JP2014150745A - Mutant microorganism of genus trichoderma and use of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a mutant microorganism of the genus Trichoderma, a method for producing cellulase by using the mutant and a method for preparing the mutant.SOLUTION: The invention relates to a mutant microorganism of the genus Trichoderma of which β-glucosidase differs from the wild type β-glucosidase in that at least one amino acid residue is substituted.

Description

  The present invention relates to a mutant strain of a microorganism belonging to the genus Trichoderma with improved cellulase production, a method for producing cellulase using the mutant strain, and a method for producing the microorganism.

  Many attempts have been made to develop chemicals such as ethanol from cellulosic biomass such as agricultural waste or wood that do not compete with food and feed. For example, a method has been developed in which wood is saccharified with sulfuric acid to form a monosaccharide such as glucose or xylose, and then the monosaccharide is fermented with yeast or bacteria to obtain ethanol (Non-Patent Documents 1 to 3). However, this method has a problem in that it is difficult to recover the used strong acid and reuse it, or in terms of the environment in the case of waste treatment.

  Therefore, a method using an enzyme has been developed as a milder and more environmentally friendly method for saccharifying biomass. In this method, cellulase or hemicellulase produced by various microorganisms is generally used. Cellulase is composed of enzyme proteins having various mechanisms of action.

  Enzymes that act on cellulose in biomass include endoglucanase (EG) that acts on amorphous cellulose and breaks the cellulose chain, and sugars in cellobiose units from the ends of crystalline and amorphous cellulose fibers. There is cellobiose hydrolase (CBH) to excise. Moreover, there exists (beta) -glucosidase (BGL) which hydrolyzes the cellobiose produced | generated by the effect | action of these enzymes, and produces | generates glucose. On the other hand, examples of hemicellulases that act on hemicellulose, which is a biomass component, include xylanase and β-xylosidase.

  Cellulose biomass saccharifying enzymes include the above-mentioned enzymes, but in reality, each of these enzymes is further composed of a number of components classified by the carbohydrate hydrolase family. Is very complex.

  As cellulase-producing bacteria, microorganisms belonging to the genus Trichoderma, for example, Trichoderma reesei or Trichoderma violet, are well known and have been used industrially (Non-patent Document 4). However, as research on biomass saccharification using cellulase produced by a strain belonging to the genus Trichoderma proceeds, β-glucosidase activity has other enzyme activities (endoglucanase activity, cellobiohydrolase activity) as the biggest drawback of the cellulase. It was found to be relatively low compared to Accordingly, Cellic CTec, Cellic CTec2 (Novozyme) and Accelerase 1500 (Genencor) have been developed as improved products with enhanced β-glucosidase activity.

  Moreover, in order to improve the protein production amount of Trichoderma reesei, the high expression or the defect | deletion of the factor which is concerned in protein production, such as a transcriptional regulatory factor and a signal transduction factor, is performed (patent documents 1-3).

International Publication No. 2011/151512 International Publication No. 2011/151513 International Publication No. 2011/151515

Jonathan R. Mielenz, (2001) Current Opinion in Microbiology, p324-329. Daisuke Taneda, November issue of OHM 2006, Ohmsha, p42-45 Daisuke Taneda, Industrial Forefront of Biorefinery Technology, 2008, CMC Publishing, Chapter 2, 3. Concentrated sulfuric acid bioethanol production process Yasushi Morikawa, Cutting Edge of Cellulose Utilization Technology, 2008, CMC Publishing, p362-376

  However, even with the amount of cellulase produced by microorganisms belonging to the genus Trichoderma, an additional amount of cellulase is required to completely saccharify the cellulosic biomass of the insoluble solid substrate. Further, from the viewpoint of enzyme production cost, it is desired to improve cellulase productivity of microorganisms belonging to the genus Trichoderma. However, microorganisms belonging to the genus Trichoderma have undergone complicated expression control and activity control, and it has been difficult to improve the expression level using conventional techniques.

  Therefore, an object of the present invention is to provide a mutant strain of a microorganism belonging to the genus Trichoderma having improved cellulase productivity, a method for producing cellulase using the mutant strain, and a method for producing the mutant strain.

  The present inventors have found that by introducing a mutation into a wild-type β-glucosidase of a microorganism belonging to the genus Trichoderma, the obtained mutant strain has improved cellulase productivity as compared to the wild strain. Completed the invention.

