JP2002272477A - Gene encoding transcription factor controlling expression of ferrichrome biosynthesis gene of aspergillus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide confirmation that a transcription control factor negatively controlling ferrichrome biosynthesis is encoded in a gene fragment sreO cloned in the invention, possibility of enhanced production or non-production of the ferrichrome biosynthesis of Aspergillus under the limitation of iron using the gene, possibility of modification in molecular level of ferrichrome production in response to usage, and applicability of the gene in various industrial fields. SOLUTION: A gene encoding transcription factor controlling expression of ferrichrysin biosynthesis gene of Aspergillus is cloned and the base sequence is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a transcription factor protein that suppresses the expression of a ferrichrome biosynthesis gene of Aspergillus, a gene encoding the protein, a recombinant vector containing the gene, and a recombinant vector containing the recombinant vector. A transformant, a recombinant Aspergillus containing the gene, a method of breeding a non-ferrichrome-producing Aspergillus oryzae by producing a transcription factor protein using the transformant, and suppressing ferrichrome by suppressing the expression of the protein in the Aspergillus oryzae. It relates to a method of mass production.

[0002] Ferrichrome is one type of iron ion chelating substance (siderophore) produced by filamentous fungi, and its main component is ferricrysin. According to the present invention, the DNA sequence of a gene encoding a transcription factor protein that suppresses the expression of this ferrichrome biosynthesis gene is clarified, and it becomes possible for the first time to freely manipulate the transcription factor. It is possible for the first time to freely control the synthesis of ferrichrome (which accounts for most of ferrichrome).

[0003] Therefore, in the present invention, if conditions are set such that the transcription factor protein is always expressed, the expression of the ferrichrome biosynthesis gene is suppressed, so that the synthesis of ferricrysin can be reduced or completely stopped. On the contrary, when the transcription factor gene is destroyed or its expression is suppressed or stopped, the expression of the ferrichrome biosynthesis gene is negatively suppressed (that is, the expression of the gene is inhibited). As a result, it becomes possible to synthesize or enhance ferricrysin. Then, in the former, ferricrysin non-producing strains or ferricrysin low-producing strains are bred.
In sake brewing, coloring can be suppressed and quality deterioration can be prevented. And in the latter, strains that synthesize large amounts of ferricrysin are bred, but by using these strains, ferricrysin can be produced in large quantities, and as iron chelators, for research and testing, It can be used as a reagent for analysis, as well as foods and drinks, nutritional foods, and pharmaceuticals for improving anemia.

[0004]

2. Description of the Related Art Iron ions are an essential atom for almost all living organisms except some lactic acid bacteria. Iron is used in vivo in the form of Fe (II) or Fe (III), and functions mainly as a prosthetic factor for a group of enzymes involved in redox. However, iron generally exists in the natural world as iron ore, and hardly exists as a solubilized ionic state. Furthermore, iron ions solubilized from the iron ore by the action of microorganisms are also immediately converted into insoluble oxides and hydroxides, so that very small amounts of iron ions can be used by living organisms. Fungi have a molecular weight of 150, called a siderophore, to efficiently obtain such a small amount of iron ions.
Produces low molecular weight iron ion chelates of 0 or less. By chelating iron ions to this siderophore,
The aim is to prevent the insolubilization of valuable iron ions and to preferentially use iron ions. Iron ions are essential ions for living organisms, but when present in excess, promote the generation of free radicals and, on the contrary, do harm to living organisms. Siderophores contribute to the detoxification of such iron ions at the same time as the acquisition of iron ions. Siderophores are colorless in the unchelated state, but when chelated on iron ions,
It is known to show red and absorb visible light.

Various siderophores have been identified to date, but the siderophore produced by the filamentous fungus is hydroxamat.
It is called es family and generally contains N-hydroxyornithine as a constituent amino acid derivative. Aspergillus fungi, which have been extensively studied as research model fungi, industrial microorganisms, and pathogenic fungi, are hydroxamates f
It produces siderophores called ferrichromes and fuzarinins among amily. In the former, glycine, serine, and alanine form a cyclic peptide in the tripeptide of N-hydroxyornithine. On the other hand, the latter has a feature that the N-position of some N-hydroxyornithine is acylated by mevalonic anhydride. By producing such a wide variety of siderophores, filamentous fungi
It is thought that iron ions are preferentially acquired and used for survival competition in nature.

On the other hand, in sake brewing, “Koji” is produced by growing Aspergillus oryzae on steamed rice, and is used as an auxiliary material for sake brewing. It is known that Aspergillus oryzae produces a large amount of ferrichromes (mainly ferricrysin), which chelate iron ions in sake brewing water and color sake. Therefore, in sake brewing, ferrichromes, which are siderophores, cause quality deterioration, and breeding of strains that do not produce ferrichromes as much as possible has been promoted.

