JP2004222609A - Aspergillus npgO gene - Google Patents

Abstract

Kind Code: A1 A cloning of a phosphopantothenate transferase gene (npgO gene) of Aspergillus oryzae was performed, and its nucleotide sequence was also determined.
[Effect] By overexpressing the npgO gene according to the present invention together with a nonribosomal peptide synthase (NRPS) gene (for example, a peptide synthetase gene asb2 or asb4 gene), an active form which cannot be obtained by asb2 or asb4 alone is obtained. It has become possible to produce non-ribosomal peptides (for example, Aspergillus ferrichrome) at a high level.
[Selection diagram] None

Description

【図面の簡単な説明】
【図１】ホスホパントテン酸転移酵素のアミノ酸配列を示す。
【図２】同上の続きを示す。
【図３】ホスホパントテン酸転移酵素をコードする遺伝子（ｎｐｇＯ遺伝子）の塩基配列を示す。
【図４】プライマーＰ１を示す。
【図５】プライマーＰ２を示す。
【図６】ペプチドシンテターゼ遺伝子（ａｓｂ２遺伝子）の塩基配列を示す。
【図７】同上の続きを示す。
【図８】同上の続きを示す。
【図９】同上の続きを示す。
【図１０】同上の続きを示す。
【図１１】ペプチドシンテターゼ遺伝子（ａｓｂ４遺伝子）の塩基配列を示す。
【図１２】同上の続きを示す。
【図１３】同上の続きを示す。
【図１４】同上の続きを示す。
【図１５】同上の続きを示す。
【図１６】同上の続きを示す。
【図１７】同上の続きを示す。
【図１８】同上の続きを示す。
【図１９】ｍｅｌＯプロモーターの塩基配列を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a koji mold phosphopantothenate transferase protein, a gene encoding the protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, a recombinant koji mold containing the gene, and the transformant Non-ribosomal peptide produced by the present invention, a transformant obtained by introducing a recombinant vector containing a phosphopantothenate transferase gene into a strain that highly expresses any peptide synthetase gene, a non-ribosomal peptide produced by the transformant, The present invention relates to a koji mold obtained by introducing a recombinant vector containing a phosphopantothenate transferase gene into a koji mold highly expressing the peptide synthetase gene of the present invention, and a nonribosomal peptide produced by the koji mold.
[0002]
[Prior art]
Microorganisms such as bacteria, actinomycetes, and filamentous fungi have the ability to biosynthesize a variety of small secondary metabolites, which have been used extensively industrially for a long history. Among the secondary metabolites, polyketides and nonribosomal peptides are the most industrially important secondary metabolites represented by antibiotics such as penicillin and cyclosporine. These have been conventionally produced by fermentation methods using wild strains or mutant strains, but their production amounts are very small and are often very expensive. Therefore, in order to provide these secondary metabolites at low cost, establishment of a high production method by a genetic engineering technique was required. Recent biochemical studies have revealed that polyketide and nonribosomal peptide synthases (PKS) and nonribosomal peptide synthase (NRPS) are involved in the biosynthesis of the basic skeleton of polyketides and nonribosomal peptides. .
[0003]
PKS and NRPS are complex-type giant enzymes, some of which have a total length exceeding 15,000 amino acid residues. Therefore, some of the coding genes have a total length of 20 to 50 kb. Subcloning of such a long chain gene is difficult, and for high expression using a genetic engineering technique, a technique of homologously recombining the enhancer region of the gene present in the genome with a high expression enhancer has been used. However, the transformants often do not produce the expected production of secondary metabolites, and the transformants have not been practically used.
[0004]
As the cause, the present inventors have made the following hypothesis. PKS and NRPS, which are complex-type giant enzymes, have a plurality of protein function motifs. Typical examples of NRPS include an amino acid activation domain, an acyl carrier protein, and a condensation domain. Of these, acyl carrier proteins require a covalently bound phosphopantothenic acid as a prosthetic factor for activity expression. This post-translational modification is catalyzed by phosphopantothenate transferase in Bacillus subtilis by Mootz HD et. al. (For example, see Non-Patent Document 1). In addition, the phosphopantothenate transferase gene has also been cloned in the filamentous fungus Aspergillus nidulans, and it has been shown that it can complement the Lys5 mutation of Saccharomyces cerevisiae (for example, see Non-Patent Document 2).
