Base sequence for protein expression and method for producing protein using same

Abstract

To provide a base sequence for protein expression that can increase the yield of protein such as diastatic enzyme by further activating a promoter of a particular gene. A base sequence 1 for protein expression includes: a gene 3 encoding protein 2; a promoter 4 of the gene 3, the promoter being linked upstream of the gene 3; and a cis element 5 whose activity is improved by an artificial transcription factor 6, the cis element being linked further upstream of the promoter 4. The cis element 5 is represented by SEQ ID NO: 1.

Claims

1. A combination of a base sequence for protein expression comprising: a gene encoding protein; a promoter of the gene, the promoter being linked upstream of the gene; and a cis element whose activity is improved by an artificial transcription factor, the cis element being linked upstream of the promoter, and a base sequence for artificial transcription factor expression comprising: a gene encoding the artificial transcription factor; and a promoter of the gene, the promoter being linked upstream of the gene, wherein the cis element is represented by SEQ ID NO: 1, and wherein the artificial transcription factor comprises a DNA binding domain comprising a polynucleotide sequence of upstream 1 to 118 aa of a transcription factor KojR and an active domain comprising a base polynucleotide sequence of downstream 150 to 604 aa of a transcription factor AmyR, and the active domain is linked downstream of the DNA binding domain, and is represented by SEQ ID NO: 2.

2. The base sequence for protein expression according to claim 1, wherein the cis element(s) is (are) linked upstream of the promoter of the gene encoding the protein at any number in a range of 1 copy to 10 copies.

3. An expression vector including a combination of a base sequence for protein expression, the base sequence for protein expression comprising: a gene encoding protein; a promoter of the gene, the promoter being linked upstream of the gene; and a cis element whose activity is improved by an artificial transcription factor, the cis element being linked upstream of the promoter, and a base sequence for artificial transcription factor expression comprising: a gene encoding the artificial transcription factor; and a promoter of the gene, the promoter being linked upstream of the gene, wherein the cis element is represented by SEQ ID NO: 1, and wherein the artificial transcription factor comprises a DNA binding domain comprising a polynucleotide sequence of upstream 1 to 118 aa of a transcription factor KojR and an active domain comprising a polynucleotide sequence of downstream 150 to 604 aa of a transcription factor AmyR, and the active domain is linked downstream of the DNA binding domain, and is represented by SEQ ID NO: 2.

4. The expression vector according to claim 3, wherein in the base sequence for protein expression, the cis element(s) is (are) linked upstream of the promoter of the gene encoding the protein at any number in a range of 1 copy to 10 copies.

5. A transformant including a combination of a base sequence for protein expression, the base sequence for protein expression comprising: a gene encoding protein; a promoter of the gene, the promoter being linked upstream of the gene; and a cis element whose activity is improved by an artificial transcription factor, the cis element being linked upstream of the promoter, and a base sequence for artificial transcription factor expression comprising: a gene encoding the artificial transcription factor; and a promoter of the gene, the promoter being linked upstream of the gene, wherein the cis element is represented by SEQ ID NO: 1, and wherein the artificial transcription factor comprises a DNA binding domain comprising a polynucleotide of upstream 1 to 118 aa of a transcription factor KojR and an active domain comprising a polynucleotide sequence of downstream 150 to 604 aa of a transcription factor AmyR, and the active domain is linked downstream of the DNA binding domain, and is represented by SEQ ID NO: 2.

6. The transformant according to claim 5, wherein in the base sequence for protein expression, the cis element(s) is (are) linked upstream of the promoter of the gene encoding the protein at any number in a range of 1 copy to 10 copies.

7. The transformant according to claim 5, wherein said transformant is derived from koji mold used as a host cell.

8. The transformant according to claim 7, wherein the koji mold is an Aspergillus oryzae HO2 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12, 2013, Deposition No.: NITE BP-01750).

9. The transformant according to claim 7, wherein the koji mold is an Aspergillus oryzae HO4 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980).

10. A method for producing protein, comprising culturing a transformant including a combination of a base sequence for protein expression which comprises: a gene encoding the protein; a promoter of the gene, the promoter being linked upstream of the gene; and a cis element whose activity is improved by an artificial transcription factor, the cis element being linked upstream of the promoter, wherein the cis element is represented by SEQ ID NO: 1, and a base sequence for artificial transcription factor expression comprising: a gene encoding the artificial transcription factor; and a promoter of the gene, the promoter being linked upstream of the gene, and recovering the protein encoded by the gene overexpressed by the base sequence for protein expression, from a medium or inside of the transformant after the culture, wherein the artificial transcription factor comprises a DNA binding domain comprising a polynucleotide sequence of upstream 1 to 118 aa of a transcription factor KojR and an active domain comprising a polynucleotide sequence of downstream 150 to 604 aa of a transcription factor AmyR, and the active domain is linked downstream of the DNA binding domain, and is represented by SEQ ID NO: 2.

11. The method for producing protein according to claim 10, wherein in the base sequence for protein expression, the cis element(s) is (are) linked upstream of the promoter of the gene encoding the protein at any number in a range of 1 copy to 10 copies.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is an illustrative diagram schematically showing the configuration and effect of a base sequence for protein expression of the present invention.

(2) FIG. 2 is an illustrative diagram schematically showing a predicted structure of a transcription factor KojR.

(3) FIG. 3 is an illustrative diagram schematically showing a predicted structure of a transcription factor AmyR.

(4) FIG. 4 is a graph showing a relative value of GUS activity when a transformant of the present invention was cultured for 60 hours with dextrin as a substrate.

(5) FIG. 5 is a graph showing GUS activity when the transformant of the present invention was cultured for 90 hours with dextrin as the substrate.

(6) FIG. 6 is a graph showing GUS activity when the transformant of the present invention was cultured for 60 hours with glucose as a substrate.

(7) FIG. 7 is a graph showing CBH1 production amount when the transformant of the present invention was cultured for 6 days with dextrin as a substrate

DESCRIPTION OF EMBODIMENTS

(8) Next, the embodiments of the present invention will be described further specifically with reference to the attached drawings.

(9) As shown in FIG. 1, a base sequence 1 for protein expression of the present embodiment comprises: a protein gene 3 encoding a desired protein 2; a promoter 4 linked upstream (on the 5-terminal side) of the protein gene 3; and a cis element 5 linked upstream (on the 5-terminal side) of the promoter 4.

