CN105492612B

CN105492612B - Recombinant cellulose saccharifying enzyme mixture, recombinant yeast composite strain and application thereof

Info

Publication number: CN105492612B
Application number: CN201480048306.2A
Authority: CN
Inventors: 孙廷薰; 成奉炫; 裴贞勋; 李超龙; 金美辰; 金炫辰; 龙焕雄; 林光默
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date: 2013-07-02
Filing date: 2014-07-02
Publication date: 2020-11-03
Anticipated expiration: 2034-07-02
Also published as: KR20150142666A; KR101744670B1; CN105492612A; KR101744190B1; KR20150141180A; KR20150004297A; KR101745479B1; WO2015002471A1

Abstract

The present invention relates to: an expression cassette having an increased ability to secrete a protein in yeast compared with a wild type, an expression vector comprising the expression cassette, a transformant formed by introducing the expression vector into a host cell, and a composite strain comprising two or more transformants, wherein the expression cassette comprises a polynucleotide encoding a Translational Fusion Partner (TFP) and a polynucleotide encoding a protein selected from the group consisting of TrXynII, TrBxl, TrEGL2, PaCEL1, PaCel2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, ClCBH2, and CfCex 1. Also, it relates to a method for producing hemicellulase, endoglucanase or exoglucanase, and a method for producing bioethanol, which comprise the step of culturing the transformant. Further, it relates to a cellulase mixture comprising β -glucosidase, endoglucanase and exoglucanase produced by the above method, and a method for saccharifying a biomass using the mixture. Further, it relates to a method for producing a bioenergy or useful biochemical substance from cellulosic biomass by using two or more cellulase-producing strains singly or in combination.

Description

Recombinant cellulose saccharifying enzyme mixture, recombinant yeast composite strain and application thereof

Technical Field

The present invention relates to: an expression cassette for protein expression, which has an increased secretion ability in yeast as compared with a wild-type protein, an expression vector comprising the expression cassette, a transformant obtained by introducing the expression vector into a host cell, and a composite strain comprising two or more of the transformants, wherein the expression cassette comprises a polynucleotide encoding a Translational Fusion Partner (TFP) and a polynucleotide encoding a protein selected from the group consisting of TrXynII, TrBxl, TrEGL2, PaCEL1, PaCel2, TeCBH1, NfCBH1, HgCBH1, CBCtH 1, ClCBH2 and CfCex 1. Also, the present invention relates to a method for producing hemicellulase, endoglucanase or exoglucanase, and a method for producing bioethanol, which comprise a step for culturing the above-mentioned transformant. Further, the present invention relates to a recombinant cellulase mixture comprising β -glucosidase, endoglucanase and exoglucanase produced by the above method, and a method for saccharification of biomass using the above mixture. Further, the present invention relates to a method for producing a bio-energy source or useful bio-chemical substance from cellulosic biomass at low cost by using a single strain for producing the cellulase or by using two or more strains for producing the cellulase in combination and simplifying the two processes of saccharification and fermentation into a single step.

Background

Worldwide, efforts to easily obtain bio-energy from inexpensive and renewable biomass are being made due to problems of crude oil depletion and global warming. Cellulosic biomass, the most abundant organic matter on earth, is a renewable feedstock capable of producing a variety of energy and feedstock platform compounds produced on the basis of existing petroleum (Hoffert et al, 2002, Science,298,981). At present, the process of obtaining bio-energy (in particular bioethanol) using this cellulosic biomass is technically feasible, but belongs to a high-priced project, with the problem of lack of economy compared to the current price of crude oil (Zaldivar et al, 2001, appl.microbiol.biotechnol.,56, 17).

In order to obtain bioethanol from cellulosic biomass, biomass is decomposed and the decomposed sugars (sugar) are fermented to obtain bioethanol, but since microorganisms present in nature cannot decompose and ferment biomass at the same time efficiently, the prior art has been inefficient in that decomposition and fermentation of biomass are carried out in two separate steps (Lynd et al 2002, microbiol. mol. biol. rev.66, 506). In particular, since cellulosic biomass has a very strong structure and its natural decomposition process is very slow, an expensive pretreatment process and an expensive cellulolytic enzyme treatment process are required to artificially accelerate the decomposition rate (Lynd et al, 1999, biotechnol. prog.15,777, Himmel et al, 2007, Science,315,804).

Therefore, in order to ensure the economy of bioenergy using cellulosic biomass and the production of platform compounds, a low-cost technology for producing cellulolytic enzymes (cellulases) capable of efficiently decomposing cellulosic biomass is required, and in particular, development of a combined bioprocessing (Consolidated bioprocessing) technology (Hahn-Hagerdal et al, 2006, Trends biotechnol, 24,549, Lynd et al, 2008, nat biotechnol, 26,169) capable of directly applying such recombinant enzyme-producing strains to a bioenergy production technology is required.

Cellulosic biomass has a very strong and stable structure by binding with cellulose as a glucose polymer, hemicellulose as a xylose polymer, and lignin (lignin). In order to decompose the plant efficiently by the enzyme, it is necessary to disrupt the stable structure of the plant body by physicochemical pretreatment, to allow the enzyme to approach the substrate, and to require various cellulolytic enzymes depending on the type of substrate. To decompose cellulose as a glucose polymer, endo-1,4- β -D-glucanase (endo-1,4- β -D-glucanase), exo-1,4- β -D-glucanase or cellobiohydrolase (exo-1,4- β -D-glucanase or cellobiohydrolase) and β -glucosidase (β -glucosidase or cellobiase ) are indispensable (Kubicek et al, 1992, adv. biochem. Eng. Biotechnol., 45, 1). In addition, endo-1,4- β -xylanase (endo-1,4- β -xylanase) and β -xylosidase (β -xylosidase) are typically required for the decomposition of hemicellulose, which is a xylose polymer, and various debranching (de-branching) enzymes are required for the complete decomposition. These enzymes are found in microorganisms that naturally corrode plant bodies, particularly molds, and as to commercially produced cellulolytic enzyme complexes, enzyme complexes derived from Trichoderma reesei (Trichoderma reesei) are sold by novacins (Novozymes) and Danisco (Danisco).

Currently, biomass-decomposing enzymes for bioenergy production are monopolized and sold by the above two international enterprises all over the world, but because they are quite expensive and are not optimized according to biomass, they have a problem that excess enzymes are required to be used according to circumstances (Merino and Cherry,2007, adv, biochem, eng, biotechnol.108,95, Kabel et al, 2006, bioeng, biotechnol.93, 56).

Therefore, if recombinant host systems such as bacteria and yeast (yeast) are used to produce each enzyme by recombination and the produced recombinant enzymes are combined and optimized for each biomass type, there is an advantage that the amount of the enzyme used can be reduced. As a host cell for producing such a recombinant enzyme, Saccharomyces cerevisiae (Saccharomyces cerevisiae) is excellent in ethanol fermentation ability, is widely used as an ethanol production strain, and attempts to introduce cellulolytic ability into the strain and many studies for producing recombinant cellulolytic enzymes (Lynd et al, 2002, microbiol.mol.biol.rev.,66,506) have been made.

Thus, although the conventional yeast has very excellent bioethanol fermentation ability, but has no ability to decompose cellulosic biomass at all, there is a problem that when cellulosic biomass, which is a non-grain resource, is used as a raw material to produce bioenergy, expensive cellulolytic enzymes are inevitably used, and in order to solve this problem, numerous recombinant strains into which foreign cellulolytic enzyme genes are introduced have been developed, but there is a problem that the produced enzymes cannot efficiently decompose cellulosic substrates in a culture medium due to low secretion productivity of the enzymes. Thus, the production of bioethanol using the produced sugar (sugar) is limited.

In recent years, although there have been reports of expressing cellulose glycosylase genes discovered from several types of fungi in saccharomyces cerevisiae and confirming the production of bioethanol using microcrystalline cellulose (Avicel), there still remains a problem that the amount of secreted enzymes is insufficient and the ethanol production efficiency is low, and that it is necessary to supply an external enzyme (m.ilmen et al, 2011, Biotechnol biofuels.4:30,2011).

Disclosure of Invention

Technical problem

On the other hand, the present inventors have developed a technique capable of producing various enzymes at high secretion by using the technique owned by the present inventors, i.e., the TFP technique (Korean patent Nos. 10-0626753, 10-0798894, and 10-0975596) which is a yeast protein-customized high-secretion production technique, in order to produce cellulolytic enzymes at high secretion in large amounts by using yeast. That is, a cellulolytic enzyme protein secreted in a large amount in yeast is found from a plurality of fungal genes, and produced by using the optimal TFP, thereby securing an enzyme group necessary for cellulolytic. As the group of enzymes necessary for the above-mentioned cellulose degradation, TrXynII (Trichoderma reesei xylanase II ), TrBxl (Trichoderma reesei beta-xylosidase, Trichoderma reesei beta-xylosidase), as hemicellulase (exoglucanase), PaCel1(Polyporus aculeatus Cel1, Polyporus infundinaceus Cel1), PaCel2(Polyporus aculeatus Cel2), TeCBH1(Talaromyces emersonii CBH1, Emmenomyces emersonii CBH1), NfCBH1 (Neostreatus Scheri CB 1, Fusarium oxysporum CB 1), Huggisrah 1 (Huygroscopicus CB5942), as endophytic fungi CBfungal strains CBF 2, as Trichoderma strain CBF 3655, as Trichoderma strain CBF CGi CGI 3655, Trichoderma reesei 2, as endophytic strains CBF 3655, Trichoderma strain CBF 2, Trichoderma strain CBF 2, Trichoderma reesei 2, Trichoderma strain CBF 2, Trichoderma strain CBZ 2, Trichoderma reesei 2, Trichoderma strain CBZ 2, saccharomycotina yeast BGL2) (Korean patent laid-open No. 10-2013-0027984), KCC (KRIBB cellulose cocktail, KRIBB Cellulase mixture) series, which is a mixture of cellulose biomass saccharifying enzymes, can be produced by artificially mixing each protein, improving the saccharifying efficiency of cellulose biomass, and the saccharifying cost of cellulose can be reduced by producing an optimal customized saccharifying enzyme. Further, the present invention has been completed in the following manner: by using the recombinant yeasts for secreting and producing the cellulolytic enzymes in a combined manner and applying the recombinant yeasts to the production engineering of synchronously saccharifying bioethanol by biomass, saccharification and fermentation of cellulosic biomass are carried out simultaneously, and the production engineering cost of cellulosic bioethanol can be reduced.

