Trichoderma mutants

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

[Technical Field]

[0001] The present invention relates to a mutant strain of a filamentous fungus of the genus Trichoderma that can maintain a low viscosity of the culture solution. [Background technology]

[0002] Trichoderma fungi are known to have a high protein production capacity, and protein production using Trichoderma fungi has been investigated. Trichoderma fungi use cellulose, lactose, cellobiose, and other inducers to produce cellulase, a type of protein-specific enzyme, specifically saccharifying enzymes. To increase cellulase production, numerous studies have been conducted over the years, including overexpression and deletion of factors that control cellulase production, genetic modification, and optimization of culture conditions.

[0003] On the other hand, Trichoderma fungi are aerobic fungi that require oxygen for growth and protein production. Furthermore, when Trichoderma fungi are cultured in liquid media, the viscosity of the culture solution increases as they grow. As the viscosity of the culture solution increases, the distribution of oxygen and nutrients becomes uneven. Therefore, when culturing Trichoderma fungi, it is necessary to maintain a constant culture environment by increasing the agitation rate and aeration volume. When the culture tank becomes larger, a huge amount of agitation power is required to maintain a constant culture environment, and strong agitation poses the problem of causing significant shear damage to the fungi.

[0004] Patent documents 1 to 6 disclose that by destroying or reducing the production of the Sfb3, Mpg1, Gas1, Seb1, Crz1, and Tps1 proteins of viscous Trichoderma filamentous fungi, respectively, it is possible to maintain a culture environment at a lower agitation rate than that of the parent strain.

[0005] Furthermore, Patent Document 7 describes that disrupting the BXL1 gene of filamentous fungi of the genus Trichoderma can suppress a decrease in the dissolved oxygen saturation of the culture solution. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2013-533751 [Patent Document 2] Special Publication No. 2014-513529 [Patent Document 3] Special Publication No. 2014-513530 [Patent Document 4] Special Publication No. 2014-513531 [Patent Document 5] Special Publication No. 2014-513532 [Patent Document 6] Special Publication No. 2014-513533 [Patent Document 7] International Publication No. 2017 / 170917 Summary of the Invention [Problem to be solved by the invention]

[0007] As described above, when producing proteins using Trichoderma fungi, it is extremely important to prevent a decrease in the dissolved oxygen concentration in the culture medium and maintain it at a certain level or higher. The present inventors believed that if the viscosity of the culture medium could be maintained low during protein production by liquid culture using Trichoderma fungi, it would be possible to reduce the energy required for stirring and prevent a decrease in the dissolved oxygen saturation level in the culture medium, even when the culture scale is increased. An objective of the present invention is to obtain a mutant strain of Trichoderma fungi that reduces the viscosity of the culture medium and to provide a method for producing proteins using the mutant strain of Trichoderma fungi. [Means for solving the problem]

[0008] The present inventors conducted extensive research into the selection of Trichoderma fungi that can maintain a low viscosity of the culture solution, and as a result, discovered that the viscosity of the culture solution can be maintained low by culturing a mutant strain of Trichoderma fungi in which the activity of β-glucosidase and β-xylosidase is reduced compared to the parent strain before the mutation was introduced, thereby completing the present invention.

[0009] That is, the present invention provides the following. (1) A mutant strain of Trichoderma fungi in which the activities of β-glucosidase and β-xylosidase were reduced compared to the parent strain before the mutation was introduced. (2) The mutant strain according to (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. (3) The mutant strain according to (1) or (2), wherein the filamentous fungus of the genus Trichoderma is Trichoderma reesei. (4) A method for producing a protein by culturing the mutant strain according to any one of (1) to (3). (5) The method according to (4), wherein the protein is cellulase. (6) A method for producing xylooligosaccharides, which comprises hydrolyzing biomass containing xylan with cellulase produced by the method according to (5). (7) A method for reducing the viscosity of a liquid culture medium of a filamentous fungus of the genus Trichoderma, the method comprising reducing β-glucosidase activity and β-xylosidase activity of the filamentous fungus of the genus Trichoderma. [Effects of the Invention]

[0010] According to the present invention, a mutant strain of a filamentous fungus of the genus Trichoderma, in which the activity of β-glucosidase and β-xylosidase is reduced compared to the parent strain before the mutation is introduced, is able to maintain a lower viscosity of the culture solution compared to the parent strain that retains the activity of these enzymes. DETAILED DESCRIPTION OF THE INVENTION

[0011] The filamentous fungus of the genus Trichoderma in the present invention is not particularly limited as long as it belongs to the genus Trichoderma and has the ability to produce cellulase. Trichoderma reesei is preferred. Alternatively, a mutant strain derived from the genus Trichoderma that has been subjected to a mutation treatment using a mutagen or ultraviolet irradiation to improve cellulase productivity may be used as the parent strain.

[0012] Specific examples of mutant strains used as the parent strain include Trichoderma paralesei (ATCC MYA-4777), an ancestor of Trichoderma reesei, and known mutant strains derived from Trichoderma reesei, such as QM6a strain (NBRC31326), QM9123 strain (ATCC24449), QM9414 strain (NBRC31329), PC-3-7 strain (ATCC66589), QM9123 strain (NBRC31327), RutC-30 strain (ATCC56765), CL-847 strain (Enzyme. Microbiol. Technol. 10, 341-346 (1988)), and MCG77 strain (Biotechnol. Bioeng. Symp. 8, 2008). 89 (1978)), MCG80 strain (Biotechnol. Bioeng. 12, 451-459 (1982)), and their derivative strains.

