JP2003230381A - Cluster gene involved in ferrichrome biosynthesis of aspergillus niger - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide cloned genetic fragments capable of forming a cluster with nsb1 and nsb2 genes involved in ferrichrome biosynthesis of Aspergillus niger on the chromosome thereof and to highly produce the ferrichrome of the Aspergillus niger widely utilizable in applications such as foods, drinks and medicines by using the genes. <P>SOLUTION: Proteins of ornithine monooxygenase and a peptide synthetase forming the ferrichrome biosynthesis cluster of the Aspergillus niger are elucidated and the genes encoding the proteins, i.e., the nsb1 and nsb2 are cloned to determine the base sequence thereof. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention comprises an ornithine monooxygenase and a peptide synthetase protein required for ferrichrome biosynthesis of Aspergillus niger, a gene encoding the protein, a recombinant vector containing the gene, and the recombinant vector. Transformant, recombinant koji mold containing the gene, ornithine monooxygenase using the transformant, a method for breeding koji mold with high ferrichrome production by producing a peptide synthetase protein, both ferrichrome biosynthetic genes other than koji mold The present invention relates to a method for producing by introducing into a living organism, and a method for synthesizing an arbitrary peptide using an Aspergillus niger peptide synthetase protein.

[0002]

2. Description of the Related Art Iron ion is an essential atom for almost all living organisms except some lactic acid bacteria. Iron is utilized in the form of Fe (II) and Fe (III) in the living body, and functions as a prosthetic factor of an enzyme group mainly involved in redox. Particularly in aerobic organisms, iron ions are indispensable for reactions in electron transfer systems that produce large amounts of energy. However, iron generally exists as iron ore in nature, and is rarely present as a solubilized ionic state. Furthermore, iron ions solubilized from iron ore by the action of microorganisms are immediately converted into insoluble oxides and hydroxides, so that the amount of iron ions that can be utilized by living organisms is extremely small. Bacteria, actinomycetes, and eukaryotic microorganisms produce a low-molecular weight iron ion chelating substance having a molecular weight of 1500 or less called a siderophore in order to efficiently acquire such a trace amount of iron ions. By chelating iron ions with this siderophore, we are trying to prevent insolubilization of precious iron ions and preferentially utilize iron ions. Further, iron ions are essential ions for living organisms, but when they are present in excess, they promote the generation of free radicals, which in turn harms living organisms. The siderophore contributes not only to the acquisition of iron ions but also to the detoxification of iron ions. Siderophores are colorless in the unchelated state,
It is known that chelating iron ions gives a red color and absorbs visible light.

Various siderophores have been identified so far, but the siderophore produced by filamentous fungi is hydroxamat.
It is called es family and generally contains N-hydroxyornithine as a constituent amino acid derivative. The fungal filamentous fungus of the genus Aspergillus, which has been extensively studied as a model filamentous fungus for research, an industrial microorganism, and a pathogenic filamentous fungus, is hydroxamates f.
Among the amily, it produces siderophores called ferrichromes and fusarinins. In the former, glycine, serine, and alanine form a cyclic peptide in the tripeptide of N-hydroxyornithine. On the other hand, the latter is characterized in that the N-position of a part of N-hydroxyornithine is acylated by mevalonic anhydride. Filamentous fungi produce a wide variety of such siderophores,
It is considered that iron ions are acquired preferentially and used for survival competition in the natural world.

On the other hand, in sake brewing, Aspergillus oryzae is grown on steamed rice to prepare "koji," which is used as a raw material for sake brewing. It is known that Aspergillus oryzae produces a large amount of ferrichromes (among others, the main component is ferricuricin) in the koji making, which chelate iron ions of brewing water to color sake. Therefore, in sake brewing, ferrichromes, which are siderophores, cause quality deterioration, and breeding of strains that do not produce ferrichromes has been promoted.

As described above, Aspergillus oryza
e) has long been known to produce ferrichromes in large quantities, but black koji molds related to the production of shochu and awamori, which are closely related to Aspergillus oryzae, also produce ferrichrome compounds in solid culture such as koji. It has been known. In actual distilled spirits such as shochu and awamori, ferrichrome in commercial products has not been a problem, but black koji mold is also considered to be a microorganism capable of producing ferrichrome, similar to yellow mold. Aspergillus niger has also been used as a food microorganism for many years, and its safety is highly evaluated. From these points, ferrichrome produced by Aspergillus niger is expected to be used as an anemia drug or a functional food. However, regarding the ferrichrome biosynthesis of Aspergillus niger, the genes involved in the biosynthesis have not been elucidated, so that it cannot be effectively used, such as increasing the production amount, and it has not yet been used efficiently.

[0006] If the biosynthesis genes of ferrichromes are identified, there is a possibility that the production of ferrichromes by Aspergillus niger can be freely regulated. For example, in the production of pharmaceuticals, a large amount of ferrichrome can be produced by more actively inducing the expression of a gene involved in ferrichrome biosynthesis. Furthermore, ferrichrome produced by Aspergillus niger has been ingested for many years as sake, awamori and other foods and drinks, and is an extremely safe substance. If ferrochrome can be produced in large quantities by Aspergillus niger, it will be applicable to a wide range of applications from pharmaceuticals to foods. As described above, the ferrichrome production of Aspergillus niger is expected to be applied to various fields, but its biosynthetic gene has not yet been elucidated, so that it has not yet been used efficiently.

[0007]

SUMMARY OF THE INVENTION The present invention is directed to Aspergillu
In order to freely control ferrichrome production of Aspergillus niger and other ferrichromes, it is to provide a cluster gene group involved in ferrichrome biosynthesis of Aspergillus niger. Another object of the present invention is to provide a recombinant vector containing this gene group and a transformant containing this recombinant vector.

[0008] Regarding the biosynthetic pathway of ferrichrome in Aspergillus oryzae, Ustilago maydi
Based on research on the synthetic pathway of ferrichromes in other filamentous fungi such as s and Aureobasidium pullulans, we have already investigated ornithine N5-, which is responsible for the first step of its biosynthesis.
Gene asb1 encoding oxygenase (Japanese Patent Application No. 20
01-176264) and asb2 (Japanese Patent Application No. 2001-324112), which encodes a peptide synthetase involved in the synthesis of peptide bonds in ferrichrome biosynthesis, and the asb1 and asb2 genes are ferrichrome biosynthetic gene clusters. It has also been confirmed that

On the other hand, aspergillus niger (for example, Aspergillus nige
r, Aspergillus awamori, Aspergillus kawachii, Aspe
rgillus usamii, Aspergillus shirousamii, Aspergill
us saitoi, etc.) is also the same as the Aspergillus oryzae for many years.
It is a highly safe and promising ferrichrome-producing bacterium used in awamori production. And as a result of examination from each direction,
The present inventors have noticed for the first time that it is necessary to obtain genes that form a ferrichrome biosynthetic gene cluster in order to realize the true high production of ferrichrome by Aspergillus niger. If these ferrichrome biosynthesis gene clusters of Aspergillus niger can be obtained, ferrichrome with high safety can be industrially supplied to foods and pharmaceuticals.

Therefore, it is an object of the present invention to develop a cluster gene that forms ferrichrome biosynthesis of Aspergillus niger. Another object of the present invention is to provide an Aspergillus oryzae that produces high ferrichrome under all culture conditions by enhancing the expression of ferrichrome biosynthesis cluster genes using genetic engineering techniques. Further, it becomes possible to provide the ferrichrome thus produced for addition to a reagent as an iron chelating agent and to a functional food that is expected to have an anemia improving effect. A further object is to introduce the gene clusters involved in ferrichrome biosynthesis of these koji molds into organisms other than koji mold to produce ferrichromes.

[0011]

Means for Solving the Problems The present invention has been made in order to achieve the above-mentioned object, and firstly, aspergillus niger (Aspergillus niger etc.)
Was examined.

Aspergillus niger (eg Aspergillus niger, Asp
ergillus awamori, Aspergillus kawachii, Aspergillu
s usamii, Aspergillus shirousamii, Aspergillus sai
(toi etc.) is a filamentous fungus that has been used in Japan's traditional fermentation industry, such as shochu and awamori. This strain is known to produce a very large amount of proteins and small molecules useful in the fermentation industry. Due to the high protein-producing ability of this strain and its safety as a brewing microorganism, it has attracted attention as a host for heterologous protein production. (Biotechnology, 6, 1419
(1988), Japanese Patent Laid-Open No. 62-272988). In the production of heterologous proteins using niger, it was confirmed that the productivity of genes of similar species such as Aspergillus is further increased. A. Niger has been reported to produce not only the above-mentioned proteins and enzymes, but also industrially extremely valuable low-molecular components such as organic acid fermentation of citric acid. Among the low molecular weight compounds produced by Aspergillus niger, ferrichrome is a substance that has received a great deal of attention in recent years as a functional component for iron deficiency such as anemia.

In order to utilize this ferrichrome of Aspergillus niger in pharmaceuticals and foods, it is necessary to further improve the productivity. However, existing strain breeding methods such as the mutation method have the productivity up to industrial production level. The improved mutant could not be obtained. Therefore, it was considered possible to mass-produce ferrichrome by isolating genes involved in ferrichrome biosynthesis and enhancing the expression ability of these genes.
The biosynthetic genes of ferrichrome in Aspergillus spp. Are described in Ustilago maydis and Aureobasidium p.
Based on the study of synthetic pathways of ferrichromes in other filamentous fungi such as ullulans, we have already shown that A. o
Gene a encoding ornithine N5-oxygenase responsible for the first step of ferrichrome biosynthesis from ryzae
sb1 (Japanese Patent Application No. 2001-176264) and asb2 (Japanese Patent Application No. 2001-2001) that encodes a peptide synthetase involved in the synthesis of peptide bonds in ferrichrome biosynthesis.
324112) has been identified.

The inventors of the present invention focused on this acquisition method, and if this acquisition method was applied to Aspergillus niger, asb1 and a of Aspergillus niger
A new idea was obtained regarding the possibility that the sb2 gene could also be obtained. If we could obtain these cluster genes in Aspergillus niger, we could breed genetically engineered Aspergillus niger, which could freely control ferrichrome production, and we got the idea that ferrichrome could be industrially produced. In order to mass produce iron ion chelating low-molecular-weight ferrichrome produced by Aspergillus oryzae as a functional component for anemia improvement, gene cloning of the ferrichrome biosynthetic cluster was performed, and a genetically modified black suitable for industrial production of ferrichrome. Breeding koji mold,
It was newly set as a new technical issue.

On the other hand, as a result of the study by the present inventors, Aspergillus oryzae As
In pergillus oryzae, asb1 encoding ornithine N5-oxygenase belonging to the biosynthetic pathway of ferrichrome (Japanese Patent Application No. 2001-83640) and asb2 encoding peptide synthetase involved in the synthesis of peptide bond in ferrichrome biosynthesis (Japanese Patent Application 200
1-324112) formed a cluster of about 36-kb with other related biosynthetic gene clusters (Japanese Patent Application No. 20).
01-324181). Besides, Aureobasidium pullulans
In the ferrichrome biosynthesis gene of Escherichia coli, ornithine N5-oxygenase gene, ABC of ferrichrome
It was reported that the transporter gene, a peptide synthetase gene involved in the synthesis of peptide bonds in ferrichrome biosynthesis, forms a cluster of about 30-kb. Also, in Ustilago maydis, it was reported that the ornithine N5 oxygenase gene and the peptide synthetase gene form a cluster via a 4.3-kb base (Walter MY, et al., J. Bac.
teriol., 183, pp4040-4051, 2001).

As a result of examining these, the present inventors have found that, even in Aspergillus niger, ferrichrome biosynthesis may form a cluster on the chromosome, like the ferrichrome biosynthetic genes of other filamentous fungi. For the first time, I got the idea. Then, the inventors have conducted diligent research to isolate and confirm the genes forming the ferrichrome biosynthetic cluster in Aspergillus niger, and finally completed the present invention. The method for isolating the Aspergillus niger ferrichrome biosynthesis genes will be specifically described below.

L, the first step in ferrichrome biosynthesis
-The gene encoding ornithine N5-oxygenase is Ustilago maydis, Pseudomonas sp. B10, Pseudomo
Those derived from nasaeruginosa and Burkholderia cepacia have been cloned. Therefore, based on the homology of the L-ornithine N5-oxygenase proteins involved in these siderophore biosynthesis, two highly conserved regions were found, and degenerate synthetic primers corresponding to these regions were designed. Using this primer, A. niger
As a result of performing PCR on the chromosomal DNA of
A bp gene fragment was amplified. The nucleotide sequence of this gene was determined to be homologous to the gene encoding the putative oxygenase of Aureobasidium pullulans and the L-ornithine N5-oxygenase of Pseudomonas aeruginosa.

Using the nucleotide sequence of the obtained gene fragment as a probe, A. A chromosomal DNA library of Niger was screened to obtain a positive clone. As a result of determining the entire nucleotide sequence of this clone, this gene encoded a protein consisting of 499 amino acid residues and contained one intron. The obtained gene was named nsb1. In addition, the cDNA of this gene was transformed into E. coli BL21.
It was confirmed that the transformant obtained by introducing into the (DE3) strain produced a significant amount of L-ornithine N5-oxygenase in the cells. Therefore, it was revealed that the obtained nsb1 gene encodes the L-ornithine N5-oxygenase protein that is the rate-determining factor for ferricuricin biosynthesis in Aspergillus niger.

Next, genes encoding filamentous fungal peptide synthetases essential for the biosynthesis of siderophores containing hydroxyornithine are Ustilago maydis and Aureobasidi.
Um pullulans and those derived from Trichoderma harzianum have been cloned. Therefore, based on the homology of these peptide synthetase proteins involved in siderophore biosynthesis, two highly conserved regions were found, and degenerate synthetic primers corresponding to each were designed. Using this primer, A. As a result of performing PCR on the chromosomal DNA of niger, a partial gene fragment of about 800 bp was amplified. As a result of determining the nucleotide sequence of this gene,
It showed homology with the gene encoding the peptide synthetase of aria alternata.

The resulting gene fragment was used as a probe for A.
Screening a niger genomic DNA library yielded positive clones. As a result, the full-length DNA of the obtained partial gene could be obtained. As a result of determining the entire base sequence of this clone, this gene encoded a protein consisting of 7064 amino acid residues and contained two introns. The resulting gene was named nsb2. The nsb2 gene is A. ns on the niger chromosome
It was located about 2-kb upstream of b1 and was found to form a cluster like other organisms.

The isolated gene may be used alone or in melO.
And glaB gene promoter (Japanese Patent Application No. 11-1542
71, JP-A-11-243965) and a high expression promoter. Oryzae can be used to control the production of ferrichrome. The gene transfer method is, for example, a known method using a niaD mutant strain as a host, and the target gene and the marker gene ni are used.
Introducing the aD gene simultaneously (ES Unkle et al., Mol. Gen.
Genet., 218, p. 99-104, 1989). By removing the heterologous gene such as a vector sequence during this gene introduction, a transformant of the self-cloning strain containing no heterologous gene can be obtained. niaD mutant (Koji mold mutant that cannot utilize nitrate: Nitrate Reductase-deficient strain)
For example, Aspergillus oryzae 1013-niaD (FE
RM P-17707) can be used. The details of the present invention will be described below.

First, among the microorganisms reported to produce a siderophore containing hydroxyornithine as a basic constituent amino acid under iron restriction, one in which a gene encoding L-ornithine N5-oxygenase was cloned was selected. To date Ustilago maydis, Pseudomonas
sp.B10, Pseudomonas aeruginosa, Burkholderia cep
Cloning in four strains of acia 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 Ala-Val-Ile-Gly- (Ala or Ser) -Gly-Gln-S
er- (Ser or Ala)-(Thr or Ala) -Glu- (Met or Ile) -Phe-
Met-Asn-Leu- (His or Pro) -Ser- (Arg or Gln) -Phe-Pro
(SEQ ID NO: 5: FIG. 48), the other is Ala-Leu- (Val or
Arg) -Pro-Ser-Asp-Asp- (Ser or Thr)-(Gly or Pro) -Phe
-Val-Asn- (Ser or Glu) -Ala- (Ala or Val) -Phe-Asp-Pro-
Glu-Arg-Thr-Asp (SEQ ID NO: 6: FIG. 49). Using a degenerate oligonucleotide probe prepared based on these two types of partial amino acid sequences, A. As a result of performing PCR on the chromosomal DNA of niger, about 160 bp
Was amplified. As a result of determining the nucleotide sequence of this gene, it showed high homology with the putative oxygenase gene of Aureobasidium pullulans.

Using the nucleotide sequence of the obtained gene fragment as a probe, A. The chromosomal DNA library of Niger was screened. As a result, a full-length clone of the obtained partial gene could be obtained. As a result of determining the entire nucleotide sequence of this clone, this gene encoded a protein consisting of 499 amino acid residues (SEQ ID NO: 1: FIGS. 4 and 5). The promoter region is 1 to 13 in SEQ ID NO: 2.
79 bp, the coding region is 1380 to 2936 bp of SEQ ID NO: 2, the terminator region is 2 of SEQ ID NO: 2
It is from 937 to 3482 bp. Intron is 1
2246 to 2302b of SEQ ID NO: 2
up to p. The obtained gene was named nsb1 (SEQ ID NO: 2: FIGS. 6 and 7). In order to identify the function of nsb1, we attempted large-scale expression of this gene in Escherichia coli. ns
The full-length cDNA of b1 was inserted downstream of the T7 promoter in the pET23b vector. This plasmid is used for E. coli BL
21 (DE3) strain.

As a result of IPTG-inducing culture of the obtained transformant, a significant amount of L-ornithine N5-oxygenase activity was confirmed in the cells. Therefore, the obtained nsb
It was revealed that one gene encodes the L-ornithine N5-oxygenase protein, which is the rate-determining factor for the ferricuricin biosynthesis of Aspergillus oryzae. This strain is Escherichia
Escherichia coli NSID1 and deposit number FERM P-18 at the Patent Biological Deposit Center, National Institute of Advanced Industrial Science and Technology
Deposited as 679. By using the deposited strain, the nsb1 protein can be produced and provided in Escherichia coli under the control of the T7 promoter under the culture condition of 1 mM IPTG induction.

On the other hand, the gene encoding the peptide synthetase was previously cloned in Ustilago maydi.
s, Aureobasidium pullulans, Trichoderma harzianum
Based on the homology of the derived peptide synthetase protein, at least two amino acid sequence conserved regions were found. One is Tyr- (Val or Leu) -Phe-Thr-Ser-Gly-Ser-Thr-Gly-L
ys-Pro-Lys- (Gly or Ala) -Val (SEQ ID NO: 9: Fig. 5
2), the other is Asp- (Thr or Arg) -Gln-Val-Lys-Val- (A
rg or Asn) -Gly-Gln-Arg- (Ile or Met) -Glu-Leu- (Glyor
Asp) -Glu (SEQ ID NO: 10: FIG. 53). These two
A degenerate oligonucleotide probe prepared on the basis of the partial amino acid sequences of various types was used. As a result of performing PCR on niger chromosomal DNA, a gene fragment of about 800 bp was amplified. As a result of determining the nucleotide sequence of this gene, it showed high homology with the peptide synthetase gene of Alternaria alternata.

