KR101106061B1 - A recombinant vector and a transformant with an improved productivity of bio-ethanol - Google Patents

Abstract

The present invention relates to endo-beta-1,4-glucanase-I (endo-β-1,4-glucanaseE, 1,4-β-D-glucan glucanohydrolase, carboxymethylcellulase derived from Clostridium sp.) Microorganisms. , CMCase; EC 3.2.1.4) and beta-glucosidase (EC 3.2.1.21) genes derived from Saccharomycopsis sp. It is about.

Beta-glucosidase, recombinant vector, transformant

Description

A recombinant vector and a transformant with an improved productivity of bio-ethanol}

The present invention relates to a yeast transformant capable of effectively decomposing fibrin provided as a substrate in yeast, which is a production strain, in bioethanol production. More specifically, endo-beta-1,4- separated from Clostridium thermocells. Recombinant vector into which the glucanase-I (endo-β-1,4-glucanaseE, 1,4-β-D-glucan glucanohydrolase, carboxymethylcellulase, CMCase; EC 3.2.1.4) gene is inserted and the yeast transformant to which the same is introduced It is about.

In Korea, natural resources such as biomass must be considered, considering that more than 97% of the country's energy depends on foreign countries, it is difficult to guarantee energy supply lines, the gasoline price reaches 1,600 won per liter, and the country is highly dependent on imported oil. The development of renewable energy production technology from CHF must be promoted.

As concerns about environmental pollution and resource depletion increase due to excessive use of fossil fuels, the concept of renewable energy, which coexists with nature and produces energy continuously, is becoming a hot topic. Among these, a policy to spread the spread of environmentally friendly renewable energy is being established, and among these, wood-based lignocellulose, which represents the most abundant and depleted renewable plant resources, is attracting the most attention. Lignocellulosic is a biomass in a narrow sense and is generally referred to as bioenergy by collectively receiving various fuels such as alcohol, diesel, and hydrogen produced from such biomass.

Biomass degradation, including organic matter in its natural state, is carried out through anaerobic biological processes, and anaerobic degradation processes make wood-based biomass, a polysaccharide, into fermentable sugars, or Involves several microbial populations that produce methane and carbon dioxide. Pretreatment processes, one of the methods for decomposing woody biomass, include physical processes, thermo-chemical treatments and specific enzyme cocktail treatments. At this time, since the role of the degrading enzyme to be used is very important, the system of extracellular hydrolytic enzymes produced out of the cell from microorganisms which is very effective for the degradation of wood-based biomass which has undergone the most important pretreatment process in biomass biodegradation. Research and development is the most urgent task.

Cellulase production through existing microorganisms focused more on yield than on specific activity. The representative strain used for this is Trichoderma reesei . However, such aerobic fungi produce independent cellulase that does not form complexes, which cannot be completely decomposed in the biomass of cellulose. In order to overcome this problem, biomass degradation using cellulosomes, which are 'large extracellular enzyme complexes' using anaerobic bacteria, which are easy to manipulate and have high biomass degradation efficiency, are studied. Is getting attention.

Yeast Saccharomyces cerevisiae , a representative ethanol producing strain, is used in various bioprocessing fields utilizing starch or fiber, which is a vegetable raw material. However, yeast has a disadvantage in that it can not break down the fibrous substrate at all by producing and secreting a small amount of starch degrading enzyme. Therefore, the new breeding of fibrinolytic yeast using genetic recombination technology can be applied to brewing, baking, alcohol production, probiotic development, and especially the production of bio ethanol, a renewable energy, and thus the utilization of these recombinant yeast is expected. have. Yeast is also used as an excellent strain for extracellular secretion of heterologous proteins. Currently, the research for producing a recombinant yeast secreting fibrinolytic enzymes is active or inadequate. For this purpose, it is necessary to secure a fibrinolytic gene having high thermal stability or excellent activity.

On the other hand, Clostridium thermocellum is an anaerobic high temperature bacterium, which is known to produce various cellulose and also to produce a cellulosome, an enzyme complex. Although the fibrinolytic enzymes produced by Clostridium thermocells differ greatly in substrate specificity and reaction mechanisms, they all degrade beta-1,4-glucoside (β-1,4-glycoside) bonds. It has been reported that there is a characteristic secreted into the medium (Shoseyov et al. 1990, Biochem Biophys Res Commun 169 (2), 667-672). In addition, effective degradation of crystalline fibrin includes at least three fibrinases, endoglucanase, exoglucanase (or cellobiohydrolase) and beta-glucosidase (β- glucosidase) is required simultaneously, and their synergy has been reported to significantly increase fibrin resolution (Murashima et al. 2002, J Bacteriol 184 (18), 5088-5095; Schwarz 2001, Appl Microbiol Biotechnol 56 (5-6)). 634-649, Shoham et al. 1999, Trends Microbiol 7 (7), 275-281).

The present invention solves the above problems, and the object of the present invention is to provide a recombinant vector capable of effectively decomposing the fiber provided by the yeast as a substrate in the production of bioethanol in the production of bioethanol .

Another object of the present invention is to provide a yeast transformant transformed with the recombinant vector.

In addition, another object of the present invention is to provide an endoglucanase production method using the transformant.

Another object of the present invention is to provide a method for producing bioethanol using the transformant.

In order to achieve the above object, the present invention encodes a gene encoding an endoglucanase derived from the genus Clostridium sp. And a betaglucosidase derived from the genus Saccharomycopsis sp. To provide a recombinant vector pαEgEαBG1 into which the gene is inserted.