That is, the present invention is as follows.
1. A mutant strain of a microorganism belonging to the genus Trichoderma, wherein at least one amino acid residue is substituted in the amino acid sequence of wild-type β-glucosidase.
2. The microorganism belonging to the genus Trichoderma is one selected from the group consisting of Trichoderma reesei, Trichoderma viride, Trichoderma longibratiatum, and the first variant selected from the group consisting of Trichoderma longibrachiatum. .
3. 3. The mutant strain according to item 2 above, wherein the microorganism belonging to the genus Trichoderma is Trichoderma reesei strain QM9414.
4). 4. The mutant according to any one of the preceding items 1 to 3, wherein the amino acid sequence of wild-type β-glucosidase is the amino acid sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 7.
5. 2. The mutant according to item 1 above, wherein the amino acid sequence of wild-type β-glucosidase has at least 50% or more homology with the amino acid sequence of Trichoderma reesei strain QM9414.
6). 6. The mutant according to item 5 above, wherein the amino acid sequence of wild-type β-glucosidase has at least 70% homology with the amino acid sequence of Trichoderma reesei strain QM9414.
7). 6. The mutant according to item 5 above, wherein the amino acid sequence of wild-type β-glucosidase has at least 80% homology with the amino acid sequence of Trichoderma reesei strain QM9414.
8). 8. The mutant strain according to any one of the preceding items 1 to 7, wherein at least one amino acid is substituted at the 407th to 411st amino acids from the N-terminal of the amino acid sequence of wild type β-glucosidase.
9. 9. The mutant according to any one of 1 to 8 above, wherein the amino acid residue substitution comprises at least one selected from the group consisting of D407P, G408W, V409F, N410H and V410F.
10. 10. The mutant according to item 9 above, wherein the amino acid residue substitution is V409F.
11. A method for producing cellulase, comprising producing cellulase by culturing the mutant strain according to any one of items 1 to 10.
12 11. A production method such as from cellulosic biomass, comprising a step of saccharifying cellulosic biomass with cellulase produced by the mutant strain according to any one of 1 to 10 above.
13. 13. A method for producing ethanol, comprising a step of fermenting a sugar obtained by the method according to item 12 above.
14 A method for producing a mutant strain of a microorganism belonging to the genus Trichoderma, wherein at least one amino acid substitution is introduced in the amino acid sequence of wild-type β-glucosidase.
15. A method for producing a mutant strain of a microorganism belonging to the genus Trichoderma, wherein a single-base substitution is introduced in a wild-type β-glucosidase gene.

  In the mutant strain of the microorganism belonging to the genus Trichoderma of the present invention, at least one amino acid substitution is introduced in the amino acid sequence of wild-type β-glucosidase, so that the productivity of cellulase is greatly improved as compared to the wild strain. . Ethanol can be efficiently produced by producing cellulase at high yield using the mutant strain and fermenting sugar obtained from cellulosic biomass using the cellulase.

It is a schematic diagram showing a cellulase-induced expression model of Trichoderma reesei. It is a figure which shows the expression level of the beta-glucosidase gene in Trichoderma reesei. It is a figure which shows construction | assembly of the beta-glucosidase (bgl2) mutant of Trichoderma reesei strain QM9414, and the beta-glucosidase (bgl2) destruction strain of Trichoderma reesei strain QM9414. It is an electrophoresis photograph which shows the result of having used culture supernatant of Trichoderma reesei strain QM9414, a bgl2 mutant strain, and a bgl2 destruction strain for SDS-PAGE. It is a figure which shows the result of having measured the decomposition | disassembly activity in the culture supernatant of Trichoderma reesei strain QM9414, a bgl2 mutant strain, and a bgl2 destruction strain. It is a figure which shows the result of having measured the protein concentration in the culture supernatant of Trichoderma reesei strain QM9414, a bgl2 mutant strain, and a bgl2 destruction strain.

  The present invention provides a mutant strain of a microorganism belonging to the genus Trichoderma, in which at least one amino acid substitution is introduced in the amino acid sequence of wild-type β-glucosidase.

  The microorganism belonging to the genus Trichoderma includes all microorganisms classified into the genus Trichoderma among filamentous fungi. The microorganism belonging to the genus Trichoderma may be a wild strain of a conventionally known microorganism belonging to the genus Trichoderma, or a mutant derived from the wild strain, or a mutation introduced by mutagen treatment such as UV irradiation or nitroso compound treatment. Strains may be used.

