JP7388628B2 - Cellulose decomposition-enhancing composition and cellulose decomposition-enhancing mutant strain - Google Patents

Description

The present invention relates to a cellulose decomposition-enhancing composition, a cellulose decomposition-enhancing mutant strain, a cellulose decomposition accelerator and its production method, a cellulose decomposition promotion method, and a silage production method.

In dairy farming, cows mainly feed on grass, but during the winter months when grass cannot grow, they need to feed on silage containing stored grass. "Sillage" is a livestock feed made by packing feed such as grass in a silo and storing it, and in particular, it is made possible to preserve it for a long time by fermenting the grass with lactic acid in the silo. Silage was originally a feed given during the winter period, but now it is the main feed given to cattle throughout the year.

Grass for silage preparation must be harvested during the appropriate grass period, which is approximately 1 week to 10 days from the beginning of heading to the beginning of heading. After this period, the nutritional value of the feed decreases and the fiber becomes coarse and stiff. Coarse and stiff fibers have poor digestibility, resulting in a decrease in the amount of food eaten by cattle. Feeding highly digestible fiber is an important issue for the livestock industry because a decrease in food intake leads to a decrease in milk production.

However, in recent years, situations have frequently occurred where grass cannot be harvested in a timely manner due to typhoons, long rains during the grass harvest period, poor grass growth due to lack of sunlight, etc. As a result, a decrease in the nutritional value of silage and a decrease in the amount of food eaten due to coarsening and stiffening of the fibers have become major problems in the livestock industry.

Decreased nutritional value in silage can be compensated for by additives such as supplements and feed design. However, there is currently no effective solution to the reduction in food intake caused by silage with coarse and stiff fibers and poor digestibility.

Until now, as a method to improve the digestibility of silage and prevent cows from reducing their intake, it has been known to provide enzyme mixtures containing cellulase directly to cows, including cellulases, hemicellulases, amylases, proteases, etc. Enzyme mixtures containing these are already commercially available. However, in order for these enzyme mixtures to be effective, a certain amount must be added, which poses a problem in that they are expensive.

The present invention involves adding an enzyme containing cellulase when stuffing grass into a silo, and allowing the enzyme containing cellulase to act on grass or rice straw to partially decompose the fibers during the silage storage period. This is a technology that improves digestibility in the rumen of cows after they have eaten it. Cellulases for silage addition include, for example, cellulases for silage preparation made by mixing cellulases produced by bacteria of the genus Acremonium and cellulases produced by bacteria of the genus Trichoderma in a fixed ratio. A formulation (MA enzyme) has been developed in the past. However, these only show the effect of improving the fermentation quality of silage, and the effect of improving digestibility has not been recognized (Patent Document 1).

Due to abnormal weather conditions in recent years, not only the fermentation quality but also the digestibility of silage has become a major problem, so new technology that can improve not only the fermentation quality but also the digestibility is desired.

Patent No. 3051900

The purpose of the present invention is to develop and provide a gene for enhancing cellulose decomposition in silage and a filamentous fungus mutant strain into which the gene is introduced, as well as to provide a method for producing highly digestible silage.

As a result of extensive research to solve the above problems, the present inventors have developed a Talaromyces strain into which the feruloyl esterase gene and the Glycoside Hydrolase family 10 endoxylanase gene (hereinafter referred to as GH10 endoxylanase gene) have been introduced. It has been found that an enzyme obtained from a Talaromyces cellulolyticus mutant strain enhances the decomposition of cellulose in collected livestock feed plants and is effective in improving the digestibility of silage. The present invention is based on the above new knowledge and provides the following.
(1) A first gene expression system comprising a promoter capable of inducing gene expression in filamentous fungal cells, and a feruloyl esterase gene arranged downstream of the promoter in an expressible state, and a gene expression system in filamentous fungal cells. A composition for enhancing cellulose decomposition in the genus Talaromyces, comprising a promoter capable of inducing expression, and a second gene expression system comprising a GH10 endoxylanase gene arranged downstream of the promoter in an expressible state.
(2) The composition for enhancing cellulose decomposition according to (1), wherein the promoter included in the first gene expression system is a GH3β-xylosidase gene promoter.
(3) The composition for enhancing cellulose decomposition according to (1) or (2), wherein the promoter included in the second gene expression system is a GH10 endoxylanase gene promoter.
(4) The cellulose decomposition enhancing composition according to any one of (1) to (3), wherein the promoter and the gene contained in the first and second gene expression systems are derived from the genus Talaromyces.
(5) The cellulose decomposition ability enhancing composition according to (4), wherein the Talaromyces genus is Talaromyces cellulolyticus (T. cellulolyticus).
(6) A gene expression system of Talaromyces cellulolyticus into which a gene expression system containing a promoter capable of inducing gene expression in filamentous fungal cells and a feruloyl esterase gene placed downstream of the promoter in a state capable of expression is introduced. Mutant strain with enhanced cellulose decomposition ability.
(7) The mutant strain with enhanced cellulose decomposition ability according to (6), wherein the promoter is a GH3β-xylosidase gene promoter.
(8) A cellulose decomposition-enhancing mutant strain of Talaromyces cellulolyticus into which the cellulose decomposition-enhancing composition according to any one of (1) to (5) has been introduced.
(9) A method for producing a cellulose decomposition promoter, comprising the step of culturing the cellulose decomposition-enhancing mutant strain according to any one of (6) to (8) in a medium to obtain a culture solution thereof. Method.
(10) The manufacturing method according to (9), further comprising a step of separating the obtained culture solution into bacterial cells and a culture supernatant to obtain a culture supernatant.
(11) The manufacturing method according to (10), further comprising the step of removing solid matter from the obtained culture supernatant.
(12) The manufacturing method according to (10) or (11), further comprising the step of drying the obtained culture supernatant or the solution obtained by removing solid matter.
(13) A step of culturing the cellulose decomposition-enhancing mutant strain according to any one of (6) to (8) in a medium to obtain a culture solution, and separating the obtained culture solution into bacterial cells and a culture supernatant. A cellulose decomposition promoter obtained by a method comprising the step of obtaining a culture supernatant.
(14) A method for promoting cellulose decomposition in plants, comprising the step of bringing the cellulose decomposition promoter according to (13) into contact with cellulosic biomass.
(15) A method for producing silage, comprising the step of contacting the collected livestock feed plant with the cellulose decomposition promoter described in (13).
(16) The method for producing silage according to (15), further comprising the step of adding lactic acid bacteria.

According to the cellulose decomposition-enhancing composition and cellulose decomposition-enhancing mutant strain of the present invention, it is possible to enhance the decomposition of cellulose in collected livestock feed plants.

According to the silage production method of the present invention, highly digestible silage can be produced.

It is a diagram showing the measurement results (average value of each n=2) of various enzyme activities (avicerase activity, CMCase activity, endoxylanase activity, feruloyl esterase activity) in various enzyme solutions (MA enzyme, CBF enzyme, CFX enzyme). . It is a figure showing free ferulic acid concentration (average value, n=3) in rice straw silage (excess amount addition test; first test section) prepared using MA enzyme or CBF enzyme. The combinations of lactic acid bacteria, MA enzyme, and CBF enzyme added to the silage are shown below. * indicates P<0.05, ** indicates P<0.01 (Tukey test). Error bars indicate standard deviation. It is a figure showing free ferulic acid concentration (average value, n=3) in rice straw silage prepared using MA enzyme or CBF enzyme (specified amount addition test; second test section). The combinations of lactic acid bacteria, MA enzyme, and CBF enzyme added to the silage are shown below. * indicates P<0.05 (Tukey test). Error bars indicate standard deviation. It is a figure showing free ferulic acid concentration and free coumaric acid concentration (average value, n=3) in grass (timothy) silage (specified amount addition test; 3rd test section) prepared using MA enzyme or CBF enzyme. . The combinations of lactic acid bacteria, MA enzyme, and CBF enzyme added to the silage are shown below. ** indicates P<0.01 (Tukey test). Error bars indicate standard deviation. Diagram showing free ferulic acid concentration and free coumaric acid concentration (average value, n=3) in grass (reed canary grass) silage (specified amount addition test; 3rd test section) prepared using MA enzyme or CBF enzyme. It is. The combinations of lactic acid bacteria, MA enzyme, and CBF enzyme added to the silage are shown below. * indicates P<0.05, ** indicates P<0.01 (Tukey test). Error bars indicate standard deviation. It is a figure showing free ferulic acid concentration (average value, n=3) in rice straw silage (excess amount addition test; 4th test group) prepared using GH10 enzyme. The combinations of lactic acid bacteria, MA enzyme, and GH10 enzyme added to the silage are shown below. ** indicates P<0.01 (Tukey test). Error bars indicate standard deviation. It is a figure showing free ferulic acid concentration (average value, n=3) in rice straw silage (excess amount addition test; 5th test section) prepared using GH10 enzyme or GH11 enzyme. The combinations of lactic acid bacteria, MA enzyme, GH10 enzyme, and GH11 enzyme added to the silage are shown below. * indicates P<0.05 (Tukey test). Error bars indicate standard deviation. It is a figure showing free ferulic acid concentration and free coumaric acid concentration (average value, n=3) in rice straw silage (specified amount addition test; 6th test group) prepared using MA enzyme or CFX enzyme. 0 indicates that the measured value is below the detection limit. The combinations of lactic acid bacteria, MA enzyme, and CFX enzyme added to the silage are shown below. * indicates P<0.05, ** indicates P<0.01 (Tukey test). Error bars indicate standard deviation. Showing the free ferulic acid concentration and free coumaric acid concentration (average value, n=3) in grass (timothy second grass) silage (specified amount addition test; 7th test plot) prepared using MA enzyme or CFX enzyme. It is a diagram. The combinations of lactic acid bacteria, MA enzyme, and CFX enzyme added to the silage are shown below. * indicates P<0.05, ** indicates P<0.01 (Tukey test). Error bars indicate standard deviation. Free ferulic acid concentration and free coumaric acid concentration (average value, n=3) in grass (frozen timothy first grass) silage (specified amount addition test; 8th test plot) prepared using MA enzyme or CFX enzyme. FIG. The combinations of lactic acid bacteria, MA enzyme, and CFX enzyme added to the silage are shown below. ** indicates P<0.01 (Tukey test). Error bars indicate standard deviation.

1. Cellulose degradability enhancing composition 1-1. Overview The first aspect of the present invention is a composition for enhancing cellulose decomposition.
The composition for enhancing cellulose decomposition of this embodiment is a composition containing as an active ingredient a gene expression system containing a feruloyl esterase gene and/or a GH10 endoxylanase gene. The cellulose decomposition-enhancing composition of this embodiment can improve the quality of silage fermentation and improve the digestibility after feeding even for grasses containing coarse and stiffened fibers such as rice straw.

1-2. Definition of Terms The following terms frequently used in this specification are defined.
"Cellulose" is a polysaccharide represented by the molecular formula (C ₆ H ₁₀ O ₅ ) _n , and is a glucose polymer in which glucose is linearly polymerized through β-1,4 glucosidic bonds. Cellulose exists as a constituent of all plant cell walls and plant fibers.

"Hemicellulose" is a general term for insoluble polysaccharides other than cellulose and pectin among the polysaccharides that constitute the cell walls of land plant cells. For example, xylan, mannan, glucuronoxylan, glucomannan, etc. are applicable.

"Xylan" is a sugar chain that has β-1,4-bonded D-xylose as its main chain. position), 4-methyl-D-glucuronic acid (positions 0-2) may be partially modified. Xylan is the main component of hemicellulose present in cell walls.

"Cellulase" is a general term for a group of enzymes that catalyze an enzymatic reaction system that hydrolyzes cellulose into glucose, cellobiose, and cellooligosaccharides, and is classified into exocellulases, endocellulases, cellobiases, etc. depending on their mode of action. Through the action of these enzymes contained in cellulase, cellulose is finally broken down into glucose.