As described above, A. oryzae has long been known to produce ferrichromes in large quantities, but little is known about its biosynthetic system. Even today, the breeding of low ferrichrome producing bacteria must rely mainly on screening and mutation methods. In recent years, the iron chelating ability of the siderophore has been used as a medicament for anemia. Ferriclysin produced by Aspergillus is also very excellent in iron ion binding ability, but its productivity is difficult to improve, and is not targeted for industrial-level production. If the biosynthetic system of ferrichromes, especially the mechanism of regulating the expression, is elucidated, there is a possibility that the production of ferrichromes by Aspergillus can be freely regulated. That is, in sake brewing, the expression control mechanism of the ferricrysin biosynthesis system is disrupted, and a strain that does not produce ferricrysin at all can be bred. In the production of pharmaceuticals, a large amount of ferriclysin can be produced by inducing the biosynthetic system more actively.
Furthermore, ferriclysin produced by Aspergillus oryzae has been ingested as sake or sake lees for many years and is an extremely safe substance. If ferricrysin can be produced in large quantities by koji mold,
A wide range of applications from pharmaceuticals to foods is possible.

[0008] As described above, ferricrysin production by Aspergillus is expected to be applied to various fields, but its biosynthesis control mechanism has not been elucidated yet, so that it has not been used efficiently yet.

[0009]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and has elucidated the mechanism of ferrichrome biosynthesis. For example, the present invention relates to Aspergillus oryzae.
illus oyzae), which clarifies the regulation mechanism of the biosynthesis of ferricrysin, which occupies most of ferrichrome, and suppresses the biosynthesis of ferricrysin, or conversely, produces ferricrysin in large quantities. The purpose was to develop a new system for freely controlling biosynthesis.

[0010]

Means for Solving the Problems The present invention has been made in order to achieve the above-mentioned object, and firstly, a koji mold was studied.

Aspergillus A. Oryzae is a filamentous fungus used in the traditional enzyme industry of Japan such as sake, soy sauce, and miso. The feature of this strain is that it produces very large quantities of proteins and low molecular components useful in the fermentation industry. Due to the high protein-producing ability of this strain and its safety as a brewing microorganism, it is attracting attention as a host for heterologous protein production (Bi
otechnology, 6, 1419 (1988), JP-A-62-2729.
88). From our research, A.I. In the production of a heterologous protein using oryzae, it was confirmed that if a gene of a near species such as Aspergillus was used, the productivity would be further increased. In particular, A.I. oryzae gene, It has been found that when expressed under the control of a high expression promoter of Oryzae, a very large amount of protein is produced (Japanese Patent Application Laid-Open No. H11-154271, Japanese Patent Application No.
36754). A. Oryzae has been reported to produce not only the above-mentioned proteins and enzymes, but also industrially valuable low molecular components. As such, production of kojic acid which is a melanin synthesis inhibitory component by Aspergillus or the like can be mentioned. It has also been suggested that ferriclysin, which has been a problem in sake brewing for a long time and which is produced by koji mold in solid culture, may be a functional component for iron deficiency such as anemia.

[0012] In order to mass-produce this ferricrysin, an attempt was made to obtain a mutant strain capable of high-producing ferricrysin, but the production amount did not reach an industrial production level.
Therefore, the present inventors have made a major change in the idea, and do not control the expression of the ferricrysin biosynthetic gene positively, as in the conventional common sense, but conversely, We have taken a completely new viewpoint of approaching the solution of the above problem from a mechanism that controls negatively.

[0013] The present inventors have conducted studies from various aspects. Rather than enhancing ferricrysin biosynthesis, the inventors have obtained a gene for a transcription control factor that controls ferricrysin biosynthesis. Focusing on the fact that it is possible to breed a genetically modified Aspergillus oryzae capable of freely controlling the production of ferricrysin and to enable the industrial production of ferricrysin. oryzae
It was also noted that if a method that does not contaminate a heterologous DNA other than the above is used, the strain becomes a self-cloning strain and is excluded from the regulation of recombinant microorganisms.

[0014] In view of the above, the present inventors have proposed a method of controlling the biosynthesis of ferricrisin in order to mass-produce ferricrysin, a low-molecular-weight iron ion chelate that causes the coloring of sake in sake brewing, as a functional component for improving anemia. The gene cloning of the factor was performed, and an attempt was made to breed a genetically modified Aspergillus oryzae suitable for industrial production of ferricrysin. As a result of earnest studies, the present invention was finally completed.

[0015] In recent years, genes for siderophore biosynthesis such as ferricrysin have been studied in detail in Ustilago maydis, a corn pathogen, for its detailed synthesis mechanism. L-Ornithine N5-oxygenase, which catalyzes the oxidation of ornithine, which is the first reaction in biosynthesis, is the rate-limiting factor for biosynthesis, and the gene sid encoding this
1 was cloned. Expression of a siderophore biosynthetic enzyme gene group such as sid1 is suppressed in an environment where iron is present, and is started under iron restriction.
The gene urbs1, a transcriptional regulator that negatively regulates the expression of the sid1 gene of Ustilago maydis, has been cloned. Furthermore, Penicilliun chrysogenum, Neurospora cra
In ssa and Aspergillus nidulans, urbs1
Factors that negatively regulate the expression of the siderophore biosynthetic enzyme gene group have been cloned. Therefore, it was thought that a gene having a urbs1-like function in Aspergillus oryzae could be obtained from the homology of these siderophore biosynthesis transcription factors.