In one embodiment of the invention, the endoglucanase derived from the Clostridium sp. ( Clostridium sp.) Preferably has the amino acid sequence of SEQ ID NO: 1, the Saccharomycopsis sp. The beta glucosidase derived from) preferably has an amino acid sequence as set forth in SEQ ID NO: 2, but is not limited thereto.

In one embodiment of the invention, the gene encoding the endoglucanase derived from the Clostridium sp. ( Clostridium sp.) Preferably has a nucleotide sequence of SEQ ID NO: 3, the genus Saccharomycocci The gene encoding beta glucosidase derived from ( Saccharomycopsis sp.) Preferably has the nucleotide sequence set forth in SEQ ID NO: 4, but is not limited thereto.

In addition, the present invention is a recombinant that inserts a gene encoding an endoglucanase derived from the genus Clostridium sp. And a gene encoding a betaglucosidase derived from the genus Saccharomycopsis sp. Vector pαEgEαBG1 A transformant transformed with the vector of claim 1 is provided.

In one embodiment of the present invention, the transformant has a deposit number of KCCM11006P and a microorganism deposited on May 06, 2009 at the Korea Microorganism Conservation Center, Hongje-dong, Seodaemun-gu, Seoul, Korea, but is not limited thereto.

In another aspect, the present invention provides a method for producing endoglucanase, characterized in that the transformant yeast of the present invention is cultured.

The present invention also provides a method for producing bioethanol, characterized in that the transformant yeast of claim 6 or 7 in the presence of a substrate.

In a preferred embodiment of the present invention, the substrate is preferably starch or fiber, but is not limited thereto.

In the case of using the yeast as a host as in the present invention, as the expression vector, for example, YEp13, YCp50, pRS-based, pYEX-based vectors and the like can be used. As a promoter, a GAL promoter, an AOD promoter, etc. can be used, for example. As a method of introducing recombinant DNA into yeast, for example, the electroporation method (Method Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)), lithium acetate method (J. Bacteriol., 153, 163-168 (1983)), and the like.

In the recombinant vector, fragments for suppressing expression having various functions for suppressing or amplifying or inducing expression, markers for selection of transformants, resistance genes against antibiotics, or secreted out of the cells It is also possible to have a gene for encoding a signal for the purpose of.

In the production of the endoglucanase of the present invention, for example, a transformant obtained by transforming a host with a recombinant vector having a gene encoding the same is cultured, and the product is a gene product in a culture (culture cell or culture supernatant). It is performed by generating and accumulating endoglucanase and obtaining from the culture.

As a method for culturing the transformant of the present invention, any conventional method used for culturing a host may be used.

As the culture method, any method used for culturing ordinary microorganisms, such as batch type, flow batch type, continuous culture, and reactor type, can be used. Transformants obtained by using bacteria such as Escherichia coli as a host As a medium for culturing, medium or synthetic medium such as LB medium, M9 medium and the like can be given. Incidentally, the incubation temperature is cultivated in the above-mentioned range of temperature to accumulate endoglucanase in cells and recover.

The carbon source is required for the growth of microorganisms, and for example, sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; Lower alcohols such as ethanol, propanol and butanol; Polyhydric alcohols such as glycerol; Organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid and gluconic acid; Fatty acids such as propionic acid, butanoic acid, pentanic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid can be used.

Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, as well as those derived from natural products such as peptone, meat juice, yeast extract, malt extract, caseinate and corn steep liquor. Moreover, as an inorganic substance, 1 potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, etc. are mentioned, for example. You may add antibiotics, such as kanamycin, ampicillin, tetracycline, chloramphenicol, and streptomycin, to a culture liquid.

In addition, when the promoter cultures the microorganism transformed using an inducible expression vector, an inducer suitable for the type of promoter may be added to the medium. For example, isopropyl-β-D-thiogalactopyranoside (IPTG), tetracycline, indole acrylic acid

(IAA) etc. can be mentioned as an inducer.

Acquisition and purification of endoglucanase is carried out by centrifugation of the cells or supernatant from the obtained culture, and cell combination, extraction, affinity chromatography, cation or anion exchange chromatography, gel filtration, or the like alone or as appropriately combined. This can be done by.

Confirmation that the obtained purified substance is the target enzyme can be performed by a conventional method, for example, SDS-polyacrylamide gel electrophoresis, western blotting or the like.

Incidentally, the culture of the transformant using the microorganism as a host, the production of the endoglucanase by the transformant, the accumulation in the cells, and the recovery of the endoglucanase from the cells are not limited to the above-described methods.

Hereinafter, the present invention will be described in detail.

The present inventors have developed a recombinant vector endoglucanase-derived gene derived from Clostridium thermocellum and a yeast transformant transformed with the vector, which were transformed into KCCM11006P on May 06, 2009. Deposited in (KCCM). In addition, the present invention was completed by confirming that the yeast transformant effectively secretes endoglucanase to increase ethanol production.

