WO2021153587A1

WO2021153587A1 - Filamentous fungus trichoderma mutant strain

Info

Publication number: WO2021153587A1
Application number: PCT/JP2021/002759
Authority: WO
Inventors: 拓也野口; 紳吾平松; 山田　勝成
Original assignee: 東レ株式会社
Priority date: 2020-01-28
Filing date: 2021-01-27
Publication date: 2021-08-05
Also published as: CN114981405A; JPWO2021153587A1

Abstract

Disclosed are: the acquisition of a mutant strain of filamentous fungus Trichoderma with which culture solution viscosity is reduced; and a method for producing a protein using the mutant strain of filamentous fungus Trichoderma. In the present invention, a mutant strain of filamentous fungus Trichoderma in which the activities of β-glucosidase and of β-xylosidase have been reduced is acquired, and this mutant strain is cultured to produce a protein. The mutant strain of filamentous fungus Trichoderma in which the β-glucosidase and of β-xylosidase activities have been reduced is capable of maintaining the culture solution viscosity at a lower level compared to the parent strain thereof in which the activities of the enzymes are preserved.

Description

Trichoderma filamentous fungus mutant strain

The present invention relates to a mutant strain of Trichoderma filamentous fungus that can keep the viscosity of the culture solution low.

Trichoderma filamentous fungi are known to have high protein production ability, and studies on protein production using Trichoderma filamentous fungi have been conducted. Trichoderma filamentous fungi use cellulose, lactose, cellobiose and the like as inducers to produce cellulase, which is classified as a saccharifying enzyme among proteins. In order to enhance the amount of cellulase produced, many studies have been conducted for a long time, such as overexpression of factors controlling cellulase production, modification of genes such as deficiency, and optimization of culture conditions.

On the other hand, Trichoderma filamentous fungi belong to aerobic filamentous fungi that require oxygen for growth and protein production. Further, when Trichoderma filamentous fungi are cultured in a liquid medium, the viscosity of the culture solution increases as the culture grows. As the viscosity of the culture solution increases, the distribution of oxygen and nutrients becomes non-uniform. It needs to be kept constant. When the culture tank becomes large, a huge amount of stirring power is required to keep the culture environment constant, and there is a problem that strong stirring causes a large shear damage to the cells.

In Patent Documents 1 to 6, the culture environment is set at a lower stirring rate as compared with the parent strain by reducing the disruption or production of the proteins Sfb3, Mpg1, Gas1, Seb1, Crz1 and Tps1 of the viscous Trichoderma filamentous fungi, respectively. It is disclosed that it will be possible to maintain.

Further, Patent Document 7 describes that disruption of the BXL1 gene of Trichoderma filamentous fungus can suppress a decrease in the dissolved oxygen saturation of the culture solution.

Special Table 2013-533751 Japanese Patent Application Laid-Open No. 2014-513259 Japanese Patent Publication No. 2014-513530 Japanese Patent Application Laid-Open No. 2014-513331 Japanese Patent Application Laid-Open No. 2014-513532 Japanese Patent Publication No. 2014-513533 International Publication No. 2017/170917

As mentioned above, when producing proteins using Trichoderma filamentous fungi, it is very important to suppress the decrease in dissolved oxygen concentration in the culture solution and keep it above a certain level. If the viscosity of the culture solution can be kept low during the production of protein by liquid culture using Trichoderma filamentous fungi, the present inventors can reduce the energy required for stirring even when the culture scale is increased. At the same time, it was considered that the decrease in dissolved oxygen saturation in the culture solution could be suppressed. An object of the present invention is to obtain a mutant strain of Trichoderma filamentous fungus in which the viscosity of a culture solution is lowered and to provide a method for producing a protein using the mutant strain of Trichoderma filamentous fungus.

As a result of diligent studies on the selection of Trichoderma filamentous fungi that can keep the viscosity of the culture solution low, the present inventor has reduced the activities of β-glucosidase and β-xylocidase from the parent strain before the introduction of the mutation. By culturing a mutant strain of the bacterium, it was found that the viscosity of the culture solution was kept low, and the present invention was completed.

That is, the present invention provides the following.
(1) A mutant strain of Trichoderma filamentous fungus in which the activities of β-glucosidase and β-xylosidase were lower than those of the parent strain before the introduction of the mutation.
(2) The mutant strain according to (1), wherein the specific activity of β-glucosidase is 0.02 U / mg-protein or less, and the specific activity of β-xylosidase is 0.002 U / mg-protein or less.
(3) The mutant strain according to (1) or (2), wherein the Trichoderma filamentous fungus is Trichoderma lysei.
(4) A method for producing a protein by culturing the mutant strain according to any one of (1) to (3).
(5) The production method according to (4), wherein the protein is cellulase.
(6) A method for producing a xylooligosaccharide, which hydrolyzes biomass containing xylan with cellulase produced by the method according to (5).
(7) A method for reducing the viscosity of a culture solution during liquid culture of Trichoderma filamentous fungi, which comprises reducing the β-glucosidase activity and β-xylosidase activity of Trichoderma filamentous fungi.

According to the present invention, a Trichoderma filamentous fungus mutant strain in which the activity of β-glucosidase and β-xylosidase is lower than that of the parent strain before the introduction of the mutation keeps the viscosity of the culture solution lower than that of the parent strain having the activity of the enzyme. It becomes possible.

The trichoderma filamentous fungus in the present invention is not particularly limited as long as it belongs to the genus Trichoderma and has the ability to produce cellulase. It is preferably Trichoderma reesei. In addition, a mutant strain derived from the genus Trichoderma, which has been subjected to mutation treatment with a mutant agent or ultraviolet irradiation, or which has improved cellulase productivity, may be used as the parent strain.