"Ferruloyl esterase" is an enzyme that hydrolyzes ferulic acid ester. “Ferrulic acid ester” is formed in the cell wall as an ester compound of ferulic acid and arabinose residues on the side chains of xylan chains. When the ferulic acid moiety photodimerizes, the xylan chains are crosslinked, and as a result, it becomes difficult for hydrolytic enzymes to access the xylan chains and cellulose chains that make up the cell wall. Therefore, the xylan chains and cellulose chains that make up the cell wall become difficult to decompose. Conversely, when ferulic acid ester is hydrolyzed by feruloyl esterase, xylan chains and cellulose chains are easily hydrolyzed, promoting cell wall decomposition. As used herein, examples of feruloyl esterase include a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

"Endoxylanase" is an enzyme that hydrolyzes the β-1,4 bond of D-xylose in the xylose main chain. Endoxylanases are classified into GH10 and GH11 based on amino acid sequence homology (Coutinho, P.M. et al., Recent Advances in Carbohydrate Bioengineering, 246, 3-12 (1999)). GH10 endoxylanase generally has a molecular weight of 30 kDa or more, whereas GH11 xylanase generally has a relatively small molecular weight of about 20 kDa (Beaugrand et al., Carbohydr. Res. 339, 2529-2540 (2004)). As used herein, examples of GH10 endoxylanase include a peptide consisting of the amino acid sequence shown in SEQ ID NO:2.

"Filamentous fungi" is a general term for fungi that are composed of tubular cells called hyphae. Examples of filamentous fungi include, but are not limited to, Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Myceliophthora, and Neurospora. The genus Penicillium, the genus Piromyces, the genus Talaromyces, the genus Thermoascaceae, the genus Thielavia, the genus Trichoderma, and the like.

Specific examples of the "Trichoderma genus" herein include, but are not limited to, T. reesei, T. viride, T. atroviride, or Trichoderma - Examples include T. longibrachiatum.

Specific examples of the "genus Talaromyces" in this specification include, but are not limited to, T. cellulolyticus, T. versatilis, T. verruculosus. , T. stipitatus, T. funiculosus, or T. pinophilus.

Specific examples of the "Penicillium genus" in this specification include, but are not limited to, Penicillium funiculosum (P. funiculosum).

The filamentous fungi used herein include both wild strains and mutant strains. For example, in the case of T. cellulolyticus, in addition to the wild strain, the deposited mutant strains Acremonium cellulolyticus C1 strain (FERM P-18508, Patent No. 4998991), CF-2612 strain ( FERM P-21290, Patent No. 4986038), and TN strain (FERM BP-685, Patent No. 1504657). Note that Acremonium cellulolyticus is the old name of T. cellulolyticus and means the same species (Fujii et al., FEMS Microbiol. Lett. 351, 32-41 (2014)). In this specification, A. cellulolyticus will be hereinafter referred to as T. cellulolyticus.

As used herein, the term "gene expression system" refers to a gene expression system that can express a gene when introduced into a living body. A gene expression system includes a promoter, a coding region, a terminator (transcription termination sequence) as essential components, and an enhancer, an intron, a polyadenylation signal, an origin of replication, a selection marker, etc. as optional components. Specific examples of gene expression systems include, but are not limited to, circular DNA containing an origin of replication, ie, a plasmid, and linear DNA.

As used herein, the term "selectable marker" is used for the purpose of selecting actually transformed cells from a cell population after transformation, or for cells that maintain a gene expression system from descendant cells of transformed cells. This is a gene used for the purpose of screening. Specifically, when an auxotrophic mutant strain lacking a metabolic gene is used as a transformed host, the metabolic gene can be used as a selection marker gene.

As used herein, "self-cloning" refers to a genetic modification technique in which a gene expression system consisting only of sequences derived from a host organism is introduced into the host organism. For example, when T. cellulolyticus is the host organism, a gene modification technique that involves introducing into T. cellulolyticus a gene expression system consisting only of sequences derived from T. cellulolyticus is applicable. Gene modification techniques using self-cloning are generally considered to be safer than gene modification techniques involving heterologous sequences.

As used herein, "stablely maintained within the filamentous fungal cell" means that the genetic information contained in the gene expression system is maintained in substantially the same state within the filamentous fungal cell over generations. Say something. Specifically, it means that when chromosomal DNA replicates, it is simultaneously replicated and distributed to at least some of the daughter cells, and virtually all cells that proliferate under given culture conditions have this gene expression system. It means that When the gene expression system is a plasmid, the plasmid usually needs to contain a selection marker in order to be stably maintained in filamentous fungal cells. Furthermore, when the gene expression system is a linear DNA, the gene expression system must be integrated into the chromosomal DNA in order to be stably maintained within the filamentous fungal cell.

As used herein, "identity" of amino acid sequences refers to when two amino acid sequences are aligned, gaps are introduced as necessary, and the degree of amino acid identity between both amino acid sequences is maximized. The ratio (%) of the number of identical amino acid residues in the amino acid sequences of the peptides being compared to the total number of amino acid residues in the peptides.

As used herein, "a plurality of amino acid residues" refers to 2 to 30, 2 to 14, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, Or two to three amino acid residues.

In this specification, "identity" of base sequences refers to when two base sequences are aligned, gaps are introduced as necessary, and the degree of base identity between both base sequences is maximized. The ratio (%) of the number of identical bases in the base sequences to be compared to the total number of bases in the base sequences.

As used herein, "a plurality of bases" refers to 2 to 30 bases, 2 to 14 bases, 2 to 10 bases, 2 to 8 bases, 2 to 6 bases, 2 to 5 bases, 2 to 4 bases, or 2 Refers to ~3 bases.

1-3. Configuration 1-3-1. Constituent Components The composition for enhancing cellulose decomposition of the present invention is composed of an active ingredient and other ingredients. Each component will be specifically explained below.

(1) Active Ingredients The cellulose decomposition-enhancing composition of this embodiment includes, as essential active ingredients, a first gene expression system containing a feruloyl esterase gene and/or a second gene expression system containing a GH10 endoxylanase gene.

(i) First gene expression system The "first gene expression system" consists of a promoter capable of inducing gene expression in filamentous fungal cells, and a feruloyl esterase placed downstream of the promoter in a state capable of expression. Contains genes.

A "promoter that can induce gene expression in filamentous fungal cells" (hereinafter often simply referred to as "promoter") is a gene expression system that can be used when the gene expression system of the present invention is introduced into filamentous fungal cells. Any promoter that can induce the expression of a gene of interest and/or a marker gene included in The type of promoter is not particularly limited. In general, for example, the target gene to be expressed can be expressed using an overexpression type promoter that can be overexpressed in the host, a constitutively active promoter that can be constantly expressed in the host, or a constitutively active promoter that can be expressed constitutively in the host, depending on the developmental stage of the host. Time-specific active promoters that can control expression within the host, expression-inducible promoters that can freely control expression within the host, and site-specific promoters that can selectively express in specific tissues or sites of the host are known. There is. It may be determined as appropriate in consideration of the intended use of the cellulose decomposition-enhancing composition of the present invention.

Examples of promoters that can be used in the gene expression system of the present invention include, but are not limited to, cellulase gene promoters, xylanase gene promoters, pectinase gene promoters, or promoters of other enzyme genes.

As the cellulase gene promoter, for example, gene promoters such as endocellulase (EC 3.2.1.4), exocellulase (EC 3.2.1.91, EC 3.2.1.176), or cellobiase (EC 3.2.1.21) can be used. The EC number attached to each enzyme name indicates the enzyme number assigned by the Enzyme Committee of the International Union of Biochemistry and Molecular Biology (hereinafter the same shall apply).

Examples of xylanase gene promoters include endoxylanase (EC 3.2.1.8), xylan 1,4-β-xylosidase (EC 3.2.1.37), endo-1,3-β-xylanase (EC 3.2.1.32), xylan 1 Gene promoters such as , 3-β-xylosidase (EC 3.2.1.72), or α-xylosidase can be used.

Examples of pectinase gene promoters include polygalacturonase (EC 3.2.1.15), rhamnogalacturonan acetyl esterase (EC 3.1.1.86), pectin esterase (EC 3.1.1.11), and α-L-rhamnosidase (EC 3.2). .1.40), α-L-arabinofuranosidase (EC 3.2.1.55), β-L-arabinosidase (EC 3.2.1.88), Arabinan endo-1,5-α-L-arabinosidase (EC 3.2) .1.99), mannan 1,4-mannobiosidase (EC 3.2.1.100), mannan endo-1,6-α-mannosidase (EC 3.2.1.101), α-mannosidase (EC 3.2.1.24), β-mannosidase ( EC 3.2.1.25), 1,6-α-D-mannosidase (EC 3.2.1.163), rhamnogalacturonan hydrolase (EC 3.2.1.171), rhamnogalacturonan galacturonohydrolase (EC 3.2.1.173) Gene promoters such as Rhamnogalacturonan Rhamnohydrolase (EC 3.2.1.174) can be used.

Examples of promoters of other enzyme genes include feruloylesterase (EC 3.1.1.73), tannase (EC 3.1.1.20), cutinase (EC 3.1.1.74), gluconolactonase (EC 3.1.1.17), and inulinase (EC 3.1.1.17). EC 3.2.1.7), dextrase (EC 3.2.1.11), chitinase (EC 3.2.1.14), β-galactosidase (EC 3.2.1.23), β-fructofuranosidase (EC 3.2.1.26), trehalase (EC 3.2) .1.28), endo-1,3(4)-β-glucanase (EC 3.2.1.6), α-amylase (EC 3.2.1.1.), β-amylase (EC 3.2.1.2), exo-1,4- α-glucosidase (glucoamylase) (EC 3.2.1.3), α-glucosidase (EC 3.2.1.20), isoamylase (EC 3.2.1.68), isoplulanase (EC 3.2.1.57), or endo-1,3( 4) Gene promoters such as -β-glucanase (EC 3.2.1.6) can be used.

Among the above promoters, examples include, but are not limited to, exocellulase (EC 3.2.1.91, EC 3.2.1.176) such as GH7 exocellulase or GH6 exocellulase; endocellulase (EC 3.2.1.4) such as GH7 endocellulase or GH5 endocellulase. ; as cellobiase (EC 3.2.1.21), GH3 cellobiase or GH1 cellobiase; as xylan 1,4-β-xylosidase (EC 3.2.1.37), GH3β-xylosidase or GH43β-xylosidase; as feruloyl esterase (EC 3.1.1.73); , feruloyl esterase; or endoxylanase (EC 3.2.1.8), the gene promoters of GH10 endoxylanase or GH11 endoxylanase are suitable.

The biological species from which the promoter is derived is not limited as long as it is a species included in filamentous fungi. Examples include the filamentous fungal species listed in the definitions above.

For example, examples of promoters derived from T. cellulolyticus include, but are not limited to, Cel7A as GH7 exocellulase; Cel7B as GH7 endocellulase; Bgl3A as GH3 cellobiase; Cel6A as GH6 exocellulase; Cel5A; GH3β-xylosidase; Bxy3B; feruloyl esterase; FaeB; GH10 endoxylanase; Xyl10A; GH11 endoxylanase; As a specific example of the promoter used in the first gene expression system of this embodiment, a bxy3B gene promoter consisting of the base sequence shown in SEQ ID NO: 3, 70% or more, 75% or more of the base sequence shown in SEQ ID NO: 3, Sequences with an identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and expression inducing activity equivalent to or higher than that of the bxy3B gene promoter. or a promoter comprising a base sequence in which one or more bases have been deleted, substituted, or added to the base sequence shown in SEQ ID NO: 3, and has an expression-inducing activity equal to or higher than that of the bxy3B gene promoter.