The present invention relates to endo-beta-1,4-glucanase-I (endo-β-1,4-glucanaseE, 1,4-β-D-glucan glucanohydrolase, carboxymethylcellulase derived from Clostridium sp.) Microorganisms. , CMCase; EC 3.2.1.4) and beta-glucosidase (EC 3.2.1.21) genes derived from Saccharomycopsis sp. More particularly, the present invention relates to a recombinant vector into which the gene is inserted and a yeast transformant which is transformed with the vector so that the protein is secreted from the yeast extracellularly. Endo-beta-1,4-glucanase-derived from the microorganism of the genus Clostridium of the present invention has the ability to directly degrade crystalline cellulose, thus transducing this gene into yeasts capable of producing bioethanol. The conversion can overcome the disadvantages of yeast that can not be used as a direct energy source can decompose the cellulose itself. In addition, beta-glucosidase derived from the microorganism of the genus Saccharomycocci of the present invention is an enzyme capable of breaking down cellobiose into glucose and using yeast as an energy source by using a product of endoglucanase as a substrate. Glucose may be used. In addition, according to the present invention, yeast transformants in which the endo-beta-1,4-glucanase-this gene and the beta-glucosidase gene derived from Clostridium spp. Microorganisms are introduced are simulative saccharification and fermentation. , SSF) can be usefully used in the process. As a result of fermentation using beta glucan as a substrate, the yeast transformants into which the genes were introduced produced more ethanol than the yeast strains.

Cellulose (cellulose) is a major component of the cell wall of higher plants and occupies most of the wood, and is broken down by cellulase, a fibrinolytic enzyme. Fibrinase, which is secreted by microorganisms parasitic in the stomach in the herbivore when ingesting fiber, includes endoglucanase, xylanase, exoglucanase, and beta-glucosidase. ). Among them, endoglucanase hydrolyzes β-D-glucan (β-D-glucan).

In the present invention, it was confirmed that the recombinant yeast strain prepared by introducing a gene of the fibrinolytic enzyme EgE into yeast from Clostridium spp. Known to maintain long activity even at room temperature expresses and secretes EgE at high levels.

According to an embodiment of the present invention obtained by a PCR reaction as the template the genomic DNA of Clostridium thermo selreom EgE the gene, the gene thus obtained yeast - by subcloning into E. coli shuttle vectors (yeast- E. coli shuttle vector) Recombinant vectors were constructed. Thereafter, yeast host cells were transformed using the recombinant vector into which the EgE was inserted. Then, in order to investigate that the protein expressed from the gene is endoglucanase, endoglucanase activity was measured by culturing recombinant yeast transformed with EgE. As a result, it was confirmed that the recombinant yeast transformant with EgE introduced according to the present invention showed significantly higher endoglucanase activity than the wild type Saccharomyces cerevisiae YPH499 strain. As a result, the recombinant and transformed EgE gene according to the present invention was confirmed that the expression of the endoglucanase.

In addition, as a result of examining the secretion of the recombinant yeast transformant and the CMC (carboxymethylcellulose) resolution, it was confirmed that the active secretion of EgE from yeast and the function of decomposing fibrin are made very efficiently. Therefore, this yeast transformant was deposited with the Korea Microorganism Conservation Center with accession number KCCM 11006P, and the present invention was completed by confirming that the yeast transformant effectively secretes endoglucanase to increase ethanol production efficiently.

As described above, in the present invention, the endoglucanase gene obtained from the strain of Clostridium was introduced into the yeast strain. Then, it was confirmed that the endoglucanase gene is expressed and secreted at a high level in the yeast strain. According to the present invention, the yeast transformants into which the endoglucanase gene derived from the strain of the genus Clostridium is introduced and the endoglucanase expressed from the gene may be usefully used for brewing, baking, alcohol production, feed production and probiotic development. In particular, the present invention is expected to be a very useful invention for the simultaneous saccharification and fermentation or consolidated bioprocessing (SSF) process, which breaks down cellulose and produces alcohols from degradation products.

Hereinafter, the present invention will be described in detail by way of non-limiting examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1: Amplification of Yeast Signal Peptide Gene and Endoglucanase Gene and Beta Glucosidase Gene

In order to clone the signal peptide gene for effectively secreting the enzyme out of the yeast, a forward primer is referred to by referring to the nucleotide sequence of the α-factor signal peptide portion from GD (BY4741) of Saccharomyces cerevisiae. The 5 'of the restriction enzyme EcoR Ⅰ, the reverse primer (5) of the reverse primer (Reverse primer) was designed to synthesize the primer to insert the restriction enzyme Not Ⅰ recognition sequence. Then, PCR was performed using the synthesized primers. As a result, the PCR band containing the yeast signal peptide gene of 255 bp was confirmed, and it is shown in Figure 1 (A).

In order to clone the EgE gene of Clostridium-derived thermocell, the base sequence excluding the signal peptide portion is referred to, and 5 'of the restriction primer BamH I and 5' of the reverse primer A primer ( 5'-gcgcggccgctcgggaacaaagctttt gga-3 '(SEQ ID NO: 5) and 5'-gtgcggccgctattgctttttttaagaatgcaag-3') (SEQ ID NO: 6) having a restriction enzyme Xho I recognition sequence was synthesized. PCR was performed using the PCR band including the EgE gene of the thermocell of 1452 bp Clostridium, which is shown in Figure 1 (B).

In order to clone the bgl1 gene of Saccharomycopsis- derived Bvl1 gene, the base sequence excluding the signal peptide portion was referred to, and 5 'of the forward primer was restricted to Not I and reverse primer. A primer (5'-ggcgcggccgcgtcccaattcaaa actatacc-3 '(SEQ ID NO: 7) and 5'-ggcactagtcgaatagtaaacaggacagatgtct-3' (SEQ ID NO: 8)) containing the restriction enzyme Spe I recognition sequence was synthesized. As a result, the PCR band containing the bgl1 gene of fibuligera derived from Saccharomycocci of 2577bp could be confirmed, which is shown in FIG. 1 (C).

