WO2019230860A1 - トリコデルマ属糸状菌変異株およびタンパク質の製造方法 - Google Patents
トリコデルマ属糸状菌変異株およびタンパク質の製造方法 Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Definitions
- the present invention relates to a mutant strain of Trichoderma filamentous fungi having improved protein production ability and a method for producing a protein using the mutant strain.
- Trichoderma filamentous fungi are known to have high protein-producing ability, and so far, production of proteins using the same filamentous fungi has been studied. Trichoderma filamentous fungi produce cellulases classified as saccharifying enzymes, among other proteins, using cellulose, lactose, cellobiose and the like as inducers.
- Non-Patent Document 1 among factors controlling cellulase production of Trichoderma filamentous fungi, Trichoderma filamentous fungi having high cellulase producing ability by reducing the function of Cre1 which is a transcription factor that suppresses cellulase production. Mutant strains have been acquired.
- Non-patent document 2 uses lactose or cellulose as an inducer when the sugar transporter of Trichoderma reesei is deleted. It is described that the production amount of cellulase at the time decreases.
- the transcription factor that controls the protein production of Trichoderma filamentous fungi has been elucidated, but this is considered to be only part of the control mechanism. Therefore, in the present invention, a novel mechanism for controlling the protein production of Trichoderma filamentous fungi is searched for, obtaining a mutant strain of Trichoderma filamentous fungus with enhanced protein production ability, and a protein using the Trichoderma filamentous fungus mutant strain It is an object to provide a manufacturing method.
- the present inventor believes that if a gene capable of increasing the protein production by gene modification can be identified, the production amount of the protein of Trichoderma filamentous fungus can be further improved. It is possible to improve protein productivity and ⁇ -glucosidase productivity by culturing a mutant strain of Trichoderma filamentous fungus in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 has been reduced by modification.
- the headline and the present invention were completed.
- the present invention includes the following (1) to (6).
- (1) A mutant strain of Trichoderma fungus, in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is reduced.
- (2) The mutant strain of Trichoderma filamentous fungus according to claim 1, wherein in the amino acid sequence represented by SEQ ID NO: 2, at least the amino acid residue after the 413rd amino acid from the N-terminal side is deleted.
- (3) A method for producing a protein, comprising a step of culturing the mutant strain of Trichoderma filamentous fungus according to (1) or (2).
- (4) A method for producing cellulase, comprising a step of culturing a mutant strain of Trichoderma filamentous fungus according to (1) or (2).
- the mutant strain of Trichoderma spp. Described in (1) or (2) is cultured in a medium containing at least one kind or two or more kinds of inducers selected from the group consisting of lactose, cellulose and xylan.
- the manufacturing method of the cellulase as described in (4) including a process.
- a method for producing sugar from cellulose-containing biomass comprising the following steps: Step a: Step of culturing a mutant strain of Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is reduced to produce cellulase Step b: Using the cellulase obtained in step a And the manufacturing method of saccharide
- the mutant strain of Trichoderma filamentous fungi in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 has been reduced has a protein production ability as compared with Trichoderma filamentous fungi in which the function of the polypeptide has not decreased. improves. Furthermore, when the produced protein is cellulase in particular, an unexpected effect of improving various specific activities of cellulase can be obtained.
- the present invention is characterized in that the protein production ability is further enhanced by introducing a mutation into the parent strain of Trichoderma filamentous fungi, which is originally a microorganism excellent in protein production ability. Therefore, the parent strain of Trichoderma filamentous fungi used in the present invention is not limited to a wild strain, and a mutant strain of Trichoderma filamentous fungus that has been improved so as to enhance protein production ability can be preferably used as a parent strain, for example, Trichoderma A mutant strain of a genus filamentous fungus can be used as the parent strain, which has been subjected to a mutation treatment with a mutation agent or ultraviolet irradiation to improve protein productivity.
- mutant strain used as the parent strain include Trichoderma paralysei (ATCC MYA-4777), an ancestor of Trichoderma reesei, QM6a strain (NBRC31326) and QM9123 strain (ATCC24449), which are known mutant strains derived from Trichoderma reesei. , 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)) ), MCG77 strain (Biotechnol. Bioeng. Symp.
- the QM6a strain, the QM9414 strain, and the QM9123 strain can be obtained from NBRC (NITE Biological Resource Center), and the PC-3-7 strain and the RutC-30 strain can be obtained from ATCC (American Type Culture Collection).
- the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a polypeptide possessed by Trichoderma filamentous fungi. In National Center for Biotechnology Information, Trichoderma reesei QM6a strain has also been registered as predicted protein (EGR44419). .
- the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a polypeptide whose function is unknown, but according to the Centered Domain Architecture Retrieval Tool of the National Center for Biotechnology Information, the 26th to 499th amino acids from the N-terminal side remain. The group is disclosed as having a Sugar (and other) Transporter domain.
- the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is presumed to be involved in transport of sugars at least between the inside and outside of the cells.
- that the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is reduced means that the gene encoding EGR44419 has a mutation.
- the decrease in the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is caused by the mutation of the base sequence encoding the amino acid sequence represented by SEQ ID NO: 2, resulting in a decrease in the function of the polypeptide. Or a state in which the function is lost.
- a base sequence other than the base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 is mutated to cause a decrease in the expression level or loss of expression of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2.
- the case is also included in the decrease in the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
- the base sequence mutation is caused by base substitution, deletion, insertion, duplication or the like.