[0005]
Aspergillus oryzae, a koji mold widely used as an industrial microorganism, also has the above-mentioned phosphopantothenate transferase gene, and is presumed to contribute to the production of many secondary metabolites. To date, it has been clarified that Aspergillus can produce a nonribosomal peptide capable of chelating iron ions called ferrichrome. We are a group of genes involved in ferrichrome biosynthesis of Aspergillus oryzae, a transcription factor sreO that negatively controls ferrichrome biosynthesis (see, for example, Patent Document 1), ornithine N5 oxygenase that catalyzes the first step of ferrichrome biosynthesis. (Japanese Patent Application No. 2001-176264), asb2 (Japanese Patent Application No. 2001-324112) encoding a peptide synthetase gene involved in ferrichrome biosynthesis of Aspergillus, cluster genes asb3 and asbT involved in ferrichrome biosynthesis of Aspergillus , AnkO, asbR (Japanese Patent Application No. 2001-324181) and asb4 (Japanese Patent Application No. 2002-348755) encoding a peptide synthetase gene involved in ferrichrome biosynthesis of Aspergillus oryzae have been cloned. Among them, the genes of asb2 and asb4 did not contribute to the improvement of ferrichrome productivity even when expressed at a high level.
[0006]
The inventors of the present invention have investigated the cause from various aspects, and found that only the apoenzyme that was not post-translationally modified by the above-described phosphopantothenate transferase was produced, and the active form of the holoenzyme was not produced. It was the first time that I had been in a position where I could. In addition, the present inventors have noticed for the first time that even in Aspergillus oryzae, the productivity of a target secondary metabolite can be improved to a practical level by highly expressing a phosphopantothenate transferase gene together with a peptide synthetase gene. Aspergillus oryzae A. From the genome sequence of oryzae, a new idea was obtained that a gene encoding a phosphopantothenate transferase capable of improving the productivity of various secondary metabolites of Aspergillus to a practical level could be obtained.
[0007]
[Non-patent document 1]
"Journal of Biological Chemistry (J. Biol. Chem.)", 276, 37289-37298, 2001.
[0008]
[Non-patent document 2]
Mootz HD et al., "FEMS Microbiological. Lett." 213, 51-57, 2002.
[0009]
[Patent Document 1]
JP-A-2002-272577
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a phosphopantothenate transferase gene of Aspergillus oryzae in order to freely control the production of a secondary metabolite containing ferrichrome of Aspergillus oryzae. Another object of the present invention is to provide a recombinant vector containing the present gene and a transformant containing the recombinant vector. Another object is to provide a non-ribosomal peptide produced by the transformant. It is still another object of the present invention to provide an arbitrary non-ribosomal peptide using a strain in which the above-mentioned recombinant vector has been introduced into a strain in which any NRPS or PKS is highly expressed.
[0011]
To date, we have a group of genes involved in ferrichrome biosynthesis of Aspergillus oryzae, a transcription factor sreO that negatively regulates ferrichrome biosynthesis (JP-A-2002-272377), and catalyzes the first step of ferrichrome biosynthesis. Asb1 encoding ornithine N5 oxygenase (Japanese Patent Application No. 2001-176264), asb2 encoding a peptide synthetase gene involved in ferrichrome biosynthesis in Aspergillus oryzae (Japanese Patent Application No. 2001-324112), cluster gene involved in ferrichrome biosynthesis in Aspergillus oryzae Asb3, asbT, ankO, asbR (Japanese Patent Application No. 2001-324181) and asb4 (Japanese Patent Application No. 2002-348755) encoding a peptide synthetase gene involved in ferrichrome biosynthesis of Aspergillus are cloned. Among them, the genes of asb2 and asb4 did not significantly contribute to the improvement of ferrichrome productivity even when expressed at high levels. This led to a novel viewpoint that only apoenzymes that were not post-translationally modified by phosphopantothenate transferase were produced.