[0013] Furthermore, the Trichoderma filamentous fungus used in the present invention is preferably deficient in carbon catabolite repression. Strains in which carbon catabolite repression is deficient have improved production of proteins such as cellulase, making it possible to produce larger amounts of protein. Even more preferred are strains in which carbon catabolite repression mediated by carbon catabolite repressor I is deficient. For example, introducing a mutation into the carbon catabolite repressor I gene (cre1) can deactivate carbon catabolite repression mediated by carbon catabolite repressor I. The CRE1 protein encoded by the cre1 gene is known to repress the expression of cellulase genes through catabolite repression induced by glucose (FEBS Lett., 376, 103-107, 1995). Therefore, introducing a mutation into the cre1 gene deactivates the repression of cellulase gene expression, thereby increasing cellulase production. Therefore, strains with a mutation in the cre1 gene are more suitable for producing proteins and cellulase compositions. A specific example of a cre1 gene mutation is the substitution of A at position 232 with C in the cre1 gene of the PC-3-7 strain (ATCC 66589), resulting in the substitution of threonine at position 78 with proline. This mutation is known to improve cellulase production (Biosci. Biotechnol. Biochem., 77 (3), 534-543, 2013). Furthermore, the cre1 gene is partially truncated in the RutC-30 strain (ATCC 56765), resulting in the release of carbon catabolite repression (BMC Genomics., 9, 327, 2008). Here, strains with mutations in the cre1 gene include strains with frameshifts due to deletion or insertion of bases within the cre1 gene region caused by gene mutation agents, ultraviolet irradiation, etc., or stop codon mutations due to base substitution, or base truncations, and also include strains in which all or part of the cre1 gene has been removed or replaced with another gene by recombination, etc.Specifically, strains that inherit the characteristics of PC-3-7 (ATCC 66589) or RutC-30 (ATCC 56765) are preferably used, and strains that inherit the characteristics of PC-3-7 (ATCC 66589) or RutC-30 (ATCC 56765) are even more preferred. PC-3-7 (ATCC 66589) or strains that inherit the characteristics of PC-3-7 (ATCC 66589) or RutC-30 (ATCC 56765) also include strains that have inherited the characteristics of PC-3-7 (ATCC 66589) or RutC-30 (ATCC 56765) and have been newly mutated or have been recombined to improve their functions.

[0014] The QM6a, QM9414, and QM9123 strains are available from the NITE Biological Resource Center (NBRC), and the PC-3-7 and RutC-30 strains are available from the American Type Culture Collection (ATCC).

[0015] In the present invention, "a mutant strain of a Trichoderma filamentous fungus having reduced β-glucosidase and β-xylosidase activity compared to the parent strain before the introduction of mutation" refers to a mutant strain obtained by introducing a mutation into the above-mentioned parent strain of a Trichoderma filamentous fungus, and having reduced β-glucosidase and β-xylosidase activity compared to the parent strain before the introduction of mutation, and may be referred to as "the mutant strain of the present invention" in this specification.

[0016] In the present invention, a 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 reduced compared to the parent strain before the mutation was introduced, and also includes a state in which the function of β-glucosidase and / or β-xylosidase is lost.

[0017] Specifically, it is preferable that the β-glucosidase specific activity and β-xylosidase specific activity are each reduced to less than one-tenth of that of the parent strain, more preferably less than one-fiftieth, more preferably less than one-hundredth, more preferably less than one-two hundredth, even more preferably less than one-fiftyth, even more preferably less than one-fiftyth, particularly preferably less than one-eight hundredth, and most preferably less than one-thousandth.

[0018] In terms of absolute values ​​of specific activity, the β-glucosidase specific activity and the β-xylosidase 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, and even more preferably 0.0025 U / mg-protein or less and 0.00025 U / mg-protein or less.

[0019] Methods for introducing mutations that reduce the specific activity of β-glucosidase and β-xylosidase into a parent strain of Trichoderma fungi include, but are not limited to, gene mutation treatments using gene mutators or ultraviolet irradiation, and site-directed mutagenesis. Alternatively, the BGL1 and BXL1 genes present in the parent strain of Trichoderma fungi may be disrupted. Specifically, β-glucosidase and β-xylosidase activities may be reduced by introducing mutations, insertions, deletions, or other mutations into these genes, their promoter regions, or genes encoding transcriptional regulators. Furthermore, this can be achieved by introducing frameshift mutations or inserting stop codons into these genes. Alternatively, the BGL1 and / or BXL1 genes can be disrupted by genetic recombination (such as homologous recombination with another gene) to remove or replace all or part of the genes, their promoter regions, or genes encoding transcriptional regulators. Alternatively, expression of the BGL1 gene and / or BXL1 gene can be inhibited by deleting all or part of the binding recognition sequence for a transcriptional regulatory factor located upstream of the BGL1 gene and / or BXL1 gene or replacing it with another gene. The BGL1 gene of filamentous fungi of the genus Trichoderma (Gene ID: 18488646) and the filamentous fungal BXL1 gene (Gene ID: 18483060) are publicly known, and therefore these genes can be easily disrupted by standard methods such as the above-mentioned gene mutagenesis method or site-directed mutagenesis. The BGL1 gene of filamentous fungi of the genus Trichoderma and the BGL1 gene contain binding sites for transcriptional regulatory factors between bases 1 and 500 upstream.A specific example of a transcriptional regulatory factor is Xyr1, and the putative binding recognition sequence of Xyr1 in Trichoderma filamentous fungi is disclosed in Borin, Carazzole, "Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30," Bioeng. Biotechnol., 2018, 6, doi.org / 10.3389 / fbioe.2018.00151. When random mutagenesis is performed using a gene mutagen or ultraviolet irradiation, each post-mutation strain is cloned, and β-glucosidase activity and β-xylosidase activity are measured using the methods described below. Strains with reduced β-glucosidase specific activity and β-xylosidase specific activity compared to the parent strain before mutation can be obtained by selecting strains with reduced β-glucosidase specific activity compared to the parent strain before mutation.