The "feruloyl esterase gene" used in the first gene expression system refers to a gene encoding the aforementioned feruloyl esterase or a gene encoding a peptide having feruloyl esterase activity. The species of origin of the feruloyl esterase gene used in the present invention is not limited. For example, a feruloyl esterase gene derived from a filamentous fungus, such as a gene derived from the genus Talaromyces, may be mentioned. The feruloyl esterase mentioned here is not limited to, but includes, for example, feruloyl esterase B (FaeB) derived from T. cellulolyticus and having the amino acid sequence shown in SEQ ID NO: 1. In addition, Aspergillus niger-derived feruloyl esterase has the amino acid sequence shown in SEQ ID NO: 4 (VRIES et al., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol 63, p4638-4644 (1997)), and Penicillium funiculosum has the amino acid sequence shown in SEQ ID NO: 5. feruloylesterase derived from Aspergillus niger (Kroon et al., Eur. J. Biochem. vol267, 6740-6752 (2000)), and feruloylesterase derived from Aspergillus niger consisting of the amino acid sequence shown in SEQ ID NO: 6 (Anthony et al., Protein Expression and Purification). , vol37, p126-133 (2004)), as well as 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98 % or more, or a polypeptide consisting of a base sequence having 99% or more amino acid identity and having an enzyme activity equal to or higher than that of FaeB derived from T. cellulolyticus, or represented by SEQ ID NO: 1, 4, 5, or 6. Examples include polypeptides in which one or more amino acids are deleted, substituted, or added in the amino acid sequence, and which have an enzyme activity equal to or higher than that of FaeB derived from T. cellulolyticus. Specific examples of the feruloyl esterase gene encoding such feruloyl esterase include, but are not limited to, the faeB gene consisting of the base sequence shown in SEQ ID NO: 7, and the faeB gene consisting of the base sequence shown in SEQ ID NO: 8, 9, or 10. Examples include the feruloyl esterase gene. In addition, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% of the base sequence shown in SEQ ID NO: 7, 8, 9 or 10. A polynucleotide consisting of a base sequence having a base identity of 98% or more or 99% or more, and a polypeptide encoded by the base sequence having an activity equal to or higher than that of FaeB derived from T. cellulolyticus, SEQ ID NO: 7, Consists of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown by 8, 9, or 10, and the polypeptide encoded by these base sequences is equivalent to or higher than FaeB derived from T. cellulolyticus. Active polynucleotide, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more with respect to the amino acid sequence shown in SEQ ID NO: 1, 4, 5 or 6, A polypeptide consisting of a base sequence encoding an amino acid sequence having an identity of 97% or more, 98% or more, or 99% or more, and whose polypeptide has an activity equivalent to or higher than that of FaeB derived from T. cellulolyticus. Examples include nucleotides. "Ferruloyl esterase gene" shall mean a coding region excluding the promoter region and terminator region, but the coding region may be a gene sequence containing an intron, for example, or the genomic sequence shown in SEQ ID NO: 11. Alternatively, it may be a gene sequence that does not contain an intron sequence, such as a cDNA sequence shown in SEQ ID NO: 7.

(ii) Second gene expression system The "second gene expression system" consists of a promoter capable of inducing gene expression in filamentous fungal cells, and a GH10 endoxylanase gene placed downstream of the promoter in a state capable of expression. including.

As the promoter used in the second gene expression system of this aspect, the promoter described in the first gene expression system can be used. As a specific example of the promoter used in the second gene expression system of this embodiment, a GH10 endoxylanase A (xyl10A) gene promoter consisting of the base sequence shown in SEQ ID NO: 12, 70% of the base sequence shown in SEQ ID NO: 12 GH10 endoxylanase A (xyl10A) A promoter that has an expression-inducing activity equivalent to or higher than that of the gene promoter, or a base sequence in which one or more bases are deleted, substituted, or added to the base sequence shown in SEQ ID NO: 12, and GH10 endoxylanase A ( xyl10A) gene promoter having an expression inducing activity equal to or higher than that of the gene promoter.

The "GH10 endoxylanase gene" used in the second gene expression system refers to a gene encoding the GH10 endoxylanase or a gene encoding a peptide having GH10 endoxylanase activity. The species of origin of the GH10 endoxylanase gene used in the present invention is not limited. For example, the GH10 endoxylanase gene derived from filamentous fungi, such as from the genus Talaromyces, can be mentioned. The GH10 endoxylanase referred to herein includes, but is not limited to, GH10 endoxylanase A (Xyl10A) derived from T. cellulolyticus and having the amino acid sequence shown in SEQ ID NO:2. In addition, GH10 endoxylanase derived from Penicillium funiculosum, which has the amino acid sequence shown in SEQ ID NO: 13 (Lafond et al., Microbial Cell Factories 10;20 (2011)), is 85% or more of the amino acid sequence shown in SEQ ID NO: 2 and 13. , an enzyme consisting of an amino acid sequence having an amino acid identity of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and that is equivalent to or more than Xyl10A derived from T. cellulolyticus. An active polypeptide, or a polypeptide in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2 or 13, and has an enzyme activity equivalent to or higher than that of Xyl10A derived from T. cellulolyticus. Examples include polypeptides. Specific examples of the GH10 endoxylanase gene encoding such GH10 endoxylanase include, but are not limited to, the xyl10A gene derived from T. cellulolyticus consisting of the base sequence shown in SEQ ID NO: 14, and the base sequence shown in SEQ ID NO: 15. Examples include the GH10 endoxylanase gene consisting of the sequence. In addition, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98 % or more or 99% or more of base identity, and the polypeptide encoded by them has an activity equivalent to or more than that of Xyl10A derived from T. cellulolyticus, SEQ ID NO: 14 or 15. A polynucleotide consisting of a base sequence in which one or more bases have been deleted, substituted, or added in the base sequence shown, and the polypeptide encoded by these has an activity equivalent to or higher than that of Xyl10A derived from T. cellulolyticus, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% of the amino acid sequence shown in SEQ ID NOs: 2 and 13. Examples include polynucleotides consisting of nucleotide sequences encoding amino acid sequences having % or more identity, and the polypeptides encoded by these having an activity equivalent to or higher than that of Xyl10A derived from T. cellulolyticus. "GH10 endoxylanase gene" shall mean the coding region excluding the promoter region and terminator region, but the coding region may be a gene sequence containing an intron, for example, or the genomic sequence shown in SEQ ID NO: 16, Alternatively, it may be a gene sequence that does not contain an intron sequence, such as a cDNA sequence shown in SEQ ID NO: 14.

(iii) Configuration of each gene expression system The first and/or second gene expression system of this embodiment may be circular DNA or linear DNA as long as it can be stably maintained within the filamentous fungal cell. It can also be DNA. When the first and/or second gene expression system of this embodiment is a circular DNA, it may be a plasmid that is stably maintained within filamentous fungal cells. The plasmid that is stably maintained within the filamentous fungal cells mentioned herein includes, but is not limited to, pAUR316 DNA or pPTR II DNA (Takara Bio Inc.), which are autonomously replicating vectors for the genus Aspergillus.

The first and/or second gene expression system of this embodiment may include a transposase recognition sequence.

The promoters and/or genes constituting the first and/or second gene expression systems may be derived from the same biological species or may be derived from different biological species. Further, the gene expression system may be derived from a host into which the gene expression system is introduced, or may be derived from a species different from the host. For example, when T. cellulolyticus is transformed by self-cloning using the cellulose decomposition-enhancing composition of this embodiment, the first and/or second gene expression systems are all derived only from T. cellulolyticus. The base sequence can be the following.

The cellulose decomposition-enhancing composition of this embodiment may contain only the first gene expression system or only the second gene expression system as an essential active ingredient. Alternatively, the composition for enhancing cellulose decomposition of this embodiment may contain both the first and second gene expression systems as essential active ingredients.

The cellulose decomposition-enhancing composition of this embodiment can include a selection marker as a selective component. One or more selectable markers can be included per gene system. Usually, different selection markers are used in the first gene expression system and the second gene expression system, but there is no particular restriction in the present invention. The selection markers between gene expression systems may be the same or different.

Each selection marker is linked downstream of and placed under the control of the same or different promoter. This promoter is not limited as long as it is a promoter that can induce gene expression in the above-mentioned filamentous fungal cells. For example, the promoter may be the same as the promoter that controls the expression of the feruloyl esterase gene or the GH10 endoxylanase gene, which are essential components of each gene expression system, or may be a different promoter. The promoters that control the expression of the selection marker contained in each of the first expression system and the second expression system may be the same or different. The specific structure of the promoter conforms to the promoter described in the section "(i) First gene expression system."

(2) Ingredients other than active ingredients Ingredients other than active ingredients are not particularly limited. Examples include marker gene expression systems, carriers, other enzymes, and the like. Each component will be specifically explained below.

(2-1) Marker gene expression system The cellulose decomposition enhancing composition of this embodiment can include a marker gene expression system, if necessary.

As used herein, the term "marker gene expression system" refers to a gene expression system that includes a selection marker and is independent of the first and second gene expression systems. Therefore, the marker gene expression system does not, in principle, include the feruloyl esterase gene or the GH10 endoxylanase gene.

The marker gene used in the marker gene expression system of this embodiment is not particularly limited as long as it can be used for the purpose of selecting transformants, but for example, drug resistance genes or auxotrophic complementary genes may be used. can. Examples of drug resistance genes that can be used include destomycin, benomyl, oligomycin, hygromycin, G418, bleomycin, phleomycin, and phosphinothricin. As the auxotrophic complementary genes, pyrF, pyrG, argB, trpC, niaD, etc. can be used.

The marker gene used in the marker gene expression system of this embodiment can be selected depending on the organism to which the cellulose decomposition enhancing composition of this embodiment is introduced. For example, when transforming an orotate phosphoribosyltransferase (pyrF) gene-deficient uracil auxotrophic strain with the cellulose decomposition-enhancing composition of this embodiment, the pyrF gene can be used as a marker gene. The promoter used in the marker gene expression system of this embodiment is not limited. For example, the promoters may be selected from among the promoters listed as "promoters capable of inducing gene expression in filamentous fungal cells" in "(1) Active ingredient (i) First gene expression system" above, or the pyrF gene promoter. may be used.

The marker gene expression system may be circular DNA or linear DNA.

The marker gene expression system of this embodiment may include a sequence other than the sequence derived from the same species as the species from which the sequences of the first and/or second gene expression system are derived; The gene expression system may be substantially free of sequences other than those derived from the same species as the one from which the sequence of the gene expression system is derived. For example, when T. cellulolyticus is transformed by self-cloning with the cellulose decomposition-enhancing composition of this embodiment, the marker gene expression system of this embodiment substantially eliminates sequences other than sequences derived from T. cellulolyticus. It may not be included.

(2-2) Carrier The cellulose degradability enhancing composition of this embodiment can contain a pharmaceutically acceptable carrier as necessary.
A pharmaceutically acceptable carrier refers to an excipient commonly used in the pharmaceutical art. Since the active ingredient of the cellulose decomposition-enhancing composition of this embodiment is a nucleic acid, a carrier that can stably maintain the nucleic acid during storage or transportation is suitable. Examples include solvents, excipients, fillers, emulsifiers, fluidity additives, pH adjusters, and the like.

The solvent may be, for example, water or other aqueous solution, or an organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose and other adjuvants, phosphate buffers, sodium acetate buffers, and TE buffers. Examples of the adjuvant include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.

Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronic, kaolin, silicic acid, or combinations thereof. It will be done.

Examples of fillers include petrolatum, the aforementioned sugars and/or calcium phosphate.

Examples of the emulsifier include sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, and propylene glycol fatty acid ester.

Examples of flow additives include silicates, talc, stearates or polyethylene glycols.

Such a carrier is mainly used to facilitate the formation of a dosage form, to maintain the dosage form and drug effect, and to prevent or avoid decomposition of the first and second gene expression systems, which are active ingredients, by enzymes, etc. It is used, and may be used appropriately as necessary.

Furthermore, the composition for enhancing cellulose decomposition ability of this embodiment may contain a component for transforming filamentous fungal cells. Components for transforming filamentous fungal cells include, for example, D-sorbitol, calcium chloride, Tris-HCl buffer, polyethylene glycol 400, and the like.

1-3-2. Method of Use The method of using the composition for enhancing cellulose decomposition of this embodiment is not particularly limited. For example, the composition for enhancing cellulose decomposition ability of this embodiment may be introduced into a host filamentous fungus to obtain a transformant with enhanced cellulose decomposition ability. The host filamentous fungus is not limited. Examples include cellulase-producing bacteria.

When the cellulase-producing bacterium is, for example, a T. cellulolyticus mutant strain, it may be, but is not limited to, the C1 strain, CF-2612 strain, TN strain described above, or a further mutant strain derived from these mutant strains. Note that the cellulase-producing bacteria and the transformant with enhanced cellulose decomposition ability will be described in detail in the next chapter, "2. Cellulose decomposition ability-enhanced mutant strain," so their explanation will be omitted here.

1-4. Effects By introducing the cellulose decomposition ability enhancing composition of this embodiment into a filamentous fungus, a transformant that produces a large amount of feruloyl esterase and/or GH10 endoxylanase can be obtained. The culture solution and culture supernatant obtained from the transformant have high cellulose decomposition ability, and when added to silage, promote the decomposition of cellulose, etc. that make up the plant body, and obtain silage with improved digestibility. be able to.

2. Mutant strain with enhanced cellulose decomposition ability 2-1. Overview The second aspect of the present invention is a mutant strain with enhanced cellulose decomposition ability.
The cellulose decomposition-enhancing mutant strain of this embodiment is a mutant strain of a cellulase-producing bacterium into which the cellulose decomposition-enhancing composition of the first embodiment has been introduced.

