JP3792467B2 - A novel promoter from the genus Aspergillus - Google Patents

Description

【００２３】
【実施例４】
（スモール Ｖ−ＡＴＰａｓｅ（small-V-ATPase）遺伝子、アクチン（Actin）遺伝子、ヒストンＨ２（Histone-H2）遺伝子のプロモーターの発現能の検討）
表２に示すように、スモール Ｖ−ＡＴＰａｓｅ（small-V-ATPase）遺伝子（１１９６塩基：配列番号４）、アクチン（Actin）遺伝子（１００１塩基：配列番号５）、ヒストンＨ２（Histone-H2）遺伝子（６８５塩基：配列番号８）のいずれのプロモーターによっても、ＧＵＳタンパクを生産することが可能であった。またＣｚａｐｅｋ−Ｄｏｘ培養（ツァペック−ドックス培地での培養）とＤＲＹ培養（デキストリン−ペプトン−酵母エキス培地での培養）を比較した場合、アクチン（Actin）遺伝子、ヒストンＨ２（Histone-H2）遺伝子のプロモーターでは、ＤＲＹ培養の方が、またスモール Ｖ−ＡＴＰａｓｅ（small-V-ATPase）遺伝子プロモーターの場合は、Ｃｚａｐｅｋ−Ｄｏｘ培養のほうが発現能が高いことが示された。
【００２４】

【００２５】
【実施例５】
（Ｐ−４５０遺伝子のプロモーターの発現能の検討）
表３に示すように、チトクロームＰ−４５０遺伝子のプロモーター（１２１４塩基：配列番号３）を利用してＧＵＳタンパクを生産することが可能であった。またＰ−４５０遺伝子プロモーターは、培地の窒素源の種類により、誘導や抑制を受けることが明らかとなった。硝酸や亜硝酸を単一窒素源とした場合、非常に高いＧＵＳ生産性が認められたが、アンモニアを窒素源とした場合は生産性の著しい低下が認められた。このように窒素源によってプロモーター活性が誘導、抑制を受けることは、発現能をコントロールできることになり、有効である。
【００２６】

【００２７】
【実施例６】
（カタラーゼ（catalase）遺伝子のプロモーターの発現能の検討）
表４に示すように、カタラーゼ遺伝子のプロモーターを利用してもＧＵＳタンパクを生産することが可能であった。カタラーゼＡ、Ｂ遺伝子プロモータ（５５０塩基：配列番号６）、（６６３塩基：配列番号７）は過酸化水素や鉄イオンなどの添加により、さらに発現能が増加した。
【００２８】

【００２９】
【発明の効果】
本発明によって、アスペルギルス・オリゼのマンガン・スーパーオキサイドデスムターゼ遺伝子その他７種類の遺伝子について、それぞれ、プロモーター領域及びその塩基配列が決定された。そして、これらの新規プロモーターは、レポーター遺伝子を利用するプロモーター解析の結果、強力なプロモーターであることがはじめて確認された。
【００３０】
このように本発明によって新たに開発された強力なプロモーターを利用することにより、タンパク質の新規高発現システムの構築が可能となった。この新規プロモーターを用いる新規高発現システムを利用することにより、麹菌その他の糸状菌、あるいはそれ以外の微生物を宿主菌として、同種のタンパク質はもとより異種のタンパク質も効率的に生産することができる。
【００３１】
【配列表】

