JP6103584B2 - Filamentous fungal mutants in which the cre1 gene does not function - Google Patents

Description

  The present invention relates to an Acremonium / Talaromyces (Penicillium) genus fungus mutant strain in which the cre1 gene does not function and a method for producing cellulase using the same.

  Microorganisms are used industrially as hosts for various enzyme production. Recent advances in gene recombination technology have made it possible to produce strains that produce useful substances more efficiently.

  In the development of high-producing strains of enzymes, many cellulase-producing strains have been acquired in the past. As microorganisms that produce cellulases, filamentous fungi such as the filamentous fungus Trichoderma reesei and the filamentous fungus Acremonium cellulolyticus are industrially used. Furthermore, mutant strains that produce a large number of cellulases at high levels by introducing random gene mutations have been obtained for these filamentous fungi.

As for Acremonium cellulolyticus, multiple cellulase high-producing mutants have been acquired by the inventors' group, and further, decoding of whole genome information and development of genetic recombination systems have made it more efficient. The improvement of a typical strain was attained.
However, there is still a need in the art for cellulase-producing bacteria having high cellulase producing ability.

  An object of the present invention is to provide a cellulase-producing bacterium having a high cellulase-producing ability. Furthermore, an object of the present invention is to provide a new technique capable of more efficiently performing cellulase production, cellulose degradation, and saccharification.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have found that a filamentous fungus mutant in which the cre1 gene does not function in a filamentous fungus belonging to the genus Acremonium / Talaromyces (Penicillium). Was found to have higher cellulase production ability than the wild type strain, and the present invention was completed.

That is, the present invention has the following features.
[1] The above-mentioned filamentous fungus mutant strain, wherein the cre1 gene does not function, and the filamentous fungus belongs to the genus Acremonium / Talaromyces (Penicillium).
[2] The filamentous fungus mutant of [1], wherein the cre1 gene is deleted.
[3] The filamentous fungus mutant according to [1] or [2], wherein the filamentous fungus is Acremonium cellulolyticus.
[4] A method for producing cellulase, comprising culturing the filamentous fungus mutant according to any one of [1] to [3] in a medium containing a raw material containing cellulose.
[5] The method according to [4], wherein the medium further contains glucose.
[6] A cellulase obtained by the method of [4] or [5].
[7] A method for decomposing or saccharifying cellulose, comprising decomposing or saccharifying cellulose using the cellulase obtained by the method of [4] or [5].

  According to the present invention, a cellulase-producing bacterium having a high cellulase-producing ability can be provided. Furthermore, according to the present invention, it is possible to provide a new method capable of more efficiently performing cellulase production, cellulose degradation, and saccharification.

FIG. 1 is obtained by culturing the cre1 gene deletion strain and the Y-94 strain in a medium in which the ratio of cellulose to glucose (mass ratio) is 5: 0, 4: 1, 2.5: 2.5, 1: 4. Cellulase activity in the culture broth. FIG. 2 shows the results of analyzing the expression level of the cbhI gene of the cre1 gene-deficient strain and the Y-94 strain when cultured in a cellulose medium or a cellulose + glucose medium. Lane 1 is when Y-94 strain is cultured in cellulose medium, Lane 2 is when Y-94 strain is cultured in cellulose + glucose medium, Lane 3 is when cre1 gene deletion strain is cultured in cellulose medium Lane 4 shows the expression of cbh1 when the cre1 gene deletion strain was cultured in cellulose + glucose medium.

(1. Acremonium / Talaromyces (Penicillium) genus fungus mutants in which the cre1 gene does not function)
In the present specification, “the cre1 gene does not function” means that the cre1 gene does not have a normal function. The “state having no normal function” means that the activity of the protein encoded by the gene is normal because it includes deletion, substitution, addition or insertion of one or more bases in the gene. Less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or no activity as compared to the activity of any protein. Preferably, “the cre1 gene does not function” means that a part or the whole of the cre1 gene is deleted.

