Yeast with cellobiose-decomposing ability

Abstract

To provide a thermostable yeast that is mutated by breeding methods not based on the DNA recombinant technique so that the original enzymes for saccharification can function, or particularly, a thermostable mutant having the ability to decompose cellobiose.SOLUTION: The present invention discloses a mutant having the ability to decompose cellobiose and belonging to Kluyveromyces marxianus or Kluyveromyces lactis, where the mutant can be grown at least under the condition of 40°C.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a yeast having the ability to decompose cellobiose. [Background technology]

[0002] 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 cost and a shortage of biomass as a raw material, its production is almost nonexistent in Japan. Even overseas, using second-generation biomass as a raw material is considered to require ultra-large-scale facilities for profitability. In order to achieve the SDGs, innovative technology development that can reduce costs is required.

[0003] To produce ethanol from cellulose, a second-generation biomass, three types of cellulase enzymes are essential: cellobiohydrolase (CBH), endoglucanase (EG), and β-glucosidase (BGL). CBH hydrolyzes the cellulose chain from its terminal end, while EG hydrolyzes the cellulose chain from its interior. Meanwhile, BGL hydrolyzes cellooligosaccharides, such as cellobiose (a compound consisting of two glucose units bonded together), produced by the decomposition of CBH and EG, to produce glucose. These cellulolytic enzymes are several times more expensive than the amylolytic enzymes used for first-generation biomass, and the cellulase used in the saccharification reaction is required in larger quantities than the amylase used in the enzymatic saccharification reaction of starch-based biomass, so the enzyme costs are high and they have not yet been put to practical use. Thus, in ethanol production from second-generation cellulosic biomass, there is a need to reduce the cost of hydrolytic enzymes in particular.

[0004] If the amount of commercially available cellulase BGL used were reduced to solve the above-mentioned problems in ethanol production from cellulosic biomass, the product glucose would inhibit BGL, resulting in the accumulation of cellobiose. The accumulated cellobiose would inhibit CBH and EG, resulting in a strong inhibition of the saccharification reaction. Furthermore, if homemade cellulase were used to reduce costs, the cellulase produced by filamentous fungi (mainly Trichoderma reesei) would have low BGL activity, resulting in a low cellulose saccharification rate and a lower final ethanol production. The common thread 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 facilities to prevent the spread of the recombinant yeast, as well as the cost of waste disposal after production, and therefore has not been used for ethanol fermentation production. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 146540 Brochure Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above problems, an objective of the present invention is to provide a yeast strain that has been mutated to have a saccharifying enzyme function, which can be used for ethanol production from cellulosic biomass, by a breeding method that does not rely on DNA recombinant DNA technology. In particular, an objective of the present invention is to provide a mutant strain that has the ability to decompose cellobiose. [Means for solving the problem]

[0007] The present inventors have been developing thermotolerant yeast, 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 temperatures between 40 and 43°C, demonstrating stable fermentation at temperatures approximately 10°C higher than the yeast (Saccharomyces cerevisiae) commonly used for ethanol production worldwide. Fermentation heat increases the temperature of the fermenter, which inhibits the growth and fermentation ability of Saccharomyces cerevisiae, making cooling essential. However, using thermotolerant yeast eliminates this cooling requirement, leading to reduced cooling energy (costs) and CO2 emissions. Furthermore, in parallel fermentation where saccharification and fermentation are performed simultaneously, thermotolerant yeast can ferment at a temperature approximately 10°C higher, so the amount of saccharification enzyme can be reduced by half compared to Saccharomyces cerevisiae, thereby reducing costs. Like Saccharomyces cerevisiae, thermotolerant yeasts also require three enzymes for saccharification reactions: cellobiohydrolase (CBH), endoglucanase (EG), and β-glucosidase (BGL) when using cellulosic biomass. However, if thermotolerant yeasts can produce these enzymes, the cost of ethanol production can be further reduced.