[0020] The specific activity of β-glucosidase and the specific activity of β-xylosidase of Trichoderma fungi are measured by the following method.

[0021] First, Trichoderma fungi are cultured, and the resulting culture solution is centrifuged at 20,000 × g for 10 minutes to recover the supernatant. The recovered supernatant is diluted to an appropriate concentration to prepare an enzyme dilution, and the specific activity of the enzyme is measured using the following method.

[0022] The β-glucosidase specific activity is measured as follows. First, 10 μL of the enzyme dilution is added to 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside (Sigma-Aldrich Japan), and the mixture is allowed to react at 30°C for 10 minutes. Next, 10 μL of 2 M 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 as 1 U, which is the activity that liberates 1 μmol of p-nitrophenol per minute.

[0023] The specific activity of β-xylosidase is measured as follows. First, 10 μL of the enzyme dilution is added to 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside (Sigma-Aldrich Japan), and the mixture is allowed to react at 30°C for 30 minutes. Next, 10 μL of 2 M 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 as 1 U, which is the activity that liberates 1 μmol of p-nitrophenol per minute.

[0024] The protein concentration required to calculate specific activity is measured as follows: 5 μL of the enzyme dilution solution is added to 250 μL of Quick Start Bradford Protein Assay (Bio-Rad), and after leaving it to stand at room temperature for 15 minutes, the absorbance at 595 nm is measured. Using bovine serum albumin solution as the standard solution, the protein concentration contained in the saccharification enzyme solution is calculated based on the calibration curve.

[0025] The "mutant strain of Trichoderma fungi in which β-glucosidase and β-xylosidase activities are reduced compared to the parent strain before mutation" of the present invention has a reduced viscosity of the culture solution compared to the parent strain. This reduces the energy and rotation speed required for aeration and agitation. Furthermore, the agitation speed can be set lower, which reduces shear damage to the mycelium. This is particularly effective when culturing on a large scale, as it reduces the capacity of the blower and agitation motor required for aeration and the agitation energy.

[0026] The culture method is not particularly limited as long as it allows for culturing Trichoderma fungi to produce protein, and can be performed by a well-known method using a well-known medium. For example, liquid culture can be performed using centrifuge tubes, flasks, jar fermenters, tanks, etc. Because Trichoderma reesei is an aerobic microorganism, among these culture methods, submerged culture in a jar fermenter or tank, in which culture is performed while aerating and stirring, is particularly preferred. The aeration rate is preferably approximately 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 approximately 25°C to 35°C, more preferably 25°C to 31°C. The pH condition during culture is preferably pH 3.0 to 7.0, more preferably pH 4.0 to 6.0. The culture is continued under conditions conducive to protein production until a recoverable amount of protein is accumulated. Usually, it is about 24 to 288 hours, and more preferably 36 to 240 hours.

[0027] In the present invention, the viscosity of the culture solution is measured under the following conditions, and the maximum values ​​of the viscosity measured under the following conditions are compared. First, spores of the Trichoderma fungus to be evaluated are added to 1.0 × 10 spores per mL of pre-culture medium. 5The spores were inoculated into a pre-culture medium (an example of the medium composition is shown in Table 1 in the Examples) and cultured in a shaker at 28°C and 120 rpm until the bacterial mass reached approximately 11 g / L. Next, the main culture medium shown in Table 2, to which Arbocel B800 (product name, cellulose fiber, manufactured by Rettenmeyer) and bagasse powder had been added at 100 g / L (w / v), was inoculated with the pre-culture medium at 10% (v / v). Submerged culture was performed in a 5 L jar fermenter. After inoculation of the pre-culture medium into the main culture medium, submerged culture was performed at 28°C, 700 rpm, and 100 mL / min aeration, while maintaining a pH of 5.0. The viscosity of the culture medium was measured using a digital rotational viscometer. The digital rotational viscometer was calibrated to the zero point in advance. Approximately 20 mL of the culture medium was sampled periodically from the start of culture until 96 hours had elapsed. Immediately after collection, the culture fluid is placed in a designated container, and a spindle is immersed in the culture fluid and rotated at 0.3 rpm. The torque, which is the viscous resistance acting on the spindle, is measured at room temperature to determine the viscosity of the culture fluid. The unit of viscosity is centipoise (cP). 1 centipoise is the viscosity of a fluid moving 1 cm in the direction of velocity on a plane perpendicular to the direction of the velocity gradient when there is a velocity gradient of 1 cm / sec per 1 cm in the fluid. 2 It is defined as the viscosity at which a stress of 1 dyne occurs per unit of pressure. A digital rotational viscometer such as the DV2T (BROOKFIELD) can be used, with a spindle such as the ULA (BROOKFIELD).

