Enhancers for promoters of filamentous fungi and their applications

Abstract

To realize a novel promoter enhancer for filamentous fungi. [Solution] An enhancer for a filamentous fungus promoter containing a polynucleotide having the base sequence shown in sequence A below: Sequence A: TXXXXCCAC; (wherein sequence A, X represents any base).

Description

[Technical Field]

[0001] This invention relates to an enhancer for a promoter of filamentous fungi and its use. [Background technology]

[0002] Conventionally, a technique using filamentous fungi as hosts for protein production by genetic engineering is known. In this technique, various useful promoters have been sought to achieve high protein expression. For example, Patent Document 1 discloses a promoter for filamentous fungi in which a promoter sequence derived from the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA), an enhancer sequence, and a promoter sequence derived from the alcohol dehydrogenase gene (adh) from Aspergillus oryzae are linked in that order.

[0003] As an enhancer used in promoters for filamentous fungi, for example, the enhancer described in Patent Document 2 is known. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2022-086783 [Patent Document 2] Japanese Patent Application Publication No. 9-9968 [Overview of the project] [Problems that the invention aims to solve]

[0005] One aspect of the present invention aims to realize a novel promoter enhancer for filamentous fungi. [Means for solving the problem]

[0006] As a result of diligent research, the inventors have discovered a novel promoter enhancer for filamentous fungi. In other words, the present invention includes the following configuration. <1>An enhancer for a filamentous fungus promoter, comprising: an enhancer comprising a polynucleotide containing the nucleotide sequence shown in the following Array A: Array A: TXXXXCCAC (In the above Array A, X means any base.). <2>A filamentous fungus promoter comprising the enhancer according to <1>. <3>A vector comprising the filamentous fungus promoter according to <2>. A vector in which a gene encoding a target protein is linked downstream of a filamentous fungus promoter contained in the vector according to <3>. <5>A transformation method, characterized by transforming a filamentous fungus using the vector according to <4>. <6>A transformant obtained by the transformation method according to <5>. <7>A method for producing a protein, comprising the step of culturing the transformant according to <6>.

Advantages of the Invention

[0007] According to one aspect of the present invention, a novel enhancer for a filamentous fungus promoter is provided.

Brief Description of the Drawings

[0008] [Figure 1] It is a schematic diagram showing the outline of a control vector and an enhancer evaluation vector according to an embodiment of the present invention. [Figure 2] It is a diagram showing the measurement results of the CALB relative activity of each vector according to Example 1 of the present invention. [Figure 3] It is a diagram showing the measurement results of the CALB relative activity of each vector according to Example 2 of the present invention. [Figure 4] It is a schematic diagram showing the outline of an evaluation vector according to Example 3 of the present invention. [Figure 5] It is a diagram showing the measurement results of the CALB relative activity according to Example 3 of the present invention. [Figure 6]This figure shows the measurement results of the CALB relative activity according to Example 4 of the present invention. [Figure 7] This figure shows the measurement results of mRNA levels according to Example 5 of the present invention. [Figure 8] This figure shows the measurement results of the CALB relative activity according to Example 6 of the present invention. [Figure 9] This figure shows the measurement results of the CALB relative activity according to Example 7 of the present invention. [Modes for carrying out the invention]

[0009] One embodiment of the present invention is described below, but the present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. All academic and patent documents cited herein are incorporated herein by reference. Unless otherwise specified herein, "A to B" representing a numerical range is intended to mean "greater than or equal to A and less than or equal to B".

[0010] This specification describes polynucleotides based on the case where the polynucleotide is DNA. If the polynucleotide is RNA, simply replace "T (thymine)" with "U (uracil)" in the following description.

[0011] In this specification, the 5' end of the DNA strand of interest may be referred to as "upstream," and the 3' end as "downstream." For example, "upstream of sequence XX" means "to the 5' end of sequence XX." "Downstream of sequence XX" means "to the 3' end of sequence XX." "From upstream to downstream" means "from the 5' end to the 3' end."

[0012] In this specification, "polynucleotide" includes RNA and DNA. An example of a polynucleotide in the form of RNA is mRNA. Examples of polynucleotides in the form of DNA include various DNA fragments, cDNA, and genomic DNA. RNA and DNA can have any structure (double-stranded or single-stranded, for example).

[0013] [1. Enhancer] An enhancer according to one embodiment of the present invention (hereinafter also referred to as "this enhancer") is an enhancer for a filamentous fungus promoter and is a polynucleotide containing the base sequence shown in the following sequence A: Sequence A: TXXXXCCAC. Here, in sequence A, X means any base. In this specification, "enhancer" means a DNA region that positively regulates the expression level of a protein by a promoter, and "enhancer sequence" means the base sequence contained in the polynucleotide contained in the enhancer.