[0008] The present inventors were able to obtain a mutant yeast strain capable of decomposing cellobiose by subjecting the thermotolerant yeast to ultraviolet irradiation and, optionally, 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 cellobiose-utilization ethanol fermentation. 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] This study concerns mutant strains of Kluyveromyces marxianus or Kluyveromyces lactis that have the ability to decompose cellobiose. wherein 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 cellobiose-degrading ability 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 was obtained by mutation using the Kluyveromyces marxianus DMKU3-1042 strain as a parent strain, and is characterized by having an increased ability to decompose cellobiose 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 irradiating the Kluyveromyces marxianus DMKU3-1042 strain with ultraviolet light 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 by being SY13-1-4 strain (accession number: P-03284), SY15-2-12 strain (accession number: P-03286), SY15-1-43 strain (accession number: P-03285), or SY15-1-19 strain (accession number: P-03253), or a mutant thereof. In another aspect, the present invention provides [6] 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 provides [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 provides [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 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, cellobiohydrolase, or endoglucanase. [Effects of the Invention]

[0009] The mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis according to the present invention having the ability to decompose cellobiose can reduce the cost of hydrolytic enzymes during the saccharification reaction of cellulosic biomass and solve the problem of BGL inhibition due to cellobiose accumulation. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 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 and EG enzymes of cellulase-producing fungi, such as filamentous fungi. Cellobiose is then decomposed into glucose by the BGL enzyme and used as a resource for ethanol production by the thermotolerant fermentation yeast. On the other hand, when a mutant strain of thermotolerant fermentation yeast with the ability to decompose cellobiose provided by the present invention is used, ethanol production becomes possible using cellobiose directly as a resource. [Figure 2]FIG. 2 is a flow chart showing the process of isolating a thermotolerant mutant yeast having the ability to decompose cellobiose by breeding Kluyveromyces marxianus DMKU3-1042 strain as a parent strain through ultraviolet irradiation and cultivation in a drug-containing medium, as described in the following Examples. [Figure 3] Figure 3 shows the results of cell growth tests, cellobiose degradation tests, and ethanol production tests for the SY13-1-4 mutant of Kluyveromyces marxianus DMKU3-1042. The parent strain, DMKU3-1042, was used as a control. [Figure 4] Figure 4 shows the results of cell growth tests, cellobiose degradation tests, and ethanol production tests for the SY14-3-7 mutant of Kluyveromyces marxianus DMKU3-1042. The parent strain, DMKU3-1042, was used as a control. [Figure 5] Figure 5 shows the results of cell growth tests, cellobiose degradation tests, and ethanol production tests for the SY15-1-19 strain, a mutant of the Kluyveromyces marxianus DMKU3-1042 strain. The parent strain, DMKU3-1042, was used as a control. [Figure 6] Figure 6 shows the results of cell growth tests, cellobiose degradation tests, and ethanol production tests for the SY15-1-43 strain, a mutant of the Kluyveromyces marxianus DMKU3-1042 strain. The parent strain, DMKU3-1042, was used as a control. [Figure 7] Figure 7 shows the results of cell growth tests, cellobiose degradation tests, and ethanol production tests for the SY15-2-12 strain, a mutant of the Kluyveromyces marxianus DMKU3-1042 strain. The parent strain, DMKU3-1042, was used as a control. DETAILED DESCRIPTION OF THE INVENTION

[0011] One aspect of the present invention provides a mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having the ability to decompose cellobiose. The mutant strain of the present invention has the ability to decompose cellobiose, thereby enabling the saccharification of cellobiose and the ethanol fermentation of cellulosic biomass.