(10) The protein 2 is, for example, a diastatic enzyme. The protein gene 3 may be any gene which encodes the protein 2.

(11) The cis element 5 is composed of a base sequence comprising enhancer DNA located at a promoter of kojT gene, and the base sequence is gacggaaaagtcgggtagat (SEQ ID NO: 1). In the base sequence 1 for protein expression, 1 to 10, for example, 8 cis elements 5 are linked upstream of the promoter 4.

(12) The base sequence 1 for protein expression also comprises a base sequence 9 for artificial transcription factor expression comprising: an artificial transcription factor gene 7 encoding an artificial transcription factor 6; and a promoter 8 linked upstream (on the 5-terminal side) of the artificial transcription factor gene 7. The activity of the cis element 5 is improved by the artificial transcription factor 6.

(13) The artificial transcription factor 6 is prepared from a transcription factor KojR 11 shown in FIG. 2 and a transcription factor AmyR 21 shown in FIG. 3. The transcription factors KojR 11 and AmyR 21 are transcription factors both classified into Cys6 cysteine-Zinc cluster type, among transcription factors having a zinc-coordinating DNA binding domain (Zn_Cluster).

(14) As shown in FIG. 2, the transcription factor KojR 11 comprises upstream (5-terminal side) Zn_Cluster 12 and comprises downstream (3-terminal side) MHR 13 which is a highly homologous region common in transcription factors classified in Cys6 cysteine-Zinc cluster type. In this context, the transcription factor KojR 11 is composed of a base sequence of 555 aa in full length. The Zn_Cluster 12 is composed of a base sequence of 15 to 45 aa. The MHR 13 is composed of a base sequence of 148 to 281 aa.

(15) In the transcription factor KojR 11, a DNA binding domain associated with binding to the cis element 5 is predicted to reside in a region 14 comprising the upstream Zn_Cluster 12. Examples of a candidate region of the DNA binding domain can include a region composed of a base sequence of 1 to 118 aa, a region composed of a base sequence of 1 to 195 aa, and a region composed of a base sequence of 1 to 239 aa.

(16) On the other hand, as shown in FIG. 3, the transcription factor AmyR 21 comprises upstream (5-terminal side) Zn_Cluster 22 and comprises a downstream (3-terminal side) region 23 comprising an active domain. In this context, the transcription factor AmyR 21 is composed of a base sequence of 604 aa in full length. The Zn_Cluster 22 is composed of a base sequence of 13 to 52 aa.

(17) Examples of a candidate region of the active domain in the transcription factor AmyR 21 can include a region composed of a base sequence of 113 to 604 aa, a region composed of a base sequence of 150 to 604 aa, a region composed of base sequence of 219 to 604 aa, and a region composed of a base sequence of 257 to 604 aa.

(18) Accordingly, the artificial transcription factor of the present embodiment has a configuration (SEQ ID NO: 2) in which an active domain comprising a base sequence of downstream 150 to 604 aa of the transcription factor AmyR is linked downstream of a DNA binding domain comprising a base sequence of upstream 1 to 118 aa of the transcription factor KojR.

(19) According to the base sequence 1 for protein expression of the present embodiment, as shown in FIG. 1, the artificial transcription factor 6 encoded by the artificial transcription factor gene 7 whose activity has been improved by the promoter 8 in the base sequence 9 for artificial transcription factor expression is produced, and the produced artificial transcription factor 6 binds to the cis element 5. The activity of the cis element 5 is improved by the binding to the artificial transcription factor 6. The activity of the promoter 4 is improved by the cis element 5 whose activity has been improved.

(20) Then, the activity of the protein gene 3 is improved by the promoter 4 whose activity has been improved, so that the protein 2 encoded by the protein gene 3 whose activity has been improved, is produced. As a result, the base sequence 1 for protein expression of the present embodiment can increase the yield of the protein 2.

(21) Next, Examples of the present invention will be shown.

EXAMPLE 1

(22) (Construction of Transformant Introduced with Artificial Transcription Factor Gene (1))

(23) In this Example, first, the genomic DNA gene of an Aspergillus oryzae HO2 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12, 2013, Deposition No.: NITE BP-01750) was used as a template in PCR to amplify an upstream sequence of tppA gene using primers 1 and 2, its downstream sequence using primers 3 and 4, a tef1 promoter gene using primers 5 and 6, an agdA terminator gene using primers 7 and 8, and a gene fragment for marker recycling using primers 9 and 10, while the genomic DNA gene of an Aspergillus awamori HA1 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12, 2013, Deposition No.: NITE BP-01751) was used as a template in PCR to amplify a gene cassette for pyrG gene expression using primers 11 and 12. DNA polymerase (manufactured by Toyobo Co., Ltd., product name: KOD FX neo) was used in each PCR amplification. The amplification products were each purified using a purification kit (manufactured by Qiagen N.V., product name: QIAquick PCR purification kit) to obtain a total of 6 gene fragments.

(24) Next, an E. coli-derived plasmid pMD20 (manufactured by Takara Bio Inc.) was used as a template in PCR to amplify a gene fragment derived from the plasmid using primers 13 and 14 and the DNA polymerase. The amplification product was purified using the purification kit to obtain the gene fragment.

(25) Next, these 7 gene fragments were sequentially treated with a cloning kit (manufactured by Takara Bio Inc., product name: In-Fusion HD Cloning kit) and used in the transformation of an E. coli HST08 strain (manufactured by Takara Bio Inc.) to construct a plasmid pPT.

(26) Next, the plasmid pPT was treated with a restriction enzyme SmaI (manufactured by Takara Bio Inc.) at 30 C. and purified using the purification kit to obtain the restriction treatment product of the plasmid (gene fragment).

(27) Next, the genomic DNA gene of an Aspergillus oryzae HO2 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12, 2013, Deposition No.: NILE BP-01750) was used as a template in PCR to amplify a DNA binding domain of a transcription factor KojR using primers 15 and 16 and an active domain of a transcription factor AmyR using primers 17 and 18. The DNA polymerase was used in each PCR amplification. The amplification products were each purified using the purification kit to obtain the DNA binding domain and the active domain.

(28) Next, the DNA binding domain and the active domain were treated with the cloning kit and used in the transformation of an E. coli HST08 strain to construct a plasmid carrying an artificial transcription factor gene in which the DNA binding domain and the active domain were joined together.