[0014] As a result of diligent research by the inventors, we have uniquely discovered that an enhancer containing the enhancer sequence shown in sequence A acts as an enhancer for filamentous fungal promoters. Such an enhancer sequence as shown in sequence A is previously unknown, and this effect by the enhancer represents a surprising finding that could not have been predicted from prior art.

[0015] The portion corresponding to X in sequence A may contain any combination of base sequences. That is, any enhancer containing any base sequence will act as an enhancer for the filamentous fungus promoter, as long as it contains at least the T at position 1 and CCAC at positions 6-9 shown in sequence A.

[0016] In order to distinguish between X in array A, X 1 ~X 4 This is how it is written. This is for convenience, intended to distinguish X contained in sequence A, and is not intended to imply any limitation. In this case, sequence A is TX 1 X 2 X3 X 4 It is represented as CCAC.

[0017] In the said array A, X 1 , X 2 , X 3 , and X 4 are arbitrary bases and can be any of A (adenine), T (thymine), G (guanine), and C (cytosine). X 1 is preferably C or G, more preferably G. X 2 is preferably A or G, more preferably G. X 3 is preferably T, G, or C, more preferably T or G, and even more preferably T. X 4 is preferably G or C, more preferably G. With the said configuration, the expression level of the protein by the promoter for filamentous fungi containing this enhancer can be improved.

[0018] In one embodiment, the enhancer sequence preferably contains any one or more of the following sequences A1 to A3. With the said configuration, the expression level of the protein by the promoter for filamentous fungi containing this enhancer can be further improved. Sequence A1: TGGTGCCAC Sequence A2: TCACCCCAC Sequence A3: TGGGGCCAC In one embodiment, the base sequence contained in the enhancer sequence may be the following sequences B to E. Sequence B: TX 1 X 2 X 3 X 4 CCACX 5 Sequence C: TX 1 X 2 X 3 [[ID=�4]]X 4 [[ID=�6]]CCACX 5 G Sequence D: TX 1 X 2 X 3 X 4 CCACX5 GX 6 Sequence E:TX 1 X 2 X 3 X 4 CCACX 5 GX 6 X 7 Among the aforementioned sequences B to E, X 5 , X 6 , and X 7 X is any base, and can be any of A (adenine), T (thymine), G (guanine), or C (cytosine). 5 is preferably T or G, and more preferably T. 6 is preferably T or C, and more preferably T. 7 is preferably A or C, and more preferably A. In one embodiment, the enhancer sequence may include one or more sequences selected from any of the following: Array B1:TGGTGCCACT (Sequence ID 6) Array C1:TGGTGCCACTG (Sequence ID 5) Array D1:TGGTGCCACTGT (Sequence ID 4) Sequence E1:TGGTGCCACTGTA (Sequence ID 3) Sequence E2:TCACCCCACGGCC (Sequence ID 8) Array E3:TGGGGCCACGGTC (Sequence ID 9) This enhancer may contain any base sequence other than sequence A, as long as it includes the aforementioned sequence A. That is, any base sequence may be added downstream of sequence A, or any base sequence may be added upstream of sequence A. Furthermore, the number of bases in this enhancer may be 9 bp to 103 bp, more preferably 9 bp to 63 bp, and even more preferably 9 bp to 13 bp.

[0019] [2. Promoter for filamentous fungi] A promoter for filamentous fungi according to one embodiment of the present invention (hereinafter also referred to as "the promoter") includes the enhancer. In other words, the promoter includes a promoter sequence and an enhancer sequence. The promoter may include only one type of enhancer sequence or multiple types. Furthermore, the enhancer sequence may be inserted in reverse. That is, a sequence obtained by swapping the 5' and 3' ends of the enhancer sequence exemplified above may be adopted as the enhancer sequence. The position in which the enhancer sequence is introduced is not particularly limited, and the enhancer sequence may be introduced at any position in the promoter sequence.

[0020] This promoter may have a repeating structure of enhancer sequences. In other words, in this promoter, multiple enhancer sequences may be linked in tandem. The number of repeats of the enhancer sequence in this promoter is preferably two or more, more preferably four or more, even more preferably six or more, and particularly preferably ten or more. If the number of repeats of the enhancer sequence is within the above range, the amount of protein expression by this promoter can be improved. In addition, linker sequences may be included between enhancer sequences as needed in order to link multiple enhancer sequences in tandem.