[0012] "Cellobiose" refers to a disaccharide formed by two glucose molecules linked by a β1,4 bond. In ethanol production using cellulosic biomass, cellobiose is produced as a cellulose degradation product by the action of cellobiohydrolase (CBH) and endoglucanase (EG). As used herein, "a mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis capable of cellobiose degradation" refers to a mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis that has the ability to decompose cellobiose contained in the culture medium into glucose. Bacteria belonging to the genus Kluyveromyces marxianus or Kluyveromyces lactis are substantially unable to decompose cellobiose. Therefore, the mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis capable of cellobiose degradation according to the present invention is a mutant with enhanced cellobiose degradation ability. In one embodiment, the mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis having the ability to decompose cellobiose has an increased ability to decompose cellobiose compared to a wild-type strain. Examples of wild-type strains of Kluyveromyces marxianus include the DMKU3-1042 strain, and examples of wild-type strains of Kluyveromyces lactis include the NBRC 0433 strain. Furthermore, one embodiment of the "mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis capable of cellobiose decomposition" is a strain obtained by mutation of the Kluyveromyces marxianus DMKU3-1042 strain as a parent strain, and has an increased ability to decompose cellobiose compared to the Kluyveromyces marxianus DMKU3-1042 strain. Specific examples of the "mutant strain capable of cellobiose decomposition" include, but are not limited to, a mutant strain of Kluyveromyces marxianus or Kluyveromyces lactis capable of decomposing cellobiose, which is obtained by mutating the strain of Kluyveromyces marxianus DMKU3-1042 using a cellobiose-containing medium (e.g., a mixed medium of 0.17% Yeast Nitrogen base (amino acid-free), 0.08% Ammonium sulfate, 0.5% yeast extract, and 3% cellobiose) to a turbidity (OD ). 660 ) = 0.1, and the mixture is cultured at 40°C for a certain period of time (e.g., 48 to 60 hours) with shaking (160 rpm). Examples of such mutants include those that decompose 30% or more, preferably 40% or more, 50% or more, 60% or more, and more preferably 90% or more of the cellobiose contained in the medium. Furthermore, for example, when 100 ml of the above cellobiose-containing medium containing 3% cellobiose is added, the turbidity (OD 660 ) = 0.1, and the resulting mixture is cultured at 40°C with shaking (160 rpm) for a certain period of time (e.g., 20 to 40 hours). When this is done, the mutant strain can decompose 1% (W / V) or more, preferably 2% (W / V) or more, and more preferably 2.5% (W / V) or more of cellobiose. As described above, the mutant strain can be cultured in a cellobiose-containing medium for a certain period of time, and then the concentration of cellobiose remaining in the medium after culture can be measured to evaluate its cellobiose-degrading ability. The cellobiose concentration in the medium can be measured by any known method, such as the dinitrosalicylic acid method (DNSA method) or high-performance liquid chromatography (HPLC method) described in the Examples below.

[0013] Furthermore, the mutant strain of the present invention is capable of cellobiose-utilizing ethanol fermentation. The mutant strain of the present invention can decompose cellobiose into glucose by saccharification, and the resulting glucose can be used for ethanol fermentation. In one embodiment, the mutant strain of the present invention can be said to have enhanced cellobiose-utilizing ethanol fermentation capabilities compared to the Kluyveromyces marxianus DMKU3-1042 strain. More specifically, but not limited to, a "mutant strain capable of cellobiose-utilizing ethanol fermentation" refers to a mutant strain capable of cellobiose-utilizing ethanol fermentation that has a turbidity (OD ) of 100 ml in 100 ml of a cellobiose-containing medium (for example, a mixed medium of 0.17% Yeast Nitrogen base (amino acid-free), 0.08% Ammonium sulfate, 0.5% yeast extract, and 3% cellobiose). 660 When the mutant-containing culture solution is transferred so that the ethanol content becomes 0.1, and the mixture is cultured at 40°C with shaking (160 rpm) for a certain period of time (e.g., 48 to 60 hours), examples of mutants that can produce ethanol at 1% (W / V) or more, preferably 2% (W / V) or more, and more preferably 2.5% (W / V) or more can be given. As described above, the ethanol production ability in cellobiose-utilizing ethanol fermentation can be evaluated by culturing the mutant in a cellobiose-containing medium for a certain period of time and then measuring the concentration of ethanol produced in the medium after the culture. The ethanol concentration can be measured by any known method, such as high performance liquid chromatography as shown in the Examples below.