[0028] The "mutant strains of the present invention, which have reduced β-glucosidase and β-xylosidase activities compared to the parent strain before mutation introduction," have a lower viscosity of the culture solution compared to the parent strain when cultured under the same conditions, with the maximum viscosity during culture being reduced to preferably 70% or less, more preferably 60% or less, and even more preferably 50% or less of that of the parent strain. Furthermore, in absolute terms, the maximum viscosity during culture of the mutant strains of the present invention is lower than that of the parent strain by preferably 50 cP or more, more preferably 100 cP or more, more preferably 150 cP or more, more preferably 200 cP or more, even more preferably 250 cP or more, even more preferably 300 cP or more, even more preferably 350 cP or more, even more preferably 400 cP or more, and particularly preferably 500 cP or more.

[0029] The protein produced in the present invention is not particularly limited, but it is possible to efficiently produce proteins that are secreted outside the bacterial cell, and among these, enzymes are preferred, more preferably saccharifying enzymes such as cellulase, amylase, invertase, chitinase, and pectinase, and even more preferably cellulase.

[0030] The cellulase produced by the present invention contains various hydrolases, including enzymes with decomposition activity against xylan, cellulose, and hemicellulose. Specific examples of hydrolases generally contained in cellulases include cellobiohydrolase (EC 3.2.1.91), which produces cellobiose by hydrolyzing cellulose chains; endoglucanase (EC 3.2.1.4), which hydrolyzes cellulose chains starting from the central portion; β-glucosidase (EC 3.2.1.21), which hydrolyzes cellooligosaccharides and cellobiose; xylanase (EC 3.2.1.8), which is characterized by acting on hemicellulose and particularly on xylan; and β-xylosidase (EC 3.2.1.37), which hydrolyzes xylooligosaccharides. The protein concentration of cellulase can be calculated using the method described above.

[0031] The method for culturing the mutant strain of the present invention is not particularly limited, and for example, it can be cultured by liquid culture using a centrifuge tube, a flask, a jar fermenter, a tank, etc. Since filamentous fungi of the genus Trichoderma are aerobic microorganisms, among these culture methods, submerged culture in a jar fermenter or a tank in which aeration and agitation are performed is particularly preferred.

[0032] The medium composition during cultivation is not particularly limited as long as it allows Trichoderma fungi to produce proteins, and well-known medium compositions for Trichoderma fungi can be used. Examples of nitrogen sources that can be used include polypeptone, meat juice, corn steep liquor (CSL), and soybean meal. Inducers for protein production may also be added to the medium. Examples of carbon sources that can be used include sugars such as glucose, sucrose, fructose, galactose, and lactose; starch saccharification liquids containing these sugars; sweet potato molasses, sugar beet molasses, and high-test molasses; organic acids such as acetic acid; alcohols such as ethanol; and glycerin. In addition to these, it is preferable to use inducers, as described below, as a carbon source, particularly when producing cellulase.

[0033] When producing cellulase according to the present invention, the cellulase can be cultured in a medium containing at least one or more inducers selected from the group consisting of lactose, cellulose, and xylan. Furthermore, biomass containing cellulose or xylan may be added as an inducer for cellulose or xylan. Specific examples of biomass containing cellulose or xylan include seed plants, ferns, mosses, algae, and aquatic plants, as well as waste building materials. Spermatophytes are classified into gymnosperms and angiosperms, and both are preferably used. Angiosperms are further classified into monocotyledons and dicotyledons. Specific examples of monocotyledons include bagasse, switchgrass, napier grass, erianthus, corn stover, corncob, rice straw, and wheat straw. Specific examples of dicotyledons include beet pulp, eucalyptus, oak, and birch.

[0034] Furthermore, biomass containing cellulose and xylan may be pretreated. The pretreatment method is not particularly limited, and known techniques 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 biomass containing cellulose and xylan that has been pretreated in this way.

[0035] Furthermore, when the culture medium in which the mutant strain of the present invention has been cultured is used as a protein solution without removing the bacterial cells, it is preferable to treat the culture medium so that the mutant strain of the present invention cannot grow in the medium, for example, by heat treatment, chemical treatment, acid / alkali treatment, UV treatment, etc.

[0036] When the protein is an enzyme, the culture medium from which the cells have been removed or treated to prevent growth as described above can be used as an enzyme solution as is.

[0037] The method for using the cellulase composition produced by the present invention is not particularly limited, but it is preferably used for the production of sugars, more preferably for the production of xylooligosaccharides and / or cellooligosaccharides, and even more preferably for the production of xylobiose and / or cellobiose.

[0038] In the present invention, xylooligosaccharide refers to a xylooligosaccharide in which at least two xylose units are linked by β-glycosidic bonds. The degree of polymerization of the xylooligosaccharide is not particularly limited, but it is preferably a highly water-soluble disaccharide (xylobiose) to hexasaccharide (xylohexaose). Most preferably, it contains xylobiose, xylotriose, and xylotetraose, which are easily assimilated as carbon sources by enterobacteria.