[0021] The promoter into which the enhancer sequence is introduced is not particularly limited, but examples include the promoter of the A. oryzae-derived enoA gene (SEQ ID NO: 1), the promoter of the A. oryzae-derived tef1 gene (SEQ ID NO: 12), the fusion promoter of the gpdA gene and the adh gene described in Japanese Patent Application Publication No. 2022-086783, promoters of hydrolytic enzyme genes such as α-amylase, glucoamylase, α-glucosidase, protease, lipase, cellulase, cellobiohydrase, and acetamidase, and promoters of glycolytic enzyme genes such as 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase.

[0022] [filamentous fungi] The filamentous fungi to which this promoter is applied are not particularly limited. That is, the filamentous fungi to which the transformation method according to one embodiment of the present invention is applied, and the filamentous fungi that serve as hosts for the transformants according to one embodiment of the present invention, are not particularly limited.

[0023] Examples of filamentous fungi include Aspergillus, Penicillium, Trichoderma, Rhizopus, Mucor, Monascus, and Fusarium. Among these, this promoter is particularly effective in Aspergillus fungi. This is because the promoter is composed of a nucleotide sequence derived from Aspergillus oryzae.

[0024] Examples of filamentous fungi of the genus Aspergillus include Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Aspergillus usamii, Aspergillus kawachii, Aspergillus sojae, Aspergillus nidulans, Aspergillus aculeatus, Aspergillus terreus, Aspergillus phoenicis, Aspergillus japonicus, and Aspergillus tubingensis. (Tubingensis) is one example.

[0025] Examples of filamentous fungi belonging to the genus Penicillium include Penicillium camemberti, Penicillium notatum, Penicillium multicolor, Penicillium purpurogenum, and Penicillium roqueforti.

[0026] Examples of filamentous fungi belonging to the genus Trichoderma include Trichoderma reesei and Trichoderma viride.

[0027] Examples of filamentous fungi of the genus Rhizopus include Rhizopus oryzae and Rhizopus japonicus.

[0028] An example of a filamentous fungus of the genus Mucor is Mucor sp.

[0029] An example of a filamentous fungus belonging to the genus Monascus is Monascus purpureus.

[0030] An example of a filamentous fungus belonging to the genus Fusarium is Fusarium oxysporum.

[0031] [3. Vector] A vector according to one embodiment of the present invention (hereinafter also referred to as "this vector") includes this promoter. This vector may have a gene encoding the target protein downstream of this promoter. When the gene encoding the target protein is included in the vector, a transformant that highly expresses the target protein can be obtained by recombining filamentous fungi with this vector.

[0032] The specific form of this vector is not particularly limited, but a plasmid is one example. Plasmid vectors may be circular or chain-like in shape.

[0033] The origin of the polynucleotides constituting this vector is not particularly limited. For example, when applying the present invention to food products, self-cloning is preferable. When self-cloning is performed, each polynucleotide constituting the vector originates from the same organism (same species) as the host into which the vector is introduced. Since the promoter for filamentous fungi is derived from Aspergillus oryzae, in the above case, the polynucleotides constituting the vector are preferably derived from Aspergillus filamentous fungi, and more preferably from Aspergillus oryzae.

[0034] Each polynucleotide constituting this vector can be obtained by known techniques. For example, the desired sequence can be obtained using DNA amplification methods such as PCR based on known sequence information. Alternatively, it can be obtained by cloning from a chromosomal DNA library of filamentous fungi.

[0035] [Target protein] The type of target protein is not particularly limited. Examples of target proteins include intracellular proteins and secretory proteins derived from filamentous fungi, proteins derived from prokaryotes, and proteins derived from eukaryotes. More specific examples include enzymes (α-amylase, glucoamylase, α-glucosidase, β-galactosidase, cellulase, chitinase, protease, aminopeptidase, carboxypeptidase, lipase, phospholipase, phytase, nuclease, catalase, glucose oxidase, glucose dehydrogenase, pectinase, esterase, etc.), ribosomal proteins, transporters, chaperones, transcription factors, lectins, milk proteins, antimicrobial proteins, antibodies, short-chain antibodies, etc. Proteins with unknown functions may also be used as target proteins. Reporters may also be used as target proteins.

[0036] This vector contains one or more expression units for expressing the target protein in host filamentous fungal cells. The expression unit includes a promoter region (such as this promoter), an open reading frame of the gene encoding the target protein, and a terminator region.

[0037] [Other arrangements] This vector can be modified to include terminators, splicing signals, poly(A) addition signals, selection markers, origins of replication, restriction enzyme recognition sequences used to introduce target genes, and other features known in this art. Furthermore, the gene encoding the target protein may be linked to a gene encoding another protein (such as glutathione S-transferase or protein A). Such a configuration allows for the expression of a fusion protein of the target protein and the other protein. This fusion protein can be separated into its individual proteins by cleavage using a suitable protease.

