JP2007104937A - Utilization of drug-resistant gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism that has excellent lignin-degrading potential, low cellulase activity and shows high processing speed. <P>SOLUTION: The Coriolus hirsutus transformant organism supports a cellulase expression-suppressing gene and a drug-resistant gene prepared by introducing mutation into the gene relating to the drug sensitivity of Coriolus hirsutus. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an Arakawaratake transformant into which a cellulolytic enzyme gene expression is suppressed and a drug resistance gene derived from Arakawaratake is introduced, and a method for producing a fiber component using the transformant.

  More specifically, using wild-type medaka anthracnose, which has not been exposed to artificial mutations such as auxotrophy, as a host, it suppresses the expression of the cellulolytic enzyme gene and introduces an anthropomorphic mutation into the gene derived from medusa agaricus. It is related with the transformant which suppressed the cellulolytic enzyme activity by introduce | transducing the drug resistance gene produced in this way. Further, the present invention relates to a method for producing a resource-saving or energy-saving fiber, characterized in that a lignocellulosic material is pretreated with the transformant, and then a fiber component is taken out through a fiberizing step. In particular, the present invention relates to a resource-saving or energy-saving paper pulp manufacturing method in the manufacture of paper pulp from paper pulp raw materials.

  In the process of extracting the fiber component from the lignocellulosic material, the structure of lignin and hemicellulose involved in bonding between fibers is broken by mechanical force, or the lignin and hemicellulose are selectively dissolved or decomposed chemically. Extracting the ingredients is done.

  Also in the manufacturing process of a paper pulp, the fiber which is a fiber component is prepared by cut | disconnecting or decomposing | disassembling the interfiber bonding material from wood and other lignocellulose materials by mechanical and chemical treatment. Such a paper pulp manufacturing process is a process that requires energy, and various measures have been implemented for many years to save energy. However, in order to solve environmental problems, development of technology for further energy saving is required.

  Pulp manufactured in the paper pulp industry is divided into mechanical pulp and chemical pulp depending on the manufacturing method. Mechanical pulp is produced by physically grinding wood fibers using mechanical energy. Since the wood component is contained almost as it is, it can be produced in a high yield, and a thin and highly opaque paper can be produced. However, large power is required for attrition.

  In order to suppress the consumption of such a large amount of power as described above, studies have been made to reduce energy by treating wood chips in advance with microorganisms, particularly basidiomycetes having a lignin-degrading ability called white rot fungi. For example, prior to the production of alder, the primary defibration thermomechanical pulp (TMP), Phanerochaete chrysosporium, together with glucose, is added to the wood chips and left for 2 weeks. Has been reported to be reduced by 25-30% (Non-patent Document 1). In addition, it has been reported that the strength of paper strength increases as compared with control when an aspen material is treated using Fanerocaete chrysosporium and Dichomitus squalens (Non-patent Document 2). .

  Akamatsu et al. Used 10 white-rot fungi containing Coralus hirsutus, cultured them on poplar wood, and examined the pulp yield, fibrillation energy, and pulp strength. As a result, it was shown that, among the strains used, Aragekawaratake is preferable as a pretreatment fungus for chips, for example, treatment with Aragekawaratake reduces fibrillation energy and increases crystallinity. However, the yield at this time decreased by about 7% (Non-patent Document 3).

  Nishibe et al. Also used 10 types of white-rot fungi preliminarily selected from 85 and 85 strains to microbially decompose Sawaguru-mi pieces and coniferous secondary disaggregation TMP, selectively delignifying, and pulp fiber It was shown that the decrease in the strength of paper strength was suppressed in the presence of glucose and urea by selecting Coprinus cinereus and Phanerocaete chrysosporium, which have less decay. However, the decrease in pulp yield was 6.3% and 9.7%, respectively, after 14 days of treatment at 30 ° C. In the case of using Arakawakawaratake, the yield reduction was 7.5% (Non-patent Document 4).

  Kashino et al. Screened the white-rot fungus IZU-154 from nature, selectively decomposed lignin from Phanerocaete chrysosporium and Trametes versicolor, and treated hardwood for 7 days. It was confirmed that it decreased by 2 to 2/3. In addition, the pulp strength increased about twice. When conifers were treated for 10-14 days, the defibration energy decreased by 1/3 and the strength increased. When the medium was added, equivalent results could be obtained in 7 days (Non-patent Document 5).

  Furthermore, in the US, a bio-pulping consortium consisting of several research institutes and several pulp and paper industry companies led by the USDA Forest Products Laboratory group was screened to screen for strains with high lignin-degrading ability and low cellulose-degrading ability. A new selection was made of Ceriporiopsis subvermispora. Using this stock, we can consider reducing the power of mechanical pulp.For example, we can reduce nearly 40% of the production energy of TMP, and the yield decline at this time is about 3 to 5%. It is reported that there is no adverse effect on the water and rather the strength is increased (Non-patent Document 6).

  The USDA Forest Products Laboratory has already built a pilot plant and is conducting a demonstration test of the isolated seripolyopsis subvermis polara. Assuming a factory in the United States, we are thinking of performing microbial treatment at the chip yard of the factory. However, Seripoliopsis subvermispolla has a low temperature suitable for growth and can only be effective at temperatures up to 32 ° C. However, since the inside of the pile storing the chips becomes hot, ventilation for cooling is not possible. Energy and cost are great and unsuitable for use in warm areas.

  In addition, microorganisms obtained by screening so far do not necessarily have high selectivity for lignin degradation, and decompose not only lignin but also cellulose, resulting in a decrease in pulp yield and paper strength. Accordingly, there is a demand for obtaining and producing microorganisms with enhanced lignin degradation selectivity, that is, microorganisms that are excellent in lignin degradation ability but have suppressed cellulose degradation.

  Mutants with enhanced lignin degradation selectivity have been produced by Ander et al. They have developed a mutant strain Cel44 with weak cellulolytic enzyme activity by mutating Sporotrichum pulverulentum by UV irradiation. Decomposition of birch wood pieces using a wild strain and this cellulolytic enzyme-suppressed strain Cel44, the former decomposes lignin and xylan well, while the latter decomposes lignin and xylan well, but glucan It hardly decomposed (Non-Patent Document 7). When birch material was treated for 6 weeks using this Cel44 strain and mechanical pulp was produced, an increase in paper strength was observed (Non-patent Document 8). Moreover, it experimented using birch material and a pine material, and it reported that the energy of fiber fibrillation and refining decreased 30% by increasing processing time (nonpatent literature 9). In the same manner, Phlerbia radiata produced a strain Cel26 with weak cellulolytic enzyme activity. After processing pine chips and pulp with this Cel26 strain, when mechanical pulp was produced, in all cases, improvement in paper strength was not seen, but a decrease in fibrillation energy was observed. Further, the weight reduction was 2% or less (Non-patent Document 10).

  On the other hand, chemical pulp is produced by a method of extracting cellulose and hemicellulose by eluting lignin in wood using chemicals. At present, kraft pulp that delignifies using sodium hydroxide and sodium sulfide is the mainstream. In kraft pulp, microbial treatment is performed in the same manner as mechanical pulp, and delignification is performed before cooking to reduce production energy and improve pulp quality.

  For example, using Fanerocaete chrysosporium to treat red oak or aspen for 30 days increases yield at the same Ka value, reduces beating energy, and increases tensile strength and burst strength. There is a report (Non-Patent Document 11). Similarly, in experiments using Fanerocaete chrysosporium, an increase in burst strength and tear strength has been reported (Non-patent Document 12). Molina et al. Report that production of Radiata pine can be reduced by 11 to 14% when treated with Pleurotus ostreatus. However, in the case of Kawatake, a decrease in pulp strength was observed (Non-Patent Document 13 and Non-Patent Document 14).

  Bajpai et al. Reported that in experiments using Seripolyopsis subvermispolla, active alkali was reduced by 18%, cooking time was reduced by 33%, and sulfidity in white liquor was reduced by 30% ( Non-patent document 15).

  As mentioned above, microbial treatment in kraft pulp improves digestibility and reduces energy, but may reduce yield and paper strength, and to put microbial treatment to practical use in the same way as mechanical pulp, There is a need to obtain and prepare microorganisms with enhanced lignin degradation selectivity, which are superior in ability to degrade lignin but have a reduced ability to degrade cellulose. In contrast to this, mutant strains with weak cellulolytic enzyme activity have been prepared as described above, and studies are being made on their use in mechanical pulp treatment. However, these mutant strains are mutated by ultraviolet irradiation. However, there is a problem that the growth rate of the mutant strain is slow and the degradation process takes a long time. For this reason, it is desired to produce a mutant strain that suppresses only the cellulolytic activity without affecting the growth rate.