Specific examples of the mutant strain used as the parent strain include Trichoderma paralyse (ATCC MYA-4777), which is the ancestor of Trichoderma lysei, QM6a strain (NBRC31326), QM9123 strain (ATCC24449), and QM9414 strain, which are known mutant strains derived from Trichoderma lysei. (NBRC31329), PC-3-7 strain (ATCC66589), QM9123 strain (NBRC31327), RutC-30 strain (ATCC56765), CL-847 strain (Enzyme.Microbiol.Technol.10,341-346 (1988)), MCG77 Examples thereof include a strain (Biotechnol. Bioeng. Symp. 8, 89 (1978)), an MCG80 strain (Biotechnol. Bioeng. 12, 451-459 (1982)) and derivative strains thereof.

Further, it is preferable that the Trichoderma filamentous fungus used in the present invention has the carbon catabolite repression released. Strains from which carbon catabolite repression has been released can produce more proteins due to the increased production of proteins such as cellulase. More preferably, it is a strain in which the carbon catabolite repression formed via the carbon catabolite repressor I is released. For example, when the carbon catabolite repressor I gene (cre1) is mutated, the carbon catabolite repression made via the carbon catabolite repressor I is released. The CRE1 protein encoded by the cre1 gene is known to suppress the expression of the cellulase gene by catabolite suppression due to glucose (FEBS Lett., 376, 103-107, 1995). Therefore, when the cr1 gene is mutated, the suppression of cellulase gene expression is released and the amount of cellulase produced increases. Therefore, a strain in which the cre1 gene is mutated is more suitable for producing a protein or cellulase composition. As a specific example of mutation of the cre1 gene, in the cre1 gene of the PC-3-7 strain (ATCC66589), the 232nd A is replaced with C, and as a result, the 78th threonine of the amino acid sequence is replaced with proline. It can be mentioned that. It is known that the inclusion of this mutation improves the production of cellulase (Bioscii. Biotechnol. Biochem., 77 (3), 534-543, 2013). Further, it is known that the cr1 gene is partially cleaved in the RutC-30 strain (ATCC56765) and the carbon catabolite repression is released (BMC Genomics., 9, 327, 2008). Here, a strain containing a mutation in the cre1 gene is a frame shift due to deletion or insertion of a base in the cre1 gene region due to a gene mutation agent, ultraviolet irradiation, etc., or a stop codon mutation due to base substitution, or a base. A strain containing a cleavage is included, and a strain in which all or part of the cre1 gene is removed or replaced with another gene by recombination or the like is also included. Specifically, a strain that inherits the characteristics of the PC-3-7 strain (ATCC66589), the RutC-30 strain (ATCC56765), the PC-3-7 strain (ATCC66589), and the RutC-30 strain (ATCC56765) is preferably used. More preferably, it is a strain that inherits the characteristics of the PC-3-7 strain (ATCC66589) or the PC-3-7 strain (ATCC66589). Here, the strain that inherited the characteristics of the PC-3-7 (ATCC66589) and RutC-30 strain (ATCC56765) includes the strain that inherited the characteristics of the PC-3-7 strain (ATCC66589) and the RutC-30 strain (ATCC56765). Includes strains that have been newly mutated and strains whose functions have been improved by recombination.

The QM6a strain, QM9414 strain, and QM9123 strain can be obtained from NBRC (NITE Biological Resource Center), and the PC-3-7 strain and RutC-30 strain can be obtained from ATCC (American Type Culture Collection).

In the present invention, "a mutant strain of Trichoderma filamentous fungus in which the activity of β-glucosidase and β-xylocidase is lower than that of the parent strain before the introduction of the mutation" is defined by introducing a mutation into the parent strain of Trichoderma filamentous fungus which is the above-mentioned parent strain. , Β-Glucosidase and β-xylosidase are referred to as mutant strains in which the activity of β-glucosidase and β-xylosidase is lower than that of the parent strain before the introduction of the mutation, and may be referred to as “mutant strain of the present invention” in the present specification.

In the present invention, the decrease in the activity of β-glucosidase and β-xylosidase means that the specific activity of β-glucosidase and the specific activity of β-xylosidase are decreased as compared with the parent strain before the mutation was introduced. It also includes a state in which the function of β-glucosidase and / or β-xylosidase is deficient.

Specifically, the β-glucosidase specific activity and the β-xylosidase specific activity are preferably reduced to 1/10 or less, more preferably 1/50 or less, respectively, as compared with the parent strain. It is preferably reduced to 1/100 or less, more preferably 1/200 or less, further preferably 1/500 or less, particularly preferably 1/800 or less, and most preferably 1/1000 or less.

As the absolute value of the specific activity, the β-glucosidase specific activity and the β-xylocidase specific activity are preferably 0.02 U / mg-protein or less and 0.002 U / mg-protein or less, more preferably 0.01 U. / Mg-protein or less and 0.001 U / mg-protein or less, more preferably 0.005 U / mg-protein or less and 0.0005 U / mg-protein or less, still more preferably 0.0025 U / mg-protein or less and , 0.00025 U / mg-protein or less.