In the cellulose decomposition-enhanced mutant strain of this embodiment, when cultured under predetermined culture conditions, the expression level of feruloyl esterase and/or GH10 endoxylanase significantly increases compared to before introduction of the composition of the first embodiment. ing.

2-2. Structure The cellulose decomposition-enhancing mutant strain of the present embodiment is a mutant strain of a cellulase-producing bacterium into which the cellulose decomposition-enhancing composition of the first embodiment has been introduced.

The composition of the cellulose decomposition-enhancing composition introduced in this embodiment is substantially the same as the content described in "1-3. Configuration" of the cellulose decomposition-enhancing composition of the first embodiment. Therefore, a detailed explanation thereof will be omitted here.

As used herein, the term "cellulase-producing microorganism" refers to a microorganism capable of producing a microorganism capable of producing cellulase within its cells. It may also be a microorganism that can further produce hemicellulase that decomposes hemicellulose. For example, it includes microorganisms belonging to the genus Aspergillus, Penicillium, etc. in addition to T. cellulolyticus, T. reesei, and T. viride.

In the cellulose decomposition-enhancing mutant strain of this embodiment, the introduction mode of the gene expression system constituting the cellulose decomposition-enhancing composition is not particularly limited. For example, it may be introduced as a plasmid and stably maintained in the cells of cellulase-producing bacteria. On the other hand, the gene expression system may be inserted into the chromosome of a cellulase-producing bacterium and stably maintained within the filamentous fungal cells. For each introduced gene expression system, one copy or multiple copies may be inserted into the chromosome of the cellulase-producing bacterium. Although the insertion position of the gene expression system is not limited, it is preferable that it does not impair the growth or enzyme productivity of the created transformant.

2-3. Production method The composition for enhancing cellulose decomposition ability can be introduced into the host according to a conventional method. For example, electroporation method, polyethylene glycol method, Agrobacterium method, etc. can be used. Although not limited, the polyethylene glycol method (Fujii et al., Biosci. Biotechnol. Biochem. 76, 245-9 (2012)) is suitable.

The transformed strain into which the cellulose decomposition-enhancing composition has been introduced can first be selected by a method depending on the selection marker contained in the cellulose-degrading ability-enhancing composition. For example, transformed strains can be selected using a medium containing a drug corresponding to a drug resistance gene used as a selection marker, or a medium containing no nutrient component corresponding to an auxotrophic complementary gene. For example, when using the pyrF gene as a marker gene, use MM agar medium that does not contain uridine or uracil (1% glucose, 10 mM ammonium chloride, 10 mM potassium dihydrogen phosphate, 7 mM KCl, 2 mM magnesium sulfate, 0.001% A strain that grows in ZnSO ₄ , 0.001% MnSO ₄ , 0.001% CuSO ₄ , 1 M sucrose, 1.5% agar, pH 6.5) can be selected as a transformed strain.

Next, in order to confirm that the first and/or second gene expression system has been introduced into the obtained transformed strain, Southern blot hybridization analysis (Southern blot analysis). For Southern blot analysis, genomic DNA extracted from transformed strains was fragmented by restriction enzyme treatment, and the resulting genomic DNA fragments were separated according to fragment size by agarose electrophoresis, and subsequently transferred to nitrocellulose or nylon membranes. Thereafter, the sequences of the first and/or second gene expression systems are used as probes to detect DNA fragments derived from the first and/or second gene expression systems. In addition, in Southern blot analysis, sequences introduced at different positions on the genome become DNA fragments of different sizes by restriction enzyme treatment, and therefore are detected as bands with different electrophoretic mobilities. Therefore, it is also effective as a means for confirming the copy number of the introduced first and/or second gene expression system.

To confirm that the first and/or second gene expression system has been transformed, a PCR detection method may be used in which PCR amplification is performed using the genome of the transformed strain as a template. PCR detection can be used as a simpler selection method than Southern blot analysis. In the PCR detection method, a DNA region included in the first and/or second gene expression system but not included in the host genome may be selected as a target for PCR amplification, and a primer sequence may be designed. For example, if either the promoter or the coding region contained in the first and/or second gene expression system is derived from a species different from the host, those skilled in the art can easily design appropriate detection primers. can. Alternatively, even if both the promoter and the coding region contained in the first and/or second gene expression system are sequences derived from the host, if the combination does not exist in the host genome, the promoter and the coding region A person skilled in the art can easily design an appropriate detection primer. On the other hand, in cases other than the above, that is, when both the promoter and the coding region contained in the first and/or second gene expression system are derived from the host and the combination is present in the host genome, PCR Detection is difficult. In this case, an enzyme solution can be prepared from the transformant according to the third embodiment below, and a strain whose enzyme activity is significantly enhanced compared to that of the host before transformation can be selected as a transformant.

2-4. Effects According to the cellulose decomposition-enhancing mutant strain of the present embodiment, by culturing under predetermined culture conditions, compared to the strain before introduction of the cellulose decomposition-enhancing composition of the first embodiment, feruloyl esterase and/or GH10 Mutant strains in which the expression level of endoxylanase is significantly increased can be obtained.

3. Method for producing cellulose decomposition accelerator 3-1. Overview The third aspect of the present invention is a method for producing a cellulose decomposition promoter.
According to the method for producing a cellulose decomposition accelerator of this embodiment, a cellulose decomposition accelerator that can promote the decomposition of cellulose contained in silage can be easily and relatively inexpensively produced.

3-2. Method The method for producing a cellulose decomposition accelerator of this embodiment includes a culturing step as an essential step, and includes a separation step, a removal step, a drying step, etc. as necessary. Each step will be specifically explained below.

3-2-1. Cultivation Step The “cultivation step” is a step of culturing the cellulose decomposition-enhanced mutant strain according to the second aspect in a medium to obtain a culture solution thereof.

The cellulose decomposition-enhancing mutant strain cultured in the culturing step of this embodiment is substantially the same as that described in "2-2. Configuration" of the cellulose decomposition-enhancing mutant strain of the second embodiment. Therefore, a detailed explanation thereof will be omitted here.

The medium used in the main culture step is not particularly limited. The medium contains powdered cellulose (including Avicel), cellobiose, filter paper, general paper, waste paper, wood, bran, wheat straw, rice straw, rice husk, bagasse, soybean meal, soybean okara, and coffee grounds as carbon sources. , rice bran, lactose, lactose hydrate, whey, dairy products and mixtures thereof, glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal and vegetable oils, etc. can be used. Nitrogen sources include inorganic ammonium salts such as ammonium sulfate and ammonium nitrate, urea, amino acids, meat extract, yeast extract, polypeptone, soybean flour, wheat germ, corn steep liquor, cottonseed meal, and organic nitrogen containing products such as protein decomposition products. and inorganic salts such as magnesium sulfate, potassium dihydrogen phosphate, potassium tartrate, zinc sulfate, manganese sulfate, copper sulfate, calcium chloride, iron chloride, manganese chloride, and the like. If necessary, a medium containing organic micronutrients may be used. The medium can be a solid medium that has been solidified by adding agar or gelatin, a semi-liquid medium that has added a low concentration of agar, or a liquid medium that contains only medium components (also called bouillon or broth). is preferred.

Specific examples of media include cellulose media (5% Solka Floc 40, 0.1% CSL, 0.24% KH ₂ PO ₄ , 0.05% (NH ₄ ) ₂ SO ₄ , 0.047% Potassium tartrate, 0.1% tween80, 0.12% MgSO ₄ , 0.2% Urea, 0.001% ZnSO ₄ , 0.001% MnSO ₄ , 0.001% CuSO ₄ , pH 4.0), medium in which only the Urea concentration was changed from the above cellulose medium to 0.4%, GY medium (3% glucose, 2% Bacto Yeast Extract) or PD medium (2.4% Difco Potato Dextrose Broth) may be used.

The culture conditions for the main culture step are not particularly limited. The mutant strain with enhanced cellulose decomposition ability may be cultured under optimal growth conditions. For example, the culture temperature may be within the optimal temperature range for the mutant strain with enhanced cellulose decomposition ability. For example, culture can be carried out within a temperature range of 20-40°C, 25-35°C, 25-30°C, 26-30°C, 28-30°C, for example, 28°C, 29°C, or 30°C.

The pH of the culture solution may also be adjusted to the optimum pH for the cellulose decomposition-enhancing mutant strain. For example, it can be cultured at a pH within the range of pH 2-8, pH 3-7, or pH 4-6, such as pH 4 or pH 5.

In addition, regarding air permeability, it is sufficient that the cellulose decomposition-enhancing mutant strain to be used can proliferate well. Since the cellulose decomposition-enhancing mutant strain of the present invention is usually aerobic, it is preferable to dissolve oxygen in the medium by aeration during culturing. The main culture method is not limited as long as it is a method that can culture a mutant strain with enhanced cellulose decomposition ability. For example, the main culture method may be static culture, but flow culture, which imparts fluidity to the medium, is preferred. Flow culture includes, for example, shaking culture, rotation culture, or stirring culture.

The main culture period is not limited. Culture is carried out until the cellulose decomposition-enhanced mutant strain reaches a sufficient concentration and the enzyme concentration in the culture solution reaches a sufficient concentration. Whether the enzyme concentration in the culture solution has reached a sufficient concentration can be confirmed by, for example, using bovine BSA as a calibration curve and checking the protein concentration in the culture solution using Bradford reagent. If the protein concentration reaches, for example, 1500 μg/mL to 2500 μg/mL in the preculture and 2000 μg/mL to 5000 μg/mL in the main culture, it is confirmed that the enzyme concentration has reached a sufficient concentration. For example, culture may be carried out for 1 to 10 days, 3 to 7 days, for example 5 days. The culture solution obtained in the main culture step can be used as it is as a cellulose decomposition promoter.

3-2-2. Separation Step The "separation step" is a step of separating the culture fluid obtained in the culture step into bacterial cells and a culture supernatant to obtain a culture supernatant, and is a selection step in the present production method.

The culture supernatant separation method that can be used in this separation step is not particularly limited as long as it can remove bacterial cells from the culture solution, but examples include centrifugation, precipitation, filtration, and these methods. Examples include combinations. When using the centrifugation method, the enzymes (feruloyl esterase, GH10 endoxylanase, etc.) secreted by the cellulose-degrading mutant strain in the culture solution are not inactivated, for example, by centrifugation at 4°C, 3500 rpm, 15 minutes, The culture supernatant may be obtained by separating the bacterial cells from the culture solution. The culture supernatant separated from the bacterial cells by centrifugation or precipitation can be further isolated by decantation or the like. Further, when using a filtration method, for example, a method of passing through a filter paper or a membrane filter can be mentioned.

The culture supernatant obtained in this separation step can be used as a cellulose decomposition promoter.

3-2-3. Removal Step The "removal step" is a step of removing solid matter from the obtained culture supernatant, and is a selection step in the present production method. This step is a step performed after the separation step, in which solid matter such as bacterial cells and other impurities contained in the culture supernatant that could not be separated in the separation step is removed.

The "solid matter" here refers to particles larger than a predetermined size contained in the culture solution, for example, particles larger than 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, or 10 μm, such as Particles larger than 0.2 μm. For example, cells of cellulase-producing bacteria remaining as contaminants after the separation process, other microbial species mixed in during the culture process, fibers remaining in the culture solution, or crystals of medium components generated in the culture solution. .

The solid matter removal method used in this step is not particularly limited, but similarly to the separation step described above, includes a filtration method, a centrifugation method, a precipitation method, a lysis method, and a combination thereof. However, in order to more completely remove solids from the culture supernatant, it is preferable to use a method with more stringent conditions than the separation step. For example, in the case of a filtration method, filtration is performed using a filter with a smaller pore size than the filter used in the separation process, and in the case of a centrifugation method, the number of rotations is higher than the rotation speed and rotation time used in the separation process. For example, a method of centrifugation with a rotation time of 100 mL, and a method of performing precipitation for a longer time than the precipitation time used in the separation step, and a method of aspirating only the supernatant. More specifically, a method of passing the cellulase-producing bacteria through a filter, a method of lysing the bacterial cells by surfactant treatment, enzyme treatment, etc., or a combination thereof may be used. For example, when using filtration, a method of passing through a membrane filter having a pore size of, but not limited to, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm or 10 μm can be used.