Means for solving the problems

The invention aims to provide an expression vector for mass production of hemicellulases TrXynII, TrBxl and endoglucanase TrEGL2, beta-glucosidase SfBGL2, exoglucanase PaCel1, PaCel2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, CfCex1 and ClCBH2 in yeast.

It is still another object of the present invention to provide a transformant transformed with the above expression vector.

Still another object of the present invention is to provide a method for producing a recombinant cellulase, comprising the steps of: the above transformants were cultured, and hemicellulases trxynII, TrBxl, endoglucanase TrEGL2, exoglucanase PaCel1, PaCel2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, ClCBH2, CfCex1 and β -glucosidase SfBGL2 were recovered from the culture or culture supernatant.

It is still another object of the present invention to provide a method for complex culture of a production strain using produced hemicellulase having xylan (xylan) decomposing ability.

It is yet another object of the present invention to mix the cellulases produced in suitable ratios to provide a biomass-customized cellulase mixture.

It is still another object of the present invention to provide a Simultaneous Saccharification (SSF) or Combined Bioprocessing (CBP) bioethanol fermentation process using a complex strain obtained by mixing the above-described transformants.

ADVANTAGEOUS EFFECTS OF INVENTION

The cellulase expression vector of the present invention can be used to produce cellulase in a large amount in Saccharomyces cerevisiae, and the strain can be applied to simultaneous saccharification or combined bioprocessing, and thus can be widely used in industrial processes from lignocellulosic biomass to bioethanol production.

Drawings

FIG. 1 is a schematic diagram showing the Polymerase Chain Reaction (PCR) and intracellular recombination process of a yeast TFP vector for introducing into Pythium infundii, Eimeria, Neosartorya fischeri, Humicola grisea, Chaetomium thermophilum, filamentous fungi, and an exocellulase gene derived from Cellulomonas coprinus, and a hemicellulase and endoglucanase gene derived from Trichoderma reesei.

FIG. 2 is an electrophoresis photograph showing the results of analyzing culture supernatants of 24 transformants obtained by introducing TrXynII (A), TrBxl (B) (FIG. 2a), TrEGL2(C), PaCel1(D) (FIG. 2b), PaCel2(E), TeCBH1(F) (FIG. 2C), NfCBH1(G), HgCBH1(H) (FIG. 2D), CtCBH1(I), ClCBH2(J) or CfCex1(K) (FIG. 2E) into yeast Y2805 strain by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

FIG. 3 is an electrophoretogram showing the results of analysis by SDS-PAGE after treatment of endoglycosidase H (Endo-H) as a sugar chain-removing enzyme for removing the sugar chain of TrXynII (A), TrBxl (B), TrEGL2(C), PaCel1(D) (FIG. 3a), PaCel2(E), TeCBH1(F), NfCBH1(G), HgCBH1(H) (FIG. 3b), CtCBH1(I), ClCBH2(J) or CfCex1(K) (FIG. 3C) proteins expressed in selected transformants.

FIG. 4 shows the results of expression of TrXynII (A) and TrBxl (B) expressed in selected transformants by MF or self-signal (self-signal) and SDS-PAGE analysis and activity analysis for comparison of relative protein expression amounts and activities.

FIG. 5 shows the results of staining TrEGL 2-expressing transformants with Congo-red (congo-red) in YP (2% yeastextract + 2% peptone ) medium containing carboxymethylcellulose (CMC) and comparing endoglucanase activity with the size of the loop.

FIG. 6 is a graph showing the results of pNPC, pNPL and microcrystalline cellulose activity analyses (C, D of FIG. 6B) performed in order to compare the relative protein expression amounts of the finally selected individual transformants of ST13-PaCel1, ST19-TrEGL2, ST13-PaCel1, ST8-TeCBH1, ST13-NfCBH1, ST13-HgCBH1, self signal-CtCBH1 and ST13-ClCBH2 with the transformants having MF or self signal (A, B of FIG. 6 a), and in order to compare the relative activities of the transformants of the individual transformants of ST8-TeCBH1, ST13-NfCBH1, ST13-HgCBH1, self signal-CtCBH1 and ST13-ClCBH2 with the transformants having MF or self signal.

FIG. 7 is a diagram showing expression vectors for genes ST5-TrXynII (A), ST4-TrBxl (B), ST19-TrEGL2(C), ST13-PaCel1(D) (FIG. 7a), ST13-PaCel2(E), ST8-TeCBH (F), ST13-NfCBH1(G), ST13-HgCBH1(H) (FIG. 7b), self-signal-CtCBH 1(I), ST13-ClCBH2(J), and CfCex1(K) (FIG. 7C).

FIG. 8 is a graph showing batch PAGE of Saccharomyces cerevisiae strains transformed by recombinant vectors containing genes ST5-TrXynII (A), ST4-TrBxl (B) (FIG. 8a), ST19-TrEGL2(C), ST13-PaCel1(D) (FIG. 8b), ST13-PaCel2(E), ST8-TeCBH (F) (FIG. 8C), ST13-NfCBH1(G), ST13-HgCBH1(H) (FIG. 8D), self-signal (self-signal) -CtCBH1(I), ST13-ClCBH2(J), ST19-SfBGL2(K), and CCex 1(L) (FIG. 8E) in a 5L fermentor, and electrophoresis of the results of analysis of the culture medium taken over time using SDS-and showing the results of the enzyme activity and the results of each cell concentration and the results of the time of the measurement.

FIG. 9 shows the results of comparing the cell growth and enzyme activity of each finally selected transformant ST5-TrXynII, ST4-TrBxl in the case of multiplex culture (A in FIG. 9 a) with those in the case of single culture and in the case of multiplex culture, and analyzing the medium of the xylanase-producing strain and the substances in the medium in the case of multiplex culture by HPLC (high performance liquid chromatography) (B in FIG. 9B).

FIG. 10 is a result of comparing the growth of recombinant composite strains KCC-1 and KCC-2 according to carbon sources (A) and analyzing secreted proteins after the culture of the composite strains using SDS-PAGE (B).

FIG. 11 shows the results of reaction for 144 hours for comparison of saccharification efficiency of complex strains KCC-1 and KCC-2 at 50 ℃ and confirmation of the produced glucose by HPLC analysis.

FIG. 12 shows the results of ethanol fermentation using 10% pretreated straw as a substrate and adding the complex strain KCC-2 and a control strain (Y2805. delta. gal80/CYH) after saccharification with cellulase in an amount of 2FPU/g, 5FPU/g and 10 FPU/g.

FIG. 13 shows the results of ethanol fermentation using pretreated straws 3% and 5% as substrates and adding complex strain KCC-3 and control strain (Y2805. delta. gal80/CYH) to cellulase at a mass of 2FPU/g and 5 FPU/g.

FIG. 14 shows the results of ethanol fermentation using 6% of straw pretreated in a 5L fermentor as a substrate, and adding complex strains KCC-3 (ST 19-BGL, ST19-EGL2, self signal-CtCBH1, ST13-ClCBH2 in Y2805. DELTA. gal 80) and a control strain (Y2805. DELTA. gal80/CYH) to cellulase of 2FPU/g cellulose.

Detailed Description

As an embodiment, the invention provides recombinant hemicellulases trxyni, TrBxl and recombinant exoglucanases PaCel1, PaCel2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, ClCBH2, CfCex1, recombinant endoglucanase TrEGL2, recombinant β -glucosidase SfBGL 2.

Preferably, the hemicellulases trxyni, TrBxl and endoglucanase TrEGL2 of the invention may be derived from Trichoderma reesei (Trichoderma reesei), the exoglucanases PaCel1, PaCel2 may be derived from Trichoderma funnelosum (polyporussarcocarpus), and the β -glucosidase SfBGL2 may be derived from saccharomyces cerevisiae (saccharomyces cerevisiae). In addition, the exoglucanases HgCBH1, TeCBH1, NfCBH1, CtCBH1, ClCBH2, CfCe x1 may be derived in their turn from Humicola grisea, Talaromyces emersonii, Neosartorya fischeri, Chaetomium thermophilum, filamentous fungi (Chrysosporium lucknowense) and Cellulomonas faecalis (Cellulomonas fimi).

In one embodiment of the invention, the genes were amplified from Trichoderma reesei (Trichoderma reesei) on the basis of the sequence information TrXynII, TrBxl, TrEGL2 (example 1), the exoglucanase PaCel1, PaCel2 genes were amplified from plasmids pCEL1 and pCEL2 containing the two exocellulase genes Cel1 and Cel2 of Polyporus infusorianus (Polyporus lactis), and the exoglucanase HgH 4, TegH 8295, TegH 4835, Clostrich 1, the sequence information of the genes was optimized by means of the codon synthesis of the genes CBCbyf 5, CBcfh 1, CB5, and CBF 466 from Humicola grisea, Talaromyces emersonii, Neosartorya fistulosa (Neosartorya fistulosa). The CfCex1 gene from Cellulomonas faecalis (Cellulomonas fimi) was amplified from KCTC1436 strain (example 1). Genes encoding trxynil represented by

sequence numbers

1 and 2, TrBxl represented by

sequence numbers

3 and 4, TrEGL2 represented by

sequence numbers

5 and 6, PaCel1 represented by

sequence numbers

7 and 8, PaCel2 represented by

sequence numbers

9 and 10, TeCBH1 represented by

sequence numbers

11 and 12, NfCBH1 represented by

sequence numbers

13 and 14, HgCBH1 represented by

sequence numbers

15 and 16, CtCBH1 represented by

sequence numbers

17 and 18, ClCBH2 represented by

sequence numbers

19 and 20, and CfCex1 represented by

sequence numbers

21 and 22 were cloned (example 2), and genes encoding the above-mentioned hemicellulase, endoglucanase, and exoglucanase were expressed by introducing strains into saccharomyces cerevisiae, and as a result, extracellular secretion of each enzyme was confirmed (example 2).