(29) The plasmid carrying the artificial transcription factor gene was used as a template in PCR to amplify a gene fragment for koji mold transformation using primers 19 and 20 using DNA polymerase (manufactured by Toyobo Co., Ltd., product name: KOD-plus-neo). The amplification product was purified using the purification kit to obtain the gene fragment for koji mold transformation.

(30) Next, an Aspergillus oryzae HO2 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12, 2013, Deposition No.: NITE BP-01750) was transformed with the gene fragment for koji mold transformation according to the standard method of the PEG-calcium technique. Subsequently, the obtained transformants were screened for a strain capable of growing in a CD plate medium to obtain a transcription factor-producing strain.

(31) Next, the transcription factor-producing strain was inoculated at 110.sup.6 cells/plate to a CD medium supplemented with fluoroorotic acid monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) (final concentration: 1 mg/mL) and uridine (manufactured by Sigma-Aldrich Inc.) (final concentration: 20 mM) and screened for a strain capable of growing therein to obtain a uridine-auxotrophic transcription factor-producing strain.

(32) The base sequences of the primers 1 to 20 are shown in Table 1.

(33) TABLE-US-00001 TABLE1 SEQ Primer ID No. Basesequence5.fwdarw.3 NO Remarks 1 ccggctcgtatgttgctggaccaaccgccaaggttag 3 Upstreamsequenceof tppAgene 2 actgaattgcaattaatggcggacaatg 4 Upstreamsequenceof tppAgene 3 tgtctcggaccttacgtgtcttagatgcgactcaatacaactgttc 5 Downstreamsequence oftppAgene 4 tgggtaacgccagggttgaggctgaagacttaaatacgacattgc 6 Downstreamsequence oftppAgene 5 ctgttacgcttccccgggtttgaaggtggtgcgaactttgtagttc 7 tef1promotergene 6 gtaaggtccgagacagtaagggattgatc 8 tef1promotergene 7 taattgcaattcagtagtaacccattcccggttctctagctg 9 agdAterminatorgene 8 gtaacgccagggcccggggaagcgtaacaggatagcctagacccac 10 agdAterminatorgene 9 ctgcaggatgattagcgtgcaaaccaagcaaacaagcatc 11 Genefragmentfor markerrecycling 10 actgaattgcaattaatggcggacaatg 12 Genefragmentfor markerrecycling 11 taattgcaattcagtgcaagctcgagcatccaactaaactag 13 GenecassetteforprG geneexpression 12 tgggtaacgccagggcccgggctaatcatcctgcagctccgtcattg 14 GenecassetteforprG geneexpression 13 ccctggcgttacccaacttaatcg 15 Plasmid-derivedgene fragment 14 caacatacgagccggaagcataaagtg 16 Plasmid-derivedgene fragment 15 cgcaccaccttcaaaatgtcgttgaataccgacgattccggtc 17 DBDoftranscription factorkojR 16 acctaggttccagctaaacccgtacac 18 DBDoftranscription factorkojR 17 atcctgttacgcttctcaaaacgaaatctcctccccagcc 19 ADoftranscription factorAmyR 18 agctggaacctaggtgcactccccgcgccac 20 ADoftranscription factorAmyR 19 cagtgagcgcaacgcaattaatgtgagttag 21 Genefragmentforkoji moldtransformation 20 gggatgtgctgcaaggcgattaagttg 22 Genefragmentforkoji moldtransformation
[Construction of GUS-producing Strain with Cis Elements Linked]

(34) First, a first gene fragment in which: 4 cis elements of SEQ ID NO: 1 were linked in tandem; restriction enzyme sites SphI and BamHI were added on the 5-terminal side thereof; and BglII and NcoI sites were added on the 3-terminal side thereof was prepared by oligo synthesis.

(35) Next, the first gene fragment and a plasmid pPEA2 containing an Aspergillus oryzae-derived enoA promoter were each fragmented by treatment with restriction enzymes SphI and NcoI. These fragments were subjected to ligation reaction, and E. coli was then transformed with the ligation product to construct a plasmid pEA4K.

(36) Next, the gene fragment was treated with a restriction enzyme BamHI, while the plasmid pEA4K was treated with restriction enzymes BglII and NcoI. These two treatment products were subjected to ligation reaction, and E. coli was then transformed with the ligation product to construct a plasmid pEA8K.

(37) Next, the plasmid pEA8K was used as a template in PCR amplification using primers 21 and 22 and DNA polymerase (manufactured by Toyobo Co., Ltd., product name: KOD-plus-). The amplification product was purified using a purification kit (manufactured by Promega Corp., product name: Wizard SV Gel and PCR Clean-Up System) to obtain a second gene fragment.

(38) Next, the genomic DNA of Aspergillus oryzae was used as a template in PCR amplification using primers 23 and 24 and DNA polymerase (manufactured by Toyobo Co., Ltd., product name: KOD-plus-). The amplification product was purified using a purification kit (manufactured by Promega Corp., product name: Wizard SV Gel and PCR Clean-Up System) to obtain a third gene fragment.

(39) Next, the second gene fragment and the third gene fragment were used as a template in fusion PCR using primers 22 and 24 to prepare a fourth gene fragment in which the second gene fragment and the third gene fragment were joined together.

(40) Next, a restriction enzyme-treated plasmid pPPG introduced with an E. coli-derived plasmid pMD20 (manufactured by Takara Bio Inc.) carrying upstream 1000 bp of Aspergillus oryzae-derived pyrG gene, an Aspergillus oryzae-derived pyrG expression cassette, and an E. coli-derived uidA gene was subjected to ligation reaction with a gene fragment for marker recycling obtained by PCR-amplifying a plasmid pPPG as a template using primers 25 and 26 and DNA polymerase (manufactured by Toyobo Co., Ltd., product name: KOD-plus-) and purifying the amplification product using a purification kit (manufactured by Promega Corp., product name: Wizard SV Gel and PCR Clean-Up System). Then, E. coli was transformed with the ligation product to construct a plasmid pPPRG.

(41) Next, the plasmid pPPRG was used as a template in PCR amplification using primers 27 and 28 and DNA polymerase (manufactured by Toyobo Co., Ltd., product name: KOD-plus-). The amplification product was purified using a purification kit (manufactured by Promega Corp., product name: Wizard SV Gel and PCR Clean-Up System) to obtain a fifth gene fragment.