  Examples of the microorganisms belonging to the genus Trichoderma, e.g., Trichoderma aggressivum, Trichoderma atroviride, Trichoderma asperellum, Trichoderma aureoviride, Trichoderma austrokoningii, Trichoderma brevicompactum, Trichoderma candidum, Trichoderma caribbaeum var. aequatoriale, Trichoderma caribbaeum var. Caribbaeum, Trichoderma catoptron, Trichoderma cremeum, Trichoderma ceramicum, Trichoderma cerinum, Trichoderma chlorosporum, Trichoderma chromospermum, Trichoderma cinnamomeum, Trichoderma citrinoviride, Trichoderma crassum, Trichoderma cremeum, Trichoderma dingleyeae, Trichoderma dorotheae, Trichoderma effusum, Trichoderma erinaceum, Trichoderma est nicum, Trichoderma fertile, Trichoderma gelatinosus, Trichoderma ghanense, Trichoderma hamatum, Trichoderma harzianum, Trichoderma helicum, Trichoderma intricatum, Trichoderma konilangbra, Trichoderma koningii, Trichoderma koningiposis, Trichoderma longibrachiatum, Trichoderma longipile, Trichoderma minutisporum, Trichoderma oblongisporum, Trichoderm ovalisporum, Trichoderma petersenii, Trichoderma phyllostahydis, Trichoderma, Trichoderma, Trichoderma, Trichoderma, Trichoderma, Trichoderma, Trichoderma, Trichoderma piluliferum, Trichoderma pleuroticola, Trichoderma pleurotum, Trichoderma polysporum, Trichoderma pseudokoningii, Trichoderma pubescens, Trichoderma reesei, Trichoderma rogersonii, Tric oderma rossicum, Trichoderma saturnisporum, Trichoderma sinensis, Trichoderma sinuosum, Trichoderma sp. MA 3642, Trichoderma sp. PPRI 3559, Trichoderma spirale, Trichoderma stramineum, Trichoderma strigosum, Trichoderma stromaticum, Trichoderma surrotundum, Trichoderma taiwanense, Trichoderma thailandcum, Trichoderma thelephorucolum, Trichoderma theobromicola, Trichoderma tomentosum, Trichoderma, Trichoderma, Trichoderma, Trichoderma velutinum, Trichoderma virens, Trichoderma vi Ride and Trichoderma viridecens. Among these, Trichoderma reesei, Trichoderma viride, Trichoderma atrobilide, or Trichoderma longibratiatum, preferably Trichoderma longibratumi, from the viewpoint of the secretory production ability of cellulase. Is more preferable.

  Examples of Trichoderma reesei include Trichoderma reesei strain QM9414 and Trichoderma reesei strain QM6a.

  Examples of wild-type β-glucosidase include intracellular β-glucosidase and secreted (extracellular) β-glucosidase, and intracellular β-glucosidase is preferable. Examples of the intracellular β-glucosidase include BGLII (or Cel1A), Cel1b, Cel3c, Cel3d, and Cel3h as Trichoderma reesei intracellular β-glucosidase. Among these, BGLII is preferable.

  As shown in FIG. 1, Trichoderma reesei, which is a microorganism belonging to the genus Trichoderma, recognizes cellulose and cellobiose (disaccharide) by cellulase (extracellular β-glucosidase) that is constitutively produced outside the cell. ) To produce soluble cellooligosaccharides. It is estimated that the cellooligosaccharide is taken into the microbial cells and inducers such as Soforus are produced, and cellulases outside the microbial cells are produced in large quantities by the inducers (FIG. 1).

  Trichoderma reesei produces five types of β-glucosidase [BGLII (also called Cel1A), Cel1b, Cel3c, Cel3d, Cel3h] in the cell. As shown in FIG. 2, among these intracellular β-glucosidases, β-glucosidase II (BGLII) has a higher expression level in the cells than other β-glucosidases.

  Therefore, β-glucosidase II (BGLII) is presumed to be a very important enzyme involved in cellobiose degradation, that is, the growth of bacterial cells, and such an enzyme is not usually targeted for introduction of amino acid mutations. In such a situation, in the present invention, it was found that by introducing an amino acid substitution into the intracellular β-glucosidase II, the production of extracellular enzymes important for enzymatic saccharification of cellulosic biomass increased. This result is an unpredictable result.

  Examples of the amino acid sequence of wild-type β-glucosidase include the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 7.

  The β-glucosidase shown in SEQ ID NO: 1 is the amino acid sequence of β-glucosidase derived from Trichoderma reesei strain QM9414 (ATCC26921).

  SEQ ID NO: 2 is the amino acid sequence of β-glucosidase derived from Trichoderma longibratiatum. SEQ ID NO: 3 is the amino acid sequence of β-glucosidase derived from Trichoderma viriens. SEQ ID NO: 4 is the amino acid sequence of β-glucosidase derived from Trichoderma atroviride. SEQ ID NO: 5 is the amino acid sequence of β-glucosidase derived from Trichoderma herjanum. SEQ ID NO: 6 is the amino acid sequence of β-glucosidase derived from Trichoderma viride. SEQ ID NO: 7 is the amino acid sequence of β-glucosidase derived from Trichoderma herjanum.