As a further embodiment, the present invention provides an expression cassette for expression of a protein having an increased secretion ability in yeast as compared to a wild-type protein, the expression cassette comprising a polynucleotide encoding a Translational Fusion Partner (TFP) and a polynucleotide encoding a protein selected from the group consisting of trxynil, TrBxl, TrEGL2, PaCEL1, PaCEL2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, ClCBH2, and CfCex 1.

In the expression cassette of the present invention,

i) in the case where trxynil is selected as the protein, the translational fusion partner may be selected from the group consisting of TFP5 having an amino acid sequence of sequence No. 53, TFP9 having an amino acid sequence of sequence No. 57, TFP13 having an amino acid sequence of sequence No. 61, and TFP19 having an amino acid sequence of sequence No. 67;

ii) in the case where TrBxl is selected as the protein, the translational fusion partner may be TFP4 having the amino acid sequence of seq id No. 52;

iii) in the case where TrEGL2 is selected as the protein, the translational fusion partner may be TFP19 having an amino acid sequence of SEQ ID NO. 67;

iv) in the case where PaCEL1 is selected as the protein, the translational fusion partner may be TFP13 having the amino acid sequence of SEQ ID NO. 61;

v) in the case of selecting PaCel2 as the protein, the translational fusion partner may be TFP13 having the amino acid sequence of SEQ ID NO. 61;

vi) in the case of selecting TeCBH1 as the protein, the translational fusion partner may be selected from TFP4 having an amino acid sequence of seq id No. 52, TFP6 having an amino acid sequence of seq id No. 54, TFP7 having an amino acid sequence of seq id No. 55, and TFP8 having an amino acid sequence of seq id No. 56;

vii) in the case where NfCBH1 is selected as the protein, the translational fusion partner may be TFP13 having the amino acid sequence of seq id No. 61;

viii) in the case where HgCBH1 is selected as the protein, the translational fusion partner may be TFP13 having the amino acid sequence of seq id No. 61;

ix) in the case where CtCBH1 is selected as the protein, the translational fusion partner may be selected from TFP7 having an amino acid sequence of sequence No. 55, TFP19 having an amino acid sequence of sequence No. 67, a self-signal having a base sequence of sequence No. 47, and a self-signal having an amino acid sequence of sequence No. 48;

x) in the case where ClCBH is selected as the protein, the translational fusion partner may be one of TFP8 having an amino acid sequence of sequence No. 56 or TFP13 having an amino acid sequence of sequence No. 61; and

xi) in the case where CfCex1 is selected as the protein, the translational fusion partner may be one of TFP8 having an amino acid sequence of sequence No. 56, TFP11 having an amino acid sequence of sequence No. 59, TFP13 having an amino acid sequence of sequence No. 61, or TFP19 having an amino acid sequence of sequence No. 67.

The term, i.e., a translational fusion partner in the present invention refers to a peptide that induces secretory production of a protein having a low expression rate by fusion with a gene encoding the protein having a low expression rate, and a gene encoding the peptide, and translational fusion partners usable in the present invention are disclosed in korean patent nos. 10-0626753, 10-0798894, and 10-0975596.

In particular, it may be a translational fusion partner selected from the group consisting of SEQ ID Nos. 49 to 72, which may be represented by TFP1 to TFP24 in the present invention (Table 3).

In a specific example of the present invention, in order to express hemicellulase, endoglucanase, exoglucanase and β -glucosidase, the expression of each enzyme was compared and analyzed using the above-mentioned 24 TFPs in a vector derived from YEp352 as a general yeast expression vector having a GAL10 promoter and GAL7 terminator. As a result, as each fusion partner, when TFP5, TFP9, TFP13 and TFP19 were selected in the case of trxyniii, TFP4 was selected in the case of Bxl, TFP2 was selected in the case of TrEGL2, TFP2 was selected in the case of PaCel2, TFP2 and TFP2 were selected in the case of TeCBH 2, TFP2 and its own signal peptide were selected in the case of NfCBH 2, TFP2 and TFP2 were selected in the case of HgCBH 2, TFP2 and its own signal peptide were selected in the case of ClCBH2, tfc 2, TFP 363, TFP 36. Reflecting this, as representative fusion partners, an expression cassette was prepared by selecting TFP5 in the case of trxyniii, TFP4 in the case of Bxl, TFP19 in the case of TrEGL2, TFP13 in the case of PaCel1, TFP13 in the case of PaCel2, TFP8 in the case of TeCBH1, TFP13 in the case of NfCBH1, TFP13 in the case of HgCBH1, self-signal peptide in the case of CtCBH1, TFP13 in the case of ClCBH2, and TFP13 in the case of CfCex1, and an expression vector was prepared using the expression cassette.

In another embodiment, the present invention provides an expression vector comprising the above-described expression cassette.

The term vector in the present invention refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of plasmid as a vector is a circular double-stranded DNA loop that enables the additional ligation of a DNA block into the plasmid.

The vector of the present invention can indicate the expression of a gene encoding a protein of interest operably linked, and such a vector is referred to as an expression vector. Generally, in the use of recombinant DNA technology, expression vectors are in the form of plasmids.

The type of expression vector that can be used can be determined depending on the host cell, and when yeast is used as the host, for example, YEpl3 vector, YCp50 vector, pRS-based vector, pYEX-based vector, and the like can be used as the expression vector. Examples of the promoter include GAL promoter and AOD promoter.

As a Method for introducing the recombinant DNA into yeast, for example, electroporation (Method enzymol.,194, 182-.

The expression vector may further contain an expression-suppressing fragment having various functions for suppressing, amplifying or inducing expression of a target gene, a marker for selecting a transformant or a resistance gene to an antibiotic, a gene encoding a signal for the purpose of secretion to the outside of the bacterial cell, a customized fusion partner suitable for a protein difficult to express, and the like. In particular, as a customized fusion partner suitable for the above-mentioned hardly-expressible protein, a Translation Fusion Partner (TFP) is preferably used.

In still another embodiment, the present invention provides a transformant obtained by introducing the expression vector of the present invention into a host cell.

The term "transformation" in the present invention means that DNA can be replicated by introducing the DNA into a host cell and utilizing an element other than a chromosome or by accomplishing chromosome integration.

The host cell used in the transformation according to the present invention may be any one of those well known in the art, and hosts having high introduction efficiency and expression efficiency of the hemicellulase, cellulase and β -glucosidase genes of the present invention may be used, and may include, for example, bacterial, fungal, yeast, plant or animal (e.g., mammalian or insect) cells.

Preferably, the above host cell may use a cell having ethanol fermentation ability, and more preferably, the cell having ethanol fermentation ability may be Zymomonas, yeast and Bacillus. Even more preferably, the cell having the above-mentioned ethanol fermentation ability may be Y2805(Mat a pep4:: HIS3 prb1can 1HIS3-200ura3-52) strain.

The yeast includes, but is not particularly limited to: candida (Candida), Debaryomyces (Debaryomyces), Hansenula (Hansenula), Kluyveromyces (Kluyveromyces), Pichia (Pichia), Schizosaccharomyces (Schizosaccharomyces), Yarrowia (Yarrowia), Saccharomyces (Saccharomyces), Saccharomyces mulberrylides (Saccharomyces), Schizosaccharomyces (Schwanniomyces), and Arxula species, more preferably Candida utilis (Candida utilis), Candida albicans (Candida albicans), Kluyveromyces lactis (Kluyveromyces lactis), Pichia pastoris (Pichia pastoris), Pichia stipitis (Pichia stipitis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), yeast strain of Saccharomyces cerevisiae, etc., Saccharomyces cerevisiae, yeast strain of the same species, yeast strain (Saccharomyces cerevisiae, and yeast strain (Saccharomyces cerevisiae, preferably, Saccharomyces cerevisiae, and yeast strain, Saccharomyces cerevisiae, and yeast strain, Saccharomyces cerevisiae, and yeast strain, such as a strain, yeast strain, and yeast strain, saccharomyces cerevisiae and Saccharomyces cerevisiae.

In one example of the present invention, it was confirmed that each enzyme was produced from the above transformant after Saccharomyces cerevisiae was transformed into Y2805(Mat apep 4:: HIS3 prb1can 1HIS3-200ura3-52) strain using an expression vector comprising the polynucleotides encoding hemicellulase, endoglucanase and exoglucanase of the present invention (example 2).

As still another embodiment, the present invention provides a method for producing a hemicellulase, an endoglucanase, an exoglucanase, or a β -glucosidase, which comprises a step for culturing the above-described transformant. Preferably, there is provided a method for producing hemicellulase, endoglucanase or exoglucanase, which comprises a step for culturing the above-mentioned transformant.

The medium and culture conditions for culturing the transformant of the present invention can be appropriately selected and utilized according to the host cell. The nutrient medium preferably comprises a carbon source, an inorganic nitrogen source or an organic nitrogen source required for the growth of the host cell. As the carbon source, glucose, dextran, soluble starch, sucrose, methanol and the like are exemplified. As the inorganic nitrogen source or the organic nitrogen source, ammonium salts, nitrates, amino acids, corn steep liquor (corn steep liquor), peptone, casein, bovine extract, soybean meal, potato extract and the like are exemplified. Other nutrients may be contained as required, and for example, inorganic salts such as sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc., vitamins, antibiotics (tetracycline, neomycin, ampicillin, and kanamycin), etc. may be contained. In the culture, the temperature, pH of the medium, culture time, and the like may be appropriately adjusted so as to be suitable for cell growth and mass production of proteins.

One example of the present invention cultured the above-mentioned transformant under the condition of 30YPDG (1% yeast extract, 2% peptone, 1% glucose, 1% galactose). The protein of the above-mentioned transformant was confirmed by SDS-PAGE (Sodium Dodessufacte Polyacyl Amide Gel Electrophoresis) method (FIG. 2).