(42) The fourth gene fragment and the fifth gene fragment were used as a template in fusion PCR using primers 24 and 27 to prepare a cis element-linked GUS (-glucuronidase) production cassette gene fragment in which the fourth gene fragment and the fifth gene fragment were joined together.

(43) Next, the uridine-auxotrophic transcription factor-producing strain was transformed using the cis element-linked GUS production cassette gene fragment according to the standard method of the PEG-calcium technique. Subsequently, the obtained transformants were screened for a strain capable of growing in a CD plate medium to obtain a GUS-producing strain with 8 cis elements linked in tandem.

(44) The base sequences of the primers 21 to 28 are shown in Table 2.

(45) TABLE-US-00002 TABLE2 Primer SEQID No. Basesequence5.fwdarw.3 NO 21 ccgctgctaggcgcgccgtgcactatagggcgaattgggc 23 22 tggggtttctacaggacgtaacattttgacgagctgcggaatt 24 23 cacggcgcgcctagcagcgggtagtggtggatacgtactcctt 25 24 ttcaggtcacgttctaagcttatcag 26 25 cccccctccggatgatgtagaagttgctcggtagctg 27 26 cccccctccggacaattgccgcgaaaaattaaattg 28 27 ccagaggtgactttatccaagatt 29 28 caattccgcagctcgtcaaaatgttacgtcctgtagaaacccca 30
[GUS Activity Measurement Method]

(46) The GUS-producing strain with 8 cis elements linked in tandem was cultured in a CD plate medium for 1 week to form spores. The spores were recovered using 0.01% POLYSORBATE 20 (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a spore suspension.

(47) Next, 50 mL of a PD medium (2 mass/volume % of dextrin, 1 mass/volume % of polypeptone, 0.1 mass/volume % of casamino acid, 0.5 mass/volume % of potassium dihydrogen phosphate, 0.05 mass/volume % of magnesium sulfate, and 0.1 mass/volume % of sodium nitrate) was placed in a 200 mL Erlenmeyer flask, to which the spores were then inoculated at a final spore concentration of 110.sup.5/mL.

(48) Next, liquid culture was performed at 30 C. for 60 hours. After the completion of the culture, the bacterial cells were disrupted, and the disrupted powder was suspended in a buffer for intracellular protein extraction having the composition given below to obtain an extract.

(49) [Composition of Buffer for Intracellular Protein Extraction]

(50) NaH.sub.2PO.sub.4.2H.sub.2O (MW=156.01) (pH 7) 1.56 g (50 mM)

(51) 0.5 M EDTA 4 mL (10 mM)

(52) Nonionic surfactant (manufactured by Sigma-Aldrich Inc., product name: Triton X-100) 0.2 g (0.1%)

(53) N-Laurylsarcosinate Na 0.2 g (0.1%)

(54) -mercaptoethanol (MW=78.13) 142 L (10 mM)

(55) Distilled water 200 mL

(56) Next, the extract was added to a buffer for GUS activity measurement having the composition given below and reacted at 37 C. for 15 minutes. Then, the absorbance was measured at a wavelength of 415 nm to calculate an activity value (U). 1 U means the amount of the enzyme necessary for forming 1 mM PNP from PNP-Glucuronide (purine nucleoside phosphorylase-glucuronic acid inclusion) at 37 C. for 1 minute.

(57) [Composition of Buffer for GUS Activity Measurement]

(58) NaH.sub.2PO.sub.4.2H.sub.2O (MW=156.01) (pH 7) 1.56 g (50 mM)

(59) -mercaptoethanol (MW=78.13) 142 L (10 mM)

(60) Nonionic surfactant (manufactured by Sigma-Aldrich Inc., product name: Triton X-100) 0.2 g (0.1%)

(61) p-Nitrophenyl -D-glucuronic acid inclusion (MW=315.23) 63 mg (1 mM)

(62) Distilled water 200 mL

(63) Next, the amount of the protein contained in the extract was measured using protein assay CBB solution (manufactured by Nacalai Tesque, Inc.), and the activity value was divided by the amount of the protein to calculate GUS activity (U/mg). The results are shown as a relative value of GUS activity in FIG. 4.

(64) Also, GUS activity (U/mg) when the liquid culture was performed at 30 C. for 90 hours is shown in FIG. 5.

COMPARATIVE EXAMPLE 1

(65) In this Comparative Example, a GUS-producing strain was constructed in totally the same way as in Example 1 except that the artificial transcription factor gene was not introduced and no cis element was linked.

(66) Next, GUS activity was measured in totally the same way as in Example 1 except that the GUS-producing strain obtained in this Comparative Example was used.

(67) A relative value of GUS activity (U/mg) when the liquid culture was performed at 30 C. for 60 hours is shown in FIG. 4. GUS activity (U/mg) when the liquid culture was performed at 30 C. for 90 hours is shown in FIG. 5.

(68) From FIG. 4, when the GUS activity (U/mg) in which the liquid culture was performed at 30 C. for 60 hours with dextrin as a substrate is defined as 1 for the GUS-producing strain of Comparative Example 1 in which the artificial transcription factor gene was not introduced and no cis element was linked, it is obvious that 25.1 times GUS activity can be obtained in the GUS-producing strain of Example 1.

(69) From FIG. 5, when the GUS activity (U/mg) when the liquid culture was performed at 30 C. for 90 hours with dextrin as a substrate is defined as 1 for the GUS-producing strain of Comparative Example 1 in which the artificial transcription factor gene was not introduced and no cis element was linked, it is obvious that 28 times GUS activity can be obtained in the GUS-producing strain of Example 1.

EXAMPLE 2

(70) In this Example, GUS activity (U/mg) was calculated in totally the same way as in Example 1 except that 50 mL of a PG medium (2 mass/volume % of glucose, 1 mass/volume % of polypeptone, 0.1 mass/volume % of casamino acid, 0.5 mass/volume % of potassium dihydrogen phosphate, 0.05 mass/volume % of magnesium sulfate, and 0.1 mass/volume % of sodium nitrate) was placed in a 200 mL Erlenmeyer flask, to which the spores of the GUS-producing strain harboring 8 cis elements linked in tandem obtained in Example 1 were then inoculated at a final spore concentration of 110.sup.5/mL, followed by liquid culture at 30 C. for 60 hours. The results are shown in FIG. 6.