The solution obtained by this solid matter removal step may be sterilized.
The solution obtained in this step can be used as a cellulose decomposition promoter.

3-2-4. Drying process The "drying process" refers to drying the culture supernatant or solution obtained after the culture process, separation process, and/or removal process to reduce the water content by evaporation or remove water to form solids. This is a selection step in the present manufacturing method.

This step is a step performed on the liquid (culture supernatant or solution) obtained after any of the above steps in the present production method, and is performed last in the steps described in this specification.

The drying method is not particularly limited as long as it can reduce the water content in the liquid and does not deactivate the enzyme secreted by the cellulose decomposition-enhanced mutant strain. For example, natural drying method where you are exposed to the outside air, dehumidification drying method where you are left in a sealed space with a dehumidifier for a certain period of time, sun drying method where you are exposed to sunlight and outside air to dry, air drying method where you are exposed to hot or cold air using a blower, etc. Thermal drying method uses a heat source such as a heater to dry, the reduced pressure drying method uses a vacuum pump etc. to degas and evaporate the liquid inside a container, and the freeze drying method uses drying the liquid while it remains frozen. , or a combination thereof.

The removal of water in the liquid may be partial or complete removal. In the case of partial removal, the enzyme concentration in the liquid can be concentrated. Moreover, in the case of complete removal, the weight of the cellulose decomposition accelerator of the present invention can be reduced. The amount of water to be removed in this step may be appropriately determined as necessary. The solidified cellulose decomposition accelerator can also be processed into blocks, flakes, granules, and powders.

By reducing the amount of water contained in the cellulose decomposition accelerator, transportation efficiency can be increased and storage space can be reduced.

4. Cellulose decomposition accelerator 4-1. Overview The fourth aspect of the present invention is a cellulose decomposition promoter. The cellulose decomposition accelerator of this aspect is a cellulose decomposition accelerator obtained by the method for producing a cellulose decomposition accelerator of the third aspect. According to the cellulose decomposition accelerator of this embodiment, it has the ability to decompose cellulose when brought into contact with plants, and when added to silage, its digestibility can be improved.

4-2. Structure The cellulose decomposition accelerator of this embodiment is obtained from a culture solution of cellulase-producing bacteria into which the cellulose decomposition ability enhancing composition of the present invention has been introduced. The culture solution of cellulase-producing bacteria contains various enzyme components that have excellent cellulase activity related to cellulase decomposition. However, all components contained in the culture solution and their contents are determined by the manufacturing method and cannot be specifically specified.

5. Method for promoting cellulose decomposition of cellulosic biomass 5-1. Overview The fifth aspect of the present invention is a method for promoting cellulose decomposition of cellulosic biomass. According to the method for promoting cellulose decomposition of this embodiment, the decomposition of cellulose in cellulosic biomass can be promoted.

5-2. Method The method for promoting cellulose decomposition of this embodiment includes a contact step as an essential step.
The "contact step" is a step of bringing a cellulose biomass into contact with a cellulose decomposition promoter.

In this embodiment, "cellulose biomass" refers to an organic resource containing cellulose as a main component. For example, plants such as herbaceous biomass and woody biomass, herbaceous waste, and woody waste fall under this category. Examples of herbaceous biomass include grass, plant agricultural waste (e.g., stems, leaves, grain husks, etc.), specifically rice straw, wheat straw, rice husk, wheat husk, cotton seed husk, bran, and corn. sorghum stems, leaves and cobs, and sorghum stems and leaves). Examples of woody biomass include forest residues of coniferous or broad-leaved trees, thinned wood, unused trees, short-cycle cultivated wood, roadside trees, and pruned wood from parks and the like. Examples of herbaceous wastes include plant juice residues (including beer lees, bagasse, beet pulp, okara, cottonseed lees, and oil lees). Examples of wood-based waste include construction debris, lumber residues (including bark, sawdust, planer scraps, and short lumber), and paper resources (e.g., newspapers, magazines, waste office paper, and cardboard). The "cellulose decomposition accelerator" used in this step is the cellulose decomposition accelerator produced by the manufacturing method of the third aspect or described in the fourth aspect. The state of the cellulose decomposition accelerator does not matter. It may be a liquid or a solid (for example, a powdered solid or a granular solid).

"Contact" in this process refers to direct physical contact between the object and the target object. In the case of the present invention, this means that the cellulose decomposition accelerator, which is the object of the present invention, comes into contact with the cellulosic biomass, which is the object. The contact method is not particularly limited. Examples include a method of spraying, scattering, dipping, or applying a liquid, powder, or granular cellulose decomposition promoter containing the cellulose decomposition promoter of the present invention onto cellulosic biomass, or a combination of these methods. Contact may be repeated multiple times if necessary.

According to the method for promoting cellulose decomposition of this embodiment, cellulosic biomass derived from plants can be partially decomposed. This makes it possible to utilize cellulosic biomass more easily.

6. Silage production method 6-1. Overview The sixth aspect of the present invention is a method for producing silage. According to the silage production method of this embodiment, silage with improved digestibility can be produced.

6-2. Method The silage manufacturing method of this embodiment includes a contacting step as an essential step, and optionally includes a lactic acid bacteria addition step. Note that the "contact step" in this embodiment is substantially the same as the content described in "5-2-1. Contact step" in the fifth embodiment. Therefore, the specific explanation thereof will be omitted here, and the lactic acid bacteria addition step, which is a characteristic step of this step, will be specifically explained below.

The "lactic acid bacteria addition step" is a step of adding lactic acid bacteria to the collected livestock feed plants. In this case, this step may be performed before, after, or simultaneously with the cellulose decomposition accelerator contact step. Alternatively, lactic acid bacteria may be mixed with a cellulose decomposition promoter in advance and brought into contact with livestock feed plants together.

As used herein, the term "plant for livestock feed" refers to any plant that can be provided as feed to livestock. Examples include feed plants used for silage. Specifically, for example, grass, feed crops, etc. fall under this category. For example, grasses include timothy, reed canary grass, orchard grass, Italian rye grass, perennial rye grass, festlorium, hybrid rye grass, Kentucky bluegrass, red top, meadow fescue, guinea grass, tall fescue, smooth loam grass, Examples include dalli grass, bahia grass, leguminous grasses such as alfalfa, red clover, and white clover. Examples of feed crops include corn, sorghum, sudan grass, barley, rye, and triticale. Preferably, grass or rice (rice straw) is used, and timothy or orchard grass is preferable as the grass.

As used herein, "livestock" refers to animals that are bred or raised to obtain livestock products such as dairy products, meat, eggs, and leather. Examples include, but are not limited to, cows, horses, pigs, goats, sheep, chickens, buffalo, yaks, donkeys, mules, camels, llamas, alpacas, wild boars, guinea pigs, rabbits, ducks, turkeys, and quail.

As used herein, "collected (plant for livestock feed)" refers to a plant that has been propagated in nature and is artificially collected, regardless of whether it is a natural product or a cultivated product. A plant that is separated from the growing environment by collection and therefore does not, in principle, grow or multiply over a long period of time unless it is placed in the growing environment again. Therefore, plants growing in fields etc. are excluded.

"Lactic acid bacteria" is a general term for bacteria that metabolize sugars and produce lactic acid. The lactic acid bacteria in this embodiment are not limited as long as they are bacteria that metabolize glucose, cellobiose, cellooligosaccharides, xylose, xylobiose, xylooligosaccharides, etc. produced from livestock feed plants to produce lactic acid, acetic acid, etc. Examples include species belonging to the genus Bifidobacterium (hereinafter, the genus Bifidobacterium will be collectively referred to as "Bifidobacteria") and species belonging to the order Lactobacillus. Species belonging to the order Lactobacillus include, for example, the genus Lactobacillus, genus Lactococcus, genus Enterococcus, genus Leuconostoc, genus Weissella, and genus Pediococcus ( Examples include the genus Pediococcus. As the Lactobacillus genus, for example, Lactobacillus plantarum (L. plantarum) may be used, and more specifically, for example, Lactic Acid Bacterium Kuso No. 1 (Snow Brand Seed Co., Ltd.) may be used. The bifidobacterium may be any species. For example, B. Bifidum (B. bifidum), B. longum (B. longum), B. Brave (B. brave), B. B. infantis and B. infantis. An example is B.adolescentis. The species belonging to the order Lactobacillus may be any species. Examples include L. paracasei, L. lactis, and E. faecium. "Addition" in this step is not particularly limited as long as it is a method that involves bringing lactic acid bacteria into contact with livestock feed plants. For example, apart from the above-mentioned contacting step with the cellulose decomposition accelerator, methods of spraying, scattering, dipping, or coating livestock feed plants, or a combination of these methods may be mentioned.

The lactic acid bacteria added through this process consume glucose produced by cellulose decomposition and produce lactic acid, thereby lowering the silage pH and suppressing the growth of harmful microorganisms, which are caused by butyric acid bacteria during sealed storage. It has the effect of suppressing butyric acid fermentation and the effect of suppressing secondary fermentation, which causes heat generation and deterioration due to the increase of yeast etc. after silage is opened.

Hereinafter, the present invention will be explained in more detail using Examples. However, the technical scope of the present invention is not limited to these Examples.

<Example 1: Production of CBF strain and CFX strain, preparation of various enzyme solutions and activity measurement>
(the purpose)
A T. cellulolyticus mutant strain is created, and various enzyme solutions are prepared from the culture fluid. Additionally, enzyme activity is measured.

(Method)
(1) Method for producing CBF strain
Using T. cellulolyticus genomic DNA as a template, PCR amplification was performed using the primer set shown in SEQ ID NO: 17 and SEQ ID NO: 18 targeting the GH3β-xylosidase B (bxy3B) gene promoter region (SEQ ID NO: 3). The pANC701 plasmid was constructed by inserting the amplified bxy3B gene promoter fragment into the plasmid pbs-pyrF (Fujii et al., Biosci. Biotechnol. Biochem. 76, 245-9 (2012)). On the other hand, using the genomic DNA of T. cellulolyticus as a template, primers shown in SEQ ID NO: 19 and SEQ ID NO: 20 were targeted to the feruloyl esterase B (faeB) gene from the coding region (including introns) to the terminator region. PCR amplification was performed using the DNA fragment, and the amplified DNA fragment was treated with SalI restriction enzyme. A Pbxy3B-faeB expression plasmid (pANC749) was constructed by inserting this into pANC701 plasmid treated with EcoRV/SalI restriction enzymes by ligation.

A 1/2 volume of ammonium acetate and 2 volumes of 100% ice-cold ethanol were added to a DNA solution in which 12 μg of plasmid had been dissolved, and the DNA was precipitated by standing at -20°C for 2 hours or more. After centrifugation at 14,000 rpm and 4°C for 10 minutes, the supernatant was removed, and 1.1 times the volume of the initial DNA solution of 70% ice-cold ethanol was added to wash the DNA. After washing, the mixture was centrifuged again at 14,000 rpm and 4°C for 10 minutes, the supernatant was discarded, and the DNA was dried by suction, and the DNA was dissolved in 10 μL of EB buffer. After using 1-2 μL and measuring the concentration using Nanodrop, the DNA concentration was adjusted to 800-850 ng/μL using EB buffer. This DNA solution was used for the next transformation.

The protoplast-PEG method was used for transformation (Fujii et al., Biosci. Biotechnol. Biochem. 76, 245-9 (2012)). The host used was the CFP3 strain, which had UV mutations added to the CF-2612 strain to make it auxotrophic for uracil. 50 μl (1x10 ⁷ cells) of the CFP3 strain, which had been protoplasted and suspended in a dedicated buffer (1.2 M sorbitol, 10 mM Tris-HCl buffer pH 7.5, 10 mM CaCl ₂ ), was dispensed into sterilized Eppendorf tubes. 4 μl of plasmid and 25 μl of PEG solution (40% polyethylene glycol, 10 mM Tris-HCl buffer pH 7.5, 10 mM calcium chloride) were added thereto, and the mixture was left standing on ice for 30 minutes. After 30 minutes, 500 μl of PEG solution was slowly added on a clean bench and suspended by gentle pipetting, and the entire amount was put into a disposable tube and left at 30° C. for 15 minutes. After standing, add 30 ml of MM-sucrose soft agar medium (MM agar medium with 1M sucrose added, containing 0.7% agar) pre-warmed to 45°C to the disposable tube, and immediately add MM-sucrose agar medium (MM agar medium). The cells were plated on a medium (with 1M sucrose added) and allowed to acclimate. After being allowed to stand and harden, it was cultured at 30°C for 5 days, and the colonies that grew were transplanted. The transformants that were sub-planted and found to grow were used for colony PCR and measurement of feruloyl esterase activity, etc., and strains were selected. The obtained strain will be referred to as the "CBF strain," an acronym for CF-Pbxy3B-faeB expression strain, and the enzyme solution obtained from the CBF strain will be referred to as "CBF enzyme."