  Enzymes that break down cellulose (also called cellulase) are generic names for several enzymes that hydrolyze the β-1,4-glucopyranosyl bond of β-1,4-glucan (cellulose) or its derivatives. It consists of glucanase (EG), cellobiohydrolase (CBH) I, II, etc. EG breaks the cellulose chain by hydrolyzing the β-1,4-glucopyranosyl bond of the cellulose main chain into an endo-type, and then breaks CBH in cellobiose units sequentially from the break point, and is a highly crystalline part Are also known to decompose. CBH I cuts cellobiose residues from the reducing end of the cellulose chain, and CBH II cuts from the non-reducing end. In addition, cellobiose dehydrogenase that oxidizes the generated cellobiose and β-glucosidase that hydrolyzes the β bond from the end of the cellooligosaccharide are known. These hydrolases act synergistically to lower the molecular weight of the cellulose substrate to produce cellobiose, and further, β-glucosidase is involved to break down to glucose units. However, as cellulolytic enzyme research progresses, cellobiohydrolase shows activity like EG and vice versa, and enzymes that cannot be explained by conventional classification methods have been found.

  To date, a large number of cellulolytic enzymes have been isolated and purified, and gene sequences have been elucidated. It has been clarified that cellulolytic enzymes can be grouped into glycoside hydrolase families by two-dimensional analysis based on the cluster structure of hydrophobic amino acids called hydrophobic cluster analysis (Non-patent Document 16). The classification of these enzymes indicates that the difference in cellulolytic properties of CBH and EG is based on the presence or absence of the peptide loop that covers each active center from the top and the state of its existence. The Trichoderma genus has been reported to have a tunnel structure in which the active center is covered with a peptide loop as a feature of the three-dimensional crystal structure of CBHI and CBHII. On the other hand, the endo-type enzyme has a groove called cleft at the active center, and after acting to capture and cleave the cellulose chain, it once separates from the cellulose chain and acts again in a non-processive cleavage mode that captures the cellulose chain again. (Non-patent document 17).

  Cellobiose dehydrogenase is an oxidoreductase that oxidizes cellobiose and cellooligosaccharide to produce cellobionolactone, and simultaneously reduces metal complexes such as quinone and iron, phenoxy radical, oxygen, and the like. This enzyme is produced simultaneously with the cellulolytic enzyme when the microorganism decomposes cellulose (Non-patent Document 18), and the inhibition of cellulolytic enzyme activity by cellobiose, that is, the inhibition of the product is canceled (Non-patent Document 19). For this reason, it is considered that cellulose degradation is promoted by conjugation with a cellulolytic enzyme.

  Cellobiose dehydrogenase is thought to be deeply involved in cellulose degradation because it causes a Fenton reaction that generates hydroxy radicals that strongly degrade cellulose. This is also suggested by the construction of mutant strains with suppressed cellobiose dehydrogenase activity by Dumonceaux et al. They made cellobiose dehydrogenase-deficient strains by homologous recombination using the antibiotic ferromycin as an index, and cellobiose dehydrogenase-deficient strains had growth rates that were higher than wild strains when amorphous cellulose was used as the carbon source. Although it does not change, the growth rate is significantly slow when cultured on crystalline cellulose, and even in cellobiose dehydrogenase-deficient strains, the hardwood unbleached pulp lignin and synthetic lignin 14C-DHP are degraded to the same extent as the wild strain (Non-patent document 20).

  According to a review of cellobiose dehydrogenase (Non-patent Document 21) and the like, microorganisms that produce cellobiose dehydrogenase include wood decay fungi such as Fanerocaete chrysosporium, Kawaratake, Schizophyllum commune, Coneophora puteana, Molds such as Myceliophthora thermophila and Humicola insolens are known (Non-patent Document 21).

  As for a gene encoding cellobiose dehydrogenase (hereinafter referred to as cellobiose dehydrogenase gene), for example, in Fanerocaete chrysosporium, cDNA of K3 strain (Non-patent Document 22), cDNA of OGC101 strain (Non-patent Document 23) Chromosomal genes have been cloned (Non-patent Document 24). Cellobiose dehydrogenase genes have also been reported in Kawaratake (Non-patent Document 25) and Statake (Pycnoporus cinnabarinus) (Non-patent Document 26).

  Elucidation of cellulolytic enzymes and cellulolytic enzyme genes such as cellobiose dehydrogenase derived from Mushroom agaricus necessary for the acquisition and production of microorganisms with enhanced lignin degradation selectivity in the treatment of microorganisms in mechanical pulp and chemical pulp production, Patent Document 1 and Patent Document 2 show a gene recombination technique using a gene and an effective pulp processing method using a transformant obtained by such a technique. However, cellulolytic enzyme activity-suppressed strains produced so far are transformed microorganisms using auxotrophic mutants such as amino acids as hosts. Therefore, when the bacterial treatment is performed using the transformed microorganism, a nutrient source that complements the requirements such as amino acids must be added in advance. Therefore, development of an effective vector system using a wild strain as a host is desired.

  Development of a vector system using wild strains as a host in basidiomycetes is the presumed autonomous replication initiation region derived from Phanerocaete chrysosporium ME446 and Phanerocaete chrysosporium BKM-F. Development of a transformation system using a vector pRR12 in which a kanamycin resistance gene derived from Escherichia coli has been incorporated into YIp5 of yeast containing the yeast has been reported (Non-patent Document 27). However, the transformation efficiency was as low as several per μg. Furthermore, it is necessary to confirm the safety of introducing DNA of heterologous microorganisms into basidiomycetes, and there is a possibility that the bacterial treatment plant may be restricted due to legal problems. Therefore, development of a wild type vector system using an endogenous gene is desired.

  Koen et al. Prepared a chromosomal library from a carboxin-resistant mutant of the tilinomycota Ustilago maydis, transformed into wild-type Kurobo, and obtained a transformant exhibiting carboxin resistance. And succinate dehydrogenase Ip subunit gene was found to be involved (Non-patent Document 28). Furthermore, Broomfield et al. Found that His257 in the succinate dehydrogenase Ip subunit gene was replaced with Leu257 (Non-patent Document 29). Honda et al. Cloned the oyster mushroom succinate dehydrogenase Ip subunit gene, and a mutant gene in which the His residue in a region well conserved in other microorganisms was replaced with a Leu residue functions as a carboxin resistance gene (Patent document 3). Sato et al. Have reported that a transformation system using shiitake has been developed by taking out a succinate dehydrogenase Ip subunit gene newly extracted from shiitake (Lentinus edodes) and introducing mutations (Patent Document 4). .

WO 03/070939, WO 03/070940 JP2001-69987 JP2003-189855 Bar-Lev and K.T.Kirk, Tappi J., 65, (10), 111, 1982 Myers., Tappi J., 71, (5), 105, 1988 Akamatsu et al., Mokuzai Gakkaishi, 30 (8) 697-702, 1984 Nishibe et al., Japan Tappi, 42 (2), 1988 Kashino et al., Tappi J., 76 (12), 167, 1993 Scott et al., Tappi J., 81. (12). 153, 1998 Ander and Eriksson, Svensk Papperstid., 18, 643, 1975 Ander and Eriksson, Svensk Papperstid., 18, 641, 1975 Eriksson and Vallander., Svensk Papperstid, 85, R33, 1982 Samuelsson et al., Svensk Papperstid., 8, 221, 1980 Oriaran et al., Tappi J., 73, (7), 147, 1990 Chen et al., Wood Fiber Sci., 27. 198, 1995 Molina, 50th Appita Annual General Conference, pp.57-63, 1996 Molina, 51th Appita Annual General Conference, pp.199-206 1997 P. Bajpai et al. J. Pulp and Paper Science: 27 (7), 235-239, 2001 Henrissat, B., Bairoch, A., Biochem. J., 293, 781 (1993) Henrissat, B., Cellulose. Commun., 5, 84-90 (1998) Eriksson et al., FEBS Lett., 49, 282-285, 1974 Igarashi et al., Eur. J. Biochem., 253, 101, 1998 Dumonceaux, Enzyme Microb. 29, 478-489, 2001 G. Henriksson et al., J. Biotechnol. 78 (2000) 93-113 Raices et al., FEBS Letters, 69, 233-238, 1995 Li et al., Appl. Environ. Microbiol., 62 (4), 1329-1335, 1996 Li et al. Appl. Environ. Microbiol., 63 (2), 796-799, 1997 T.J.Dumonceaux et al., Gene. 210. 211-219 (1998) S.M.Moukha et al., Gene, 234, 23-33, 1999 T. Randall et. Al., Biochem. Biophys. Res. Commun. 161 (2), 720-725, 1989 J. P. R. Keon et.al., Curr. Genet. 19, 475-481, 1991 P. L. E. Broomfield et.al., Curr. Genet. 22, 117-121, 1992