[0012]
The present inventors have focused on the novel viewpoint described above, and for the first time focused on the fact that even in Aspergillus oryzae, the productivity of the target secondary metabolite can be improved to a practical level by highly expressing the phosphopantothenate transferase gene together with the peptide synthetase gene. Aspergillus A., partially disclosed in 2002. From the genome sequence of oryzae, a new idea was obtained that a gene encoding a phosphopantothenate transferase capable of improving the productivity of various secondary metabolites of Aspergillus to a practical level could be obtained.
[0013]
For example, the obtained phosphopantothenate transferase gene is transformed into a gene encoding a NRPS gene involved in ferrichrome biosynthesis (a peptide synthase (peptide synthetase) involved in ferrichrome biosynthesis which is a kind of non-ribosomal peptide: When expressed together with the NRPS gene alone, the peptide synthetase encoded by the NRPS gene is produced not as an inactive apoenzyme but as an active holoenzyme, As a result, a new idea that the productivity of ferrichrome can be improved was obtained. By realizing such a new idea, ferrichrome produced efficiently can be effectively used as a functional food or drug itself that can be expected to improve anemia, for example, as a reagent as an iron chelating agent. However, it can also be used as a component to be added to these, and its effectiveness is very large.
[0014]
[Means for Solving the Problems]
Aspergillus A. Oryzae is a filamentous fungus used in the traditional fermentation industry in Japan such as sake, soy sauce, and miso. The strain is to produce proteins and small molecules useful in the fermentation industry in very large quantities. Due to the high protein-producing ability of this strain and its safety as a brewing microorganism, it is attracting attention as a host for heterologous protein production. (Biotechnology, 6, 1419 (1988), JP-A-62-272988). From the inventors' research, A.I. In the production of a heterologous protein using oryzae, it was found that if a gene of a near species such as Aspergillus was used, its production ability was further increased. In particular, A. oryzae gene is It has been found that when expressed under the control of a high expression promoter of oryzae, a very large amount of protein is produced (JP-A-11-154271, Japanese Patent Application No. 2000-36754). A. Oryzae has been reported to produce not only the above-mentioned proteins and enzymes, but also low-molecular-weight components which are secondary metabolites which are very valuable industrially. Among them, ferrichrome produced by Aspergillus oryzae in solid culture is a substance that has recently attracted much attention as a functional component for iron deficiency such as anemia.
[0015]
In order to use this koji mold ferrichrome in medicines and foods, it is necessary to further improve the productivity.However, with existing strain breeding methods such as the mutation method, a mutant strain having improved productivity up to the industrial production level is required. Could not be obtained. Therefore, the present inventors have considered that it is possible to mass-produce ferrichrome by isolating genes involved in ferrichrome biosynthesis and enhancing the expression ability of these genes.
[0016]
Regarding the ferrichrome biosynthesis pathway in Aspergillus oryzae, the first step of the biosynthesis is the hydroxylation of ornithine by ornithine N5 oxygenase, followed by the tripeptide of N-hydroxyornithine by peptide synthetase to the cyclic peptide of glycine, serine and alanine. It is thought that it is. Up to now, we have a group of genes involved in ferrichrome biosynthesis in Aspergillus oryzae, a transcription factor sreO that negatively regulates ferrichrome biosynthesis (Japanese Patent Application No. 2001-83640) and ornithine that catalyzes the first step of ferrichrome biosynthesis. Asb1 encoding N5 oxygenase (Japanese Patent Application No. 2001-176264), asb2 encoding a peptide synthetase gene involved in koji mold ferrichrome biosynthesis (Japanese Patent Application No. 2001-324112), and cluster gene asb3 involved in koji mold ferrichrome biosynthesis , AsbT, ankO, asbR (Japanese Patent Application No. 2001-324181), and asb4 (Japanese Patent Application No. 2002-348755) encoding a peptide synthetase gene involved in ferrichrome biosynthesis of Aspergillus oryzae. Among them, the genes of asb2 and asb4 did not significantly contribute to the improvement of ferrichrome productivity even when expressed at high levels.