COMPARATIVE EXAMPLE 2

(71) In this Comparative Example, GUS activity was measured in totally the same way as in Example 2 except that the GUS-producing strain obtained in Comparative Example 1 was used. GUS activity (U/mg) when the liquid culture was performed at 30 C. for 60 hours is shown in FIG. 6.

(72) From FIG. 6, when the GUS activity (U/mg) in which the liquid culture was performed at 30 C. for 60 hours with glucose as a substrate is defined as 1 for the GUS-producing strain of Comparative Example 2 in which the artificial transcription factor gene was not introduced and no cis element was linked, it is obvious that 14.8 times GUS activity can be obtained in the GUS-producing strain of Example 2.

EXAMPLE 3

(73) (Construction of Transformant Introduced with Artificial Transcription Factor Gene (2))

(74) In this Example, first, the genomic DNA gene of an Aspergillus oryzae HO4 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980) was used as a template in PCR to amplify an upstream sequence of ligD gene using primers 29 and 30, its downstream sequence using primers 31 and 32, a marker recycling sequence using primers 33 and 34, and a pyrG gene using primers 35 and 36. DNA polymerase (manufactured by Toyobo Co., Ltd., product name: KOD FX neo) was used in each PCR amplification. The amplification products were each purified using a purification kit (manufactured by Qiagen N.V., product name: QIAquick PCR purification kit) to obtain a total of 4 gene fragments.

(75) Next, an E. coli-derived plasmid pMD20 (manufactured by Takara Bio Inc.) was used as a template to obtain a gene fragment derived from the plasmid using primers 13 and 14.

(76) Next, gene fragment of upstream sequence of ligD gene, gene fragment of its downstream sequence, and gene fragment of plasmid pMD20 were treated with a cloning kit (manufactured by Takara Bio Inc., product name: In-Fusion HD Cloning kit) and used in the transformation of an E. coli HST08 strain (manufactured by Takara Bio Inc.) to construct a plasmid pM-Ao ligD.

(77) Next, the plasmid pM-Ao ligD was treated with the restriction enzyme SmaI (manufactured by Takara Bio Inc.) at 30 C. and purified using the purification kit to obtain the restriction treatment product of the plasmid pM-Ao ligD (gene fragment).

(78) Next, gene fragment of plasmid pM-Ao ligD and gene fragment of pyrG gene were treated with the cloning kit and used in the transformation of an E. coli HST08 strain (manufactured by Takara Bio Inc.) to construct a plasmid pM-Ao ligD::pyrG in which pyrG gene was introduced between the upstream sequence of ligD gene and its downstream sequence.

(79) Next, the plasmid pM-Ao ligD::pyrG was treated with the restriction enzyme SmaI (manufactured by Takara Bio Inc.) at 30 C. and purified using the purification kit to obtain the restriction treatment product of the plasmid pM-Ao ligD::pyrG (gene fragment).

(80) Next, gene fragment of plasmid pM-Ao ligD::pyrG and gene fragment of the marker recycling sequence were treated with the cloning kit and used in the transformation of an E. coli HST08 strain (manufactured by Takara Bio Inc.) to construct a plasmid pM-Ao ligD::pyrGR in which the marker recycling sequence was introduced between the downstream sequence of ligD gene and the pyrG gene.

(81) Next, the plasmid pM-Ao ligD::pyrGR was treated with the restriction enzyme SmaI (manufactured by Takara Bio Inc.) at 30 C. and purified using the purification kit to obtain the restriction treatment product of the plasmid pM-Ao ligD::pyrGR (gene fragment).

(82) Next, the genomic DNA gene of an Aspergillus oryzae HO4 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980) was used as a template in PCR to amplify an enoA promoter gene using primers 37 and 38, and a plasmid introduced with the artificial transcription factor gene in which the DNA binding domain and the active domain were joined together prepared in Example 1 was used as a template in PCR to amplify an agdA terminator gene and an artificial transcription gene using primers 39 and 40. The DNA polymerase was used in each PCR amplification. The amplification products were each purified using the purification kit to obtain the DNA binding domain and the active domain.

(83) Next, gene fragment of plasmid pM-Ao ligD::pyrGR, gene fragment of enoA promoter gene, agdA terminator gene, and artificial transcription gene were treated with the cloning kit and used in the transformation of an E. coli HST08 strain (manufactured by Takara Bio Inc.) to construct a plasmid pM-Ao ligD::pyrGR-TF1 in which artificial transcription factor gene was introduced between the upstream sequence of ligD gene and its downstream sequence.

(84) The plasmidpM-Ao ligD::pyrGR-TF1 carrying the artificial transcription factor gene was used as a template in PCR to amplify a gene fragment for koji mold transformation using primers 19 and 20 using the DNA polymerase. The amplification product was purified using the purification kit to obtain the gene fragment for koji mold transformation.

(85) Next, an Aspergillus oryzae HO4 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980) was transformed with the gene fragment for koji mold transformation according to the standard method of the PEG-calcium technique. Subsequently, the obtained transformants were screened for a strain capable of growing in a CD plate medium to obtain a transcription factor-producing strain.

(86) Next, the transcription factor-producing strain was inoculated at 110.sup.6 cells/plate to a CD medium supplemented with fluoroorotic acid monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) (final concentration: 1 mg/mL) and uridine (manufactured by Sigma-Aldrich Inc.) (final concentration: 20 mM) and screened for a strain capable of growing therein to obtain a uridine-auxotrophic transcription factor-producing strain.

(87) The base sequences of the primers 29 to 40 are shown in Table 3.