The step of recovering the above hemicellulase, endoglucanase, exoglucanase and β -glucosidase may be isolated and purified according to a usual biochemical isolation technique or by an immunoaffinity method using an antibody against one part or another of the protein. As another method, purification is performed according to an affinity method using another substance having suitably high affinity for an antibody or such a tag by adding a tag (tag) to a protein sequence. Typical biochemical separation techniques include various kinds of chromatography such as treatment with a protein precipitant (salting-out method), centrifugal separation, ultrasonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like, and proteins with high purity can be usually separated by using these in combination.

As still another embodiment, the present invention provides a method for producing bioethanol, comprising the steps of: culturing the transformant obtained by the transformation; and recovering bioethanol from the culture or culture supernatant obtained by the above steps. In the present invention, the procedure for culturing the transformant is the same as that described above.

The term "bioethanol" in the present invention refers to ethanol obtained by fermentation after conversion of carbohydrates in biomass to glucose (glucose). Unlike fossil fuels, bioethanol is completely free of environmental pollutants and obtains fuel from plants, and thus is a renewable energy source. As a fuel additive for vehicles and the like, biodiesel has been widely used for a long time, and studies on mass production have been continued.

The step of recovering bioethanol in the present invention may use any method widely used in the art. For example, distillation or the like may be used.

As still another embodiment, the present invention provides a complex strain comprising two or more of the above transformants. The two or more types refer to transformants containing two or more types of expression vectors encoding different proteins.

In particular, the complex strain may comprise more than one strain secreting hemicellulase. The above hemicellulase may be endo-xylanase and beta-xylosidase. In particular, the endoxylanase may be TrXynII and the β -xylosidase may be TrBxl. The complex strain of the present invention may be a strain having xylanolytic ability.

In one example of the present invention, in order to confirm whether xylan (xylan) can be used as a carbon source in complex culture of yeast strains for producing endoxylanase and β -xylosidase, two strains, a strain for expressing TrXynII and a strain for expressing TrBxl, were complex cultured. As a result, it was confirmed that xylose could be secured from xylan by the stepwise action of two enzymes produced by complex culture of two strains (example 5).

In yet another embodiment, the present invention provides a cellulase mixture (KCC) comprising β -glucosidase and recovered endoglucanases and exoglucanases. In particular, the endoglucanase may be an endoglucanase, and the exoglucanase may be an exocellulase.

The β -glucosidase may have an amino acid sequence of seq id No. 74. In particular, the enzyme may have an amino acid sequence encoded by the base sequence of SEQ ID NO. 73.

In one embodiment of the present invention, the present inventors compared the decomposition ability of each substrate by using cellulose substrate as a carbon source after fermentation and concentration of exocellulase using an exocellulase secretion-producing yeast developed in the present invention. All cellulase mixtures for activity improvement were prepared by fixing the ratio of exocellulase type 1(CBH1), exocellulase type 2(CBH2), endo cellulase (EGL) and β -glucosidase (BGL) to 3.5: 3.5: 2: 1, the EGL used in the mixture was ST19-TrEGL2 enzyme, and the ST19-SfBGL2 enzyme was used for BGL. ST13-PaCel1 and ST13-PaCel2, which have good activity under the condition of mixing water-insoluble cellulose in EGL, BGL in the above-mentioned ratio, are named KCC-1, ST13-HgCBH1 and ST13-ClCBH2 in EGL, BGL in the above-mentioned ratio, are named KCC-2, self-signals-CtCBH 1 and ST13-ClCBH2 in EGL, BGL in the above-mentioned ratio, are named KCC-3, and ST13-CfCex1 and ST13-ClCBH2 in EGL, BGL in the above-mentioned ratio, are named KCC-4, thereby producing a cellulase mixture.

According to the present invention, there is provided a method for applying simultaneous saccharification or combined bioprocessing by complex culture of a yeast transformant transformed by an expression vector.

In still another embodiment, the present invention provides a cellulase mixture comprising the above hemicellulase. The mixture may comprise an endoxylanase and a beta-xylosidase, and in particular, the endoxylanase may be a trxynil protein and the beta-xylosidase may be TrBxl.

In yet another embodiment, the invention provides a method for saccharification of biomass using the cellulase enzyme mixture of the invention. The biomass in the present invention may be cellulose, and may also be xylan.

In one embodiment of the present invention, it was confirmed that cellulose can be saccharified with a cellulase mixture containing the β -glucosidase and the recovered endoglucanases and exoglucanases (example 6).

As still another embodiment, the present invention provides a method for simultaneous saccharification or combined bioprocessing by complex culturing of the strain of the present invention.

In a specific example of the present invention, it was confirmed that the four recombinant cellulase secretion-producing yeast strain complexes KCC-1 and KCC-2 can be used as a composite strain capable of directly producing bioethanol from biomass (example 7), and it was confirmed that ethanol can be produced from biomass by simultaneous saccharification engineering using the minimum amount of commercial cellulase in the case of applying the KCC3 composite strain to ethanol production (example 8).

The present invention relates to a method for producing a mixture of cellulose saccharifying enzymes using recombinantly produced hemicellulases and cellulases and simultaneously saccharifying the mixture by effectively using a recombinant yeast complex strain for producing each enzyme, and more particularly, to the following: trxynil and TrBxl were used as the hemicellulases, PaCel1, PaCel2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, ClCBH2, and CfCex1 were used as exoglucanases, TrEGL2 was used as an endoglucanase, and SfBGL2 was used as a beta-glucosidase, and recombinant secretion production was performed, and the secreted proteins were artificially mixed to produce a cellulose biomass-customized glucoamylase mixture, thereby improving saccharification efficiency of cellulose biomass and producing optimal glucoamylase, and thus cellulose saccharification cost could be reduced. In addition, the recombinant yeast strain secreting and producing cellulolytic enzyme is used as a composite strain and applied to the production engineering of biomass simultaneous saccharification bioethanol, thereby improving the biomass saccharification and fermentation efficiency and reducing the production engineering cost of bioethanol.

The present invention will be described in more detail below with reference to examples. However, these examples are intended to schematically illustrate the present invention, and therefore the scope of the present invention is not limited to these examples.

Example 1: using strains and experimental materials

Ensuring yeast-based hemicellulase, endoglucanase, exoglucanase and beta-glucosidase genes

In order to effectively utilize endoxylanase (endo-xylanase) trxynil (SEQ ID NOS: 1, 2), beta-xylosidase (beta-xylosidase) TrBxl (SEQ ID NOS: 3, 4) and endoglucanase (endo-xylosidase) TrEGL2 (SEQ ID NOS: 5, 6) genes derived from Trichoderma reesei (Trichoderma reesei), the base sequence information of the National Center for Biotechnology Information (NCBI) was utilized. With respect to Trichoderma reesei, a strain distributed from a center of biological resources (KCTC 696) was cultured at 25 ℃ for 5 days in Potato dextrose agar medium (Difco), and after filtration and recovery of only the completely cultured mold mycelia, T.H.Al-Samarrai mold genomic DNA extraction method (T.H.Al-Sammari et al, 2000, Letters in Applied Microbiology, 30, 53-56) was used.

For cloning of TrXynII gene, polymerase chain reaction was performed using the extracted genomic DNA sample 1 as a template (5 minutes 1 at 94 ℃, 94 ℃ C., 30 seconds at 55 ℃ C., and 3 minutes 25 at 72 ℃ C., and 7 minutes 1 at 72 ℃ C.) using the forward primer Trxyn _ F (SEQ ID NO: 23) and the backward primer Trxyn _ R (SEQ ID NO: 24), and for cloning of TrBxl gene, polymerase chain reaction was performed using the extracted genomic DNA sample 1 as a template (5 minutes 1 at 94 ℃, 94 ℃ C., 30 seconds at 55 ℃ C., and 30 seconds 25 at 72 ℃ C., and 7 minutes 1 at 72 ℃ C.) using the forward primer Trbxl _ F (SEQ ID NO: 25) and the backward primer Trbxl _ R (SEQ ID NO: 26) and a section of about 2.4kb and about 600bp was recovered by agarose gel electrophoresis.

To clone the TrEGL2 gene, the distributed trichoderma reesei strain was cultured at 150rpm for 3 days at 25 ℃ in a medium in which 1% carboxymethylcellulose (CMC) was added to YM (Difco, medium), and total RNA was isolated using a total RNA extraction kit (RNeasy plantanmi kit, Qiagen) after filtering and recovering only the completely cultured mold mycelia. Polyadenylated mRNA was isolated from the total RNA isolated using an mRNA purification Kit (Oligotex mRNA Mini Kit, Qiagen). The mRNA isolated as described above was amplified by RACE using SMARTERTM RACE cDNA amplification kit (Clontech). Using the synthesized cDNA sample 1 as a template, a polymerase chain reaction (5 minutes at 94 ℃ C. 1 time; 94 ℃ C. 30 seconds, 55 ℃ C. 30 seconds and 72 ℃ C. 1 minute at 30 seconds 25 times; 7 minutes at 72 ℃ C. 1 time) was carried out using the upstream primer TrEGL2_ F (SEQ ID NO: 27) and the downstream primer TrEGL2_ R (SEQ ID NO: 28), and a section of about 1200bp was recovered by agarose gel electrophoresis. The PCR products were cloned into pGEM-T Easy vector (Promega) and the nucleotide sequences were analyzed, and as a result, the sequences of TrXynII, TrBxl, and TrEGL2 genes (SEQ ID NOS: 1, 3, and 5) were confirmed.

[ Table 1]

Primer and method for producing the same	Sequence of	Serial number
			Trxyn_F	5'-CTCGCCTTAGATAAAAGATCTTGCCGTCCCGCCGCC-3'	23
Trxyn_R	5'-CACTCCGTTCAAGTCGACTTAGCTGACGGTGATGGA-3'	24
			Trbxl_F	5'-CTCGCCTTAGATAAAAGACAGAACAATCAAACATAC-3'	25
Trbxl_R	5'-CACTCCGTTCAAGTCGACTTATGCGTCAGGTGTAGC-3'	26
			TrEGL2_F	5'-CTCGCCTTAGATAAAAGACAGCAGACTGTCTGGGGC-3'	27
TrEGL2_R	5'-CACTCCGTTCAAGTCGACCTTTCTTGCGAGACACGAG-3'	28

In order to use NCBI base sequence information to apply PaCel1 (SEQ ID NOS: 7, 8) and PaCel2 (SEQ ID NOS: 9, 10) genes derived from Porphyra funnelorum (Polyporus arcularius), polymerase chain reactions (5 minutes 1 time at 94 ℃, 30 seconds at 55 ℃,1 minutes 30 seconds at 72 ℃ and 25 times at 72 ℃ and 7 minutes 1 time at 72 ℃) were performed using 1. mu.L of the vectors pCEL1 and pCEL2 as templates using upstream/downstream primers Cel1_ F (SEQ ID NO: 29)/Cel1_ R (SEQ ID NO: 30) and Cel2_ F (SEQ ID NO: 31)/Cel2_ R (SEQ ID NO: 32), respectively, and sections of about 1.3kb and about 1.3kb were secured by agarose gel electrophoresis.