[0017]
As the cause, the present inventors have made the following hypothesis. PKS and NRPS, which are complex-type giant enzymes, have a plurality of protein function motifs. Typical examples of NRPS include an amino acid activation domain, an acyl carrier protein, and a condensation domain. Of these, acyl carrier proteins require a covalently bound phosphopantothenic acid as a prosthetic factor for activity expression. This post-translational modification is catalyzed by phosphopantothenate transferase in Bacillus subtilis by Mootz HD et. al. (J Biol Chem, 276, 37289-37298, 2001). In addition, the phosphopantothenate transferase gene was also cloned in the filamentous fungus Aspergillus nidulans and was shown to be able to complement the Lys5 mutation of Saccharomyces cerevisiae (Mootz HD et. Al., FEMS Microbiol Lett, 213, 571-51). .
[0018]
That is, the genes of asb2 and asb4 did not significantly contribute to the improvement of ferrichrome productivity even when expressed at a high level, because NRPS, which is an active holoenzyme post-translationally modified by phosphopantothenate transferase, was produced. The present inventors considered that this was not done. Therefore, A. A novel idea was obtained that a phosphopantothenate transferase gene was cloned from oryzae and that it was highly expressed together with the asb2 and asb4 genes, thereby contributing to a significant increase in ferrichrome-producing ability. As a result, the present inventors have obtained a new idea that a genetically modified koji mold capable of freely controlling ferrichrome production can be bred and industrial production of ferrichrome becomes possible. Hereinafter, among ferrichromes produced by Aspergillus, the production of ferricrisin with the highest content will be mainly described.However, the present invention is a technology applicable to all ferrichromes, and is limited to only ferricrysin. It does not do.
[0019]
We highly express the NRPS genes asb2 and asb4 involved in ferricrysin biosynthesis and efficiently activate their gene products after translation by phosphopantothenate transferase to obtain genes suitable for industrial production of ferricrysin. We tried to breed the recombinant koji mold.
[0020]
We thought that it would be possible to search for the above phosphopantothenate transferase gene from the genome gene sequence of Aspergillus oryzae, which was partially disclosed in 2002.
[0021]
Genes considered to be phosphopantothenate transferase genes were extracted from the Blast search results among the contig sequences contained in the koji mold genome data. Among them, Gene Finding Con1616-JUNK-1 is A.I. The protein showed 57% homology with npgA of the C. nidulans phosphopantothenate transferase gene. The gene was found to be free of introns by cDNA analysis using poly A + RNA prepared from Aspergillus oryzae cells liquid-cultured in an iron-restricted medium as a template. This gene encoded a protein consisting of 343 amino acid residues. The obtained gene was designated as npgO.
[0022]
In order to identify the function of npgO, we attempted to overexpress this gene in Aspergillus oryzae. First, a koji mold overexpressing asb2 encoding NRPS among ferrichrome biosynthesis genes under the control of a melO promoter (Japanese Patent Application No. 11-154271) that highly expresses in a liquid culture was bred, and the resulting transformant was melO promoter. Plasmids expressing npgO under promoter control were further transformed. The transformant which overexpressed both asb2 and npgO produced 8.5-fold ferrichrome as compared to the control strain which overexpressed only asb2. Therefore, it was shown that npgO converts NRPS gene products such as asb2 into an active form. The melO gene is known as a tyrosinase gene of Aspergillus oryzae (Biochem. Biophys. Acta., 1261 (1), p. 151, 1995), and a transformant (Aspergillus oryzae MEL-P450nor) into which a melO promoter-containing plasmid has been introduced is known. Deposited with FERM P-17942 at the Biotechnology Research Institute of Technology (currently the Patent Organism Depositary). FIG. 19 shows the base sequence of the melO promoter.
[0023]
The gene thus isolated may be used alone or together with a high expression promoter such as the melO or glaB gene promoter (Japanese Patent Application No. 11-154271, Japanese Patent Application Laid-Open No. 11-243965). It can be expressed in oryzae to control NRPS activity. In the gene transfer method, for example, a target gene and a niaD gene as a marker gene are simultaneously introduced by a known method using a niaD mutant strain (for example, Aspergillus oryzae 1013-niaD: FERM p-17707) as a host. (S. Uncle et al., Mol. Gen. Genet., 218, pp. 99-104, 1989) At the time of this gene transfer, a self-cloning strain containing no heterologous gene at all was eliminated by eliminating the heterologous gene such as a vector sequence. A transformant can be obtained.