  A method for obtaining fiber components from lignocellulosic materials, especially in the manufacture of paper pulp, when microbial treatment is performed prior to the manufacture of mechanical pulp and chemical pulp for the purpose of saving resources and energy, the cellulose-degrading enzyme produced by the microorganisms makes the paper Cellulose, which is the main component, is decomposed, resulting in a decrease in pulp yield and paper strength. In addition, a long treatment time is required according to the growth rate of microorganisms. As a method for solving these problems, there is a demand for the production of microorganisms having excellent lignin degrading ability, low cellulolytic enzyme activity, and high processing speed. The present inventors have so far succeeded in producing useful strains that are excellent in lignin degrading ability and have a plurality of cellulolytic enzyme activities suppressed. However, cellulolytic enzyme activity-suppressed strains produced so far are transformed microorganisms using auxotrophic mutants such as amino acids as hosts. Therefore, when the bacterial treatment is performed using this transformed microorganism, it is necessary to add in advance a nutrient source that complements the requirements such as amino acids. Therefore, development of an effective transformed microorganism produced using a wild strain as a host has been desired.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies and found that a drug resistance gene can be created by introducing a mutation into a gene involved in drug susceptibility of Aragekawaratake. Then, by using the gene as a drug resistance marker together with a gene that suppresses the expression of the cellulolytic enzyme gene, it is found that a microorganism having a low cellulolytic enzyme activity and a high growth ability can be obtained, and the present invention is completed. It came to.

That is, the present invention includes the following inventions.
(1) An Arakawaratake transformant carrying a cellulose-degrading enzyme expression-suppressing gene and a drug resistance gene obtained by introducing a mutation into a gene involved in the drug sensitivity of Arakawaaratake.
(2) The cellulolytic enzyme expression suppressing gene is an antisense DNA encoding an antisense RNA having a sequence substantially complementary to all or a part of mRNA transcribed from the cellulolytic enzyme gene. (1) The Arakawaaratake transformant according to the above.
(3) The Arakawa aratake transformant according to (2), wherein the antisense DNA is linked under the control of a promoter of the cellobiose dehydrogenase gene or glyceraldehyde-3-phosphate dehydrogenase gene.
(4) The jellyfish oyster mushroom transformant according to any one of (1) to (3), which carries a cellulolytic enzyme expression suppression gene for six or more types of cellulolytic enzyme genes.
(5) The cellulolytic enzyme expression-suppressing gene for at least one gene selected from the group consisting of an endoglucanase gene, a cellobiohydrolase gene, and a cellobiose dehydrogenase gene is carried. Alage Kawaratake transformant.
(6) A method for producing a fiber component by taking out the fiber component from the lignocellulosic material, wherein the lignocellulosic material is treated with the Allage Kawaratake transformant according to any one of (1) to (5), and the fiber Said method comprising separating and obtaining the components.
(7) The method according to (6), wherein the fiber component is pulp.
(8) The method according to (7), wherein the pulp is paper pulp.
(9) The method according to (8), wherein the method for separating and obtaining paper pulp is a chemical pulping method, a mechanical pulping method, or a semichemical pulping method.

  According to the present invention, a microorganism having excellent lignin degrading ability, low cellulolytic enzyme activity, and high processing speed is provided. According to the present invention, in the production of mechanical pulp, it is possible to eliminate the problem of the prior art such as a decrease in yield, and it is possible to reduce energy and improve paper strength. Further, in the production of chemical pulp, it is possible to suppress the yield and paper strength reduction.

  The present inventors isolated a succinic acid dehydrogenase Ip subunit gene, which is a gene whose function is suppressed by carboxin, from an artificial moss, and introduced an artificial mutation into the gene, thereby producing a carboxin resistance gene. Succeeded in creating. The present inventors have found that wild-type larvae can be transformed by using the gene as a drug resistance gene. As a result of using the drug resistance gene as a drug resistance marker and suppressing the expression of the cellulolytic enzyme gene by the antisense method, a transformed strain with fast growth and suppressed cellulolytic enzyme activity was produced. Succeeded in doing. The present invention is based on the above findings.

  In one embodiment, the present invention relates to an Arakawa aratake transformant carrying a cellulolytic enzyme expression-suppressing gene and a drug resistance gene obtained by introducing a mutation into a gene involved in the drug susceptibility of Arakawa aratake.

  The cellulolytic enzyme expression-suppressing gene refers to a gene having a function of suppressing the expression of a cellulolytic enzyme in the host, that is, the expression of the cellulolytic enzyme gene when the gene is transcribed in the host. In the present invention, the cellulolytic enzyme expression-suppressing gene preferably suppresses the expression of the cellulolytic enzyme gene by a mechanism based on the antisense method, co-suppression method, ribozyme method or RNA interference method.

  Cellulolytic enzyme gene encodes an enzyme that has cellulolytic activity, ie, an activity that hydrolyzes β-1,4-glucan (cellulose) or its derivatives β-1,4-glucopyranosyl bond, and suppresses its expression The cellulose-degrading enzyme gene to be the target is a cellulose-degrading enzyme gene possessed by wild-type agaricus. Examples thereof include cellobiose dehydrogenase (CDH) gene, endoglucanase (EG) gene (eg, EG5 gene, EG12 gene, EG61 gene), cellobiohydrolase gene (eg, CBHI-27 gene, CBHI gene, CBHII gene). Preferably, by suppressing the expression of two or more types of cellulolytic enzyme genes, more preferably six or more types, it is possible to obtain a microorganism with enhanced suppression of cellulose decomposing ability.

  In one embodiment, the cellulolytic enzyme expression suppressing gene is an anti-virus encoding an RNA (antisense RNA) having a sequence substantially complementary to all or a part of mRNA transcribed from the cellulolytic enzyme gene. Sense DNA. When present in the host, antisense RNA, which is a transcript of antisense DNA, binds to its complementary strand, the cellulolytic enzyme gene mRNA, thereby inhibiting cellulolytic enzyme gene translation and Suppress.

  Moreover, substantially, this antisense RNA hybridizes with mRNA to form a double strand, and as long as it inhibits translation of mRNA into protein, it is deleted in a sequence complementary to this mRNA, It means that there may be mutation such as substitution or addition. More specifically, it means that the antisense RNA hybridizes with the subject cellulolytic enzyme gene mRNA under stringent conditions. Here, the stringent condition means a condition where a specific hybrid is formed and a non-specific hybrid is not formed, that is, high homology (homology is 80% or more, preferably 90% or more, more preferably Is 95% or higher). More specifically, such conditions include: after performing hybridization at 42-68 ° C in the presence of 0.5-1 M NaCl, or 42 ° C in the presence of 50% formamide, or 65-68 ° C in aqueous solution. , It can be achieved by washing at a room temperature to 68 ° C. with an SSC solution having a concentration of 0.1 to 2 times.

  The length of the base sequence of the antisense DNA encoding the antisense RNA can be appropriately set as long as it can suppress the expression of the target cellulolytic enzyme gene. It does not necessarily have to be the same length as the whole. The part is a length corresponding to usually 30%, preferably 50%, more preferably 80%, and still more preferably 90% of the entire base sequence of the cellulolytic enzyme gene mRNA.

  Preparation of antisense DNA encoding antisense RNA and introduction into a host can be carried out by conventional methods known to those skilled in the art. Specifically, antisense DNA for cellulolytic enzyme gene mRNA is obtained by PCR amplification so that the exon part of the base sequence of the cellulolytic enzyme gene can be obtained and digested with appropriate restriction enzymes. Can do. It can also be obtained from cellulolytic enzyme gene cDNA. Furthermore, this antisense DNA may be a synthetic DNA artificially produced based on the base sequence information of the cellulolytic enzyme gene.