  The identity (homology) in amino acid sequences between these β-glucosidases is 91% between SEQ ID NO: 1 and SEQ ID NO: 2, 90% between SEQ ID NO: 1 and SEQ ID NO: 3, SEQ ID NO: 1 and 89% between SEQ ID NO: 4, 87% between SEQ ID NO: 1 and SEQ ID NO: 5, 86% between SEQ ID NO: 1 and SEQ ID NO: 6, and 86% between SEQ ID NO: 1 and SEQ ID NO: 7.

  In the mutant strain of the present invention, as the amino acid sequence of wild-type β-glucosidase, one or more amino acids were deleted, substituted or added in the β-glucosidase amino acid sequence (SEQ ID NO: 1) of Trichoderma reesei strain QM9414. A polypeptide comprising an amino acid sequence and having a β-glucosidase function, and the amino acid sequence represented by SEQ ID NO: 1, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, and still more preferably A polypeptide comprising an amino acid sequence having a homology of 80% or more, particularly preferably 90% or more, most preferably comprising an amino acid sequence having a homology of 95%, 96%, 97%, 98% and 99% or more, A polypeptide having the function of β-glucosidase is also included. In particular, those that can be aligned with β-glucosidase of Trichoderma reesei strain QM9414 are preferred.

  The mutant strain of the present invention preferably has amino acids 2 to 453, more preferably amino acids 380 to 440, still more preferably amino acids 400 to 416, particularly preferably amino acids from the N-terminus of the amino acid sequence of wild-type β-glucosidase. Is preferably substituted with at least one amino acid residue in the 407th to 411st amino acids, and most preferably the 409th amino acid residue is substituted.

  As specific examples of amino acid residue substitution, for example, substitution of V409F / P / W, A409F / P / W, and C409F / P / W is preferable, V409F / P / W is more preferable, and V409F is more preferable. In addition to the above-described substitutions, one to several other amino acid substitutions, deletions, and / or as required, as long as the cellulase productivity of the mutant strain of the present invention is not substantially adversely affected. Insertion can be included.

  In the present invention, the nomenclature used to define the substitution of amino acid residues is identified by the amino acid before substitution and its position, and the amino acid after substitution. That is, “V number F” indicates that the [number] -th V is replaced with F, and the substituted amino acid delimited by a slash indicates that the amino acid is replaced with any of the amino acids. That is, “V number F / T” indicates a mutation in which the [number] -th V is replaced with F or T.

  The mutant strain of the present invention can be obtained by any method known in the art such as gene targeting by homologous recombination [for example, Dan Burke et al. (Translated by Junichi Ohya et al.) Yeast Gene Experiment Manual, Maruzen Co., Ltd. Issued by Zinnia Rahman et al., Appl Microbiol Biotechnol (2009) 82, p899-908].

As a specific example, a method of gene targeting by homologous recombination will be specifically described. Gene targeting by homologous recombination is performed by the following procedures (1) to (4).
(1) An upstream region and a downstream region of a wild-type β-glucosidase gene (bgl) of a microorganism belonging to the genus Trichoderma are cloned into an E. coli plasmid vector, and a mutation is introduced into the target region.
As a method for transforming a plasmid into Escherichia coli, a method commonly used in the field of genetic engineering can be used. For example, Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory (1989) Can be mentioned.
Examples of E. coli plasmid vectors include pBluescript® II, pT7BlueT, pAG, pUC and the like. The introduction of the mutation into the target region into the wild-type β-glucosidase gene can be performed using, for example, QuikChange II Site-Directed Mutagenesis Kit manufactured by Stratagene. As the mutation, substitution of at least one base is preferable, and substitution of one base is more preferable.
(2) A selection marker gene for transforming a microorganism belonging to the genus Trichoderma is introduced into the Escherichia coli plasmid vector into which bgl introduced with the mutation obtained in (1) has been cloned.
Examples of selectable marker genes include genes that confer resistance to drugs such as hygromycin, zeocin, kanamycin, chloramphenicol, and G418, and nutrients such as uracil synthase, leucine synthase, adenine synthase, or lysine synthase Examples include genes that complement requirements.
(3) A fragment of “bgl upstream region-mutant bgl-bgl downstream region-selection marker-bgl downstream region” is excised from the plasmid by restriction enzyme treatment.
(4) Using the excised fragment, a microorganism belonging to the genus Trichoderma is transformed to obtain a mutant in which at least one amino acid residue is substituted in the amino acid sequence of wild-type β-glucosidase.

  The mutant strain produced in this way exhibits excellent cellulase productivity. Culturing the mutant strain for producing cellulase can be carried out by those skilled in the art under commonly used culture conditions.