[0039] In the present invention, cellooligosaccharide refers to a cellooligosaccharide in which at least two glucose units are linked by β-glycosidic bonds. The degree of polymerization of the cellooligosaccharide is not particularly limited, but it is preferably a highly water-soluble disaccharide (cellobiose) to hexasaccharide (cellohexaose). Most preferably, it contains cellobiose, cellotriose, and cellotetraose, which are easily assimilated as carbon sources by enterobacteria.

[0040] In the present invention, the cellulase composition is obtained by culturing a filamentous fungus of the genus Trichoderma and is used in the saccharification reaction of biomass. The method for preparing the cellulase composition is not particularly limited, but it is preferable that the fungal cells of the filamentous fungus of the genus Trichoderma contained in the culture solution are removed or not grown. This is to prevent the consumption of glucose and xylooligosaccharides produced during the saccharification reaction of the cellulase composition and biomass by the fungal cells. Examples of methods for removing the fungal cells include centrifugation and membrane separation. Examples of treatment methods for preventing the growth of the fungal cells include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment.

[0041] The method for producing sugar using a cellulase composition obtained by culturing a filamentous fungus of the genus Trichoderma is not particularly limited, but biomass can be saccharified using the cellulase composition. Biomass containing cellulose and / or xylan can be used for the saccharification reaction. The cellulose and / or biomass used in the saccharification reaction may be pretreated in advance. The pretreatment method is not particularly limited, but known techniques such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, pulverization, and steaming can be used. The reaction pH is also not particularly limited, but a pH of approximately 3 to 7 is preferred, more preferably 4 to 6, and even more preferably approximately 5. The reaction temperature is also not particularly limited, but a temperature of 40 to 70°C is preferred.

[0042] The saccharification reaction described above can yield xylooligosaccharides, preferably xylobiose, from biomass containing xylose, cellooligosaccharides, preferably cellobiose, from biomass containing cellulose, and xylooligosaccharides and cellooligosaccharides, preferably xylobiose and cellobiose, from biomass containing cellulose and xylose. Furthermore, the sugars obtained by the saccharification reaction described above may include, in addition to xylooligosaccharides and cellooligosaccharides, monosaccharides such as mannose, arabinose, and galactose produced by the hydrolases contained in the cellulase composition, as well as oligosaccharides such as cellotriose, cellotetraose, mannobiose, and galactobiose.

[0043] The post-reaction solution produced from the saccharification reaction of the present invention may contain impurities such as inorganic salts, amino acids, proteins, and lignin, and may be subjected to a purification procedure to remove these impurities. Known methods such as ion exchange, membrane separation, crystallization, and desalination can be used as the purification procedure.

[0044] The monosaccharide fraction (glucose, xylose, etc.) and oligosaccharide fraction (xylooligosaccharides, cellooligosaccharides, etc.) produced by the present invention are preferably separated in a subsequent process. Glucose is preferably used as a fermentation feedstock in the production of chemical products, and xylooligosaccharides are preferably used for feed, food, and cosmetics. Specific examples of chemical products include alcohols such as ethanol, 1,3-propanediol, 1,4-butanediol, and glycerol; organic acids such as acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, and citric acid; nucleosides such as inosinic acid and guanosine; nucleotides such as inosinic acid and guanylic acid; and amine compounds such as cadaverine. On the other hand, the microorganisms that can be used as a fermentation feedstock for xylose are limited. Furthermore, when xylose is fed to pigs or other animals as feed, approximately half of it is excreted in the urine. For this reason, it is preferable to minimize the decomposition of xylan and xylooligosaccharides into xylose and improve the xylooligosaccharide yield.

[0045] In the present invention, the method for separating the monosaccharide fraction and the oligosaccharide fraction is not particularly limited, and known methods can be used. For example, membrane separation as described in WO 2017 / 110975 is preferably used. In this case, the lower the proportion of xylose, the lower the amount of xylose contaminating the oligosaccharide fraction, which is advantageous in the membrane separation process. [Example]

[0046] The present invention will be specifically described below with reference to examples.

[0047] <Reference Example 1> Cultivation of Trichoderma fungi (preculture) 1.0 × 10 spores of various mutant strains of Trichoderma reesei 7 The spores were diluted with saline to a concentration of 1 / mL, and 2.5 mL of the diluted spore solution was inoculated into 250 mL of pre-culture medium placed in a 1 L baffled flask shown in Table 1, and cultured in a shaking incubator at 28°C and 120 rpm for 72 hours. As a control, Trichoderma longibrachiatum strain PC-3-7 was used, and the same experimental procedures were performed.

[0048] [Table 1] *1 Mandels is 7g / L (NH4)2SO4, 10g / L KH2PO4, 3g / Contains 3g / L CaCl2 and 3g / L MgSO4·7H2O (same below). *2 Trace element solution: 0.3g / L H3BO3, 1.3g / L (NH4)6Mo7O 24 ·4H2O, 5g / L FeCl3·6H2O, 2g / L CuSO4·5H2O, 0.4g / L MnCl2·4H2O, 10g / L ZnCl2 (same below).

[0049] (main culture) Arbocel B800 (trade name, Rettenmeyer, powdered cellulose) was added to the main culture medium shown in Table 2, and submerged culture was examined using a 5 L jar fermenter (Bioit).

[0050] 250 mL of preculture solution of Trichoderma reesei strain PC-3-7 and various mutant strains of Trichoderma reesei was inoculated into 2.5 L of main culture medium supplemented with Arbocel B800 (trade name).