[0014] As used herein, the term "mutant" refers to a strain that has been altered in a given trait from a parent strain, and includes, but is not limited to, mutant strains that have newly acquired traits not possessed by the parent strain, and mutants in which the function of a trait possessed by the parent strain has been improved or changed. Furthermore, "mutants" include natural mutants, mutants that have been subjected to mutagenesis using ultraviolet irradiation, X-ray irradiation, or a mutagen (e.g., N-methyl-N-nitro-N-nitrosoguanidine), and mutants obtained by screening using a drug-containing medium. ,ohand polyploids. In a preferred embodiment, the mutant strain is a mutant strain that can be selected by ultraviolet irradiation and / or culture in a drug-containing medium. Unlike mutant strains produced using DNA recombinant technology, mutant strains selected by ultraviolet irradiation or culture in a drug-containing medium are preferable in that they do not require the cost of containment equipment to prevent the spread of yeast or the disposal of waste after production.

[0015] The mutant strains listed above can be produced according to known methods. For example, ultraviolet treatment methods for producing yeast mutant strains are known, and bacterial cells can be irradiated with ultraviolet light using an ultraviolet light irradiation device for microbial breeding. The ultraviolet light irradiation dose is preferably set to a level that allows approximately 10% of the target bacterial strain to survive. Specifically, in a clean bench, a glass petri dish with an outer diameter of 10 cm is placed in a cell culture medium until the cell turbidity reaches OD . 660 Cells can be added so that the pH becomes 1.0, and ultraviolet light can be irradiated from a germicidal lamp (GL-15, manufactured by Hotalux Co., Ltd., wavelength = 250 nm) at a distance of 20 cm for 5 minutes.

[0016] Furthermore, a method for producing a mutant strain using a drug-containing medium can be carried out, for example, as follows. 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. Examples of acetaldehyde inhibitors include, but are not limited to, disulfiram (DSF). The concentration of the acetaldehyde inhibitor in the medium can be appropriately set depending on conditions such as the drug used. When DSF is used as the acetaldehyde inhibitor, the concentration can be, for example, in the range of 0.1 μM to 10 μM, but is not limited to the range. 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 5 μM to 50 μM for MCZ. Examples of growth inhibitors include, but are not limited to, 2-deoxyglucose (2-DG), a glucose analog. The concentration of the growth inhibitor in the medium can be appropriately determined depending on the conditions, such as the drug used. When 2-DG is used as the growth inhibitor, the concentration can 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 cell membrane permeability may also be used. The medium to which the agent is added may be any known medium suitable for culturing yeast, preferably containing cellobiose, such as, but not limited to, a mixed medium containing 0.17% yeast nitrogen base (amino acid-free), 0.08% ammonium sulfate, 0.5% yeast extract, and 3% cellobiose, to which the desired agent can be added at a desired concentration.

[0017] The method of selecting mutant strains using a drug-containing medium involves culturing the bacterial cells in the drug-containing medium for a certain period of time under culture conditions that include, but are not limited to, 30 to 41°C for 48 to 60 hours.

[0018] It is also preferable to perform long-term subculture using a cellobiose-containing medium after ultraviolet irradiation treatment or culture using a drug-containing medium. Long-term subculture allows mutations adapted to cellobiose degradation or cellobiose assimilation to be established. Examples of subculture are not limited as long as they allow mutations to be established. For example, the cultured mutant strain can be plated on a cellobiose-containing medium and subcultured at 37°C at 24-hour, 48-hour, 74-hour, or 96-hour intervals for 240 to 432 hours.