Example 2: Cloning of Signal Peptides with EgE Gene

The signal peptide and the EgE amplification product obtained in Example 1 were electrophoresed on a 0.8% agarose gel, and DNA fragments on the agarose gel were recovered using a Qiaex II gel extraction kit (Qiagen).

Then, in the case of a yeast signal peptide is EcoR Ⅰ, Not Ⅰ, e gE and bgl1 is Not Ⅰ, was cut with Spe Ⅰ yeast - E. coli shuttle vectors (yeast- E. coli shuttle vector) of pESC-trp (Clontech) Ligation was performed to transform Escherichia coli ( E. coli ) DH5α (RBC Co.). The ligation of recombinant plasmid DNA was then isolated from the transformants. The recombinant vectors were named pESC-α-EgE and pESC-α-Bgl1, respectively. Subsequently, the enzyme signal peptide and the endoglucanase were gene amplified using pESC-α-EgE as a template and ligated to BamH I and Xho I sites of pESC-α-Bgl1 to transform Escherichia coli DH5α. The ligated recombinant plasmid DNA was then isolated from the transformants. The recombinant vector was named pαEgEαBG1 and is shown in FIG. 2. Then, the recombinant vectors were used to transform yeast host cells according to the experimental method provided by Clontech's yeast maker yeast transformation kit (YEASTMAKER yeast transformation kit2).

Saccharomyces cerevisiae YPH499 ( S. cerevisiae YPH499, ura3-52lys2-801amberade2-101ochretrp1-Δ63 his3-Δ200 leu2-Δ1) was used as the yeast host cell. Subsequently, the transformants were selected using tryptophan deficient SD medium (0.67% yeast nitrogen base, 2% glucose, 0.067% yeast nigrogen base w / o trp, 2% agar), which were then transformed into Saccharomyces cerevisiae YPH499 /. It was named pαEgEαBG1.

Example 3: Measurement of Endoglucanase Activity of Yeast Transformants

In order to confirm the expression of the enzyme protein of the transformant obtained in Example 2, enzyme activity band (halo) measurement, SDS-PAGE and Zymogram were performed on the medium.

By inducing the expression of YPH499 / pαEgEαBG1 to galactose to secrete EgE enzyme protein into the culture solution, the culture supernatant was prepared by condensation at 30 ° C for 48 hours, centrifuged and concentrated (Millipore, amicon 10kDa cut off) EgE enzyme Obtained protein.

To confirm the enzyme activity (halo), 0.3% CMC (carboxy methyl cellulose) was dissolved in YPG agar medium and used as a substrate for the enzyme reaction. Colonies of transformed yeast were inoculated in YPG agar medium (yeast extract, peptone, galactose) and incubated at 30 ° C. for 48 hours, and 5 μl of the culture medium incubated at 30 ° C. for 48 hours in YPG agar medium (CMC, Carboxymethylcellulose, Sigma) was added to the agar containing the medium and the reaction was carried out at 50 ℃ for 24 hours. After staining with 0.25% Congo-Red (Sigma) for 20 minutes and rinsing several times with 1 mol sodium chloride (halo chloride) was confirmed the enzyme activity (halo) and the results are shown in Figures 3a and 3b, respectively.

 In order to confirm the expression of EgE and Bgl1, SDS-PAGE electrophoresed the enzyme protein using a 10% poly-acrylamide gel and a poly-acrylamide gel in which 0.1% CMC (carboxymethylcellulose) was dissolved. As a result, protein bands were identified at 53kDa and 95kDa positions. This result is shown in FIG.

Zymogram was performed to confirm the enzyme expression at the correct position. To do this, the renature buffer (100mM Succiniate, 10mM CaCl2, 1mM dTT) to which the substrate was added was changed every 1 hour for 2 hours at 50 ℃. The reaction was carried out for 3 hours. After completion of the reaction, the gel was stained with 0.25% Congo-Red for 30 minutes and rinsed several times with 1M NaCl to confirm the enzyme activity (halo) at the same position as the EgE protein size. This result is shown in FIG.

Example 4 Production of Bioethanol Using Yeast Transformants

YG499 / pαEgEαBG1 and YPH499 / pESC-trp were expressed in galactose-induced SG medium for 24 hours, followed by 48 hours of shaking culture. 50g / l cellobiose, 20g / l CMC (carboxymethylcellulose) and 20g / l β-glucan were substrates. When the absorbance at 600nm wavelength was added to the fermentation broth added to make 5 and 20 respectively. The fermentation broth was incubated at 30 ° C. and 100 rpm, and the ethanol produced was measured by performing gas chromatography by taking a culture solution at a predetermined time. As a result of the above method, the ethanol production yield of YPH499 / pαEgEαBG1 into which endoglucanase and betaglucosidase were inserted was determined to be higher than that of YPH499 / pESC-trp.

Figure 1 (A) is a figure confirming the PCR product of the signal peptide gene of yeast by performing agarose gel electrophoresis in the present invention,

Figure 1 (B) is a figure confirming the PCR product of the endo-beta-1,4-glucanase-gene by performing agarose gel electrophoresis in the present invention,

Figure 1 (C) is a figure confirming the PCR product of the beta glucosidase gene by performing agarose gel electrophoresis in the present invention, Figure 1 (A) Lane 1, 100bp DNA marker; Lane 2, alpha factor signal sequence PCR product, (B) Lane 1, 1 kb DNA marker; Lane 2, egE PCR product, (C) Lane 1, 1 kb DNA marker; Lane 2, bgl1 PCR product is shown.