  The filamentous fungal mutant strain of the present invention may be caused by spontaneous mutation, or a filamentous fungus belonging to the genus Acremonium / Talaromyces (Penicillium) having cellulase-producing ability may be irradiated with ultraviolet rays or a mutagenic agent (for example, N-methyl). -N'-nitro-N-nitrosoguanidine, ethylmethanesulfonic acid, etc.).

  Alternatively, the filamentous fungal mutant of the present invention can be produced by a genetic recombination technique using a filamentous fungus belonging to the genus Acremonium / Talaromyces (Penicillium) having cellulase producing ability as a host. The filamentous fungus belonging to the genus Acremonium / Talaromyces (Penicillium) is Acremonium cellulolyticus. In the present invention, Acremonium cellulolyticus Y-94 strain and mutants thereof can be used. As a mutant of Acremonium cellulolyticus Y-94, a uracil-requiring mutant (YP4 strain) can be used. In addition, Acremonium cellulolyticus Y-94 strain was registered as FERM BP-5826 at the National Institute of Advanced Industrial Science and Technology (NIST) Biotechnology Institute, National Institute of Advanced Industrial Science and Technology on January 12, 1983. It has been deposited.

  In addition, it is hard to say that genetic information is prevailing at the beginning of Acremonium cellulolyticus isolation, and it is classified as Acremoniumu genus filamentous fungus from the results of morphological observation and has been used up to now. However, since subsequent gene sequence analysis of ribosomal DNA revealed that Acremonium cellulolyticus was most closely related to the genus Talaromyces (Penicillium), the genus name change to the genus Talaromyces (Penicillium) is scheduled to be announced. .

  The cre1 gene of the genus Acremonium / Talaromyces (Penicillium) can be obtained by a known technique. That is, screening is performed against a cDNA library of the genus Acremonium / Talaromyces (Penicillium) based on the sequence of a known cre1 gene (for example, the cre1 gene derived from Penicillium marneffei (registered as XM_002152098 in GenBank)) The gene can be identified as a screening method by hybridization under low, moderate or high stringent conditions using methods well known in the art for nucleic acid hybridization and cloning. BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) for ORF databases derived from Acremonium / Talaromyces (Penicillium) genus Perform a search, etc., c A homologue to the re1 gene can also be identified, in which case appropriate PCR primers are prepared to amplify the entire gene based on the searched sequence, and the resulting PCR product is used for appropriate cloning. The target gene can also be cloned by inserting it into the vector of 1. The identified gene is subcloned into an appropriate cloning vector, and the sequence is confirmed. The target cre1 gene can be obtained by PCR using a primer designed based on the base sequence and using genomic DNA or cDNA extracted from Acremonium / Talaromyces (Penicillium) genus as a template.

In the present invention, “cre1 gene” includes a gene encoding the amino acid sequence represented by SEQ ID NO: 2.
The “cre1 gene” in the present invention has a sequence in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 2, Examples thereof include a gene encoding a protein having a function of suppressing the expression of a cellulase gene in the presence. Here, “plurality” means 1 to 30, preferably 1 to 20, and more preferably 1 to 10.

  Furthermore, the “cre1 gene” in the present invention is 80% or more, more preferably 90% when calculated using the amino acid sequence shown in SEQ ID NO: 2, BLAST, etc. (for example, default or default parameters). As mentioned above, the gene which codes the protein which has the sequence which has 95% or more of identity most preferably, and has the function to suppress the expression of a cellulase gene in presence of glucose is mentioned.

  Examples of such a “cre1 gene” include DNA consisting of the 4001 to 5248th bases in the base sequence represented by SEQ ID NO: 1. In the present invention, when preparing a mutant strain in which the cre1 gene does not function, the sequence information shown in SEQ ID NO: 1 can be used.