(88) TABLE-US-00003 TABLE3 SEQ Primer ID No. Basesequence5.fwdarw.3 NO Remarks 29 tcgagctcggtacccggttactgctctcccttgatgatg 31 Upstreamsequenceof ligDgene 30 taggtagtgaacctatttcgagagcag 32 Upstreamsequenceof ligDgene 31 taggttcactacctagcggccgcacaggcaccttgcatcatcatc 33 Downstream sequenceofligDgene 32 ctctagaggatccccggaccgacgattcgttgaagag 34 Downstream sequenceofligDgene 33 aggtatcgaattcccgacgagctcgtacagatctttg 35 markerrecycling sequence 34 ccatgggaaatgcccgggagagcagagtgcatggaatactag 36 markerrecycling sequence 35 gcactctgctctcccggtggtgggaaatcttgtatataattgtgattg 37 pyGgene 36 ccatgggaaatgcccgggcgacactggaagaactgcttgaagag 38 pyGgene 37 cctgccgcgagatctgggcatttcccatgggcctaacccaaatc 39 enoApromotergene 38 ccatgggaaatgcccagatctcgcggcagggttgacacagttgac 40 enoApromotergene 39 gcactctgctctcccagtaacccattcccggttctctagc 41 *1 40 atgtcgttgaataccgacgattccggtc 42 *1 *1: agdA terminator gene, artificial transcription gene
[Construction of CBH1 Producing Strain with Cis Elements Linked]

(89) First, the genomic DNA gene of an Aspergillus oryzae HO4 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980) was used as a template in PCR to amplify an upstream sequence of pyrG gene using primers 41 and 42, its downstream sequence using primers 43 and 44. A cis element-linked GUS (-glucuronidase) production cassette gene fragment obtained in Example 1 was used as a template in PCR to amplify a cis element linked promoter gene using primers 45 and 46, agdA terminator gene using primers 47 and 48. The genomic DNA gene of an Acremonium cellulolyticus H1 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Sep. 5, 2011, Deposition No.: FERM BP-11508) was used as a template in PCR to amplify a cellobiohydrolase (cbh1) gene using primers 49 and 50. The genomic DNA gene of an Aspergillus awamori HA1 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12, 2013, Deposition No.: NITE BP-01751) was used as a template in PCR to amplify a gene cassette for pyrG gene expression using primers 51 and 52. DNA polymerase was used in each PCR amplification. The amplification products were each purified using the purification kit to obtain a total of 6 gene fragments.

(90) Next, plasmid pMD20 (manufactured by Takara Bio Inc.) was treated with a restriction enzyme SmaI (manufactured by Takara Bio Inc.) at 30 C. and purified using the purification kit to obtain the restriction treatment product of the plasmid pMD20 (gene fragment).

(91) Next, gene fragment of the upstream sequence of pyrG gene, gene fragment of its downstream sequence, gene fragment of the cis element linked promoter gene, gene fragment of the agdA terminator gene, gene fragment of the bch1 gene, gene fragment of the gene cassette for pyrG gene expression, and gene fragment of the plasmid pMD20 were sequentially treated with the cloning kit and used in the transformation of an E. coli HST08 strain (manufactured by Takara Bio Inc.) to construct a plasmid pPPeA8-CBH1.

(92) Next, the plasmid pPPeA8-CBH1 was used as a template in PCR amplification using primers 19 and 20 and using DNA polymerase. The amplification product was purified using the purification kit to obtain a gene fragment for koji mold transformation (pyrG-CBH1 fragment).

(93) Next, the Aspergillus oryzae HO4 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980) was transformed using the gene fragment for koji mold transformation (pyrG-CBH1 fragment) according to the standard method of the PEG-calcium technique. Subsequently, the obtained transformants were screened for a strain capable of growing in a CD plate medium to obtain a transformant which corresponds to the transformant of Example 1.

(94) The transformant is introduced with cellobiohydrolase (cbh1) gene in the chromosome, and is capable of producing cellobiohydrolase. Hereinafter, a transformant introduced with cbh1 gene in the chromosome and capable of producing cellobiohydrolase is abbreviated as CBH1 producing strain.

(95) The base sequences of the primers 41 to 52 are shown in Table 4.

(96) TABLE-US-00004 TABLE4 SEQ Primer ID No. Basesequence5.fwdarw.3 NO Remarks 41 ggatatcggatccccccagaggtgactttatccaagattccttc 43 Upstreamsequenceof pyrGgene 42 caattgccgcgaaaaattaaattgaatctatg 44 Upstreamsequenceof pyrGgene 43 gtagtggtggatacgtactccttttatg 45 Downstreamsequence ofpyrGgene 44 tcgagctcggtacccttcaggtcacgttctaagcttatcagctg 46 Downstreamsequence ofpyrGgene 45 cgtatccaccactaccactatagggcgaattgggcccgac 47 PeA8promotorgene 46 gttcaaggcagacattttgacgagctgcggaattggtcag 48 PeA8promotorgene 47 ccaccactaccccgggaagcgtaacaggatagcctagacc 49 agdAterminatorgene 48 ctgcaggatgattagagtaacccattcccggttctctagctg 50 agdAterminatorgene 49 atgtctgccttgaactctttcaatatgtacaag 51 cbh1gene 50 atcctgttacgcttcctacaaacattgagagtagtaagggttcacg 52 cbh1gene 51 ctaatcatcctgcagctccgtcattg 53 CassetteforpyrGgene expression 52 ttttcgcggcaattggcaagctcgagcatccaactaaactag 54 CassetteforpyrGgene expression
[Enzyme Production Amount Measurement Method]

(97) In order to measure the enzyme (cellobiohydrolase) production amount by the CBH1 producing strain, first, the CBH1 producing strain with 8 cis elements linked in tandem was cultured in a CD plate medium for 1 week to form spores. The spores were recovered using 0.01% POLYSORBATE 20 (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a spore suspension.

(98) Next, 30 mL of a PD medium (2 mass/volume % of dextrin, 1 mass/volume % of polypeptone, 0.1 mass/volume % of casamino acid, 0.5 mass/volume % of potassium dihydrogen phosphate, 0.05 mass/volume % of magnesium sulfate, and 0.1 mass/volume % of sodium nitrate) was placed in a 100 mL Erlenmeyer flask, to which the spores were inoculated at a final spore concentration of 110.sup.4/mL.

(99) Next, liquid culture was performed at 30 C. for 6 days, to obtain a culture solution of CBH1 producing strain in which cellobiohydrolase (CBH1) was secreted and expressed in the culture medium.

(100) Next, the CBH1 concentration in the culture solution was measured by SDS-PAGE analysis. BSA of 0.25 g, 0.5 g, and 2 g were migrated at the same time as the reference of the protein, and the CBH1 concentration in the culture solution 10 L was calculated by image analysis using an image automatic detection system (manufactured by BIO-RAD Corporation, product name: ChemiDoc XRS+system). The result is shown in FIG. 7.

COMPARATIVE EXAMPLE 3

(101) In this Comparative Example, a CBH1 producing strain was constructed in totally the same way as in Example 3 except that the artificial transcription factor gene was not introduced and no cis element was linked.

(102) Next, the CBH1 concentration in the culture solution was measured in totally the same way as in Example 3 except that the CBH1 producing strain obtained in this Comparative Example was used. The result is shown in FIG. 7.