Using the obtained gene fragment as a probe,
A. The Niger lambda EMBL3 genomic DNA library was screened. As a result, a clone encoding the full-length gene of the obtained partial gene could be obtained. As a result of determining the entire nucleotide sequence of this clone, this gene was found to have 7064 amino acid residues (SEQ ID NO: 3: FIGS. 8 to 3).
It encodes a protein consisting of 5) and contains 2 introns.
Included one. The promoter region is 1 to 1725 bp of SEQ ID NO: 4, and the coding region is 172 of SEQ ID NO: 4.
6 to 23024 bp, and the terminator region is from 23025 to 23114 bp of SEQ ID NO: 4. The introns were 11191 to 11240 bp and 19170 to 19223 bp of SEQ ID NO: 4. The obtained gene was designated as nsb2 (SEQ ID NO: 6: FIGS. 36 to 47). The nsb2 gene is A. ns on the niger chromosome
It was located about 2-kb upstream of b1 and was found to form a cluster like other organisms.

Based on the above results, the cloned gene fragments nsb1 and nsb2 were obtained from Aspergillus niger. It was revealed to be a niger ferrichrome biosynthesis gene, and both genes formed a cluster. Using this gene, it is possible to highly produce or non-produce ferrichrome of Aspergillus niger, and it is possible to modify ferrichrome production according to the application at the molecular level, and it can be applied to various industrial fields. .

[0028]

Example 1 Cloning of nsb1 gene and determination of nucleotide sequence Aspergillus niger In order to perform gene cloning of L-ornithine N5-oxygenase which catalyzes the first step of niger ferrichrome biosynthesis, comparison of amino acid sequence conserved regions of other microorganism-derived L-ornithine N5-oxygenase which has already been reported to be cloned did. from now on
Ustilago maydis, Pseudomonas sp. B10, Pseudomonas
Cloning in four strains of aeruginosa and Burkholderia cepacia 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 amino acid sequence conserved region (1) has the amino acid sequence shown in SEQ ID NO: 5 (FIG. 48) (in the sequence, the first Xaa from the left is Ala or Ser, and the second Xaa is Ser. Or Ala, the third Xaa is Th
r or Ala, 4th Xaa is Met or Ile, 5
The 6th Xaa is His or Pro, and the 6th Xaa is A
rg or Gln are shown respectively. ), And the other amino acid sequence conserved region (2) had the amino acid sequence shown by SEQ ID NO: 6 (FIG. 49) (the first Xaa from the left in the sequence).
Is Val or Arg, the second Xaa is Ser or Th
r, 3rd Xaa is Gly or Pro, 4th Xa
a is Ser or Glu and the fifth Xaa is Ala or V
al is shown respectively. ).

Based on these conserved sequences (1) and (2),
Two degenerate oligonucleotide DNA synthesis primers P
1 and P2 were synthesized respectively. The nucleotide sequence of primer P1 is shown in SEQ ID NO: 7 (FIG. 50), and the nucleotide sequence of primer P2 is shown in SEQ ID NO: 8 (FIG. 51). In these sequences, n (I in the figure) is a deoxyinosine residue and s is G
Or C, w is A or T, r is A or G, y is T or C,
m represents a degenerate base according to the I or U code of A or C.

Using the degenerate primers P1 and P2 described above, PCR was performed using the genomic DNA prepared from Aspergillus niger IFO 4067 strain as a template. An example of the reaction conditions is as follows.

(PCR conditions) -96 ° C (5 minutes), 1 cycle-96 ° C (20 seconds), 45 ° C (1 minute), 72 ° C (3
Min), 30 cycles, 72 ° C (7 minutes), 1 cycle

As a result of analysis of the reaction solution by agarose gel electrophoresis, amplification of a fragment of about 160 bp was observed. As a result of determining the nucleotide sequence of this gene amplification product,
It showed homology with the putative oxygenase of Aureobasidium pullulans. The partial gene 160-bp thus obtained was used as a probe for Aspergillus niger. The lambda EMBL3 genomic DNA library of Niger IFO 4067 strain was screened. Gene made by Amersham Pharmacia
Fluorescein labeling of the probe was performed using the Image Labeling Kit, and about 5000 phage clones were screened. The entire nucleotide sequence contained in the obtained positive clone was determined by the dideoxy method.

As a result, this gene encoded a protein consisting of 499 amino acid residues. The promoter region is from 1 to 1379 bp of SEQ ID NO: 2, the coding region is from 1380 to 2936 bp of SEQ ID NO: 2, and the terminator region is from 2937 to 3482 bp of SEQ ID NO: 2. One intron is included, SEQ ID NO: 2
2246 to 2302 bp. The obtained gene was named nsb1. Its base sequence is SEQ ID NO: 2
(FIGS. 6 and 7), and the corresponding amino acid sequence is shown in SEQ ID NO: 1 (FIGS. 4 and 5).

[0035]

Example 2 Cloning of nsb2 gene and determination of nucleotide sequence Aspergillus niger Comparison of amino acid sequence conserved regions of peptide synthetases involved in siderophore biosynthesis from filamentous fungi, which have already been reported for cloning, for gene cloning of peptide synthetases involved in the synthesis of peptide bonds in niger ferrichrome biosynthesis did. To date, genes encoding filamentous fungal peptide synthetases essential for the biosynthesis of siderophores containing hydroxyornithine have been identified by Ustilago maydis, Aureobasidium.
Those derived from pullulans and Trichoderma harzianum have been cloned. Therefore, it was considered that the gene responsible for the peptide synthetase activity in Aspergillus oryzae could be obtained from the homology of these peptide synthetase proteins involved in siderophore biosynthesis. 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 amino acid sequence conserved region (3) has the amino acid sequence shown in SEQ ID NO: 9 (FIG. 52) (in the sequence, the first Xaa from the left is Val or Leu, and the second Xaa is Gly. Or Ala, respectively, and the other amino acid sequence conserved region (4) had the amino acid sequence shown in SEQ ID NO: 10 (FIG. 53) (the first Xaa from the left in the sequence is Thr or Arg). 2nd Xaa is Arg or Asn, 3rd Xaa is Ile
Alternatively, Met and the fourth Xaa represent Gly or Asp, respectively. ).

Based on these conserved sequences (3) and (4),
Two degenerate oligonucleotide primers P3 and P4 were synthesized respectively. The nucleotide sequence of primer P3 is shown in SEQ ID NO: 11 (FIG. 54), and the nucleotide sequence of primer P4 is shown in SEQ ID NO: 12 (FIG. 55). In these sequences, n (I in the figure) is a deoxyinosine residue, S is G or C, r is A or G, and y is a degenerate base according to the IUB code of T or C.

PCR was performed on the chromosomal DNA prepared from Aspergillus niger IFO 4067 strain using the above-mentioned degenerate primers P3 and P4. The reaction conditions were the same as for nsb1.

As a result of analysis of the reaction solution by agarose gel electrophoresis, amplification of a fragment of about 800 bp was observed. As a result of determining the nucleotide sequence of this gene amplification product,
Indeed, it contained the above P3 and P4 sequences, and as a result of searching for homology, it showed homology with the gene encoding the peptide synthetase of Alternaria alternata. The partial gene 800-bp thus obtained was used as a probe for Aspergillus niger. nig
er lambda EMBL3 genomic DNA library was screened. Fluorescein labeling of the probe was performed using a Gene Image labeling kit manufactured by Amersham Pharmacia, and about 5000 phage clones were screened. As a result, one positive clone was obtained. The entire nucleotide sequence contained in the obtained clone was determined by the dideoxy method.

As a result, this gene encodes a protein consisting of 7064 amino acid residues, and has two introns.
Included one. The promoter region is 1 to 1725 bp of SEQ ID NO: 4, and the coding region is 172 of SEQ ID NO: 4.
6 to 23024 bp, and the terminator region is from 23025 to 23114 bp of SEQ ID NO: 4. The introns were 11191 to 11240 bp and 19170 to 19223 bp of SEQ ID NO: 4. The resulting gene was named nsb2. The nsb2 gene is shown in FIG.
As shown in FIG. It was found to be located approximately 2-kb upstream of nsb1 on the niger chromosome and to form a cluster like other organisms.

The nucleotide sequence of the nsb2 gene is shown in SEQ ID NO: 4.
(FIGS. 36 to 47), and the corresponding amino acid sequence is shown in SEQ ID NO: 3 (FIGS. 8 to 35). This gene is
After labeling with fluorescein as a probe, lambda EMBL3 of Aspergillus niger IFO 4067 strain
It could be obtained by screening a genomic gene library using phage. Further, regarding this gene, A. Since the position on the Niger chromosome has been specified, and the nucleotide sequence and the amino acid sequence of the protein encoded by it have been clarified by the present inventors, A. This gene can be obtained by separating it from the niger chromosome, and the present invention is easy to carry out.

[0042]

[Example 3] Large-scale expression of nsb1 gene in E. coli and enzyme assay In order to identify the function of the nsb1 gene, large-scale expression of this gene in E. coli was performed. Attempts were first made to obtain the nsb1 cDNA. Specifically, iron-restricted medium (2% glucose, 0.6% asparagine, 0.1% dipotassium hydrogen phosphate, 0.1% magnesium sulfate, 30%, 4 days)
Aspergillus flavus A. cultivated in liquid with 0.04% calcium chloride, pH 6.0). Niger to Nippon Gene ISOGE
Total RNA was extracted using N. MRNA was extracted from the total RNA using the Takara Shuzo Oligotex-dT30 <Super> mRNA purification kit. Obtained mRNA 0.5μ
Based on g, a full-length cDNA region (1380 to 2933 bp of SEQ ID NO: 2) was amplified by RT-PCR, and then a blunt-ended fragment was prepared using T4 polynucleotide kinase and T4 DNA polymerase. This fragment is made by Novagen
The ET-23b vector was subcloned into the HincII site in the forward direction with the T7 promoter.

This plasmid was designated as nsb1 Escherichia coli high expression plasmid pENS1 (FIG. 2). After transforming this plasmid into Escherichia coli BL21 (DE3) of Novagen, a transformant showing ampicillin resistance was selected. After culturing the obtained transformant in LB medium at 37 ° C. to OD 0.6,
The final 1 mM IPTG was added and the cells were further cultured at 30 ° C. for 3 hours. From the obtained intracellular protein of E. coli, Toyobo's Ma
Purified using g ExtractorHis-Tag kit (Fig. 3). The obtained purified protein was extracted with 100 mM sodium phosphate buffer (pH 6), the final 1 mM sodium pyruvate, 2 mM L-ornithine was added to the supernatant, and the mixture was aerobically incubated at 37 ° C. for about 2 hours. After stopping the reaction with 10% trichloroacetic acid, hydroxyamine in the centrifugal supernatant was subjected to an iodination method (Gillam et al. Anal. Chem. 5: 841-84.
4, 1981). The specific activity is 1 enzyme that produces 1 nmol of hydroxyamine per minute.
As a unit, the amount of production per mg of bacterial protein was calculated and shown in Table 1 below.

[0044] (Table 1)   Ornithine N5-monooxygenase activity of nsb1 gene transformant ──────────────────────────────────   Plasmid ornithine N5-monooxygenase activity                                         (U / mg) ──────────────────────────────────   Control ND   pENS1 cell-free extract 5.32   pENS1 purified protein 102.1 ──────────────────────────────────

As shown in Table 1, L-ornithine N5-
Oxygenase activity was observed. Therefore, it was revealed that nsb1 encodes L-ornithine N5-oxygenase that catalyzes the first step of ferrichrome biosynthesis. This strain was designated as Escherichia co
li) Named NSID1 and deposited at the Patent Biological Depository Center, National Institute of Advanced Industrial Science and Technology, deposit number FERM P-1
Deposited as 8679. By using the deposited strain, the nsb1 protein can be produced and provided in Escherichia coli under the control of the T7 promoter under the culture condition of 1 mM IPTG induction.

[0046]

According to the present invention, it was confirmed that the cloned gene fragment nsb1 encodes L-ornithine N5-oxygenase which catalyzes the first step of ferrichrome biosynthesis. In addition, the cloned gene fragment nsb2 encoded a peptide synthetase involved in the synthesis of peptide bonds in ferrichrome biosynthesis. nsb1 and nsb2 formed a cluster. Further, using this gene, ferrichromes can be highly produced or non-produced, and ferrichromes can be produced according to the intended use. The ferrichromes that can be mass-produced by the present invention are very safe and can be used in various industrial fields such as foods, pharmaceuticals, and research reagents. It is also possible to design any peptide by recombining each domain of the peptide synthetase protein encoded by nsb2 by protein engineering.

[0047]