[0038] This vector may contain one or more selection markers. Commonly used markers for selecting filamentous fungal transformants can be used as selection markers. Specific examples include niaD, sC, argB, adeA, ptrA, and pyrG. Alternatively, drug resistance genes used for bacterial culture, such as those for E. coli (e.g., tetracycline resistance genes, ampicillin resistance genes), may be used as selection markers. The selection markers can be used to confirm whether or not the vector has been introduced into host cells.

[0039] The target protein may be expressed as a fusion polypeptide with a selected marker. For example, green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria may be used as the selected marker, and the target protein may be expressed as a GFP fusion polypeptide.

[0040] This vector may contain a restriction enzyme recognition sequence. A restriction enzyme recognition sequence that is present only once in the vector and not within the target sequence can be used when introducing the target sequence into the vector. Considering the versatility of the vector, the restriction enzyme recognition sequence is preferably a recognition sequence of a restriction enzyme that recognizes 6 or more bases, which have a low frequency of recognition site occurrence, and more preferably a recognition sequence of a restriction enzyme that recognizes 8 or more bases. Examples of restriction enzyme recognition sequences that recognize 6 bases include GGGCCC (ApaI), GGATCC (BamHI), ATCGAT (ClaI), AAGCTT (HindIII), CCATGG (NcoI), CATATG (NdeI), CACGTG (PmlI), GCATGC (SphI), TCTAGA (XbaI), and CTCGAG (XhoI). Examples of restriction enzyme recognition sequences that recognize 8 or more bases include GGCGCGCC(AscI), GCGATCGC(AsiSI), GGCCGGCC(FseI), GCGGCCGC(NotI), TTAATTAA(PacI), GTTTAAAC(PmeI), CCTGCAGG(SbfI), GCCCGGGC(SrfI), and ATTTAAAT(SwaI).

[0041] This vector may contain polynucleotides derived from the host fungus necessary for homologous recombination. The length of the host fungus-derived polynucleotides is not particularly limited. Considering the efficiency of homologous recombination, a length of 200 bp to 20,000 bp is preferred, and 500 bp to 10,000 bp is more preferred. A length of 200 bp or more allows for efficient homologous recombination. A length of 20,000 bp or less allows for efficient uptake of the vector into the host fungal cells.

[0042] [4. Method for transformation, transformant, and protein production] A transformation method according to one embodiment of the present invention (hereinafter also referred to as "this transformation method") transforms a filamentous fungus with the vector described in Section [3. Vectors]. This transformation method yields a transformant according to one embodiment of the present invention (hereinafter also referred to as "this transformant"). Furthermore, a protein production method according to one embodiment of the present invention (hereinafter also referred to as "this protein production method") can be carried out by culturing this transformant. According to these embodiments, the target protein can be produced with high efficiency.

[0043] In this transformation method, known methods are appropriately employed for transforming filamentous fungi. Specific examples include the calcium-PEG method and the electroporation method.

[0044] Examples of filamentous fungi that can serve as hosts in this transformed organism include the filamentous fungi mentioned above.

[0045] In the method for producing this protein, the culture method for the transformed organism can be any medium and culture conditions that are commonly used for culturing filamentous fungi. The transformed organism may be cultured in solid or liquid form. Examples of culture media used include: Modified dextrin-peptone (DPY) medium (4% dextrin, 2% polypeptone, 2% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O), Modified glucose-peptone (GPY) medium (4% glucose, 2% polypeptone, 2% yeast extract, 0.5% KH2PO4, 0.05% MgSO4, 7H2O), Modified Czapek-dox medium (0.2% NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.2% KCl, 0.002% CuSO4·5H2O, 0.001% FeSO4·7H2O, 0.0001% ZnSO4·7H2O, 0.0001% MnSO4·5H2O, 0.0001% AlCl3, 2%) Examples of culture media include glucose (pH 5.5) and wheat bran medium (wheat bran, moisture content 40%). The culture temperature can be any temperature within the range in which the transformants can grow. An example of a culture temperature is 20-37°C.

[0046] The culture of this transformed organism contains the target protein. If necessary, the target protein may be subjected to qualitative or quantitative analysis. Alternatively, the target protein may be isolated or purified from the culture.

[0047] If the transformed organism is cultured in liquid and the target protein is secreted outside the cell, the culture medium itself can be recovered and used for analysis, isolation, or purification of the target protein. Alternatively, if the transformed organism is cultured in solid form and the target protein is secreted outside the cell, a solution containing the target protein can be extracted from the culture using a suitable buffer solution and used for analysis, isolation, or purification of the target protein. If the transformed organism is cultured in solid form and the target protein accumulates within the cell, the cell can be disrupted by known means, and the target protein can be extracted using a suitable buffer solution and used for analysis, isolation, or purification of the target protein. In protein production systems using filamentous fungi, the target protein is often secreted outside the cell.