[0019] Mutant strains that have undergone mutation treatment such as ultraviolet irradiation or culture in a drug-containing medium, or that have undergone long-term culture after mutation treatment, can be selected by evaluating the size of colonies formed by plate culture, growth ability, cellobiose-degrading ability, or ethanol production ability.

[0020] The step of isolating mutant strains by ultraviolet irradiation or the step 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.

[0021] In one embodiment, the mutant strain of the present invention is a thermotolerant mutant strain. As used herein, "thermostable" means that it can grow at a temperature of at least 40°C. "Able to grow at a temperature 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, this means that the mutant strain can grow at 40°C, preferably 43°C, and more preferably 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.

[0022] To obtain a mutant strain that has the ability to decompose cellobiose and can grow at temperatures of at least 40°C, a thermotolerant strain capable of growing at temperatures of at least 40°C can be used as a parent strain for breeding to obtain a mutant strain with cellobiose decomposition ability. Known thermotolerant strains capable of growing at temperatures of at least 40°C can be used, for example, Kluyveromyces marxianus DMKU3-1042.

[0023] 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 irradiating the Kluyveromyces marxianus DMKU3-1042 strain with ultraviolet light and culturing it in a drug-containing medium containing at least one of an acetaldehyde inhibitor, an erigosterol synthesis inhibitor, or a growth retardant. The Kluyveromyces marxianus DMKU3-1042 strain is a thermotolerant yeast strain disclosed in Japanese Patent No. 5051727 (Accession Number: 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 SY13-1-4 strain, SY15-2-12 strain, SY15-1-43 strain, and SY15-1-19 strain are, respectively, deposited under accession numbers NITE P-03284, NITE P-03286, NITE P-03285, and NITE P-03253 at the Patent Microorganisms Deposit Center (NITE, Independent Administrative Institution, National Institute of Technology and Evaluation, Room 122, Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on August 5, 2020, or September 15, 2020, and are available for distribution under certain conditions.

[0024] In another aspect, the present invention provides a composition for decomposing cellobiose, which comprises the mutant strain of the present invention or a lysate thereof. The cellobiose-degrading composition of the present invention can be used for the degradation of cellobiose. In one embodiment, the cellobiose-degrading composition of the present invention can contain a lysate of the mutant strain of the present invention, thereby containing a cellobiose-degrading enzyme derived from the mutant strain of the present invention. The lysis method for preparing the lysate is not particularly limited, and examples thereof include an enzymatic hydrolysis method in which an enzyme preparation obtained from a microorganism or plant is added to solubilize the lysate, a hydrolysis method in which an acid or alkali is used to solubilize the lysate, and a method in which the cell wall is physically broken down by ultrasonication or the like to release the cellobiose-degrading enzyme from the bacterial cells. The cellobiose-decomposing composition of the present invention may further contain additives such as a culture medium for maintaining the mutant strain and the enzyme, and a stabilizer.

[0025] Another aspect of the present invention provides a method for decomposing cellobiose, which uses the mutant strain of the present invention or the 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-decomposing ability of the present invention or the composition for decomposing cellobiose of the present invention is used. Furthermore, 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-degrading ability or the composition for cellobiose degradation of the present invention may be added to a solvent containing cellobiose, followed by culturing or reaction for a certain period of time. The solvent is not limited as long as it allows the mutant strain or the composition for cellobiose degradation to decompose cellobiose. When cellobiose is decomposed using the mutant strain, yeast culture media 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% L-amino acid-free yeast nitrogen base), and YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose) can 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, spinning culture, shaking and spinning culture, continuous culture, stationary culture, and combinations thereof, with shaking culture and spinning culture being preferred. Examples of the culture time include 1 to 10 days, preferably 2 to 7 days, and more preferably 2 to 3 days.