[Sequence list]                         SEQUENCE LISTING <110> Gekkeikan Inc., Ltd. <120> Ferrichrome Biosynthetic Gene Cluster of Black Koji-mold <130> 6553 <141> 2002-2-6 <160> 12 <210> 1 <211> 499 <212> PRT <213> Aspergillus niger <400> 1 Met Glu Pro Ala Val Arg Lys Pro Glu Val Ser Phe His Ser Gln Arg  1 5 10 15 Asn Met Pro Ser Lys Gln Gln Arg Val Pro Ser Lys Leu Lys Ala Thr              20 25 30 Pro Lys Asp Glu Leu His Asp Leu Leu Cys Val Gly Phe Gly Pro Ala          35 40 45 Ser Leu Ala Ile Ala Ile Ala Leu His Asp Ala Leu Asp Pro Cys Leu      50 55 60 Asn Lys Ser Ala Pro Ala Pro Gly Ser Gln Pro Lys Val Cys Phe Val 65 70 75 80 Glu Arg Gln Lys Gln Phe Ala Trp His Ser Gly Met Leu Val Pro Gly                  85 90 95 Ser Arg Met Gln Ile Ser Phe Ile Lys Asp Leu Ala Thr Leu Arg Asp             100 105 110 Pro Arg Ser Ser Phe Thr Phe Leu Asn Tyr Leu His Gln Lys Asp Arg         115 120 125 Leu Ile His Phe Thr Asn Leu Gly Thr Phe Leu Pro Ala Arg Leu Glu     130 135 140 Phe Glu Asp Tyr Met Arg Trp Cys Ala Gln Gln Phe Ser Asp Val Val 145 150 155 160 Ser Tyr Gly Glu Glu Val Val Asp Val Met Pro Gly Lys Thr Asp Pro                 165 170 175 Thr Ser Ser Val Val Asp Phe Phe Thr Val Arg Ser Arg Asn Val Glu             180 185 190 Thr Gly Glu Ile Thr Ala Arg Arg Ala Arg Lys Val Val Thr Ala Leu         195 200 205 Gly Gly Ser Ala Lys Met Pro Pro Gly Leu Pro Gln Asp Pro Arg Ile     210 215 220 Met His Ser Ser Lys Tyr Cys Thr Asn Leu Pro His Leu Leu Lys Asn 225 230 235 240 Pro Asn Glu Pro Tyr Asn Ile Ala Val Leu Gly Ser Gly Gln Ser Ala                 245 250 255 Ala Glu Ile Phe His Asp Leu Gln Lys Arg Tyr Pro Asn Ser Lys Thr             260 265 270 Thr Leu Ile Met Arg Asp Ser Ala Met Arg Pro Ser Asp Asp Ser Pro         275 280 285 Phe Val Asn Glu Val Phe Asn Pro Glu Arg Val Asp Lys Phe Tyr Asn     290 295 300 Leu Ser Ala Glu Glu Arg Gln Arg Ser Leu Lys Ala Asp Lys Ala Thr 305 310 315 320 Asn Tyr Ser Val Val Arg Leu Glu Leu Ile Glu Glu Ile Tyr His Asp                 325 330 335 Met Tyr Val Gln Arg Val Lys Asn Pro Asp Glu Lys Gln Trp Gln His             340 345 350 Arg Ile Leu Pro Gly Arg Lys Ile Thr Arg Val Glu His His Gly Pro         355 360 365 Gln Ser Arg Met Arg Ile His Val Arg Ala Thr Lys Asp Gly Ser Asp     370 375 380 Ser Leu Val Gly Asp Gly Lys Glu Ile Leu Glu Val Asp Ala Leu Met 385 390 395 400 Val Ala Thr Gly Tyr Tyr Arg Asn Ala His Glu Gln Leu Leu Ser Asn                 405 410 415 Val Gln His Leu Arg Pro Ala Gly Gln Glu Glu Trp Lys Pro Ser Arg             420 425 430 Asp Tyr Arg Val Glu Met Asp Ala Ser Lys Val Ser Pro Tyr Ala Gly         435 440 445 Ile Trp Leu Gln Gly Cys Asn Glu Lys Thr His Gly Leu Ser Asp Ser     450 455 460 Leu Leu Ser Val Leu Ala Ala Arg Gly Gly Glu Met Val Glu Ser Ile 465 470 475 480 Phe Gly Glu Gln Leu Ala Gly Ala Thr Val Pro Val Thr Lys Leu Arg                 485 490 495 Ala Met Leu         499 <210> 2 <211> 3482 <212> DNA <213> Aspergillus niger <400> 2 ctgtattgat ttcatttgag tgagggaaag cttatcaaga ttggtacctg aacaatccaa 60 ccggttacag gaggtaatgt aagttgggcg tcaatacgat acggataccg ccattcagcc 120 cagtcttgga aacgttgtga aatttcccga catcgggcga ctgcggatct aatctaagcg 180 tccatcatcc cacttttcct ggattaattt gcattgcaca ccggatcatg ctgcggggct 240 cctggtcggc tcttcgggct cggagactga gtaggcagct tggcttattg tttgctttct 300 ttgctatttt gggttcaatt tttatgaagc aaattgaagt gttcgtgggt acaactggtt 360 gatatgcctc gatgaatttt cactggtgct aatggtgatg ggggtggtgg ttgttgtggc 420 gtgatcctca acttcccagg atgtgaacgg atctggggcg agcaaaggga agaagggagg 480 ggctatggtt agcacttcgt ttacaagttc ataccacaag tgggctcttc tggacctgat 540 aaaacatcat cttatctccg aggaaacagc catctatgct tggaatttcg acatatggcg 600 ttgggtttcc tcagtcacac aactactaat atgtgaacac ttccctctcc ttcaagccca 660 acgttaacgc ggagacgata caaatcctcg cgaaatcgcc tgctagatca agcttcagta 720 ttgttaatac ctattctaca taccttgcca accaccaaga gcctcggtgc cccagcatta 780 aaccagtacg taaaaaaaga ggagcagcaa ccacgtttct catgggcagg tggtggaacc 840 gaggagtgcc tatcaagtag gccattgccg gcccgctgtc agaagaacta cggccggcta 900 ctgcttttgc tcggtggcct ggaaaaaaac ccgtccttaa cggatccgct gcttcagaaa 960 aaaaaaaaaa aaagaagaag ggttcggcgc tgataccctt ttctttttta tgttacactc 1020 gtcataaatt ctgaattttt ttttattatt aatattttag tcgaatttac ctttttggta 1080 cctagttttt attttaacat atttttattt ttctctcccc cttacggcgc aaaggtctcc 1140 agtgtgtact aaagttggaa gaaatggtcc ttccccccat ctgtttatcc accaggggat 1200 ataactacga ccatcgctcc caccatttgc cgcggttctc tcccaccttc gttcagaatc 1260 ccatcatcat tgcgtcttcc ttccatcttc ctgctcccca aactctgagc acggaatctc 1320 tgcctcttct ttcattcaca cactttctac tccttatatt cttctcctag tctgtatcaa 1380 tggaacctgc ggtacggaag cccgaagtga gcttccacag ccagcgcaac atgccctcca 1440 agcagcagcg ggtgccttcg aagctcaagg ccacccccaa ggatgagctt cacgatttac 1500 tttgcgtggg tttcggtcct gcctctttgg cgatcgcaat tgccttgcac gatgctctgg 1560 acccttgtct gaacaagtcg gcacctgcgc ctggatcgca gcccaaggtc tgctttgtcg 1620 agcgtcagaa gcagtttgct tggcattcgg gcatgttggt ccccggttca aggatgcaga 1680 tctccttcat caaggatttg gcgactctcc gggatcctcg cagcagcttc accttcctca 1740 actatttgca ccagaaggat cgtttgatcc atttcacaaa cctgggaaca ttccttccgg 1800 ctcgcttgga gttcgaggac tatatgcggt ggtgtgcgca acaattctct gatgtcgtgt 1860 cttatggaga ggaagtggtc gatgtgatgc ccggaaagac ggatcctacc agctcggtgg 1920 tcgacttctt caccgtccgc tcccgcaacg ttgagacggg tgagattact gcgagaagag 1980 cgcgcaaggt ggtgactgca ctcggtggct ctgcgaagat gcccccaggc ctgccccagg 2040 acccccgcat tatgcactcg tcgaagtact gcaccaatct gcctcacctg ctgaagaacc 2100 cgaacgaacc ctacaacatc gccgtgcttg gaagtggcca gagtgctgct gagatcttcc 2160 acgacctgca aaagagatac cccaactcaa agacgacact gatcatgaga gattcggcta 2220 tgcggcctag tgatgactct cctttgtgag tataataccc cttgcacgcc cgacaggtcg 2280 caactaacag caaacaatat agtgttaacg aggtcttcaa ccccgaacgt gtcgacaaat 2340 tctataacct ctctgccgaa gagcgccaac gttccctcaa ggctgacaag gccaccaact 2400 acagtgtggt ccgtcttgaa ctcattgagg agatttacca cgacatgtac gtccagcggg 2460 tcaagaaccc cgacgagaag caatggcagc accgcatcct ccccggacgc aagatcacgc 2520 gagtcgaaca ccatggacct cagagtcgga tgcggattca cgtgagggcg accaaggatg 2580 gatcggacag ccttgtcggc gacggcaagg agatattgga ggtggatgca ctcatggtgg 2640 cgaccggcta ctaccgcaac gcacatgaac agctcttgag caacgtgcag catctgcgac 2700 cggcaggcca ggaggaatgg aagccgagtc gggattaccg ggtcgaaatg gatgccagca 2760 aggtcagccc ctatgctgga atctggctgc agggctgtaa cgagaagaca cacgggctaa 2820 gcgacagtct tctgtcggtg ctggcagcac gcggtggaga gatggtggag tcgatctttg 2880 gcgagcagct tgcgggtgca acggtgccgg tcacgaagtt gcgcgctatg ctgtaaaggc 2940 ttttcgccgg atcggagacc caggaggggt tccgctttct agacgcgtga aatgatggcg 3000 ctagggcgga cattttaatt tccttgggac aaaagaatgc cccggaaaaa tgcatgcata 3060 tccccaaaca aattggtagg gggtgatatc ttcttttgcg atcatctact ctactgtatc 3120 tgtgttcatt tctgtctcac caccttatac tttgttacta ttgttattac cccatcatct 3180 gattccccaa gtacccccat cgcgagttat atagccagcc acattggaat tttataacga 3240 atagagatcg gaatgcatcg agtctgtcct tgttgatttt aattccaccg ctcattgtgt 3300 taggaagaag gccatgctaa tggcgcacgt ttcttttttt cattcgctgc agatgtaatt 3360 cattctcctt aaacttatcc ggcccctgac acgtgcctgt agccgacagg aggacgaaaa 3420 taagtgccta ggaaagaaaa agcgagtggc tggtcgcaca accgcagtct agcacggacc 3480 cg 3482 <210> 3 <211> 7064 <212> PRT <213> Aspergillus niger <400> 3 Met Arg Ser Pro Asn Asp Leu Thr Thr Ser Ala Thr Met Glu Asp Ile   1 5 10 15 His Gln Ile Trp Ser Trp Asn Ala Asn Val Pro Glu Ala Gly Glu Thr              20 25 30 Cys Val His Thr Leu Ile Thr Asp Lys Ala Leu Gln Gln Pro Asp Ala          35 40 45 Leu Ala Val Asp Ala Trp Asp Gly Arg Trp Thr Tyr Gly Glu Leu Glu      50 55 60 Thr Thr Ser Thr Lys Leu Ala Leu Arg Leu Leu Asp Leu Gly Val Gly  65 70 75 80 Pro Gly Thr Asn Val Val Ile Cys Phe Glu Lys Ser Lys Tyr Thr Pro                  85 90 95 Leu Ala Met Leu Ala Val Met Lys Ala Gly Gly Ala Ser Ile Ala Leu             100 105 110 Asp Thr Ser Gln Pro Gln Thr Arg Leu Gln Ser Ile Ile Asn Gln Val         115 120 125 Asp Pro Val Val Ile Leu Cys Ser Ala Ser Lys Ser Gln Leu Ala Lys     130 135 140 Ser Ile Ile Thr Glu Ser Ala Val Ala Leu Thr Ile Asp Glu Asn Ser 145 150 155 160 Leu Ser Glu Met Asn Phe Glu Pro Asp Ser Val Ala Arg Leu Pro Asp                 165 170 175 Val Ser Leu Asp Asn Asn Leu Tyr Val Val Phe Thr Ser Gly Ser Thr             180 185 190 Gly Thr Pro Lys Gly Val Val Val Thr His Leu Asn Tyr Ser Thr Ala         195 200 205 Ile Leu His Gln Gln Glu Ala His Gly Phe Lys Ser Thr Ser Arg Val     210 215 220 Tyr Asp Phe Ala Ser Tyr Ala Phe Asp Val Ser Trp Ser Asn Leu Ile 225 230 235 240 His Thr Leu Thr Ile Gly Ala Cys Leu Cys Ile Pro Ser Glu Gln Asp                 245 250 255 Arg Lys Asp Asn Leu Ile Glu Ser Ile Arg Ser Leu Cys Ala Thr His             260 265 270 Ile Asp Val Thr Pro Ser Val Ala Arg Leu Ile Pro Asp Ser Leu Leu         275 280 285 Cys Lys Ile Glu Thr Leu Val Leu Gly Gly Glu Lys Leu Pro Ala Glu     290 295 300 Leu Ala Arg His Leu Ser Ser Leu Val Thr Leu Lys Asn Pro Tyr Gly 305 310 315 320 Pro Ser Glu Cys Thr Pro Thr Ser Thr Ile Ala Thr Ile Arg Pro Asp                 325 330 335 Asp Asp Asp Ser Lys Ile Ser Ser Ile Gly Arg Gly Leu Gly Val Asn             340 345 350 Thr Trp Val Val Asp Ser Glu Asn Glu Glu Ile Leu Val Pro Ile Gly         355 360 365 Gln Val Gly Glu Leu Leu Leu Glu Gly His Leu Leu Gly Asn Gly Tyr     370 375 380 Leu Asn Asp Gln Thr Lys Thr Thr Ala Ala Phe Val Asn Asn Pro Leu 385 390 395 400 Phe Leu Leu Asn Gly Gly Asp Gly Pro Gly Gln Pro Gly Arg Arg Gly                 405 410 415 Arg Leu Tyr Lys Thr Gly Asp Leu Val Arg Tyr Glu Lys Asp Gly Ser             420 425 430 Leu Thr Ile Ile Gly Arg Lys Asp Thr Gln Ser Arg Leu Arg Pro Arg         435 440 445 Val Glu Leu Gly Asp Ile Glu His His Ile Tyr Arg His Ile Pro His     450 455 460 Gly Thr Val Ser Val Arg Gln Ile Ala Ala Glu Ile Ile Ser Pro Lys 465 470 475 480 Thr Gly Ser Asn Ala Val Leu Ala Ala Phe Leu Glu Val Asp Leu Gly                 485 490 495 Val Glu Asp Thr Gly Ile Ala Glu Gln Leu Phe Ser Lys Thr Glu Lys             500 505 510 Met Met Ser Asn Leu Arg Ser Asn Leu Ala Arg Asp Val Pro Ser Tyr         515 520 525 Met Val Pro Ala Val Phe Ile Pro Leu Arg Asn Phe Pro Leu Ser Pro     530 535 540 Thr Gly Lys Thr Asp Arg Arg Gln Leu Arg Ala Ile Gly Glu Ser Met 545 550 555 560 Asp Leu Thr Val Leu Ala Gly Phe Gly Ala Ala Pro Asn Glu Ala Arg                 565 570 575 Ile Pro Leu Thr Leu Arg Glu Lys Gln Leu Arg Arg Leu Trp Gly Ser             580 585 590 Ile Leu Arg Ile Asp Glu Asn Leu Ile Ala Leu Asp Asp Asn Phe Leu         595 600 605 Gln Arg Ala Gly Asn Pro Asn Ala Ala Met Lys Leu Val Thr Ala Ala      610 615 620 Arg Arg Glu Gly Phe Ser Leu Ser Val Ala Asn Val Leu Lys Tyr Pro 625 630 635 640 Arg Leu Gln Asp Met Ala Gln Val Val Gly Thr Val Glu Gln Glu Gln                 645 650 655 Ala His Glu Ile Met Pro Phe Glu Leu Leu Ser Asp Asp Ile Asn Leu             660 665 670 Asp Leu Ala Leu Arg Glu Ala Ala Ala Ser Cys Asn Val Gln Gly Asn         675 680 685 Gln Ile Gln Asp Ile Tyr Pro Cys Thr Pro Leu Gln Glu Gly Met Met     690 695 700 Ser Leu Ser Ala Lys Arg Glu Gly Asp Tyr Ile Met Gln Tyr Thr Leu 705 710 715 720 Glu Leu His His Arg Cys Asp Ile Glu Arg Leu Gly Lys Ala Trp Ala                 725 730 735 Thr Val Val Ala Thr Thr Pro Ile Leu Arg Thr Arg Ile Val Asp Ile             740 745 750 Thr Ser Gln Gly Leu Val Gln Ala Val Leu Asp Glu Gln Trp Ser Ser         755 760 765 Ser Ser Ile Gln Arg Arg Thr Leu Ser Gln Ala Arg Asp His Lys His     770 775 780 Gln Phe Gly Leu Gly Met Pro Leu Gly Arg Phe Glu Ile Val Thr Gly 785 790 795 800 Asp Ser Ser Asp Phe Lys His Tyr Phe Val Trp Thr Leu His His Ala                 805 810 815 Leu Tyr Asp Gly Trp Ser Leu Gln Leu Leu Leu Glu Lys Leu Glu Asn             820 825 830 Glu Tyr Ala Gly Lys Ala Asp Ala Gln Ser Asn Ser Pro Asp Phe Lys         835 840 845 Arg Phe Ile Lys Tyr Ile Ser Thr Arg Asp Gly Glu Lys Thr His Ser     850 855 860 Phe Trp Thr Glu Gln Phe Gln Asp Val Glu Ala Gln Ile Phe Pro Ser 865 870 875 880 Leu Pro Ser Val Asp Tyr Gln Pro Arg Ser Asp Lys Leu Tyr Thr His                 885 890 895 Ser Val Gly Gly Ile Gln Trp Pro Lys Asn Gly Ile Thr Pro Ser Thr             900 905 910 Thr Ile Arg Ala Ala Tyr Ser Ile Leu Ile Ser Ser Leu Thr Asn Ser         915 920 925 Pro Asp Val Val Phe Gly Ser Ile Thr Thr Gly Arg Gln Ala Ala Val     930 935 940 Asp Glu Val Glu Glu Leu Ile Ala Pro Thr Ile Ala Thr Val Pro Val 945 950 955 960 Arg Val Ser Ile Asp Ser Lys Asp Glu Leu Gly Gln Phe Leu Gln Arg                 965 970 975 Ile Gln Ser Gln Ala Ala Asp Met Ile Glu Phe Glu Gln Thr Gly Leu             980 985 990 His Gln Ile Arg His Ile Asn Ala Asp Ala Glu Arg Ala Cys Gln Phe         995 1000 1005 Gln Thr Leu Leu Val Val Gln Pro Ala Glu Gly Ser Gly Thr Ala Pro    1010 1015 1020 Ser Asp Ile Phe Thr Asn Ile Pro Asp Asp Ile Arg Lys Gly Asp Gly 1025 1030 1035 1040 Asn Ser Ala Ala Glu Leu Gly Thr Tyr Ala Leu Thr Met Glu Cys Leu                1045 1050 1055 Leu Lys Lys Asp Gly Leu Asp Leu His Met Asn Tyr Cys Ser Ala Val            1060 1065 1070 Ile Ser Glu His Gln Val Arg Arg Leu Ser Gln Gln Phe Glu His Val        1075 1080 1085 Leu Gly Gln Ile Cys His Leu Thr Met Ile Ile His Arg Gln Arg Thr   1090 1095 1100 Thr Leu Glu Ser Leu Arg Gln Pro Thr Thr Glu Thr Glu Arg Gln Met 1105 1110 1115 1120 Gln Arg Ile Trp Ala Gln Val Leu Asn Leu Asn Gln Ala Ser Ile Gly                1125 1130 1135 Leu Asp Tyr Ser Phe Phe Gln Leu Gly Gly Asp Ser Ile Ala Ala Met            1140 1145 1150 Glu Val Val Thr Glu Ala Arg Lys Leu Gly Leu Lys Leu Ala Val Ser        1155 1160 1165 Asp Ile Phe Arg Arg Pro Lys Leu Gln Asp Val Ala Lys Lys Ala Cys    1170 1175 1180 Asp Ser Gly Leu Gln Leu Tyr Gly Glu Glu Gln Leu Met Asn Asp Ser 1185 1190 1195 1200 Glu Val Gln Val Lys Gly Gly Thr Glu His Thr Lys Leu Pro Asn Asp                1205 1210 1215 Asp Lys Ala Val Ala Gly His Glu Thr Gln Gln Val Met Val Trp Glu            1220 1225 1230 Gly Met Phe Asp Lys Glu Val Tyr Gly Thr Ile Asn Asp Val Gln Leu        1235 1240 1245 Glu Lys Ile Gly Arg Asp Phe Ile Gly Trp Thr Ser Met Tyr Asn Gly    1250 1255 1260 Asn Gln Ile Asp Asn Val Glu Leu Asn Glu Trp Leu Asp Asp Thr Ile 1265 1270 1275 1280 Ala Thr Ile Arg Ser Ser Gly Ser Thr Ala Asn Ile Leu Glu Leu Gly                1285 1290 1295 Ser Gly Ser Gly Met Ile Leu Phe Asn Leu Val Asn Gly Leu His Ser            1300 1305 1310 Tyr Val Gly Leu Asp Pro Ser Glu Lys Ala Val Asp Phe Val Cys Ser        1315 1320 1325 Thr Val Lys Ser Ile Pro Gln Leu Ala Asp Arg Val Tyr Asn Ile Lys    1330 1335 1340 Gly Thr Ala Asp Asn Ile Asn Ser Leu Gly Val Pro Ile Ser Ala Asn 1345 1350 1355 1360 Met Val Ile Val Asn Ser Val Val Gln Tyr Phe Pro Ser Gln Asp Tyr                1365 1370 1375 Leu Leu Lys Val Ile Glu Asp Leu Val Gln Leu Glu Thr Val Arg Thr            1380 1385 1390 Ile Phe Phe Gly Asp Ile Arg Ser Tyr Ala Leu Phe Arg Glu Phe Gln        1395 1400 1405 Val Thr Arg Ala Leu His Ile Ala Gly Asp Thr Ala Thr Glu Asp Glu    1410 1415 1420 Ile Arg Gln Met Met Ala Asn Met Glu Gln Val Glu Leu Glu Leu Leu 1425 1430 1435 1440 Val Asp Pro Ala Phe Phe Thr Ser Leu Val Asp Arg Phe Pro