  Examples of the sugar source used for the culture include various celluloses such as Avicel, filter paper powder, and biomass containing cellulose, and examples of the nitrogen source include polypeptone, gravy, CSL, and soybean meal.

  In addition, components required for producing the target cellulase can be added to the medium. Further, xylanase can be increased by adding various xylan components to the medium.

  For the culture of these strains, various culture methods such as shaking culture, stirring culture, stirring shaking culture, stationary culture, and continuous culture can be adopted, and shaking culture or stirring culture is preferred. The culture temperature is usually 20 ° C. to 35 ° C., preferably 25 ° C. to 31 ° C., and the culture time is usually 4 to 10 days, preferably 4 to 9 days.

  The cellulase produced in the mutant strain in this way can be recovered from outside the mutant strain. Cellulase can be measured by directly analyzing a cell lysate or culture supernatant sample by dodecyl sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, which are known in the art.

  Cellulase produced during cell culture is secreted in the medium and purified or separated, for example, by removing unwanted components from the cell medium. Cellulase purification methods include, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, ethanol precipitation, reverse phase HPLC, cation exchange chromatography on silica or DEAE, chromatofocusing, SDS-PAGE. And ammonium sulfate precipitation method and gel filtration method. These methods can be used alone or in combination.

  Cellulose biomass can be saccharified using the cellulase produced in the mutant strain of the present invention. The cellulosic biomass may be a herbaceous plant, a woody plant, or a processed product or waste thereof.

  Examples of herbaceous plants include, but are not limited to, rice, Elianthus, wheat, sugarcane, reed, Japanese pampas grass, corn, sorghum, napiergrass, switchgrass, and Miscanthus.

  Examples of woody plants include cedars, eucalyptus, cypress, pine, rice eel, poplar, birch, willow, scallop, oak, oak, shii, beech, acacia, bamboo, sasa, oil palm and sago palm. However, it is not limited to these.

  Generally, biomass is difficult to undergo saccharification by cellulase as it is. For this reason, before performing saccharification by a cellulase, it is preferable to perform the process for changing a cellulosic biomass into the form which is easy to receive the saccharification by an enzyme.

The pretreatment method for cellulosic biomass is not particularly limited. For example, mechanochemical pulverization method, hydrothermal treatment, alkali treatment [for example, caustic soda (NaOH), KOH, Ca (OH) ₂ , Na ₂ SO ₃ , NaHCO _{3 3} , NaHSO ₃ , Mg (HSO ₃ ) ₂ , Ca (HSO ₃ ) ₂ , ammonia (NH ₃ , NH ₄ OH), etc.], dilute sulfuric acid treatment, steam explosion treatment, solvolysis treatment and microbial treatment, and composites thereof Processing.

  By performing such pretreatment, the cellulose present in the raw material biomass remains on the solid residue side depending on the treatment method and treatment conditions, or it is hydrolyzed and transferred to the solubilized fraction. To do.

  A method of hydrolyzing cellulose and collecting it in a solubilized fraction as oligosaccharides or monosaccharides by setting pretreatment methods and conditions has been studied, but generally when pretreatment conditions become severe, Various components are excessively decomposed, and as a result, inhibitors in the alcohol fermentation process such as acetic acid, formic acid, furfural, hydroxymethylfurfural, and lignin decomposition products are generated, which is not preferable.

  Therefore, in order to recover the sugar component constituting the cellulose in the biomass as a monosaccharide in a high yield in the saccharified solution, it is necessary to leave as much cellulose as possible on the solid residue side by the pretreatment, and to make the remaining cellulose as enzyme as possible. It is preferable to balance the form of being easily subjected to saccharification.

  Thus, after pre-processing raw material biomass, it solid-liquid-separates by filtration, centrifugation, etc., and a pre-processing solid substance and filtrate are obtained. Here, various methods can be adopted as a method for quantitatively analyzing the raw material biomass and cellulose in the pretreated solid, but the National Analytical Laboratory Procedure of the US NREL (National Renewable Energy Laboratory), which is used as a standard formulation worldwide ( A method in conformity with (LAP) Determination of Structural Carbohydrates and Lignin in Biomass is preferred.

  In the above method, the raw material biomass and the pretreated solid are hydrolyzed with sulfuric acid, and then the content of constituent sugars such as glucose, xylose, mannose, galactose, etc. contained in these samples are analyzed and quantified by the HPLC method to determine the content rate. It is. In this method, the obtained glucose is regarded as derived from cellulose, and other components such as xylose, mannose, galactose and the like are derived from hemicellulose, and each content is determined.