- a specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is the base sequence represented by SEQ ID NO: 1.
- a method for reducing the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 there is a complete deletion of the Sugar (and other) Transporter domain, a partial deletion of the Sugar (and other) Transporter domain, and a Sugar (and other) Examples thereof include a method for introducing a mutation that causes a change in the three-dimensional structure of the Transporter domain or a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 as a whole.
- the function of the polypeptide can also be reduced by introducing a mutation that reduces the expression level of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or eliminates the expression.
- Sugar (and other) Transporter domain deficiency refers to the loss of the domain, the disappearance of a part, the change of all to a different amino acid, the change of a part to a different amino acid, or a combination thereof. More specifically, the amino acid sequence represented by SEQ ID NO: 2 indicates that the sequence identity with the amino acid sequence of the Sugar (and other) Transporter domain shown above is 80% or less, preferably 50% or less. More preferably 20% or less, further preferably 10% or less, further preferably 5% or less, further preferably 3% or less, further preferably 1% or less, and most preferably 0%.
- the function of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 is reduced by mutation such as deletion, substitution, or addition in the amino acid sequence located in the Sugar (and other) Transporter domain.
- mutation such as deletion, substitution, or addition in the amino acid sequence located in the Sugar (and other) Transporter domain.
- Specific examples include a frameshift mutation in which 11 bases are inserted at the 1415th position in the base sequence represented by SEQ ID NO: 1. It is presumed that due to this mutation, translation ends at the 419th amino acid sequence represented by SEQ ID NO: 2, thereby shortening the amino acid sequence constituting the Sugar (and other) Transporter domain and reducing the original function.
- the entire deletion of the Sugar (and other) Transporter domain, the partial deletion of the Sugar (and other) Transporter domain, and the total deletion of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 are the amino acid sequence represented by SEQ ID NO: 2.
- the gene sequence encoding the polypeptide consisting of is performed by frameshifting by base deletion, insertion, substitution or the like, or by stop codon mutation.
- the decrease in the expression level or loss of expression of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is carried out by mutation of the promoter or terminator region of the gene encoding the amino acid sequence represented by SEQ ID NO: 2.
- a promoter and terminator region correspond to a region of several hundred bases before and after a gene involved in transcription, and a base comprising a promoter and terminator involved in transcription of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
- a specific example of the sequence is the base sequence represented by SEQ ID NO: 7.
- the mutant strain of Trichoderma filamentous fungus of the present invention has improved protein production ability compared to Trichoderma filamentous fungus in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is not reduced.
- the mutant strain of Trichoderma filamentous fungus of the present invention is cultured, the protein concentration increases as compared with the culture solution of Trichoderma filamentous fungus in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is not reduced.
- the protein is an enzyme
- the specific activity of the enzyme increases.
- the increase rate of the protein concentration and the increase rate of the specific activity of the enzyme are not particularly limited as long as they increase, but are preferably 20% or more.
- the mutant strain of Trichoderma spp. Of the present invention may have a genetic mutation that improves the protein production amount in addition to the functional degradation of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
- a gene mutation that reduces the function of the polypeptide represented by SEQ ID NO: 8 can be mentioned.
- the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 is a polypeptide possessed by Trichoderma reesei, and is registered as EGR50654 of predicted protein possessed by the Trichoderma reesei QM6a strain in National Center for Biotechnology Information.
- the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 is a polypeptide whose function is unknown, but according to the Centered Domain Architecture Retrieval Tool of National Center for Biotechnology Information, the 95th to 277th amino acid residues from the N-terminal side remain.
- the group is “Middle domain of eukaryotic initiation factor 4G domain” (hereinafter referred to as MIF4G domain), and the 380th to 485th amino acid residues from the N-terminal side are disclosed to have MA-3 domain. Both domains of MIF4G and MA-3 are known to have a function of binding to DNA or RNA (Biochem. 44, 12265-12272 (2005), Mol. Cell. Biol. 1, 147-156 (2007). )). Based on these descriptions, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 is presumed to have at least a function of binding to DNA and / or RNA.
- a specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 is the base sequence represented by SEQ ID NO: 9.
- the gene mutation in which the function of EGR50654 is reduced includes all deletion of MIF4G domain and / or MA-3 domain, partial deletion of MIF4G domain and / or MA-3 domain, and MIF4G domain and MA-3 domain Examples include gene mutations that change the configurational relationship.
- the function of the polypeptide can also be reduced by introducing a mutation that reduces or eliminates the expression level of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8.
- the present invention also relates to a method for producing a protein comprising a step of culturing a mutant strain of Trichoderma filamentous fungus having a reduced function of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2.
- the protein produced in the present invention is not particularly limited, but it can efficiently produce a protein that is secreted outside the cell body.
- an enzyme is preferable, and a cellulase, amylase, invertase, chitinase, pectinase is more preferable. And particularly preferably cellulase.
- the cellulase produced in the present invention includes various hydrolases, including enzymes having a degrading activity for xylan, cellulose, hemicellulose, and the like.
- specific examples include cellobiohydrase (EC 3.2.1.91) that produces cellobiose by hydrolysis of cellulose chains, and endoglucanase (EC 3.2.1.4) that hydrolyzes from the central part of cellulose chains.