  A mutant strain in which the cre1 gene does not function can be produced by a known technique using a homologous recombination technique. For example, double crossover by homologous recombination method and the like can be mentioned. Briefly, the host cre1 gene is cloned into a common vector known to those skilled in the art for gene transfer. Thereafter, a foreign gene (eg, drug resistance gene) is inserted into the cloned cre1 gene sequence to prepare a DNA construct containing the cre1 gene disrupted by the foreign gene (eg, drug resistance gene). By introducing this DNA construct into the host, homologous recombination occurs between the fragmented cre1 gene on the DNA construct and the cre1 gene on the host chromosome so that the host cre1 gene does not function. Alternatively, instead of inserting a foreign gene into the cre1 gene and dividing it, prepare a DNA construct containing the cre1 gene in which a part or all of the nucleotide sequence of the cre1 gene has been deleted and introduce it into the host. Thus, the cre1 gene having a partial or total deletion on the DNA construct and the cre1 gene on the host chromosome can undergo homologous recombination, so that the host cre1 gene does not function.

  Examples of the method for introducing a DNA construct into a host include a calcium phosphate method or a calcium chloride / rubidium chloride method, an electroporation method, an electroinjection method, a method using chemical treatment such as PEG, and a method using a gene gun. .

  The filamentous fungus mutant of the present invention has a higher cellulase production ability than the wild type strain. Moreover, the filamentous fungal mutant of the present invention has a higher cellulase production ability than the wild type strain even under the condition where saccharides (eg, glucose) are present in the medium.

  The filamentous fungal mutant of the present invention may be immobilized on a solid support (eg, polymer, mineral, metal, gel, polysaccharide, etc.). The filamentous fungus can be immobilized on a solid support using a covalent bond or a non-covalent bond.

(2. Culture of filamentous fungal mutants)
In the medium, as a carbon source, powdered cellulose (including Avicel), waste paper, straw paper, general paper, wood, straw, rice straw, bran, rice bran, and bagasse, nitrogen sources such as ammonium sulfate and ammonium nitrate Organic ammonium content such as inorganic ammonium salt, urea, amino acid, meat extract, yeast extract, polypeptone, and protein degradation product, and inorganic salts include magnesium sulfate, potassium dihydrogen phosphate, potassium tartrate, zinc sulfate, magnesium sulfate, Copper sulfate, calcium chloride, iron chloride, manganese chloride and the like can be included. If necessary, a medium containing organic micronutrients may be used. The medium can be a solid medium solidified by adding agar or gelatin, a semi-fluid medium added with low-concentration agar, or a liquid medium (also referred to as bouillon or broth) containing only medium components. Is preferred.

  The medium can further include sugars. Examples of sugars added to the medium include monosaccharides, disaccharides, and polysaccharides. Examples of the saccharide include glucose, and the amount of cellulose and glucose in the medium can be included at 4: 1 (mass ratio).

  The culture is performed at 25 to 35 ° C. for 48 hours to 10 days. The culture temperature and culture time can be appropriately adjusted according to the type of genetically modified filamentous fungus and cellulase production ability. Examples of the fermenter that can be used for the culture include an aeration stirring type, a bubble column type, a fluidized bed type, and a packed bed type.

(3. Recovery of cellulase)
After completion of the culture, the culture solution is collected. The obtained culture solution can be directly used as a crude enzyme solution of cellulase without further treatment.

  In addition, microbial cells can be removed from the obtained culture solution by a known method such as centrifugation or filtration, and the supernatant can be collected. This supernatant can be used as a crude enzyme solution of cellulase without further treatment.

  Cellulase is a known method used for protein purification from the above supernatant, for example, ammonium sulfate salting out, precipitation separation with an organic solvent (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectric point chromatography, gel Filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrates or antibodies, chromatography such as reverse phase column chromatography, filtration treatment such as microfiltration, ultrafiltration, reverse osmosis filtration, etc. , Can be purified using one or more in combination.