  Preferably, at least two kinds of such antisense DNA are linked in the direction of antisense in the vector. The linkage may be direct or via a linker. The gene whose expression is to be suppressed may be a cellulolytic enzyme gene having different activities, or may be an isozyme gene having the same activity. It is preferable to bind as many kinds of antisense DNA as possible. More preferably, expression suppression genes for 6 or more types of cellulolytic enzyme genes are linked.

  When antisense DNA is transcribed in the host, it is introduced into the host so that antisense RNA that hybridizes to mRNA that is a transcription product of the cellulolytic enzyme gene is generated. Usually, antisense DNA is ligated to a vector so that antisense RNA is produced, and the vector is introduced into a host. In order to link to the vector so that antisense RNA is generated, the antisense DNA is linked downstream of the DNA fragment having the promoter sequence in the antisense direction (reverse direction) and transcribed into RNA by the operation of the promoter. Like that. The RNA obtained is an antisense RNA to the mRNA of the cellulolytic enzyme gene.

  The promoter is not particularly limited as long as it is a gene fragment having a promoter action, and promoters of all genes can be used. For example, cellobiose dehydrogenase gene promoter, GPD (glyceraldehyde-3-phosphate dehydrogenase) gene promoter, ras gene promoter, lignin degradation-related gene promoter and the like can be mentioned. These promoters can also be obtained by well-known genomic cloning or PCR methods based on sequences registered in Genebank, sequences described in literature, and the like. Or about the gene deposited, what can be obtained by the request for distribution can be utilized.

  The promoter and antisense DNA of the cellulolytic enzyme gene can be ligated using an appropriate DNA ligase after introduction of a restriction site, blunt end or cohesive end, if necessary. Recombinant DNA techniques including cloning, ligation, PCR, etc. are described in, for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989 and Short Protocols In Molecular Biology, Third Edition. A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, Inc. can be used.

  Suppression of cellulolytic enzyme gene expression can also be carried out using a DNA encoding a ribozyme as a cellulolytic enzyme expression suppression gene. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type and a size of 400 bases or more like M1RNA contained in RNaseP, but some have an active domain of about 40 bases called hammerhead type or hairpin type. DNA encoding a ribozyme designed to cleave the target is ligated to a promoter and transcription termination sequence so that it is transcribed in the host. However, at that time, if an extra sequence is added to the 5 ′ end or 3 ′ end of the transcribed RNA, the ribozyme activity may be lost. In such a case, in order to accurately cut out only the ribozyme part from the RNA containing the transcribed ribozyme, another trimming ribozyme that acts in cis for trimming is placed on the 5 'side or 3' side of the ribozyme part. (K. Taira et al., Protein Eng., 3: 733 (1990); AMDzianott and JJ Bujarski, Proc. Natl. Acad. Sci. USA., 86: 4823 (1989); CAGrosshans And RTCech, Nucleic Acids Res., 19: 3875 (1991); K. Taira et al., Nucleic Acids Res., 19: 5125 (1991)). In addition, such a structural unit can be arranged in tandem so that a plurality of sites in the target mRNA can be cleaved, thereby further enhancing the effect (N. Yuyama et al., Biochem. Biophys. Res. Commun., 186: 1271, 1992). Such a ribozyme can be used to specifically cleave the transcript of the cellulolytic enzyme gene and suppress the expression of the gene.

  Suppression of cellulolytic enzyme gene expression can be further achieved by using a DNA encoding RNA that suppresses cellulolytic enzyme gene expression by co-suppression as a cellulolytic enzyme expression inhibitory gene, that is, a sequence identical or similar to the target gene sequence. It can also be achieved by using a DNA having the same. Co-suppression is a phenomenon in which the expression of both the introduced foreign gene and the target cellulolytic enzyme gene is suppressed when a DNA having the same or similar sequence as the target cellulolytic enzyme gene is introduced into the host by transformation. Say. The gene used for co-suppression need not be completely identical to the target gene, but has at least 70% or more, preferably 80% or more, more preferably 90% or more sequence identity.

  Suppression of cellulolytic enzyme gene expression is a cellulolytic enzyme expression-suppressing gene, DNA encoding RNA that suppresses cellulolytic enzyme gene expression by RNA interference, that is, the same or similar sequence as the target cellulolytic enzyme gene sequence Can also be achieved by using DNA placed in inverted repeats. RNA interference refers to the expression of a target gene by introducing double-stranded RNA derived from foreign DNA into a plant by introducing DNA with the same or similar sequence as the target cellulolytic enzyme gene into inverted repeats by transformation. This is a phenomenon in which is suppressed. As the mechanism of RNA interference, as the first step, complementary RNA is synthesized using the target gene mRNA and the double-stranded RNA derived from the introduced sequence as a complex and the associated sequence is synthesized, and the second step is endogenous. When this complex is fragmented by RNase and double-stranded RNA fragmented to 20-30 base pairs as a third step functions as a secondary RNA interference signal, the target gene mRNA is degraded again. (Curr. Biol., 7: R793, 1997; Curr. Biol., 6: 810, 1996). The length of the DNA used for RNA interference may be the full length of the target gene, but may be at least 20 bases, preferably 30 bases or more, more preferably 50 bases or more.

  A drug resistance gene refers to a gene that confers resistance to a specific drug to a host. In the present invention, a drug resistance gene derived from the host agaricus mushroom was successfully produced by introducing a mutation into a gene involved in drug sensitivity of the mushrooms. The drug resistance gene can be used as a selection marker at the time of transformation. Since such a drug resistance gene is a gene derived from the medaka aratake, which is the host, even if it is introduced into the medaka cultivar as a selection marker, the proliferation ability of the medaka aratake does not decrease. Therefore, an Arakawaaratake transformant having a high growth ability and a high treatment speed can be obtained. Further, unlike a transformant using an auxotrophic mutant as a host, it is not necessary to previously add a nutrient source that complements the requirement.

  The gene involved in drug sensitivity for introducing a mutation is not particularly limited as long as it is present in Aragekawaratake, for example, a gene encoding a succinate dehydrogenase Ip subunit. In addition, a drug resistance gene that imparts resistance to a benzimidazole drug can be obtained by single base substitution at codon 198 of the β-tubulin gene. In addition, a drug resistance gene that imparts resistance to a strobilurin-based drug can be obtained by a single nucleotide mutation in the cytochrome b gene. Preferably, a mutation is introduced into the gene encoding the succinate dehydrogenase Ip subunit. By introducing a mutation into the gene encoding the succinate dehydrogenase Ip subunit, it is possible to create a mutant succinate dehydrogenase Ip subunit that is resistant to carboxin, that is, a carboxin resistance gene. it can.

  The mutation introduced in the present invention causes deletion, substitution or addition of one or several amino acids in the amino acid sequence originally encoded by the gene. The number of amino acids to be mutated is not limited as long as the gene of the present invention is effective as a target drug resistance gene.

  An example of a carboxin resistance gene obtained by introducing a mutation into a gene encoding a succinate dehydrogenase Ip subunit is a DNA comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 33. An example of such a base sequence is the base sequence of SEQ ID NO: 32. The drug resistance gene of the present invention includes DNA functionally equivalent to the DNA consisting of the nucleotide sequence of SEQ ID NO: 32. Methods well known to those skilled in the art for preparing DNA functionally equivalent to certain DNA include hybridization techniques (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press (1989)).

  Examples of DNA functionally equivalent to the DNA consisting of the base sequence of SEQ ID NO: 32 include DNA that hybridizes with a DNA consisting of a base sequence complementary to the base sequence of SEQ ID NO: 32 under stringent conditions. The DNA has a function of carboxin resistance gene. The stringent condition refers to a condition in which a specific hybrid is formed and a non-specific hybrid is not formed, that is, high homology (homology is 80% or more, preferably 90% or more, more preferably 95). % Or higher)). Examples of hybridization conditions include low stringency conditions. Low stringent conditions are, for example, conditions of 42 ° C., 5 × SSC, and 0.1% SDS, and preferably 50 ° C., 5 × SSC, and 0.1% SDS in washing after hybridization. More preferable hybridization conditions include highly stringent conditions. Highly stringent conditions are, for example, conditions of 65 ° C., 0.1 × SSC and 0.1% SDS.