- ⁇ -glucosidase that hydrolyzes cellooligosaccharide and cellobiose (EC 3.2.1.21), xylanase (EC 3.2.1.8), xylooligosaccharide characterized by acting on hemicellulose and especially xylan And ⁇ -xylosidase (EC 3.2.1.37) that hydrolyzes.
- the confirmation of the improvement in the specific activity of cellulase for confirming the improvement in the protein production ability of the mutant strain of Trichoderma of the present invention is that any of these hydrolases has an improved specific activity.
- the cellulase produced by the present invention particularly improves ⁇ -glucosidase activity.
- ⁇ -glucosidase specific activity is measured by the following method. First, 10 ⁇ L of enzyme diluted solution is added to 90 ⁇ L of 50 mM acetate buffer containing 1 mM p-nitrophenyl- ⁇ -glucopyranoside (manufactured by Sigma Aldrich Japan GK), and reacted at 30 ° C. for 10 minutes. Next, 10 ⁇ L 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 activity to release 1 ⁇ mol of p-nitrophenol per minute is defined as 1 U, and the specific activity is calculated by dividing this by the amount of protein.
- ⁇ -xylosidase specific activity is measured by the following method. First, 10 ⁇ L of enzyme dilution is added to 90 ⁇ L of 50 mM acetate buffer containing 1 mM p-nitrophenyl- ⁇ -xylopyranoside (manufactured by Sigma Aldrich Japan GK), and reacted at 30 ° C. for 30 minutes. Next, 10 ⁇ L 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 with 1 U as the activity that liberates 1 ⁇ mol of p-nitrophenol per minute.
- the cellobiohydrolase specific activity is measured by the following method. First, 10 ⁇ L of enzyme dilution solution is added to 90 ⁇ L of 50 mM acetate buffer containing 1 mM p-nitrophenyl- ⁇ -lactopyranoside (manufactured by Sigma Aldrich Japan GK), and reacted at 30 ° C. for 60 minutes. Next, 10 ⁇ L 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 activity to release 1 ⁇ mol of p-nitrophenol per minute is defined as 1 U, and the specific activity is calculated by dividing this by the amount of protein.
- the culture medium composition in the culturing step is not particularly limited as long as Trichoderma reesei can produce a protein, and a well-known medium composition of Trichoderma bacteria can be employed.
- a nitrogen source for example, polypeptone, gravy, CSL, soybean meal and the like can be used.
- the cellulase When producing cellulase according to the present invention, the cellulase can be cultured in a medium containing at least one kind or two or more kinds of inducers selected from the group consisting of lactose, cellulose and xylan.
- Cellulose and xylan may be added with biomass containing cellulose or xylan as an inducer.
- biomass containing cellulose or xylan in addition to plants such as seed plants, fern plants, moss plants, algae and aquatic plants, waste building materials and the like can also be used.
- Seed plants are classified into gymnosperms and angiosperms, and both can be preferably used.
- Angiosperms are further classified into monocotyledonous plants and dicotyledonous plants.
- monocotyledonous plants include bagasse, switchgrass, napiergrass, eliansus, corn stover, corn cob, rice straw, and straw.
- dicotyledonous plants beet pulp, eucalyptus, oak, birch and the like are preferably used.
- pretreated biomass may be used as the biomass containing cellulose and xylan.
- the pretreatment method is not particularly limited, and known methods such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, fine pulverization treatment, and steaming treatment can be used. Pulp may be used as biomass containing cellulose or xylan that has been subjected to such pretreatment.
- Non-Patent Document 2 it is described that when lactose is used as an inducer when cultivating a mutant strain lacking the sugar transporter of Trichoderma reesei, the production amount of cellulase decreases. In the mutant strain of Trichoderma spp., When the lactose is used as an inducer, the amount of protein produced is improved and various specific activities of cellulase are also improved.
- the method for culturing the mutant of Trichoderma filamentous fungus of the present invention is not particularly limited. For example, culturing in liquid culture using a centrifuge tube, flask, jar fermenter, tank or the like, or solid culture using a plate or the like. Can do. Trichoderma reesei is preferably cultured under aerobic conditions. Among these culture methods, jar fermenter and deep culture where aeration and agitation are carried out in the tank are particularly preferable.
- the ventilation rate is preferably about 0.1 to 2.0 vvm, more preferably 0.3 to 1.5 vvm, and particularly preferably 0.5 to 1.0 vvm.
- the culture temperature is preferably about 25 to 35 ° C, more preferably 25 to 31 ° C.
- the pH condition in the culture is preferably pH 3.0 to 7.0, more preferably pH 4.0 to 6.0.
- the culture time is performed under the conditions for producing the protein until a recoverable amount of protein is accumulated. Usually, it is about 24 to 240 hours, more preferably 36 to 192 hours.
- the method for recovering the protein contained in the culture solution obtained by culturing the mutant of Trichoderma spp. Is not particularly limited, but the protein can be recovered by removing the Trichoderma spp.
- Examples of the method for removing the cells include a centrifugal separation method, a membrane separation method, and a filter press method.
- the cells of Trichoderma filamentous fungus should not grow in the culture solution. It is preferable to process. Examples of the method for treating cells so that they cannot grow include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment.
- the culture solution treated so that the cells are not removed or grown as described above can be used as an enzyme solution as it is.
- the cellulase obtained by culturing a mutant strain of Trichoderma filamentous fungus in which the function of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 of the present invention has been reduced is not reduced in the function of the polypeptide.