The method for introducing a mutation that reduces the specific activity of β-glucosidase and the specific activity of β-xylosidase into the parent strain of Trichoderma filamentous fungus is not particularly limited, but the gene mutation treatment by a gene mutagen, ultraviolet irradiation, or the like is not particularly limited. Examples thereof include a method and a site-specific mutation method. In addition, the BGL1 gene or BXL1 gene possessed by the parent strain of Trichoderma filamentous fungus may be disrupted. Specifically, mutations such as mutations, insertions, and deletions are introduced into both of these genes, the promoter region, or the gene encoding the transcriptional regulatory factor of the gene to reduce β-glucosidase and β-xylosidase activities. You may let me. Furthermore, it can be performed by introducing a frame shift mutation into both of the above genes or by inserting a stop codon, and by gene recombination (homologous recombination with another gene, etc.), the gene or promoter region. Or, the BGL1 gene and / or the BXL1 gene can be disrupted by removing or replacing all or part of the gene encoding the transcriptional regulatory factor of the gene with another gene. Alternatively, the expression of the BGL1 gene and / or the BXL1 gene is inhibited by removing all or part of the binding recognition sequence of the transcriptional regulatory factor located upstream of the BGL1 gene and / or the BXL1 gene or replacing it with another gene. be able to. Since the BGL1 gene (Gene ID; 18488646) of Trichoderma filamentous fungus and the filamentous fungus BXL1 gene (Gene ID; 18483060) are known, disruption of these genes can be carried out by the above-mentioned gene mutation introduction treatment method, site-specific mutation method, or the like. It can be easily done by the conventional method of. The binding sites of transcriptional regulators are present at 1 to 500 bases upstream of the BGL1 gene and the BGL1 gene of Trichoderma filamentous fungi. Specific examples of transcriptional regulators include Xyr1, and the putative binding recognition sequence of Xyr1 in Trichoderma filamentous fungi is Borin, Carazzole. "Gene Co-expression Network Revels Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30" Biogen. Biotechnol. , 2018, 6, doi. org / 10.3389 / fbioe. It is disclosed in 2018.00151. In the case of a random mutation introduction method such as gene mutagenesis or ultraviolet irradiation, each strain after the mutation treatment is cloned, β-glucosidase activity and β-xylosidase activity are measured by the method described later, and these activities are measured. By selecting a strain in which the specific activity of β-glucosidase is lower than that of the parent strain before the introduction of the mutation, a strain in which the specific activity of β-glucosidase and the specific activity of β-xylosidase are lower than those of the parent strain before the introduction of the mutation can be obtained.

The specific activity of β-glucosidase and β-xylosidase of Trichoderma filamentous fungi are measured by the following methods.

First, the culture solution obtained by culturing Trichoderma filamentous fungi is centrifuged at 20,000 xg for 10 minutes, and the supernatant is collected. The recovered supernatant is diluted to an appropriate concentration to prepare an enzyme diluent, and the specific activity of the enzyme is measured by the following method.

The β-glucosidase specific activity is measured by the following method. First, 10 μL of an enzyme diluent is added to 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside (manufactured by Sigma-Aldrich Japan) and reacted at 30 ° C. for 10 minutes. Next, 10 μL of 2M sodium carbonate is added and mixed well to stop the reaction, and the increase in absorbance at 405 nm is measured. Finally, the specific activity is calculated assuming that the activity of liberating 1 μmol of p-nitrophenol per minute is 1 U.

The β-xylosidase specific activity is measured by the following method. First, 10 μL of an enzyme diluent is added to 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside (manufactured by Sigma-Aldrich Japan) and reacted at 30 ° C. for 30 minutes. Next, 10 μL of 2M sodium carbonate is added and mixed well to stop the reaction, and the increase in absorbance at 405 nm is measured. Finally, the specific activity is calculated assuming that the activity of liberating 1 μmol of p-nitrophenol per minute is 1 U.

The protein concentration required to calculate the specific activity is measured as follows. Add 5 μL of the enzyme diluent to 250 μL of the Quick Start Bloodford protein assay (manufactured by Bio-Rad), and measure the absorbance used at 595 nm after standing at room temperature for 15 minutes. Using bovine serum albumin solution as a standard solution, calculate the protein concentration contained in the saccharifying enzyme solution based on the calibration curve.

The "variant strain of Trichoderma filamentous fungus in which β-glucosidase and β-xylocidase activity is lower than that of the parent strain before the introduction of the mutation" of the present invention has a lower viscosity of the culture solution than the parent strain. As a result, the energy required for aeration and agitation and the number of rotations can be reduced. Further, since the rotation speed of stirring can be set low, shear damage to the hyphae can be reduced. In particular, when culturing on a large scale, it is more effective because it leads to reduction of the capacity of the blower and the stirring motor required for ventilation and the stirring energy.

The culturing method is not particularly limited as long as it is a method capable of culturing Trichoderma filamentous fungi to produce a protein, and can be carried out by a well-known method using a well-known medium. For example, it can be cultured in a liquid culture using a centrifuge tube, a flask, a jar fermenter, a tank, or the like. Since Trichoderma lysei is an aerobic microorganism, among these culture methods, a jar fermenter or a deep culture in which the culture is carried out while aeration or stirring is performed in a tank is particularly preferable. The air volume is preferably about 0.1 vvm to 2.0 vvm, more preferably 0.3 vvm to 1.5 vvm, and particularly preferably 0.5 vvm to 1.0 vvm. The culture temperature is preferably about 25 ° C. to 35 ° C., more preferably 25 ° C. to 31 ° C. The pH conditions in the culture are preferably pH 3.0 to 7.0, and more preferably pH 4.0 to 6.0. Incubation time is carried out under the condition that protein is produced until a recoverable amount of protein is accumulated. Usually, it is about 24 to 288 hours, more preferably 36 to 240 hours.