【図面の簡単な説明】
【図１】プロモーター活性測定用プラスミドｐＮＧＳ１を示す。
【図２】Ｍｎ−ＳＯＤ遺伝子プロモーターの塩基配列を示す。
【図３】Ｃｕ、Ｚｎ−ＳＯＤ遺伝子プロモーターの塩基配列を示す。
【図４】チトクローム Ｐ−４５０遺伝子プロモーターの塩基配列を示す。
【図５】スモール−Ｖ−ＡＴＰａｓｅ遺伝子プロモーターの塩基配列を示す。
【図６】アクチン遺伝子プロモーターの塩基配列を示す。
【図７】カタラーゼＡ遺伝子プロモーターの塩基配列を示す。
【図８】カタラーゼＢ遺伝子プロモーターの塩基配列を示す。
【図９】ヒストン−Ｈ２遺伝子プロモーターの塩基配列を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel promoter of Aspergillus filamentous fungi. More specifically, the present invention clones a novel promoter sequence derived from Aspergillus oryzae and uses a plasmid having DNA fragments necessary for expressing useful proteins and peptides using Aspergillus oryzae as a host. The present invention relates to a method for producing useful proteins and peptides.
[0002]
[Prior art]
With the recent development of genetic recombination technology, it has become possible to produce useful human proteins using E. coli and yeast. However, it has been pointed out that when a gene derived from a eukaryote such as human is expressed using E. coli as a host, normal processing is not performed and sugar chains are not attached. In addition, when secretory production of a heterologous protein is carried out by yeast, there is a disadvantage that although the sugar chain is bound, the amount of secretion is very small. Thus, filamentous fungi with high protein secretion ability have attracted attention as hosts for eukaryotic protein expression. Among them, Aspergillus oryzae is actively used for heterogeneous gene expression because it has been used for a long time in the brewing industry such as sake and miso. Mucor miehei's acidic protease has already been industrially produced using A. oryzae as a host.
[0003]
Currently, the amylase gene promoter is often used for the production of such heterologous proteins by Aspergillus. Examples include the promoter of Aspergillus niger glucoamylase gene (Biotechnology, 5 , 368 (1983)), the promoter of Aspergillus oryzae α-amylase gene (Biotechnology, 6 , 1419 (1987)), and the like. .
[0004]
[Problems to be solved by the invention]
Amylase gene promoters, particularly α-amylase gene promoters, show very high expression even in liquid culture and have been found useful in the production of heterologous proteins (Japanese Patent Laid-Open No. 7-51067). It is necessary to add inducers such as. In addition, when a protein that produces glucose such as glucoamylase or glucosidase is expressed as a heterologous protein, a large amount of glucose is produced by the expressed protein, and thus a phenomenon in which the production amount decreases due to glucose repression is observed. Therefore, in order to broaden the possibility of producing recombinant proteins using Aspergillus oryzae, it is desirable to search for a promoter having high expression ability other than the amylase gene control system.
[0005]
In addition, as a promoter for producing a heterologous protein of Aspergillus oryzae, a target is mainly applied to a gene of a protein having a large production amount such as a secretory enzyme of Aspergillus oryzae. This is the idea that the gene promoter of such proteins is highly expressed because gonococci secrete large amounts of proteins outside the cells. However, when we look at the expression of all proteins of Neisseria gonorrhoeae, the genes for extracellular proteins are not necessarily highly expressed, and it is thought that some of the genes for intracellular proteins have high expression ability. . Therefore, when searching for a high expression promoter of Neisseria gonorrhoeae, a wider range of screening methods are required, including intracellular protein genes.
[0006]
[Means for Solving the Problems]
The present invention has been made in view of the above points, and searches for a high expression promoter of Aspergillus oryzae, isolates a clone having high expression ability in liquid culture, and produces a heterologous protein using this expression system. It was made for the purpose of providing a method.
The present invention has succeeded in achieving the above object as follows.
[0007]
Among the proteins in the cell, the protein that constitutes the cell component can be considered as a protein with a large production amount. However, the expression of these proteins is often constant regardless of the culture conditions and the culture time, and is not suitable for controlling the expression level. On the other hand, the protein corresponding to the change (stress) in the external culture condition was refocused on the fact that the expression inducing factor is clear and efficiently expressed in a short time. Therefore, in view of these circumstances, the inventors obtained the idea that a high-expression promoter effective for heterologous protein production exists in the gene of the protein corresponding to stress, and Aspergillus oryzae (RIB-40) (ATCC 42149) ) Was extracted from the EST library, and the promoter region was isolated.
[0008]
An example of the isolation method is a PCR method using aspergillus genomic DNA as a template. Genomic DNA is extracted from Neisseria gonorrhoeae, fully digested with each restriction enzyme that recognizes 6 bp such as EcoRV, ScaI, DraI, PvuII, or SspI and cuts into blunt ends, and appropriate adapters are ligated to both ends of the genomic DNA fragment. To do. PCR is carried out using a genomic DNA fragment with adapters linked to both ends as a template, a primer synthesized from the base sequence of cDNA, and a primer having the single-stranded portion of the adapter. The amplified product is confirmed by electrophoresis, and a DNA fragment having a length of about 1 to 2 kb is isolated and purified by electrophoresis. The base sequence of this fragment is determined, and the upstream of the start codon estimated from the EST sequence is used as the promoter sequence.
[0009]
Furthermore, the expression ability of the obtained promoter fragment was examined using the promoter analysis system described below. For the promoter analysis, a method was adopted in which a reporter gene was linked to the downstream region of the promoter to be examined and the activity of the reporter gene product was used as an indicator of promoter expression. As a result of examining the promoters of various genes, a suitable promoter was discovered, and as a result of further research, a high gene expression system was successfully created, and the present invention was completed.
[0010]