- cellulase Compared with cellulase obtained by culturing Trichoderma filamentous fungi, cellulase has a high specific activity, especially ⁇ -glucosidase, so it efficiently decomposes cellulose-containing biomass and saccharifies with high glucose concentration. Liquid can be obtained, and more sugar can be obtained.
- the method of saccharifying cellulose containing biomass and manufacturing sugar is not specifically limited. The saccharification reaction may be performed batchwise or continuously.
- the conditions for the saccharification reaction are not particularly limited, but the temperature for the saccharification reaction is preferably in the range of 25 to 60 ° C, more preferably in the range of 30 to 55 ° C.
- the saccharification reaction time is preferably in the range of 2 hours to 200 hours.
- the pH of the saccharification reaction is preferably in the range of pH 3.0 to 7.0, more preferably in the range of pH 4.0 to 6.0. In the case of Trichoderma-derived cellulase, the optimum pH for the reaction is 5.0. Furthermore, since a change in pH occurs during the hydrolysis, it is preferable to add a buffer solution to the reaction solution or to maintain a constant pH using an acid or alkali.
- the used enzyme composition can be separated and recovered from the saccharified solution obtained by saccharification of the cellulose-containing biomass.
- the method for separating and recovering the enzyme composition is not particularly limited, but the saccharified solution can be filtered through an ultrafiltration membrane or the like and recovered to the non-permeating side. You may remove solid content from a saccharified liquid as a pre-process of filtration as needed. The recovered cellulase can be used again for the saccharification reaction.
- the saccharification reaction was performed as follows. Arbocel (registered trademark) B800 or bagasse powdered to an average particle size of 100 ⁇ m and sodium acetate buffer (pH 5.2) were added to a final concentration of 0.1 M in a 2 mL tube, and the solid content concentration started. Sometimes pure water was added to 8 wt% when using Arbocel (registered trademark) B800 and 10 wt% when using bagasse. Furthermore, an enzyme solution was added, and the reaction was started under a reaction condition of 50 ° C. using a heat block rotator.
- the sample after saccharification reaction for 24 hours is centrifuged at 10,000 ⁇ g for 10 minutes, the supernatant is collected, and 1N aqueous 1N sodium hydroxide solution is added, which is 1/10 of the volume of the supernatant.
- the saccharification reaction was stopped.
- the sugar concentration in the saccharified solution after the reaction was stopped was subjected to saccharide analysis by UPLC shown below.
- the enzyme solution used for the saccharification reaction was used by calculating the addition amount so as to meet the conditions of each Example or Comparative Example from the protein concentration of the culture solution and the specific activity.
- Glucose, xylose, and cellobiose were quantitatively analyzed using the ACQUITY (registered trademark) UPLC system (Waters) under the following conditions. Quantitative analysis was performed on the basis of a calibration curve prepared with samples of glucose, xylose and cellobiose. When cellobiose was a value lower than 1 g / L, it was set below the detection limit.
- Trichoderma reesei QM9414 mutant I in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was reduced The function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was reduced
- a mutant strain of Trichoderma reesei was prepared as follows. The gene sequence represented by SEQ ID NO: 3 as a DNA fragment containing the gene encoding the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 with reduced function of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 The DNA fragment was prepared and transformed into Trichoderma reesei QM9414 strain.
- a mutant strain of Trichoderma reesei having a polypeptide in which 11 bases are inserted at the 1415th position in SEQ ID NO: 1 and translation is completed at the 419th position in SEQ ID NO: 2 is obtained.
- Acetamide and an acetamidase (AmdS) gene (amdS) capable of degrading acetamide were used as selectable markers for DNA fragment introduction.
- AmdS acetamidase
- amdS acetamidase
- a DNA fragment obtained by treating the synthesized DNA fragment represented by SEQ ID NO: 4 with restriction enzymes AflII and NotI was used as an upstream DNA fragment.
- PCR was performed using genomic DNA extracted from Trichoderma reesei QM9414 strain according to a conventional method and the oligo DNAs represented by SEQ ID NOs: 5 and 6, and the resulting amplified fragment was treated with restriction enzymes MluI and SwaI.
- a downstream DNA fragment was introduced, and the upstream and downstream DNA fragments were introduced into the plasmid into which amdS had been inserted using restriction enzymes AflII and NotI, and MluI and SwaI, respectively, to construct a mutation introduction plasmid.
- Trichoderma reesei QM9414 strain (NBRC # 31329) was transformed with the obtained DNA fragment represented by SEQ ID NO: 3.
- Molecular biological techniques were performed as described in Molecular cloning, laboratory manual, 1st, 2nd, 3rd (1989). Further, transformation was performed using a standard method, protoplast-PEG method, specifically as described in Gene, 61, 165-176 (1987). The obtained Trichoderma reesei mutant strain was used as the QM9414 mutant strain I in the following experiments.
- Example 2 Protein production test using QM9414 mutant I (flask culture)
- the spores of QM9414 variant I prepared in Example 1 were diluted with physiological saline to 1.0 ⁇ 10 7 / mL, and 0.1 mL of the diluted spore solution was added to a 50 mL baffle shown in Table 1 or Table 2.
- a 10 mL flask medium placed in a flask was inoculated and cultured in a shaking incubator at 28 ° C. and 120 rpm for 120 hours.
- the protein concentration contained in the culture solution was measured by the method described in Reference Example 1, and various specific activities of cellulase were measured by the method described in Reference Example 2.