[0051] The culture conditions were as follows: after inoculating the pre-culture medium into the main culture medium, submerged culture was carried out at 28°C, 700 rpm, and an aeration rate of 100 mL / min, while controlling the pH at 5.0.

[0052] [Table 2]

[0053] (Collection of culture fluid) The culture medium was sampled in 20 mL increments over time from the start of the culture until the end of the culture at 74 hours. 16 mL of the sampled culture medium was used for the viscosity measurement described below. The remaining culture medium was centrifuged at 20,000 × g and 4°C for 10 minutes to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter, and the filtrate was used as a cellulase solution in the subsequent saccharification test.

[0054] <Reference Example 2> Measurement of viscosity of culture medium The viscosity (cP) of 16 mL of the culture medium was measured at 0, 24, 48, and 74 hours after the start of cultivation using a digital rotational viscometer DV2T with a spindle ULA (manufactured by Brookfield) at a rotation speed of 0.3 rpm.

[0055] <Reference Example 3> Protein concentration measurement conditions Protein concentration measurement reagent used: Quick Start Bradford Protein Assay, Bio-Rad Measurement conditions Measurement temperature: room temperature Protein concentration measurement reagent: 250 μL Filamentous fungal culture medium: 5 μL Reaction time: 5 minutes Absorbance: 595nm Standard product: BSA.

[0056] <Reference Example 4> Conditions for measuring the specific activity of cellulase (Measurement conditions for β-glucosidase specific activity) Substrate: p-nitrophenyl-β-glucopyranoside (Sigma-Aldrich Japan) Reaction solution: 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside Enzyme dilution solution: 10 μL Reaction temperature: 30℃ Reaction time: 10 minutes Reaction stopper: 10 μL of 2 M sodium carbonate Absorbance: 405nm.

[0057] (Measurement conditions for β-xylosidase specific activity) Substrate: p-nitrophenyl-β-xylopyranoside (Sigma-Aldrich Japan) Reaction solution: 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside Enzyme dilution solution: 10 μL Reaction temperature: 30℃ Reaction time: 30 minutes Reaction stopper: 10 μL of 2 M sodium carbonate Absorbance: 405nm.

[0058] <Reference Example 5> Saccharification test Amount of enzyme solution used: 8mg / g (amount of enzyme used per 1g of biomass) Biomass: Arbocel B800 (product name, Rettenmeyer), bagasse powder Biomass feed amount: 5% (w / v) Reaction temperature: 50℃ Response time: 24 hours Reaction pH: 5.0 The reaction mixture was centrifuged at 8,000 × g for 5 minutes to obtain a supernatant, which was then filtered through a 0.22 μm filter, and the filtrate was subjected to the analysis of various sugars by the method described in Reference Example 6.

[0059] <Reference Example 6> Measurement of sugar concentration Xylooligosaccharides, glucose, and xylose were quantitatively analyzed using a Hitachi high-performance liquid chromatograph LaChrom Eite (HITACHI) under the following conditions.

[0060] Quantitative analysis was performed based on calibration curves prepared using standards of xylooligosaccharides (xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose), cellooligosaccharides (cellobiose and cellotriose), and monosaccharides (glucose and xylose). Note that the term "xylooligosaccharide" used in this example refers to a xylooligosaccharide in which 2 to 6 xylose units are linked by β-glycosidic bonds. Also, the term "cellooligosaccharide" used in this example refers to a cellooligosaccharide in which 2 to 6 glucose units are linked by β-glycosidic bonds.

[0061] Column: KS802, KS803 (Shodex) Mobile phase: water Detection method: RI Flow rate: 0.5mL / min Temperature: 75℃

[0062] <Comparative Example 1> Preparation of a mutant strain (PC-3-7-ΔBGL / BXL strain) with reduced BGL activity using Trichoderma reesei PC-3-7 as a parent strain 1.0 x 10 spores of Trichoderma reesei PC-3-7 7The spores were diluted with saline to a concentration of 0.1 mL / mL, and 0.1 mL of the diluted spore solution was inoculated into 10 mL of the preculture medium shown in Table 1 in a 50 mL baffled flask and cultured for 4 hours at 28°C and 120 rpm in a shaker. The culture was then subjected to a 90-minute mutagenesis treatment with NTG (1-methyl-3-nitro-1-nitrosoguanidine). Mutant spores were collected from the suspension by centrifugation and washed. They were then inoculated into a medium containing 2 wt% Arbocel B800 (Rettenmeyer) and cultured at 28°C for 120 hours at 125 rpm. To select mutants with reduced β-glucosidase activity, the supernatant obtained after centrifugation of the culture was assayed for β-glucosidase activity using the method described in Reference Example 4. Enzyme activity (U) was calculated as the amount of enzyme liberating 1 mol of p-nitrophenol per minute. As a result, a strain was obtained in which β-glucosidase activity was reduced to approximately 1 / 25 of that of the parent strain, PC-3-7. The results are shown in Table 3. Furthermore, analysis of the gene sequence of the PC-3-7-ΔBGL / BXL strain revealed that a frameshift mutation had occurred due to the deletion of thymine (T) at position 1226 of the BGL1 gene.