Gly Leu                1445 1450 1455 Val Glu His Val Glu Ile Leu Pro Lys Arg Leu Lys Ser Thr Asn Glu            1460 1465 1470 Leu Ser Ala Tyr Arg Tyr Ala Ala Val Val His Leu Lys Asp Ser Asn        1475 1480 1485 Gln Leu Ala Gln Pro Leu Gln Val His Asp Ile Gln Lys Glu Ser Trp    1490 1495 1500 Ile Asn Tyr Ser Gly Arg Gln Leu Asn Arg Gln Ser Leu Leu Gln Leu 1505 1510 1515 1520 Val Gln Asp Ser Phe Leu Arg Asp Pro Ser Pro Ser Ser Val Val Ala                1525 1530 1535 Val Cys Asn Ile Pro Tyr Ser Met Thr Val Tyr Glu Arg His Val Ile            1540 1545 1550 Glu Trp Leu Asp Ser Gly Leu Thr Ala Gly Pro Asp Ala Glu Asp Trp        1555 1560 1565 Leu Ser Ser Ile Arg Gln Thr Ser Gln Glu Cys Ser Ser Leu Ser Ala    1570 1575 1580 Leu Asp Leu Gln Gln Ile Ala His Gln Thr Gly Trp Gln Val Glu Ile 1585 1590 1595 1600 Ser Trp Ser Arg Gln Phe Ser Gln Arg Gly Gly Leu Asp Ala Ile Phe                1605 1610 1615 His Arg His His Ser Arg Gly Asp Arg Thr Ser Arg Ala Leu Phe Asn            1620 1625 1630 Phe Pro Thr Asp Tyr Gln Gly Arg Pro Phe Gln Ser Leu Ser Lys Trp        1635 1640 1645 Pro Leu Gln Arg Lys Arg Gln Gly Glu Glu Lys Gln Ile Thr Leu Gly    1650 1655 1660 Asp Val Ser Thr Val Cys Lys Glu Asp Leu His Asp Ile Trp Thr Trp 1665 1670 1675 1680 Asn Glu Val Val Pro Asp Ala Leu Glu Ala Cys Val His Asp Leu Ile                1685 1690 1695 Ser Asp Thr Val Arg Ala Gln Pro Gln Ser Pro Ala Ile Cys Ala Trp            1700 1705 1710 Asp Gly Glu Trp Ser Tyr Ile Glu Leu Asp Asp Leu Ser Ser Arg Leu        1715 1720 1725 Ala His Ala Leu Ala Pro Phe Gly Val Ala Asn Thr Val Val Pro Ile    1730 1735 1740 Cys Phe Glu Lys Ser Lys Trp Thr Pro Val Ala Thr Leu Ala Val Met 1745 1750 1755 1760 Lys Ala Gly Ala Ala Ser Val Thr Leu Asp Ala Ser Gln Pro Leu Glu                1765 1770 1775 Arg Leu Arg Ser Ile Ile Ser Gln Thr Asp Pro Arg Val Ile Leu Ser            1780 1785 1790 Ser Ala Ser Lys Gln Gly Leu Gly Ala Gln Leu Thr Lys Ala Pro Asn        1795 1800 1805 Leu Val Val Asp Gln His Ser Ile Ser Thr Met His Ile Thr Ala Glu    1810 1815 1820 Pro Leu Pro Thr Val Asp Pro Ser Ser Lys Leu Tyr Ile Val Phe Thr 1825 1830 1835 1840 Ser Gly Thr Thr Gly Val Pro Lys Gly Val Ile Ile Thr His Ser Asn                1845 1850 1855 Phe Ser Ser Ala Ile Arg His Gln Gln Lys Ala His Gly Phe Lys Ser            1860 1865 1870 Thr Ser Arg Ile Tyr Asp Phe Ala Ser Tyr Ala Phe Asp Val Ser Trp        1875 1880 1885 Ser Asn Phe Ile His Ala Leu Thr Val Gly Ala Cys Leu Cys Ile Pro    1890 1895 1900 Ser Asp Glu Asp Arg Arg Asp Asp Leu Ala Gly Ser Leu Glu Arg Phe 1905 1910 1915 1920 Gly Ala Thr His Val Asp Met Thr Pro Ser Ala Ala Ser Leu Leu Pro                1925 1930 1935 Glu Lys Ser Phe Lys Arg Leu Glu Thr Val Val Leu Gly Gly Glu Lys            1940 1945 1950 Leu Ser Val Glu Ser Ala Gln Arg Trp Ser Ser Leu Val Ser Leu Lys        1955 1960 1965 Asn Pro Tyr Gly Pro Ser Glu Cys Thr Pro Thr Ala Thr Ile Ala Thr    1970 1975 1980 Val Thr Pro Thr Asp Glu Tyr Lys Ser Ser Ile Gly Arg Gly Leu Gly 1985 1990 1995 2000 Leu Asn Thr Trp Ile Val Asn Thr Val Thr Asp Ser Leu Val Pro Val                2005 2010 2015 Gly Gly Val Gly Glu Leu Leu Leu Glu Gly Pro Leu Val Gly Ala Gly            2020 2025 2030 Tyr Leu Gly Asp Asp Thr Lys Thr Ala Ala Ser Phe Val Glu Asp Pro        2035 2040 2045 Gln Phe Leu Leu Gln Ile Cys Pro Gln Gly Gln Ala Arg His Thr Arg    2050 2055 2060 Met Tyr Lys Thr Gly Asp Leu Val His Tyr Asn Pro Asp Gly Ser Leu 2065 2070 2075 2080 Ser Phe Val Gly Arg Lys Asp Ala Gln Val Lys Ile His Gly Gln Arg                2085 2090 2095 Val Glu Leu Thr Glu Ile Glu Ser His Ile Arg Arg Thr Ser Lys Thr            2100 2105 2110 Ile Gln Val Ala Val Leu Phe Thr Lys Ser Gly Leu Cys Ala Asn Arg        2115 2120 2125 Val Val Ala Phe Val Cys Ile Gln Gly Thr Gly Gln Thr Gln Thr Ala    2130 2135 2140 Ala Asp Gln Ile Arg Leu Ile Asp Pro Lys Tyr Ser Thr Leu Val Thr 2145 2150 2155 2160 Ala Tyr Thr Glu Ser Ala Lys Ser Ser Leu Ser Asp Thr Leu Pro Ala                2165 2170 2175 Tyr Met Ile Pro Ser Ile Trp Ile Pro Leu Gln His Val Pro Leu Ser            2180 2185 2190 Thr Ser Gly Lys Leu Asp Tyr Lys Ala Leu Lys Ser Trp Leu Asp Ser        2195 2200 2205 Met Asp Ala Lys Thr Phe Ala Asn Ile Leu Thr Ala Ser Asp Gly Asp    2210 2215 2220 Val Lys Leu Arg Lys Ala Glu Thr Glu Leu Glu Gln Val Ile Val Glu 2225 2230 2235 2240 Ala Cys Ala Lys Ile Leu Asn Ile Thr Ala Ser Lys Val Asn Leu Asp                2245 2250 2255 Arg Ser Phe Ile Ala Asn Gly Gly Asp Ser Ile Ser Ala Met Arg Leu            2260 2265 2270 Val Ala His Cys Arg Ala Asp Asn Val Val Phe Ser Val Ala Lys Leu        2275 2280 2285 Leu Lys Ser Lys Thr Leu Ala Ala Leu Ala Ser Ser Ser Lys Ile Lys    2290 2295 2300 Ser Ala Ser Asn Val Leu Gly Phe Tyr Glu Glu Lys Ser Asp Ser Phe 2305 2310 2315 2320 Ala Leu Ser Pro Ile Gln Gln Trp Phe Phe Glu Gln Gly Leu Tyr Lys                2325 2330 2335 Arg Ser Asn Asp Asn Phe Asp Asn Gln Gly Phe Tyr Leu Lys Val Lys            2340 2345 2350 Arg Pro Leu Leu Thr Lys Asp Ile Asp Ser Ala Ile Ser Lys Val Val        2355 2360 2365 Gln His His Ser Met Leu Arg Ala Arg Phe His Arg Asn Gly Asp Glu    2370 2375 2380 Trp Thr Gln Lys Thr Leu Lys Pro Asp Thr Asn Gly Leu Tyr His Phe 2385 2390 2395 2400 Gly Val His His Thr Cys Leu Pro Ala Asp Ile Glu Arg Leu Ala Leu                2405 2410 2415 Ser Arg His Gln Met Ile Asp Ile Glu Lys Gly Pro Val Phe Ser Ala            2420 2425 2430 Asp Ile Cys His Asn Ala Phe Gly Glu Gln Tyr Leu Ile Leu Ile Ala        2435 2440 2445 His His Leu Val Val Asp Leu Val Ser Trp Arg Val Ile Leu Glu Asp    2450 2455 2460 Ile Glu Ser Leu Leu Gly Gly Ser Asn Leu Gln Pro Ser Leu Pro Phe 2465 2470 2475 2480 Gln Val Trp Asn Asp Met Gln Ile Glu Arg Ala Lys Glu Ser Ser Leu                2485 2490 2495 Leu Asp Pro Glu Asn Val Leu Ser Thr Thr Gly Ile Asn Asn Asn Leu            2500 2505 2510 Asp Phe Trp Gln Ala Thr Ala Glu Thr Lys Asn Thr Val Glu Asp His        2515 2520 2525 Leu Asn Phe Cys Thr Lys Ile Asp Ser Ser Lys Gln Ser Ser Ser Ser    2530 2535 2540 Lys Thr Gln Ile Thr Arg Ser Thr Leu Glu Pro Val Asp Leu Leu Leu 2545 2550 2555 2560 Ala Ala Val Trp His Ala Phe Phe Lys Thr Phe Pro Gln Arg Asp Gly                2565 2570 2575 Leu Thr Ile Phe Ile Glu Gly His Gly Arg Glu Pro Trp Ser Ser Asp            2580 2585 2590 Ile Asp Leu Ser Arg Thr Val Gly Trp Phe Thr Thr Ile Ser Pro Ile        2595 2600 2605 His Val Ser Lys Ser Asp Val His Lys Ser Val Ala Ser Leu Val Arg    2610 2615 2620 Val Val Lys Asp Ala Arg Arg Leu Leu Pro Ala Asn Gly Trp Ala Tyr 2625 2630 2635 2640 Phe Ala Ser Arg Tyr Phe Asn Glu Ser Gly Lys Ser Ala Phe Lys Ser                2645 2650 2655 His Asp Ser Ile Met Glu Ile Thr Phe Asn Tyr His Gly Gln Phe Gln            2660 2665 2670 Gln Leu Glu Asn Glu Lys Ala Met Phe Glu Asn Val Thr Leu Ser Gly        2675 2680 2685 Val Cys Glu Gln Gly Pro Ala Leu Pro Ala Ser Ser Leu Ile Ala Val    2690 2695 2700 Glu Val Ser Ile Asp Arg Gly Gln Val Thr Phe Asp Val Ser Ala Asn 2705 2710 2715 2720 Arg Tyr Ile Asn His Gln Asp Cys Ile Ser Asn Trp Ile Lys Ala Ile                2725 2730 2735 Ser Gln Ser Leu Glu Thr Ile Ser Asn Glu Leu Val Ser Thr Glu Ile            2740 2745 2750 Ser His Arg Thr Leu Cys Asp Tyr Glu Phe Leu Ser Leu Gly Tyr Thr        2755 2760 2765 Glu Leu Asp Arg Leu Gln Glu Ser Val Ile Pro Glu Ile Glu Lys Leu    2770 2775 2780 Asn Asn Ser Thr Val Glu Cys Ile Tyr Arg Cys Leu Pro Thr Val Asp 2785 2790 2795 2800 Gly Ile Leu Ile Ser Gln Phe Lys Asp Pro Glu Ser Tyr Lys Thr Val                2805 2810 2815 Gln His Phe Glu Ile Thr Ser His Ile Asp Asp Gln Ile Asp Leu Glu            2820 2825 2830 His Leu Ser Leu Ala Trp Gln Lys Val Val Ala Asn Gln Pro Ala Leu        2835 2840 2845 Arg Thr Val Phe Ile Pro Gly Met Asp Lys Ala Ala Ala Phe Asn Gln  2850 2855 2860 Val Val Leu Ser Gln Tyr His Ala Glu Leu Ile Ile Leu His Thr Ala 2865 2870 2875 2880 Ser Asp Glu Tyr Thr Glu Ala Leu Glu Met Phe Lys Asn Leu Ile Pro                2885 2890 2895 Ile Asn Tyr Gln Ser Phe Lys Pro Pro His Arg Ala Ala Ile Cys Arg            2900 2905 2910 Ile Ser Pro Ser Arg Val Leu Cys Gln Val Glu Met Ser His Ala Ile        2915 2920 2925 Thr Asp Gly Ala Ser Thr Ser Ile Leu Ala Asn Asp Leu Leu Gln Ala    2930 2935 2940 Tyr Asn Gly Asn Ser Met Pro Ile Asn Leu Met Asp Thr Ala Cys Glu 2945 2950 2955 2960 Phe Ala Arg Ala Gln Leu Thr Ser Ser Phe Gly Glu Lys Leu Ser Tyr                2965 2970 2975 Trp Lys Lys Lys Leu Arg Glu Met Asp Pro Cys His Phe Pro Lys Ile            2980 2985 2990 Ser Gly Ala Ser Thr Gln Gly Thr Gly Thr Ser Val Cys Lys Ile Arg        2995 3000 3005 Gly Ala Leu Phe Ser Lys Ile Gln Asp Tyr Cys Asn Ser Val Glu Val    3010 3015 3020 Thr Thr Ala Ser Leu Phe Gln Thr Ile Trp Ala Leu Thr Leu Ala Ala 3025 3030 3035 3040 Tyr Thr Gly Asn Asp Ser Thr Cys Phe Gly Tyr Leu Ala Ser Gly Arg                3045 3050 3055 Asp Leu Pro Ile Ala Gly Ile Asp Lys Ser Ile Gly Ala Phe Thr Asn            3060 3065 3070 Met Leu Val Cys Arg Val Asn Ile Asn Arg Glu Thr Glu Ile Leu Gln        3075 3080 3085 Phe Val Gln Thr Val His Asp Gln Val Met Gln Asp Leu Glu His Gln    3090 3095 3100 His Cys Ser Leu Ala Ser Ile Gln His Glu Leu Gly Ile Asn Ser Asp 3105 3110 3115 3120 Asn Pro Leu Phe Asn Ser Ile Leu Ser Tyr Gln Lys Gln Asp Asp Glu                3125 3130 3135 Pro Ala Gly Asp Glu Gly Leu Val Ile Lys Ala Leu Asp Gly Gln Asp            3140 3145 3150 Pro Thr Glu Tyr Asp Ile Val Leu Asn Ile Gly His Ala Thr Asp His        3155 3160 3165 Ile Glu Ile Val Phe Asp Tyr Lys His Ala Cys Leu Ser Ser Ile Gln    3170 3175 3180 Ala Glu Ser Val Leu Ser Leu Met Gln Ser Thr Ala Ala Ala Leu Val 3185 3190 3195 3200 Gln His Ala Ser Gly Asp His Gln Thr Leu Arg Ser Val Asn Met Val                3205 3210 3215 Ser Thr Glu Asp Ile Ser Asp Ile Trp Gln Trp Asn Ser Asp Val Pro            3220 3225 3230 Val Thr Val Asp Asp Cys Val His His Ile Ile Thr Arg Thr Cys His        3235 3240 3245 Lys Arg Pro Gln Ala Pro Ala Ile Cys Ala Trp Asp Gly Asp Trp Thr    3250 3255 3260 Tyr Ala Glu Val Asn Lys Leu Ser Asp Lys Leu Ala His Leu Leu Val 3265 3270 3275 3280 Ser Tyr Gly Val Gly Pro Gly Val Val Val Pro Leu Cys Phe Glu Lys                3285 3290 3295 Ser Lys Trp Thr Pro Ile Ala Met Met Ala Val Met Lys Ala Gly Gly            3300 3305 3310 Ala Ser Val Ala Met Asp Ser Thr Gln Pro Glu Glu Arg Leu Arg Ala        3315 3320 3325 Ile Val Asn Gln Val Lys Ser Pro Ile Ile Leu Ser Ser Phe Ala Asn    3330 3335 3340 Glu Gln Leu Ala Ser Arg Leu Ile Ser Glu Leu Pro Ala His Gln Gly 3345 3350 3355 3360 Pro His Asn Lys Arg Gln Arg Ser Gly Lys Phe Glu Cys Ser Glu Trp                3365 3370 3375 Lys Pro Leu Pro His Val Asn Pro Ser Asp Thr Leu Tyr Val Val Phe            3380 3385 3390 Thr Ser Gly Ser Thr Gly Val Pro Lys Gly Val Ala Val Thr His Ser        3395 3400 3405 Asn Ile Ala Ser Ala Ile Lys His Gln Arg His Leu Leu Gly Phe Thr    3410 3415 3420 Ser Glu Ser Arg Val Phe Asp Phe Ser Ser Tyr Met Phe Asp Val Val 3425 3430 3435 3440 Trp Cys Asn Leu Leu Gln Gly Leu Ser Ala Gly Ser Cys Val Cys Ile                3445 3450 3455 Pro Ser Asp Asn Glu Arg Lys Thr Asp Phe Met Ala Ala Ile Val Lys            3460 3465 3470 Met Arg Ala Asn Leu Val Ile Leu Thr Pro Ser Ala Ile Arg Gly Leu        3475 3480 3485 Lys Leu Asp Ala Leu Asn Ser Leu Cys Asn Val His Phe Ile Gly Glu    3490 3495 3500 Pro Leu His Val Asp Thr Phe Arg Ser Val Asp Glu Ser Val Thr Ile 3505 3510 3515 3520 Ser Asn Leu Tyr Gly Pro Thr Glu Cys Thr Thr Phe Ser Thr Val Gln                3525 3530 3535 Thr Ile Cys Gly Arg Gln His Gln Ser Ile Thr Ile Gly Lys Gly Ala            3540 3545 3550 Gly Leu Asn Thr Trp Val Ala Asp Ile Ala Thr Gly Thr Ala Leu Val        3555 3560 3565 Pro Ile Gly Ser Ala Gly Glu Leu Leu Leu Glu Gly Pro Leu Val Ala    3570 3575 3580 Ala Gly Tyr Arg Gly Asp Ala Val Lys Thr Ala Ala Ala Phe Val Tyr 3585 3590 3595 3600 Asp Pro Pro Phe Leu Leu Arg Gly Ser Val Gly His Pro Gly Arg Arg                3605 3610 3615 Gly Arg Leu Tyr Lys Thr Gly Asp Ile Val Arg Tyr Asn Ser Asn Gly            3620 3625 3630 Thr Leu Thr Phe Leu Gly Arg Lys Asp Ser Gln Val Lys Ile Asn Gly        3635 3640 3645 Gln Arg Val Glu Phe Gly Asp Ile Glu Ser His Ile Asn Gly Ala Leu    3650 3655 3660 Leu Pro Asp Phe Ser Glu Gly Gln Ala Leu Val Asp Phe Val Thr Pro 3665 3670 3675 3680 Gln Gly Ser Ser Arg Pro Met Leu Val Ala Phe Val Tyr Phe Gly Pro                3685 3690 3695 Thr Val Thr Glu Gly Met Asp Glu Ala Asp Leu Leu Ser Leu Ala Lys            3700 3705 3710 Arg Thr Ala Ile Ser Leu Asp Glu Ser Leu Ala Ala Arg Ile Pro Ala        3715 3720 3725 Phe Met Ile Pro Ser Ala Tyr Ile Pro Leu Gln Lys Ile Pro Val Thr    3730 3735 3740 Ala Thr Gly Lys Thr Asp Arg Arg Arg Leu Arg Glu Met Ala Lys Asp 3745 3750 3755 3760 Val Thr Trp Asp Gln Leu Ile Lys Ala Asp Ser His Gly Pro Asp Arg                3765 3770 3775 Cys Gln Pro Gly Thr Glu Met Glu Ile Gln Leu Gln Ile Leu Trp Gly            3780 3785 3790 Thr Val Leu Gly Val Glu Ser Ser Leu Ile Gly Ala His Asp Asn Phe        3795 3800 3805 Met Arg Val Gly Gly Asp Ser Val Gly Ala Ile Arg Leu Ala Ser Ser    3810 3815 3820 Ala Arg Glu Leu Gly Phe Thr Leu Asn Val Ala Asp Ile Leu Lys Asn 3825 3830 3835 3840 Pro Lys Leu Ser Asp Met Ala Lys Leu Met Ile Arg Thr Glu Pro Ser                3845 3850 3855 Gln Asp Ile Ser Ile Lys Glu Phe Ser Leu Leu Lys Pro Gly Ser Asp            3860 3865 3870 Val Asn Trp Ala Val Ala Glu Thr Ser Ala Leu Cys Gly Val Asp Gly        3875 3880 3885 Asn Gln Val Glu Asp Leu Tyr Pro Cys Thr Pro Leu Gln Glu Gly Leu    3890 3895 3900 Leu Ala Leu Thr Thr Lys Arg Pro Gly Asp Tyr Ile Ile Arg Cys Ile 3905 3910 3915 3920 Leu Glu Leu Lys Arg Ser Thr Asp Val Lys Lys Phe Cys Ala Ser Trp                3925 3930 3935 Glu Ala Val Leu Glu Ser Thr Pro Ile Leu Arg Thr Arg Ile Val Asp            3940 3945 3950 Ile Ala Glu Gln Gly Leu Val Gln Ala Val Ile Lys Gln Pro Ala Gln        3955 3960 3965 Trp Thr Ser Ala Glu Ala Ser Ser Leu Val Asp Phe Val Ala Ala Asp    3970 3975 3980 Asn Glu Lys Thr Thr Gly Leu Gly Met Pro Leu Val Arg Phe Gly Leu 3985 3990 3995 4000 Val Gln Glu Thr Asn Lys His Phe Phe Val Leu Thr Leu His His Ala                4005 4010 4015 Val Tyr Asp Gly Trp Ala Leu Asn Leu Val Phe Glu Lys Leu Glu Asn            4020 4025 4030 Phe Tyr Ala Gly Ser Ser Arg His Glu Ser Pro Asp Phe Arg His Phe        4035 4040 4045 Val Lys His Ile Ser Ser Leu Asp Asn Asp Ala Ala Ala Lys Phe Trp    4050 4055 4060 Lys Asp Gln Leu Gln Gly Ser Glu Ala Pro Thr Phe Pro Ser Leu Pro 4065 4070 4075 4080 Thr Ala Thr Phe Val Pro Lys Ser Glu Lys Thr Ile Leu His Thr Val                4085 4090 4095 Glu Glu Leu Gln Trp Pro Lys Thr Asn Val Thr Ala Phe Thr Leu Val            4100 4105 4110 Arg Ala Ala Leu Ser Leu Leu Thr Ala Ala Tyr Thr Asn Ser Glu Asp        4115 4120 4125 Val Cys Phe Gly Val Thr Ser Asn Gly Arg Gln Val Gly Leu Pro Gly    4130 4135 4140 Val Glu Arg Met Ile Gly Pro Thr Ile Ala Thr Val Pro Val Arg Val 4145 4150 4155 4160 Arg Ile Asp Arg Glu Gln Arg Leu Gln Ala Phe Leu Thr Gln Met