  That is, in the entire length of the base sequence of SEQ ID NO: 32, various artificial treatments, for example, site-directed mutagenesis, random mutation by mutagen treatment, mutation of nucleic acid fragments by restriction enzyme cleavage, deletion, ligation, etc. Even if the sequence is altered, these mutant DNAs hybridize under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of SEQ ID NO: 32, and function as a carboxin resistance gene. As long as it is different from the nucleotide sequence of SEQ ID NO: 32, it is included in the drug resistance gene of the present invention.

  In addition, the drug resistance gene of the present invention has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 99%, with the base sequence set forth in SEQ ID NO: 32. DNA comprising a nucleotide sequence having the same identity is also included.

  The above-mentioned cellulolytic enzyme expression-suppressing gene and drug resistance gene can be introduced into Arakawakawatake according to a general gene recombination technique. Typically, these genes are ligated to an appropriate vector, and an Arakawaratake transformant of the present invention can be obtained by transforming an Arakawaratake with the vector. The cellulolytic enzyme expression-suppressing gene and the drug resistance gene may be linked to the same vector or may be linked to separate vectors.

  The type of vector is not particularly limited, and a vector capable of autonomous replication or capable of homologous recombination in the chromosome can be used. Examples include plasmids, phage-containing viruses, and cosmids. The vector can appropriately include a selection marker, a replication origin, a terminator, a polylinker, an enhancer, a ribosome binding site, and the like. Various vectors are commercially available or are described in the literature.

  The ligation of the gene to the vector can be performed, for example, using the technique described in J. Sambrook et al. (Supra). In addition, when linking antisense DNA encoding antisense RNA to a vector, as described above, ligation is performed so that antisense RNA against cellulolytic enzyme gene mRNA is generated by transcription. It is also possible to use the recombinant DNA for transformation in a circular form. Moreover, in order to avoid simultaneously transforming genes derived from other organisms, it is also possible to cut out only necessary regions and use them for transformation.

  The host cell, Aragekawaratake, is not particularly limited, and for example, a strain that can be obtained as an NBRC4917 strain from the National Institute for Product Evaluation Technology can be used.

  Examples of transformation methods include, but are not limited to, calcium chloride / PEG method, electroporation method, protoplast method, lipofection method and the like.

  The present invention is also a method for producing a fiber component by removing the fiber component from the lignocellulosic material, wherein the lignocellulosic material is treated with the Arakawakawatake transformant of the present invention, and the fiber component is separated and obtained. Comprising said method.

  Lignocellulosic materials targeted in the present invention are wood, bamboo, cotton, linter, corn cob, bagasse, beer lees, straws, rice husks and other agricultural wastes, old newspapers, magazines, cardboard, office waste paper, pulp And waste pulp discharged from a paper manufacturer. The method of the present invention is particularly effective for wood having high lignin content and agricultural waste.

  Further, as the fiber component, preferably pulp is separated and more preferably paper pulp is separated and obtained. When taking out pulp as a raw material for paper as a fiber component, a wood chip obtained by mechanically cutting wood into a size of 2 to 3 cm and a thickness of about 5 mm can be used as the lignocellulosic material. For example, it is obtained from coniferous trees such as pine, cedar, fir, spruce, douglas fir and radiatapine and wood containing hardwood such as beech, hippopotamus, alder, maple, eucalyptus, poplar, acacia, lawan, rubber, etc. Any type of chip can be used as long as it can be used. As methods for separating and obtaining paper pulp, chemical pulping, mechanical pulping, semi-chemical pulping, etc. can be used (Paper Pulp Manufacturing Technology Series, Volume 1 Kraft Pulp, Volume 2 Mechanical Pulp, Paper Pulp (Technical Association).

  In the step of treating the lignocellulose material with the transformant of the present invention, the lignocellulosic material can be used as it is without any pretreatment, as long as the transformant of the present invention with suppressed cellulose decomposing ability grows sufficiently. However, if it is easier for the transformant of the present invention to grow if pretreatment for sterilizing other microorganisms is performed, it is preferable to pretreat the lignocellulose material by a treatment such as autoclave or steaming. .

  The temperature at which the lignocellulose material is treated with the transformant of the present invention is preferably 10 to 60 ° C, more preferably 20 to 30 ° C. The water content in the lignocellulosic material is 20 to 80%, preferably 30 to 50%. The amount of air supplied to the lignocellulosic material after inoculation is not necessary if the microorganisms with suppressed cellulose decomposing ability can grow sufficiently, but the amount of air supplied per 1 liter of chip or lignocellulosic material volume is usually 0.001 per minute To 1 L / (L · min) (hereinafter, unit of air supply L / (L · min) is referred to as vvm), preferably 0.01 vvm to 0.1 per volume of chip or lignocellulosic material vvm.

  The inoculation amount of the transformant of the present invention into the lignocellulosic material can be appropriately set as long as the pulp yield and paper strength are not reduced. The transformant of the present invention can be pulverized with sterilized water, inoculated with respect to the lignocellulosic material, and cultured, but may be treated by adding a medium to the lignocellulosic material. Any medium can be used as long as the transformant of the present invention can grow. For example, glucose, cellobiose, starch, amorphous cellulose, etc. can be used as the carbon source. As the nitrogen source, nitrogen compounds such as yeast extract, peptone, various amino acids, soybean meal, corn steep liquor, urea and various inorganic nitrogen can be used. Furthermore, various salts, vitamins, minerals, and the like can be used as needed.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1 Production of Carboxin Resistance Gene a. A carrageenan-derived carboxin resistance gene was obtained by the following steps.
1) Preparation of genomic DNA library of Mushroom agaricum 2) Isolation of succinate dehydrogenase Ip subunit 3) Determination of nucleotide sequence 4) Introduction of mutation

Hereinafter, each process is demonstrated in order.
1) Preparation of a genomic DNA library of Arakawaratake mushroom Agar pieces (diameter 5 mm in diameter) were punched out with a cork borer from plate agar culture of Arakawaaratake (IFO 4917 strain), and glucose peptone medium (glucose 2%, polypeptone 0.5%, yeast extract) 0.2%, KH ₂ PO ₄ 0.1%, MgSO ₄ .7H ₂ O 0.05%, adjusted to pH 4.5 with phosphoric acid) was inoculated into 200 ml, and subjected to rotary shaking culture at 28 ° C. for 7 days. The cells were collected by filtration, washed with 1 L of sterilized water, and frozen with liquid nitrogen.

  5 g of this frozen cell was pulverized with a mortar. Transfer the crushed cells to a centrifuge tube, add 10 ml of lysis buffer (100 mM Tris (pH 8), 100 mM EDTA, 100 mM NaCl, and proteinase K added to 100 μg / mL). Incubated for 3 hours at ° C. After incubation, phenol treatment and chloroform treatment were performed, and ethanol was gradually added to the aqueous layer portion. When DNA was precipitated, the chromosomal DNA was wound and suspended in the TE solution.

  The obtained genomic DNA (100 μg) was partially digested with the restriction enzyme Sau3AI and fractionated by 5-20% sucrose density gradient ultracentrifugation (30,000 rpm, 18 hours) to collect 20-40 kbp fragment sections. This fragment segment was ligated to a Toyobo phage λEMBL3-BamHI arm using T4 DNA ligase, and the resulting phage DNA was packaged with STRATAGENE Gigapack Gold, and then infected with E. coli strain P2392 to obtain a genomic DNA library.

2) Isolation of the succinate dehydrogenase Ip subunit gene from the genomic DNA library The succinate dehydrogenase Ip subunit gene involved in the desired carboxin sensitivity is obtained from a known method (Plaque High). Screening can be performed by a hybridization method, a PCR method, or the like. That is, based on the highly conserved amino acid sequence in the succinate dehydrogenase Ip subunit gene, a degenerate sense primer of SEQ ID NO: 1 and a degenerate antisense primer of SEQ ID NO: 2 were synthesized and used to degenerate. As a result of PCR, a 233 base pair DNA fragment was amplified. The base sequence of the obtained DNA amplified fragment was determined by a known method, and compared with known succinate dehydrogenase Ip subunits in other basidiomycetes such as oyster mushrooms and shiitake mushrooms, they had homology. Subsequently, screening was performed from the genomic DNA library obtained in 1) above using the Amarsham AlkPhos Direct Labering and Detection System with the DNA fragment as a probe. As a result, 2 positive clones were selected from about 40,000 plaques. Recombinant phage DNA prepared from positive clones according to a conventional method was digested with various restriction enzymes, and Southern hybridization was performed using the above synthetic DNA. As a result, both hybridized to a Sal I fragment of about 3 kb. This fragment was subcloned into pBluescriptsII SK (-).