As a promoter analysis system, for example, in addition to pNGUS (Japanese Patent Laid-Open No. 11-243965), a promoter analysis plasmid (plasma activity measurement plasmid pNGS-1) as shown in FIG. 1 is used. This plasmid contains a niaD gene (S. Unkles et al., Mol. Gen. Genet., 218, p.99-104, 1989) of A. oryzae, which is a marker for transformation, and β-glucuronidase of Escherichia coli, which is a reporter gene. GUS) encoding uidA gene (RA Jefferson et al., Proc. Natl. Acad. Sci., P. 8447-8451, 1986), which is novel. Various gene promoters to be examined or a part thereof are inserted into the upstream region (for example, SalI, PstI sites) of the uidA gene of this plasmid, and the constructed plasmid for promoter analysis is used as a gonococcal host, for example, A. oryzae (Aspergillus oryzae GLB-01: This strain was introduced into a niaD mutant (nitrate deficiency deficient strain, Nitrate Reductase deficient) of FERM P-15826 already deposited at the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. Was named Aspergillus oryzae 1013-niaD. This strain has been deposited as FERM P-17707 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. A transformant in which only one copy of the introduced plasmid has been introduced at niaD loci on the host chromosome (a strain in which only one copy has undergone homologous recombination) is selected. Then, these transformants (gene-introduced strains) were cultured under various conditions, and GUS activity in the cells was measured to obtain promoter expression ability (promoter activity index).
[0011]
As a result of various examinations by the above screening methods, it was found that the promoters of the following genes are strongly expressed in liquid culture.
Manganese superoxide desmutase (Mn-SOD) gene, copper zinc superoxide desmutase (Cu, Zn-SOD) gene, cytochrome P-450 gene, small V-ATPase (small-V- ATPase) gene, actin gene, catalase A gene, catalase B gene, and human sun H2 (Histone-H2) gene.
[0012]
The nucleotide sequences of the promoters of the above genes according to the present invention are shown in SEQ ID NOs: 1 to 8 and FIGS.
[0013]
The promoter of the present invention is hybridized under stringent conditions with respect to the base sequence determined as described above, for example, 0.1% SDS (60 ° C., 0.3 M NaCl, 0.03 M sodium citrate). The base sequence to be included is also included.
[0014]
The promoter of the present invention can produce a significant amount of useful protein by constructing a vector linked with a desired useful protein gene downstream thereof, transforming a host with the vector, and culturing the vector. As the host, microorganisms such as Aspergillus oryzae and other Aspergillus genus, Neurospora genus, Penicillium genus, Trichoderma genus and Rhizopus genus can be used. In particular, from the viewpoint of expression efficiency, it is preferable to use Aspergillus oryzae as a host.
[0015]
The useful protein gene can be linked to the obtained promoter and inserted into the vector by a method known per se. The useful protein gene is not particularly limited. Examples of the vector include those containing auxotrophic complementary genes such as arginine requirement, carbon such as acetamide assimilation, nitrogen source utilization genes, drug resistance genes such as oligomycin resistance, and the like. Transformation of the host can also be performed by a method known per se. Further, the transformant can be cultured according to a conventional method by appropriately selecting a medium and culture conditions suitable for the desired protein, and the obtained protein can be collected and purified by a known method. it can.
[0016]
Hereinafter, the present invention will be described in detail with reference to examples, but the plasmids and the like used in the examples are given as examples, and are not limited to these as long as they can be used in the present invention. .
[0017]
[Example 1]
(Method for isolating promoter fragments of useful genes)
A template DNA library for obtaining a promoter fragment was constructed using Genome Walker manufactured by Clontech. First, A. oryzae genomic DNA is cleaved with 5 types of restriction enzymes (DraI, EcoRV, PvuII, ScaI, StuI), and asymmetric adapters are ligated to both ends of the genomic DNA cleaved with each restriction enzyme for PCR amplification. As a genomic template. Next, using the primer designed from the antisense sequence of the gene obtained from the EST information and the primer designed from the asymmetric part of the adapter, a PCR reaction is performed on the previously prepared genomic template. An example of the reaction conditions is as follows.
[0018]
(PCR conditions)
• 95 ° C. (1 minute), 1 cycle • 94 ° C. (5 seconds), 72 ° C. (3 minutes), 70 ° C. (3 minutes), 7 cycles • 94 ° C. (5 seconds), 67 ° C. (3 minutes), 70 ℃ (3 minutes), 32 cycles · 67 ℃ (4 minutes), 1 cycle
After PCR reaction for each of the five types of templates, the amplified 1-kb DNA fragment was isolated using an electrophoresis apparatus. The base sequences of these fragments were determined, and the upstream of the start codon was used as the promoter portion.
[0020]
[Example 2]
(Method for measuring promoter activity of useful genes)
First, the target promoter fragment is inserted into a promoter activity measuring plasmid. Using a promoter DNA fragment isolated by Genome Walker as a template, PCR reaction is performed with primers designed so that a SalI site is generated downstream of the promoter and a PstI site is generated upstream. Next, the amplified DNA fragment is digested with SalI PstI and then inserted upstream of the GUS gene (that is, the udiA gene) of the plasmid pNGS1 (FIG. 1) developed for measuring the promoter activity. As a result, the GUS gene which is a reporter gene is expressed under the control of the inserted promoter, and the expression ability of the promoter can be measured by GUS activity. A. oryzae is transformed with this plasmid, and a transformant in which only one copy of the plasmid has been introduced into niaD locus of the host genome is selected. This transformant was cultured under various culture conditions, GUS production ability was measured, and each promoter was expressed. Since the plasmid pNGS1 is deposited with a microorganism that carries the plasmid pNGS1, for example, by removing it from Aspergillus oryzae 1013-niaD (FERM P-17707) and inserting the desired promoter into the SalI and PstI sites (these Since the nucleotide sequence of the promoter is determined according to the present invention, it can be easily obtained by a conventional method such as synthesis or extraction)), and an expression plasmid can be easily constructed. When introduced into an Aspergillus oryzae niaD mutant, a transformant can be easily prepared. Thus, by treating the obtained transformant by a conventional method such as culturing, a large amount of GUS can be obtained, and the present invention can be easily carried out. Moreover, if another useful protein gene is introduced instead of the GUS gene, this useful protein can be efficiently expressed.
[0021]
[Example 3]
(Examination of promoter expression of manganese superoxide desmutase (Mn-SOD) gene and copper, zinc superoxide demutase (Cu, Zn-SOD) gene) As shown in Table 1, isolated promoters Large amounts of GUS protein were produced under control, indicating the effectiveness of this promoter. Further, regarding Mn-SOD gene promoter (1038 bases: SEQ ID NO: 1), oxidative stress and culture temperature due to linoleic acid, H ₂ O _{2 and the} like, and Cu, Zn-SOD gene promoter (900 bases: SEQ ID NO: 2) are cultured. It was shown to be induced at temperature.
[0022]