  The conditions for saccharifying the cellulosic biomass thus pretreated with the cellulase produced from the mutant of the present invention are as follows. The substrate concentration is usually about 1 to 20 (w / v)%, preferably about 5 to 10 (w / v)%. The pH is usually in the range of 3-9, preferably 4-6. The temperature is 10 to 80 ° C, preferably 40 to 60 ° C. The enzyme concentration is 1 to 20 mg / g-biomass weight, preferably 1 to 10 mg / g-biomass weight, based on the amount of protein per dry matter weight of the biomass pretreatment product.

  Under these conditions, for example, the saccharification reaction can proceed under shaking or standing. In this saccharification reaction, a bactericidal agent such as sodium azide may be added for the purpose of preventing contamination with various bacteria. In this case, it is preferable to select a compound and a concentration that do not adversely affect the subsequent fermentation process of the saccharified solution. The saccharification rate can be determined by analyzing the product in the saccharified solution by various reducing sugar quantification methods and HPLC methods.

  By fermenting the saccharified solution thus obtained, ethanol can be produced. A general method can be applied to the production of ethanol from the saccharified solution. Examples of the microorganism used for producing ethanol include, but are not limited to, microorganisms belonging to the genus Saccharomyces, the genus Zymomonas, the genus Pichia, the genus Zymobacter, the genus Corynebacterium, the genus Kluyveromyces, and the genus Escherichia.

  It is also possible to use a microorganism incorporating a gene related to a metabolic system for converting sugar to ethanol, or a mutant thereof. By inoculating and culturing these microorganisms in a saccharified solution, ethanol can be produced.

  The saccharified solution used preferably has a sugar concentration of 3 to 15% and a pH of 3 to 7. The culture temperature is preferably 20 to 40 ° C. Ethanol fermentation can be carried out either batchwise or continuously. Ethanol fermentation can be performed simultaneously with the saccharification reaction of the above-mentioned cellulosic biomass (parallel double fermentation). In this case, the conditions such as the sugar concentration, temperature, pH, etc. that give the highest productivity as a whole may be appropriately selected by combining the conditions for the saccharification reaction and the conditions for ethanol fermentation.

[Example 1]
As shown in FIG. 3, a bgl2 mutant strain and a bgl2 disruption strain of Trichoderma reesei strain QM9414 were prepared.
(1) Preparation of vector containing bgl2 mutant gene β-glucosidase gene (bgl2) (SEQ ID NO: 8) derived from Trichoderma reesei QM9414 strain and chromosomal DNA (SEQ ID NO: 9) containing its upstream and downstream regions were transformed into plasmid vector pUC118. Inserted into. Using the obtained plasmid vector as a template, by using the oligonucleotide primers described below, by PCR, the 1298th base G from the 5 ′ end of bgl2 was replaced with base T, and a single base mutation was introduced to obtain bgl2 A mutant gene was prepared. This mutation is a mutation that substitutes F, which is the 409th amino acid from the N terminus of QM9414 β-glucosidase (BGLII) (SEQ ID NO: 1). The nucleotide sequence of the obtained DNA fragment containing the bgl2 mutant gene was sequenced to confirm that there were no PCR errors. For determination of the base sequence, CEQ ^™ 2000XL DNA Analysis System (BECKMAN COULTER) was used, and details of the operation were in accordance with the attached instruction manual.

For bgl2 mutation introduction (sense primer):
5′-ggacgggttcaacgtcaaggggtactttgcctg-3 ′ (SEQ ID NO: 10)
For bgl2 mutation introduction (antisense primer):
5′-acgttgaacccgtccagctccacggcggtaacc-3 ′ (SEQ ID NO: 11)

  The PCR reaction conditions for amplifying the DNA fragment consisted of a three-stage reaction at 98 ° C. for 10 seconds, 50 ° C. for 15 seconds, and 72 ° C. for 5 minutes and 20 seconds, and this was repeated 30 cycles. For PCR, PrimeSTAR (registered trademark) HS DNA polymerase (Takara Bio Inc.) was used. The PCR reaction conditions for mutagenesis consisted of a three-stage reaction of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 45 seconds, which was repeated 30 cycles.

(2) Introduction of selectable marker gene The pyr4 derived from Trichoderma reesei, which is the selectable marker gene, and the plasmid pUpyr4 containing its upstream and downstream regions were excised by SalI treatment. Thereafter, pyr4 was inserted into the restriction enzyme site ApaI in the downstream region of bgl2 of the DNA fragment containing the bgl2 mutant gene cloned in (1). For determination of the base sequence, CEQ ^™ 2000XL DNA Analysis System (BECKMAN COULTER) was used, and details of the operation were in accordance with the attached instruction manual.