The Ustilago m from which the gene has been isolated to date
As a result of comparing the homology at the amino acid level of transcription factors of aydis, Penicillium chrysogenum, Neurospora crassa and Aspergillus nidulans, two highly conserved regions were found. Degenerate synthetic primers corresponding to each of these regions were designed. Using this primer, A.I. or
As a result of performing PCR on yzae genomic DNA, a gene fragment of about 500 bp was amplified. As a result of determining the nucleotide sequence of this gene, sre encoding a transcription factor that negatively regulates ferrichrome biosynthesis in Aspergillus nidulans was determined.
It showed the highest homology with the A gene. As a result of Northern analysis of this gene, its expression was observed in the presence of iron, and its expression was suppressed under iron restriction.

The obtained gene fragment was labeled with fluorescein as a probe. The oryzae chromosomal DNA library was screened. As a result, it was confirmed that the above fragment was contained in the obtained positive clone.
Revealed by the sequence. As a result of determining the entire nucleotide sequence of this clone, this gene encoded a protein consisting of 566 amino acid residues and had two introns. A. s from nidulans
It had 65% homology at the amino acid level with the reA gene. The obtained gene was named sreO. Structurally, GATA is similar to other filamentous fungal transcription factors.
Belongs to the GATA family that binds to the gene sequence of

In order to identify the function of sreO, the gene was disrupted. The partial sequence of the open reading frame (ORF) of sreO was inserted into a vector containing the arginine requirement marker argB. This plasmid was used as a plasmid for disrupting the sreO gene. or
yzae was introduced into an arginine-requiring strain. The resulting transformants were subjected to Southern analysis, and three strains in which the sreO gene was disrupted were selected. The obtained disrupted strain was subjected to liquid culture in the presence of iron, and as a result, significant ferriclysin productivity was observed as compared with the control strain. Therefore, it was revealed that sreO encodes a transcription factor that negatively regulates ferriclysin biosynthesis.

The isolated gene can be used alone or in melO
And glaB gene promoter (Japanese Patent Application No. 11-1542)
71, JP-A-11-243965). Oryzae can be expressed to control ferricrysin production. The gene can be introduced by, for example, a known method using a niaD mutant strain as a host, and the target gene and the marker gene ni.
The aD gene is introduced simultaneously. (S. Uncle et al., M.
ol. Gen. Genet. 218, p. 99-10
4, 1989) By excluding a heterologous gene such as a vector sequence during the gene transfer, a transformant of a self-cloning strain containing no heterologous gene can be obtained. niaD mutant (Koji mold mutant that cannot assimilate nitrate: Nitrate Reduced deficient strain)
As, for example, Aspergillus oryz
ae 1013-niaD (FERM P-1770
7) can be used.

That is, the present invention provides an Aspergillus oryzae
In order to freely control ferricrysin biosynthesis of yzae,
It is intended to provide a DNA encoding a transcriptional regulator that negatively regulates the expression of these genes. The present invention also provides a recombinant vector containing the gene and a transformant containing the recombinant vector. Is provided. The present invention also provides a method for producing a transcription factor protein using the above transformant, and a koji mold that does not produce ferricrysin in solid culture using rice koji by expressing the gene under iron restriction in koji mold. The obtained koji mold can be advantageously used in Japan's unique brewing industry such as sake, miso, soy sauce, and mirin. The present invention also provides an Aspergillus oryzae capable of highly producing ferriclysin under all culture conditions by constitutively suppressing the expression of the gene using a genetic engineering technique. In addition, the ferricrisin thus produced can be provided as a reagent as an iron chelating agent and as an addition to a functional food that can be expected to improve anemia.

The details of the present invention are described below.

First, among filamentous fungi for which siderophore production was reported under iron restriction, those in which the gene of a factor that negatively regulates siderophore biosynthesis was cloned were selected. Ustilago maydis, Penicillium chr to date
ysogenum, Neurospora crassa and Aspergillus nidulan
In S, a gene for a factor that negatively regulates siderophore biosynthesis has been reported. As a result of comparing the homology of these genes at the amino acid level, at least two amino acid sequence conserved regions were found. One is Cys-Ser-Asn
-Cys-Gly- (Thor Val)-(Ly
s or Thr)-(Ser or His) -Th
r-Pro-Leu-Trp-Arg-Arg- (Se
r or Ala) -Pro, and the other is Gly-
Cys-Pro-Ala-Tyr-Asn-Asn-
(Arg or Asn) -Trp-Arg-Arg-
Asp-Glu. Using a degenerate oligonucleotide probe prepared based on these two types of partial amino acid sequences, P. oryzae chromosomal DNA
CR was performed. As a result, amplification of a fragment of about 500 bp was observed. As a result of determining this base sequence, As
Pergillus nidulans showed the highest homology with the sreA gene, which encodes a transcription factor that negatively regulates ferrichrome biosynthesis. As a result of Northern analysis of this gene, its expression was observed in the presence of iron, and its expression was suppressed under iron restriction.