[0023]
[Example 4]
(Examination of promoter expression of small V-ATPase (small-V-ATPase) gene, actin gene, and histone H2 gene)
As shown in Table 2, small V-ATPase (small-V-ATPase) gene (1196 bases: SEQ ID NO: 4), actin gene (1001 bases: SEQ ID NO: 5), histone H2 (Histone-H2) gene It was possible to produce the GUS protein with any promoter of (685 bases: SEQ ID NO: 8). In addition, when comparing Czapek-Dox culture (culture on Czapek-Dox medium) and DRY culture (culture on dextrin-peptone-yeast extract medium), promoters of actin (Actin) gene and histone H2 (Histone-H2) gene In the case of DRY culture, and in the case of a small V-ATPase (small-V-ATPase) gene promoter, it was shown that Czapek-Dox culture has higher expression ability.
[0024]

[0025]
[Example 5]
(Examination of P-450 gene promoter expression ability)
As shown in Table 3, it was possible to produce a GUS protein using the cytochrome P-450 gene promoter (1214 bases: SEQ ID NO: 3). It was also revealed that the P-450 gene promoter is induced and repressed depending on the type of nitrogen source in the medium. When nitric acid or nitrous acid was used as a single nitrogen source, very high GUS productivity was observed, but when ammonia was used as a nitrogen source, a significant decrease in productivity was observed. Thus, induction and suppression of promoter activity by a nitrogen source is effective because the expression ability can be controlled.
[0026]