In the present invention, the viscosity of the culture solution uses the values measured under the following conditions, and the viscosity comparison is made by comparing the maximum values among the values measured under the following conditions. First, to be a pre-culture medium 1.0 × 10 ⁵ spores per 1mL spores Trichoderma filamentous fungus to be evaluated pre-culture medium (an example of a specific medium composition, shown in Table 1 in the Examples) Inoculate to and incubate in a shaking incubator under the conditions of 28 ° C. and 120 rpm until the amount of bacterial cells reaches about 11 g / L. Next, 10% (v / v /) of the main culture medium shown in Table 2 to which Arbocel B800 (trade name, cellulose fiber, manufactured by Rettenmeier) and bagasse powder were added so as to be 100 g / L (w / v). Inoculate the pre-culture medium so that it becomes v), and perform deep culture using a 5 L jar fermenter. As for the culture conditions, after inoculating the preculture medium into the main culture medium, deep culture is performed under the culture conditions of 28 ° C., 700 rpm, and an aeration rate of 100 mL / min while controlling the pH to 5.0. A digital rotational viscometer is used to measure the viscosity of the culture solution. The digital rotational viscometer performs zero-point calibration in advance. About 20 ml of the culture solution from the start of the culture to the time when 96 hours have passed is collected over time. Immediately after collection, put the culture solution in the designated container, immerse the spindle in the culture solution, rotate it at a rotation speed of 0.3 rpm, and measure the torque, which is the viscous resistance acting on the spindle at this time, under room temperature conditions. By doing so, the viscosity of the culture solution is measured. The unit of viscosity is centipores (cP). One centimeter pores is defined as the viscosity at which when there is a velocity gradient of 1 cm / sec per cm in a fluid, a stress of the magnitude of a force of 1 dyne per ^{cm 2 of the direction of velocity occurs in a plane perpendicular to the direction of the velocity gradient.} NS. DV2T (BROOKFIELD) can be used for the digital rotational viscometer, ULA (BROOKFIELD) or the like can be used for the spindle.

In the "mutant strain in which β-glucosidase and β-xylosidase activity was lower than that in the parent strain before the introduction of the mutation" of the present invention, the viscosity of the culture solution was lower than that in the case where the parent strain was cultured under the same conditions, and the culture solution was in culture. The maximum value of the viscosity is preferably 70% or less of the parent strain, more preferably 60% or less, and further preferably 50% or less. In terms of absolute value, the maximum value of the viscosity of the mutant strain of the present invention in culture is preferably 50 cP or more, more preferably 100 cP or more, more preferably 150 cP or more, more preferably 200 cP or more, and further, as compared with the parent strain. It is preferably 250 cP or more, more preferably 300 cP or more, still more preferably 350 cP or more, still more preferably 400 cP or more, and particularly preferably 500 cP or more.

The protein produced in the present invention is not particularly limited, but a protein secreted outside the cells can be efficiently produced, and among them, an enzyme is preferable, and cellulase, amylase, invertase, chitinase, and pectinase are more preferable. Etc., and more preferably cellulase.

The cellulase produced in the present invention contains various hydrolases, such as enzymes having degrading activity against xylan, cellulose, and hemicellulose. Specific examples of the hydrolyzing enzyme generally contained in cellulase include cellobiose hydrase (EC 3.21.91), which produces cellobiose by hydrolyzing a cellulose chain, and hydrolyzes from the central portion of the cellulose chain. Endoglucanase (EC 3.2.1.4), β-glucosidase (EC 3.2.1.21) that hydrolyzes cellooligosaccharides and cellobiose, xylanase (EC) characterized by acting on hemicellulose and especially xylan. 3.21.8), β-xylossidase (EC 3.21.37) that hydrolyzes xylooligosaccharide, and the like. The protein concentration of cellulase may be calculated by the method described above.

The culture method for culturing the mutant strain of the present invention is not particularly limited, and for example, it can be cultured in a liquid culture using a centrifuge tube, a flask, a jar fermenter, a tank, or the like. Since Trichoderma filamentous fungi are aerobic microorganisms, among these culture methods, a jar fermenter or a deep culture in which the cells are cultured while being aerated or stirred in a tank is particularly preferable.

The medium composition at the time of culturing is not particularly limited as long as the medium composition is such that Trichoderma filamentous fungi can produce proteins, and a well-known medium composition of Trichoderma filamentous fungi can be adopted. As the nitrogen source, for example, polypeptone, gravy, corn steep liquor (CSL), soybean meal and the like can be used. In addition, an inducer for producing a protein may be added to the medium. As carbon sources, sugars such as glucose, sucrose, fructose, galactose, and lactose, starch saccharified liquid containing these sugars, molasses, sugar beet molasses, high test molasses, organic acids such as acetic acid, ethanol, etc. Alcohols, glycerin and the like can be used. In addition to these, especially when producing cellulase, it is preferable to use an inducer described later as a carbon source.

When cellulase is produced according to the present invention, it can be cultured in a medium containing at least one or two or more inducers selected from the group consisting of lactose, cellulose and xylan. Further, as the cellulose or xylan, biomass containing cellulose or xylan may be added as an inducer. Specific examples of the biomass containing cellulose and xylan include plants such as seed plants, fern plants, moss plants, algae, and aquatic plants, as well as waste building materials. Seed plants are classified into gymnosperms and angiosperms, both of which can be preferably used. Angiosperms are further classified into monocotyledonous plants and dicotyledonous plants, and specific examples of monocotyledonous plants include bagus, switchgrass, napiergrass, elianthus, corn stover, corn cob, rice straw, and straw. As specific examples of dicotyledonous plants, beet pulp, eucalyptus, nara, white birch and the like are preferably used.