- the results when cultured in the medium of Table 1 are shown in Table 3, and the results when cultured in the medium of Table 2 are shown in Table 4.
- Example 3 Production of Trichoderma reesei QM9414 variant II in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was reduced The function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was reduced A mutant strain of Trichoderma reesei was prepared by preparing a DNA fragment comprising the gene sequence represented by SEQ ID NO: 10 and transforming the DNA fragment into the Trichoderma reesei QM9414 strain. By this method, a mutant strain of Trichoderma reesei in which amdS is inserted between positions 435 and 436 in SEQ ID NO: 1 and the function of SEQ ID NO: 2 is reduced is obtained.
- PCR was performed using the genomic DNA extracted from Trichoderma reesei QM9414 strain according to a conventional method and the oligo DNAs represented by SEQ ID NOs: 11 and 12, and the resulting amplified fragments were treated with restriction enzymes AflII and NotI. The fragment was taken as the upstream fragment.
- PCR was performed using genomic DNA and oligo DNAs represented by SEQ ID NOs: 13 and 14, and the obtained amplified fragment was treated with restriction enzymes MluI and SphI as a downstream fragment, and upstream and downstream DNA fragments were designated as AflII.
- Trichoderma reesei QM9414 strain was transformed as described in Example 1 with the obtained DNA represented by SEQ ID NO: 10.
- the obtained Trichoderma reesei mutant was used as QM9414 mutant II in the following experiment.
- Example 4 Protein Production Test Using QM9414 Mutant II Culture was performed under the same procedures and conditions as in Example 2 except that QM9414 Mutant II was used instead of QM9414 Mutant I prepared in Example 1. The protein concentration contained in the culture solution and various specific activities of cellulase were measured. The results are shown in Tables 3 and 4.
- Trichoderma reesei Protein production test using QM9414 strain The same procedures and conditions as in Example 2 except that Trichoderma reesei QM9414 strain was used instead of QM9414 mutant I prepared in Example 1.
- the protein concentration contained in the culture medium and various specific activities of cellulase were measured.
- the results when cultured in the medium of Table 1 are shown in Table 3, and the results when cultured in the medium of Table 2 are shown in Table 4.
- Example 4 and Comparative Example 1 in the culture using the medium shown in Table 1, when the protein concentration contained in the culture solution obtained by culturing Trichoderma reesei QM9414 strain is 1, the QM9414 mutant strain The relative value of the protein concentration contained in the culture solution of I was 1.5, and the relative value of the protein concentration contained in the culture solution of QM9414 variant II was 1.3. From these results, the amount of protein produced can be reduced by culturing Trichoderma reesei in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is reduced, compared to the case in which the function of the polypeptide is not reduced. It turns out that it can improve.
- cellulase obtained by culturing a Trichoderma reesei mutant in which the function of the polypeptide represented by the amino acid sequence represented by SEQ ID NO: 2 has been reduced does not decrease the function of the polypeptide.
- the specific activity of cellulase was measured for each of the obtained culture solutions by the method described in Reference Example 2.
- the various specific activities of the culture solution obtained by culturing the QM9414 strain are 1, the ⁇ -glucosidase specific activities are QM9414 mutant I: 4.3, QM9414 mutant II: 1.6, and ⁇ -xylosidase Specific activities were QM9414 mutant I: 2.5, QM9414 mutant II: 1.2, and cellobiohydrolase specific activities were QM9414 mutant I: 3.5 and QM9414 mutant II: 1.1. .
- cellulase obtained by culturing a Trichoderma reesei mutant in which the function of the polypeptide represented by the amino acid sequence represented by SEQ ID NO: 2 has been reduced does not decrease the function of the polypeptide.
- QM9414 mutant I in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was reduced was cultured in a medium to which lactose was added, the culture was performed in a medium to which cellulose was added. It was found that various specific activities of cellulase were further improved as compared with the case where it was performed.
- Example 5 Saccharification reaction test using cellulase of QM9414 mutant I Using the culture solution at 120 hours after the start of culture using the medium shown in Table 1 of QM9414 mutant I obtained in Example 2, According to the procedures and conditions described in Table 5 and Reference Example 3, a saccharification reaction test of cellulose-containing biomass was performed. Arbocel (registered trademark) B800 or powdered bagasse was used as the cellulose-containing biomass. The results are shown in Table 6.
- Example 6 Saccharification reaction test 1 using cellulase of QM9414 mutant II Of the culture solution of QM9414 variant II obtained in Example 4, the culture solution at 120 hours from the start of culture when cultured in the medium described in Table 1 was used. Thus, a saccharification reaction test of cellulose-containing biomass was conducted. Table 7 shows the saccharification reaction of Arbocel (registered trademark) B800, and Table 8 shows the saccharification reaction of powdered bagasse. The results are shown in Table 9.
- a saccharification reaction test of cellulose-containing biomass was conducted under the same procedures and conditions. The results are shown in Tables 6 and 9.
- Example 7 Saccharification reaction test 2 using cellulase of QM9414 mutant II Among the culture solutions of QM9414 variant II obtained in Example 4, the culture solution at 120 hours after the start of culture when cultured in the medium shown in Table 2, was used as described in Table 6 and Reference Example 3. A saccharification reaction test of cellulose-containing biomass was performed according to the operation and conditions. Table 10 shows the saccharification reaction for Arbocel (registered trademark) B800, and Table 11 shows the saccharification reaction for powdered bagasse. The results are shown in Table 12.