(3) Preparation of vector containing bgl2 disruption gene β-glucosidase gene (bgl2) (SEQ ID NO: 8) of Trichoderma reesei strain QM9414 and a chromosomal DNA (SEQ ID NO: 9) containing the upstream and downstream regions thereof were transferred to plasmid vector pUC118. Inserted. In the obtained plasmid, the selection marker pyr4 obtained in (2) above was inserted between the restriction enzyme sites XhoI present in the bgl2 gene and in the downstream region of bgl2.

(4) Transformation of Trichoderma reesei Each constructed vector was introduced into Trichoderma reesei QM9414 strain (WT, wild type) by protoplast PEG method. The transformant was selected with a selective medium for acetamide utilization ability. The composition of the medium was as follows.

20 mg / mL glucose, 1 M sorbitol, 2% agar, 15 mg / mL KH ₂ PO ₄ (pH 5.5), 0.6 mg / mL CaCl ₂ · 2H ₂ O, 12.5 mM CsCl ₂ , 0.6 mg / mL MgSO ₄ 7H ₂ O, 10 mM Acetamide, 5 μg / mL FeSO ₄ .7H ₂ O, 1.6 μg / mL MnSO ₄ .H ₂ O, 1.4 μg / mL ZnSO ₄ .7H ₂ O, 2 μg / mL CoCl ₂

  Confirm that the bgl2 gene mutation vector and the bgl2 gene disruption vector constructed in (1), (2) and (3) are homologously integrated into the bgl2 conformation on the chromosome of the obtained transformant. Therefore, colony PCR was performed using the oligonucleotide primers described below, and a strain that showed correct DNA amplification was used as a transformant. For the bgl2 gene mutant, direct sequencing was performed to confirm whether or not the mutation was introduced into the bgl2 gene on the chromosome.

To confirm the expression of the resulting transformants β- glucosidase (BglII), 10 ⁷ spores were inoculated into 300mL Erlenmeyer flask containing Trichoderma reesei liquid medium 50 mL. The composition of the medium was as follows.
1% Avicel, 0.14% (NH ₄ ) ₂ SO ₄ , 0.2% KH ₂ PO ₄ , 0.03% CaCl ₂ .2H ₂ O, 0.03% MgSO ₄ .7H ₂ O, 0.1 % Bacto Polypepton, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element, 50 mM tartrate buffer (pH 4.0)

The composition of the trace element was as follows.
6 mg H ₃ BO ₃ , 26 mg (NH ₄ ) ₆ MO ₇ O ₂₄ · 4H ₂ O, 100 mg FeCl ₃ · 6H ₂ O, 40 mg, CuSO ₄ · 5H ₂ O, 8 mg MnCl ₂ · 4H ₂ O, 200 mg ZnCl ₂ Made up to 100 ml with distilled water.

  The mycelium was collected by filtering the culture medium cultured at 220 rpm and 28 ° C. for 3 days, and treated with 2000 rpm for 30 seconds at 4 ° C. in the presence of zirconia beads using a multi-bead shocker (manufactured by Yasui Machinery Co., Ltd.). Repeated once (interval 1 minute). The supernatant obtained by centrifuging the obtained cell disruption solution at 4 ° C. was used as a cell-free extract. It was confirmed that the BGL2 activity of the cell-free extract was cellobiose degrading activity and the activity was lost in the transformant. Cellobiose decomposition activity was performed by detecting glucose produced by a 30-minute reaction with a substrate concentration of 21 mM, pH 6.0 using a glucose kit test wako manufactured by Wako Pure Chemicals, using celloose manufactured by Sigma.

[Example 2]
Trichoderma reesei QM9414 strain and (WT) prepared in Example 1, planted in 300mL Erlenmeyer flask containing QM9414 Bgl2 mutants and QM9414 Bgl2 respective spores 10 ⁷ to Trichoderma reesei liquid medium (1% Avicel) destruction strain 50mL Fungus. After culturing at 220 rpm and 28 ° C. for 7 days, the culture solution was centrifuged at 3000 rpm for 15 minutes, the separated culture supernatant was further filtered with Miracloth (Cosmo Bio), and the obtained filtrate was used as an enzyme solution. FIG. 4 shows 20 μL of the obtained enzyme solution subjected to SDS-PAGE.

  About the obtained enzyme liquid, CMC decomposition | disassembly activity (CMCase activity) was measured. For CMC degradation activity, carboxymethylcellulose (Low viscosity) manufactured by Sigma is used, and the reducing sugar produced by the reaction at a substrate concentration of 1 (w / v)%, pH 5.0, 50 ° C. for 15 minutes is used by reduction of copper oxide. It was quantified and measured with glucose as a standard by a quantitative method (Somogy Nelson method). In addition, the amount of protein was measured using Bio-rad Laboratories, Inc. Was quantified using Bovine Gamma Globulin as a standard using the Quick Start Bradford Protein Assay kit. These enzyme activity values and protein amounts are shown in FIG. 5 and FIG.