(103) From FIG. 7, when the liquid culture was performed at 30 C. for 6 days with dextrin as a substrate, according to the CBH1 producing strain of Example 3, it is obvious that 24.3 times enzyme (cellobiohydrolase, CBH1) can be produced compared to the CBH1 producing strain of Comparative Example 3 in which the artificial transcription factor gene was not introduced and no cis element was linked.

REFERENCE SIGNS LIST

(104) 1 base sequence for protein expression 2 protein 3 gene 4 promoter 5 cis element 6 artificial transcription factor

(105) TABLE-US-00005 SequenceListing <110> HONDAMOTORCO.,LTD. <120> Basesequenceforproteinexpressionandproductionofprotein <130> PCT160077 <160> 30 <170> PatentInversion3.5 <210> 1 <211> 20 <212> DNA <213> Aspergillusoryzae <400> 1 gacggaaaagtcgggtagat 20 <210> 2 <211> 1872 <212> DNA <213> Aspergillusoryzae <400> 2 atgtcgttgaataccgacgattccggtcggataaggacccggcaacgcgccaaaagagcg 60 tgcgaaacgtgcaaactgcgcaagaggaaatgtgacggccatgagccctgcacttactgc 120 ttgcgatacgaatatcagtgcactttcaagcctcatccacggagaaagcctgcagcttcc 180 aaatcttccgcacggcccagcgaggaagaagactcaccaaagtttctcgacagagttgat 240 gctaaccaagaacacatggaggccaactcaggcaccgctttcccccatctcctagggatg 300 aggttgaacccgcagggtgctcccaaggtgtacgggtttagctggaacctaggtgcactc 360 cccgcgccacgccgtctgtcgacgccaaaccttctagcccatgtcaatgtcttcctcaag 420 tacctgttcccgatcatgcccgtcgtgagacaggaccagctgcagcaggactgccaccag 480 ccggagcgcttgtctccccaacgctacgctttcattgccgctctatgcgcggccacgcac 540 atccaactgaagctggacggtgcagcaccgggtcccgaggcggcttccgcgcgagccagc 600 ctcgacggacatcctatgttgtcgggagaagaactcctggctgaagccgtgcgcgcaaga 660 aaggaatacaacgtggtcgacgaaattaacatggaaaacctcctaacctccttctttctc 720 ttcgccgcctacggaaacctagacagacaggatcaggcctggttctacctatgtcagacc 780 acgtccatggtcttcacactaggcctacaacgggaatccacatactcgaaactaagcgtc 840 gaggaagcagaagagaaaaggagagtattctggctcttattcgtcacagaaaggtaagaa 900 aagaaaaaactctactttcccaatcaccaccacgtaccaaaaataacacgaaaaaccaga 960 ggctacgcattacaacaagcaaaaccagtcatgctccgcaactccatccacaaaccacag 1020 gtcctgtgctcagacgacccaatcctagcctacggcttcatcaacctcatcaacgtcttc 1080 gaaaagctcagcccaaatctctacgactgggtctccgccggcggcagcagcgcagacggc 1140 gaccccccgcctacttcttctatccaatccagtctcgccaagcaaatctccctcgagggc 1200 gtctccgagatccagaaagtagacatcctcatcactcagcaatggctacaaaccatgatg 1260 tggaaactctccatgacccacgtcacacagcccggctctcgcgatgacgccgttctcccc 1320 ttccacctgcccgtgctagtcggcaaggccgtcatgggcgtcatcgccgcggcatcccaa 1380 ggtgctgttgacgctcatggtatcggaatggtaagaaagcgaccttacctcatcacaccc 1440 tccctcatcagtcactccccatcatctatacccgcaatctaacaaaaaccgcaggaacaa 1500 aaactctacgacctcggcacctccgtagccgacgtctcccgctccctaagcacaaaagcc 1560 gcccaccacctcgccgaatcgaccatcgacccccgagaactcctctggggcattctcaca 1620 accctatcccgaatccgcggttcccaatcatacctcttcccagcgctcgtcgagcaaagt 1680 cgaggcatcatcagtttcgactgttcgctttccatcagtgactttctgccttcgtttggt 1740 gggccgccggctattatgtggcggacgggtgaatctgggtttgatttattggggatcgcg 1800 gatgatttgcaagagagggagaatgagggtggggaggggattgtggtggctggggaggag 1860 atttcgattga 1872 <210> 3 <211> 37 <212> DNA <213> Aspergillusoryzae <400> 3 ccggctcgtatgttgctggaccaaccgccaaggttag 37 <210> 4 <211> 28 <212> DNA <213> Aspergillusoryzae <400> 4 actgaattgcaattaatggcggacaatg 28 <210> 5 <211> 46 <212> DNA <213> Aspergillusoryzae <400> 5 tgtctcggaccttacgtgtcttagatgcgactcaatacaactgttc 46 <210> 6 <211> 45 <212> DNA <213> Aspergillusoryzae <400> 6 tgggtaacgccagggttgaggctgaagacttaaatacgacattgc 45 <210> 7 <211> 46 <212> DNA <213> Aspergillusoryzae <400> 7 ctgttacgcttccccgggtttgaaggtggtgcgaactttgtagttc 46 <210> 8 <211> 29 <212> DNA <213> Aspergillusoryzae <400> 8 gtaaggtccgagacagtaagggattgatc 29 <210> 9 <211> 42 <212> DNA <213> Aspergillusoryzae <400> 9 taattgcaattcagtagtaacccattcccggttctctagctg 42 <210> 10 <211> 46 <212> DNA <213> Aspergillusoryzae <400> 10 gtaacgccagggcccggggaagcgtaacaggatagcctagacccac 46 <210> 11 <211> 40 <212> DNA <213> Aspergillusoryzae <400> 11 ctgcaggatgattagcgtgcaaaccaagcaaacaagcatc 40 <210> 12 <211> 28 <212> DNA <213> Aspergillusoryzae <400> 12 actgaattgcaattaatggcggacaatg 28 <210> 13 <211> 42 <212> DNA <213> Aspergillusawamorii <400> 13 taattgcaattcagtgcaagctcgagcatccaactaaactag 42 <210> 14 <211> 47 <212> DNA <213> Aspergillusawamorii <400> 14 