  The purified cellulase can be immobilized and used. Immobilization is effective in that it can be stabilized and used repeatedly continuously. Cellulase can be immobilized using a carrier binding method, a crosslinking method, or a comprehensive method. In the carrier binding method, cellulase can be bound to a water-insoluble carrier (eg, polyacrylamide gel, polystyrene resin, porous glass, metal oxide, etc.) using physical adsorption, ionic bonding, and covalent bonding. In the crosslinking method, cellulase and cellulase are immobilized by crosslinking using a reagent having two or more functional groups. Cross-linking reagents include glutaraldehyde that forms Schiff bases, isocyanic acid derivatives that form peptide bonds, N, N'-ethylenemaleimide, bisdiazobenzines that perform diazo coupling, or N, N'-polymethylene bisiodo that performs alkylation. Acetamide or the like can be used. The inclusion method uses a lattice type in which cellulase is incorporated into a fine lattice of a polymer gel and a microcapsule type in which cellulase is coated with a semipermeable polymer film. In the lattice-type method, a high molecular compound such as a polyacrylamide gel, polyvinyl alcohol, or a photocurable resin, which is a synthetic polymer substance, can be used. In the microcapsule type method, hexamethylenediamine, sebacoyl chloride, polystyrene, lecithin, etc. can be used (Saburo Fukui, Ichiro Chibata, Shuichi Suzuki, “Enzyme Engineering”, published by Tokyo Chemical Dojin, 1981).

  “Cellulase” is a general term for an enzyme group that catalyzes an enzyme reaction system that hydrolyzes cellulose into glucose, cellobiose, and cellooligosaccharide, such as exo-β-glucanase, endo-β-glucanase, and β-glucosidase. As long as it has cellulolytic activity, it is included in the cellulase of the present invention.

(4. Measurement of cellulase activity)
Cellulase activity occurs after adding a substrate such as filter paper, carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel), salicin and cellobiose to the supernatant or purified cellulase and allowing the enzyme reaction to proceed for a certain period of time. The reduced sugar can be colored by the Somogy-Nelson method, DNS method, etc., and colorimetrically determined at a predetermined wavelength for measurement.

  In the Somogy-Nelson method, the reaction is stopped by adding a Somogy copper reagent (Wako Pure Chemical Industries, Ltd.) to the reaction solution reacted for a certain period of time. Then boil for about 20 minutes, and rapidly cool with tap water after boiling. After cooling, a Nelson reagent is injected to dissolve the reduced copper precipitate to develop color, and after standing for about 30 minutes, distilled water is added and the absorbance is measured.

  When using the DNS method, the enzyme reaction is performed for a certain period of time and then stopped by boiling or the like. Dinitrosalicylic acid is added to this reaction solution, boiled for 5 minutes, and after cooling, the absorbance is measured (Yutaki Yutaka “Enzyme of Fermented Filamentous Fungi”, Microbial Genetic Resource Use Manual (16), published by National Institute of Agrobiological Sciences) Published February 29, 2004).

(5. Decomposition or saccharification of cellulose)
“Decomposition or saccharification of cellulose” refers to decomposition of cellulose into oligosaccharides, disaccharides, monosaccharides, and mixtures thereof. That is, “degradation or saccharification of cellulose” refers to hydrolysis of a glycosidic bond of a polysaccharide by cellulase.

A known method can be used for the decomposition or saccharification of cellulose. For example, suspending a cellulose-containing substance in an aqueous medium, adding a culture solution or supernatant containing the cellulase or a purified cellulase, and heating or heating while stirring or shaking to decompose or saccharify the cellulose-containing substance. Can do. As the cellulose-containing substance, those listed as “carbon sources” included in the medium can be used. However, in order to make it possible to decompose or saccharify cellulose by cellulase, a cellulose-containing substance that has been pretreated by a known method is used as necessary. In the decomposition or saccharification of cellulose, the pH and temperature of the reaction solution may be within the range where cellulase is not inactivated. In general, when the reaction is performed at normal pressure, the temperature is 5 to 95 ° C., and the pH is 1 to 1. A range of 11 may be used.
The step of decomposing or saccharifying cellulose may be performed batchwise or continuously.

EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these.
In the following examples, Acremonium cellulolyticus Y-94 strain (FERM BP-5826) (hereinafter referred to as “Y-94 strain”) and Y-94 as cellulase-producing bacteria. A uracil-requiring mutant strain derived from a strain (hereinafter referred to as “YP4 strain”) was used.

(Example 1: Extraction of genomic DNA)
Acremonium cellulolyticus genomic DNA was extracted and isolated by the following method.
The Y-94 strain was cultured overnight in PD (potato dextrose) medium, and the resulting culture was collected by centrifugation. Next, suspend the cells in 2 to 3 times the amount of TE (containing 2% SDS) buffer, incubate at 50 ° C for 1 hour, and add 5 M potassium acetate solution to 1/10 of the total amount. After adding an amount and cooling on ice for 1 hour, the supernatant was collected by centrifugation. The resulting supernatant was treated twice with phenol / chloroform, 2.5 times the amount of 100% ethanol was added, and the mixture was cooled at −20 ° C. for 1 hour or longer, and then nucleic acid was precipitated by centrifugation. The obtained precipitate was washed twice with 70% ethanol, suspended in an appropriate amount of TE buffer, RNase A was added, and the mixture was incubated at 37 ° C. for 1 hour.

(Example 2: Preparation of cre1 gene deletion plasmid)
From the genome database of Acremonium cellulolyticus, using IMC (in silico molecular cloning, in silico biology inc.) Based on the sequence information of the known cre1 gene (Penicillium marneffei ATCC 18224, XM_002152098) The cre1 gene of Acremonium cellulolyticus was searched.

  Based on the obtained sequence information (SEQ ID NO: 1), primers for amplifying the upstream region and the downstream region of the cre1 gene were designed, and PCR was performed using the genomic DNA obtained in Example 1 as a template. Primer sequences used in the PCR are as follows.

(Upstream region) (consisting of the 2191th to 4580th bases of the base sequence shown in SEQ ID NO: 1)
forward primer 5'-GGGTCGACTGACGGAAACGAGAATGCCG-3 '(SEQ ID NO: 3)
Reverse primer 5'-GGGAATTCGCGAGGTGTAGTTGGTGTAA-3 '(SEQ ID NO: 4)
(Downstream region) (consisting of the 4601st to 6960th bases of the base sequence shown in SEQ ID NO: 1)
forward primer 5'-CCTCTAGACCTTATTCACGCAACAGCGA-3 '(SEQ ID NO: 5)
Reverse primer 5'-CCGCGGCCGCGTCTGGCCGAACACGTGATT-3 '(SEQ ID NO: 6)
It was.

  The upstream region and the downstream region obtained by PCR were classified into the SalI-EcoRI site and XbaI-NotI of a known plasmid pbs-pyrF (Fujii T. et al., Biosci. Biotechnol. Biochem. Vol 76, pp245-249, 2012). Each was introduced into the site and used as a plasmid for cre1 gene deletion.

(Example 3: Preparation of cre1 gene deletion strain)
The YP4 strain cultured overnight using PD medium was recovered by centrifugation and washed with 0.8 M NaCl.
To the obtained cells, 0.2% Yatalase (Takara Bio Inc.) suspended in 10 mM KH ₂ PO ₄ and 0.8 M NaCl was added, and gently shaken at 30 ° C. for 1 hour. After confirming that the cells were protoplasted under a microscope, the cells were collected by centrifugation, and then washed with 0.8 M NaCl. The obtained cells were suspended in solution A (1.2 M Sorbitol, 10 mM Tris-HCl (pH 7.5)) to obtain a protoplast solution.