The obtained gene fragment was labeled with fluorescein as a probe. oryzae lambda EMBL
A three chromosome DNA library was screened. As a result, DNA sequencing revealed that the obtained positive clone contained the above fragment.
As a result of determining the entire nucleotide sequence of this clone,
It consisted of 66 amino acid residues and had two introns. The promoter region corresponds to 1 to 141 of SEQ ID NO: 2.
2. The coding region is from 1413 to 3249, and the terminator region is from 3250 to 3687 bp.
A. Nidulans-derived sreA gene had 65% homology at the amino acid level. The obtained gene was named sreO. In the promoter region of the present gene, a sequence characteristic of a eukaryotic promoter such as TATA-box was present at -182 bp.
The transcription factor structurally belongs to the GATA family that binds to the GATA gene sequence.

The sreO according to the present invention determined as described above
The nucleotide sequence of the gene is shown in SEQ ID NO: 2 (FIGS. 4 to 6),
The amino acid sequence of the protein deduced from this nucleotide sequence is shown in SEQ ID NO: 1 (FIGS. 1 to 3).

The DNA of the sreO gene according to the present invention is
The plasmid was ligated to plasmid pUC118 using T4 DNA ligase (Takara Shuzo), and the recombinant vector (pPIN54) was ligated.
7) was obtained. The ligated DNA as a recombinant vector was transformed into Escherichia coli JM109. The cells were cultured in an ampicillin-containing medium, ampicillin-resistant strains were selected, and the resulting transformants were transformed into Escherichia coli IN
No. 547 and deposited as FERM P-18255 with the National Institute of Advanced Industrial Science and Technology, Ministry of Economy, Trade and Industry.

In order to identify the function of sreO, the gene was disrupted. The 440 bp region from the partial coding region 1413 to 1852 of the sreO was replaced with the arginine-requiring marker A. nidulans
Inserted into vector containing gB. Sre
As a plasmid for disrupting the O gene, oryzae arginine-requiring strain M-2-3. The resulting transformants were subjected to Southern analysis, and three strains in which the sreO gene was disrupted were selected. The obtained disrupted strain was subjected to liquid culture in the presence of iron, and as a result, significant ferriclysin productivity was observed as compared with the control strain. Therefore, it was revealed that sreO encodes a transcription factor that negatively regulates ferriclysin biosynthesis.

From the above results, it was confirmed that the cloned gene fragment sreO encodes a transcriptional regulator that negatively regulates ferriclysin biosynthesis.
It is also possible to use this gene to produce or not produce ferricillin biosynthesis in Aspergillus under iron limitation, and to modify ferricrysin production according to the application at the molecular level. It was also confirmed that application was possible. Hereinafter, examples of the present invention will be described.

Example 1 A transcriptional regulator (s) that negatively regulates ferricrysin biosynthesis
gene cloning of Aspergillus oryzae A. reO) In order to clone a gene for a transcriptional regulator that negatively regulates oryzae ferricrysin biosynthesis, the amino acid sequence conserved regions of the present gene derived from other filamentous fungi were compared. Ustilago maydis, Penicillium to date
chrysogenum, Neurospora crassa and Aspergillus nid
A gene for a factor that negatively regulates siderophore biosynthesis in ulans has been reported. As a result of comparing the homology of these genes at the amino acid level, at least two amino acid sequence conserved regions were found. One is Cys-Ser-Asn-Cys-Gly- (Thr
or Val)-(Lys or Thr)-(Se
r or His) -Thr-Pro-leu-Trp
-Arg-Arg- (Ser or Ala) -Pro
And the other is Gly-Cys-Pro-Ala-T
yr-Asn-Asn- (ArgoAsn) -Tr
p-Arg-Arg-Asp-Glu.

The amino acid sequence of the region is shown in SEQ ID NO: 3 (FIG. 7). In the same sequence, the first Xaa is Th
r or Val, the second Xaa is Lys or Thr,
3rd Xaa is Ser or His, 4th Xaa
a represents Ser or Ala. The amino acid sequence of the region is shown in SEQ ID NO: 4 (FIG. 8). In the same sequence, Xaa represents Arg or Asn.

Based on the above sequence, two degenerate oligonucleotide primers P1 and P2 were synthesized. The nucleotide sequence of primer P1 is shown in SEQ ID NO: 5 (FIG. 9), and the nucleotide sequence of primer P2 is shown in SEQ ID NO: 6 (FIG. 10). In these sequences, n (I in the figure) is a deoxyinosine residue, s is G + C, y is C + T, m is A + C, r
Represents a degenerate base according to the IUB code of A + G.