[0048] If the target protein recovered as described above needs to be further purified, for example, the protein-containing solution can be purified by known methods. Examples of such methods include ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (HPLC) is preferably used for protein purification. [Examples]

[0049] [Example 1. Confirmation of the enhancer's effect] [Reporter protein] Lipase B (CALB) derived from Candida antarctica was used as the reporter protein.

[0050] [Vector generation method] Schematic diagrams of the control vector and the enhancer evaluation vector are shown in Figure 1. As shown in Figure 1, the control vector has the PenoA sequence (sequence number 1). On the other hand, the enhancer evaluation vector has the enhancer sequence between positions 155 and 156 of the PenoA sequence. The manufacturing methods for the control vector and the enhancer evaluation vector are described below.

[0051] (Control vector) To use the upstream 512 bp (SEQ ID NO: 1) of the enoA gene derived from Aspergillus oryzae RIB40 as the promoter sequence (PenoA), PCR was performed using the genomic DNA of A. oryzae RIB40 as a template to amplify the gene fragment containing the sequence of SEQ ID NO: 1. Furthermore, using the CALB expression control vector described in Example 1 of Patent 7540709 as a template, a fragment (CALB-T2512-sC-pUC118) with only the promoter portion removed was amplified by PCR. Each of the two gene fragments amplified by PCR was subjected to agarose gel electrophoresis, and the target bands were extracted and purified. The purified gene fragments were ligated together by seamless cloning to form a control vector.

[0052] (Enhancer evaluation vector) Using primers with enhancer sequences A1-E1 and W1-Z1 added downstream of positions 1-155 of SEQ ID NO: 1, gene fragments containing positions 1-155 of SEQ ID NO: 1 and either A1-E1 or W1-Z1 were amplified by PCR using A. oryzae RIB40 genomic DNA as a template. Furthermore, gene fragments containing positions 156-512 of SEQ ID NO: 1 were amplified by PCR using A. oryzae RIB40 genomic DNA as a template. The two types of gene fragments amplified by PCR were subjected to agarose gel electrophoresis, and the target bands were extracted and purified. The two purified gene fragments and a CALB expression control vector fragment with only the promoter portion removed were ligated by seamless cloning to obtain an enhancer evaluation vector.

[0053] [Preparation of transformed organisms] Using the prepared vector, Aspergillus oryzae NS4 strain (a double knockout strain of niaD and sC obtained by mutagenesis) was transformed using the protoplast-PEG method. Transformants were selected based on complementation of nutritional requirements by the sC gene contained in the vector. Genomic DNA was extracted from the obtained transformants, and transformants with only one copy of the expression cassette were selected by real-time PCR. The selected transformants were cultured at 30°C in modified DPY medium (4% dextrin, 2% polypeptone, 2% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O), and the culture supernatant was used as the crude enzyme solution.

[0054] [Measurement of CALB activity] 5 μL of p-nitrophenyl butyrate was added to 250 μL of 100% ethanol and suspended using a pipette. This suspension was made up to 50 mL with distilled water to obtain the PNPB solution. Next, 150 μL of the PNPB solution was mixed with 50 μL of 20 mM phosphate buffer (pH 7.0) to obtain the substrate solution. To the obtained substrate solution, 2 μL of crude enzyme solution, diluted as appropriate, was added to initiate the reaction. The reaction temperature was 37°C and the reaction time was 3 minutes. CALB activity was measured by measuring the increase in absorbance at 400 nm. The results of the CALB activity measurement are shown in Figure 2. In Figure 2, the activity value of the enhancer evaluation vector is shown as relative activity, with the activity with the control vector introduced set to 1.00.

[0055] As shown in Figure 2, transformants containing the enhancer sequence A1:TGGTGCCAC(9bp) exhibited significantly higher CALB activity compared to transformants containing the sequence Z1:TGGTGCCA(8bp). Furthermore, transformants containing any of the enhancer sequences B1, C1, D1, or E1 showed higher CALB activity than those containing sequence Z1. This indicates that the "C" at position 9 in the enhancer sequence plays an important role in CALB activity.

[0056] On the other hand, transformants containing sequence W1, which lacks the T at position 1 of sequence E1, showed lower CALB activity than the control. This indicates that the T at position 1 of the enhancer sequence plays an important role in CALB activity. These results suggest that the T at position 1 and the C at position 9 of the nucleotide sequence contained in this enhancer are at least necessary for improving protein production efficiency.