2 is a schematic diagram showing the recombination process with the recombinant vector pαEgEαBG1 in which the endo-beta-1,4-glucanase-this gene and the betaglucosidase gene are inserted in both directions.

Figure 3a is a diagram showing the enzyme secretion by showing the enzymatic activity in the colony of the recombinant vector pαEgEαBG1 inserted yeast transformant presented in the present invention,

Figure 3b is a diagram showing the enzyme secretion into the culture medium showing the enzyme activity in the culture medium of the yeast transformant inserted recombinant vector pαEgEαBG1 presented in the present invention,

4 is a diagram confirming the expression of the enzyme expressed in the present invention by the SDS-PAGE technique and Zymogram technique, Lane 1 in the figure is a protein marker (Elpis); Lane 2 is a band of EgE and Bgl1 obtained from coma blue stained pαEgEαBG1 SDS-PAGE, and Lane 3 is an EgE halo showing the activity of endoglucanase on SDS-PAGE to which 0.1% CMC was added as a substrate by zymogram technique.

5 is a graph confirming that the recombinant vector pαEgEαBG1 introduced in the present invention produces ethanol using CMC and β-glucan as substrates. closed circles, pαEgEαBG1; closed squares, pESC-trp; Closed diamond, pαEgE; lines, CMC; dotted lines, β-glucan.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> A recombinant vector and a transformant with an improved          productivity of bio-ethanol <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 440 <212> PRT <213> Clostridium sp. <400> 1 Ser Gly Thr Lys Leu Leu Asp Ala Ser Gly Asn Glu Leu Val Met Arg   1 5 10 15 Gly Met Arg Asp Ile Ser Ala Ile Asp Leu Val Lys Glu Ile Lys Ile              20 25 30 Gly Trp Asn Leu Gly Asn Thr Leu Asp Ala Pro Thr Glu Thr Ala Trp          35 40 45 Gly Asn Pro Arg Thr Thr Lys Ala Met Ile Glu Lys Val Arg Glu Met      50 55 60 Gly Phe Asn Ala Val Arg Val Pro Val Thr Trp Asp Thr His Ile Gly  65 70 75 80 Pro Ala Pro Asp Tyr Lys Ile Asp Glu Ala Trp Leu Asn Arg Val Glu                  85 90 95 Glu Val Val Asn Tyr Val Leu Asp Cys Gly Met Tyr Ala Ile Ile Asn             100 105 110 Leu His His Asp Asn Thr Trp Ile Ile Pro Thr Tyr Ala Asn Glu Gln         115 120 125 Arg Ser Lys Glu Lys Leu Val Lys Val Trp Glu Gln Ile Ala Thr Arg     130 135 140 Phe Lys Asp Tyr Asp Asp His Leu Leu Phe Glu Thr Met Asn Glu Pro 145 150 155 160 Arg Glu Val Gly Ser Pro Met Glu Trp Met Gly Gly Thr Tyr Glu Asn                 165 170 175 Arg Asp Val Ile Asn Arg Phe Asn Leu Ala Val Val Asn Thr Ile Arg             180 185 190 Ala Ser Gly Gly Asn Asn Asp Lys Arg Phe Ile Leu Val Pro Thr Asn         195 200 205 Ala Ala Thr Gly Leu Asp Val Ala Leu Asn Asp Leu Val Ile Pro Asn     210 215 220 Asn Asp Ser Arg Val Ile Val Ser Ile His Ala Tyr Ser Pro Tyr Phe 225 230 235 240 Phe Ala Met Asp Val Asn Gly Thr Ser Tyr Trp Gly Ser Asp Tyr Asp                 245 250 255 Lys Ala Ser Leu Thr Ser Glu Leu Asp Ala Ile Tyr Asn Arg Phe Val             260 265 270 Lys Asn Gly Arg Ala Val Ile Ile Gly Glu Phe Gly Thr Ile Asp Lys         275 280 285 Asn Asn Leu Ser Ser Arg Val Ala His Ala Glu His Tyr Ala Arg Glu     290 295 300 Ala Val Ser Arg Gly Ile Ala Val Phe Trp Trp Asp Asn Gly Tyr Tyr 305 310 315 320 Asn Pro Gly Asp Ala Glu Thr Tyr Ala Leu Leu Asn Arg Lys Thr Leu                 325 330 335 Ser Trp Tyr Tyr Pro Glu Ile Val Gln Ala Leu Met Arg Gly Ala Gly             340 345 350 Val Glu Pro Leu Val Ser Pro Thr Pro Thr Pro Thr Leu Met Pro Thr         355 360 365 Pro Ser Pro Thr Val Thr Ala Asn Ile Leu Tyr Gly Asp Val Asn Gly     370 375 380 Asp Gly Lys Ile Asn Ser Thr Asp Cys Thr Met Leu Lys Arg Tyr Ile 385 390 395 400 Leu Arg Gly Ile Glu Glu Phe Pro Ser Pro Ser Gly Ile Ile Ala Ala                 405 410 415 Asp Val Asn Ala Asp Leu Lys Ile Asn Ser Thr Asp Leu Val Leu Met             420 425 430 Lys Lys Tyr Leu Leu Arg Ser Ile         435 440 <210> 2 <211> 859 <212> PRT <213> Saccharomycopsis sp. <400> 2 Val Pro Ile Gln Asn Tyr Thr Gln Ser Pro Ser Gln Arg Asp Glu Ser   1 5 10 15 Ser Gln Trp Val Ser Pro His Tyr Tyr Pro Thr Pro Gln Gly Gly Arg              20 25 30 Leu Gln Asp Val Trp Gln Glu Ala Tyr Ala Arg Ala Lys Ala Ile Val          35 40 45 Gly Gln Met Thr Ile Val Glu Lys Val Asn Leu Thr Thr Gly Thr Gly      50 55 60 Trp Gln Leu Asp Pro Cys Val Gly Asn Thr Gly Ser Val Pro Arg Phe  65 70 75 80 Gly Ile Pro Asn Leu Cys Leu Gln Asp Gly Pro Leu Gly Val Arg Phe                  85 90 95 Ala Asp Phe Val Thr Gly Tyr Pro Ser Gly Leu Ala Thr Gly Ala Thr             100 105 110 Phe Asn Lys Asp Leu Phe