[0024]
The details of the present invention are described below.
[0025]
It was suggested that a novel phosphopantothenate transferase gene was present in the aspergillus genomic gene contig. From the Blast search results among the contig sequences included in the koji mold genome data, A gene having homology with nidlans phosphopantothenate transferase gene npgA was extracted. Con1616-JUNK-1 (11199-12552), Gene Finding in Contig1616 (57457 bp), The protein showed 57% homology with npgA of the C. nidulans phosphopantothenate transferase gene. As a result of cloning the cDNA amplified by RT-PCR using poly A + RNA prepared from Aspergillus oryzae cells liquid-cultured in an iron-restricted medium as a template and determining the nucleotide sequence, an accurate coding region different from Gene Finding was revealed. Was found not to exist. This gene encodes a protein consisting of 343 amino acid residues shown in SEQ ID NO: 1 and does not contain any intron. The coding region is from 1 to 1032 bp of SEQ ID NO: 2. The obtained gene was designated as npgO. That is, the amino acid sequence of the phosphopantothenate transferase of Aspergillus oryzae according to the present invention is shown in SEQ ID NO: 1 (FIGS. 1 and 2), and the nucleotide sequence of the gene encoding the same (npgO gene) is shown in SEQ ID NO: 2 (FIG. 3). It was shown to.
[0026]
Furthermore, expression analysis in Aspergillus was performed for functional analysis of npgO. Asb2, which encodes NRPS involved in ferrichrome biosynthesis, is controlled by melO promoter and sC is used as a marker for Aspergillus niger. oryzae M-NS4. The resulting transformant was transformed with npgO under the control of the melO promoter using niaD as a marker. The obtained transformant produced 8.5 times as much ferrichrome as the control strain in which only asb2 was highly expressed in an iron-limited liquid culture at 30 ° C. for 7 days. Thus, npgO was shown to convert NRPS gene products such as asb2 to the post-translationally active form.
[0027]
The present invention has been made based on these useful new findings, and relates to a protein having a phosphopantothenate transferase activity, a gene encoding the same, and use thereof, examples of which are shown below. .
[0028]
(Aspect 1) The following protein of (a) or (b):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or (b) consisting of an amino acid sequence in which amino acids are deleted, substituted or added in the amino acid sequence (a), and having a phosphopantothenate transferase activity protein.
[0029]
(Aspect 2) The gene encoding the protein (a) according to claim 1, which is represented by the nucleotide sequence of SEQ ID NO: 2, or hybridizes with the gene under stringent conditions, and has a phosphopantothenate transferase activity. DNA of a gene encoding the protein (b) having
[0030]
In the present invention, the specific amino acid sequence described above includes an amino acid sequence that is substantially the same as this. An amino acid sequence that is substantially identical to a specific amino acid sequence in the present invention is, on average, about 30% or more, preferably about 50% Above, more preferably about 80% or more, particularly preferably about 90% or more amino acid sequences are identical. Therefore, a protein consisting of an amino acid sequence substantially identical to a certain amino acid sequence is considered to have substantially the same function as a protein consisting of the amino acid sequence.
[0031]
In addition, the amino acid sequence in which some amino acids are deleted, substituted or added in the specific amino acid sequence of the protein of the present invention is preferably as long as it has a function substantially equivalent to that of the protein comprising the amino acid sequence. About 1 to 20, more preferably about 1 to 10, and even more preferably several amino acids are deleted, substituted or added, or an amino acid sequence obtained by combining them. The “function” of a protein means a biological function or activity that the protein exhibits inside and / or outside a cell (cell).
[0032]
Proteins consisting of an amino acid sequence substantially identical to the specific amino acid sequence described above, or proteins consisting of an amino acid sequence in which some amino acids have been deleted, substituted or added are, for example, site-directed mutagenesis, gene homology It can be easily prepared by appropriately combining methods known to those skilled in the art, such as a recombination method, a primer extension method, and a PCR method.
At this time, in order to have substantially the same function, among the amino acids constituting the protein, homologous amino acids (polar / non-polar amino acids, hydrophobic / hydrophilic amino acids, positive / negative charged amino acids, aromatic (Such as group amino acids) are considered possible. In order to maintain substantially the same function, it is desirable that amino acids in the functional domains contained in each protein of the present invention be retained.