To 0.2 ml of protoplast solution, add cre1 gene deletion plasmid (about 10 μg) previously treated with NotI and 50 μl of PEG solution (40% PEG 4000, 10 mM Tris-HCl, 10 mM CaCl ₂ : pH 7.5), After standing on ice for 30 minutes, 1 ml of PEG solution was added, mixed well, and allowed to stand at room temperature for 30 minutes. Next, 0.2 ml of the obtained solution was applied to an MM agar medium supplemented with 1M sucrose, and colonies that had grown after 5 to 7 days were selected.
Total DNA of the obtained colonies was collected, and a strain lacking the transcription factor gene was selected by Southern analysis.

(Example 4: Evaluation of cellulase activity)
The cre1 gene-deficient strain and Y-94 strain obtained in Example 3 were each 48% in a medium in which the ratio of cellulose to glucose (mass ratio) was 5: 0, 4: 1, 2.5: 2.5, 1: 4. After culturing for a time, the culture broth was separated by centrifugation, and cellulase activity was measured to confirm enzyme production.

  Enzymatic activity was determined by colorimetric quantification of the released glucose using the DNS method, using filter paper (Whatman No. 1) as a substrate, mixing and incubating the enzyme solution at 50 ° C. for 60 minutes. Calculated.

The enzyme activity under each culture condition is shown in FIG.
As shown in FIG. 1, the cre1 gene-deficient strain has a higher cellulase production ability than the Y-94 strain, and the culture solution of the cre1 gene-deficient strain has a higher capacity than the Y-94 strain. It was revealed that cellulase activity was high. In addition, even when cultured in the presence of glucose in the medium, it is clear that the culture solution of the cre1 gene deletion strain shows higher cellulase activity than the culture solution of the Y-94 strain. became.

(Example 5: Gene expression analysis)
The cre1 gene-deficient strain and Y-94 strain obtained in Example 3 were cultured in a medium in which the ratio of cellulose to glucose (mass ratio) was 5: 0 (cellulose medium) and 1: 4 (cellulose + glucose medium). Each was cultured for 24 hours, and the cultured cells were collected by centrifugation. Total RNA was collected from the obtained bacterial cells, and cDNA was synthesized using oligo-dT primer (Toyobo). The expression of the cellobiohydrolase I (cbhI) gene was analyzed by PCR using the obtained cDNA as a template.

The results of cbhI expression analysis are shown in FIG.
As shown in FIG. 2, the expression level of cbhI significantly decreased in the Y-94 strain when glucose was present in the medium. On the other hand, the expression level of cbhI was not significantly reduced in the cre1 gene deletion strain even when glucose was present in the medium.

  From the above, it has been clarified that the cre1 gene-deficient strain has a markedly higher cellulase production ability than the wild-type strain.

  According to the filamentous fungus mutant in which the cre1 gene of the present invention does not function, cellulase can be produced more efficiently than the wild type. Moreover, according to the filamentous fungal mutant strain in which the cre1 gene of the present invention does not function, cellulase can be efficiently produced even under the condition where glucose is present in the medium. Therefore, by using the filamentous fungal mutant strain in which the cre1 gene of the present invention does not function, it can be expected that the cost of cellulase production will be reduced and the yield of monosaccharides in lignocellulosic biomass saccharification will be increased.

Claims (2)

Cellulase comprising culturing an Acremonium cellulolyticus mutant in which the cre1 gene does not function in a medium containing a raw material containing cellulose and glucose in a ratio of 4: 1 (mass ratio) Manufacturing method. Cellulose is degraded or saccharified using a culture solution of a mutant strain of Acremonium cellulolyticus in which the cre1 gene does not function in a medium containing a raw material containing cellulose and glucose at a ratio of 4: 1 (mass ratio) A method for decomposing or saccharifying cellulose.

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