(2) Method for producing CFX strain
Three types of linear DNA cassettes derived from the T. cellulolyticus genome sequence: (i) The first gene cassette (SEQ ID NO: 21) consisting of the bxy3B gene promoter and the faeB gene coding region (including introns) and terminator region; Pbxy3B-faeB-TfaeB), (ii) a marker gene cassette consisting of the pyrF gene promoter, coding region (including introns), and terminator region (PpyrF-pyrF-TpyrF shown in SEQ ID NO: 22), (iii) GH10 endoxylanase A (xyl10A) gene promoter, xyl10A gene coding region (including intron), and terminator region. CFP3 strain made uracil auxotrophic was transformed. Transformation was performed by the protoplast-PEG method.

In the primary screening, the success or absence of transformation was determined using the return from uracil auxotrophy due to the introduction of the marker gene cassette as an indicator. In the secondary screening, the introduction of the first gene cassette was confirmed by colony PCR that amplified the sequence between the bxy3B gene promoter and the faeB gene coding region. In the tertiary screening, the endoxylanase activity of the transformed candidate strain was measured, and introduction of the second gene cassette was confirmed by an increase in activity. This is because the second gene cassette cannot be distinguished from the endogenous xyl10A gene by PCR. In the fourth screening, we confirmed whether the faeB gene was expressed as a protein by measuring feruloyl esterase activity.

In the obtained strain, the gene was introduced by self-cloning without using a vector or the like. It was confirmed by Southern analysis that approximately 1 to 2 copies of each introduced gene were located at non-homologous sites on the chromosome. These genes are stably maintained in the genome.

The obtained strain will be referred to as the "CFX strain" by taking the initials of the CF-faeB-xyl10A strain, and the enzyme solution obtained from the CFX strain will be referred to as the "CFX enzyme."

(3) Preparation method of CBF enzyme and CFX enzyme Cut a 1cm square cut of the CBF strain and CFX strain grown sufficiently in potato dextrose agar (PDA) medium, and add to 10 ml of cellulose medium (5% Solka Floc 40, 0.1% CSL, 0.24% KH ₂ PO ₄ , 0.05% (NH ₄ ) ₂ SO ₄ , 0.047% Potassium tartrate, 0.1% tween80, 0.12% MgSO ₄ , 0.2% Urea, 0.001% ZnSO ₄ , 0.001% MnSO ₄ , 0.001% CuSO ₄ , pH 4.0) and precultured at 28°C at 230 rpm for 5 days. After culturing, 0.5 mL of the culture solution was inoculated into a new cellulose medium (only the Urea concentration was changed to 0.4%), and cultured with shaking at 230 rpm and 28°C for 5 days. Subsequently, the culture solution was centrifuged at 4° C. and 3500 rpm for 15 minutes, and the supernatant was filtered using a 0.2 μm filter, and the filtrate was used as an enzyme solution for various activity measurements and for the preparation of various silages. Enzyme solutions derived from CBF strain and CFX strain are referred to as CBF enzyme and CFX enzyme, respectively.

(4) Avicelase activity measurement method As an indicator of the ability to decompose crystalline cellulose, the avicelase activity of each enzyme solution prepared using crystalline cellulose (avicel) as a substrate was measured.

Transfer 2.5 mL of 2% Avicel suspension (2% Avicel, 0.04% glucose in deionized water) and 2.0 mL of 0.05 M acetate buffer to an L-shaped test tube, and place in a tabletop shaking constant temperature bath (PERSONAL-11). , Taitec Co., Ltd.) at 50°C for 10 minutes with shaking at a rotational speed of 60 rpm. After 10 minutes, 0.5 mL of an appropriately diluted enzyme solution was added and reacted at 50°C for 30 minutes with shaking. After 30 minutes, 0.5 mL of 0.5N NaOH was added to stop the reaction. The entire sample was transferred to a centrifuge tube, and after centrifugation (3000 rpm, 10 minutes), 0.5 mL of supernatant was taken, 1.5 mL of DNS reagent was added thereto, and the mixture was stirred. After stirring, the reaction mixture was heated in a boiling water bath for 5 minutes, and the reaction was immediately stopped with ice water. After the reaction, 4.0 mL of deionized water was added to the reaction solution, and the absorbance at 540 nm was measured. A blank was used in which 0.5 mL of enzyme solution was first added to a test tube, followed by 0.5 mL of 0.5 N NaOH, 2.5 mL of 2% Avicel suspension, and 2.0 mL of 0.05 M acetate buffer. Operations after centrifugation were performed in the same manner as for the sample. The calibration curve was prepared by adding 1.5 mL of DNS to 0.5 mL of glucose solution (4 types: 0.2, 0.4, 0.6, and 0.8 mg/mL). In order to match the DNS reaction conditions with the sample and blank, the sample, blank, and calibration curve sample were heated and reacted at the same timing. After the reaction, 4.0 mL of deionized water was similarly added, the absorbance at 540 nm was measured, and a calibration curve was created. Avicelase activity was calculated based on the assumption that 1 U is the amount of enzyme that releases 1 μmol of glucose per minute. The calculation formula is shown in Formula 1 below.

(5) CMCase activity measurement method CMCase activity was measured using carboxymethylcellulose (CMC) as a soluble cellulose substrate. In carboxylmethylcellulose, some of the hydroxyl groups contained in cellulose are carboxylmethylated.

0.25 mL of a substrate solution (1% carboxymethyl cellulose (CMC), 0.04% glucose dissolved in 0.1 M acetate buffer solution (pH 4.5)) was added to the test tube, and the tube was preheated at 50°C for 10 minutes. After preheating, 0.25 mL of an appropriately diluted enzyme solution was added, and the mixture was left standing at 50°C for 30 minutes to perform an enzyme reaction. After 30 minutes of enzymatic reaction, the enzymatic reaction was stopped by adding 1.5 mL of DNS solution. After stopping the enzymatic reaction, the sugar and DNS reagent were allowed to chemically react in a boiling water bath for 5 minutes, and immediately after 5 minutes, the chemical reaction was stopped by placing them in ice water. After the chemical reaction had stopped, 4.0 mL of distilled water was added, and after stirring, the absorbance at 540 nm was measured. A blank was used in which 0.25 mL of enzyme solution, 1.5 mL of DNS reagent, and 0.25 mL of substrate solution were added in this order. The calibration curve was prepared by adding 1.5 mL of DNS to 0.5 mL of glucose solution (4 types: 0.2, 0.4, 0.6, and 0.8 mg/mL), as in the case of Avicelase activity measurement. In order to match the chemical reaction conditions of DNS and sugar, the sample, blank, and calibration curve sample were reacted simultaneously in a boiling water bath. After the reaction was completed, the absorbance wavelength was measured at 540 nm. Similarly to Avicelase activity, CMCase activity was calculated based on the assumption that 1U is the amount of enzyme that releases 1 μmol of glucose per minute. The calculation formula is shown in Formula 2 below.

(6) Endoxylanase activity measurement method Add 0.25 mL of substrate solution (2% xylan, 0.04% xylose dissolved in 0.1 M acetate buffer solution (pH 4.5)) to a test tube and preheat at 50℃ for 10 minutes. did. After preheating, 0.25 mL of an appropriately diluted enzyme solution was added, and the mixture was left standing at 50°C for 30 minutes to perform an enzyme reaction. After 30 minutes of enzymatic reaction, the enzymatic reaction was stopped by adding 1.5 mL of DNS solution. After stopping the enzymatic reaction, the sugar and DNS reagent were allowed to chemically react in a boiling water bath for 5 minutes, and immediately after 5 minutes, the chemical reaction was stopped by placing them in ice water. After the chemical reaction was stopped, 4.0 mL of distilled water was added, centrifuged at 3000 rpm for 10 minutes, and the absorbance at 540 nm was measured using the supernatant. A blank was used in which 0.25 mL of enzyme solution, 1.5 mL of DNS reagent, and 0.25 mL of substrate solution were added in this order. The calibration curve was prepared by adding 1.5 mL of DNS to 0.5 mL of xylose solution (4 types: 0.2, 0.4, 0.6, and 0.8 mg/mL). In order to match the chemical reaction conditions of DNS and sugar, the sample, blank, and calibration curve sample were reacted simultaneously in a boiling water bath. After the reaction was completed, the sample was used for absorbance wavelength measurement at 540 nm. The activity was calculated based on the assumption that 1 U is the amount of enzyme that releases 1 μmol of xylose per minute. The calculation formula is shown in Equation 3 below.

(7) Method for measuring feruloyl esterase activity Add 0.25 mL of substrate solution (0.01% methyl ferulate dissolved in 0.1M acetate buffer (pH 4.5)) to an Eppendorf tube and incubate in a dry bath set at 50℃ for 10 minutes. Preheated for minutes. After preheating, 0.25 mL of an appropriately diluted enzyme solution was added, and an enzyme reaction was performed at 50°C for 30 minutes. After the enzyme reaction, 0.5 mL of methanol acetic acid solution (98% methanol, 2% acetic acid) was added to stop the reaction. After stopping the reaction, the mixture was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was filtered through a 0.2 μm filter. After filtration, the amount of ferulic acid released was measured by HPLC. For HPLC, LC-30AD, SIL-30AC, CTO-20A, and SPD-M20A manufactured by Shimadzu Corporation were used. YMC-Triart C18 was used as the column, and 40% methanol and 1% acetic acid were used as the mobile phase. The HPLC conditions were a flow rate of 0.6 mL/min, a column temperature of 40°C, an absorption wavelength of 320 nm, and isolactic conditions. Ferulic acid solutions of 0.0003125%, 0.000625%, 0.00125%, 0.0025%, 0.005%, and 0.01% were used for the calibration curve. The activity was calculated based on the assumption that 1 U is the amount of enzyme that releases 1 μmol of ferulic acid per minute. The calculation formula is shown in Formula 4 below.

(8) Method for quantifying total protein concentration In order to standardize the activity measurements in each enzyme solution with the total protein concentration of each enzyme solution, the total protein concentration was measured for each enzyme solution.

WAKO's Protein Assay Bradford Reagent was used to measure the total protein concentration of the enzyme solution. After diluting the enzyme sample to an appropriate concentration, 100 μL of the enzyme dilution solution and 100 μL of Bradford reagent were added in this order to the wells of a microplate reader, and the reaction was performed for 10 minutes at room temperature. The reaction was performed using a 96-well microplate reader, and after the reaction, absorbance at 595 nm was measured using a microplate reader Varioskan Flash manufactured by Thermo Scientific. A calibration curve was created using six types of bovine serum albumin BSA 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 (μg/mL) manufactured by WAKO. As with the samples, 100 μL of BSA solution of each concentration and 100 μL of Bradford reagent were added to the microplate reader in this order, and the reaction was carried out for 10 minutes at room temperature. After the reaction, absorbance at 595 nm was measured and used to create a calibration curve.

(result)
(1) Enzyme activity of various enzyme solutions The results of activity measurements of various enzyme solutions are shown in FIG.
The activity measurement value for each enzyme solution was standardized by the total protein concentration of each enzyme solution, and is shown as a relative value with MA enzyme activity as 1. The CBF enzyme and CFX enzyme had increased endoxylanase activity and feruloyl esterase activity compared to the MA enzyme conventionally used by Snow Brand Seed Co., Ltd. (Figure 1).

<Example 2: Evaluation of fermentation quality in silage prepared using CBF enzyme>
(the purpose)
Evaluate the fermentation quality of silage prepared using CBF enzymes.

(Method)
(1) Rice straw silage preparation method The rice straw used for silage preparation was obtained from a farmer in Hokkaido after harvesting edible rice. First, rice straw was dried at 60°C for more than 2 days. This drying treatment was performed to adjust the moisture content before silage preparation.

Next, it was passed through a 1 mm mesh in a pulverizer to obtain dried rice straw powder.