[0033]
In the present specification, “under stringent conditions” means that the degree of homology between nucleotide sequences is, for example, about 80% or more, preferably about 90% or more, more preferably about 95% or more on average. As described above, it means the conditions under which a hybrid is specifically formed only between base sequences having high homology. Specifically, for example, at a temperature of 60 ° C. to 68 ° C., conditions such as a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and a pH of 6 to 8 can be mentioned.
Hybridization can be performed by a method known in the art, such as a method described in Current protocols in molecular biology (edited by Frederic M. Ausubel et al., 1987), or the like. It can be carried out according to a method corresponding thereto. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
[0034]
As described above, the npgO gene according to the present invention is a novel gene, which encodes a novel phosphopantothenate transferase different from the conventionally known phosphopantothenate transferase, and has the activity of the novel phosphopantothenate transferase. It indicates at least one selected from a gene encoding a protein and a gene containing at least one of these genes (hereinafter, sometimes simply referred to as the npgO gene, and its base sequence (1032 bp) is represented by a sequence in the sequence listing. This is shown in number 2 and in FIG. 3 of the drawings.)
[0035]
The DNA of the gene (npgO gene) encoding the phosphopantothenate transferase according to the present invention has been clarified by the present inventors as shown in SEQ ID NO: 2 (FIG. 3). It can be prepared by methods well known to those skilled in the art. For example, each DNA represented by a specific nucleotide sequence in the present specification can be prepared, for example, by the shotgun cloning method described in the Examples using the genome of Aspergillus oryzae as a starting material. At this time, each fragmented chromosomal DNA is ligated to an appropriate cloning vector such as a plasmid vector or a phage depending on its length and the like, and Escherichia coli or the like is then used by an appropriate method such as electroporation using this. And a clone library for cloning each fragmented chromosomal DNA can be prepared.
Furthermore, the nucleotide sequence of each fragmented chromosomal DNA obtained from such a clone library can be determined according to a known method such as a chemical decomposition method (Maxam-Gilbert method) and a dideoxy method.
[0036]
Alternatively, it can also be prepared by chemical synthesis well-known to those skilled in the art based on information on the nucleotide sequence or amino acid sequence of the DNA of the present invention described in the present specification, or by amplification using PCR using the primers of the present invention. I can do it.
[0037]
As an example of the genome of Aspergillus oryzae used as the DNA source according to the present invention, the genome of Aspergillus oryzae RIB40 strain can be used. This strain has been deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary as FERM P-18273. In addition, since the genome sequence of Aspergillus oryzae has also been partially disclosed, it may be used as a DNA source, and the preparation of the npgO gene DNA according to the present invention is easy. At that time, according to the specific examples described later, cloning of the present gene is even easier.
[0038]
Thus, not only is the preparation of the phosphopantothenate transferase gene (eg, npgO gene) easy, but also this gene and the non-ribosomal peptide synthase (NRPS) gene (eg, asb2, asb4 genes) It is also easy for those skilled in the art to prepare a novel transformant that is co-expressed to efficiently produce a nonribosomal peptide (eg, ferrichrome).
[0039]
That is, since the melO promoter and niaD mutant are both known and have been deposited (FERM P-17942, FERM P-17707), there is no difficulty in obtaining them and, as already described, , Asb2 and asb4 have already been filed (Japanese Patent Application Nos. 2001-324112 and 2002-248755). The nucleotide sequence of asb2 gene DNA is shown in SEQ ID NO: 5 (FIGS. 6 to 10), the nucleotide sequence of asb4 gene DNA is shown in SEQ ID NO: 6 (FIGS. 11 to 18), and a transformant Escherichia into which asb2 gene was introduced -Escherichia coli ASB2 has been deposited (FERM P-18541), and their preparation is easy.
[0040]
And further, the above A.I. oryzae M-NS4 is a strain that can be obtained from the National Institute of Liquor Research, and the transformation of the present invention may be performed using a conventional method of the PEG calcium method. According to the embodiment of the present invention, breeding can be easily performed. By breeding this strain, large-scale production of koji mold ferrichrome becomes possible.