Further, as the biomass containing cellulose and xylan, pretreated biomass may be used. The pretreatment method is not particularly limited, but known methods such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, pulverization treatment, and steaming treatment can be used. Pulp containing xylan may be used as the biomass containing such pretreated cellulose and xylan.

In addition, when the mutant strain of the present invention is used as a protein lysate without removing the cells from the culture solution in which the mutant strain of the present invention is cultured, it is possible to treat the mutant strain of the present invention so that it cannot grow in the culture solution. preferable. Examples of the method for treating the cells so that they cannot grow include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment.

When the protein is an enzyme, the culture solution treated as described above so that the cells are not removed or grown can be used as it is as the enzyme solution.

The method of using the cellulase composition produced in the present invention is not particularly limited, but it is preferably used for producing sugar. More preferably it is used in the production of xylobiose and / or cellobiose, and even more preferably it is used in the production of xylobiose and / or cellobiose.

The xylooligosaccharide in the present invention refers to a xylooligosaccharide in which at least two or more xyloses are linked by a β-glycosidic bond. The degree of polymerization of the xylooligosaccharide is not particularly limited, but it is preferably a disaccharide (xylobiose) to a hexasaccharide (xylohexaose) having high water solubility. Most preferably, it contains xylobiose, xylobiose, and xylotetraose, which are easily assimilated as a carbon source by the intestinal bacteria.

The cellooligosaccharide in the present invention refers to a cellooligosaccharide in which at least two or more glucoses are linked by a β-glycosidic bond. The degree of polymerization of the cellooligosaccharide is not particularly limited, but it is preferably a disaccharide (cellobiose) to a hexasaccharide (cellobiose) having high water solubility. Most preferably, it contains cellobiose, cellotriose, and cellotetraose, which are easily assimilated as a carbon source by the intestinal bacteria.

In the present invention, the cellulase composition is obtained by culturing Trichoderma filamentous fungi and is used for the saccharification reaction of biomass. The method for preparing the cellulase composition is not particularly limited, but it is preferable that the cells of Trichoderma filamentous fungi contained in the culture solution are not removed or grown. This is to prevent glucose and xylooligosaccharides produced during the saccharification reaction between the cellulase composition and the biomass from being consumed by the cells. Examples of the method for removing the bacterial cells include centrifugation and membrane separation. Examples of the treatment method for preventing the growth of bacterial cells include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment.

The method for producing sugar using the cellulase composition obtained by culturing Trichoderma filamentous fungi is not particularly limited, but the biomass can be saccharified with the cellulase composition. Biomass containing cellulose and / or xylan can be used as the biomass used for the saccharification reaction. Further, the cellulose and / or biomass used in the saccharification reaction may be pretreated in advance. The pretreatment method is not particularly limited, but specifically, known methods such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, pulverization treatment, and steaming treatment can be used. can. The reaction pH is also not particularly limited, but the pH is preferably around 3 to 7, more preferably around 4 to 6, and even more preferably around 5. The reaction temperature is also not particularly limited, but is preferably 40 to 70 degrees.

By the above-mentioned saccharification reaction, xylobiose from the biomass containing xylose, preferably xylobiose, cellobiose from the biomass containing cellulose, preferably cellobiose, xylobiose and cellooligosaccharide from the biomass containing cellulose and xylose, preferably xylobiose. You can get cellobiose. In addition to xylooligosaccharides and cellooligosaccharides, the sugars obtained by the above-mentioned saccharification reaction include monosaccharides such as mannose, arabinose, and galactose produced by hydrolases contained in the cellulase composition, and cellotriose and cellotetraose. Oligosaccharides such as mannose and galactose may be contained.

The post-reaction liquid produced from the saccharification reaction of the present invention may also contain inorganic salts, amino acids, proteins, lignin and the like as impurities, and in order to remove these impurities, a purification operation is performed. May be good. As the purification operation, known methods such as ion exchange, membrane separation, crystallization, and desalting can be applied.

It is preferable that the monosaccharide fraction (glucose, xylose, etc.) and the oligosaccharide fraction (xylooligosaccharide, cellooligosaccharide, etc.) produced by the present invention are separated by a subsequent step. Glucose is preferably used as a fermentation raw material in the production of chemical products, and xylooligosaccharide is preferably used in feed, food and cosmetic applications. Specific examples of chemical products include alcohols such as ethanol, 1,3-propanediol, 1,4-butanediol and glycerol, and organic substances such as acetic acid, lactic acid, pyruvate, succinic acid, malic acid, itaconic acid and citric acid. Examples thereof include nucleosides such as acid, inosin and guanosine, nucleotides such as inosic acid and guanylate, and amine compounds such as cadaberin. On the other hand, xylose has a limited number of microorganisms that can be used as a fermentation raw material. Furthermore, when xylose is given to pigs as feed, about half of it is excreted as urine. Therefore, it is preferable to minimize the decomposition of xylan and xylooligosaccharides into xylose and improve the yield of xylooligosaccharides.

In the present invention, the method for separating the monosaccharide fraction and the oligosaccharide fraction is not particularly limited, and a known method can be used. For example, membrane separation as described in WO 2017/11975 is preferably used. At this time, the smaller the proportion of xylose, the lower the amount of xylose mixed in the oligosaccharide fraction, which is advantageous in the membrane separation step.

The present invention will be specifically described below with reference to examples.

<Reference example 1> Culturing of Trichoderma filamentous fungi (preculture)
The spores of various trichodermarysei mutants ^{were diluted with physiological saline to 1.0 × 10 7} / mL, and 2.5 mL of the diluted spore solution was placed in a flask with a 1 L baffle shown in Table 1 in 250 mL. The culture medium was inoculated and cultured in a shaking incubator at 28 ° C. and 120 rpm for 72 hours. As a control, Trichoderma lysei PC-3-7 strain was used, and the same experimental operation was performed.