tgggtaacgccagggcccgggctaatcatcctgcagctccgtcattg 47 <210> 15 <211> 24 <212> DNA <213> Escherichiacoli <400> 15 ccctggcgttacccaacttaatcg 24 <210> 16 <211> 27 <212> DNA <213> Escherichiacoli <400> 16 caacatacgagccggaagcataaagtg 27 <210> 17 <211> 43 <212> DNA <213> Aspergillusoryzae <400> 17 cgcaccaccttcaaaatgtcgttgaataccgacgattccggtc 43 <210> 18 <211> 27 <212> DNA <213> Aspergillusoryzae <400> 18 acctaggttccagctaaacccgtacac 27 <210> 19 <211> 40 <212> DNA <213> Aspergillusoryzae <400> 19 atcctgttacgcttctcaaaacgaaatctcctccccagcc 40 <210> 20 <211> 31 <212> DNA <213> Aspergillusoryzae <400> 20 agctggaacctaggtgcactccccgcgccac 31 <210> 21 <211> 31 <212> DNA <213> Aspergillusoryzae <400> 21 cagtgagcgcaacgcaattaatgtgagttag 31 <210> 22 <211> 27 <212> DNA <213> Aspergillusoryzae <400> 22 gggatgtgctgcaaggcgattaagttg 27 <210> 23 <211> 40 <212> DNA <213> Aspergillusoryzae <400> 23 ccgctgctaggcgcgccgtgcactatagggcgaattgggc 40 <210> 24 <211> 44 <212> DNA <213> Aspergillusoryzae <400> 24 tggggtactacaggacgtaacattttgacgagctgcggaattg 44 <210> 25 <211> 43 <212> DNA <213> Aspergillusoryzae <400> 25 cacggcgcgcctagcagcgggtagtggtggatacgtactcctt 43 <210> 26 <211> 26 <212> DNA <213> Aspergillusoryzae <400> 26 ttcaggtcacgttctaagcttatcag 26 <210> 27 <211> 37 <212> DNA <213> Aspergillusoryzae <400> 27 cccccctccggatgatgtagaagttgctcggtagctg 37 <210> 28 <211> 36 <212> DNA <213> Aspergillusoryzae <400> 28 cccccctccggacaattgccgcgaaaaattaaattg 36 <210> 29 <211> 24 <212> DNA <213> Aspergillusoryzae <400> 29 ccagaggtgactttatccaagatt 24 <210> 30 <211> 44 <212> DNA <213> Aspergillusoryzae <400> 30 caattccgcagctcgtcaaaatgttacgtcctgtagaaacccca 44 <210> 31 <211> 39 <212> DNA <213> Aspergillusoryzae <400> 31 tcgagctcggtacccggttactgctctcccttgatgatg 39 <210> 32 <211> 27 <212> DNA <213> Aspergillusoryzae <400> 32 taggtagtgaacctatttcgagagcag 27 <210> 33 <211> 45 <212> DNA <213> Aspergillusoryzae <400> 33 taggttcactacctagcggccgcacaggcaccttgcatcatcatc 45 <210> 34 <211> 37 <212> DNA <213> Aspergillusoryzae <400> 34 ctctagaggatccccggaccgacgattcgttgaagag 37 <210> 35 <211> 37 <212> DNA <213> Aspergillusoryzae <400> 35 aggtatcgaattcccgacgagctcgtacagatcatg 37 <210> 36 <211> 42 <212> DNA <213> Aspergillusoryzae <400> 36 ccatgggaaatgcccgggagagcagagtgcatggaatactag 42 <210> 37 <211> 48 <212> DNA <213> Aspergillusoryzae <400> 37 gcactctgctctcccggtggtgggaaatcttgtatataattgtgattg 48 <210> 38 <211> 44 <212> DNA <213> Aspergillusoryzae <400> 38 ccatgggaaatgcccgggcgacactggaagaactgcttgaagag 44 <210> 39 <211> 44 <212> DNA <213> Aspergillusoryzae <400> 39 cctgccgcgagatctgggcatacccatgggcctaacccaaatc 44 <210> 40 <211> 45 <212> DNA <213> Aspergillusoryzae <400> 40 ccatgggaaatgcccagatctcgcggcagggttgacacagttgac 45 <210> 41 <211> 40 <212> DNA <213> Aspergillusoryzae <400> 41 gcactctgctctcccagtaacccattcccggttctctagc 40 <210> 42 <211> 44 <212> DNA <213> Aspergillusoryzae <400> 42 atgtcgttgaataccgacgattccggtc 28 <210> 43 <211> 44 <212> DNA <213> Aspergillusoryzae <400> 43 ggatatcggatccccccagaggtgactttatccaagattccttc 44 <210> 44 <211> 32 <212> DNA <213> Aspergillusoryzae <400> 44 caattgccgcgaaaaattaaattgaatctatg 32 <210> 45 <211> 28 <212> DNA <213> Aspergillusoryzae <400> 45 gtagtggtggatacgtactccattatg 28 <210> 46 <211> 44 <212> DNA <213> Aspergillusoryzae <400> 46 tcgagctcggtacccttcaggtcacgttctaagcttatcagctg 44 <210> 47 <211> 40 <212> DNA <213> Aspergillusoryzae <400> 47 cgtatccaccactaccactatagggcgaattgggcccgac 40 <210> 48 <211> 40 <212> DNA <213> Aspergillusoryzae <400> 48 gttcaaggcagacanttgacgagctgcggaattggtcag 40 <210> 49 <211> 40 <212> <213> Aspergillusoryzae <400> 49 ccaccactaccccgggaagcgtaacaggatagcctagacc 40 <210> 50 <211> 42 <212> DNA <213> Aspergillusoryzae <400> 50 ctgcaggatgattagagtaacccattcccggttctctagctg 42 <210> 51 <211> 33 <212> DNA <213> Acremoniumcellulolyticus <400> 51 atgtctgccttgaactcntcaatatgtacaag 33 <210> 52 <211> 46 <212> <213> Acremoniumcellulolyticus <400> 52 atcctgttacgcttcctacaaacattgagagtagtaagggttcacg 46 <210> 53 <211> 26 <212> DNA <213> Aspergillusoryzae <400> 53 ctaatcatcctgcagctccgtcattg 26 <210> 54 <211> 42 <212> DNA <213> Aspergillusoryzae <400> 54 tatcgcggcaattggcaagctcgagcatccaactaaactag 42