[0027]
[Example 6]
(Examination of catalase gene promoter expression ability)
As shown in Table 4, it was possible to produce a GUS protein even by using a catalase gene promoter. The expression ability of the catalase A and B gene promoters (550 bases: SEQ ID NO: 6) and (663 bases: SEQ ID NO: 7) was further increased by the addition of hydrogen peroxide or iron ions.
[0028]

[0029]
【The invention's effect】
According to the present invention, the promoter region and its nucleotide sequence were determined for the Aspergillus oryzae manganese superoxide desmutase gene and seven other genes, respectively. As a result of promoter analysis using a reporter gene, these novel promoters were confirmed for the first time to be strong promoters.
[0030]
Thus, the use of a strong promoter newly developed according to the present invention has made it possible to construct a novel high expression system for proteins. By using a novel high expression system using this novel promoter, it is possible to efficiently produce not only the same kind of protein but also a heterogeneous protein using aspergillus or other filamentous fungi or other microorganisms as host fungi.
[0031]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a plasmid pNGS1 for measuring promoter activity.
FIG. 2 shows the base sequence of the Mn-SOD gene promoter.
FIG. 3 shows the nucleotide sequence of a Cu, Zn-SOD gene promoter.
FIG. 4 shows the base sequence of cytochrome P-450 gene promoter.
FIG. 5 shows the base sequence of the small-V-ATPase gene promoter.
FIG. 6 shows the base sequence of the actin gene promoter.
FIG. 7 shows the base sequence of the catalase A gene promoter.
FIG. 8 shows the base sequence of the catalase B gene promoter.
FIG. 9 shows the nucleotide sequence of a histone-H2 gene promoter.

Claims (6)

  DNA represented by at least one base sequence of SEQ ID NOs: 1 to 8 in the sequence listing. A DNA that hybridizes with the DNA of claim 1 under stringent conditions and has promoter activity .   A promoter derived from the genus Aspergillus represented by the nucleotide sequences of SEQ ID NOs: 1 to 8 in the Sequence Listing.   The promoter according to claim 3, wherein the Aspergillus genus is Aspergillus oryzae.   Aspergillus oryzae manganese superoxide desmutase (Mn-SOD) gene, copper zinc superoxide desmutase (Cu, Zn-SOD) gene, cytochrome P-450 gene, catalase A (catalase A) ) Gene, catalase B (catalase B) gene, small V-ATPase (small-V-ATPase) gene, actin (Actin) gene, histone H2 (Histone-H2) gene promoter. High expression methods useful protein gene is characterized by utilizing the promoter according to claim 4 or 5.

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