Using the above primers, A. oryzae
A PCR reaction was performed on the genomic DNA. The reaction conditions are as follows. PCR conditions • 96 ° C (5 minutes), 1 cycle • 96 ° C (20 seconds), 50 ° C (30 seconds), 72 ° C (3 seconds)
Min), 35 cycles ・ 72 ° C (7 minutes), 1 cycle

The reaction solution was analyzed by agarose gel electrophoresis. As a result, amplification of a fragment of about 500 bp was observed. As a result of determining the base sequence of this gene amplification product,
Certainly, it contains the above-mentioned P1 and P2 sequences. It showed the highest homology with the sreA gene of Nidulans.

The amplified product was labeled with fluorescein and used as a probe for Aspergillus oryzae O-1.
Lambda EMBL3 genomic DNA library of about 3000 phage clones of strain 013 (FERM P-16528) was screened by plaque hybridization. As a result, positive clones were obtained. This clone was confirmed to encode the above probe sequence.

[Example 2] Determination of the entire base sequence of sreO and homology search The above-mentioned positive clone was subjected to the dideoxy method,
The entire nucleotide sequence of the reO gene was determined. The sequence of 3687 bp was determined by combining the promoter region, the coding region, and the terminator region (SEQ ID NO: 2; FIGS. 4 to 6). Of the above sequences, the promoter region is from position 1 to position 1412, and the coding region is 1413.
And the terminator region extends from the position 3250 to the position 3687 bp. A. It had 65% homology at the amino acid level with the sreA gene from Nidulans. The obtained gene was named sreO. In the promoter region of the present gene, a sequence characteristic of a eukaryotic promoter such as TATA-box was present at -182 bp. The transcription factor structurally belongs to the GATA family that binds to the GATA gene sequence. In addition, this gene had two introns, and it was revealed that the protein deduced from the nucleotide sequence had 566 amino acid residues (SEQ ID NO: 1; FIGS. 1 to 3).

[Example 3] Construction of sreO gene-disrupted strain In order to identify the function of sreO, the gene of this gene was disrupted. partial coding region 1413 of sreO
After amplification of a 440 bp region from to 852 bp by PCR, the arginine auxotrophy marker A. nidulans
ArgB was inserted into the BamHI site of the vector pRBG1 and the plasmid pRSE for sreO gene disruption was inserted.
1 was constructed (FIG. 11). This plasmid was used as a plasmid for disrupting the sreO gene. oryzae arginine-requiring strain M-2-3. The resulting transformants were subjected to Southern analysis, and three strains in which the sreO gene was disrupted were selected. The obtained disrupted strain was transformed into an iron-containing medium (glucose 2%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.1%, potassium chloride 0.05%, ammonium sulfate 0.2%).
%, Ferric sulfate 0.00001 M, pH 6.3). As a result, significant ferriclysin productivity was observed as compared with the control strain as shown in FIG. The determination of ferricrysin was performed according to the method of Sato et al.
Vol. 8, No. 875-880 (1967)). Therefore, sr
eO was found to encode a transcriptional regulator that negatively regulates ferricrysin biosynthesis.

[0036]

According to the present invention, it was confirmed that the gene fragment sreO, which has been successfully cloned for the first time, encodes a transcriptional regulator that negatively regulates ferrichrome biosynthesis. The transformant Escherichia coli IN547 containing the sreO gene is
Since it has been deposited with Life Research Institute as ERM P-18255, it can be appropriately obtained by extracting a plasmid retaining the sreO gene. As described above, this strain contains a plasmid obtained by inserting the gene sreO encoding the siderophore biosynthesis control factor of Aspergillus oryzae into plasmid pUC118 using Escherichia coli JM109 as a host. This plasmid is sre
Genes can be supplied to various applications using O genes.

Therefore, aspergillus oryzae can be transformed by a conventional method such as electroporation or protoplast-polyethylene glycol method using this plasmid to obtain a non-synthetic ferricrisin strain. It can be used to prevent quality deterioration. Further, by disrupting the sreO gene as in the above-described example, a ferricrisin high-producing strain can be obtained.

As described above, the ferrichrome biosynthesis of Aspergillus oryzae can be highly produced or non-produced under iron limitation by using this gene, and the ferrichrome production can be modified at the molecular level according to the intended use. It was also confirmed that it could be applied to various industrial fields.

[0039]