For the application of the TeCBH1 (seq id nos. 11, 12), NfCBH1 (seq id nos. 13, 14), HgCBH1 (seq id nos. 15, 16), CtCBH1 (seq id nos. 17, 18) as each CBH1 derived from the molds Talaromyces emersonii, neoseto freudenreichii (Neosartorya fischeri), Humicola grisea (Humicola grisea), chaetomium thermophilum (chaetomium thermophilum), and ClCBH2 (seq id nos. 19, 20) as CBH2 derived from filamentous fungi (Chrysosporium lucknowense), base sequence information of NCBI was used. Five kinds of exoglucanase genes were artificially synthesized by the company Bai, Bioneer through the codon optimization process of yeast. Using each of TeCBH1, NfCBH1, HgCBH1, CtCBH1, and ClCBH2 synthetic gene samples contained in pGEM-T Easy vectors as a template, polymerase chain reactions (5 minutes 1 at 94 ℃, 30 seconds at 55 ℃,1 minutes 30 seconds at 72 ℃, and 1 minute 25 at 72 ℃ for 25 times; 7 minutes 1 at 72 ℃) were carried out using each of the upstream/downstream primers CR244 (SEQ ID NO: 33)/CR246 (SEQ ID NO: 34), CR247 (SEQ ID NO: 35)/CR249 (SEQ ID NO: 36), CR250 (SEQ ID NO: 37)/CR252 (SEQ ID NO: 38), CR253 (SEQ ID NO: 39)/CR255 (SEQ ID NO: 40), and CR256 (SEQ ID NO: 41)/CR258 (SEQ ID NO: 42), and sections of about 1.3kb, about 1.5kb, and about 1.4kb, respectively, were secured by agarose gel electrophoresis. In order to use the gene CfCex1 (SEQ ID NOS: 21, 22) as CBH1 derived from Cellulomonas faecalis (Cellulomonas fimi), based on the NCBI base sequence and using genomic DNA (genomic DNA) of KCTC1436 strain as a template, polymerase chain reaction (5 minutes 1 time at 94 ℃ C.; 30 seconds at 94 ℃ C., 30 seconds at 55 ℃ C., 1 minute 30 seconds at 72 ℃ C., and 1 minute 25 times at 72 ℃ C.; 7 minutes 1 time at 72 ℃ C.) was performed using the upstream/downstream primer CR158 (SEQ ID NO: 43)/CR159 (SEQ ID NO: 44) and a section of about 1.3kb was secured by agarose gel electrophoresis.

Beta-glucosidase derived from Saccharomyces cerevisiae fibuligera was directly expressed and used as ST19-SfBGL2 (Korean patent publication: No. 10-2013-0027984) strain introduced into Y2805. delta. gal80 strain by modifying existing genes.

[ Table 2]

Example 2: hemicellulases, endoglucanases and exo-cellulases by introduction of a Translational Fusion Partner (TFP) Secretory production of endoglucanase

In order to express the two hemicellulase, one endoglucanase and seven exoglucanase genes ensured by example 1 in a s.cerevisiae strain, 24 protein secretion fusion partners (TFP 1 to TFP24, Korean patent No. 10-0975596) which facilitate protein secretion expression were introduced into Y2805(Mat a pep4:: HIS3 prb1can 1HIS3-200ura3-52) strain by in vivo recombination (in vivo recombination) after amplification using primers LNK39 (SEQ ID NO: 45) and GT50R (SEQ ID NO: 46) from the respective genes. The sequences of TFP1 through TFP24 are shown in table 3 below.

[ Table 3]

Since the polymerase chain reaction product amplified using the LNK39 (seq id No. 45) and GT50R (seq id No. 46) primers contains the same sequence as the vector of 40base or more, when the product is introduced into yeast cells together with the linearized vector, the product is crossed inside the cells to generate a circular plasmid vector (fig. 1). Each transformant was selected from among selective medium lacking uracil (0.67% yeast matrix lacking amino acids, 0.77% nutritional supplement lacking uracil, 2% glucose), cultured in YPDG (1% yeast extract, 2% peptone, 1% glucose, 1% galactose) medium for 40 hours, and then subjected to SDS-PAGE analysis after precipitating 0.6ml of the supernatant with 0.4ml of acetone for 2 hours, to thereby screen out strains having a large secretion amount of endoxylanase, β -xylosidase, endoglucanase and exoglucanase at a time (FIGS. 2a to 2 e).

From the results, it was found that strong bands having different protein sizes were observed in the culture supernatants of the cells containing the respective carriers. However, the bands shown on SDS-PAGE have a considerable difference in size from proteins deduced from the size classes of TrXynII, TrBxl, TrEGL2, PaCEL1, PaCel2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, ClCBH2 and CfCex1 genes, which is presumed to be a difference due to the addition of sugar chains since ten, two, one, two, one, three, one and four N-glycosylation-inducing sequences are included in the above-mentioned order in these protein sequences, respectively. Among the 24 transformants, transformants which were estimated to have a size of the hemicellulase, endoglucanase and exoglucanase proteins and which had a high band expression were selected, respectively, and in order to remove sugars added to the proteins by N-glycosylation of the selected transformants, each protein was subjected to Endo-H enzyme treatment, and then, SDS-PAGE (FIGS. 3a to 3c) was performed again. As expected, it was confirmed that the expressed protein was N-glycosylated by confirming that the molecular weight was normal after the Endo-H treatment.

Example 3: hemicellulases, endoglucanases and exo-enzymes by secretory expression in Saccharomyces cerevisiae strains Confirmation of Glucan cleavage Activity

In order to confirm the activity of the protein secreted from each transformant, the activity analysis method for each protein was used as described below.

3-1, confirming endoxylanase activity of secreted TrXynII protein

To confirm the activity of the secreted TrXynII protein, DNS (dinitrosalicylic acid) quantification (Miller, G.L., anal. chem, 55:952-959, 1959) was used. To 100. mu.l of the enzyme solution was added 100. mu.l of a substrate solution (100mM potassium phosphate solution containing 1% oat xylan (opt spelt xylan), pH5.0), reacted at 60 ℃ for 30 minutes, then 700. mu.l of a DNS (3,5-Dinitrosalicylic acid, 3,5-Dinitrosalicylic acid) solution was added and treated at 100 ℃ for 5 minutes, and then, measured at an absorbance of 540nm after cooling with cold water. As a result of analyzing endoxylanase activity (table 4), the activity of TrXynII enzyme expressed by TFP5 was 74.61 units/ml, showing the highest activity. 1 unit (U) of enzyme was set as the amount of enzyme that released 1. mu. mol of reducing sugar in 1 minute.

[ Table 4]

TFP No.	Endoxylanase Activity (units/ml)
		1	58.92
5	74.61
		8	65.74
9	72.63
		13	73.80
19	72.56

3-2. confirmation of the beta-xylosidase Activity of the secreted TrBxl protein

To confirm the activity of the secreted TrBxl protein and to confirm the activity of the secreted protein, 0.1mL of the enzyme solution was reacted with 0.9mL of a base solution (100mM sodium phosphate buffer solution (pH7.0), 1mM of P-Nitrophenyl- β -D-Xylopyranoside (P-Nitrophenyl- β -D-Xylopyranoside; pNPX) at 50 ℃ for 10 minutes, an amount of 0.4M sodium carbonate equivalent to the reaction solution was added and the reaction was stopped, and the absorbance was measured at 405nm, as a result (Table 5), the activity of the TrBxl enzyme expressed by TFP4 was 25.38 units/mL and was the highest.

[ Table 5]

TFP No.	Beta-xylosidase activity (units/ml)
		4	25.38
6	10.77
		8	18.46
9	11.54
		13	9.23
19	11.54

The Saccharomyces cerevisiae strains ST5-TrXynII and ST4-TrBxl, in which the protein expression rate and activity of each transformant were the highest under the control of each fusion partner, were finally selected by comparing the protein expression rate and the activity according to the expression rate, and SDS-PAGE analysis and activity analysis were performed on each transformant in order to compare the relative protein expression amounts and activities of transformants having MF or self-signaling (FIG. 4).

3-3, confirming endoglucanase Activity of the secreted TrEGL2 protein

In order to confirm the activity of the secreted endoglucanase TrEGL2 protein, the transformed cells were cultured in YP (2% yeast extract + 2% peptone) medium containing 2% carboxymethylcellulose (CMC) at 30 ℃ for 3 days, and after staining the plates with 0.1% congo-red (congo-red) for one hour, washed several times with 1M sodium chloride (NaCl), and then the endoglucanase activity in the transformed cells was confirmed by the ring size around the cells (fig. 5), and for quantitative confirmation, a colorimetric method using DNS (3,5-dinitrosalicylic acid) was used. After 50ul of the culture supernatant dilution was added to a reaction solution in which 100mM phosphate buffer solution (pH6.0) and 1% CMC150ul were combined, the reaction was carried out at 45 ℃ for 30 minutes, 700ul of DNS was added thereto, the mixture was boiled for 10 minutes, and then cooled, and the absorbance was measured at 550nm to carry out a colorimetric analysis, the results of which are shown in Table 6. Since the value of reducing sugar at 0 hour of the TFP13-TrEGL2 is higher than that after the reaction and the activity cannot be measured, the TFP19-TrEGL2 shows the highest activity at 0.38 unit/ml. In this case, the enzyme activity was determined to be 1 unit (U) of enzyme capable of producing 1g of reducing sugar in 1 minute.