[0026] The glucose derived from cellobiose obtained in the above embodiment may be used as a raw material for combining with another fermentation process. Furthermore, the mutant strain of the present invention is also capable of cellobiose-utilization ethanol fermentation. Therefore, another aspect of the present invention provides a method for producing ethanol, comprising the 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 the step of saccharifying glucose from cellobiose using the mutant strain having the cellobiose-degrading ability of the present invention or the composition for decomposing cellobiose of the present invention. In this embodiment, the mutant strain of the present invention having the ability to decompose cellobiose can perform ethanol fermentation using glucose obtained by saccharification of cellobiose, thereby enabling ethanol production by culturing the mutant strain in a medium containing cellobiose. In one embodiment, a microorganism capable of glucose-utilizing ethanol fermentation may be further used to promote alcoholic fermentation to produce ethanol from glucose. Such microorganisms capable of glucose-utilizing ethanol fermentation can be known, including yeasts such as Saccharomyces and Kluyveromyces, and bacteria such as Zymomonas and Zymobacter. Preferred examples include thermotolerant yeasts (e.g., Kluyveromyces marxianus DMKU3-1042 strain) and thermotolerant Zymomonas mobilis.

[0027] 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 Bacillus bacteria such as Bacillus licheniformis can be used. When cellulose is saccharified into cellobiose using cellobiohydrolase or endoglucanase, a preferred embodiment is one in which cellulose is saccharified into cellobiose using cellobiohydrolase and endoglucanase.

[0028] In the method for producing ethanol according to the present invention, any conventionally known method can be used to recover the ethanol produced from the culture medium. For example, there can be mentioned a method in which a liquid layer containing ethanol is separated from a solid layer containing yeast and solid components by solid-liquid separation, and then the ethanol contained in the liquid layer is separated and purified by distillation to recover the ethanol. [Example]

[0029] (Identification of 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 grown in a clean bench in a glass petri dish with an outer diameter of 10 cm, under conditions where 10% of the heat-resistant yeast cells survived. 660Cells were added so that the β-actin concentration became 1.0, and the yeast was irradiated with UV light (GL-15, Hotalux, Inc., wavelength 250 nm) from a distance of 20 cm for 5 minutes (mutation treatment). The mutated yeast was then inoculated into 10 mL of cellobiose-containing medium (a mixture 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 and 160 rpm for 24 hours with shaking. The cultured mutant strain was then plated on the cellobiose-containing medium and cultured at 37°C for 48–72 hours. Large colonies (96–144) were selected from the resulting colonies, and a strain with high cellobiose consumption and ethanol production was further selected (SY5-1-17). The cellobiose and ethanol concentrations in the medium after culturing the mutant strain were measured using HPLC. Specifically, the culture medium was centrifuged (1,400 rpm, 1 minute), and the supernatant was filtered through a membrane filter (Nihon Pall Corporation) to obtain a test solution. The concentrations were then measured using high-performance liquid chromatography (Hitachi High-Technologies Corporation). The high-performance liquid chromatography analysis conditions for measuring the ethanol concentration in the medium were as follows: a Gelpack® GL-C610-S column (Hitachi High-Technologies Corporation), an oven temperature of 60°C, a flow rate in the column of 0.3 ml / min, and ion-exchanged water as the mobile phase.

[0030] Next, the SY5-1-17 strain was subjected to the same UV irradiation mutation treatment as above, followed by shaking and plate culture in the cellobiose-containing medium. A strain with high cellobiose consumption and ethanol production was selected (SY5-1-17-3-40 strain). The resulting SY5-1-17-3-40 strain was then subjected to the same UV irradiation mutation treatment as above, followed by long-term subculture in the cellobiose-containing medium (subculture at 48-hour intervals for 384 to 432 hours). 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 UV irradiation mutation treatment as above, followed by shaking and plate culture in cellobiose-containing medium additionally containing 1 μM DSF (disulfiram). By selecting the larger colonies from the colonies that appeared and then selecting strains with high cellobiose consumption and ethanol production, we finally succeeded in isolating a strain (SY13-1-4) that not only had the ability to decompose cellobiose but also the ability to utilize cellobiose to produce ethanol (Figure 2).