* 1 Mandels is 7g / L (NH ₄ ) ₂ SO ₄ , 10g / L KH ₂ PO ₄ , 3g /
L containing _{_{CaCl 2, 3g / L MgSO 4}} · 7H 2 O ( hereinafter the same).
* 2 trace element _{_{solution, 0.3g / L H 3 BO 3}} , 1.3g / L (NH 4) 6 Mo 7 O 24 · 4H 2 O, 5g / L FeCl 3 · 6H 2 O, 2g / L CuSO _{_{_{_{4 · 5H 2 O, 0.4g /}}}} L MnCl 2 · 4H 2 O, 10g / L containing ZnCl ₂ (hereinafter the same).

(Main culture)
Arbocel B800 (trade name, Rettenmeier Co., Ltd., powdered cellulose) was added to the main culture medium shown in Table 2, and a deep culture study was conducted using a 5 L jar fermenter (manufactured by Biot Co., Ltd.).

250 mL of pre-culture solution of Trichoderma Risei PC-3-7 strain and various Trichoderma Risei mutant strains was inoculated into 2.5 L of the main culture medium to which Arbocel B800 (trade name) was added.

As for the culture conditions, after inoculating the preculture medium into the main culture medium, deep culture was performed under the culture conditions of 28 ° C., 700 rpm, and an aeration rate of 100 mL / min while controlling the pH to 5.0.

(Collecting culture medium)
From the start of the culture to 74 hours after the end of the culture, 20 mL of the culture solution was collected over time. Of the collected culture broth, 16 mL was used for measuring the viscosity described later. The remaining culture broth was centrifuged under the conditions of 20,000 × g and 4 ° C. for 10 minutes to obtain a supernatant. The supernatant was filtered through a 0.22 μm filter, and the filtrate was used as a cellulase solution for the subsequent saccharification test.

<Reference Example 2> Measurement of Viscosity of Culture Solution 16 mL of the culture solution after 0, 24, 48, and 74 hours from the start of culture was used with a digital rotational viscometer DV2T and a spindle ULA (manufactured by BROOKFIELD) to set the rotation speed to 0. The viscosity (cP) when set to 3 rpm was determined.

<Reference Example 3> Protein concentration measurement conditions Protein concentration measurement reagent used: Quick Start Blade protein assay, Bio-Rad measurement conditions Measurement temperature: Room temperature Protein concentration measurement reagent: 250 μL
Filamentous fungus culture: 5 μL
Reaction time: 5 minutes Absorbance: 595 nm
Standard product: BSA.

<Reference Example 4> Measurement conditions for cellulase specific activity (Measurement conditions for β-glucosidase specific activity)
Substrate: p-nitrophenyl-β-glucopyranoside (manufactured by Sigma-Aldrich Japan)
Reaction: 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside
Enzyme diluent: 10 μL
Reaction temperature: 30 ° C
Reaction time: 10 minutes Reaction terminator: 2M sodium carbonate 10 μL
Absorption: 405 nm.

(Measurement conditions for β-xylosidase specific activity)
Substrate: p-nitrophenyl-β-xylopyranoside (manufactured by Sigma-Aldrich Japan)
Reaction: 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside
Enzyme diluent: 10 μL
Reaction temperature: 30 ° C
Reaction time: 30 minutes Reaction terminator: 2M sodium carbonate 10 μL
Absorption: 405 nm.

<Reference Example 5> Amount of saccharification test enzyme solution used: 8 mg / g (amount of enzyme used per 1 g of biomass)
Biomass: Arbocel B800 (trade name, Rettenmeier), bagasse powder biomass charge: 5% (w / v)
Reaction temperature: 50 ° C
Reaction time: 24 hours Reaction pH: 5.0
The reaction solution was centrifuged under the condition of 8,000 × g for 5 minutes to obtain a supernatant. The supernatant was filtered through a 0.22 μm filter, and the filtrate was subjected to analysis of various sugars by the method shown in Reference Example 6.

<Reference Example 6> Measurement of sugar concentration Xylooligosaccharide, glucose, and xylose were quantitatively analyzed under the following conditions using Hitachi High Performance Liquid Chromatograph LaChrom Eite (HITACHI).

Based on the calibration curve prepared from the xylobiose, xylotiose, xylotetraose, xylopentaose, xylhexaose, cellobiose and cellotriose monosaccharides, and glucose and xylose monosaccharides. , Quantitative analysis. The xylooligosaccharide described in this example refers to a xylooligosaccharide in which 2 to 6 xylose units are bound by β-glycosidic bond. Further, the sero-oligosaccharide described in this example refers to a sero-oligosaccharide in which 2 to 6 glucose units are bound by β-glycosidic bond.