Leu Gln Arg Gly Gln Ala Leu Gly His Glu         115 120 125 Phe Asn Ser Lys Gly Val His Ile Ala Leu Gly Pro Ala Val Gly Pro     130 135 140 Leu Gly Val Lys Ala Arg Gly Gly Arg Asn Phe Glu Ala Phe Gly Ser 145 150 155 160 Asp Pro Tyr Leu Gln Gly Thr Ala Ala Ala Ala Thr Ile Lys Gly Leu                 165 170 175 Gln Glu Asn Asn Val Met Ala Cys Val Lys His Phe Ile Gly Asn Glu             180 185 190 Gln Glu Lys Tyr Arg Gln Pro Asp Asp Ile Asn Pro Ala Thr Asn Gln         195 200 205 Thr Thr Lys Glu Ala Ile Ser Ala Asn Ile Pro Asp Arg Ala Met His     210 215 220 Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ser Val Arg Ala Gly Val Gly 225 230 235 240 Ser Val Met Cys Ser Tyr Asn Arg Val Asn Asn Thr Tyr Ala Cys Glu                 245 250 255 Asn Ser Tyr Met Met Asn His Leu Leu Lys Glu Glu Leu Gly Phe Gln             260 265 270 Gly Phe Val Val Ser Asp Trp Gly Ala Gln Leu Ser Gly Val Tyr Ser         275 280 285 Ala Ile Ser Gly Leu Asp Met Ser Met Pro Gly Glu Val Tyr Gly Glu     290 295 300 Trp Asn Thr Gly Thr Ser Phe Trp Gly Gln Asn Leu Thr Lys Ala Ile 305 310 315 320 Tyr Asn Glu Thr Val Pro Ile Glu Arg Leu Asp Asp Met Ala Thr Arg                 325 330 335 Ile Leu Ala Ala Leu Tyr Ala Thr Asn Ser Phe Pro Thr Glu Asp His             340 345 350 Leu Pro Asn Phe Ser Ser Trp Thr Thr Lys Glu Tyr Gly Asn Lys Tyr         355 360 365 Tyr Ala Asp Asn Thr Thr Glu Ile Val Lys Val Asn Tyr Asn Val Asp     370 375 380 Pro Ser Asn Asp Phe Thr Glu Asp Thr Ala Leu Lys Val Ala Glu Glu 385 390 395 400 Ser Ile Val Leu Leu Lys Asn Glu Asn Asn Thr Leu Pro Ile Ser Pro                 405 410 415 Glu Lys Ala Lys Arg Leu Leu Leu Ser Gly Ile Ala Ala Gly Pro Asp             420 425 430 Pro Ile Gly Tyr Gln Cys Glu Asp Gln Ser Cys Thr Asn Gly Ala Leu         435 440 445 Phe Gln Gly Trp Gly Ser Gly Ser Val Gly Ser Pro Lys Tyr Gln Val     450 455 460 Thr Pro Phe Glu Glu Ile Ser Tyr Leu Ala Arg Lys Asn Lys Met Gln 465 470 475 480 Phe Asp Tyr Ile Arg Glu Ser Tyr Asp Leu Ala Gln Val Thr Lys Val                 485 490 495 Ala Ser Asp Ala His Leu Ser Ile Val Val Val Ser Ala Ala Ser Gly             500 505 510 Glu Gly Tyr Ile Thr Val Asp Gly Asn Gln Gly Asp Arg Lys Asn Leu         515 520 525 Thr Leu Trp Asn Asn Gly Asp Lys Leu Ile Glu Thr Val Ala Glu Asn     530 535 540 Cys Ala Asn Thr Val Val Val Val Thr Ser Thr Gly Gln Ile Asn Phe 545 550 555 560 Glu Gly Phe Ala Asp His Pro Asn Val Thr Ala Ile Val Trp Ala Gly                 565 570 575 Pro Leu Gly Asp Arg Ser Gly Thr Ala Ile Ala Asn Ile Leu Phe Gly             580 585 590 Lys Ala Asn Pro Ser Gly His Leu Pro Phe Thr Ile Ala Lys Thr Asp         595 600 605 Asp Asp Tyr Ile Pro Ile Glu Thr Tyr Ser Pro Ser Ser Gly Glu Pro     610 615 620 Glu Asp Asn His Leu Val Glu Asn Asp Leu Leu Val Asp Tyr Arg Tyr 625 630 635 640 Phe Glu Glu Lys Asn Ile Glu Pro Arg Tyr Ala Phe Gly Tyr Gly Leu                 645 650 655 Ser Tyr Asn Glu Tyr Glu Val Ser Asn Ala Lys Val Ser Ala Ala Lys             660 665 670 Lys Val Asp Glu Glu Leu Pro Glu Pro Ala Thr Tyr Leu Ser Glu Phe         675 680 685 Ser Tyr Gln Asn Ala Lys Asp Ser Lys Asn Pro Ser Asp Ala Phe Ala     690 695 700 Pro Ala Asp Leu Asn Arg Val Asn Glu Tyr Leu Tyr Pro Tyr Leu Asp 705 710 715 720 Ser Asn Val Thr Leu Lys Asp Gly Asn Tyr Glu Tyr Pro Asp Gly Tyr                 725 730 735 Ser Thr Glu Gln Arg Thr Thr Pro Ile Gln Pro Gly Gly Gly Leu Gly             740 745 750 Gly Asn Asp Ala Leu Trp Glu Val Ala Tyr Lys Val Glu Val Asp Val         755 760 765 Gln Asn Leu Gly Asn Ser Thr Asp Lys Phe Val Pro Gln Leu Tyr Leu     770 775 780 Lys His Pro Glu Asp Gly Lys Phe Glu Thr Pro Ile Gln Leu Arg Gly 785 790 795 800 Phe Glu Lys Val Glu Leu Ser Pro Gly Glu Lys Lys Thr Val Glu Phe                 805 810 815 Glu Leu Leu Arg Arg Asp Leu Ser Val Trp Asp Thr Thr Arg Gln Ser             820 825 830 Trp Ile Val Glu Ser Gly Thr Tyr Glu Ala Leu Ile Gly Val Ala Val         835 840 845 Asn Asp Ile Lys Thr Ser Val Leu Phe Thr Ile     850 855 <210> 3 <211> 1320 <212> DNA <213> Clostridium sp. <400> 3 tcgggaacaa agcttttgga tgcaagcgga aacgagcttg taatgagggg catgcgtgat 60 atttcagcaa tagatttggt taaagaaata aaaatcggat ggaatttggg aaatactttg 120 gatgctccta cagagactgc ctggggaaat ccaaggacaa ccaaggcaat gatagaaaag 180 gtaagggaaa tgggctttaa tgccgtcaga gtgcctgtta cctgggatac gcacatcgga 240 cctgctccgg actataaaat tgacgaagca tggctgaaca gagttgagga agtggtaaac 300 tatgttcttg actgcggtat gtacgcgatc ataaatcttc accatgacaa tacatggatt 360 atacctacat atgccaatga gcaaaggagt aaagaaaaac ttgtaaaagt ttgggaacaa 420 atagcaaccc gttttaaaga ttatgacgac catttgttgt ttgagacaat