3) Determination of base sequence The DNA fragment obtained in 2) above is subjected to PCR reaction using a base sequence analysis reagent manufactured by Applied Biosystems, and the reaction product is subjected to a DNA sequencer ABI PRISM 310 automatic base sequencer manufactured by Applied Biosystems. The nucleotide sequence was determined.

The result is shown in SEQ ID NO: 3. More specifically, this Sal I fragment consisted of a promoter region of about 1.7 kb, a structural gene region of 1 kb, and a terminator region of about 100 bp. Compared with other basidiomycete succinate dehydrogenase gene amino acid sequences, the structural gene region was composed of 268 amino acid residues and was thought to be divided by five introns. In the comparison the amino acid sequence of the Coriolus hirsutus SEQ ID NO: 4 of the succinate dehydrogenase Ip subunit from (Cbx ^S) and succinic acid derived from Pleurotus of SEQ ID NO: 5 dehydrogenase Ip subunit (Cbx ^S) A homology of 81.4% was observed. In addition, three cystein-rich clusters, I to III, were recognized as highly conserved regions (Fig. 1).

4) Introduction of mutation The carboxin resistance gene is carboxin resistant by introducing a site-specific mutation into Leu at the His residue contained in Cys-rich clusterIII, which is a characteristic structure of the succinate dehydrogenase Ip subunit. Gene is obtained. Therefore, in the base sequence shown in SEQ ID NO: 3, a sense primer of SEQ ID NO: 6 having a base sequence in which the 2834th adenine (A) is replaced with thymine (T) when the 5 ′ upstream end is the first is prepared. TAKARA Mutan-super Express Km Kit was used for mutagenesis. In order to introduce mutations, the 3 kb Sal I fragment was subcloned into the Sal I site of the cloning vector pKF19k.

  The resulting plasmid was subjected to 30 cycles of PCR reaction at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 4 minutes with the composition shown in Table 1 using the mutation-introducing sense primer and the selection primer included in the kit. .

  25 μl of 4M ammonium acetate and 100 μl of ethanol were added to the obtained PCR product and left at −20 ° C. for 30 minutes. Then, it centrifuged for 10 minutes with the microcentrifuge, and collect | recovered precipitation. The precipitate was washed twice with 70% ethanol, ethanol was removed, and the precipitate was dried by vacuum drying. The precipitate was suspended in 5 μl of sterile distilled water. 2 μl of DNA solution recovered by ethanol precipitation was mixed with 100 μl of E. coli MV1184 competent cell, and allowed to stand at 0 ° C. for 30 minutes, 42 ° C. for 45 seconds, and 0 ° C. for 2 minutes. Thereafter, LB medium at 37 ° C. was added to make 1 ml, and the mixture was shaken at 37 ° C. for 1 hour. 20 μl of a shaking solution was applied on an LB-Km plate containing 100 μg / ml of kanamycin, and left at 37 ° C. overnight. DNA was prepared from the appearing colonies, sequenced, and it was confirmed that His at the 239th residue was mutated to Leu to obtain plasmid pCHCBX1.

Example 2 Transformation of Aragekawa Ratake with Carboxin Resistance Gene 1) Mononuclear mycelium culture SMY medium (1% sucrose, malt extract 1) in a 500 ml Erlenmeyer flask containing about 30 glass beads with a diameter of about 6 mm After sterilizing by dispensing 100 ml of yeast extract 0.4%), agar plates with a diameter of 5 mm were punched out from a flat plate agar plate of Arakawakawatake IFO4917 with a cork borer and inoculated into SMY medium, and allowed to stand at 28 ° C for 7 days. Incubated (preculture). However, to subdivide the mycelium, it was shaken once or twice a day. Next, dispense 200 ml of SMY medium into a 1 L Erlenmeyer flask, add a rotor, sterilize, collect the precultured mycelia with a nylon mesh (pore size 30 μm), inoculate the whole volume, and incubate at 28 ℃ Incubated with The mycelium was subdivided by stirring with a stirrer for about 2 hours a day. This culture was carried out for 4 days.

2) Preparation of Protoplast The above liquid culture mycelium was collected by filtration with a nylon mesh (pore size 30 μm) and washed with an osmotic pressure adjusting solution (0.5 M MgSO ₄ , 50 ml maleate buffer (pH 5.6)). Next, the suspension was suspended in 1 ml of cell wall degrading enzyme solution per 100 mg of wet cells and incubated at 28 ° C. for 3 hours with gentle shaking to release protoplasts. The following commercially available enzyme preparations were used in combination as cell wall lytic enzymes. That is, 5 mg of cellulase ONOZUKA RS (manufactured by Yakult) and 10 mg of yatalase (manufactured by Takara Shuzo) were dissolved in 1 mg of the osmotic pressure adjusting solution and used as an enzyme solution.

3) Purification of protoplasts After removing mycelial fragments from the enzyme reaction solution with a nylon mesh (pore size 30 μm), in order to increase the protoplast recovery rate, the mycelial fragments remaining on the nylon mesh and the protoplasts were added with the above osmotic pressure control solution. Washed twice. The resulting protoplast suspension is centrifuged (1,000 × g, 5 minutes), the top is removed, resuspended in 20 mM MOPS buffer (pH 6.3) containing 4 ml of 1M sucrose, and centrifuged. The operation was repeated and washed twice with the above 1M sucrose solution. The precipitate was suspended in 500 μl of a 20 mM MES buffer (pH 6.4) containing 1 M sorbitol and 40 mM calcium chloride added to form a protoplast suspension. This suspension was stored at 4 ° C. The protoplast concentration was determined by direct microscopy using a hemocytometer. All centrifugation operations were performed at 1,000 × g for 5 minutes at room temperature on a swing rotor.

4) against the protoplast suspension 100 [mu] l of transformed about ^106/100 [mu] l, cut with restriction enzyme SalI of plasmid pCHCBX1 prepared in Example 1, DNA fragments were recovered only DNA segment from Coriolus hirsutus from agarose gel 2 μg was added and cooled on ice for 30 minutes. Next, an equal amount of PEG solution (20 mM MOPS buffer (pH 6.4) containing 50% PEG 3,400) was added to the protoplast DNA mixture, and the mixture was further ice-cooled for 30 minutes. Next, the mixture was gently mixed in 10 ml of a minimum agar medium (1% agar) containing 0.5 M sucrose, and cultured at 28 ° C. The petri dish was cultured at 28 ° C. for several days. Three days later, 10 ml of a minimum agar medium containing 2 μg / ml carboxin was overlaid, and the culture was further continued. After overlaying, the transformants that grew were selected. At this time, no growth of the cells was observed in the transformation system in which pCHCBX was not mixed.

Example 3 Construction of a Vector Containing Cellulolytic Enzyme Expression Suppressing Gene In Example 2, the mutation was introduced by mutating the His residue at the 239th residue of the succinate dehydrogenase Ip subunit derived from Pseudomonas aeruginosa to Leu residue. The type gene was found to be resistant to carboxin. By using this drug resistance gene as a selection marker and using it together with the cellulolytic enzyme expression-suppressing gene, useful strains that grow rapidly and have cellulolytic enzyme activity suppressed were selected.

1) Preparation of a vector plasmid having a promoter that produces antisense RNA
Any promoter may be used as long as it functions in the agaric bamboo shoot, but it is preferable to use a lignin degradation-related gene promoter or a cellulolytic enzyme-related gene promoter in order to function on woody biomass. Therefore, in order to amplify the cellobiose dehydrogenase gene promoter region, which has a high enzymatic activity when cultivated on a pulp, the primer shown in SEQ ID NO: 7 and the primer shown in SEQ ID NO: 8 PCR reaction was carried out. The obtained DNA fragment of about 2.2 kb was cloned using TOPO TA Cloning Kit (manufactured by Invitrogen) to obtain pTACDHP. Since an NcoI site was present approximately 800 bp upstream of the translation start point in the promoter region, pTACDHP was digested with the restriction enzyme NcoI, followed by blunting reaction and ligation reaction again. This plasmid was digested with restriction enzymes BamHI and restriction enzyme NotI, and then introduced into a vector digested with pBluescriptII SK + plasmid with restriction enzymes BamHI and restriction enzyme NotI to obtain pCDHP.