[0041]
From the above results, it was confirmed that the cloned gene fragment npgO encodes a phosphopantothenate transferase gene. If the obtained phosphopantothenate transferase gene is expressed together with the NRPS genes asb2 and asb4 involved in ferrichrome biosynthesis, the productivity of ferrichrome can be improved. In addition, the ferrichrome thus produced can be provided as a reagent as an iron chelating agent and added to a functional food that can be expected to improve anemia.
Hereinafter, examples of the present invention will be described.
[0042]
Embodiment 1
Cloning of phosphopantothenate transferase gene from Aspergillus genomic sequence contig
Aspergillus A. In order to clone a novel phosphopantothenate transferase gene involved in the post-translational activation of oryzae NRPS, a search was made for Aspergillus genomic sequence contigs. Genes considered to be phosphopantothenate transferase genes were extracted from the Blast search results among the contig sequences contained in the koji mold genome data. Con 1616-JUNK-1 (11199-12552) in Gene Finding Contig 1616 (57457 bp) The protein showed 57% homology with npgA of the C. nidulans phosphopantothenate transferase gene.
[0043]
RT-PCR was performed to obtain this full-length cDNA. Liquid in iron-limited medium (2% glucose, 0.6% asparagine, 0.1% dipotassium hydrogen phosphate, 0.1% magnesium sulfate, 0.04% calcium chloride, pH 6.0) at 30 ° C for 4 days Total RNA was extracted from the cultured Aspergillus oryzae cells using Nippon Gene's ISOGEN. MRNA was extracted from the total RNA by using Takara Shuzo Oligotex-dT30 <Super> mRNA purification kit. Based on 0.5 μg of the obtained mRNA, Takara RNA LA PCR kit (AMV) ver. RT-PCR was attempted according to 1.1. The primers used were P1: SEQ ID NO: 3 (FIG. 4) and P2: SEQ ID NO: 4 (FIG. 5). The reaction conditions were as follows.
[0044]
PCR conditions
● 30 ° C (10 minutes), 42 ° C (60 minutes), 99 ° C (5 minutes), 5 ° C (5 minutes), 96 ° C (5 minutes), 1 cycle
● 96 ° C (20 seconds), 45 ° C (30 seconds), 72 ° C (3 minutes), 30 cycles 72 ° C (7 minutes), 1 cycle
● 72 ° C (7 minutes), 1 cycle
[0045]
The reaction solution was analyzed by agarose gel electrophoresis. As a result, amplification of a fragment of about 1-kb was observed. As a result of determining the base sequence of this amplification product, it was 11199-12552 bp (Con1616-JUNK-1) in Contig1616 (57457 bp) that was GeneFinded in the genomic sequence, but actually 10599-11630 bp Was the coding area. Also, no intron was present. This gene encodes a protein consisting of 343 amino acid residues shown in SEQ ID NO: 1 and does not contain any intron. The coding region is from 1 to 1032 bp of SEQ ID NO: 2. A. The protein showed 57% homology with npgA of the C. nidulans phosphopantothenate transferase gene. The obtained gene was designated as npgO.
[0046]
Embodiment 2
High expression of npgO gene in Aspergillus oryzae
High expression was attempted to determine the function of npgO in Aspergillus. First, various plasmids were constructed. Plasmid A is located in PstI-HindIII of pUC119. Oryzae 4.5-kb niaD, melO promoter 1-kb and npgO 1-kb SalI were sequentially subcloned. Plasmid B is located in BamHI of pUC118 with A. Oryzae 3-kb sC BamHI-BglII, melO promoter 1-kb and asb26-kb BamHI were subcloned in this order. Plasmid C used as a control contains A. coli in PstI-HindIII of pUC119. oryzae 4.5 kb niaD was subcloned and plasmid D was inserted into BamHI of pUC118 by A. Oryzae 3-kb sC BamHI-BglII was subcloned. Transformation was performed using A. cerevisa provided by the National Research Institute of Brewing. Oryzae M-NS4 (niaD, sC) was used as a host. The desired transformant was transformed into M-NS4 strain in the order of plasmid B and plasmid A. As control strain 1, a strain obtained by transforming M-NS4 strain in the order of plasmid D and plasmid A was used. As control strain 2, a strain obtained by transforming M-NS4 strain in the order of plasmid B and plasmid C was used.