Subsequently, a set amount of distilled water was added to the dried rice straw powder. This set amount was calculated using two levels (70% and 80% moisture content) and assuming the moisture content after drying as 0%. The moisture content was set at 80% in the 1st, 4th, and 5th test plots, and 70% in the 2nd and 6th test plots. The reason why we set the moisture setting value at two levels is because the moisture content after cutting is generally 70% (i.e., the common sense moisture condition for preparing silage is 70%), but the higher the moisture content, the faster the enzyme reaction (i.e. This is because the dry matter loss rate after silage storage tends to improve.

Thereafter, a standard amount (1×10 ⁵ cfu/g fresh grass grass) of lactic acid bacterium No. 1 (Snow Brand Seedlings) and two standard amounts (excess amount and specified amount) of the test enzyme were added by sprayer.

The 1st, 4th, and 5th test sections were tests for excessive amounts added, and the 2nd and 6th test sections were tests for adding specified amounts. "Specified amount" refers to the amount of test enzyme added that corresponds to the avicelase activity relative to the product recommended amount of cellulase MA enzyme for silage used by Snow Brand Seed Co., Ltd. (150 g of MA enzyme product per 10 tons of pasture). It is. Avicelase activity (approximately 13.85 U / kg fresh grass grass) contained in 150 g of MA enzyme product may vary slightly depending on the production lot, so the amount of test enzyme added may vary from test to test due to differences in the production lot of MA enzyme product used as a control. It can be different. In addition, "excess amount added" or "excess amount" refers to the amount of test enzyme added that contains the avicelase activity of 55.4 U/kg of fresh grass grass. The corresponding amount of MA enzyme added may also vary slightly depending on the manufacturing lot.

In the fourth test group, GH10 endoxylanase, which has an endoxylanase activity equivalent to twice that of MA enzyme, was added.

Pack 100g or 200g of rice straw silage to which lactic acid bacteria and test enzymes have been sprayed into pouch bags (Asahi Kasei Packs Co., Ltd., standard bag Hiryu N-9) and deaerate using a commercial tabletop sealing packaging machine (SQ-303W). It was sealed. After degassing and sealing, store at a temperature of 37℃ or 25℃ for 2 weeks to 1 month, and after opening, measure organic acid content, pH, moisture content, and amount of ferulic acid/coumaric acid released, fiber analysis, and dry matter disappearance. The rate was measured. Coumaric acid, like ferulic acid, is a type of hydroxycinnamic acid and mediates ether bonds between lignin and cell wall polysaccharides. In this test, ferulic acid was mainly measured, but it was confirmed that not only ferulic acid but also coumaric acid was significantly liberated during the measurement process, so it was decided to perform quantitative analysis. The results are expressed as weight percentage of fiber content relative to fresh matter (FM). For example, if 0.0001 g of ferulic acid is liberated in 100 g of rice straw containing 70% moisture, the concentration of ferulic acid liberated in the original material is expressed as 0.0001% FM.

(2) Grass (timothy 1st grass, reed canary grass, timothy 2nd grass) silage preparation method Timothy, reed canary grass, timothy 2nd grass are cut by hand using a sickle in the meadow on the day of silage preparation. The one collected in was used. The growth stage was harvested at the appropriate time. The appropriate period refers to the beginning of heading, and specifically, the period when approximately 3 ears/m ² of ears are observed. In this test, timothy, reed canary grass, and grasshopper were cut from early to mid-June, and timothy second grass was cut in mid-August.

The cut grass was chopped into pieces with a cutting length of 1 cm using a cutting machine, and after stirring well by hand, it was used for silage preparation.

For silage preparation, standard amounts (1 x 10 ⁵ cfu / g of raw grass) of the SBS0001s strain (Lactococcus lactis) and SBS0003 strain (Lactobacillus paracasei), which are used in Cymaster AC (Snow Brand Seedling Sales), were used as lactic acid bacteria. ) and a specified amount (according to (1) above) of the test enzyme were added by spraying with a sprayer.
The preparation method other than the above points was in accordance with (1).

(3) Organic acid analysis method and pH measurement method Weigh 30g of silage after opening into a stoma filter (manufactured by Central Scientific Trading Co., Ltd.), and use sterilized distilled water using a graduated cylinder that has been autoclaved (121℃, 15 minutes). After adding 90 mL of the mixture, the mixture was rubbed for 1 minute using a masticator S type (manufactured by Central Scientific Trading Co., Ltd.). Thereafter, the soluble components were transferred to the water fraction by allowing the sample to stand overnight at 4°C. After standing overnight, the sample was filtered using ADVANTEC No. 5A filter paper and used for pH measurement and organic acid analysis. For organic acid analysis, the filtrate was diluted 10 times with pure water and filtered through a 0.22 μm filter (Millex GP, manufactured by Merck Millipore). For organic acid analysis, Shimadzu LC-20AD, LC-30AD, SIL-30AC, CTO-20A, and SPD-M20A were used. 0.75mM sulfuric acid was used as the mobile phase (liquid A), and a BTB solution (0.1mM BTB, 7.5mM Na ₂ HPO ₄ ) was used as the reaction solution (liquid C). Analysis was performed using a post-column method. TOSOH's TSKgel OApak-A was used as the column, the column temperature was 40°C, and the flow rate was 0.8 mL/min for both liquids A and C. Lactic acid, acetic acid, and butyric acid were used for the calibration curve. ×2 and ×10 solutions were prepared using 0.1%, 0.05%, and 0.01% as ×1 solution, respectively, and a calibration curve was created at three points.

(4) Ferulic acid/coumaric acid analysis method Mix 500 μL of the silage extract prepared in the same manner as in (3) above and 500 μL of methanol acetic acid solution (98% methanol, 2% acetic acid), and centrifuge at 14,000 rpm for 10 minutes. , samples filtered through a 0.22 μm filter were used for HPLC analysis. For HPLC, LC-30AD, SIL-30AC, CTO-20A, and SPD-M20A manufactured by Shimadzu Corporation were used, and synegi Hydro-RP manufactured by phenomenex was used as the column. A 40% methanol/1% acetic acid solution was used as the mobile phase. The HPLC conditions were a flow rate of 1 mL/min, a column temperature of 40°C, an absorption wavelength of 320 nm, and isolactic conditions. For the calibration curve, ferulic acid and coumaric acid solutions of 0.0003125%, 0.000625%, 0.00125%, 0.0025%, 0.005%, and 0.01% were used.

(5) Moisture measurement method First, the entire amount of silage after opening was put into a paper bag, and the weight was measured. Next, the paper bag was closed and dried at 60°C for 2 days, and the weight was measured again. The weight of the paper bag is the "bag weight", the weight of the silage before drying (excluding the weight of the paper bag) is the "silage weight", and the weight of the silage after drying, including the paper bag, is the "dry weight". The moisture content was calculated using the formula shown.

The remaining dry matter of the silage was used for fiber analysis and dry matter loss test.

(6) Fiber analysis method Three items were analyzed: total fiber content (OCW) using enzyme fractionation, highly digestible fiber content (Oa), and low digestible fiber content (Ob). Details of the analysis method are described in the "Roughage Quality Evaluation Guidebook" published by the Japan Grassland Association. Highly digestible fiber content (Oa) is evaluated as the fiber fraction that is immediately degraded in the rumen, and low digestible fiber content (Ob) is evaluated as the fiber fraction that takes time to digest in the rumen (grass and Horticulture (1985, December issue)). In particular, Ob has a large effect on the digestibility of forage, with 97% of Oa being digested in the rumen, while 37% of Ob is said to be digested. Results are expressed as weight percent fiber content relative to dry matter (DM). For example, if there is 24 g of fiber (OCW) in 100 g of rice straw containing 70% moisture, 24 g of the 30 g of dry matter excluding 70% moisture is the fiber fraction, which is 80% DM. .

(result)
(1) Fermentation quality evaluation of rice straw silage prepared using CBF enzyme (excess amount addition test; 1st test group)
1.214 [mg protein/100g rice straw] of enzyme was added to dry rice straw powder. Silage was stored at 37°C for 2 weeks.
As a result, in rice straw silage prepared with the addition of CBF enzyme, the amount of free ferulic acid in the silage extract was increased compared to those without additive, with addition of lactic acid bacteria only, and with MA enzyme (Figure 2) . With the CBF enzyme, as a result of the increased expression level of feruloyl esterase, the relative avicelase activity and CMCase activity relative to the MA enzyme contained in the unit protein weight tended to decrease (Figure 1), but the fermentation quality was It was comparable to MA enzyme (Table 1, Figure 2).

As a result of fiber analysis, the total fiber content (OCW) of the CBF enzyme group showed a tendency to decrease significantly compared to the addition of lactic acid bacteria only and the MA enzyme group (Table 2). The increase in feruloyl esterase activity decreased the total fiber content (OCW) regardless of the decrease in avicelase activity and CMCase activity, so the ratio of various enzyme activities contained in the enzyme solution is important in silage preparation. It was thought that there was.

(2) Fermentation quality evaluation of rice straw silage prepared using CBF enzyme (specified amount addition test; 2nd test section)
The amount of test enzyme added in the preparation of rice straw silage was a specified amount (approximately one-fourth of the excess amount added in (1) above). 0.239 [mg protein/100g rice straw] of enzyme was added to dry rice straw powder. Silage was stored at 37°C for 1 month.

The results showed a similar trend to the excess addition test (Figure 3, Table 3). In the silage extract prepared by adding CBF enzyme, the amount of free ferulic acid was significantly increased compared to the case without addition or the addition of lactic acid bacteria only (Figure 3).

(3) Fermentation quality evaluation of grass silage prepared using CBF enzyme (specified amount addition test; 3rd test area)
The effect of a defined amount of CBF enzyme on grass was investigated. Grass is a feed with lower coarse stiffness as a fiber source than rice straw. Timothy and reed canary grass were used as grass species for silage preparation.

0.264 [mg protein/100g fresh grass] of enzyme was added to grass (timothy or reed canary grass), and the silage was stored at 25°C for one month.

As a result, in both timothy silage and reed canary grass silage, the CBF enzyme tended to have an increased total organic acid content and a lower pH compared to the MA enzyme (Tables 4 and 5). Therefore, CBF enzyme is more effective in improving fermentation quality than MA enzyme in grass. Furthermore, in the CBF enzyme, compared to the MA enzyme, the amount of ferulic acid released and the amount of coumaric acid released were significantly improved (FIGS. 4 and 5).

<Example 3: Fermentation quality evaluation of silage prepared using GH10 enzyme>
(the purpose)
Endoxylanases are classified into the glycoside hydrolase family (GH family) GH10 and GH11. It is known that between GH10 and GH11 endoxylanases, GH11 endoxylanase has higher endoxylanase activity than GH10 when xylan with fewer side chains is used as a substrate. Compared to GH11 endoxylanase, GH10 endoxylanase has a larger enzyme molecular weight and a huge substrate pocket, so it is said to have a higher enzymatic activity ability on miscellaneous substrates such as plant biomass. GH11 has a low molecular weight and a small substrate pocket, and is not good at degrading xylan with many side chains, whereas GH10, which has a large substrate pocket, does not consider some side chains to be a problem and can degrade xylan. It has the ability to cut chains. In the present invention, we focused on GH10 endoxylanase as an endoxylanase that can effectively act on miscellaneous biomass such as grass and rice straw.

In order to examine the effect of adding GH10 endoxylanase on silage fermentation quality, silage was prepared by adding a mixed enzyme solution in which GH10 enzyme was added to MA enzyme, and the silage fermentation quality was evaluated.

(Method)
(1) Method for preparing GH10 enzyme and GH11 enzyme
An expression plasmid containing the GH10 endoxylanase A (xyl10A) gene derived from T. cellulolyticus under the control of the glucoamylase promoter was introduced into the T. cellulolyticus YP-4 host strain (pyrF gene-deficient strain), and starch Using a recombinant T. cellulolyticus strain Y208 that can specifically induce production of Xyl10A as a carbon source (Kishishita et al., Protein. Expr. Purif. 94, 40-45 (2014)), and a similar method. Recombinant T. cellulolyticus strain Y223 (Watanabe et al., AMB Express 4,27 (2014)), which can induce production of GH11 endoxylanase C (Xyl11C) derived from T. cellulolyticus, was cultured in starch liquid medium (2% Starch, 0.1% CSL, 0.24% KH ₂ PO ₄ , 0.05% (NH ₄ ) ₂ SO ₄ , 0.047% Potassium tartrate, 0.1% tween80, 0.12% MgSO ₄ , 0.2% Urea, 0.001% ZnSO ₄ , 0.001% MnSO ₄ , 0.001% CuSO ₄ , pH 4.0) and cultured with shaking at 28° C. at 230 rpm for 5 days. The culture solution cultured with shaking for 5 days was centrifuged at 4° C. and 3500 rpm for 15 minutes to remove bacterial cells, and the supernatant was filtered through a 0.2 μm filter, and the filtrate was used as an enzyme solution. The enzymes obtained from the GH10-producing strain and the GH11-producing strain were referred to as GH10 enzyme and GH11 enzyme, respectively, and were used for various activity measurements and for the production of various silages.