  Furthermore, in order to introduce the 3 ′ terminal region (0.8 kb) containing the intron of the manganese peroxidase gene (MnP) downstream of pCDHP, the plasmid pBSMPOG1 containing the MnP gene derived from Pseudomonas aeruginosa was used as a template and the primer shown in SEQ ID NO: 9 PCR reaction was performed using the primer shown in SEQ ID NO: 10. The obtained 0.8 kb DNA fragment was cloned using the TOPO TA Cloning Kit, the resulting plasmid was digested with restriction enzymes NcoI and NotI, and then 0.8 kb DNA fragment was obtained by agarose gel electrophoresis. The plasmid pCDHP was collected and introduced into the vector pCDHP digested with the restriction enzymes NcoI and NotI to obtain a plasmid pCDHP-Mnpter.

2) Preparation of a plasmid containing antisense DNA SEQ ID NO: 11 shows a cDNA library prepared from the agaric agaricus growing on a chip as a template for amplifying a DNA fragment of the endoglucanase family 61 gene (EG61). Amplify by PCR using the primer and the primer shown in SEQ ID NO: 12, clone the 430 bp fragment using the TOPO TA Cloning Kit, and use the resulting plasmid using the M13 forward (-20) primer shown in SEQ ID NO: 13. Analysis was performed, and a clone inserted in the sense direction with respect to the lacZ gene (β-galactosidase gene) present on pCR-TOPO was selected and designated as pTA-EG61.

  Next, in order to amplify the third exon region (620 bp) of the cellobiohydrolase II gene, PCR was carried out using genomic DNA as a template, a primer shown in SEQ ID NO: 14 with a restriction enzyme site added, and a primer shown in SEQ ID NO: 15. Went. The obtained DNA fragment was digested with restriction enzymes HindIII and HincII to obtain an insert fragment. This DNA fragment was introduced into a vector obtained by digesting the plasmid pTA-EG61 with the restriction enzymes HindIII and HincII to obtain pTA-CBHII-EG61.

  Next, in order to amplify the third exon region (750 bp) of the cellobiohydrolase I-27 gene, genomic DNA was used as a template, and the primer shown in SEQ ID NO: 16 and the primer shown in SEQ ID NO: 17 were added. Amplified by PCR. The obtained DNA fragment was digested with restriction enzyme SacI and restriction enzyme XbaI to obtain an insert fragment. This DNA fragment was introduced into a vector obtained by digesting the plasmid pTA-CBHII-EG61 with the restriction enzymes SacI and XbaI to obtain a plasmid pTA-CBHII-EG61-CBHI.

  Furthermore, in order to amplify a 630 bp DNA fragment of the cellobiose dehydrogenase gene, amplification was performed by PCR using genomic DNA as a template and primers shown in SEQ ID NO: 18 and SEQ ID NO: 19 with restriction enzyme sites added. did. The obtained DNA fragment was digested with the restriction enzyme SacI to obtain an inserted DNA fragment. This DNA fragment was introduced into a vector obtained by digesting the above plasmid pTA-CBHII-EG61-CBHI with SacI, and the resulting plasmid was subjected to PCR using the primer shown in SEQ ID NO: 17 and the primer shown in SEQ ID NO: 18 to obtain about 1.4 kb. A clone in which the DNA fragment was amplified was selected as pTA-CBHII-EG61-CDH-CBHI.

  Furthermore, in order to obtain a 500 bp DNA fragment of the endoglucanase family 5 gene (EG5), the primer and SEQ ID NO: 20 shown in SEQ ID NO: 20 were used as a template with a cDNA library prepared from the mushrooms grown on the chip. PCR was performed using the primers shown in 21 to amplify the DNA fragment. The obtained DNA fragment was cloned using TOPO TA Cloning Kit, and as a result of analysis using M13 primer, a clone inserted in the sense direction with the β-galactosidase gene was designated as pTA-EG5.

  In addition, in order to obtain a 500 bp DNA fragment of the endoglucanase family 12 gene (EG12), a primer and SEQ ID NO. Amplification was carried out by PCR using the primers shown below. The obtained fragment was digested with restriction enzymes XhoI and XbaI, and then introduced into the vector obtained by digesting plasmid pTA-EG5 obtained above with restriction enzymes XhoI and restriction enzyme XbaI to obtain pTA-EG5-EG12.

  In order to prepare a DNA fragment containing EG5 and EG12, PCR was performed using the primer shown in SEQ ID NO: 24 and the primer shown in SEQ ID NO: 25. The obtained DNA fragment was digested with the restriction enzyme KpnI to obtain an approximately 1 kb fragment having KpnI sites at both ends. This fragment was introduced into the KpnI site at the CDH (cellobiose dehydrogenase) gene site of plasmid pTA-CBHII-EG61-CDH-CBHI, which has four types of cellulolytic enzyme gene fragments, and the six gene fragments were linked in the same direction. Plasmid pTA-CBHII-EG61-EG5-EG12-CDH-CBHI having the prepared DNA sequence was prepared.

  Next, an operation was performed in which the DNA fragment of the cellulolytic enzyme gene was inserted in the antisense direction into the plasmid pCDHP-Mnpter containing the CDH gene promoter region. In order to amplify a region containing DNA fragments of six types of cellulolytic enzyme genes from the plasmid, the plasmid pTA-CBHII-EG61-EG5-EG12- was prepared using the primer shown in SEQ ID NO: 26 and the primer shown in SEQ ID NO: 27. PCR reaction was performed using CDH-CBHI as a template to obtain a DNA fragment of about 3.4 kb. The obtained DNA fragment was digested with NcoI and then inserted in the antisense direction with respect to the cellobiose dehydrogenase gene promoter at the NcoI site at the junction of the promoter region of pCDHP-Mnpter and the 3 'end region of Mnp gene. The CBHI-27, CDH, EG12, EG5, EG61, and CBHII gene fragments are sequentially linked downstream of the promoter region. By the above operation, a plasmid pCDHP-T6 containing 6 types of antisense DNA, which generates antisense RNA for 6 types of cellulolytic enzyme genes by the promoter of CDH gene, was obtained (FIG. 2).

Example 4 Selection of transformants in which cellulolytic enzyme activity was suppressed Example 2 was carried out using pCHCBX1 (0.2 μg) obtained in Example 1 and pCDHP-T6 (2.0 μg) obtained in Example 3. Algae agaricus was transformed by the method described. Transformants isolated by the above transformation method were oxygen bleached hardwood pulp (LOKP) and peptone medium (LOKP 1%, polypeptone 1%, KH ₂ PO ₄ 0.15%, MgSO ₄ 0.05%, pH with phosphoric acid In a 300 ml Erlenmeyer flask containing 100 ml each, and cultured with shaking at 28 ° C. and 120 rpm. Cellobiohydrolase I activity, cellobiose dehydrogenase activity, and CMC degradation activity were measured over time. As a result, a transformant having cellobiohydrolase I activity of 80%, cellobiose dehydrogenase activity of 90%, and CMC degradation activity of 30% was obtained on the fifth day of culture. On the other hand, this activity was not observed in the culture supernatant of Aragekawaratake OJI-1078 strain (FERM BP-4210) not containing pCHCBHI27PMP cultured under the same conditions.

Example 5 Production of mechanical pulp from wood chips treated with transformants with suppressed cellulolytic enzyme activity Transformed strains with reduced cellulolytic enzyme activity selected according to Example 4 were prepared on potato dextrose agar medium. After culturing at 0 ° C., it was stored at 4 ° C. Glucose and peptone media (5% glucose, 1% polypeptone, KH ₂ PO ₄ 0.15%, MgSO ₄ 0.05%, adjusted to pH 5.0 with phosphoric acid) were cut into 5 pieces from this plate with a 5 mm diameter cork borer. A 300 ml Erlenmeyer flask containing 100 ml was inoculated and cultured with shaking at 28 ° C. and 100 rpm for 1 week. After the cultivation, the cells were filtered off, and the medium remaining in the cells was washed with sterilized water. The bacterial cells were treated with sterilized water in a Waring blender for 45 seconds, and inoculated so that the dry weight of the bacterial cells was 5 mg on a softwood chip having an absolutely dry weight of 1 kg. After inoculation, the mixture was stirred well so that the bacteria spread throughout. The culture was performed at 28 ° C. with aeration for 2 weeks. At this time, saturated water vapor was aerated as needed so that the moisture content of the chip was 40 to 65%. In addition, the air flow when venting was set to 0.01 vvm per chip.