[0047]
The obtained transformant was transformed into an iron-limited medium (2% glucose, 0.6% asparagine, 0.1% dipotassium hydrogen phosphate, 0.1% magnesium sulfate, 0.04% calcium chloride, pH 6.0). At 30 ° C. for 7 days. The target transformant secreted 46.1 mg / 1 L-broth ferrichrome outside the cells. On the other hand, the control strain 1 produced 3.5 mg / 1L-broth, and the control strain 2 produced 5.4 mg / 1L-broth. The transformant secreted extra-ferrichrome 8.5 times that of the control strain 2.
[0048]
From the above results, it was confirmed that the cloned gene fragment npgO encodes a phosphopantothenate transferase gene. If the obtained phosphopantothenate transferase gene is expressed together with the NRPS genes asb2 and asb4 involved in ferrichrome biosynthesis, the productivity of ferrichrome can be improved. Further, the ferrichrome thus produced can be provided as a reagent as an iron chelating agent and added to a functional food which can be expected to improve anemia.
[0049]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the phosphopantothenate transferase gene npgO of Aspergillus oryzae could be provided in order to freely control the production of the secondary metabolite containing ferrichrome of Aspergillus oryzae. By overexpressing this gene together with NRPS, the ability to produce nonribosomal peptides biosynthesized by NRPS such as ferrichrome could be greatly improved. By overexpressing this gene in koji molds overexpressing various NRPS genes, it was possible to breed koji molds capable of producing nonribosomal peptides at an industrial use level.
[0050]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the amino acid sequence of phosphopantothenate transferase.
FIG. 2 shows a continuation of the above.
FIG. 3 shows the nucleotide sequence of a gene (npgO gene) encoding phosphopantothenate transferase.
FIG. 4 shows primer P1.
FIG. 5 shows primer P2.
FIG. 6 shows the nucleotide sequence of a peptide synthetase gene (asb2 gene).
FIG. 7 shows a continuation of the above.
FIG. 8 shows a continuation of the above.
FIG. 9 shows a continuation of the above.
FIG. 10 shows a continuation of the above.
FIG. 11 shows the nucleotide sequence of the peptide synthetase gene (asb4 gene).
FIG. 12 shows a continuation of the above.
FIG. 13 shows a continuation of the above.
FIG. 14 shows a continuation of the above.
FIG. 15 shows a continuation of the above.
FIG. 16 shows a continuation of the above.
FIG. 17 shows a continuation of the above.
FIG. 18 shows a continuation of the above.
FIG. 19 shows the nucleotide sequence of the melO promoter.

Claims (11)

The following (a) or (b) protein:
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or (b) consisting of an amino acid sequence in which amino acids are deleted, substituted or added in the amino acid sequence (a), and having a phosphopantothenate transferase activity protein. A DNA comprising the nucleotide sequence represented by SEQ ID NO: 2, which is a gene encoding the protein (a) according to claim 1, or which hybridizes with said gene under stringent conditions, and is a phosphopantothenate transferase DNA of a gene encoding an active protein (b). A recombinant vector containing the gene according to claim 2. A transformant comprising the recombinant vector according to claim 3. A koji mold comprising the recombinant vector according to claim 3. A non-ribosomal peptide produced by the transformant according to claim 4. A transformant obtained by introducing the recombinant vector according to claim 3 into a microorganism that highly expresses any peptide synthetase gene. The transformant according to claim 7, wherein the microorganism is a koji mold. A non-ribosomal peptide produced by the transformant according to claim 7. 10. The non-ribosomal peptide according to claim 9, wherein a peptide synthetase related to ferrichrome biosynthesis of Aspergillus oryzae is used as the non-ribosomal peptide synthase gene, and the non-ribosomal peptide is ferrichrome. By transforming a host with a recombinant vector containing a DNA encoding a nonribosomal peptide synthetase, and a recombinant vector containing a DNA encoding a phosphopantothenate transferase, and culturing the host, A method for producing an active non-ribosomal peptide, comprising simultaneously expressing both of the above DNAs.

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