Column: KS802, KS803 (Shodex)
Mobile phase: Water detection method: RI
Flow velocity: 0.5 mL / min
Temperature: 75 ° C

<Comparative Example 1> Preparation of mutant strain (PC-3-7-ΔBGL / BXL strain) having reduced BGL activity using Trichoderma Risei PC-3-7 as a parent strain 1.0 spores of Trichoderma Risei PC-3-7 diluted with physiological saline to × become ¹⁰ 7 / mL, were inoculated with the diluted spore solution 0.1mL previous culture medium 10mL was placed into 50mL baffled flask shown in Table 1, in shaker The cells were cultured at 28 ° C. and 120 rpm for 4 hours. The culture medium was mutated with NTG (1-Methyl-3-nitro-1-nitrosoguanidine) for 90 minutes. The mutant spores collected and washed by centrifugation from this suspension were inoculated into a medium containing 2 wt% Arbocel B800 (trade name, Rettenmeier) and cultured at 28 ° C. for 120 hours at 125 rpm. For the selection of mutant strains having decreased β-glucosidase activity, the β-glucosidase activity was evaluated by the method described in Reference Example 4 using the supernatant obtained by centrifuging the culture solution. The enzyme activity (U) was calculated as the amount of enzyme liberating 1 mol of p-nitrophenol per minute. As a result, a strain in which the β-glucosidase activity was reduced to about 1/25 as compared with the parent strain PC-3-7 was obtained. The results are shown in Table 3. In addition, as a result of analyzing the gene sequence of the PC-3-7-ΔBGL / BXL strain, it was clarified that a frameshift mutation occurred as a result of deletion of thymine (T) at position 1226 of the BGL1 gene.

<Comparative Example 2> Preparation of mutant strain (PC-3-7BGL / ΔBXL strain) having reduced BXL activity using Trichoderma Risei PC-3-7 as a parent strain 1.0 × 10 spores of Trichoderma Risei PC-3-7 ^{Dilute with physiological saline to 7} / mL, inoculate 0.1 mL of the diluted spore solution into 10 mL of preculture medium in a flask with a 50 mL baffle shown in Table 1, and use a shaking incubator at 28 ° C. , 120 rpm for 4 hours. The culture medium was mutated with NTG (1-Methyl-3-nitro-1-nitrosoguanidine) for 90 minutes. The mutant spores collected and washed by centrifugation from this suspension were inoculated into a medium containing 2 wt% Arbocel B800 (trade name, Rettenmeier) and cultured at 28 ° C. for 120 hours at 125 rpm. For the selection of mutant strains having decreased β-xylosidase activity, the β-xylosidase activity was evaluated by the method described in Reference Example 4 using the supernatant obtained by centrifuging the culture solution. The enzyme activity (U) was calculated as the amount of enzyme liberating 1 mol of p-nitrophenol per minute. As a result, a strain in which β-xylosidase activity was reduced to about 1/210 as compared with the parent strain PC-3-7 was obtained. The results are shown in Table 3. In addition, as a result of analyzing the gene sequence of the PC-3-7-BGL / ΔBXL strain, it was clarified that the 1640th glycine (G) of the BXL gene was mutated to adenine (A).

<Example 1> Preparation of mutant strain (PC-3-7ΔBGL / ΔBXL) strain having reduced BGL and BXL activity using PC-3-7BGL / ΔBXL as a parent strain. Dilute with physiological saline to 0 × 10 ⁷ / mL, inoculate 0.1 mL of the diluted spore solution into a 10 mL preculture medium in a flask with a 50 mL baffle shown in Table 1, and put it in a shaking incubator. The cells were cultured at 28 ° C. and 120 rpm for 4 hours. The culture medium was mutated with NTG (1-Methyl-3-nitro-1-nitrosoguanidine) for 90 minutes. Mutant spores collected and washed by centrifugation from this suspension were cloned on PDA agar medium. The obtained mutant spores were ^{diluted with physiological saline to 1.0 × 10 7} / mL and inoculated with 1% (v / v) in a medium containing 2 wt% Arbocel B800 (trade name, Rettenmeier). Then, the cells were cultured at 28 ° C. for 120 hours at 125 rpm. The mutant strain having decreased β-glucosidase activity was selected by evaluating the β-glucosidase activity of the supernatant obtained by centrifuging the culture solution by the method described in Reference Example 4. The enzyme activity (U) was calculated as the amount of enzyme liberating 1 mol of p-nitrophenol per minute. As a result, we obtained a strain in which the β-glucosidase activity was reduced to about 1/22, respectively, as compared with the parent strain PC-3-7BGL / ΔBXL in which the β-glucosidase activity was not reduced. The results are shown in Table 3. In addition, as a result of analyzing the gene sequence of the PC-3-7-ΔBGL / ΔBXL strain, it was found that adenine (A), which is presumed to be the Xyr1 binding site 198 bases upstream from the translation initiation site of the BGL gene, was deleted. Do you get it.

<Example 2> Preparation of mutant strain (PC-3-7ΔBGL / ΔBXL-B) strain having reduced BGL and BXL activity using PC-3-7ΔBGL / BXL as a parent strain. Dilute with physiological saline to 1.0 × 10 ⁷ / mL, inoculate 0.1 mL of the diluted spore solution into a 10 mL preculture medium in a flask with a 50 mL baffle shown in Table 1, and shake culture. The cells were cultured on the machine at 28 ° C. and 120 rpm for 4 hours. The culture medium was mutated with NTG (1-Methyl-3-nitro-1-nitrosoguanidine) for 90 minutes. Mutant spores collected and washed by centrifugation from this suspension were cloned on PDA agar medium. The obtained mutant spores were ^{diluted with physiological saline to 1.0 × 10 7} / mL and inoculated with 1% (v / v) in a medium containing 2 wt% Arbocel B800 (trade name, Rettenmeier). Then, the cells were cultured at 28 ° C. for 120 hours at 125 rpm. The mutant strain having decreased β-xylosidase activity was selected by centrifuging the culture solution and evaluating the β-xylosidase activity of the supernatant obtained by the method described in Reference Example 4. The enzyme activity (U) was calculated as the amount of enzyme liberating 1 mol of p-nitrophenol per minute. As a result, a mutant strain lacking β-xylosidase activity was obtained. The results are shown in Table 3. Further, as a result of analyzing the gene sequence of the PC-3-7-ΔBGL / ΔBXL-B strain, the 2018 A from the translation start point of the BXL gene was replaced with T, and the 2065 A was replaced with T. It turned out.