[0057] [Example 2. Confirmation of enhancer effect in the same region of other Aspergillus species] We also confirmed whether proteins could be expressed using nucleotide sequences derived from species other than A. oryzae. Sequences in the same region as the A. oryzae sequence identified in [Example 1. Confirmation of Enhancer Effect] were identified in A. niger, A. terreus, and A. nidulans. Using the method described in [1. Confirmation of Enhancer Effect], enhancer evaluation vectors were prepared by inserting sequences derived from A. niger (sequences E2, A2, and Z2), A. terreus (sequences E3, A3, and Z3), and A. nidulans (sequences E4, A4, and Z4) into the PenoA promoter, respectively, and the CALB activity was examined. The preparation of transformants and measurement of CALB activity were performed in the same manner as in [1. Confirmation of Enhancer Effect]. The results are shown in Figure 3.

[0058] As shown in Figure 3, transformants containing sequences derived from A. niger and A. terreus all exhibited higher CALB activity than the control, PenoA. Furthermore, sequence A2 showed higher activity than sequence Z2, and sequence A3 showed higher activity than sequence Z3. On the other hand, transformants containing sequences derived from A. nidulans all exhibited CALB activity similar to or lower than the control.

[0059] As shown in Figure 3, the base sequences common to sequences A2 and A3 are T at position 1 and CCAC at positions 6-9. These base sequences are also common to sequence A1 shown in Figure 2. On the other hand, sequence A4, derived from A. nidulans, which has low CALB activity, does not have these common base sequences. Furthermore, sequences Z2 and Z3 lack the C that is present at position 9 of sequence A. Therefore, it has been shown that in order to improve the efficiency of protein production, the base sequence included in the enhancer must have T at position 1 and CCAC at positions 6-9, i.e., the base sequence TXXXXCCAC. In addition, since the base included in the X portion of sequence A differs between sequences A1-A3, it has been shown that it can be any type of base.

[0060] [Example 3. Confirmation of the effect when enhancers are connected in tandem] [Method for generating evaluation vectors] The change in protein productivity was confirmed when multiple enhancer sequences (sequence A1) identified above were tandem-linked. The 800 bp upstream of the Accession number: AO090011000202 gene was used as the promoter (Native). Within this promoter, the sequence of Sequence ID No. 11 (103 bp) is present, and within the Sequence ID No. 11 sequence, the enhancer sequence A1 is present. Figure 4 schematically shows the evaluation vector obtained when one or more Sequence ID No. 11 sequences are introduced. As shown in Figure 4, in the case of the Native sequence, the number of Sequence ID No. 11 sequences in the vector is 1. When two Sequence ID No. 11 sequences are introduced, the number of Sequence ID No. 11 sequences in the vector becomes 3. In this way, genes were synthesized so that there were 3, 5, 8, and 13 Sequence ID No. 11 sequences. Each of the synthesized genes was ligated with a CALB expression control vector fragment, excluding only the promoter portion described in [Example 1. Confirmation of Enhancer Effect], using seamless cloning. This allowed us to create an evaluation vector containing multiple sequences of sequence number 11, as shown in Figure 4.

[0061] [CALB Productivity Evaluation] CALB activity was measured for each evaluation vector. The preparation of transformants and measurement of CALB activity were carried out in the same manner as in [Example 1. Confirmation of Enhancer Effect]. The results are shown in Figure 5. In Figure 5, "Native" shows the results when a vector containing one sequence of sequence number 11 was introduced. "3t," "5t," "8t," and "13t" show the results when vectors containing 3, 5, 8, and 13 sequences of sequence number 11 were introduced, respectively. Figure 5 shows that CALB activity increases as the number of sequence repeats increases. It also shows that when the number of sequence repeats exceeds 5, the increase in CALB activity with increasing repeats becomes more gradual. Therefore, it was shown that the higher the number of sequence repeats of this enhancer, the more the protein production efficiency can be improved.

[0062] [Example 4. Comparison of protein productivity with multiple promoters] We confirmed that the enhancer sequence A1 of the present invention exhibits activity in promoters other than PenoA. As promoters, we used the promoter of the A. oryzae-derived enoA gene (PenoA), the promoter of the A. oryzae-derived tef1 gene (Ptef1: SEQ ID NO: 12), and the fusion promoter of the gpdA and adh genes (PgpdA+adh). We compared the activity of the enhancer sequence A1 with that of the known enhancer sequence Region III (SEQ ID NO: 5 in Patent Document 1). As a CALB expression vector containing the Region III sequence in PenoA, we used the CALB expression control vector described in Example 1 of Patent Document 1. As a CALB expression vector containing Region III in PgpdA+adh, we used the vector described in Example 1-1 of Patent Document 1. For the promoter containing Region III in Ptef1, we designed the sequence so that the Region III sequence is introduced directly below position 531 of the Ptef1 sequence described in SEQ ID NO: 12, and synthesized the full-length promoter.