[0063] Comparative Example 2: Preparation of a mutant strain (PC-3-7BGL / ΔBXL strain) with reduced BXL activity using Trichoderma reesei PC-3-7 as a parent strain 1.0 x 10 spores of Trichoderma reesei PC-3-7 7The spores were diluted with saline to a concentration of 0.1 mL / mL. 0.1 mL of the diluted spore solution was inoculated into 10 mL of the preculture medium shown in Table 1 in a 50 mL baffled flask and cultured for 4 hours at 28°C and 120 rpm in a shaker. The culture was then subjected to a 90-minute mutagenesis treatment with NTG (1-methyl-3-nitro-1-nitrosoguanidine). Mutant spores were collected from the suspension by centrifugation and washed. They were then inoculated into a medium containing 2 wt% Arbocel B800 (Rettenmeyer) and cultured at 28°C for 120 hours at 125 rpm. To select mutants with reduced β-xylosidase activity, the supernatant obtained after centrifugation was assayed for β-xylosidase activity using the method described in Reference Example 4. Enzyme activity (U) was calculated as the amount of enzyme liberating 1 mol of p-nitrophenol per minute. As a result, a strain was obtained in which β-xylosidase activity was reduced to approximately 1 / 210 of that of the parent strain, PC-3-7. The results are shown in Table 3. Furthermore, analysis of the gene sequence of the PC-3-7-BGL / ΔBXL strain revealed that glycine (G) at position 1640 of the BXL gene had been mutated to adenine (A).

[0064] Example 1: Preparation of a mutant strain (PC-3-7ΔBGL / ΔBXL) with reduced BGL and BXL activities using PC-3-7BGL / ΔBXL as a parent strain 1.0 × 10 spores of the PC-3-7BGL / ΔBXL strain 7 The spores were diluted with physiological saline to a concentration of 1.0 × 10 / mL, and 0.1 mL of the diluted spore solution was inoculated into 10 mL of pre-culture medium placed in a 50 mL baffled flask shown in Table 1, and cultured for 4 hours at 28°C and 120 rpm in a shaking incubator. The culture solution was subjected to a mutagenesis treatment with NTG (1-Methyl-3-nitro-1-nitrosoguanidine) for 90 minutes. The mutated spores were collected from this suspension by centrifugation, washed, and cloned on PDA agar medium. The resulting mutated spores were collected at a concentration of 1.0 × 10 7The culture medium was diluted with saline to a concentration of 1 / mL and inoculated at 1% (v / v) into a medium containing 2% by weight of Arbocel B800 (Rettenmeyer). The mixture was then cultured at 28°C for 120 hours at 125 rpm. Mutants with reduced β-glucosidase activity were selected by centrifuging the culture medium, and the supernatant was assayed for β-glucosidase activity using 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, strains with reduced β-glucosidase activity were obtained, approximately 1 / 22 of that of the parent strain PC-3-7BGL / ΔBXL, which had no reduced β-glucosidase activity. The results are shown in Table 3. Gene sequence analysis of the PC-3-7-ΔBGL / ΔBXL strain revealed a deletion of an adenine (A) 198 bases upstream from the translation start site of the BGL gene, which is thought to be the Xyr1 binding site.

[0065] <Example 2> Preparation of a mutant strain (PC-3-7ΔBGL / ΔBXL-B) with reduced BGL and BXL activities using PC-3-7ΔBGL / BXL as a parent strain 1.0 × 10 spores of the PC-3-7ΔBGL / BXL strain 7 The spores were diluted with physiological saline to a concentration of 1.0 × 10 / mL, and 0.1 mL of the diluted spore solution was inoculated into 10 mL of pre-culture medium placed in a 50 mL baffled flask shown in Table 1, and cultured for 4 hours at 28°C and 120 rpm in a shaking incubator. The culture solution was subjected to a mutagenesis treatment with NTG (1-Methyl-3-nitro-1-nitrosoguanidine) for 90 minutes. The mutated spores were collected from this suspension by centrifugation, washed, and cloned on PDA agar medium. The resulting mutated spores were collected at a concentration of 1.0 × 10 7The culture medium was diluted with saline to a concentration of 1 / mL and inoculated at 1% (v / v) into a medium containing 2% by weight of Arbocel B800 (Rettenmeyer). The mixture was then cultured at 28°C for 120 hours at 125 rpm. Mutants with reduced β-xylosidase activity were selected by centrifuging the culture medium, and the resulting supernatant was assayed for β-xylosidase activity using the method described in Reference Example 4. Enzyme activity (U) was calculated as the amount of enzyme liberating 1 mol of p-nitrophenol per minute. Mutants lacking β-xylosidase activity were identified. The results are shown in Table 3. Gene sequence analysis of the PC-3-7-ΔBGL / ΔBXL-B strain revealed a substitution of A at position 2018 with T and A at position 2065 with T from the translation start site of the BXL gene.

[0066] [Table 3]

[0067] Comparative Example 3: Viscosity measurement of Trichoderma reesei PC-3-7 strain Using the method described in Reference Example 2, the viscosity of the culture medium of Trichoderma longibrachiatum PC-3-7 was measured over time. The maximum viscosity during cultivation was 226 cP. The results are shown in Table 4. This value was set as 100% and compared with the viscosity of the culture medium of the mutant strain.