Gln                4165 4170 4175 His Gln Ser Ile Asp Met Ile Ala Phe Glu Gln Phe Gly Leu Gln Gln            4180 4185 4190 Ile Arg Lys Ser Ser Pro Asp Ala Glu Arg Ala Cys Asn Phe Gln Ser        4195 4200 4205 Leu Leu Ile Val Gln Pro Ala Glu Glu Thr Ala Gln Trp Gln Ser Asp    4210 4215 4220 Ile Ile Ala Arg Asp Ile Gly Glu Gly Ala Asp Asp Pro Met Gly Ile 4225 4230 4235 4240 Gln Glu Ile Gly Thr Tyr Ala Leu Thr Leu Glu Cys His Leu Gly Pro                4245 4250 4255 Asp Ser Leu Leu Ile Lys Ala Asn Phe Asp Ser Asn Val Ile Asp Glu            4260 4265 4270 Leu Gln Val Lys Arg Phe Thr Lys Gln Phe Glu His Val Leu Arg Gln        4275 4280 4285 Ile Cys Cys Ser Gly Ser Gly Leu Val Val Ser Asp Ile Asp Thr Thr    4290 4295 4300 Ser Arg Gln Asp Met Glu Asp Ile Trp Lys Trp Asn Ala Val Val Pro 4305 4310 4315 4320 Gln Ser Val Asn Thr Pro Val His Glu Leu Ile Ser Ser Val Ala Arg                4325 4330 4335 Arg Leu Pro His Val Gln Ala Val Cys Ala Trp Asp Gly Asn Trp Thr            4340 4345 4350 Tyr Arg Gln Leu Asp Asp Leu Ser Asn Tyr Val Ala His His Leu Val        4355 4360 4365 Asp Leu Gly Val Gly Ser Gln Asp Ile Val Pro Leu Leu Phe Glu Lys    4370 4375 4380 Ser Lys Trp Met Pro Ile Ala Met Leu Gly Val Met Lys Ala Gly Ala 4385 4390 4395 4400 Ala Ser Val Ala Val Asp Thr Ser Gln Pro Lys Asp Arg Leu Arg Met                4405 4410 4415 Ile Ile Asp Gln Ala Asn Pro Thr Val Ala Leu Ser Ser Ala Asp Lys            4420 4425 4430 Leu Pro Leu Val Arg Ser Leu Thr Lys Ala Gln Ser Phe Val Val Ser        4435 4440 4445 Gly Gln Gly Ile Asp Arg Leu Leu Lys Pro Ser Leu Asn Ala Thr Leu    4450 4455 4460 Pro Val Val Asp Pro Ser Ser Arg Leu Tyr Leu Val Phe Thr Ser Gly 4465 4470 4475 4480 Ser Thr Gly Val Pro Lys Gly Val Ile Ile Arg His Cys Asn Phe Ala                4485 4490 4495 Ser Ala Ile Lys His Gln Lys Glu Val Gln Gly Ile Leu Pro Thr Ser            4500 4505 4510 Arg Val Tyr Asp Phe Ala Ser Tyr Ala Phe Asp Val Ala Trp Ala Asn        4515 4520 4525 Ala Leu Leu Thr Phe Glu Ser Gly Ala Cys Leu Cys Ile Pro Ser Asp    4530 4535 4540 Ala Asp Arg Lys Asn Asp Leu Asn Gly Ser Ile Ala Arg Leu Lys Pro 4545 4550 4555 4560 Thr His Ala Asp Leu Thr Pro Ser Ala Ala Leu Val Leu Ser Lys Glu                4565 4570 4575 Ser Leu Gln Gln Leu Asp Thr Leu Thr Leu Gly Gly Glu Arg Leu Leu            4580 4585 4590 Ala Glu Tyr Ala Thr Lys Trp Ser Gln Phe Val Thr Val Lys Asn Ser        4595 4600 4605 Tyr Gly Pro Ser Glu Cys Thr Pro Thr Ala Thr Phe Thr Glu Ala Ile    4610 4615 4620 Gly Arg Gly Tyr Asp Leu Gly Ala Ser Ile Gly Lys Pro Ala Gly Leu 4625 4630 4635 4640 Asn Thr Trp Val Val Asp Pro Val Thr Gly Gln Ser Leu Val Pro Ile                4645 4650 4655 Gly Gly Val Gly Glu Leu Phe Leu Glu Gly Pro Leu Val Gly Ala Gly            4660 4665 4670 Tyr Leu Asp Asp Ala Glu Lys Thr Asn Ala Ala Phe Ile His Asp Pro        4675 4680 4685 Pro Phe Leu Leu Arg Gly Asn Val Val Ala Gln Pro Gly Arg Arg Gly    4690 4695 4700 Thr Leu Tyr Lys Thr Gly Asp Ile Val Arg Tyr Asn Ser Asp Gly Ser 4705 4710 4715 4720 Leu Thr Phe Val Trp Arg Lys Asp Thr Gln Val Lys Ile Asn Gly Gln                4725 4730 4735 Arg Val Glu Leu Ala Glu Ile Glu Ser His Ile Ala Leu Tyr Thr Ala            4740 4745 4750 Thr Arg Gln Val Ala Thr Leu Leu Pro Ser Thr Gly Leu Cys Ala Asn        4755 4760 4765 Lys Leu Val Ala Met Ile Ser Leu Thr Asp Val Asn Tyr Asp Val Ser    4770 4775 4780 Glu Asp Leu Ala Glu Asn Lys Ile Glu Leu Ala Ser Ser Glu His Asp 4785 4790 4795 4800 Gln Leu Ile Asn Glu His Ile Glu Ala Leu Gln Ser Leu Leu Arg Glu                4805 4810 4815 Ser Leu Pro Gln Tyr Met Ile Pro Ser Leu Trp Val Val Leu Tyr Asn            4820 4825 4830 Leu Pro Met Thr Ala Ser Gly Lys Gln Asp Asn Lys Ala Leu Lys Ser        4835 4840 4845 Trp Leu Glu Asn Met Asp Glu Thr Leu Phe Ser Lys Ile Asn Asn Ala    4850 4855 4860 Asn Gly Ser Asp Ile Ile Arg Lys Pro Asp Thr Glu Asp Glu Arg Val 4865 4870 4875 4880 Leu Ser Gln Lys Cys Ser Ile Val Leu Asn Met Pro Val Asp Lys Ile                4885 4890 4895 Asn Leu Asp Lys Ser Phe Ile Ala Asn Gly Gly Asp Ser Ile Ser Ala            4900 4905 4910 Met Arg Leu Ala Ser His Tyr Arg Thr Val Gly Ile Ser Ile Ser Val        4915 4920 4925 Ser Thr Leu Leu Gln Ser Lys Thr Leu Ala Asp Phe Ala Ala Phe Ser    4930 4935 4940 Gly Ala Thr Ala Ile Ser Gly Val Ser Gln Glu Glu His Thr Asp Val 4945 4950 4955 4960 Pro Phe Glu Leu Ser Pro Ile Gln Gln Trp Phe Phe Asp Gln Ser Pro                4965 4970 4975 Phe Met Ser Gln Gln Lys His Asp Arg Phe Tyr Asn Gln Gly Phe Tyr            4980 4985 4990 Val Arg Leu Arg Arg Thr Val Arg Ile Asn Asp Leu Glu Ser Ala Phe        4995 5000 5005 Leu Ser Leu Val Asn Arg His Ala Met Leu Arg Ser Arg Phe Gln His    5010 5015 5020 His Gly Gly Lys Trp Lys Gln Ile Ile Leu Ser His Ser Lys Arg Ala 5025 5030 5035 5040 Leu His Leu Asn Val Ser Gln His Leu Ser Met Ser Glu Ile Ala Ser                5045 5050 5055 Leu Ala Gln Glu Arg His Arg Gln Ile Asp Ile Glu Lys Gly Pro Val            5060 5065 5070 Phe Ser Val Asp Ile Cys Leu Leu Gly Gln Gln Gln His Leu Val Met        5075 5080 5085 Ile Ala His His Leu Val Thr Asp Leu Val Ser Trp Arg Ile Ile Leu   5090 5095 5100 Asp Asp Leu Glu Thr Ile Leu Asn Gly His Ser Leu Thr Ala Ala Leu 5105 5110 5115 5120 Pro Phe Gln Val Trp Ser Arg Leu Gln Ala Glu Arg Ala Val Ser Ser                5125 5130 5135 Thr Leu Lys Pro His Asn Leu Leu Ser Thr Asp Gly Val His Asn Asn            5140 5145 5150 Leu Lys Phe Trp Lys Tyr Thr His Asp Thr Pro Asn Cys Leu Ala Asp        5155 5160 5165 His Arg Leu Arg Ser Val Thr Ile Asp Arg Glu Thr Thr Ala Val Leu    5170 5175 5180 Leu Gly Glu Ala Asn Asn Ala Met Asn Thr Glu Pro Val Glu Ile Leu 5185 5190 5195 5200 Leu Ser Ala Val Trp Asp Ala Phe Phe Arg Thr Phe Ser Gln Arg Asn                5205 5210 5215 Ser Leu Thr Ile Phe Asn Glu Gly His Gly Arg Glu Ala Trp Ser Asp            5220 5225 5230 Glu Ile Asp Leu Ser Ser Thr Val Gly Trp Phe Thr Thr Leu Ser Pro        5235 5240 5245 Ile Asn Ile Tyr Arg Asn Asn Ala Thr Ser Glu Thr Asp Met Val Arg    5250 5255 5260 Leu Val Lys Asp Ala Arg Arg Ser Leu Pro Ala Asn Gly Trp Ser Tyr 5265 5270 5275 5280 Phe Thr Ser Arg Tyr Leu Asn Pro Asp Gly Gln Arg Ala Phe Glu Ser                5285 5290 5295 His Asn Thr Val Ser Glu Val Val Phe Asn Tyr His Gly Gln Phe Gln            5300 5305 5310 Gln Leu Glu Ser His Gln Ala Leu Phe Glu Asp Ile Asp Leu Val Gly        5315 5320 5325 Val Arg Val Gln Gly Arg Ser Ile Ser Ala Gly Ser Leu Phe Asn Ile    5330 5335 5340 Glu Val Ala Ile Glu Ala Met Gln Ala His Phe Glu Phe Ser Val Asn 5345 5350 5355 5360 Gln Asn Ile Ala His Gln Ser Leu Ile Asn Gln Trp Ile Asp Gln Ile                5365 5370 5375 Gln Pro Ser Leu Glu Arg Ile Cys Leu Val Leu Leu Glu Ala Asn Pro            5380 5385 5390 Thr His Thr Leu Cys Asp Phe Lys Phe Ile Ser Leu Asp Tyr Gln Arg        5395 5400 5405 Leu Asp Asp Leu Thr Ser Arg Leu Leu Pro Glu Ile Glu Ser Ile Asn    5410 5415 5420 Gln Ser Thr Val Glu Glu Ile Phe Ser Cys Ser Pro Ile Val Asp Gly 5425 5430 5435 5440 Met Leu Leu Ser Gln Ile Lys Gln Pro Glu Ser Tyr Lys Thr Leu Gln                5445 5450 5455 Arg Tyr Glu Val Leu Ser Ser His Asp His Pro Ile Cys Leu Asp Thr            5460 5465 5470 Leu Lys Ile Ala Trp Gln Arg Val Ile Ser Arg Gln Pro Ala Leu Arg        5475 5480 5485 Thr Val Phe Ile Ala Gly Leu Asp Gly Ser Thr Ala Phe Tyr Gln Ala    5490 5495 5500 Leu Leu Lys Gln Cys Ser Gly Asp Val Ile Val Val Glu Ala Lys Thr 5505 5510 5515 5520 Glu Glu Glu Ala Leu Lys Ala Phe Ser Ser Leu Pro Lys Val Asp Tyr                5525 5530 5535 Gln Gln Ala Lys Pro Pro His Arg Leu Thr Leu Cys Gln Thr Pro Asp            5540 5545 5550 Asp Lys Val Phe Cys Gln Ile Glu Met Ser His Ala Ile Thr Asp Gly        5555 5560 5565 Ala Ser Ser Thr Ile Leu Ile Lys Asp Leu Ile Asp Ala Tyr Gly Asp    5570 5575 5580 Arg Leu Ser Ser Thr Asp Leu Val Lys Thr Thr Arg Glu Phe Ala Ser 5585 5590 5595 5600 His Leu Leu Ala Lys Pro Gln Ser Gln Lys Ile Ser Tyr Trp Asn Thr                5605 5610 5615 Lys Leu Lys Gly Leu Glu Pro Cys Arg Phe Pro Ser Leu Ser Ser Met            5620 5625 5630 Ser Arg Glu Lys His Glu Cys Ser Ser Glu Ile Gly Val Phe Val Glu        5635 5640 5645 Asp Lys Met Phe Ala Gln Ile Gln Asp Phe Cys Ser Ile Asn Gln Val    5650 5655 5660 Thr Pro Ala Ser Leu Leu Lys Ser Ala Trp Ala Leu Thr Leu Ser Thr 5665 5670 5675 5680 Tyr Val Gln Asn Gln Ser Val Cys Phe Gly Tyr Leu Ala Ser Gly Arg                5685 5690 5695 Asp Leu Pro Ile Ala Gly Met Asp Glu Ser Val Gly Ala Tyr Thr Asn            5700 5705 5710 Ile Met Val Cys Arg Ala Asp Leu Asp Gly Gln Gln Pro Gly Val Ala        5715 5720 5725 Leu Val Arg Gln Leu Gln Asn Gln Leu Met Gln Asp Leu Ser Phe Gln    5730 5735 5740 His Ile Ser Leu Ala Ser Ile Gln His Glu Leu Gly Leu Ala Ser Asp 5745 5750 5755 5760 Gln Gln Leu Phe Asn Ser Ile Val Ser Phe Gln Arg Ser Gly Asp Asp                5765 5770 5775 Asn Glu Gln Ser Ala Glu Glu Gly Lys Leu Arg Phe Lys Asn Ile Asp            5780 5785 5790 Gly Leu Asp Pro Thr Glu Tyr Asp Ile Val Leu Gly Ile Asn Gln Gly        5795 5800 5805 Thr Arg Ser Ile Glu Ile Asp Leu Glu Phe Ser His Ser Cys Leu Thr    5810 5815 5820 Ser Asn Gln Ala Lys Arg Ile Leu Glu His Leu Gln Ser Asn Ile Ala 5825 5830 5835 5840 Ala Ile Leu His Asn Glu Pro Pro Ala Leu Ile Ser Pro Gln Asp Glu                5845 5850 5855 Gln Asp Ile Trp Ser Trp Asn Ser Thr Val Pro Asp Met Val Asn Ile            5860 5865 5870 Cys Val His Asp Leu Ile Ser Lys Ile Val Phe Arg Gln Pro Asp Ala        5875 5880 5885 Pro Ala Val Cys Ser Trp Asp Gly Asp Phe Thr Tyr Ala Glu Leu Asp    5890 5895 5900 Asn Leu Ala Thr Arg Leu Ala Asn Ser Leu Ser Lys Met Gly Ile Gly 5905 5910 5915 5920 Arg Gly Ser Ile Val Pro Leu Cys Phe Glu Lys Ser Lys Trp Thr Pro                5925 5930 5935 Val Ala Met Leu Ala Val Met Lys Thr Gly Ala Ala Ser Val Thr Met            5940 5945 5950 Asp Thr Ser Gln Pro Glu Glu Arg Leu Gln Ser Ile Val Ala Gln Val        5955 5960 5965 Asp Ala Lys Leu Val Ile Ser Ser Thr Leu Lys Val Glu Leu Ala Ala    5970 5975 5980 Arg Leu Thr Thr Ala Pro Val Leu Ala Ile Asp Lys Ala Ser Met Lys 5985 5990 5995 6000 Ala Met Ala Asp Asp Thr Pro Leu Ala Ala Val Asp Pro Ala Asn Ser                6005 6010 6015 Ile Tyr Ile Val Phe Thr Ser Gly Ser Thr Gly Thr Pro Lys Gly Val            6020 6025 6030 Ile Ile Thr His Thr Asn Tyr Ser Ser Ala Ile Lys His Gln Gln Ser        6035 6040 6045 Glu His Gly Phe Lys Pro Thr Ser Arg Val Phe Asp Phe Ala Ser Tyr    6050 6055 6060 Ala Phe Asp Val Ser Trp Ser Asn Phe Leu His Thr Leu Thr Ile Gly 6065 6070 6075 6080 Ala Cys Leu Cys Ile Pro Ser Asp His Asp Arg Lys Asn Asp Pro Ala                6085 6090 6095 Gly Ala Ile Asp Arg Leu Arg Cys Thr His Val Asp Met Thr Pro Ser            6100 6105 6110 Ala Ala Ser Val Leu Pro Ala Ser Thr Leu Ala Lys Leu Asp Thr Ile        6115 6120 6125 Val Leu Gly Gly Glu Lys Leu Ser Leu Glu Tyr Ala Gln Arg Trp Ser    6130 6135 6140 Ala Leu Thr Ser Val Arg Asn Pro Tyr Gly Pro Ser Glu Cys Thr Pro 6145 6150 6155 6160 Thr Ser Thr Ile Thr Glu Ile Asn Ser Ala Glu Ile Ser Lys Gly Lys                6165 6170 6175 Val Ser Ile Gly Lys Gly Val Gly Leu Asn Thr Trp Ile Val Asp Pro            6180 6185 6190 Ala Thr Ala Gln His Leu Met Pro Ile Gly Ile Pro Gly Glu Leu Leu        6195 6200 6205 Leu Glu Gly Pro Leu Val Gly Ala Gly Tyr Leu Gly Asp Pro Val Lys    6210 6215 6220 Thr Ala Ser Ala Phe Ile Glu Asp Pro Glu Phe Leu Val Lys Gly Ala 6225 6230 6235 6240 Ser Pro Gly Ile Pro Gly Arg Arg Gly Arg Leu Tyr Arg Thr Gly Asp                6245 6250 6255 Leu Val Thr Tyr Asn Thr Asp Gly Ser Leu Ser Phe Val Gly Arg Arg            6260 6265 6270 Asp Ser Gln Ile Lys Ile Asn Gly Gln Arg Val Glu Leu Gly Asp Ile        6275 6280 6285 Glu Ser His Val Ser Ala Asn Leu Val Ser His Gly Ser Ala Gln Val    6290 6295 6300 Ala Val Glu Val Val Ser Pro Gln Ala Ser Ser Asn Asn Ile Leu Val 6305 6310 6315 6320 Ala Phe Val Ser Phe Asp Asp Leu Asn Ser Ile Asn Leu Asn Asp Glu                6325 6330 6335 Lys Leu Leu Ala Arg Thr Lys Ala Ala Thr Glu Gly Ile Arg Glu Lys            6340 6345 6350 Leu Ala Thr Gln Ile Pro Ser Tyr Met Ile Pro Ser Val Tyr Ile Pro        6355 6360 6365 Val Thr Val Phe Pro Thr Thr Ala Thr Gly Lys Thr Asp Arg Arg Arg    6370 6375 6380 Leu Arg Glu Met Ala Ser Ser Leu Thr Leu Glu Gln Leu Thr Ser Ile 6385 6390 6395 6400 Asn Gln Ala Gln Gln Gln Tyr Gln Pro Pro Thr Thr Pro Leu Glu Val                6405 6410 6415 Ala Leu Arg Glu Leu Trp Ile Ser Val Leu Lys Leu Gly Ser Arg Lys            6420 6425 6430 Ile Ser Thr Thr Asn Asn Phe Phe Glu Leu Gly Gly Asp Ser Ile Gly        6435 6440 6445 Ala Ile Arg Leu Val Gly Ala Ala Arg Asp His Gly Leu Ser Leu Ser  6450 6455 6460 Val Val Asp Ile Phe Lys His Pro Lys Phe Ser Glu Met Ala Ala Leu 6465 6470 6475 6480 Leu Arg Ser Val Asp Lys Pro Gln Leu Glu Glu Pro Arg Val Phe Gln                6485 6490 6495 Pro Thr Ser Leu Leu Ser Lys Asp His Asn Lys Asp Gln Ile Leu Ser            6500 6505 6510 Arg Leu Phe Asp Phe Gly Ile Asp Leu Glu Asn Val Glu Asp Ile Leu        6515 6520 6525 Pro Val Thr Asp His Gln Ala Arg Ser Ile Ala Met Thr His Ser Ala   6530 6535 6540 Ser Arg Asp Leu Leu Leu Tyr Pro Thr Leu Asp Ser Lys Gly Val Pro 6545 6550 6555 6560 Asn Met Arg Lys Met Arg Ala Val Cys Asn Glu Leu Val Asn Arg Tyr                6565 6570 6575 Asp Leu Met Arg Thr Leu Phe Ile Ala His Lys Asp Ser Phe Leu Gln            6580 6585 6590 Val Val Leu Lys Ala Phe Pro Val Asp Ile Thr Val Leu Arg Ile Glu        6595 6600 6605 Asn Ala Ser Leu Glu Glu Cys Thr Glu Glu Leu Arg Leu Arg Asp Arg    6610 6615 6620 Asp Asp Glu Leu Arg Tyr Gly Ser Leu Leu Thr Lys Ile Ala Ile Leu 6625 6630 6635 6640 His Gln Ile Arg Asp Asn Glu Tyr Arg Leu Val Val Arg Ile Ser His                6645 6650 6655 Ala Gln His Asp Gly Met Ser Leu Met Lys Met Trp Asn Ala Phe Glu            6660 6665 6670 Glu Met Tyr Gly Asp Gly Ser Asp Asp Ser Phe His Ile Pro Ser Asp        6675 6680 6685 Thr Ser Phe Gln Glu Lys Ser Lys Ala Ser Phe Ser Asn Tyr Met His    6690 6695 6700 Ala Val Ala Gly Thr Asn Arg Glu Gln Ala Lys Ser His Trp Arg Arg 6705 6710 6715 6720 Leu Leu Lys Gly Ser Ser Met Thr Asn Leu Lys Pro His Ala Ser Tyr                6725 6730 6735 Ala Leu Thr Phe Gly Glu Gly Pro Cys Val Ala Arg His Val Pro Lys            6740 6745 6750 Ser Ile Ala Gln Gly Thr Gly Phe Thr Phe His Thr Val Leu Lys Ala        6755 6760 6765 Ala Trp Ala Tyr Val Leu Ala Lys His Leu Ala Asn Asp Asp Val Val    6770 6775 6780 Phe Cys Ser Leu Thr His Gly Arg Gly Leu Pro Gly Thr Gln Asp Val 6785 6790 6795 6800 Phe Gly Asp Cys Val Asn Ile Ile Pro Thr Arg Val Ser Phe Thr Asp                6805 6810 6815 Gly Trp Thr Val Arg Asp Leu Leu Ser Ala Leu Asn Ala Gln Gln Ile            6820 6825 6830 Ala Ser Met Glu His Glu Asn Ile Gly Thr Arg Glu Ile Val Arg Asp        6835 6840 6845 Cys Thr Thr Trp Pro Lys Trp Thr Tyr Ala Gly Ser Ile Val Tyr His    6850 6855 6860 His Asp Phe Asp Asp Gly Glu His Ile Ala His Asn Arg Ser Met His 6865 6870 6875 6880 Val Glu Gln Glu Leu Asn Leu Ser His Gly Lys Val Asp Met Thr Asp                6885 