-7 strain also showed ethanol production. These results show that the SY14-3-7 strain has a superior growth ability compared to the DMKU3-1042 strain, and further has improved cellobiose decomposition ability and cellobiose assimilation ethanol production ability compared to the SY13-1-4 strain.

The measurement results of the turbidity (OD ₆₆₀ ), cellobiose concentration, and ethanol concentration of the culture solutions of the SY15-1-19 strain, SY15-1-43 strain, and SY15-2-12 strain are shown in FIGS. 5 to 7, respectively.
Compared with the DMKU3-1042 strain, all of the SY15-1-19 strain, SY15-1-43 strain, and SY15-2-12 strain showed better growth from the start of culture. In addition, all of the SY15-1-19 strain, SY15-1-43 strain, and SY15-2-12 strain showed cellobiose differentiation ability, and the cellobiose decomposition ability was improved compared to the SY13-1-4 strain and the SY14-3-7 strain. Furthermore, all of the SY15-1-19 strain, SY15-1-43 strain, and SY15-2-12 strain also showed ethanol production. Ethanol production could be observed immediately after the start of culture, which indicates that they have better ethanol production ability than the SY13-1-4 strain and the SY14-3-7 strain. These results show that the SY15-1-19 strain, the SY15-1-43 strain, and the SY15-2-12 strain all have superior growth ability compared to the DMKU3-1042 strain, and further have improved cellobiose decomposition ability and cellobiose-utilization ethanol production ability compared to the SY13-1-4 strain and the SY14-3-7 strain.

In recent years, technological developments aimed at utilizing non-edible cellulosic biomass have been conducted both in Japan and overseas, but they have not been put to practical use due to the high production costs. Cellulose fermentation using the yeast of the present invention is expected to improve the saccharification rate of cellulase and reduce costs, so it can be used for bioethanol production (biorefinery technology) using waste cellulosic biomass.

Claims (7)

A mutant strain obtained by irradiating Kluyveromyces marxianus DMKU3-1042 strain with ultraviolet light and culturing the strain in a cellobiose-containing medium without using DNA recombination technology, the mutant having an increased ability to decompose cellobiose compared to the Kluyveromyces marxianus DMKU3-1042 strain,
The OD was adjusted to 100 ml of a mixed medium of 0.17% yeast nitrogen base (containing no amino acids), 0.08% ammonium sulfate, 0.5% yeast extract, and 3% cellobiose. ₆₆₀ The mutant strain is capable of producing 1.5% (W/V) or more of ethanol when the mutant-containing culture solution is transferred to the culture medium so that the ratio of the total volume of the culture medium to the total volume of the mutant strain is 0.1 and the culture medium is subjected to shaking culture (160 rpm) at a temperature of 40° C. for 48 hours. The mutant strain according to claim 1, which is SY13-1-4 strain (accession number: NITE P-03284), SY15-2-12 strain (accession number: P-03286), SY15-1-43 strain (accession number: NITE P-03285), or SY15-1-19 strain (accession number: NITE P -03253). A composition for decomposing cellobiose, comprising the mutant strain according to claim 1 or 2 or a lysate thereof. A method for decomposing cellobiose, which comprises using the mutant strain according to claim 1 or 2 or the composition for decomposing cellobiose according to claim 3 . 1. A method for producing ethanol, comprising:
A method for producing ethanol, comprising a step of saccharifying cellobiose using the mutant strain according to claim 1 or 2 or the composition for decomposing cellobiose according to claim 3 . 6. The method for producing ethanol according to claim 5 ,
A method for producing ethanol, further comprising using a glucose-utilizing, ethanol-fermenting microorganism in the step of saccharifying cellobiose. 7. A method for producing ethanol according to claim 5 or 6 , comprising:
A method for producing ethanol, further comprising a step of saccharifying cellulose into cellobiose using a cellulase-producing bacterium, or cellobiohydrolase or endoglucanase.