  Use wood lab refiner (manufactured by Kumagai Riki Kogyo Co., Ltd.) to beat the wood chips after the fungus treatment to make the Canadian Standard Freeness 80 ml, and then prepare the handsheets for pulp physics testing using the JIS test method. In accordance with (P8209), the physical test of the pulp handsheet was conducted according to the Tappi method T220 om-83. The electric energy used was a watt meter (Hiokidenki model 3133) and an integrator (model 3141). For the chip yield measurement, 1 kg of moisture-containing wood chips were collected in a container in a dry weight, the chip dry weight before and after the treatment was measured, and the chip yield was calculated using the following formula.

(Absolute dry weight after treatment) / (Absolute dry weight before treatment) x 100
The results are shown in Table 2.

Comparative Example 1 Production of mechanical pulp using the wild strain of Arakawaratake In Example 5, except that a wild strain of Arakawakaratake was used instead of the transformed strain in which the cellulolytic enzyme activity was suppressed, the same method was used. . The results are shown in Table 2.

Comparative Example 2 Production of mechanical pulp from chips not subjected to fungus treatment In Example 5, the same method was carried out without inoculating the fungus. The results are shown in Table 2.

  As shown in Table 2, the transformant of Example 4 in which the cellulolytic enzyme activity was suppressed was able to reduce the fibrillation energy as compared with the case where the fungus treatment shown in Comparative Example 2 was not performed. The wild type strain of Comparative Example 1 can reduce the fibrillation energy compared with the case of no bacterial treatment of Comparative Example 2, but both the chip yield and paper strength (specific tear strength and specific burst strength) are greatly reduced. It was. The transformed strain of Example 4 can improve the decrease in yield compared to the wild strain shown in Comparative Example 1, and both the tear strength and burst strength can be improved compared to the wild strain of Comparative Example 1. did it.

Example 6 Production of Kraft Pulp from Chip Treated with Transformant with Cellulolytic Enzyme Activity Suppressed After chipping hardwood eucalyptus wood instead of radiata pine (coniferous chip) according to Example 5, Weigh chips with an absolute dry weight of 400 g and add cooking white liquor so that the liquid ratio is 5, the sulfidity is 30%, and the effective alkali is 16% (as Na ₂ O) in the autoclave, and the cooking temperature is between 160 ° C. Kraft cooking was done so that the kappa number after cooking was 16. After completion of kraft cooking, the black liquor was separated, and the obtained chips were defibrated with a high-concentration disintegrator, and then centrifugal dehydration and water washing were repeated three times with a filter cloth. Subsequently, undistilled straw was separated with a screen, and centrifugally dehydrated to obtain unbleached pulp. The soot was dried at 105 ° C. and then measured for absolute dry weight. Further, a portion of unbleached pulp was taken and the dry weight was measured to obtain the yield from the chip (selection yield). The pulp kappa number was measured in accordance with JIS P 8211. The results are shown in Table 3.

Next, 2.0% by mass of NaOH was added to the pulp obtained by kraft cooking, oxygen gas was injected, and treatment was performed at 100 ° C. and oxygen gauge pressure of 0.49 MPa (5 kg / cm ² ) for 60 minutes. . Subsequently, as shown below, the obtained pulp was subjected to a four-stage bleaching treatment of chlorine dioxide treatment (D) -alkali extraction treatment (E) -hydrogen peroxide treatment (P) -chlorine dioxide treatment (D). The first chlorine dioxide treatment (D) was prepared so that the pulp concentration was 10% by mass, 0.4% by mass of chlorine dioxide was added, and the treatment was performed at 70 ° C. for 40 minutes. Next, after washing with deionized water and dehydration, the pulp concentration was adjusted to 10% by mass, 1% by mass of caustic soda was added, and alkali extraction treatment (E) was performed at 70 ° C. for 90 minutes. Next, after washing with deionized water and dehydration, the pulp concentration is adjusted to 10% by mass, 0.5% by mass of hydrogen peroxide and 0.5% by mass of caustic soda are sequentially added, and hydrogen peroxide treatment (P ) Next, after washing with deionized water and dehydration, the pulp concentration was adjusted to 10%, 0.25% by mass of chlorine dioxide was added, and chlorine dioxide treatment (D) was performed at 70 ° C. for 180 minutes. Finally, after washing with deionized water and dehydration, a bleached pulp having a whiteness of 86.0% according to JIS P 8123 was obtained.

  The pulp slurry having a pulp concentration of 4% by mass obtained above was beaten with a refiner so that the freeness was 410 ml (CSF). Preparation of a handsheet for pulp physical test was performed in accordance with JIS test method (P8209), and physical test for pulp handsheet was performed in accordance with Tappi method T220 om-83. The results are shown in Table 3.

Comparative Example 3 Production of kraft pulp using a wild strain of Arakawaratake In Example 6, except that a wild strain of Arakawakaratake was used instead of the transformed strain in which cellulolytic enzyme activity was suppressed, the same method was used. It was. The results are shown in Table 3.

Comparative Example 4 Production of kraft pulp from chips not subjected to fungus treatment In Example 6, the same method was carried out without inoculating fungi. The results are shown in Table 3.

  As shown in Table 3, the selected yield of pulp when digesting wood chips treated with the transformed strain (Example 6) and wood chips treated with the wild strain (Comparative Example 3) to a kappa number of 16 As compared with the case where no treatment was carried out with bacteria (Comparative Example 4), both increased and the wrinkle rate decreased. In addition, in any of the bacterial treatments (Example 6, Comparative Example 3), the number of rotations of beating by the PFI mill until the target freeness reached 410 ml could be reduced as compared with the case where no treatment was performed (Comparative Example 4).

  When treated with wild strains (Comparative Example 3), the specific tear strength, tear length, specific burst strength, and bending strength all decreased compared to when not treated with bacteria (Comparative Example 4). However, when treated with the transformant (Example 6), the strength of specific tear strength and tear length was reduced compared to the case where the fungus was not treated (Comparative Example 4). Was smaller than that in the case of the wild strain (Comparative Example 3), and the specific burst strength was rather higher than that in the case of no bacterial treatment.

  Lignin in wood is obtained by inoculating a transformant with reduced cellulolytic enzyme activity obtained according to the present invention into wood chips, aerating and keeping warm, thereby reducing pulp yield and paper strength. In the mechanical pulp manufacturing process, beating energy that consumes a large amount of electric energy can be reduced. In the chemical pulp manufacturing process, it is possible to improve the digestibility and increase the yield, which is advantageous in the paper pulp manufacturing process.

Shows the results of comparison between the Coriolus hirsutus derived succinic acid dehydrogenase Ip subunit (Cbx ^S) and Pleurotus derived succinate dehydrogenase Ip subunit (Cbx ^S). The plasmid containing 6 types of antisense DNA which produces the antisense RNA with respect to 6 types of cellulolytic enzyme genes is shown.

Claims (9)

  An Arakawaratake transformant carrying a cellulolytic enzyme expression-suppressing gene and a drug resistance gene obtained by introducing a mutation into a gene involved in the drug sensitivity of Arakawaaratake.   The cellulolytic enzyme expression-suppressing gene is an antisense DNA encoding an antisense RNA having a sequence substantially complementary to all or a part of mRNA transcribed from the cellulolytic enzyme gene. The described Arakawakawatake transformant.   The antibacterial bamboo shoot transformant according to claim 2, wherein the antisense DNA is linked under the control of the promoter of the cellobiose dehydrogenase gene or the glyceraldehyde-3-phosphate dehydrogenase gene.   The jellyfish oyster mushroom transformant according to any one of claims 1 to 3, which carries a cellulolytic enzyme expression suppression gene for six or more types of cellulolytic enzyme genes.   The cellulase transformation agent according to any one of claims 1 to 4, which carries a cellulolytic enzyme expression suppression gene for at least one gene selected from the group consisting of an endoglucanase gene, a cellobiohydrolase gene, and a cellobiose dehydrogenase gene. body.   A method for producing a fiber component by taking out the fiber component from the lignocellulosic material, wherein the lignocellulosic material is treated with the Aragekawaratake transformant according to any one of claims 1 to 5 to separate the fiber component. Said method comprising obtaining.   The method according to claim 6, wherein the fiber component is pulp.   The method according to claim 7, wherein the pulp is paper pulp.   The method according to claim 8, wherein the method for separating and obtaining paper pulp is a chemical pulping method, a mechanical pulping method, or a semichemical pulping method.

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