[0031] In order to obtain a strain with improved cellobiose-decomposing ability or cellobiose-utilizing ethanol-producing ability from the SY13-1-4 strain obtained above, the following procedures were carried out. The SY13-1-4 strain was further subjected to UV irradiation and then cultured on a cellobiose-containing medium containing 1 μM DSF and 0.1% 2DG (2-deoxyglucose) in a shaking culture and on a plate. By selecting the larger colonies from the resulting colonies and further selecting strains with high cellobiose consumption and ethanol production, we successfully isolated a strain (SY14-3-7) with improved cellobiose-degrading and cellobiose-utilizing ethanol production capabilities (Figure 2). Furthermore, we further mutated strain SY14-3-7 by UV irradiation. After mutagenesis, we performed shaker and plate cultures on three media: (i) cellobiose-containing medium supplemented with 1 μM DSF and 0.5% 2DG; (ii) cellobiose-containing medium supplemented with 1 μM DSF, 0.5% 2DG, and 4 μg CTZ; and (iii) cellobiose-containing medium supplemented with 10 μM DSF and 0.1% 2DG. By selecting larger colonies from the resulting colonies and further selecting strains with higher cellobiose consumption and ethanol production, we successfully isolated strains with improved cellobiose-degrading and cellobiose-utilizing ethanol production capabilities (strains SY15-2-12, SY15-1-43, and SY15-1-19) (Figure 2).

[0032] (Growth test, cellobiose degradation test, and ethanol production test) The thermotolerant mutant yeasts with cellobiose-degrading ability obtained as described above (SY13-1-4, SY14-3-7, SY15-2-12, SY15-1-43, and SY15-1-19) were cultured and assayed as follows to evaluate their growth, cellobiose-degrading, and ethanol production abilities. Kluyveromyces marxianus DMKU3-1042 (parent strain) was used as a control for the mutant strains. The culture and assay for the growth test were performed independently of the culture and assay for the cellobiose differentiation and ethanol production tests. One loopful of 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, and the mixture was cultured at 37°C for 18 hours with shaking (160 rpm) to obtain a preculture solution. Next, the mixture was added to 30 ml of cellobiose-containing medium (cellobiose content: approximately 3-4% (w / v)) in an Erlenmeyer flask until the OD 660 The preculture solution was transferred so that the β-glucan content became 0.1, and the mixture was cultured at 40°C with shaking (160 rpm).

[0033] The growth of the thermotolerant mutant yeast and Kluyveromyces marxianus DMKU3-1042 capable of differentiating from cellobiose was measured 12, 24, 36, and 48 hours after the start of cultivation, as well as the cellobiose and ethanol concentrations in the medium. The growth of the thermotolerant mutant yeast and Kluyveromyces marxianus DMKU3-1042 capable of differentiating from cellobiose was measured by the degree of turbidity (OD) of the culture medium. 660 ) was measured using 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 medium (1,400 rpm, 1 minute), filtering the supernatant through a membrane filter (manufactured by Nippon Pall Corporation) to obtain a test solution, and measuring it using 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 column (manufactured by Hitachi High-Technologies Corporation) 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.

[0034] The turbidity (OD) of the culture medium of the SY13-1-4 strain 660 The measurement results of the ethanol concentration, cellobiose concentration, and ethanol concentration are shown in FIG. The SY13-1-4 strain showed superior growth after approximately 10 hours of culture compared to the DMKU3-1042 strain. The SY13-1-4 strain also demonstrated cellobiose differentiation. On the other hand, the DMKU3-1042 strain did not substantially decompose cellobiose. Furthermore, the SY13-1-4 strain also produced ethanol. On the other hand, the DMKU3-1042 strain did not produce ethanol. These results indicate that the SY13-1-4 strain has superior growth ability compared to the DMKU3-1042 strain, and also has the ability to decompose cellobiose and utilize cellobiose to produce ethanol.