6890 6895 Val His Ile Thr Ser Lys Pro Asp Asn Asn Met Phe Arg Ile Glu Leu            6900 6905 6910 Asp Phe Ala His Gly Val Val Ser Glu Arg Asp Ala Glu Leu Leu Ala        6915 6920 6925 Ala Lys Leu Thr Glu Ser Ile Ile Val Phe Cys Asn Val Met Asp Gln    6930 6935 6940 Pro Leu Leu Ser Pro Asp Glu Ile Arg Tyr Leu Arg Thr Thr Thr Leu 6945 6950 6955 6960 Leu Pro Ser Glu Glu Pro Leu Ser Ala Thr Pro Thr Asn Glu Gln Leu                6965 6970 6975 Met Val Ala Ser Ile Ser Pro Thr Glu Met Gln Trp Ala Leu Glu Ser            6980 6985 6990 Ala Trp Lys Asp Thr Phe Asn Cys Pro Leu Ser Pro Glu Val Lys Ala        6995 7000 7005 Gly Lys Thr Ile Phe Asp Leu Gly Gly Asp Leu Ile Ser Ala Ser Leu    7010 7015 7020 Ile Ser Ala His Met Glu Arg Gln Gly Tyr Val Leu Ser Val Glu Asp 7025 7030 7035 7040 Val Leu Gly Asn Pro Thr Trp Phe Ser Gln Leu Thr Leu Leu Thr Lys                7045 7050 7055 Arg Thr Leu Arg Asp Val Asp Val            7060 7064 <210> 4 <211> 23114 <212> DNA <213> Aspergillus niger <400> 4 aaacccatat ataaagataa tatatattaa taaaataata gtaaataacc taacaaacct 60 caccctaacc ggctaattat atgatacttt tatttgatta tcaataattt ttaataacct 120 tactactagc ttatataaac cataaatatc tttaatatta gcctcatcac gcacgccctc 180 gggcccctat gtaccgcgta agtttgacat ttgagattga attatatata tctctgccaa 240 tactcagaac aatcttccta tactttacta gtagattaga aatactatct atactatatg 300 cactcaatat catatcctga atggatatgg ttgttgaagt atgaaaatgt aatttatcgc 360 atttagtatg atgttttagg gtgaccgaga cggccgggaa tgttacgtta gagccgggta 420 tatatgacta agcgctagat gataactagt atcttacgtc ggtagaacgt accaatagca 480 atatctttag catgtccatc ttattccgta acatactact ctgacactgc cgcctgttgc 540 gctgctacca aggacggcag cagttttcta ataggggcag ttgtcacggc agtagcctat 600 agttaactag agccggggtt atatgactaa gcactagtgc agttcccacg ggtccactgg 660 tcattcaagc ttgtggattc ggctcggcgc ggcccccaac catagttgga gacagcccta 720 aatgtgatgg gaatgatcta cggcttgagc gagagcgatc ctggggtctt gggggctatg 780 gagtaagctc aattgcttct ccaaatagta atcaatacgc gcaaccctcg aatttgaaat 840 ttcttcgatt tttcagcctt ccaactacct caatgcctca tttccggttt tcattgatct 900 tccaaaaatt aaatttatcc taaacccatc caagactctg ataagatttc ctacgtttgc 960 tttagagatg atcatcattg taatattcat tagaagcctc ataatcacgt tacgtaccca 1020 ctgcacattg cctgatgtgc cacggagaac cagggatcct aagcctccac tcttgtatct 1080 gtttgtctca ctcgtaaggc aaccacctgt cggatgcaaa tctaggcttc cgctgaggta 1140 gtatcggatt gttgcaagat ccacagtgga gccaaaaaga acacgaaaag cacaaagaaa 1200 gggctttgaa agttcatatg ctttgttgtt ttttgcgttt ttagtcttca aatcttcctt 1260 gagatgtttc agacatctat gtataatgag atgacggaga gcaactttgg agatatttcg 1320 tcagaaacga cgcctcgcac tgacgagcgg ttttagagac aatgaccctg cagatcaggg 1380 tcattgtctc taatggattt ggccccttag cacatgtttg ctggggaaaa cttgctttgg 1440 cgctgtagga ttcagatgca acagtatact gcatatttgc ttatgtgctt tccgactaag 1500 cccaagatga cgacctcgga aattcattgc ccgtcggggg agtctggcct taatggcgtt 1560 ttactagcat agttacagtg ttggtactat agctggcaca tcttcatata ttctgctgat 1620 gccccgtcca aacatcttaa aatgagaaga gtccacagaa taccatcaat tgcttcataa 1680 caatacactc atcaatccct caaacgatca ataaatctta tcaaaatgcg atctcccaat 1740 gatcttacaa catcagcaac catggaagat attcatcaaa tatggtcttg gaacgctaac 1800 gtgcctgagg cgggtgagac atgcgttcac accctcatca ccgacaaagc tctccagcag 1860 ccagatgcac ttgccgtcga cgcttgggac gggaggtgga cgtatggtga gcttgagact 1920 acttctacca aactggcatt gcgtttgctt gaccttggag ttggccctgg aaccaatgtt 1980 gtcatatgtt ttgaaaagtc gaagtatacg cctctggcta tgctggccgt catgaaggcg 2040 ggtggagcct ccattgcgct cgatacaagc caaccacaga cacggttaca gtctatcatc 2100 aaccaggtcg accctgtagt gatactatgc tccgcgtcca agagtcagtt ggccaagtca 2160 atcatcaccg agtctgcagt agcattgaca attgatgaaa attctctatc cgaaatgaat 2220 ttcgagccgg actcagtcgc ccgtctccct gacgtcagtc tggacaacaa tctctacgtg 2280 gtctttacct ccggaagtac aggaactccc aagggcgtcg tcgtcacaca tctcaattac 2340 tccactgcca ttctccacca acaagaagcg catggcttca agtctacttc aagagtgtat 2400 gactttgctt cgtatgcttt tgatgtgagt tggtcaaatc tcatccacac cctcaccatc 2460 ggcgcctgtc tgtgtattcc atcagagcag gaccgtaagg acaatctcat cgagtccata 2520 cgctcactgt gtgctacaca tatcgatgtg accccgtctg ttgctcgcct tatccctgat 2580 tccttgctct gcaagattga aacacttgtt ttaggtggcg aaaagctgcc tgcagagctt 2640 gccagacatt tgtcatcgct ggtaacattg aaaaatccgt atggaccgag tgaatgtacg 2700 cctacatcaa ctatcgctac aattcgaccg gatgatgatg attcaaaaat tagcagcatt 2760 ggccgaggct tgggtgtcaa cacctgggtt gtagacagtg agaatgaaga aattttggtg 2820 cctattggac aggttggaga gctattactt gaagggcatc tgcttggcaa tggctacttg 2880 aacgaccaga cgaagaccac tgcggccttt gtcaacaatc cgctttttct gctcaatggc 2940 ggcgatggac ctgggcagcc tgggaggcgc gggcgtctct acaagaccgg cgacctggtc 3000 cgttacgaga aagacggtag cttgaccatt attggccgta aggacacgca gtcaagatta 3060 cggccacgag ttgagctggg tgatattgag catcatattt accgccacat cccacatggc 3120 accgtgtctg tgcgacaaat tgcagcagaa attatttcac caaagactgg ttccaatgct 3180 gtcctggcag catttctcga ggtcgacctt ggtgtagagg atacgggaat agcggaacag 3240 ttgttttcaa aaaccgagaa gatgatgtct aacttgagaa gcaatcttgc tcgagatgtc 3300 ccttcatata tggttcctgc tgtattcatt ccgttgagga acttcccatt gtctccgaca 3360 ggcaagacag accgacgcca actgcgggcc attggtgaat caatggactt gactgtcttg 3420 gcaggctttg gcgcagcacc taatgaggct cggatccctc tcactctgcg agagaaacag 3480 ttgagaagac tctggggctc tatcctcagg attgacgaaa accttattgc tctcgacgat 3540 aactttttgc aaagagccgg caacccaaat gcagccatga aactcgtcac tgcagctcga 3600 agagaaggct tctccctgag cgtggccaac gtgctgaagt atccgcgact tcaggacatg 3660 gcacaggtgg ttggaacagt ggagcaagag caggcacatg aaattatgcc atttgagctt 3720 ctgagcgatg atattaatct tgatctagcc ctcagagagg ctgcagcatc gtgcaacgta 3780 caagggaatc aaattcaaga catttatccc tgcacaccgc tacaggaagg catgatgtcc 3840 ctatccgcaa aacgcgaggg tgattacatc atgcagtata cgctggagct acaccatcga 3900 tgcgacatag agcgcctagg taaagcctgg gcgacagtag ttgctacgac gcccattttg 3960 agaacacgaa ttgtcgacat cactagccag gggttggtac aggctgttct agacgagcag 4020 tggtcaagtt cgtcgatcca aaggaggacc ttgagccagg cgagagacca caagcatcaa 4080 ttcgggctag gcatgccatt aggcagattt gaaattgtca ccggcgactc gtctgatttt 4140 aagcactact tcgtctggac actgcaccat gccctgtatg acgggtggtc acttcagctt 4200 ttactcgaaa agctcgagaa cgagtacgcc ggaaaggcag acgcgcagtc aaactcaccc 4260 gattttaaac gttttatcaa gtatatttca acgagggatg gcgagaagac tcattcgttc 4320 tggaccgaac agttccaaga cgtcgaagcc cagatctttc cgagtttgcc atctgtcgac 4380 taccagccta gatcggacaa actgtacact cattctgtgg gaggaataca atggccaaaa 4440 aatggaatca ccccatcgac gacaattcgt gcggcttact ctatcttgat atccagtctg 4500 accaacagcc cagatgttgt cttcggcagc ataacgacgg gcagacaagc cgccgtggat 4560 gaggttgaag agcttattgc accaactata gccactgtac ctgtgcgcgt ttcgatcgac 4620 agcaaagacg agttgggaca gttcttacag cgaattcaat ctcaggccgc agatatgatt 4680 gagtttgagc agactgggct gcatcagatt cgacatatca atgcagatgc ggagcgagca 4740 tgccagtttc agacactgct tgttgttcag cccgcggagg gctcgggcac agcaccgagc 4800 gacattttta ccaacattcc cgacgacata aggaaaggag atggaaacag cgccgcagag 4860 ctggggactt atgccttgac tatggagtgt ctgctcaaga aagatggcct tgacctccat 4920 atgaactatt gttccgcagt catatcggag catcaagtgc gccgtttgtc acagcagttt 4980 gagcatgtcc ttggtcaaat atgtcatctg acgatgataa ttcacagaca gagaacaaca 5040 cttgaatcgc tgcggcagcc aacaacggaa actgagcggc agatgcagcg aatctgggca 5100 caggtactca accttaatca agcatcaatt gggctagatt acagcttttt ccaactcggc 5160 ggcgactcta ttgctgccat ggaagttgtg acagaagctc gtaagcttgg cttgaaactg 5220 gctgtgtcag acatattccg tcgcccgaaa ttgcaagacg ttgcaaagaa ggcttgtgac 5280 agtggcctgc agctttatgg agaagagcag ctcatgaacg actcagaagt ccaagttaaa 5340 ggaggtaccg agcacactaa gctacccaat gatgacaagg cagttgctgg ccatgaaacg 5400 cagcaagtca tggtatggga gggaatgttc gataaggagg tctatggaac tattaatgat 5460 gtccaattgg agaagattgg gcgcgacttc atcggatgga cgtccatgta caatggcaac 5520 caaatagaca atgtcgaatt gaacgagtgg ctagacgata ctattgccac aatacgcagc 5580 agcggatcga cagccaacat actcgagctg gggtccggca gtggaatgat cttgttcaat 5640 cttgtcaacg gcctgcacag ttatgttgga cttgacccgt cagaaaaggc cgtggacttc 5700 gtttgttcca cagttaaatc cattccccag ttggccgacc gtgtctataa tataaaggga 5760 acagcagaca acatcaacag cctaggcgta cccatctctg ctaacatggt catcgtgaac 5820 tctgtcgtcc agtactttcc cagtcaagac tatctgctca aagtaattga ggacctagtt 5880 cagctggaaa cagtccgcac gatctttttc ggcgacattc gctcctacgc gctgtttcga 5940 gagttccagg ttaccagggc gctgcatatt gcaggagata ctgcaaccga ggacgagatc 6000 cgacagatga tggccaacat ggagcaagtc gagctggagt tgctggtaga tcccgctttt 6060 ttcacatcct tagtcgaccg gttccctgga ctcgtcgaac atgtggagat tcttccaaaa 6120 agattgaagt cgacgaatga gctgagtgcc tatcgatacg ctgccgtcgt gcatctcaaa 6180 gactcaaacc aactagctca gccattacaa gtccacgata tccagaaaga gagctggatt 6240 aactattccg ggcgtcaact caatcgtcag tcgctgttac aacttgtaca ggattctttc 6300 ttacgagacc cgtccccctc atcagttgtg gccgtatgca atatacctta tagcatgaca 6360 gtctacgaga ggcacgtgat tgaatggctt gatagtggcc tcactgccgg tcccgatgct 6420 gaggactggc tctcttctat ccgccaaaca tcacaagaat gctcatcact ctcggctctc 6480 gatttgcaac aaatagctca ccagacagga tggcaagtgg agattagctg gagtcggcaa 6540 ttttctcagc gcggtggctt ggatgccata ttccaccgac accattcacg aggagaccga 6600 accagcaggg ctttgttcaa cttccccaca gattatcaag gacggccatt tcaatcgttg 6660 agcaaatggc ctttgcagcg caagcgacag ggagaggaaa agcaaataac tttgggagat 6720 gtttctaccg tctgcaaaga ggacttgcat gacatctgga cttggaatga agttgtccca 6780 gacgccctcg aggcctgtgt ccacgatctc atctcggaca cagtaagagc tcagcctcaa 6840 tcccctgcta tttgtgcttg ggatggtgag tggagttaca tcgagcttga tgatttgtct 6900 agccgtcttg cacacgcgct tgctccgttt ggcgttgcca acacagttgt acctatatgc 6960 tttgaaaagt caaaatggac accagtggca acgctggctg tgatgaaggc cggggcagcg 7020 tcagtcaccc tcgatgcctc tcagcctctg gagagattac ggtcgattat ctcccagact 7080 gaccctcgag tgatcttgtc atcggcgtct aagcagggct tgggtgccca actcactaaa 7140 gctccaaacc ttgttgttga ccaacattcc atttctacta tgcacatcac cgccgagcct 7200 cttcccaccg ttgacccctc cagcaagcta tatattgttt tcacatctgg caccacgggt 7260 gttcctaagg gcgttattat cacacactct aactttagca gtgccatccg acaccaacaa 7320 aaagctcatg gtttcaaatc gacatctcgg atttatgact ttgcctcata cgcgtttgac 7380 gttagctggt ctaacttcat acatgcactt actgttggtg cctgcttgtg tattccgtca 7440 gatgaggacc gccgtgatga cttggccggg tcgttggaga ggttcggcgc tacccatgtt 7500 gacatgacgc cttccgcagc aagcttgctc ccagagaagt cattcaaaag gcttgagacg 7560 gttgtactcg gaggcgagaa gctttctgtc gagagcgcac agcgctggag ctcactagtc 7620 agcctcaaga acccatacgg tcccagtgaa tgcacgccca cggctaccat tgcgacggta 7680 acacccaccg atgagtacaa atccagtatt ggaagaggac taggtctgaa tacatggatt 7740 gtaaatactg ttacagactc tttagttcca gtcggcgggg tcggagagct cctgctcgag 7800 gggcctctcg ttggcgcagg gtacctcggc gatgacacga agacggccgc ttcctttgtg 7860 gaagatcctc agttcctatt gcaaatatgc cctcaaggtc aagcaagaca taccagaatg 7920 tataagactg gcgatctggt tcattacaac ccggacggaa gtcttagttt cgttgggcgc 7980 aaggacgctc aggtcaagat tcatggtcag cgtgtcgagt tgaccgagat tgagagccat 8040 attcgtcgca cctctaagac tatccaggtg gctgttttgt ttaccaagtc agggctgtgt 8100 gcaaataggg tagttgcatt tgtctgcatc cagggaactg gccaaactca gacagccgcc 8160 gatcaaattc gactcatcga tcccaagtat tcgacccttg ttacggctta caccgagtcc 8220 gcaaagtcta gtctcagcga cactcttcct gcttatatga tcccctcaat ttggattcca 8280 ctccagcatg ttccgttgtc aacgtccgga aagctcgatt acaaagcttt gaaatcatgg 8340 cttgatagca tggatgccaa gacgtttgcc aatattttaa ctgcgtctga tggcgacgtg 8400 aagcttcgca aggctgagac agaattggaa caggtcatag tagaggcttg cgctaaaatt 8460 ctcaacatta cagcatcgaa ggtgaacctg gatcggtcgt tcattgcgaa tggtggtgat 8520 tccatctccg ccatgaggct cgttgctcat tgtcgcgcgg acaacgtcgt attttcggtg 8580 gctaaattgc taaagagcaa aactttggcc gctttggcct catcttcaaa aatcaagtca 8640 gcttccaacg tgttgggctt ctatgaggaa aaaagtgact cttttgcgtt gtcgccgatc 8700 caacagtggt tctttgaaca aggtctctac aagagatcca atgacaactt cgacaatcaa 8760 ggattttatc tcaaggttaa gcgcccatta ttgacaaagg atattgactc ggcgatttcc 8820 aaagttgttc agcatcactc tatgttgaga gctcgctttc atcggaacgg cgacgagtgg 8880 acccaaaaaa cgcttaagcc tgacaccaat ggtttatatc actttggtgt ccaccacacg 8940 tgccttccag ctgatataga gcgactagct ctatcacgcc accagatgat cgacatcgaa 9000 aaggggccag tattctctgc tgacatttgc cataatgcgt ttggagaaca gtacctgatc 9060 ctgattgctc accatcttgt tgttgattta gtctcgtggc gtgtcatcct ggaggacata 9120 gaatctctac tcggtggtag taatttgcaa ccaagtcttc catttcaagt ctggaatgat 9180 atgcaaattg agcgggcgaa ggaatcgagc ctacttgatc ctgaaaatgt tctatcaacc 9240 actggaatta ataacaacct tgatttctgg caagccacag cagaaaccaa gaacaccgtc 9300 gaagatcacc taaatttctg taccaagatc gacagcagca aacagagctc atcctcaaag 9360 acgcaaatta cccgttcaac acttgaacct gtggacctcc ttcttgcggc tgtttggcat 9420 gcattcttca agacgttccc ccaaagagac ggtctcacta tttttattga aggacatggt 9480 cgcgagccgt ggtcctcgga tattgatctt tcccgcactg tcggttggtt tactaccatc 9540 agccccattc atgtgtcaaa gagcgacgta cacaagtctg tcgcaagtct agtacgcgtt 9600 gtcaaagatg ctcggcggct cctgcctgca aatggctggg cttactttgc ttctcgatat 9660 ttcaatgaat cgggaaaatc agcgttcaaa tcgcacgact ctattatgga aataaccttt 9720 aactatcacg gccaatttca acagctggag aacgagaagg cgatgtttga aaatgttacg 9780 ctcagtggag tctgcgagca aggaccagct cttcccgcct cttccttgat tgctgtcgaa 9840 gtctcgatag atcgagggca ggtcaccttc gacgtttctg ccaaccgcta cattaatcac 9900 caggattgta tttcaaattg gatcaaagca atttcacagt cattggagac catttccaat 9960 gagcttgtgt ctacagaaat ctcacatcgc acgctttgcg actacgaatt tctgagcctc 10020 gggtatacgg agctggatcg attacaggaa agtgtcatcc cagaaattga gaagctaaat 10080 aattccacag tagaatgcat ttaccgctgt cttcccacag ttgatggtat tctgatcagc 10140 cagttcaaag acccagaatc gtataaaaca gtgcaacatt tcgagattac ttcccacatc 10200 gacgaccaaa tcgacctaga gcatctttct ctagcatggc aaaaagtggt tgccaatcaa 10260 cctgctctac gaaccgtctt tatccctggc atggacaaag ccgctgcatt caatcaagtt 10320 gtgctgtctc agtaccacgc cgagctcatc atcctgcata ctgccagcga cgagtacacc 10380 gaagccttgg agatgttcaa gaatctaatc ccaatcaatt atcagagctt caagccacct 10440 catagagccg cgatctgtcg aatctcacct agtagagtac tctgccaggt tgagatgagt 10500 catgccatca cagacggtgc atcgacatct attttggcca atgatcttct ccaggcatac 10560 aacggcaact cgatgccaat aaatctcatg gacacagcat gtgagtttgc tcgggcccaa 10620 ctcacttcct cttttgggga aaaactgtca tactggaaga aaaagcttcg ggaaatggat 10680 ccctgccact tccccaaaat ctcgggtgct tcaacacaag gcaccggtac atccgtctgc 10740 aaaattcgtg gcgctctgtt tagcaaaatc caggattatt gcaattcagt agaagtcacg 10800 acggccagtt tatttcagac catatgggca cttactctgg ccgcctatac cggcaacgac 10860 tcgacatgct ttgggtatct agcatctggc cgcgacttgc ctattgctgg catcgataaa 10920 tctatcggtg cattcacaaa catgttggtt tgtcgagtca atattaaccg agaaactgaa 10980 atacttcaat ttgttcagac tgttcatgat caagttatgc aagatctgga gcaccagcac 11040 tgctcactag caagcatcca gcatgaacta ggcataaatt ccgataaccc tcttttcaac 11100 tccatcctat catatcagaa gcaggacgat gagccggcag gagacgaggg tttggtcatc 11160 aaggccttgg acggacagga tcccacagag gtacgttgta tcgcatgtca aagttcttta 11220 cggaatctga caaatcctag tacgacattg tgttgaatat tgggcacgct accgaccaca 11280 tcgaaattgt ttttgactat aaacacgcct gcctttcaag catccaggca gagagcgtac 11340 tttcactcat gcaatctaca gctgccgctc tagttcagca tgcttcggga gatcatcaga 11400 ctttaagaag cgtcaacatg gttagcactg aagatatatc tgacatatgg caatggaact 11460 cagacgttcc agtcactgtc gacgattgtg tgcatcatat cattacgcga acttgtcata 11520 aacgccccca agctccggca atctgtgcat gggatggcga ttggacctat gctgaagtca 11580 ataagctgtc agataaactt gcccaccttc tagtctccta tggtgttggc cctggagtgg 11640 ttgtcccgct ttgcttcgaa aagtcaaagt ggacacccat