[Sequence List] SEQUENCE LISTING <110> Gekkeikan Inc., Ltd. <120> Gene-encoding Transcript Factor Repressing Expression of Ferrichrome Biosynthesis-encoding Gene of Koji-mold <130> 6429 <141> 2001-3-22 <160> 6 <210> 1 <212> 566 <211> PRT <213> Aspergillus oryzae <400> 1 Met Leu Ala Ser Leu Pro Gln Met Glu Thr Val Arg Ser Leu Pro Arg 1 5 10 15 Asn Pro Asp Val Val Ala Arg His Pro Ser Ala Glu Asp Leu Asp Ala 20 25 30 Ala Gln Gln Leu Ile Ser Ser Ala Gln Ala Gly Arg Glu His Pro Val 35 40 45 Asp Arg Pro Arg Asp Glu Thr Gly Ser Arg Arg Tyr Glu Glu Met Pro 50 55 60 Ser Arg Gly Leu Tyr Glu Ala Asp Lys Phe Leu Glu Gly Ser Thr Pro 65 70 75 80 Tyr Pro Ser Glu Ile Gly Ala Asn Gln Ser Glu Lys Ala Ser Ser Pro 85 90 95 Lys Ser Gln Lys Asp Thr Ser Phe Leu Gly His Ser Cys Ser Asn Cys 100 105 110 Gly Thr Lys Ser Thr Pro Leu Trp Arg Arg Ser Pro Thr Gly Ala Met 115 120 125 Ile Cys Asn Ala Cys Gly Leu Tyr Leu Lys Ala Arg Asn Val Ala Arg 130 140 135 Pro Thr Lys Arg Asn Arg Val Gln Thr Ser Pro Glu Thr Thr Gln Pro 145 150 160 155 Pro Ser Asn Pro Ser His Pro Pro His Asp Ser Thr Ala Pro Ser Ser 165 170 175 His Glu Gly Gly Gly Cys His Gly Ser Lys Gly Ser Cys Pro Gly Gly 180 185 190 Gly Asn Cys Asn Gly Thr Gly Gly Ala Glu Gly Cys Asp Gly Cys Pro 195 200 205 Ala Tyr Asn Asn Arg Val Tyr Lys Ser Thr Pro Arg Gly Thr Val Pro 210 215 220 Val His Ala Trp Asn Arg Ala Thr Thr Ser Asp Ser Glu Lys Pro Pro 225 230 240 235 Leu Gln Glu Pro Asp Leu Ser Val Lys Asn Gly Thr Pro Ala Thr Thr 245 250 255 Thr Thr Glu Gly Asn Met Leu Val Ser Cys Gln Asn Cys Gly Thr Thr 260 265 270 Val Thr Pro Leu Trp Arg Arg Asp Glu Asn Gly His Pro Ile Cys Asn 275 280 285 Ala Cys Gly Leu Tyr Tyr Lys Leu His Gly Cys Tyr Arg Pro Thr Thr 290 295 300 Met Lys Lys Thr Ile Ile Lys Arg Arg Lys Arg Val Val Pro Ala Leu 305 310 315 320 Arg Glu His Ser Pro Thr Gly Ala Thr Gln Ser Ser Asn Gly Ser Ser 325 330 335 Ala Ser Pro Glu Ala Ser Pro Ala Ala Leu Ala Pro His Asp Asp His 340 345 350 Tyr Arg Tyr Tyr Ser Ser Glu Pro Met Asp His Tyr Lys His Met Pro 355 360 365 Gly Asp Arg Ile Ser Pro Gln Ala Pro Arg Gln Phe Gly Phe Ala Pro 370 375 380 Pro Pro Val Asp Phe Thr Gly Phe Gly Ser Ala Thr Val Ser Leu Pro 385 390 395 400 400 His His Pro Pro Pro Pro Arg Leu Leu Glu Pro Glu Arg Ile Asn Ala 405 410 415 Pro Ser His Ser Pro Val Ser Gln Phe Ala Arg Arg Ser Leu Ser Pro 420 425 430 Asn Pro Ala Asn Pro Lys Lys Arg Thr Leu Ala Glu Thr Ala Ser Ser 435 440 445 Ala Glu Ala Ala Ser Ile Pro Thr Thr Leu Glu Ser Gly Ser Asn Gln 450 455 460 Leu Pro Pro Ile Met Ser Ala Ala Asn Pro Ser Pro Pro Gly Arg Leu 465 470 475 480 Ser Ser Ile Ser Ser Ile Leu Asn His Pro Asn Thr Arg Asp Glu Ser 485 490 495 Arg Leu Asp Pro Ser Leu Ala Ala Leu Ser Arg Gln Gln His Gln His 500 505 510 Ser His Gln Ala Leu Ala Pro Pro Gln Pro Gln Ser Leu Pro Gly Val 515 520 525 Thr Glu Leu Asp Ser Met Arg Glu Glu Arg Arg Ala Gln Leu Gln Arg 530 535 540 Glu Ala Glu Glu Met Arg Glu Met Leu Arg Ala Lys Glu Arg Glu Leu 545 550 555 560 Ala Glu Leu Gly Arg Gln 565 566 <210> 2 <212> 3687 <211> DN A <213> Aspergillus oryzae <400> 2 tagcggtgga gccagatgat acgatctggg ggagaagcag agcaatcatg atggatgcaa 60 aacgaaagaa gggaaatgtg acttttgctg ttagccaaaa aaaaaaaaat taaaaagaaa 120 tttaccacaa ggcaaagttc tgtaaagtaa ccaaactcaa caaagcaatc cttccccaac 180 tttcttggga aaagacaaga cctgaacaag ccgacaacac aatccacact gcaggggaaa 240 aagcaaaaat accaacgaga aaggccggtg tagatcctcc tacccccctg gagatcgata 300 acgaaacgtc cttcgataag accacactgt tgggactcgg ggaatctgac ttgggaactt 360 cttccaggca acaagaatag aaacataagg gtaagtggtg gtctggccct tttctttttt 420 tccctttcat cttcagcagc agcagcatca ttattaccca cttcttccgc ctttcccaat 480 cgttctttat caatcaaatc tatctctcac gcctcattct tcatccttcc cacgtcgtca 540 ttccaccccc cttcctgtct cttccgcaga aaacgtgtga ttattttctt ctttttttat 600 tattattttg tgttgtattt tatttttatt ctttttttcc tgtgaacccc tggtcacctc 660 cacacatcat atctgatcgg attgattgat cgcccttccc cgtcgatctt tcttgtcgac 720 gctttcactt ttcgtcttct acattccacc ccctgcttcc acctgtcgtg