- Trichoderma reesei Saccharification reaction test 2 using cellulase of QM9414 strain Of the culture solution of Trichoderma reesei QM9414 strain obtained in Comparative Example 1, the same culture solution as in Example 7 was used except that the culture solution at 120 hours from the start of culture when cultured in the medium shown in Table 2 was used. A saccharification reaction test of cellulose-containing biomass was conducted under the operation and conditions. The results are shown in Table 12.
- Example 5 the cellulase of Trichoderma reesei QM9414 strain was used in the saccharification reaction of Arbocel (registered trademark) B800 using the culture solution obtained by culturing the medium shown in Table 1.
- the glucose concentration contained in the saccharified solution in this case was 3.3 g / L, whereas in the case of using QM9414 mutant I, it was 4.8 g / L.
- the xylose concentration contained in the saccharified solution was 3.9 g / L when QM9414 strain was used, whereas it was 4.9 g / L when QM9414 mutant I was used.
- the QM9414 strain had a glucose release of 1.4 g / L, whereas the mutant strain I had 1.7 g / L, and the xylose release was 2.3 g / L in the QM9414 strain. In L and mutant I, it was 2.5 g / L.
- Example 6 From the results of Example 6 and Comparative Example 2, in the saccharification reaction of Arbocel (registered trademark) B800 using the culture solution obtained by culturing the medium described in Table 1, the glucose release amount in the QM9414 strain was 13 g / L. Whereas QM9414 variant II was 14.3 g / L. The amount of xylose released was 8.5 g / L in the QM9414 strain, whereas it was 9.1 g / L in the mutant strain II. In the saccharification reaction of powdered bagasse, the QM9414 strain had a glucose release of 6.1 g / L, whereas the mutant strain II had 6.5 g / L. The amount of xylose released was 4.1 g / L for both QM9414 and mutant II.
- Example 7 From the results of Example 7 and Comparative Example 3, in the saccharification reaction of Arbocel (registered trademark) B800 using the culture solution obtained by culturing the medium shown in Table 2, the glucose concentration in the QM9414 strain was 5.9 g / In contrast to L, mutant II was 7.2 g / L. The xylose concentration was 3.4 g / L in the QM9414 strain, whereas it was 4.5 g / L in the mutant strain II. Moreover, in the saccharification reaction of powdered bagasse, the glucose concentration was 2.9 g / L in the QM9414 strain, whereas it was 3.9 g / L in the mutant strain II.
- Arbocel registered trademark
- the xylose concentration was 2.5 g / L in the QM9414 strain, whereas it was 3.0 g / L in the mutant strain II. From these results, the cellulase produced by a variant of Trichoderma filamentous fungus with reduced function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 can produce more sugar than the cellulase produced by the QM9414 strain. I understood it.
Abstract