  As shown in FIG. 4, it was found that the Trichoderma reeseiQM9414 bgl2 mutant strain has a stronger intensity of each protein band than the Trichoderma reeseiQM9414 strain (WT).

  Further, as shown in FIG. 5, the Trichoderma reeseiQM9414 bgl2 mutant has a CMC degrading activity of 1.5 times or more and a protein production amount of 1.5 times that of the Trichoderma reeseiQM9414 strain (WT). From the above enhancement, it was revealed that the productivity of the cellulase enzyme protein was remarkably improved.

The sequence of each SEQ ID NO is as follows.
SEQ ID NO: 1: Amino acid sequence of Trichoderma reesei derived BGL II SEQ ID NO: 2: Amino acid sequence of β-glucosidase derived from Trichoderma longibratiatum SEQ ID NO: 3: Amino acid sequence of β-glucosidase derived from Trichoderma virens SEQ ID NO: 4: Derived from Trichoderma atrobilide The amino acid sequence of β-glucosidase of SEQ ID NO: 5: The amino acid sequence of β-glucosidase derived from Trichoderma herjanum SEQ ID NO: 6: The amino acid sequence of β-glucosidase derived from Trichoderma viridae SEQ ID NO: 7: The amino acid sequence of β-glucosidase derived from Trichoderma herjanum No. 8: Base sequence of β-glucosidase gene (bgl2) derived from Trichoderma reesei QM9414 strain SEQ ID NO: 9 β-Glu derived from Trichoderma reesei QM9414 strain Oxidase gene (Bgl2) and its upstream nucleotide sequence SEQ ID NO of the downstream region 10: Bgl2 mutagenic sense primer nucleotide sequence SEQ ID NO 11: Bgl2 mutagenic nucleotide sequence of the antisense primer

  The mutant strain of the present invention is very useful industrially because cellulase productivity is enhanced in the same nutrient source as the conventional strain. Further, it is very useful in that high expression of a heterologous protein using a cellulase gene promoter can be expected with the mutant strain as a host.

Claims (15)

  A mutant strain of a microorganism belonging to the genus Trichoderma, wherein at least one amino acid residue is substituted in the amino acid sequence of wild-type β-glucosidase.   The microorganism belonging to the genus Trichoderma is selected from the group consisting of Trichoderma reesei, Trichoderma viride and Trichoderma longibrachiatum, wherein the mutation is one selected from the group consisting of Trichoderma longibrachiatum. stock.   The mutant strain according to claim 2, wherein the microorganism belonging to the genus Trichoderma is Trichoderma reesei strain QM9414.   The mutant according to any one of claims 1 to 3, wherein the amino acid sequence of wild-type β-glucosidase is the amino acid sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 7.   The mutant according to claim 1, wherein the amino acid sequence of wild-type β-glucosidase has at least 50% or more homology with the amino acid sequence of Trichoderma reesei strain QM9414.   The mutant according to claim 5, wherein the amino acid sequence of wild-type β-glucosidase has at least 70% homology with the amino acid sequence of Trichoderma reesei strain QM9414.   The mutant according to claim 5, wherein the amino acid sequence of wild-type β-glucosidase has at least 80% homology with the amino acid sequence of Trichoderma reesei strain QM9414.   The mutant strain according to any one of claims 1 to 7, wherein at least one amino acid is substituted at the 407th to 411st amino acids from the N-terminal of the amino acid sequence of wild-type β-glucosidase.   The mutant according to any one of claims 1 to 8, wherein the amino acid residue substitution comprises at least one selected from the group consisting of D407P, G408W, V409F, N410H and V410F.   The mutant according to claim 9, wherein the amino acid residue substitution is V409F.   A method for producing cellulase, comprising producing cellulase by culturing the mutant strain according to any one of claims 1 to 10.   The manufacturing method from cellulosic biomass including the process of saccharifying cellulosic biomass with the cellulase which the mutant of any one of Claims 1-10 produces.   The manufacturing method of ethanol including the process of fermenting the saccharide | sugar obtained by the method of Claim 12.   A method for producing a mutant strain of a microorganism belonging to the genus Trichoderma, wherein at least one amino acid substitution is introduced in the amino acid sequence of wild-type β-glucosidase.   A method for producing a mutant strain of a microorganism belonging to the genus Trichoderma, wherein a single-base substitution is introduced in a wild-type β-glucosidase gene.