[0035] The turbidity (OD) of the culture medium of strain SY14-3-7 660 The results of measuring the OD, cellobiose concentration, and ethanol concentration are shown in Figure 4. In the graph of cell growth, the vertical axis is OD660 The vertical axis represents the cellobiose concentration in the medium (% w / v), and the horizontal axis represents time (hours). In the graph of cellobiose concentration in Figure 4, the vertical axis represents the cellobiose concentration in the medium (% w / v), and the horizontal axis represents time. In the graph of ethanol concentration, the vertical axis represents the ethanol concentration in the medium (% w / v), and the horizontal axis represents time. The vertical and horizontal axes in Figures 5 to 7 also have the same units. The SY14-3-7 strain showed superior growth compared to the DMKU3-1042 strain after approximately 10 hours of culture. The SY13-3-7 strain also demonstrated cellobiose differentiation. Its cellobiose degradation ability was improved compared to the SY13-1-4 strain. Furthermore, the SY14-3-7 strain also produced ethanol. These results indicate that the SY14-3-7 strain has superior growth ability compared to the DMKU3-1042 strain, and also has improved cellobiose degradation and cellobiose utilization ethanol production abilities compared to the SY13-1-4 strain.

[0036] The turbidity (OD) of the culture solution of the SY15-1-19, SY15-1-43, and SY15-2-12 strains was 660 The measurement results of the ethanol concentration, the cellobiose concentration, and the ethanol concentration are shown in FIGS. 5 to 7, respectively. Strains SY15-1-19, SY15-1-43, and SY15-2-12 all showed superior growth compared to strain DMKU3-1042 from the beginning of cultivation. Furthermore, strains SY15-1-19, SY15-1-43, and SY15-2-12 all exhibited cellobiose differentiation and showed improved cellobiose degradation abilities compared to strains SY13-1-4 and SY14-3-7. Furthermore, strains SY15-1-19, SY15-1-43, and SY15-2-12 all produced ethanol. Ethanol production was observed immediately after the start of cultivation, indicating superior ethanol production compared to strains SY13-1-4 and SY14-3-7. These results indicate that the SY15-1-19, SY15-1-43, and SY15-2-12 strains all have superior growth ability compared to the DMKU3-1042 strain, and furthermore have improved cellobiose degradation ability and cellobiose-utilization ethanol production ability compared to the SY13-1-4 and SY14-3-7 strains. [Industrial Applicability]

[0037] In recent years, technological development aimed at utilizing non-food cellulosic biomass has been conducted both domestically and internationally, but has not yet 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

1. A mutant strain obtained by subjecting Kluyveromyces marxianus DMKU3-1042 strain to ultraviolet irradiation and then culturing the strain in a cellobiose-containing medium without using DNA recombination technology, the mutant strain having an increased ability to decompose cellobiose compared to the Kluyveromyces marxianus DMKU3-1042 strain, The mutant strain can produce 1.5% (W / V) or more of ethanol when the mutant-containing culture solution is transferred to 100 ml of a mixed medium containing 0.17% yeast nitrogen base (containing no amino acids), 0.08% ammonium sulfate, 0.5% yeast extract, and 3% cellobiose so that the degree of suspension (OD) is 0.1, and the culture is subjected to shaking culture (160 rpm) at 40° C. for 48 hours.

2. The mutant strain according to claim 1, which is SY13-1-4 strain (accession number: NITEP-03284), SY15-2-12 strain (accession number: P-03286), SY15-1-43 strain (accession number: NITEP-03285), or SY15-1-19 strain (accession number: NITEP-03253).

3. A composition for decomposing cellobiose, comprising the mutant strain according to claim 1 or a lysate thereof.

4. 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.

5. 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. 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. 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.

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