Description
(1)配列番号2で表されるアミノ酸配列からなるポリペプチドの機能が低下している、トリコデルマ属糸状菌の変異株。
(2)配列番号2で表されるアミノ酸配列において、少なくともN末端側から413番目以降のアミノ酸残基が欠損している請求項1に記載のトリコデルマ属糸状菌の変異株。
(3)(1)または(2)に記載のトリコデルマ属糸状菌の変異株を培養する工程を含む、タンパク質の製造方法。
(4)(1)または(2)に記載のトリコデルマ属糸状菌の変異株を培養する工程を含む、セルラーゼの製造方法。
(5)(1)または(2)に記載のトリコデルマ属糸状菌の変異株を、ラクトース、セルロースおよびキシランからなる群から選択される少なくとも1種類または2種類以上の誘導剤を含む培地で培養する工程を含む、(4)に記載のセルラーゼの製造方法。
(6)セルロース含有バイオマスから糖を製造する方法であって、以下の工程:
工程a:配列番号2で表されるアミノ酸配列からなるポリペプチドの機能が低下しているトリコデルマ・リーセイの変異株を培養し、セルラーゼを製造する工程
工程b:工程aで得られたセルラーゼを用いて、前記バイオマスを糖化する工程
を含む、糖の製造方法。
タンパク質濃度測定試薬(Quick Start Bradfordプロテインアッセイ、Bio-Rad社製)を使用した。室温に戻したタンパク質濃度測定試薬250μLに希釈した糸状菌の培養液を5μL添加し、室温で5分間静置後の595nmにおける吸光度をマイクロプレートリーダーで測定した。標準品としてBSAを使用し、検量線に照らし合わせてタンパク質濃度を算出した。
(β-グルコシダーゼ比活性測定方法)
1mMp-ニトロフェニル-β-グルコピラノシド(シグマアルドリッチジャパン合同会社製)を含有する50mM酢酸バッファー90μLに酵素希釈液10μLを添加して30℃で10分間反応させた。その後2M炭酸ナトリウム10μLを加えてよく混合して反応を停止し、405nmの吸光度の増加を測定した。1分間あたり1μmolのp-ニトロフェノールを遊離する活性を1Uと定義し、これをタンパク質の量で割ることで比活性を算出した。
1mMp-ニトロフェニル-β-キシロピラノシド(シグマアルドリッチジャパン合同会社製)を含有する50mM酢酸バッファー90μLに酵素希釈液10μLを添加し30℃で30分間反応させた。その後、2M炭酸ナトリウム10μLを加えてよく混合して反応を停止し、405nmの吸光度の増加を測定した。1分間あたり1μmolのp-ニトロフェノールを遊離する活性を1Uと定義し、これをタンパク質の量で割ることで比活性を算出した。
1mMp-ニトロフェニル-β-ラクトピラノシド(シグマアルドリッチジャパン合同会社製)を含有する50mM酢酸バッファー90μLに酵素希釈液10μLを添加し30℃で60分間反応させた。その後、2M炭酸ナトリウム10μLを加えてよく混合して反応を停止し、405nmの吸光度の増加を測定した。1分間あたり1μmolのp-ニトロフェノールを遊離する活性を1Uと定義し、これをタンパク質の量で割ることで比活性を算出した。
糖化対象のバイオマスとしては、木材原料粉末セルロースArbocel(登録商標)B800(レッテンマイヤー社製)または平均粒径100μmに粉末化したバガスを使用した。酵素液としては、トリコデルマ・リーセイまたはトリコデルマ・リーセイの変異株の培養液を1mL採取して遠心分離し、菌体を除去した上清を回収し、さらに0.22μmのフィルターでろ過したろ液を用いた。なお、木材原料粉末セルロースArbocel(登録商標) B800(レッテンマイヤー社製)は、以降Arbocel B800と記載する場合がある。
糖化反応は以下のようにして行った。2mLチューブの中にArbocel(登録商標)B800もしくは平均粒径100μmに粉末化したバガスと、酢酸ナトリウムバッファー(pH5.2)を終濃度0.1Mになるように添加し、固形分濃度が反応開始時にArbocel(登録商標)B800を用いた際には8重量%、バガスを用いた際には10重量%となるように純水を加えた。さらに、酵素液を添加し、ヒートブロックローテーターを用いて、50℃の反応条件で反応を開始した。24時間糖化反応後のサンプルを10,000×gの条件下で10分間遠心分離を行い、上清を分取し、上清のボリュームの10分の1量の1N 水酸化ナトリウム水溶液を添加し、糖化反応を停止させた。反応停止後の糖化液中の糖濃度を下記に示すUPLCによる糖分析に供した。糖化反応に用いる酵素液は、培養液のタンパク質濃度と、比活性から各実施例または比較例の条件に合うように添加量を算出して用いた。
グルコース、キシロース、セロビオースは、ACQUITY(登録商標)UPLC システム(Waters)を用いて、以下の条件で定量分析した。
グルコース、キシロース、セロビオースの標品で作製した検量線をもとに、定量分析した。セロビオースが1g/Lより低い値の場合は、検出限界以下とした。
カラム:AQUITY(登録商標)UPLC BEH Amide 1.7μm2.1×100mm Column
分離法:HILIC
移動相:移動相A:80%アセトニトリル、0.2%TEA水溶液、移動相B:30%アセトニトリル、0.2%TEA水溶液とし、下記グラジエントに従った。グラジエントは下記の時間に対応する混合比に到達する直線的なグラジエントとした。
開始条件:(A99.90%、B0.10%)、開始2分後:(A96.70%、B3.30%)、開始3.5分後:(A95.00%、B5.00%)、開始3.55分後:(A99.90%、B0.10%)、開始6分後:(A99.90%、B0.10%)。
検出方法:ELSD(蒸発光散乱検出器)
流速:0.3mL/min
温度:55℃。
配列番号2で表されるアミノ酸配列からなるポリペプチドの機能が低下したトリコデルマ・リーセイの変異株は以下のとおり作製した。配列番号2で表されるアミノ酸配列からなるポリペプチドの機能が低下した配列番号2で表されるアミノ酸配列からなるポリペプチドをコードする遺伝子を含むDNA断片として、配列番号3で表される遺伝子配列からなるDNA断片を作製し、当該DNA断片をトリコデルマ・リーセイ QM9414株に形質転換することで作製した。この方法により、配列番号1において、1415番目に11塩基が挿入し、配列番号2において、419番目で翻訳が終了するポリペプチドを有するトリコデルマ・リーセイの変異株が得られる。DNA断片導入のための選択マーカーとしてアセトアミドおよびアセトアミドを分解することができるアセトアミダーゼ(AmdS)遺伝子(amdS)を使用した。amdSを含むDNA配列の上流および下流に、上記の配列番号3で表される塩基配列からなるDNA断片を導入するために、トリコデルマ・リーセイ QM9414株の遺伝子配列と相同的な部分を付加するように変異導入用プラスミドを作製した。