The Saccharomyces cerevisiae strain ST19-TrEGL2, in which the protein expression rate and the activity of each transformant were the highest under the control of each fusion partner, was finally selected by comparing the protein expression rate and the activity according to the expression rate, and the relative protein expression amounts of the transformants having MF or self-signals were compared with respect to each transformant (A of FIG. 6 a).

[ Table 6]

TFP No.	Endoglucanase Activity (units/ml)
		5	0.17
9	0.22
		13	-
19	0.36

3-4. confirmation of the exoglucanase Activity of the secreted PaCel1 and PaCel2 proteins

In order to confirm the activity of the secreted PaCel1 and PaCel2 exoglucanase proteins, after the transformants were cultured in 3YPDG for 40 hours, the culture supernatant was used for activity confirmation. In 50mM acetic acid buffer solution (pH5.0), 900ul of reaction solution and 100ul of culture supernatant dilution were added at 0.5mg/ml by melting 1mg/ml of pNPC (p-Nitrophenyl-beta-cellobioside ) and D-gluconolactone (D-Glucono-1,5-delta-lactone, D-Glucono-1, 5-lactone), and reacted at 50 ℃ for 30 minutes. When 2% sodium carbonate was added in an amount equivalent to the reaction solution and the absorbance was measured at 410nm, it was confirmed that PaCel1 had no activity on pNPCdp, and only showed a weak activity of 1.7 units/ml in the case of TFP13-PaCel2 (Table 7). At this time, the control group was an untransformed s.cerevisiae strain Y2805. In this case, the enzyme activity was determined to be 1 unit (U) of an enzyme capable of producing 1M nitrophenol in 1 minute.

The Saccharomyces cerevisiae strains ST13-PaCel1 and ST13-PaCel2, in which the protein expression rate and the activity under the control of each fusion partner were the highest, were finally selected for each transformant by comparing the protein expression rate and the activity according to the expression rate, and the relative protein expression amounts of the transformants having MF or self-signals were compared for each transformant (A of FIG. 6 a).

[ Table 7]

3-5 Exo-glucanase Activity assay

To analyze the activity of the remaining 6 exoglucanases, 0.1ml of the enzyme solution was reacted with 0.9ml of a matrix solution (50mM sodium acetate buffer (pH 5.0)) and 1mg/ml of P-Nitrophenyl-beta-D-Cellobioside (P-Nitrophenyl-beta-D-Cellobioside; pNPC) as a water-soluble matrix at 50 ℃ for 10 minutes, 2% sodium carbonate in an amount equivalent to the reaction solution was added and the reaction was stopped, and the absorbance was measured at 410. Further, as a result of activity measurement of p-Nitrophenyl- β -D-lactobioside (pNPL) as another synthetic substrate by the same method as described above, it was confirmed that pNPC and pNPL of STFP13-CfCex1 were 2076.7 units/ml and 210 units/ml, respectively, and showed the highest activity, and the other exocellulases showed a minimal activity of 40 units/ml or less (table 8). In this case, the enzyme activity was determined to be 1 unit (U) of an enzyme capable of producing 1. mu.M nitrophenol in 1 minute.

[ Table 8]

TFP No.	pNPC _ Exo-glucanase Activity (units/ml)	pNPL _ Exo-glucanase Activity (in ml)
			8	1569.8	150.9
11	1183.7	86.3
			13	2076.7	210.0
19	1129.1	107.6

Further, in order to analyze the activity of secreted exo-cellulase using microcrystalline cellulose (Avicel) as a water-insoluble substrate, 500. mu.L of an enzyme solution was reacted with 500. mu.L of a substrate solution (2% microcrystalline cellulose pH-105 cellulose, 0.04% sodium azide, 0.3. mu.L of Novozyme lipase-188 (0.3. mu.L of Novozyme-188), 50mM sodium acetate buffer (pH5.0) at 35 ℃ and 1000rpm for 48 hours, and then the amount of sugar after decomposition was measured using DNS (3,5-dinitrosalicylic acid) at 540 nm.the result of analyzing the activity of microcrystalline cellulose (Table 9) was that ClCBH2 was 66.3 units/ml after 48 hours and the highest activity was exhibited, HgCBH1 was 27 units/ml in the type of exo-glucanase 1 and the highest activity was exhibited, CfCex1, which is the highest in the decomposition activity of the water-soluble substrate, was 1.35 units/ml, showing the lowest activity.

[ Table 9]

Five strains of s.cerevisiae (ST8-TeCBH1, ST13-NfCBH1, ST13-HgCBH1, self-signal sequence-CtCBH 1, ST13-ClCBH2) in which the protein expression rate and activity under the control of each fusion partner were the highest were finally selected for each transformant by comparing the protein expression rate and the activity according to the expression rate, and for each transformant, SDS-PAGE (B of FIG. 6 a) analysis was performed in order to compare the relative protein expression amounts of the transformants having MF or self-signal, and activity analysis was performed by using pNPC, pNPL and microcrystalline cellulose as a substrate (C, D of FIG. 6B) in order to compare the relative activities. As a result, CtCBH1 showed 2.5-fold higher activity than TFP when it used its own signal. Higher secretory expression rates were confirmed in the case where the remaining four exocellulases all utilized the TFP technique.

FIGS. 7a to 7c are schematic diagrams showing expression vectors of hemicellulase, endoglucanase and exoglucanase genes fused to TFPs, respectively. When the GAL10 promoter was used to induce protein expression, an expensive galactose was required for the expression vector containing the recombinant production strain, but when Y2805 Δ GAL80, which is a strain lacking the GAL80 gene, was used, induction of expression of the GAL10 promoter could be achieved only with glucose. Seven cellulase genes finally selected were introduced into Y2805. delta. gal80(Mat a pep4:: HIS3gal 80:: Tc190, prb1can 1HIS3-200ura3-52) strain by in vivo recombination (in vivo recombination) and used as a mass-producing strain.

Example 4: mass production of cellulase genes

In order to mass-produce each of the hemicellulase, endoglucanase, exoglucanase and β -glucosidase in a 5L fermentor by using ST5-TrXynII, ST4-TrBxl, ST19-TrEGL2, ST13-PaCel1, ST13-PaCel2, ST8-TeCBH1, ST13-NfCBH1, ST13-HgCBH1, self-signal-CtCBH 1, ST13-ClCBH2, ST13-CfCex1 producing strain and ST19-SfBGL2 (Korean patent publication: No. 10-2013-0027984), which have been introduced into the above-mentioned Y2805. DELTA. gal80, a batch culture was carried out in a 5L fermentor. Before entering the culture, after an initial culture was performed in 50 mldenb (0.67% yeast substrate lacking amino acids, 0.5% casamino acids, and 2% glucose) medium, the culture was again activated by culturing in 200mL YEPD liquid medium, inoculated into the culture solution, and cultured at 30 ℃ for 48 hours.

FIGS. 8a to 8f are photographs showing the results of the fed-batch fermentation of the strain Saccharomyces cerevisiae Y2805. DELTA. gal80 transformed by recombinant vectors comprising ST5-TrXynII, ST4-TrBxl (FIG. 8a), ST19-TrEGL2, ST13-PaCel1 (FIG. 8b), ST13-PaCel2, ST8-TeCBH1 (FIG. 8c), ST13-NfCBH1, ST13-HgCBH1 (FIG. 8d), selessignal-CtCBH 1, ST13-ClCBH2 (FIG. 8e), ST19-SfBGL2, ST13-CfCex1 (FIG. 8f) in a 5L fermentor, and the results of the analysis of the electrophoretic culture medium extracted over time by SDS-PAGE, and the results of the optical concentration or the enzyme activity per time. The amount of exoglucanase secreted varies depending on the type of enzyme, but secretory production was confirmed to be 1 to 2 g/L. With respect to each cellulase produced by fermentation, each enzyme activity was analyzed by concentration and desalting processes and then frozen and stored, so that hemicellulase was used in a complex strain culture experiment, and the remaining cellulases were used in an activity measurement experiment after preparing a mixture.

Example 5: composite strain culture of recombinant hemicellulase and xylan decomposition capacity

In order to confirm whether xylan (xylan) can be used as a carbon source in the complex culture of yeast strains for producing endoxylanase and β -xylosidase, both strains were complex cultured. For the complex culture, after initial culture in 3mL of a minimum medium (0.67% yeast substrate lacking amino acids, 0.5% casamino acids, 2% glucose), the cells were inoculated into 50mL of YPD medium containing 1% xylan, and cultured at 30 ℃ for 48 hours, and then the cell mass and enzyme activity were measured in terms of time. The endoxylanase activity was determined by DNS method, and the β -xylosidase activity was p-Nitrophenyl- β -D-xylopyranoside (pNPX). As shown in FIG. 9a, it was confirmed that each enzyme was secreted and produced even when the two strains were cultured in combination, and that endoxylanase and β -xylosidase exhibited enzyme activities of 130 units/mL and 100 units/mL at 48 hours of culture.

In order to confirm the xylanolytic activity of the recombinant strains, 2% xylan was added to YPD (yeast extract 1%, peptone 2%, glucose 2%) medium, and the respective strains were separately or compositely cultured to analyze the xylanolytic pattern in the medium (fig. 9B). The pattern of breakdown of xylan was analyzed by taking samples over time and using HPLC (Agilent, RIDedetector, Agilent technologies, Inc., differential refractometer). The HPLC solid phase was analyzed by using HPX-87H sugar column (column) (Biorad) at a temperature of 65 ℃ and a flow rate of 0.6mL on the sugar column and a RID temperature of 55 ℃. In contrast to the production of only xylobiose (xylobiose) and xylotriose (xylotriose) in the medium in which the xylanase producing yeast (TrXYL) was cultured, xylo-oligosaccharide (xylo-oligosaccharide) was produced from xylan due to the produced endo-xylanase in the medium in which complex culture was performed (TrXYL + TrBXL), and the produced xylobiose was not further decomposed (B of fig. 9B). At this time, it was confirmed that xylose, xylobiose and xylotriose were produced by converting undecomposed xylobiose and xylooligosaccharide into xylose by β -xylosidase, and xylan was hardly decomposed in the medium in which the β -xylosidase producing strain was cultured (B in fig. 9B). Thus, HPLC analysis confirmed that a total of 2.7g/L xylose, 0.9g/L xylobiose, 0.3g/L xylotriose, etc. were produced due to the decomposition of xylan by these two enzymes. Therefore, it was confirmed that, in order to use xylan as a single carbon source, xylose was secured from xylan by the stepwise action of two enzymes produced by complex culture of two strains.