tctttcagct 780 ggctcttcat tccctttaac tttattattc attgtaatcc cgattgtcgt c tccatcctc 840 acccaaaaca gaagaaaaaa aaatccagca ccgataaccg cctcaaccca ccactgagcg 900 tcacgccgga tcgccaccca gcctcatctc ggattggcca acaaaaccac cttcgtggca 960 gatcaggcgt ctctttccaa tctccttcta gtcgaatacc ttttcctctt tacttttttg 1020 atcgctcacc cttcgggaat ccgactccac cctattatct ctgagccgct gtcagggata 1080 accggtctct cttccccggc tccgcctggt tggtttagga gtctcactag ctgcagctct 1140 tttactctac cggttcgccc ttcgagttag gcgttttaac ttttcttttt ctcctgcact 1200 gtcatcttga ccgacttttg actttgtgat acaatacatt acaaccccga ctgtggtgca 1260 acctaccctc atcctcccct tatttcgacg atccgcctcg aacggccatc cgctttatct 1320 ttgggggttc tatactgccc ttccctgggt tgtcgtcacc ccggaacacc aagcaaagag 1380 aagataccat cttctgacca acagcggtgg caatgttagc gtccctcccg cagatggaga 1440 cggtacgatc gctcccccgg aatccggacg ttgtcgcgcg acacccatcc gcagaggacc 1500 ttgatgctgc gcagcagtta atatcatcag cccaggcggg tcgggaacat cctgtcgacc 1560 gacctcgcga tgaaacgggg tccagacgct acgaagaaat gcccagtcgt ggtctctatg 1620 aagcagacaa gttcttggag gggtcgacac catatccgtc ggaaattggt gcaaaccaat 1680 cagagaaggc ttcgtcgccg aagtcccaga aagacacttc atttctagga cattcgtgca 1740 ggttagtatt cggtcttctg ccttggcccg tgtgcgtcgt actgatttga gatgctttac 1800 agtaattgcg ggacaaaaag caccccatta tggcggcgtt ctccgacagg agctatgata 1860 tgcaatgctt gcggtctcta tttgaaggct cgtaacgtgg ctcgaccgac aaagcgcaac 1920 cgtgtacaga caagcccaga aactacgcaa ccaccgagca atccttcgca tcctccccac 1980 gattcaactg ctccatcatc gcacgaggga ggaggttgtc atggctcaaa agggtcgtgt 2040 cctggtggag gtaactgcaa cggtacagga ggggctgagg ggtgtgacgg atgccccgct 2100 tacaataacc gtgtgtacaa atcgactcct cggggcacgg tccctgtgca tgcatggaat 2160 cgagcaacca cctccgacag tgaaaagcct cctctgcagg agcctgattt atcggtaaag 2220 aatggcaccc cggcaacaac aacaacagaa ggcaatatgt tggtatcctg ccagaattgc 2280 ggcactacgg taactccact ttggcgacga gacgagaatg gtcacccaat ctgtaatgcg 2340 tgtggtatgt ttttttcttt ttactaaaat tttcctccta ttataccgaa agccgtaatt 2400 gtactgacaa ataatttagg tctctattat aaacttcacg gctgttatcg gcctaccacg 2460 atgaagaaga ctatcattaa acgacggaaa agggttgtgc cggcgctgcg cgagcattct 2520 ccgacggggg ccacgcagtc atcgaatggg tcttctgcct cgccggaagc gtctcctgct 2580 gcattggctc ctcacgacga tcattatcgc tattacagtt cggagcccat ggaccattac 2640 aagcatatgc cgggtgatcg tatttcaccg caggccccaa gacagtttgg tttcgcgccg 2700 ccgcctgttg atttcacggg tttcggctct gcgacagttt ccctgccgca ccaccctcct 2760 ccgccgaggc tgttggaacc agaacgcatc aacgctccca gccattctcc cgtctcgcaa 2820 tttgctcgac gatccctctc gcctaatcct gcgaatccta agaaacggac tctcgcagag 2880 accgcgtcat ctgcagaggc cgcctcgatc cccacaacgt tggaatcagg gtcgaaccaa 2940 ctcccaccca tcatgtcagc tgccaaccct tccccaccgg gtcggctcag ctccatttct 3000 tccattctca accatcctaa cacgcgggac gagtcacgct tggacccgtc gctcgctgca 3060 ctgagtcgcc agcagcacca acattcgcac caggctctgg cacctccgca gcctcagtcc 3120 ttgcccggag tcaccgaact cgacagtatg cgagaagaac gccgggctca actccagcgg 3180 gaagctgagg agatgcggga gatgctgaga gcgaaggagc gagaattggc cgagttgggg 3240 cggcaatgat cgttatttcc tttctgagac tcgatgtctg aatgcgcttt ttgctcatct 3300 ttgtccatta atacaccagg cgcagcagtt gctttctgac gagataccac cttcaagagt 3360 cgatgg atta accgggtttt gtttccatat tcagggaaaa gtggctttgg gttgtttgtg 3420 aattaccatg tgttatttga agcggactga atagcgggtt taccaggaaa agagtccagg 3480 aaggctttgt cacgatgttt gataactaca gatctctcct caaattgagg ttttaatttc 3540 ggaaacgtca tacatacata gatcgacgag atagatcggg aatcggatat ttgcatttac 3600 tgtaaccgtt gttgatatga cagacagtcg tatgtaggat gtaggatgta catatgggca 3660 gtcaatctcg tgcgttcgga acggccc 3687 <210> 3 <212> 16 <211> PRT <213> Aspergillus oryzae <400> 3 Cys Ser Asn Cys Gly Xaa Xaa Xaa Thr Pro Leu Trp Arg Arg Xaa Pro 5 10 15 <210> 4 <212> 13 <211> PRT <213> Aspergillus oryzae <400> 4 Gly Cys Pro Ala Tyr Asn Asn Xaa Trp Arg Arg Arg Glu 5 10 <210> 5 <212> 47 <211> DNA <213> Artificial Sequence <400> 5 tgnnnnaayt gyggnnnnam nnnnacnccn ytntggcgnc gnnsncc 47 <210> 6 <212> 38 <211> DNA <213> Artificial Sequence <400> 6 tcntcnctnc tccanytrtt rttrtangcn ggrcancc 38