agcaatgatg gctgtcatga 11700 aggcaggagg cgcatctgtt gccatggact caacccaacc agaggaacgc ctacgagcaa 11760 tcgtaaacca agtgaagtca cctattatct tgtcatcatt tgccaacgaa cagcttgcaa 11820 gccgactaat cagcgagctt ccagcccacc aaggtcctca caataagcga caaagaagtg 11880 gaaaatttga atgttccgag tggaagccac ttcctcatgt caaccccagt gatactctct 11940 atgtggtatt cacatccggt agtacagggg tgccaaaggg agtggcagtt actcactcca 12000 acattgccag tgcgatcaaa caccagcgac acttgcttgg attcacttct gaatctaggg 12060 tattcgactt ttcttcctat atgtttgatg ttgtctggtg caacttacta cagggtcttt 12120 ctgctggaag ctgcgtttgc atcccgagcg acaatgaaag gaagactgac tttatggccg 12180 ctattgttaa gatgagggca aaccttgtca tattgacacc ttctgctatc cgcggcctga 12240 agcttgacgc tctgaacagc ctatgcaacg tccacttcat cggcgaacct ttacatgttg 12300 acacttttag atcagttgac gaaagtgtca cgatatccaa cctatatggc cccactgaat 12360 gtacaacatt cagcacagta caaaccatct gcggcagaca gcatcagtca atcacaattg 12420 gcaagggagc gggtctgaat acctgggtcg cggacatagc cactggtacg gctcttgtac 12480 caattggcag tgcgggagag cttctacttg aaggcccatt agtcgccgcc ggctaccggg 12540 gtgatgctgt caaaaccgct gccgcattcg tatatgaccc gccatttctt ctgcgtggat 12600 cggtgggcca ccctggtaga cgtggccgcc tatacaaaac cggagatatt gttcggtata 12660 actccaacgg tactttgact ttccttggcc gaaaggattc gcaagtcaag atcaacggac 12720 agcgagttga gttcggtgat attgagtctc acataaacgg ggcgctgcta ccggacttca 12780 gtgaaggtca agctttagtc gactttgtaa cacctcaagg aagctcacgt ccaatgcttg 12840 tagcttttgt ttacttcgga cctactgtca ctgagggcat ggatgaggcc gatctgctaa 12900 gcctagccaa gcgcacagcc atatcgctgg atgagagtct tgctgctcga atccctgcat 12960 tcatgatacc atctgcttat attccattgc agaaaattcc tgtcacagcg acaggcaaga 13020 cagaccgccg tcgtttacgc gagatggcca aagatgttac ctgggaccag ctcattaaag 13080 ctgactccca cggtcctgat cgttgtcaac ctggcacaga gatggagata cagctgcaga 13140 tcctatgggg gactgtgcta ggagttgaaa gtagtttgat cggtgcacat gacaacttca 13200 tgcgcgtcgg tggtgattcc gtgggcgcaa tacgcttagc gagttctgcc cgggaacttg 13260 gtttcacgct gaacgtggct gatattctca agaacccgaa actaagtgat atggcaaaac 13320 ttatgatacg aacagagccg tcgcaggata tttcaatcaa ggaattctct ctcctcaaac 13380 ctggctctga tgtcaactgg gctgttgcag agacatccgc tttatgtggc gtggacggta 13440 accaagttga agatttatat ccttgcacac ccctgcaaga gggcttgctg gcgctgacaa 13500 caaagcgccc cggcgactat atcatccggt gcattttgga actgaagaga tcaacagacg 13560 tcaaaaagtt ctgcgcctct tgggaggcgg tgttggagag caccccaata ctacgaactc 13620 ggatcgtaga catcgcggaa caaggtttgg tgcaagctgt catcaagcaa ccagcacagt 13680 ggacatcggc agaagcctcc agtttggtcg acttcgtggc tgctgataat gagaagacaa 13740 ctggtctggg tatgcccttg gtgcgattcg gactagtaca agagacgaac aaacactttt 13800 ttgttttgac tctgcatcat gcagtatatg atggctgggc gctgaatctg gtcttcgaaa 13860 agctcgaaaa tttttatgct gggtcttcca ggcatgaaag cccggatttc agacacttcg 13920 tcaagcacat ttcaagtctc gacaacgacg cggctgccaa gttttggaaa gaccaactcc 13980 aaggctcaga ggcacccact tttccctcct taccaactgc cacgttcgtg cccaagtctg 14040 aaaagaccat tttgcacaca gttgaagagt tgcagtggcc caagactaat gtcactgctt 14100 ttacgttggt gcgtgcggca ctgtcacttt taacggcggc ctacaccaat tcagaagacg 14160 tttgctttgg cgtgacttcc aacggtcggc aggttgggct ccctggagta gaaagaatga 14220 taggcccgac tattgcgaca gtgccagttc gtgttcgcat tgaccgcgag cagcgtctcc 14280 aagccttcct cacacagatg cagcatcagt ccattgacat gatagcgttt gaacaatttg 14340 gtctgcagca aatacggaag tcaagtcctg acgccgaacg ggcctgtaac ttccagtcac 14400 ttctcattgt ccagccggca gaagagacag cccagtggca gagcgatata attgcccgtg 14460 atatcggaga aggagctgat gatcctatgg gcattcaaga aattggaaca tatgccctta 14520 ctctcgaatg ccatctcgga cccgacagtt tgctcataaa ggccaacttt gattccaatg 14580 taatcgatga actacaggtc aagcgattta caaagcagtt tgagcacgtg cttcgtcaaa 14640 tatgctgctc tggtagtggc cttgtcgttt ctgatattga taccaccagc agacaagaca 14700 tggaagatat ttggaagtgg aatgccgtag tcccccagtc agtcaacacg cctgttcatg 14760 agctcatctc ctctgtggca cgcagactgc cgcatgtcca agctgtatgc gcatgggacg 14820 gcaactggac ttaccgtcaa ctggatgacc tgtcaaatta tgttgcgcac cacctcgtcg 14880 accttggtgt tggctctcag gacatcgtac cgctgttgtt cgagaagtcc aagtggatgc 14940 cgatcgcgat gcttggtgtc atgaaagcag gggctgcgtc ggtggctgtc gataccagtc 15000 agcccaaaga ccgacttcgc atgattatcg accaggcaaa tcccacggtc gcgctaagct 15060 cagctgataa gttgcctctt gtccggagtc tgacaaaggc gcaaagcttc gttgtcagtg 15120 gccaaggtat tgaccgctta ttaaaaccta gccttaatgc tacacttcca gttgtcgatc 15180 cgtccagcag actgtatttg gtcttcacct ctggtagtac gggtgtcccg aagggtgtta 15240 taattcgaca ttgcaatttt gccagtgcaa ttaaacacca gaaagaagtt caaggcatcc 15300 ttccaacctc gcgtgtctat gattttgctt catatgcatt tgacgtcgca tgggccaatg 15360 cgctactgac ctttgagagc ggcgcttgtc tctgtatccc atccgatgct gacagaaaga 15420 atgatctaaa cggttcgatt gcgcgactga agccaactca cgctgatctt acgccttcgg 15480 cagcactggt cctgtccaaa gagtcacttc agcagcttga taccctcact ttaggtggcg 15540 aacgtctcct agcggagtac gcaacaaagt ggtcccagtt tgtgacagtt aaaaactcat 15600 acggaccaag cgagtgcact ccaactgcca catttaccga ggcaattggg cgtggatacg 15660 atcttggtgc tagcattggt aagcctgctg gtctcaacac ttgggtggtt gatcctgtga 15720 cggggcagtc gctcgttccc attggaggcg tcggcgagct gttcttggag gggccgcttg 15780 tcggtgctgg ctatcttgat gatgcagaga aaacgaatgc tgcttttatc catgatccac 15840 catttttgct ccgcggcaac gttgtcgcgc aacctggacg acgcggcacg ctgtacaaga 15900 cgggtgacat tgtgcgatac aactcagatg gcagtctcac ttttgttttt cggaaagata 15960 cacaagtcaa gatcaatggc cagagagtag agcttgctga gattgagagt catatagctc 16020 tatacacagc gacccgacaa gtggcgaccc tattgcctag cactggcctc tgtgcaaaca 16080 agctggtagc tatgatcagc ctcacagatg tgaactatga tgttagcgag gacctcgccg 16140 aaaacaagat tgagctggca tcttcggaac atgaccaact catcaatgag cacatcgagg 16200 cccttcaatc gttattgcgc gagtctttgc cgcagtacat gattccatct ctgtgggtgg 16260 tcttgtacaa cctccctatg acagcatcgg gaaagcaaga caataaggca ctcaaatcct 16320 ggctggaaaa tatggatgaa acgctctttt ctaaaatcaa caatgcgaac ggtagcgaca 16380 ttatccgaaa accagacaca gaagacgaaa gagtcctcag ccagaaatgc agtattgtcc 16440 ttaatatgcc cgtcgacaag atcaatctcg acaaatcttt tattgcgaat ggtggagatt 16500 ccatttccgc catgaggctt gcatcccact acagaactgt gggaatttcc atctcagtct 16560 caacactcct tcaaagtaaa accctcgcgg atttcgcagc attttcgggc gcaacagcca 16620 ttagcggagt cagtcaggaa gagcatactg acgttccttt cgaactatca cccatccagc 16680 agtggttctt tgaccaatcg ccatttatga gccagcagaa gcatgacaga ttctacaacc 16740 aaggattcta tgtgagactc aggcgcacag tgagaatcaa tgatctagaa tcagcctttc 16800 tttctctggt caatcgccac gcgatgttac gctcgcgatt tcagcatcat gggggcaaat 16860 ggaagcaaat aatcctcagt cacagcaaac gagctcttca tttgaacgta tcgcagcacc 16920 tgtccatgag cgagattgca tcgttagctc aagagcggca ccgacagatt gacatagaga 16980 aaggtcctgt cttctcggtt gatatttgct tactaggcca gcagcagcat ctggtcatga 17040 tagcccatca tttggttacc gacttggtct cctggcggat tatcttagat gatctggaga 17100 ctattctcaa cggccattcc ctaacggccg ctctaccctt ccaagtgtgg agcaggctgc 17160 aagctgagcg ggctgtatct tccactttga agcctcacaa cttgctgtcg actgacggcg 17220 tccataacaa tctcaagttt tggaagtata cgcacgacac gcccaactgc ttggcagacc 17280 acaggctccg atcggtcact attgatcgag aaactacggc tgtactactg ggcgaggcaa 17340 acaatgccat gaatacagag ccagtagaga tccttttatc agctgtttgg gatgcattct 17400 ttcggacgtt ttcccagcgc aacagcctga ccatattcaa cgaaggtcat ggtcgcgaag 17460 cttggtccga tgaaattgac ttgtcgagca ctgttggatg gtttaccaca ctcagtccca 17520 tcaacatcta tagaaataat gccaccagcg agaccgacat ggtgcggctc gtcaaggatg 17580 cccgccgcag ccttcccgcg aacggctggt cttactttac ctcccgatat ctgaaccccg 17640 acgggcaaag agcttttgaa agccataaca cggtgtctga agttgttttc aattatcatg 17700 gccaattcca acaactagaa agtcatcaag ccctttttga agacattgat cttgtcggtg 17760 tacgtgtgca aggtcgctca atatctgcag ggtctttgtt taacatcgag gttgccattg 17820 aagcaatgca agcgcacttt gaattctccg ttaatcaaaa tatcgctcat cagagcttga 17880 ttaaccagtg gattgaccaa attcaaccct ctttggaaag gatttgcctc gtccttctcg 17940 aggccaaccc aacgcatacg ctgtgtgact tcaagttcat cagtctcgac taccagcgcc 18000 ttgacgatct taccagtcga ctacttccgg agatcgagtc aatcaaccaa tcaactgttg 18060 aggagatttt ctcgtgctct ccgattgtcg atggaatgct cctcagtcag ataaagcagc 18120 cagaatcata caagacactt cagcgatacg aggtgttgtc atctcatgac catcctatct 18180 gtctcgacac cctcaaaatt gcttggcaaa gggtgatttc tcgccagcct gccttgagga 18240 cagttttcat cgctggcctt gacggatcta ccgcctttta ccaggccctt ctcaaacagt 18300 gctccgggga tgttatcgtt gttgaggcta aaactgaaga ggaagccctg aaagcctttt 18360 cctcgcttcc gaaagtcgat taccaacagg ccaaacctcc tcatcgcctc actctttgtc 18420 aaacgccgga tgacaaagtc ttttgtcaga ttgaaatgag ccatgctatt actgatggtg 18480 cctcttcaac cattttgatt aaggatctga ttgacgctta tggtgatagg ctgtcgtcaa 18540 cagaccttgt caaaacaaca cgcgaatttg ctagccactt gctggctaag ccacagtccc 18600 aaaagatttc gtattggaac acaaaactca agggccttga accttgccgc tttccatccc 18660 tatcgagcat gtctcgggag aagcacgagt gtagctcgga gattggagtt tttgtcgaag 18720 acaagatgtt tgcgcagatt caagacttct gtagcataaa ccaggtcacg ccagcaagtc 18780 tcctcaaaag cgcctgggct ttgacactct cgacctacgt acaaaatcaa tctgtctgct 18840 ttggctatct ggcatctggt cgagacctcc caatcgccgg gatggacgaa tctgtcggcg 18900 cttacactaa catcatggtc tgccgtgccg atttggatgg gcaacagcct ggtgtggcac 18960 tcgtgcgaca acttcagaat caattaatgc aggacttgag cttccaacat atatcgcttg 19020 ctagcattca acatgagctt ggactggcgt ccgatcagca gcttttcaac tctattgtctct 19080 cttttcagag gtcaggagac gataatgaac aatcagcaga ggagggtaaa cttcgattca 19140 agaatattga tggtttggac ccaacagaag tcagtggatt gttgagcatg agagtgtata 19200 catctggact aatgttttta tagtacgaca tcgtattggg tatcaaccaa ggaaccaggt 19260 ccatcgaaat tgaccttgaa ttctcacaca gctgtctcac tagtaatcag gcaaaacgca 19320 ttctcgagca tctgcagtca aacattgccg ctattcttca caatgagcca cctgctctga 19380 tcagccccca agatgagcaa gatatttgga gctggaactc cactgttcct gacatggtca 19440 acatctgcgt tcacgatctg atctccaaga tagtattccg ccagcctgat gcaccagctg 19500 tttgctcatg ggacggcgat ttcacctatg cggagctgga taatcttgca acacgccttg 19560 ccaatagcct cagcaagatg ggcatcggaa gaggcagcat cgtcccgcta tgttttgaaa 19620 aatccaaatg gacaccagtt gccatgctag cagttatgaa aacaggtgca gcctctgtta 19680 cgatggatac tagccagcct gaagaacggc tccagtctat tgttgcacag gtggatgcta 19740 agcttgtgat ttcttcgaca ttgaaggttg agctagcagc caggctcaca acggctcccg 19800 tcttggctat agacaaagcc agcatgaagg cgatggctga cgatacgccg ctggctgcag 19860 tcgatcccgc aaacagtatt tacattgtct tcacatcagg gagcactggc acgccaaaag 19920 gtgtcattat cactcatacc aactacagca gcgccatcaa gcatcagcag agtgaacacg 19980 gcttcaagcc aacctctagg gtctttgact ttgcctctta tgcgttcgat gtcagctggt 20040 ccaatttctt gcacaccttg accattgggg cctgcttatg cattccttct gatcatgatc 20100 gtaaaaatga cccggcgggc gcaattgacc gcttacggtg tacacacgtt gatatgaccc 20160 cctcggccgc aagcgtctta cctgccagta cgttggctaa attggatacc attgttctgg 20220 gaggcgagaa gctctcgctt gaatatgccc aacgctggtc cgccctgaca agcgtgcgta 20280 atccgtacgg gccttctgaa tgcacaccga cgtcgacaat tacggagatc aattctgcgg 20340 aaataagcaa gggcaaagtg agcatcggca aaggagtggg actcaatact tggattgttg 20400 atcctgccac tgcacaacat ttaatgccga ttggcatccc tggggagtta ttactcgaag 20460 gtccgcttgt tggtgctggc tatcttggag accctgtcaa aaccgcttca gcatttattg 20520 aagacccaga attcctagtc aaaggcgcta gtccaggaat tccaggccgc cgtggtcgtc 20580 tgtacaggac gggcgatcta gtcacctata ataccgatgg tagcttgtca tttgtgggcc 20640 gaagggactc tcaaatcaaa ataaacgggc aacgcgtcga attaggcgac atcgagtcgc 20700 acgtttctgc aaacctggtg agtcatggca gtgctcaggt tgcggtcgag gttgtgtcac 20760 cccaagctag ctccaacaac atacttgtcg ccttcgtgag ctttgacgac ctgaattcta 20820 tcaacctgaa tgatgaaaag cttcttgccc gcacgaaagc ggcgaccgag ggaattaggg 20880 agaaactcgc gacacaaatc ccatcttata tgattccttc ggtctacatc cctgttactg 20940 tttttcccac aacagctact gggaagactg atcggcgccg attacgtgaa atggcctcaa 21000 gcctcaccct ggagcagctt acctcaatca accaagctca acagcaatat caacccccca 21060 ctactccctt ggaagtggca cttcgggagc tctggatctc agtcctcaaa ttaggatcgc 21120 gaaaaattag cactacaaac aacttcttcg aacttggtgg agattccatc ggtgctatta 21180 ggctggtagg cgcggcccgt gaccacggac tatcgctttc cgttgtagat attttcaagc 21240 atcctaagtt cagcgaaatg gctgctttgc ttcgttctgt ggataagccg cagttggaag 21300 agccacgggt atttcaaccc acttcgcttc tgtccaaaga tcacaacaaa gaccagatac 21360 tctctcgact ctttgacttt ggtattgact tggaaaatgt tgaagacatc ctccctgtca 21420 cggatcatca agctcgttcc atcgcgatga ctcactctgc gtcccgcgac ttgctactct 21480 atcccacttt agatagcaag ggcgtgccaa atatgcgcaa gatgcgagca gtgtgcaatg 21540 agctcgtcaa tagatatgat ctcatgcgaa cccttttcat cgcacataaa gacagcttct 21600 tgcaggtcgt gctgaaggcc tttcctgtgg atataaccgt cttgagaatt gagaatgcca 21660 gcctagagga atgcacagaa gagctacgat tacgcgacag ggacgatgag ctccgttatg 21720 gctcgctcct aacaaagatt gctattttgc atcaaatccg cgacaacgaa taccgtcttg 21780 tggtccgcat ttcccatgct caacatgacg gaatgagctt gatgaaaatg tggaacgcat 21840 ttgaagaaat gtacggtgac gggagtgacg actcattcca cattccttcg gacactagct 21900 tccaagaaaa gtctaaggct agtttctcca actacatgca tgccgtggct ggtacaaacc 21960 gggagcaagc taagtctcac tggcgcagac tcctcaaggg ctctagcatg acgaacctca 22020 agccccatgc ttcatatgcc ctgacctttg gcgaaggacc atgtgtcgcc agacatgttc 22080 ctaagagcat tgctcaaggt actggattta catttcatac tgtactgaag gctgcttggg 22140 cgtacgttct ggcaaaacac cttgccaacg acgacgtggt tttctgtagc ctcactcacg 22200 gtcggggctt gcccgggaca caagatgtct ttggagactg cgtgaacatc attcctactc 22260 gagtatcttt taccgacgga tggactgttc gcgaccttct cagtgcattg aacgcccaac 22320 agattgcgag catggagcat gagaatatag gcacacgcga aattgtccgt gactgtacta 22380 catggccgaa gtggacatat gcaggatcca tcgtttatca ccatgacttt gacgatggag 22440 aacatattgc tcataaccgc agtatgcacg ttgagcagga gctaaatctg tctcacggca 22500 aagtggacat gaccgacgtt catatcactt ctaagcctga taacaacatg ttccgaattg 22560 agctggactt cgcacatggc gtggtgtctg agcgtgacgc tgaactgcta gctgcgaaac 22620 tgacggagtc tatcatcgtt ttctgcaatg tcatggacca gcctttgtta tctcccgacg 22680 agatcagata tctgcggaca accacattgc tgccctcaga ggagcctctc agtgcaaccc 22740 caacaaatga acagttaatg gttgctagca ttagcccgac tgaaatgcaa tgggcgcttg 22800 agagtgcgtg gaaggacact ttcaattgcc cccttagtcc tgaggtgaaa gcgggcaaga 22860 caatttttga tcttggtggt gacttgataa gcgctagtct gatatcagct cacatggaga 22920 ggcaaggata tgtccttagt gttgaggatg tcttggggaa tccgacgtgg ttctcgcaac 22980 tgacgctgtt gacgaagcgc actcttcggg atgttgatgt ctgatgaaca agaatgttta 23040 atatttagtc tgtcattcct atttggtatc atcactcagt acttcggcgt ttcttttttt 23100 ttttttttcc ccaa 23114 <210> 5 <211> 21 <212> PRT <213> Aspergillus niger <400> 5 Ala Val Ile Gly Xaa Gly Gln Ser Xaa Xaa Glu Xaa Phe Met Asn Leu   1 5 10 15 Xaa Ser Xaa Phe Pro              20 <210> 6 <211> 22 <212> PRT <213> Aspergillus niger <400> 6 Ala Leu Xaa Pro Ser Asp Asp Xaa Xaa Phe Val Asn Xaa Ala Xaa Phe   1 5 10 15 Asp Pro Glu Arg Thr Asp              20 <210> 7 <211> 62 <212> DNA <213> Artificial Sequence <400> 7 gcngtnatng gnnsnggnca nwsnnnnrcn ganatnttna tgaayntncm nnnncrntty 60 cc 62 <210> 8 <211> 66 <212> DNA <213> Artificial Sequence <400> 8 rtcngtncgn tcnggrtcra anrcngcnnn rttnacraan ssnnnrtcrt cnnnnggnnn 60 narngc 66 <210> 9 <211> 14 <212> PRT <213> Aspergillus niger <400> 9 Tyr Xaa Phe Thr Ser Gly Ser Thr Gly Lys Pro Lys Xaa Val   1 5 10 <210> 10 <211> 15 <212> PRT <213> Aspergillus niger <400> 10 Asp Xaa Gln Val Lys Val Xaa Gly Gln Arg Xaa Glu Leu Xaa Glu   1 5 10 15 <210> 11 <211> 44 <212> DNA <213> Artificial Sequence <400> 12 tayntnytnt tyacnnsngg nnsnacnggn aarccnaarg sngt 44 <210> 12 <211> 41 <212> DNA <213> Artificial Sequence <400> 12 tcnycnaryt cnatyctytg nccnytyttn acytgnstrt c 41