gaacgaaccg 480 agagaagtag gttcacctat ggaatggatg ggcggaacgt atgaaaaccg agatgtgata 540 aacagattta atttggcggt tgttaatacc atcagagcaa gcggcggaaa taacgataaa 600 agattcatac tggttccgac caatgcggca accggcctgg atgttgcatt aaacgacctt 660 gtcattccga acaatgacag cagagtcata gtatccatac atgcttattc accgtatttc 720 tttgctatgg atgtcaacgg aacttcatat tggggaagtg actatgacaa ggcttctctt 780 acaagtgaac ttgatgctat ttacaacaga tttgtgaaaa acggaagggc tgtaattatc 840 ggagaattcg gaaccattga caagaacaac ctgtcttcaa gggtggctca tgccgagcac 900 tatgcaagag aagcagtttc aagaggaatt gctgttttct ggtgggataa cggctattac 960 aatccgggtg atgcagagac ttatgcattg ctgaacagaa aaactctctc atggtattat 1020 cctgaaattg tccaggctct tatgagaggt gccggcgttg aacctttagt ttcaccgact 1080 cctacaccta cattaatgcc gaccccctcg cccacggtga cagcaaatat tttgtacggt 1140 gacgtaaacg gggacggaaa aataaattct acagactgta caatgctaaa gagatatatt 1200 ttgcgtggca tagaagaatt cccaagtcct agcggaatta tagccgctga cgtaaatgcg 1260 gatctgaaaa tcaattccac cgacttggta ttgatgaaaa aatatctact gcgctcaata 1320                                                                         1320 <210> 4 <211> 2577 <212> DNA <213> Saccharomycopsis sp. <400> 4 gtcccaattc aaaactatac ccagtctcca tcccagagag atgagagctc ccaatgggtg 60 agcccgcatt attatccaac tccacaaggt ggtaggctcc aagacgtctg gcaagaagca 120 tatgctagag caaaagccat cgttggccag atgactattg ttgaaaaggt caatttgacc 180 actggtaccg gttggcaatt agatccatgt gttggtaata ccggttctgt tccaagattc 240 ggcatcccaa acctttgcct acaagatggg ccattgggtg ttcgattcgc tgactttgtt 300 actggctatc catccggtct tgctactggt gcaacgttca ataaggattt gtttcttcaa 360 agaggtcaag ctctcggtca tgagttcaac agcaaaggtg tacatattgc gttgggccct 420 gctgttggcc cacttggtgt caaagccaga ggtggcagaa atttcgaagc ctttggttcc 480 gacccatatc tccaaggtac tgctgctgct gcaaccatca aaggtctcca agagaataat 540 gttatggctt gtgtcaagca ctttattggt aacgaacaag aaaagtacag acagccagat 600 gacataaacc ctgccaccaa ccaaactact aaagaagcta ttagtgccaa cattccagac 660 agagccatgc atgcgttgta cttgtggcca tttgccgatt cggttcgagc aggtgttggt 720 tctgttatgt gctcttataa cagagtcaac aacacttacg cttgcgaaaa ctcttacatg 780 atgaaccact tgcttaaaga agagttgggt tttcaaggct ttgttgtttc ggactggggt 840 gcacaattaa gtggggttta tagcgctatc tcgggcttag atatgtctat gcctggtgaa 900 gtgtatgggg gatggaacac cggcacgtct ttctggggtc aaaacttgac gaaagctatt 960 tacaatgaga ctgttccgat tgaaagatta gatgatatgg caaccaggat cttggctgct 1020 ttgtatgcta ccaatagttt cccaacagaa gatcaccttc caaatttttc ttcatggaca 1080 acgaaagaat atggcaataa atattatgct gacaacacta ccgagattgt caaagtcaac 1140 tacaatgtgg acccatcaaa tgactttacg gaggacacag ctttgaaggt tgctgaggaa 1200 tctattgtgc ttttaaaaaa tgaaaacaac actttgccaa tttctcccga aaaggctaaa 1260 agattactat tgtcgggtat tgctgcaggc cctgatccga taggttatca gtgtgaagat 1320 caatcttgca caaatggcgc tttgtttcaa ggttggggtt ctggcagtgt tggttctcca 1380 aaatatcaag tcactccatt tgaggaaatt tcttatcttg caagaaaaaa caagatgcaa 1440 tttgattata ttcgggagtc ttacgactta gctcaagtta ctaaagtagc ttccgatgct 1500 catttgtcta tagttgttgt ctctgctgca agcggtgagg gttatataac cgttgacggt 1560 aaccaaggtg acagaaaaaa tctcactttg tggaacaacg gtgataaatt gattgaaaca 1620 gttgctgaaa actgtgccaa tactgttgtt gttgttactt ctactggtca aattaatttt 1680 gaaggctttg ctgatcaccc aaatgttacc gcaattgtct gggccggccc attaggtgac 1740 agatccggga ctgctatcgc caatattctt tttggtaaag cgaacccatc aggtcatctt 1800 ccattcacta ttgctaagac tgacgatgat tacattccaa ttgaaaccta cagtccatcg 1860 agtggtgaac ctgaagacaa ccacttggtt gaaaatgact tgcttgttga ctatagatat 1920 tttgaagaga agaatattga gccaagatac gcatttggtt atggcttgtc ttacaatgag 1980 tatgaagtta gcaatgcaaa ggtctcggca gccaaaaaag ttgatgagga gttgcctgaa 2040 ccagctacct acttatcgga gtttagctat caaaatgcaa aagacagcaa aaatccaagt 2100 gatgcttttg ctccagcaga tttaaacaga gttaatgagt acctttatcc atatttagat 2160 agcaatgtta ccttaaaaga cggaaactat gagtatcctg atggctacag cactgagcaa 2220 agaacaacac ctaaccaacc tgggggcggc ttgggaggca acgatgcttt gtgggaggtc 2280 gcttataact ccactgataa gtttgttcca cagggtaact ccactgataa gtttgttcca 2340 cagttgtatt tgaaacaccc tgaggatggc aagtttgaaa cccctattca attgagaggg 2400 tttgaaaagg ttgagttgtc cccgggtgag aagaagacag ttgatttgag gcttttgaga 2460 agagatctta gtgtgtggga taccaccaga cagtcttgga tcgttgaatc tggtacttat 2520 gaggccttaa ttggcgttgc tgttaatgat atcaagacat ctgtcctgtt tactatt 2577 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcgcggccgc tcgggaacaa agcttttgga 30 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gtgcggccgc tattgctttt tttaagaatg caag 34 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggcgcggccg cgtcccaatt caaaactata cc 32 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggcactagtc gaatagtaaa caggacagat gtct 34  