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of the present transcription factor protein.

FIG. 2 shows a continuation of the above.

FIG. 3 shows a continuation of the above.

FIG. 4 shows the nucleotide sequence of the present transcription factor gene.

FIG. 5 shows the continuation of the above.

FIG. 6 shows a continuation of the above.

FIG. 7 shows the amino acid sequence of a conserved region.

FIG. 8 shows the amino acid sequence of a conserved region.

FIG. 9 shows primer P1. In the sequence, I represents a deoxyinosine residue, S represents G or C, Y represents C or T, M represents A or C, and R represents A or G.

FIG. 10 shows primer P2. In the sequence, I represents a deoxyinosine residue, Y represents C or T, and R represents A or G, respectively.

FIG. 11: Plasmid pRSE1 for sreO gene disruption
FIG.

FIG. 12 shows ferricrysin production of a transformant.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/04 5/10 C12R 1:19) C12P 21/04 1:69) // ( C12N 15/09 ZNA (C12N 1/15 C12R 1:19) C12R 1:69) (C12N 15/09 ZNA (C12N 1/21 C12R 1:69) C12R 1:19) (C12N 1/15 (C12P 21 / 04 C12R 1:69) C12R 1:69) (C12N 1/21 C12N 15/00 ZNAA C12R 1:19) 5/00 A (C12P 21/04 C12R 1:19) C12R 1:69) 1:69) ( 72) Inventor Shoji Kawado 24 Shimo-Toba Koyanagi-cho, Fushimi-ku, Kyoto City, Tsukubakan Co., Ltd. (72) Inventor Yasuhisa Abe 300 Katahara-cho, Fushimi-ku, Kyoto-shi, Tsukikamikan Co., Ltd. (72) Inventor Satoshi Imayasu, Fushimi, Kyoto-shi Ward Haramachi 300 laurel wreath Co., Ltd. in the F-term (reference) 4B024 AA05 BA80 CA01 EA04 GA19 4B064 AG37 CA05 CA19 CC24 DA01 DA10 DA13 4B065 AA26X AA63X AA63Y AB01 AC14 BA02 CA24 CA41 CA44 CA46 4H045 AA10 BA10 BA14 BA30 CA15 EA01 EA20 EA50 FA74

Claims (8)

[Claims] 1. A transcription factor protein having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and suppressing expression of a ferrichrome biosynthesis gene. 2. The DNA of the gene encoding the protein according to claim 1, which is represented by the nucleotide sequence of SEQ ID NO: 2. 3. A recombinant vector comprising at least the coding region of the DNA according to claim 2. 4. The recombinant vector pPIN547. A transformant into which the recombinant vector according to claim 3 or 4 has been introduced. 6. A transformant, Escherichia coli.
erichia coli) IN547 (FERM P-1825)
5). 7. A method for breeding a non-ferrichrome-producing Aspergillus oryzae by producing a transcription factor protein which suppresses the expression of a ferrichrome biosynthesis gene in the Aspergillus into which the DNA of claim 2 has been introduced. 8. A method for mass-producing ferrichrome, comprising suppressing the expression of the protein according to claim 1 in a koji mold.