(2) Rice straw silage preparation method Rice straw silage was prepared according to the method described in Example 2.

(result)
(1) Fermentation quality evaluation of rice straw silage prepared using GH10 enzyme (excess amount addition test; 4th test group)
For addition of lactic acid bacteria and MA enzyme (lactic acid bacteria/MA enzyme), 1.214 [mg protein/100 g of rice straw] of enzyme was added to dry rice straw powder. Addition of lactic acid bacteria, MA enzyme, and GH10 enzyme (lactic acid bacteria/MA enzyme/GH10 enzyme) further increases the endoxylanase activity of GH10, which is equivalent to twice the amount of MA enzyme (corresponding to about 15% of MA enzyme in protein weight ratio). Added enzyme. Rice straw silage was stored at 37°C for 2 weeks.

As a result, when GH10 enzyme was added to MA enzyme (lactic acid bacteria/MA enzyme/GH10 enzyme), the amount of ferulic acid released increased compared to MA enzyme (lactic acid bacteria/MA enzyme) (Figure 6). It was suggested that the degradation of xylan by GH10 endoxylanase may promote the release of ferulic acid by feruloyl esterase.

In addition, when GH10 enzyme was added to MA enzyme (lactic acid bacteria/MA enzyme/GH10 enzyme), the total fiber content (OCW) was significantly lower than when no additive was added or when only lactic acid bacteria were added, resulting in low digestibility. Sexual fiber (Ob) was significantly decreased compared to other test plots (Table 7). Since fiber digestibility generally improves when low-digestible fiber (Ob) decreases, it was suggested that the addition of GH10 enzyme may improve fiber digestibility.

(2) Fermentation quality evaluation of rice straw silage prepared using GH10 enzyme or GH11 enzyme (excess amount addition test; 5th test section)
In this test, a test similar to (1) was conducted using GH10 enzyme and GH11 enzyme.
Even when GH10 enzyme or GH11 enzyme was additionally added to MA enzyme, no significant difference was observed in organic acid content or pH (Table 8); In both cases, the amount of ferulic acid released was significantly increased compared to the addition of MA enzyme alone (FIG. 7).

In addition, silage to which GH10 enzyme or GH11 enzyme was added to MA enzyme showed a tendency for total fiber content (OCW) to decrease (Table 9). Furthermore, in the case of GH10 enzyme, the low digestible fiber content (Ob) showed a tendency to decrease (Table 9). From this, it became clear that the GH10 enzyme is likely to have the ability to lower the low digestible fiber content (Ob).

<Example 4: Fermentation quality evaluation of silage prepared using CFX enzyme>
(the purpose)
Evaluate the fermentation quality of silage prepared using CFX enzymes.

(Method)
(1) Method for preparing frozen timothy first grass silage The first timothy grass cut at the appropriate time was cut into pieces with a cutting length of 1 cm, stored at -20°C, thawed, and used for silage preparation.
For silage preparation, standard amounts (1 x 10 ⁵ cfu / g of raw grass) of the SBS0001s strain (Lactococcus lactis) and SBS0003 strain (Lactobacillus paracasei), which are used in Cymaster AC (Snow Brand Seedling Sales), were used as lactic acid bacteria. ), and an amount of test enzyme corresponding to the specified amount of MA enzyme (an amount of test enzyme corresponding to 150 g of MA enzyme per 10 tons of fresh grass in terms of avicelase activity) was added by spraying with a sprayer. After opening, the silage was measured for organic acid content, pH, moisture content, amount of ferulic acid and coumaric acid released, fiber analysis, and dry matter disappearance rate.

(2) Method for preparing rice straw silage and grass (timothy second grass) silage Rice straw silage and grass (timothy second grass) silage were prepared according to the method described in Example 2.

(result)
(1) Measurement of various enzyme activities contained in CFX enzyme
The CFX enzyme had overwhelmingly improved endoxylanase activity and feruloyl esterase activity compared to the MA enzyme (Figure 1).

(2) Fermentation quality evaluation of rice straw silage prepared using CFX enzyme (specified amount addition test; 6th test section)
Rice straw silage prepared using a specified amount of CFX enzyme was evaluated for silage fermentation quality. The amount of enzyme added was 0.167 [mg protein/100g of grass], and silage was prepared by storing at 25°C for one month.

As a result, no significant difference in pH and free organic acid content was detected between the enzyme sections (Table 10). On the other hand, the amounts of free coumaric acid and free ferulic acid were significantly higher when using the CFX enzyme than when using the MA enzyme (FIG. 8).

(3) Fermentation quality evaluation of grass (timothy second grass) silage prepared using CFX enzyme (specified amount addition test; 7th test area)
Grass (timothy second grass) silage was prepared using a specified amount of CFX enzyme. The amount of enzyme added was 0.167 [mg protein/100g of grass], and silage was prepared by storing at 25°C for one month.

The lactic acid content of the grass (timothy grass) silage prepared using the CFX enzyme showed a tendency to increase significantly compared to the addition of lactic acid bacteria alone, but no significant difference was observed between the enzyme intervals (Table 11). Regarding the amount of free ferulic acid, the CFX enzyme showed a tendency to be significantly higher than the addition of lactic acid bacteria only and the MA enzyme (FIG. 9).

(4) Fermentation quality evaluation of grass (frozen timothy first grass) silage prepared using CFX enzyme (specified amount addition test; 8th test area)
Grass (frozen timothy first grass) silage was prepared using a specified amount of CFX enzyme. The amount of enzyme added was 0.215 [mg protein/100g of grass], and silage was prepared by storing at 25°C for one month.

Grass (frozen timothy first grass) silage prepared using the CFX enzyme showed comparable lactic acid content compared to the MA enzyme (Table 12). Furthermore, the amounts of free coumaric acid and free ferulic acid were significantly higher in the CFX enzyme than in the MA enzyme (FIG. 10).

For total fiber content (OCW), CFX enzymes showed a tendency to be lower than MA enzymes, but no difference was observed for low digestible fiber content (Ob) (Table 13).

<Example 5: Evaluation of digestibility in cow's body of silage prepared using CBF enzyme and CFX enzyme>
(the purpose)
Evaluate the digestibility of silage prepared using CBF and CFX enzymes in cows.

(Method)
(1) Dry matter disappearance rate test An in situ method, which is used to measure the decomposition of feed in the rumen, was used. The "in situ method" involves forming a fistula, which is a hole that allows objects to be directly inserted into and taken out of the rumen from outside the cow's body.The test sample is introduced into the rumen from the outside through the fistula, and after incubation for a certain period of time, it is taken out. This is a method to check digestibility.

After pulverizing the dry silage with a pulverizer, put 2 g of the pulverized material into a pre-weighed sample bag (made of polyester, small, 5cm x 10cm, 280 mesh/opening 53 microns (±10)), Tie it tightly with a rubber band. A sample bag containing dry silage was placed in a laundry net tied to the fistula lid with a string, and the laundry net and the bag were placed directly into the rumen of the fistula cow, and the lid was tightened. Incubation time was 5-6 hours, 24 hours. After incubation, samples were removed from fistula cows, washed thoroughly with running water, and then dried at 60°C for 2 days. After drying, the weight was measured, and the dry matter disappearance rate was calculated using the formula shown in Equation 6 below.

(result)
(1) Evaluation of digestibility in cows of rice straw silage prepared using CBF enzyme (first test group)
A dry matter disappearance rate test was conducted using the rice straw silage prepared in the result (1) of Example 2 (excess amount addition test; first test section).
In the rice straw silage prepared using the CBF enzyme, the dry matter loss rate was significantly improved compared to the addition of only lactic acid bacteria and the MA enzyme (Table 14). It is thought that digestibility was improved due to the fiber being degraded during silage storage and the soluble fraction increasing.

(2) Evaluation of digestibility in cows of rice straw silage prepared using CFX enzyme (6th test group)
A dry matter disappearance rate test was conducted using the rice straw silage prepared in the result (2) of Example 4 (specified amount addition test; 6th test section).

Rice straw silage prepared using CFX enzymes showed an overall trend towards higher digestibility compared to MA enzymes (Table 15). In particular, CFX enzyme showed a tendency to be higher in initial digestibility (6 hours) compared to no additives, lactic acid bacteria only added, and MA enzyme.

(3) Evaluation of digestibility in cows of grass (Timothy 2nd grass) silage prepared using CFX enzyme (Test Group 7)
A dry matter disappearance rate test was conducted using the grass (Timothy 2nd grass) silage (specified amount addition test; 7th test area) prepared in result (3) of Example 4.
The results showed that the digestibility of silage prepared using CFX enzyme was the highest at all time points, especially after 24 hours, where CFX enzyme had a 4% increase in digestibility compared to MA enzyme (Table 16).

(4) Fermentation quality evaluation of grass (frozen timothy first grass) silage prepared using CFX enzyme (specified amount addition test; 8th test area)
A dry matter disappearance rate test was conducted using the grass (frozen timothy first grass) silage (specified amount addition test; 8th test area) prepared in result (4) of Example 4.
The results showed that the silage prepared using the CFX enzyme had the highest digestibility, with an approximately 6% increase in digestibility compared to the MA enzyme (Table 17).

Claims (15)

a first gene expression system comprising a promoter capable of inducing gene expression in filamentous fungal cells; and a feruloyl esterase gene disposed downstream of the promoter in a state capable of expression; and a second gene expression system comprising a GH10 endoxylanase gene arranged in an expressible manner downstream of said promoter , for introducing into the genus Talaromyces to enhance its cellulose decomposition ability. , a composition for enhancing cellulose decomposition. The composition for enhancing cellulose decomposition according to claim 1, wherein the promoter included in the first gene expression system is a GH3β-xylosidase gene promoter. The composition for enhancing cellulose decomposition according to claim 1 or 2, wherein the promoter included in the second gene expression system is a GH10 endoxylanase gene promoter. The cellulose decomposition-enhancing composition according to any one of claims 1 to 3, wherein the promoter and the gene included in the first and second gene expression systems are derived from Talaromyces. The cellulose decomposition-enhancing composition according to claim 4, wherein the Talaromyces genus is Talaromyces cellulolyticus (T. cellulolyticus). A mutant strain of Talaromyces cellulolyticus with enhanced cellulose decomposition ability,
A gene expression system is introduced that includes a promoter capable of inducing gene expression in filamentous fungal cells, and a feruloyl esterase gene placed downstream of the promoter in an expressible state,
The cellulose decomposition-enhancing mutant strain, wherein the promoter is a xylanase gene promoter, a cellulase gene promoter, or a pectinase gene promoter. The mutant strain with enhanced cellulose decomposition ability according to claim 6, wherein the promoter is a GH3β-xylosidase gene promoter. A cellulose decomposition-enhancing mutant strain of Talaromyces cellulolyticus, into which the cellulose decomposition-enhancing composition according to any one of claims 1 to 5 has been introduced. A method for producing a cellulose decomposition promoter, comprising:
The method comprising the step of culturing the cellulose decomposition-enhanced mutant strain according to any one of claims 6 to 8 in a medium to obtain a culture solution thereof. The manufacturing method according to claim 9, further comprising the step of separating the obtained culture solution into bacterial cells and a culture supernatant to obtain a culture supernatant. The manufacturing method according to claim 10, further comprising the step of removing solid matter from the obtained culture supernatant. The manufacturing method according to claim 10 or 11, further comprising the step of drying the obtained culture supernatant or the solution obtained by removing solid matter. A method for promoting cellulose decomposition in plants, comprising the step of contacting cellulosic biomass with a cellulose decomposition promoter produced by the production method according to any one of claims 10 to 12 . A method for producing silage, comprising the step of contacting a collected plant for livestock feed with a cellulose decomposition promoter produced by the production method according to any one of claims 10 to 12 . The method for producing silage according to claim 14 , further comprising the step of adding lactic acid bacteria.

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