[0063] A sequence (ud-20_9t) was created by tandem-linking nine sequences of Sequence ID No. 2 (ud-20:63bp), which contains the enhancer sequence A1, into PenoA, Ptef1, and PgpdA+adh. The sequence was designed to be introduced at the same position as Region III in each promoter, and the full-length promoters were synthesized. Evaluation vectors were obtained by seamless cloning by linking each synthesized promoter with a CALB expression control vector fragment, excluding only the promoter portion described in [Example 1. Confirmation of Enhancer Effect]. Transformants were prepared in the same manner as in [Example 1. Confirmation of Enhancer Effect]. Transformants were cultured in modified DPY medium or modified GPY medium (4% glucose, 2% polypeptone, 2% yeast extract, 0.5% KH2PO4, 0.05% MgSO4, 7H2O). CALB activity was measured in the same manner as in [Example 1. Confirmation of Enhancer Effect]. The results of the CALB activity measurement are shown in Figure 6.

[0064] In Figure 6, the CALB activity values ​​for each promoter are shown as relative activity, with the activity value of the Region III promoter set to 1.00. From Figure 6, it can be seen that the activity is significantly higher than when using Region III, especially from day 4 onwards. Therefore, it has been shown that this enhancer can improve protein production efficiency even when applied to promoters other than PenoA.

[0065] [Example 5. Comparison of mRNA levels] To investigate the factors contributing to the increased reporter protein productivity with this enhancer compared to the Region III promoter, the mRNA level of the reporter protein (CALB) was confirmed. The confirmation was performed using transformants containing one copy of the CALB expression cassette, either with the Region III promoter or the PagdA+adh promoter (ud-20_9t), which was created in [Example 4. Comparison of Protein Productivity with Multiple Promoters]. The transformants were cultured in modified DPY medium, and RNA was extracted from the cells on days 2 and 4. Reverse transcription and real-time PCR were then performed to confirm the amount of CALB gene mRNA. The relative amount of CALB mRNA, with the amount of Histone H4 mRNA (selected as an internal standard) set to 1, is shown in Figure 7.

[0066] Figure 7 shows that the mRNA levels in the transformants introduced with this enhancer were significantly higher than those introduced with Region III. Therefore, it was shown that the increase in mRNA levels due to this enhancer led to an increase in protein production.

[0067] [Example 6. Comparison of productivity of other reporter proteins using A. oryzae as a host] The productivity of reporter proteins other than CALB was also compared. As reporter proteins, A. oryzae-derived α-amylase and Escherichia coli-derived β-glucuronidase (GUS) were selected. For these proteins, the production yields were compared using Region III or a promoter using this enhancer. The α-amylase gene was amplified by PCR using the genomic DNA of A. oryzae RIB40 as a template. For the Region III vector, the CALB expression control vector described in Example 1 of Patent Document 1 was used as a template, and a fragment (T2512-sC-pUC118-PgpdA+adh(Region III)) with only the CALB gene portion removed was amplified by PCR. The vector using this enhancer was prepared by introducing a sequence (ud-20_9t) consisting of nine tandem copies of the sequence number 2 (ud-20:63bp) into the PgpdA+adh promoter prepared in [Example 5. Comparison of mRNA amounts]. Using this CALB expression vector as a template, a fragment (T2512-sC-pUC118-PgpdA+adh(ud-20_9t)) with only the CALB gene portion removed was amplified by PCR. The gene fragments other than the α-amylase gene and the two reporter genes amplified by PCR were subjected to agarose gel electrophoresis, and the target band was extracted and purified. These purified fragments were ligated by seamless cloning to obtain the amylase productivity evaluation vector. For the GUS expression vector, the vector using PgpdA+adh (Region III) was the GUS expression vector described in Example 2 of Patent Document 1. For the vector using PgpdA+adh(ud-20_9t), the GUS gene was amplified by PCR using a GUS expression vector as a template, subjected to agarose gel electrophoresis, and the target band was extracted and purified. The obtained GUS gene fragment and the fragment used in the preparation of the amylase expression vector (T2512-sC-pUC118-PgpdA+adh(ud-20_9t)) were ligated by seamless cloning to obtain a GUS productivity evaluation vector.