<Comparative Example 3> Viscosity measurement of Trichoderma Risei PC-3-7 strain Using the method described in Reference Example 2, the viscosity of Trichoderma Risei PC-3-7 in the culture solution over time was measured. As a result, the maximum viscosity during culturing was 226 cP. The results are shown in Table 4. This value was set to 100% and compared with the viscosity of the culture solution of the mutant strain.

<Comparative Example 4> Viscosity measurement of PC-3-7ΔBGL / BXL strain Using the method described in Reference Example 2, the viscosity of the PC-3-7ΔBGL / BXL strain prepared in Comparative Example 1 over time in the culture solution was determined. It was measured. As a result, the maximum viscosity during culturing was 230 cP. The results are shown in Table 4. It was found that there was no change compared to the Trichoderma Risei PC-3-7 strain.

<Comparative Example 5> Viscosity measurement of PC-3-7BGL / ΔBXL strain Using the method described in Reference Example 2, the viscosity of the PC-3-7BGL / ΔBXL strain prepared in Comparative Example 2 over time in the culture solution was determined. It was measured. As a result, the maximum viscosity during culturing was 164 cP. The results are shown in Table 4. It decreased by 27.4% compared to Trichoderma Risei PC-3-7 strain.

<Example 3> Viscosity measurement of PC-3-7ΔBGL / ΔBXL strain Using the method described in Reference Example 2, the viscosity of the PC-3-7ΔBGL / ΔBXL strain prepared in Example 1 over time in the culture solution was determined. It was measured. As a result, the maximum viscosity during culturing was 117 cP, which was 48.2% lower than that of the Trichoderma Risei PC-3-7 strain (Table 4).

<Example 4> Viscosity measurement of PC-3-7ΔBGL / ΔBXL-B strain P prepared in Example 2 using the method described in Reference Example 2.
The viscosity of the C-3-7ΔBGL / ΔBXL strain in the culture solution over time was measured. As a result, the maximum viscosity during culturing was 131 cP, which was 42.0% lower than that of the Trichoderma Risei PC-3-7 strain (Table 4).

<Comparative Example 6> Glycation test using a cellulase composition of Trichoderma Risei PC-3-7 strain Two types of biomass (Arbocel B800 (trade name, Rettenmeier) and bagasse powder are used by the method described in Reference Example 5). The saccharification reaction was carried out at 50 ° C. for a period of time. The saccharification reaction solution was quantitatively analyzed by the method described in Reference Example 6. Focusing on the concentration of xylobiose, Arbocel B800 (trade name) and bagasse powder were found in the PC-3-7 strain. No production was observed in any of the biomasses. The results are shown in Table 5.

<Comparative Example 7> Glycation test using cellulase composition of PC-3-7BGL / ΔBXL strain Two types of biomass (Arbocel B800 (Rettenmeier), bagasse powder) were used for 24 hours by the method described in Reference Example 5. It was subjected to a saccharification reaction at 50 ° C. The saccharification reaction solution was quantitatively analyzed by the method described in Reference Example 6. As a result, 3.15 g / L and 1.07 g / L of xylobiose were released in the PC-3-7BGL / ΔBXL strain, respectively. The results are shown in Table 5.

<Example 5> Glycation test using cellulase composition of PC-3-7ΔBGL / ΔBXL strain Two types of biomass (Arbocel B800 (Rettenmeier), bagasse powder) were used for 24 hours by the method described in Reference Example 5. It was subjected to a saccharification reaction at 50 ° C. The saccharification reaction solution was quantitatively analyzed by the method described in Reference Example 6. In the PC-3-7ΔBGL / ΔBXL strain, the amount of xylobiose released was 3.45 g / L and 1.28 g / L, respectively, which was surprisingly compared with the results of the PC-3-7BGL / ΔBXL strain, respectively. It increased by 52% and 19.6%. The results are shown in Table 5.

<Example 6> Glycation test using cellulase composition of PC-3-7ΔBGL / ΔBXL strain Two types of biomass (Arbocel B800 (Rettenmeier), bagasse powder) were used for 24 hours by the method described in Reference Example 5. It was subjected to a saccharification reaction at 50 ° C. The saccharification reaction solution was quantitatively analyzed by the method described in Reference Example 6. In the PC-3-7ΔBGL / ΔBXL-B strain, the free amounts of xylobiose were 3.45 g / L and 1.28 g / L, respectively, which were surprisingly compared with the results of the PC-3-7BGL / ΔBXL strain, respectively. It increased by 44.1% and 61.2%. The results are shown in Table 5.

Claims

A mutant strain of Trichoderma filamentous fungus in which the activities of β-glucosidase and β-xylosidase were lower than those of the parent strain before the introduction of the mutation.
The mutant strain according to claim 1, wherein the specific activity of the β-glucosidase is 0.02 U / mg-protein or less, and the specific activity of the β-xylosidase is 0.002 U / mg-protein or less.
The mutant strain according to claim 1 or 2, wherein the Trichoderma filamentous fungus is Trichoderma lysei.
A method for producing a protein by culturing the mutant strain according to any one of claims 1 to 3.
The production method according to claim 4, wherein the protein is cellulase.
A method for producing a xylooligosaccharide, which hydrolyzes biomass containing xylan with cellulase produced by the method according to claim 5.
A method for reducing the viscosity of a culture solution during liquid culture of Trichoderma filamentous fungi, which comprises reducing the β-glucosidase activity and β-xylosidase activity of Trichoderma filamentous fungi.