Example 6: biomass saccharification mixture manufacture and activity modification

The exocellulase-producing yeast developed by the present invention was used to ferment and concentrate exocellulase, and then cellulose substrates were used as a carbon source, and the decomposition ability of each substrate was compared. All cellulase mixtures for activity improvement were prepared by fixing the ratio of exocellulase type 1(CBH1), exocellulase type 2(CBH2), endo cellulase (EGL) and β -glucosidase (BGL) to 3.5: 3.5: 2: 1 for the analysis, the EGL used in the mixture was ST19-TrEGL2 enzyme, and the BGL used was ST19-SfBGL2 (Korean patent laid-open No. 10-2013-0027984) enzyme.

In addition, to minimize the cost of enzyme purification, the enzymes used in the manufacture of the mixture are used in the following manner: four cellulolytic enzymes produced by fermentation culture were recovered from the fermentation medium, desalted and 10-fold concentrated, and each enzyme was directly added to the mixture composition without purification. ST13-PaCel1 and ST13-PaCel2, which were highly active under the condition of mixing water-insoluble cellulose in the above-described ratio, were mixed in EGL and BGL, and named KCC-1, ST13-HgCBH1 and ST13-ClCBH2, and named KCC-2, and self signals (self signal) -CtCBH1 and ST13-ClCBH2, and named KCC-3, were mixed in the above-described ratio in EGL and BGL, and thus compared in activity with a mixture, as a commercial enzyme, in which 0.5% Novozyme188 (0.5% Novozyme188) (Sigma) was added to cellulase (Celluclast).

The activity of each mixture was compared by an activity measuring method (B.Adney et al, 1996) using a filter paper (filter paper) as a water-insoluble cellulose as a matrix, in which the enzyme activity required for saccharifying 4% of the filter paper matrix per hour was expressed by FPU. Table 10 below shows the results of the activities of preparing a mixture by four main enzymes for decomposing cellulose and decomposing filter paper by FPU per gram of protein, and the results of comparing the relative activities of the mixtures using the pretreated EFB (empty fruit bunch) as a substrate.

[ Table 10]

It was confirmed that when HgCBH1 and ClCBH2(KCC-2) or CtCBH1 and ClCBH2(KCC-3) were added, the activity was improved compared to the original version of KCC-1 mixture (CBH1 and CBH2 derived from Polyporus archaeus). As for the filter paper activity, it was confirmed that KCC-3 was improved 8.8 times as much as KCC-1, KCC-3 was improved 2.65 times as much as KCC-2, and when 0.5% Novozyme188 (0.5% Novozyme188) (BGL) was added to Novozyme cellulase (Celluclast), the activity was 2.62 times as much as KCC-3, and the activity of KCC-3 was slightly higher than before.

Regarding the EFB activity after pretreatment, it was confirmed that KCC-3 was improved about 2.2 times as compared with KCC-1, about 1.5 times as compared with KCC-2, and about 2.7 times as compared with the sample in which 0.5% Novozyme188 (0.5% Novozyme188) was added to cellulase (Celluclast).

The decomposition ability of microcrystalline cellulose (Avicel) matrix was low in the CBH1 type, but the activity was analyzed by mixing the ST13-CfCex1 fermentation concentrate, which had the highest activity among the water-soluble matrices, with EGL, BGL and ST13-ClCBH2 in the above ratio, named KCC-4, and using pretreated biomass EFB as a target. As a result, it was confirmed that KCC-4 had an activity improved by 130% as compared with KCC-3 (table 11), and it was considered that this effect is that the enzyme ST13-CfCex1 added additionally to KCC-4 has not only exo-cellulase activity but also endo-xylanase (endo-xylanase) activity as a hemicellulolytic enzyme, and thus it is predicted that the activity higher than KCC-3 in pretreated biomass is related to xylanase (xylanase) activity.

[ Table 11]

To confirm this effect, ST5-TrXynII and ST4-TrBxl, which were recombinantly mass-produced using yeast, were added to each enzyme mixture and activities of straw pretreated with sodium hydroxide (NaOH) were compared, and as a result, the activity was improved to about 600% when ST5-TrXynII was added, and the activity was improved to about 130% when ST5-TrXynII and ST4-TrBxl were added to KCC-4, which already contains xylanase activity, as compared to the case of adding ST4-TrBxl to KCC-3 (Table 12). Therefore, it is presumed that KCC-4 is affected by hemicellulase in the water-insoluble pretreated biomass and the cellulase activity is low, and therefore, in order to develop an effective cellulase mixture, it is preferable to develop a cellulase mixture containing not only cellulase but also hemicellulase.

[ Table 12]

Example 7: by using cellulolytic saccharidesDevelopment of simultaneous saccharification technology using sexual enhancement composite strain

In order to develop a complex strain that can directly produce bioethanol from biomass using the four recombinant cellulase secretion-producing yeast strain complexes KCC-1 and KCC-2 constituting KCC-1 and KCC-2, small amounts of 1% glucose and 1% microcrystalline cellulose or carboxymethylcellulose were supplied to the culture medium and cultured for 24 hours. Enzymatic saccharification was carried out at 37 ℃ for 3 hours so that the secreted cellulase produced was able to break down microcrystalline cellulose and carboxymethylcellulose and cell growth was compared up to 96 hours with the control strain (Y2805. DELTA. gal80/CYH) (FIG. 10A). It was confirmed that there was no difference in growth between the two strains in the case of supplying glucose as a single carbon source, whereas growth of KCC-1 and KCC-2 was increased as compared with the control group although slightly in the medium in which the glucose concentration was reduced and carboxymethyl cellulose and microcrystalline cellulose were supplied as carbon sources. However, there was no large difference in cell growth of the KCC-1 and KCC-2 strain complexes (FIG. 10A).

In order to analyze the secretion of cellulase from the complex strain in culture, the culture medium was subjected to SDS-PAGE analysis after 96 hours of culture, and it was found that cellulase not identified in the control group was secreted into the culture medium by the complex strains KCC-1 and KCC-2 (FIG. 10B).

Example 8: comparative analysis of cellulose decomposition ability and ethanol fermentation ability of recombinase mixture

In order to compare the temperature-induced enzymatic saccharification efficiencies of the complex strains KCC-1 and KCC-2 secreting four enzymes, after preculture of the four complex strains, the respective strains were treated in a 50mL flask containing microcrystalline cellulose as Exo (Exo): EGL: BGL 7: 2: the ratio of 1 was inoculated to each strain, and cellulase was produced by sufficient secretion through 24 hours of culture. The produced complex enzyme was allowed to react at 37 ℃ and 50 ℃ for 144 hours, and the formed glucose was confirmed by HPLC analysis. Although glucose was not produced at all in the case of the enzyme reaction at 37 ℃ and the efficiency of saccharification was very low at 50 ℃, 0.54g/L glucose was produced in the case of KCC-2 (FIG. 11). Therefore, it was confirmed that saccharification of microcrystalline cellulose (Avicel) as an insoluble cellulose (cellulose) matrix can be achieved by optimizing the composition of the enzyme mixture and the saccharification conditions.

When the yeast complex for secretory production of cellulase developed by the present invention is directly applied to ethanol production to carry out saccharification and fermentation, it is desired to realize low-cost ethanol production by minimizing the amount of cellulase to be separately added for saccharification, and thus studies on actual ethanol fermentation have been made. For ethanol fermentation, four strains of strains ST19-BGL, ST19-EGL2, ST13-HgCBH1 and ST13-ClCBH2, which produce enzymes constituting KCC-2, were first inoculated and cultured, after which the respective cell amounts were adjusted to 1: 1: 4: 4 ratios were mixed and used. With respect to biomass, the solid-to-liquid ratio was adjusted by using 1: 10 of 2% NaOH (sodium hydroxide) was used by pretreating dried straw at 120 ℃ for 1 hour. As the ethanol fermentation conditions, 10% biomass and ethanol medium (yeast extract 0.5%, peptone 0.5%, KH2PO4, ammonium sulfate 0.2%, MgSO 2)₄·H₂O0.04%, ph5.0) was mixed to prepare a fermentation medium, and then commercial cellulase (C-Tec: H-Tec ═ 7: 3) after the contents of the cells were adjusted to 2FPU/g, 5FPU/g and 10FPU/g cellulose (cellulose), respectively, the cells were subjected to initial saccharification at 50 ℃ and 150rpm for 6 hours. After 6 hours of saccharification, the strains cultured by the inoculum were mixed in an appropriate ratio (KCC-2) and inoculated on the culture. As a control fermentation strain, Y2805. delta. gal80/CYH strain (CYH) transformed with a vector not containing a cellulase gene was used, and the control fermentation strain was represented by a graph after sampling with time and confirming the production and consumption of glucose and ethanol by HPLC (FIG. 12).

As a result, although the difference was slight in the case of using 10FPU cellulase to which an enzyme was externally added as compared with the control group, the difference was as large as the ethanol productivity of the control group in the case of using 5FPU cellulase, and particularly, the difference in ethanol productivity was about 3 times or more as large as the control group in the case of using 2FPU cellulase (fig. 12A). Generally, in bioethanol production using cellulosic biomass, although there is a difference in external enzyme input depending on the type of cellulosic biomass in order to maximize productivity, enzyme costs can be significantly reduced as compared with the case of using about 10 to 50FPU/g cellulose (cellulose).