[Brief description of drawings]

FIG. 1 shows a ferrichrome biosynthesis gene cluster of Aspergillus niger in Aspergillus niger.

FIG. 2 E. coli expression plasmid pEN of nsb1 gene
It is a photograph showing S1 (drawing substitute photograph).

FIG. 3: Purification of nsb1 Escherichia coli expressed protein and SDS-P
It is a photograph showing a pattern of AGE (drawing substitute photograph).

FIG. 4 shows the amino acid sequence of ornithine monooxygenase protein.

FIG. 5 shows the continuation of the above.

FIG. 6 shows the nucleotide sequence of nsb1 gene.

FIG. 7 shows the continuation of the above.

FIG. 8 shows the amino acid sequence of the peptide synthetase protein.

FIG. 9 shows the continuation of the above.

FIG. 10 shows the continuation of the above.

FIG. 11 shows the continuation of the above.

FIG. 12 shows the continuation of the above.

FIG. 13 shows the continuation of the above.

FIG. 14 shows the continuation of the above.

FIG. 15 shows the continuation of the above.

FIG. 16 shows the continuation of the above.

FIG. 17 shows the continuation of the above.

FIG. 18 shows the continuation of the above.

FIG. 19 shows the continuation of the above.

FIG. 20 shows the continuation of the above.

FIG. 21 shows the continuation of the above.

FIG. 22 shows the continuation of the above.

FIG. 23 shows the continuation of the above.

FIG. 24 shows the continuation of the above.

FIG. 25 shows the continuation of the above.

FIG. 26 shows the continuation of the above.

FIG. 27 shows the continuation of the above.

FIG. 28 shows the continuation of the above.

FIG. 29 shows the continuation of the above.

FIG. 30 shows the continuation of the above.

FIG. 31 shows the continuation of the above.

FIG. 32 shows the continuation of the above.

FIG. 33 shows the continuation of the above.

FIG. 34 shows the continuation of the above.

FIG. 35 shows the continuation of the above.

FIG. 36 shows the nucleotide sequence of nsb2 gene.

FIG. 37 shows the continuation of the above.

FIG. 38 shows the continuation of the above.

FIG. 39 shows the continuation of the above.

FIG. 40 shows the continuation of the above.

FIG. 41 shows the continuation of the above.

FIG. 42 shows the continuation of the above.

FIG. 43 shows the continuation of the above.

FIG. 44 shows the continuation of the above.

FIG. 45 shows the continuation of the above.

FIG. 46 shows the continuation of the above.

FIG. 47 shows the continuation of the above.

FIG. 48 shows the amino acid sequence of amino acid conserved region (1).

FIG. 49 shows the amino acid sequence of the amino acid conserved region (2).

FIG. 50 shows primer P1.

FIG. 51 shows primer P2.

FIG. 52 shows the amino acid sequence of the amino acid conserved region (3).

FIG. 53 shows the amino acid sequence of the amino acid conserved region (4).

FIG. 54 shows primer P3.

FIG. 55 shows primer P4.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12P 21/02 C12N 15/00 A (72) Inventor Shoji Kawato 24 Shimotoba Koyanagi-cho, Fushimi-ku, Kyoto 24 Corporate Research Institute (72) Inventor Koji Suginami 300 Katahara-cho, Fushimi-ku, Kyoto City Gekkeikan Co., Ltd. (72) Inventor Yasuhisa Abe 300 Katahara-cho, Fushimi-ku, Kyoto City F-Term (reference) 4B024 AA01 BA08 CA02 DA06 DA11 4B050 CC03 DD03 LL01 4B064 AG01 CA05 CA19 CC24 DA01 4B065 AA26X AA62Y AA63X AB01 CA28

Claims (11)

[Claims] 1. A protein of the following (a) or (b): (a) a protein having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; (b) a deletion or substitution of an amino acid in the amino acid sequence (a) Alternatively, an ornithine monooxygenase protein having an added amino acid sequence and forming a ferrichrome biosynthetic cluster. 2. The following protein (c) or (d): (c) a protein having the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing; (d) amino acid sequence (c) having an amino acid deletion or substitution Alternatively, a peptide synthetase protein having an added amino acid sequence and forming a ferrichrome biosynthetic cluster. 3. A gene encoding the protein according to claim 1 or 2, or an ornithine monooxygenase gene (SEQ ID NO: that hybridizes with the gene under stringent conditions and forms a ferrichrome biosynthetic cluster). 2), at least 1 selected from the group consisting of peptide synthetase protein gene (SEQ ID NO: 4)
DNA of one gene. 4. A recombinant vector comprising at least a coding region of the DNA according to claim 3. 5. A recombinant vector pENS1. 6. A transformant in which the recombinant vector according to claim 4 or 5 is inserted. 7. A transformant, Escherichia coli (Es)
cherichiacoli) NSID1 (FERM
P-18679). 8. A recombinant Aspergillus oryzae containing the recombinant vector according to claim 4 or 5. 9. The transformant according to claim 6 or 8, wherein a koji mold with high ferrichrome production is bred by producing at least one of ornithine monooxygenase or peptide synthetase protein forming a ferrichrome biosynthetic cluster. , A breeding method characterized by. 10. A method for producing ferrichrome by introducing a gene cluster involved in Aspergillus niger ferrichrome biosynthesis into an organism other than Aspergillus oryzae. 11. A method for synthesizing a desired peptide by recombining at least one of each domain of Aspergillus niger peptide synthetase protein by protein engineering.