(フラスコ培養)
実施例1で作製したQM9414変異株Iの胞子を1.0×107/mLになるように生理食塩水で希釈し、その希釈胞子溶液0.1mLを表1または表2に示した50mLバッフル付フラスコへ入れた10mLのフラスコ培地へ接種させ、振盪培養機にて28℃、120rpmの条件にて120時間培養を行った。培養液中に含まれるタンパク質濃度を参考例1に記載の方法で、セルラーゼの各種比活性を参考例2に記載の方法で測定した。表1の培地で培養した際の結果を表3に、表2の培地で培養した際の結果を表4に示す。
培養開始120時間後に1mL培養液を採取した。培養液を15,000×g、4℃の条件下で10分間遠心分離を行い、上清を得た。その上清を0.22μmのフィルターでろ過し、そのろ液をセルラーゼ溶液として、以下の実験に用いた。
参考例1で記載した手法を用い、培養開始120時間目の培養液におけるタンパク質濃度を測定し、続いて参考例2に記載の方法でセルラーゼの比活性を測定した。結果を表3と表4に示す。
配列番号2で表されるアミノ酸配列からなるポリペプチドの機能が低下したトリコデルマ・リーセイの変異株は、配列番号10で表される遺伝子配列からなるDNA断片を作製し、当該DNA断片をトリコデルマ・リーセイ QM9414株に形質転換することで作製した。この方法により、配列番号1において、435番目と436番目の間にamdSが挿入され、配列番号2の機能が低下したトリコデルマ・リーセイの変異株が得られる。amdSを含むDNA配列の上流および下流に、上記の配列番号10で表される塩基配列からなるDNA断片を導入するために、トリコデルマ・リーセイQM9414株の遺伝子配列と相同的な部分を付加するように変異導入用プラスミドを作製した。
実施例1で作製したQM9414変異株Iの代わりにQM9414変異株IIを用いた以外は、実施例2と同様の操作・条件で培養を行い、培養液中に含まれるタンパク質濃度と、セルラーゼの各種比活性を測定した。結果を表3と表4に示す。
実施例1で作製したQM9414変異株Iの代わりにトリコデルマ・リーセイ QM9414株を用いた以外は、実施例2と同様の操作・条件で培養を行い、実施例2と同様の方法で培養液中に含まれるタンパク質濃度とセルラーゼの各種比活性を測定した。表1の培地で培養した際の結果を表3に、表2の培地で培養した際の結果を表4に示す。
実施例2で得られたQM9414変異株Iの表1に示す培地を用いた培養開始から120時間目の培養液を用いて、表5および参考例3に記載の操作・条件に従って、セルロース含有バイオマスの糖化反応試験を行った。セルロース含有バイオマスとして、Arbocel(登録商標)B800または粉末バガスを用いた。結果を表6に示す。
実施例4で得られたQM9414変異株IIの培養液のうち、表1に記載の培地で培養した際の培養開始から120時間目の培養液を用いて、参考例3に記載の操作・条件に従って、セルロース含有バイオマスの糖化反応試験を行った。Arbocel(登録商標)B800の糖化反応は、表7に、粉末バガスの糖化反応は表8にそれぞれ反応条件を示す。結果を表9に示す。
比較例1で得られたトリコデルマ・リーセイ QM9414株の培養液のうち、表1に記載の培地で培養した際の培養開始から120時間目の培養液を用いた以外は、実施例5または6と同様の操作・条件でセルロース含有バイオマスの糖化反応試験を行った。結果を表6と9に示す。
実施例4で得られたQM9414変異株IIの培養液のうち、表2に記載の培地で培養した際の培養開始から120時間目の培養液を用いて、表6および参考例3に記載の操作・条件に従って、セルロース含有バイオマスの糖化反応試験を行った。Arbocel(登録商標)B800の糖化反応は、表10に、粉末バガスの糖化反応は表11にそれぞれ反応条件を示す。結果を表12に示す。
比較例1で得られたトリコデルマ・リーセイ QM9414株の培養液のうち、表2に記載の培地で培養した際の培養開始から120時間目の培養液を用いた以外は、実施例7と同様の操作・条件でセルロース含有バイオマスの糖化反応試験を行った。結果を表12に示す。
実施例5と比較例2の結果から、表1に記載の培地の培養にて得られた培養液を用いたArbocel(登録商標) B800の糖化反応において、トリコデルマ・リーセイ QM9414株のセルラーゼを用いた場合の糖化液に含まれるグルコース濃度は3.3g/Lであるのに対し、QM9414変異株Iを用いた場合では4.8g/Lであった。また、糖化液に含まれるキシロース濃度は、QM9414株を用いた場合では3.9g/Lであるのに対し、QM9414変異株Iを用いた場合では4.9g/Lであった。
Claims (6)
- 配列番号2で表されるアミノ酸配列からなるポリペプチドの機能が低下している、トリコデルマ属糸状菌の変異株。
- 配列番号2で表されるアミノ酸配列において、少なくともN末端側から413番目以降のアミノ酸残基が欠損している、請求項1に記載のトリコデルマ属糸状菌の変異株。
- 請求項1または2に記載のトリコデルマ属糸状菌の変異株を培養する工程を含む、タンパク質の製造方法。
- 請求項1または2に記載のトリコデルマ属糸状菌の変異株を培養する工程を含む、セルラーゼの製造方法。
- 請求項1または2に記載のトリコデルマ属糸状菌の変異株を、ラクトース、セルロースおよびキシランからなる群から選択される少なくとも1種類または2種類以上の誘導剤を含む培地で培養する工程を含む、請求項4に記載のセルラーゼの製造方法。
- セルロース含有バイオマスから糖を製造する方法であって、以下の工程:
工程a:配列番号2で表されるアミノ酸配列からなるポリペプチドの機能が低下しているトリコデルマ・リーセイの変異株を培養し、セルラーゼを製造する工程
工程b:工程aで得られたセルラーゼを用いて、前記バイオマスを糖化する工程
を含む、糖の製造方法。
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CN201980036263.9A CN112243458A (zh) | 2018-05-31 | 2019-05-30 | 木霉属丝状菌突变株和蛋白质的制造方法 |
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JP7400468B2 (ja) | 2023-12-19 |
CN112243458A (zh) | 2021-01-19 |
BR112020024153A2 (pt) | 2021-03-30 |
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