[0068] Comparative Example 4: Viscosity measurement of PC-3-7ΔBGL / BXL strain Using the method described in Reference Example 2, the viscosity of the culture medium of the PC-3-7ΔBGL / BXL strain prepared in Comparative Example 1 was measured over time. As a result, the maximum viscosity during cultivation was 230 cP. The results are shown in Table 4. It was found that there was no change compared to the Trichoderma longibrachiatum PC-3-7 strain.

[0069] Comparative Example 5: Viscosity measurement of PC-3-7BGL / ΔBXL strain Using the method described in Reference Example 2, the viscosity of the culture medium of the PC-3-7BGL / ΔBXL strain prepared in Comparative Example 2 was measured over time. As a result, the maximum viscosity during cultivation was 164 cP. The results are shown in Table 4. This was a 27.4% decrease compared to the Trichoderma reesei PC-3-7 strain.

[0070] Example 3: Viscosity measurement of PC-3-7ΔBGL / ΔBXL strain The viscosity of the culture medium of the PC-3-7ΔBGL / ΔBXL strain prepared in Example 1 was measured over time using the method described in Reference Example 2. As a result, the maximum viscosity during cultivation was 117 cP, which was 48.2% lower than that of the Trichoderma longibrachiatum PC-3-7 strain (Table 4).

[0071] Example 4 Viscosity measurement of PC-3-7ΔBGL / ΔBXL-B strain Using the method described in Reference Example 2, the P prepared in Example 2 The viscosity of the culture medium of the C-3-7ΔBGL / ΔBXL strain was measured over time. The maximum viscosity during cultivation was 131 cP, which was 42.0% lower than that of the PC-3-7 strain (Table 4).

[0072] [Table 4]

[0073] Comparative Example 6: Saccharification test using cellulase composition of Trichoderma reesei PC-3-7 strain Using the method described in Reference Example 5, two types of biomass (Arbocel B800 (trade name, Rettenmeyer) and bagasse powder) were subjected to a saccharification reaction for 24 hours at 50°C. The saccharification reaction solution was quantitatively analyzed using the method described in Reference Example 6. Focusing on the concentration of xylobiose, no production was observed with the PC-3-7 strain in either Arbocel B800 (trade name) or bagasse powder. The results are shown in Table 5.

[0074] <Comparative Example 7> Saccharification test using cellulase composition of PC-3-7BGL / ΔBXL strain Using the method described in Reference Example 5, two types of biomass (Arbocel B800 (Rettenmeyer) and bagasse powder) were subjected to a saccharification reaction at 50°C for 24 hours. The saccharification reaction solution was quantitatively analyzed using the method described in Reference Example 6. As a result, the PC-3-7BGL / ΔBXL strain liberated 3.15 g / L and 1.07 g / L of xylobiose, respectively. The results are shown in Table 5.

[0075] <Example 5> Saccharification test using cellulase composition of PC-3-7ΔBGL / ΔBXL strain Using the method described in Reference Example 5, two types of biomass (Arbocel B800 (Rettenmeyer) and bagasse powder) were subjected to a saccharification reaction for 24 hours at 50°C. The saccharification reaction solution was quantitatively analyzed using the method described in Reference Example 6. The PC-3-7ΔBGL / ΔBXL strains exhibited xylobiose release of 3.45 g / L and 1.28 g / L, respectively, which was surprisingly a 9.52% and 19.6% increase compared to the results for the PC-3-7BGL / ΔBXL strain. The results are shown in Table 5.

[0076] <Example 6> Saccharification test using cellulase composition of PC-3-7ΔBGL / ΔBXL strain Using the method described in Reference Example 5, two types of biomass (Arbocel B800 (Rettenmeyer) and bagasse powder) were subjected to a saccharification reaction for 24 hours at 50°C. The saccharification reaction solution was quantitatively analyzed using the method described in Reference Example 6. The PC-3-7ΔBGL / ΔBXL-B strains exhibited xylobiose release of 3.45 g / L and 1.28 g / L, respectively, which was surprisingly a 44.1% and 61.2% increase, respectively, compared to the results for the PC-3-7BGL / ΔBXL strain. The results are shown in Table 5.

[0077] [Table 5]

Claims

1. A cellulase composition comprising a culture medium of a mutant strain of Trichoderma longibrachiatum in which the activities of β-glucosidase and β-xylosidase are reduced compared to the parent strain before the introduction of the mutation, the culture medium having a specific activity of β-glucosidase of 0.02 U / mg-protein or less and a specific activity of β-xylosidase of 0.002 U / mg-protein or less, and the mutation is a mutation that disrupts or inhibits the expression of each of the BGL1 gene and the BXL1 gene.

2. A method for producing xylooligosaccharides, comprising hydrolyzing biomass containing xylan with the cellulase composition according to claim 1.

3. A method for reducing the viscosity of a culture medium during liquid culture of Trichoderma longibrachiatum, comprising introducing mutations into Trichoderma longibrachiatum to reduce β-glucosidase activity and β-xylosidase activity compared to those of the parent strain before the introduction of the mutations, thereby reducing the specific activity of β-glucosidase in the culture medium to 0.02 U / mg-protein or less and the specific activity of β-xylosidase to 0.002 U / mg-protein or less, wherein the mutations disrupt or inhibit the expression of the BGL1 gene and the BXL1 gene, respectively.

4. A method for producing a protein by the method according to claim 2 or 3.

5. A method for producing a cellulase composition by the method according to claim 2 or 3.

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