To confirm ethanol produced when commercial cellulase was added to an enzyme-producing complex strain constituting a KCC-3 mixture enzyme excellent in glucoamylase activity, ethanol was purified in Y2805 Δ gal80 as 1: 1: 4: the four strains ST19-BGL, ST19-EGL2, self signal-CtCBH1 and ST13-ClCBH2 were mixed at a ratio of 4 and used. Straw and a fermentation medium were used by being produced by the same method as described above, and the amount of biomass was used at 3% and 5%, whereby cultivation was performed in a flask. After adding commercial cellulase (C-Tec: H-Tec ═ 7: 3) from Novozyme to 2FPU/g and 5FPU/g (glucose content of straw), respectively, 30% (30mL) of the initially cultured strain was mixed in YPD medium and simultaneous saccharification was carried out at 30 ℃ and 150rpm (fig. 13). It was confirmed that, in the case of simultaneous saccharification with 2FPU cellulase, ethanol was not produced in the control group, whereas, in the case of using the composite strain, 3% of the added straw (containing 21g of glucose) was saccharified and 95% or more was converted into ethanol (A in FIG. 13). When the amounts of straw and cellulase used were increased, ethanol was also produced in the control group, but the production amount was far from that of the recombinant composite strain.

Example 9: comparative analysis of ethanol fermentation Capacity of recombinase mixture in fermentor

C-Tec and H-Tec as commercial enzymes were mixed in four complex strains producing KCC-3 mixture enzymes and simultaneous saccharification and fermentation was performed in a 5L fermentor. 6% of pulverized straw used as a substrate by neutralizing and drying after treating with 2% sodium hydroxide (NaOH) at 160 ℃ for 1 hour, and using an ethanol fermentation medium (peptone 5g/L, yeast extract 5g/L, KH2PO 45 g/L, ammonium sulfate 2g/L, MgSO)₄0.4 g/L). The expression of the enzyme KCC-3 producing strain Y2805. delta. gal80 was as follows: 1: 4: ST19-BGL, ST19-EGL2, self signal-CtCBH1 and ST13-ClCBH2 were mixed at a ratio of 4 and commercial cellulase (C-Tec: H-Te) from Novozyme was added theretoc is 7: 3) this was made to be 2FPU/g cellulose. A strain (50mL) which had been once cultured in a YNB (yeast nitrogen source) medium of 6.7g/L, casamino acid (casamino acid) of 5g/L, and glucose (glucose) of 20g/L was inoculated in YPD in a secondary culture (450mL) so that the amount of the strain inoculated became 30%, mixed in respective ratios, and then simultaneously subjected to saccharification and fermentation at 30 ℃, 150rpm to 300rpm, pH5.3 to pH5.5, and 0vvm to 0.1 vvm. From the above results, it was confirmed that 4g/L of ethanol was produced in the control group, whereas ethanol was produced in the KCC-3 strain complex up to a yield of 19 g/L/close to the theoretical yield (FIG. 14A). In order to confirm that the difference is caused by cellulase secreted from the recombinant yeast cells, SDS-PAGE analysis of the medium during fermentation was performed, and as a result, it was confirmed that although the initially added external enzyme C-tec was not present any more after about 56 hours of fermentation, recombinant cellulase not confirmed in Wild-type yeast (Wild-type yeast) was continuously produced by secretion in KCC-3 during fermentation. Therefore, it was confirmed that in the case of mixing the cellulase-producing strains provided by the present invention to apply to ethanol production, ethanol can be produced from biomass through simultaneous saccharification engineering using a minimum amount of commercially available cellulase.

Those skilled in the art to which the present invention pertains will appreciate from the foregoing description that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this connection, it should be understood that the above-described embodiments are illustrative in all respects, not restrictive. The scope of the present invention should be construed that all modifications and variations derived from the meaning and scope of the claims to be described later and the concept equivalent thereto are included in the scope of the present invention as compared with the above detailed description.

Claims

1. An expression cassette for protein expression having an increased secretion capacity in yeast as compared to a wild-type protein, the expression cassette comprising a polynucleotide encoding a Translational Fusion Partner (TFP) and a polynucleotide encoding a protein selected from the group consisting of trxynil, TrBxl, TrEGL2, PaCEL1, PaCEL2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, ClCBH2, and CfCex1, wherein trxynil consists of the amino acid sequence of seq id No. 2; the TrBxl is composed of an amino acid sequence with a sequence number of 4; the TrEGL2 is composed of an amino acid sequence with a sequence number of 6; the PaCEL1 is composed of an amino acid sequence of SEQ ID NO. 8; the PaCel2 is composed of an amino acid sequence with the sequence number of 10; the TeCBH1 is composed of an amino acid sequence with the sequence number of 12; the NfCBH1 consists of an amino acid sequence with a sequence number of 14; the HgCBH1 consists of an amino acid sequence with a sequence number of 16; the CtCBH1 consists of an amino acid sequence with the sequence number of 18; the ClCBH2 consists of an amino acid sequence with the sequence number of 20; and the CfCex1 is composed of an amino acid sequence of SEQ ID NO. 22,

i) in the case where trxynil is selected as the protein, the translational fusion partner is selected from the group consisting of TFP5 consisting of the amino acid sequence of seq id No. 53, TFP9 consisting of the amino acid sequence of seq id No. 57, TFP13 consisting of the amino acid sequence of seq id No. 61, and TFP19 consisting of the amino acid sequence of seq id No. 67;

ii) in the case where TrBxl is selected as the protein, the translational fusion partner is TFP4 consisting of the amino acid sequence of SEQ ID NO. 52;

iii) in case TrEGL2 is selected as the protein, the translational fusion partner is TFP19 consisting of the amino acid sequence of seq id No. 67;

iv) in the case where PaCEL1 is selected as the protein, the translational fusion partner is TFP13 consisting of the amino acid sequence of seq id No. 61;

v) in the case of selecting PaCel2 as the protein, the translational fusion partner is TFP13 consisting of the amino acid sequence of SEQ ID NO. 61;

vi) in the case of selecting TeCBH1 as the protein, the translational fusion partner is selected from TFP4 consisting of the amino acid sequence of seq id No. 52, TFP6 consisting of the amino acid sequence of seq id No. 54, TFP7 consisting of the amino acid sequence of seq id No. 55, and TFP8 consisting of the amino acid sequence of seq id No. 56;

vii) in case NfCBH1 is selected as the protein, the translational fusion partner is TFP13 consisting of the amino acid sequence of seq id No. 61;

viii) when HgCBH1 is selected as the protein, the translational fusion partner is TFP13 consisting of the amino acid sequence of seq id No. 61;

ix) when CtCBH1 is selected as the protein, the translational fusion partner is selected from TFP7 consisting of the amino acid sequence of seq id No. 55, TFP19 consisting of the amino acid sequence of seq id No. 67, a self-signal consisting of the base sequence of seq id No. 47, and a self-signal consisting of the amino acid sequence of seq id No. 48;

x) when ClCBH2 is selected as the protein, the translational fusion partner is one of TFP8 consisting of the amino acid sequence of seq id No. 56 and TFP13 consisting of the amino acid sequence of seq id No. 61;

xi) when CfCex1 is selected as the protein, the translational fusion partner is one of TFP8 composed of an amino acid sequence of sequence No. 56, TFP11 composed of an amino acid sequence of sequence No. 59, TFP13 composed of an amino acid sequence of sequence No. 61, and TFP19 composed of an amino acid sequence of sequence No. 67.

2. The expression cassette according to claim 1, wherein,

i) the TrXynII is coded by a polynucleotide sequence with a sequence number of 1;

ii) the TrBxl is encoded by a polynucleotide sequence of SEQ ID NO. 3;

iii) said TrEGL2 is encoded by a polynucleotide sequence of seq id No. 5;

iv) said PaCEL1 is encoded by the polynucleotide sequence of SEQ ID NO. 7;

v) said PaCel2 is encoded by a polynucleotide sequence of SEQ ID NO. 9;

vi) said TeCBH1 is encoded by the polynucleotide sequence of SEQ ID NO. 11;

vii) said NfCBH1 is encoded by the polynucleotide sequence of seq id No. 13;

viii) said HgCBH1 is encoded by a polynucleotide sequence of seq id No. 15;

ix) the CtCBH1 is encoded by the polynucleotide sequence of seq id No. 17;

x) said ClCBH2 is encoded by the polynucleotide sequence of seq id No. 19; and

xi) the CfCex1 is encoded by the polynucleotide sequence of SEQ ID NO. 21.

3. An expression vector comprising the expression cassette of any one of claims 1 to 2.

4. A transformant formed by introducing the expression vector according to claim 3 into a host cell.

5. The transformant according to claim 4,

the host cell is a cell having ethanol fermentation ability.

6. The transformant according to claim 5,

the cell having ethanol fermentation ability was Y2805(Mat a pep4:: HIS3 prb1can 1HIS3-200ura3-52) strain.

7. A method for producing a hemicellulase, an endoglucanase or an exoglucanase, comprising a step for culturing the transformant according to claim 4.

8. A method for producing bioethanol, comprising the steps of:

i) culturing the transformed transformant according to claim 4; and

ii) recovering bioethanol from the culture or culture supernatant obtained in said step i).

9. The method for producing bioethanol according to claim 8, wherein,

the transformant is made of a host cell having ethanol fermentation ability.

10. A complex strain comprising two or more transformants according to claim 4.

11. The composite strain of claim 10, wherein,

the composite strain comprises more than one strain secreting hemicellulase.

12. The composite strain of claim 10, wherein,

the compound strain has xylan decomposition capability.

13. A cellulase cocktail comprising β -glucosidase and endoglucanases and exoglucanases produced according to claim 7.

14. The cellulase enzyme mixture according to claim 13,

the beta-glucosidase comprises an amino acid sequence of SEQ ID NO. 74.

15. The cellulase enzyme mixture according to claim 13,

the mixture comprises endo-cellulase, exo-cellulase and beta-glucosidase.

16. A method of saccharifying a biomass using the mixture of claim 13.

17. A cellulase enzyme mixture comprising the hemicellulase produced according to claim 7.

18. The cellulase enzyme mixture according to claim 17,

the mixture comprises an endoxylanase and a beta-xylosidase.

19. The cellulase enzyme mixture according to claim 18,

the endo-xylanase is TrXynII protein, and the beta-xylosidase is TrBxl.

20. A method of saccharifying a biomass using the mixture of claim 17.