Claims (11)

Transformed into recombinant vector pαEgEαBG1 incorporating gene encoding endoglucanase from Clostridium sp. And gene encoding betaglucosidase from Saccharomycopsis sp. Converted Saccharomyces cerevisiae KCCM11006P. The method of claim 1, wherein the endoglucanase derived from the genus Clostridium sp. (Saccharomyces cerevisiae KCCM11006P) characterized in that it has the amino acid sequence of SEQ ID NO: 1. 2. The Saccharomyces cerevisiae KCCM11006P of claim 1, wherein the betaglucosidase derived from Saccharomycopsis sp. Has the amino acid sequence of SEQ ID NO: 2. 2. The Saccharomyces cerevisiae KCCM11006P according to claim 1, wherein the gene encoding the endoglucanase derived from the genus Clostridium sp. Has the nucleotide sequence set forth in SEQ ID NO: 3. The method of claim 1, wherein the gene encoding the beta glucosidase derived from the Saccharomycopsis sp. Saccharomyces cerevisiae KCCM11006P characterized in that it has the nucleotide sequence shown in SEQ ID NO: 4. . 2. The Saccharomyces cerevisiae KCCM11006P of claim 1, wherein said recombinant vector has a cleavage map as described in FIG. delete delete delete A method for producing bioethanol, comprising culturing the transformant yeast of claim 1 in the presence of a substrate. The method of claim 10, wherein the substrate is starch or fiber.

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