[0068] [Amylase, GUS productivity evaluation] Transformants were prepared in the same manner as in [Example 1. Confirmation of Enhancer Effect]. The obtained transformants were cultured in modified DPY medium, and the amylase activity in the culture supernatant and the GUS activity in the cell lysate supernatant were measured on days 2, 4, and 7 of culture. Amylase activity was measured using an α-amylase measurement kit (Kikkoman Biochemifa Co., Ltd.). GUS activity was measured according to a known method (Reference: Proc Natl Acad Sci (1986) Vol.83, No.22, pp.8447-8451). The results are shown in Figure 8.

[0069] In Figure 8, the activity value of the ud-20_9t promoter is shown as relative activity, with the activity value of the RegionIII promoter set to 1.00. From Figure 8, the transformants introduced with this enhancer showed higher activity than the transformants introduced with RegionIII at all three culture days (2, 4, and 7). Therefore, this enhancer was shown to be able to improve the production of proteins other than CALB.

[0070] [Example 7. Comparison of reporter protein productivity using A. niger as a host] [Vector creation] To confirm that this enhancer is effective in hosts other than A. oryzae, experiments were conducted using A. niger as the host. As reporter proteins, A. niger-derived β-glucosidase (BGL) and Myrothecium verrucaria-derived bilirubin oxidase (BOD) were selected. For these proteins, production was compared between promoters using Region III and promoters using this enhancer. The BGL gene was amplified by PCR using the genomic DNA of A. niger NS48 strain (a niaD, sC double knockout strain obtained through mutation) as a template, and the target band was extracted and purified by agarose gel electrophoresis. The obtained BGL gene fragment was then ligated with a fragment prepared in [Example 6. Comparison of productivity of other reporter proteins using A. oryzae as the host], which was modified by introducing Region III or ud-20_9t, using seamless cloning to obtain a BGL productivity evaluation vector. Furthermore, a BOD productivity evaluation vector was obtained by seamlessly cloning a fragment of T2512-sC-pUC118-PgpdA+adh, which was prepared in the same manner as described above in [Example 6.A. Comparison of productivity of other reporter proteins using oryzae as a host], into which Region III or ud-20_9t had been introduced.

[0071] [BGL, BOD productivity evaluation] Transformants were prepared using A. niger NS48 strain as the host in the same manner as in [Example 1. Confirmation of Enhancer Effect]. The obtained transformants were cultured in modified DPY medium, and BGL activity and BOD activity were measured in the culture supernatant on days 2, 4, and 7 of culture. BGL activity was measured by the following method: 5 μL of appropriately diluted culture supernatant was added to a reaction solution of 1 mM p-nitrophenyl-β-D-glucopyranoside + 20 mM sodium acetate buffer (pH 4.5) to initiate the reaction. After reacting at 37°C for 5 minutes, 50 μL of 5% sodium carbonate solution was added, and the absorbance at 415 nm was measured. The obtained value was taken as the BGL activity value. BOD activity was measured by the following method: 2 μL of appropriately diluted culture supernatant was added to a reaction solution of 2 mM ABTS + 50 mM potassium phosphate buffer (pH 6.5) to initiate the reaction. After reacting at 37°C for 5 minutes, the BOD activity was measured by the increase in absorbance at 436 nm. The results are shown in Figure 9.

[0072] In Figure 9, the activity value of the ud-20_9t promoter is shown as relative activity, with the activity value of the RegionIII promoter set to 1.00. From Figure 9, the activity of the transformants introduced with this enhancer was similar to that of the transformants introduced with RegionIII at all three culture days (2, 4, and 7). Therefore, this enhancer is shown to be effective in hosts other than A. oryzae. [Industrial applicability]

[0073] This invention can be used, for example, in the production of proteins using filamentous fungi.

Claims

1. An enhancer for a filamentous fungus promoter is further introduced, which includes a polynucleotide containing the base sequence shown in the following sequences A1 to A3 or sequence C. Compared to the filamentous fungal promoter before the addition of the aforementioned enhancer, promoter activity is improved. Promoter for filamentous fungi: Sequence A1: TGGTGCAC Array A2: TCACCCCAC Sequence A3: TGGGGGCCAC Array C: TX 1 X 2 X 3 X 4 CCACX 5 G (In the sequence C above, X represents any base.)

2. The promoter for filamentous fungi according to claim 1, wherein a plurality of enhancers are introduced in addition.

3. A vector comprising the promoter for filamentous fungi according to claim 1 or 2.

4. A vector comprising a gene encoding a target protein, ligated downstream of a promoter for filamentous fungi contained in the vector according to claim 3.

5. A transformation method characterized by transforming a filamentous fungus using the vector described in claim 4.

6. A transformant obtained by the transformation method described in claim 5.

7. A method for producing a protein, comprising the step of culturing the transformant described in claim 6.

Images