JP2007189969A - Blue pigment synthesis gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To search a gene specifically existing in a D-cycloserine producing Streptomyces lavendulae, to elucidate its function and to obtain a useful gene and its use. <P>SOLUTION: The protein is (a) a protein composed of an amino acid sequence of a specific sequence or (b) a protein composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) the protein and which has indigoidine synthesis ability. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a blue pigment synthesis gene specifically present in D-cycloserine (DCS) -producing actinomycetes ( Streptomyces lavendulae ) and a method for producing a blue pigment using the same.

  Natural plant pigments contribute to the natural color development of plants, but the main substances include chlorophylls, carotenoid pigments and anthocyanin pigments. Blue coloration is due to anthocyanin dyes, but the coloration mechanism is complex and research is ongoing.

  Moreover, in plants sold at florists, there are surprisingly few varieties of blue flowers close to marine blue. For roses, the creation of blue roses has been a dream of breeders since ancient times, but it has been said that conventional breeding techniques are difficult. In recent years, the creation of blue roses using genetic engineering has been studied. There is nothing to satisfy.

On the other hand, actinomycetes belonging to Streptomyces (Streptomyces), since making a lot of secondary metabolites, including the practical antibiotics, industrially also important microorganisms. In recent years, it has been known that many peptide antibiotics are biosynthesized by non-ribosomal peptide synthetase (NRPS) (Non-Patent Document 1), but specific to actinomycetes It is thought that there are many NRPSs that have not been found yet.
Finking, R. et al., (2004) Annu. Rev. Microbiol. 58, 453-488.

  It is an object of the present invention to provide a useful gene and its use by searching for a gene specifically present in actinomycetes and elucidating its function.

As a result of searching for a gene specifically present in DCS-producing actinomycetes ( S. lavendulae ), the present inventor found a single-module non-ribosomal peptide that is rare in the non-ribosomal peptide synthase (NRPS) family. It was found that a gene encoding a synthase exists, and that the gene functions as a blue pigment indigoidine synthesis gene and is useful for the production of a blue pigment.

That is, the present invention relates to the following (1) to (9).
(1) The following protein (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1,
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) and having the ability to synthesize indigoidine.
(2) A gene encoding the protein according to (1).
(3) Gene consisting of the following DNA (a) or (b):
(A) DNA comprising the base sequence shown in SEQ ID NO: 2,
(B) A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) and encodes a protein having the ability to synthesize indigoidine.
(4) A recombinant vector containing any of the genes according to (2) or (3).
(5) A transformant comprising the recombinant vector according to (4).
(6) The method for producing a protein according to (1), wherein the transformant according to (5) is cultured, and the protein according to (1) is collected from the culture.
(7) A method for producing indigoidine characterized by culturing actinomycetes into which the gene according to (2) or (3) has been introduced and collecting indigoidine from the culture.
(8) culturing a host introduced with the gene according to (2) or (3) and a 4'-phosphopantetheinyl transferase gene, and collecting indigoidine from the culture A method for producing indigoidine.
(9) A method for producing indigoidine, characterized in that the protein according to (1) and 4′-phosphopantetheinyltransferase are allowed to act on L-glutamine.

By using the gene of the present invention, blue pigment indigoidine can be produced using Escherichia coli or the like as a host. In addition, by introducing the gene of the present invention into a plant or cocoon that does not have the pigment-producing ability, a transformant that produces indigoidine can be obtained. Creation becomes possible.
Furthermore, indigoidine having a color close to marine blue can be produced in vitro by using the protein of the present invention.

The gene consisting of the base sequence shown in SEQ ID NO: 2 of the present invention is a so-called genome obtained by subtracting the genome of S. lavendulae JCM4055, which is a non-DCS producing strain, from the genome of DCS-producing S. lavendulae ATCC11924 as shown in the Examples below. It was acquired by using the subtraction (genome subtraction) method (Example 1). The gene encodes a non-ribosomal peptide synthetase (NRPS) present in a DNA fragment specifically present in DCS-producing bacteria. Although the known NRPS has almost a multi-module structure, the gene product obtained here (protein consisting of the amino acid sequence shown in SEQ ID NO: 1) is a single module type rare in the NRPS protein family. (Oxidation) -domain is present (Example 1). In addition, the protein showed 57% identity in amino acid sequence with IndC, which was identified in Erwinia chrysanthemi as essential for the synthesis of the blue pigment indigoidine.

When this NRPS gene was introduced into S. lividans cells, the transformed strain newly produced a blue pigment. As a result of analyzing the chemical properties and structure of the blue dye by visible absorption spectrum, IR spectrum, ¹ H-NMR spectrum, EI-mass spectrum, etc., the structure is as shown below and produced by IndC. Indigoidine (1. Kuhn, R., et al., (1965) Arch. Mikrobiol. 51, 71-84, 2. Mortimer, PS, et al., (1966) Appl. Microbiol. 14, 870- 872) (Example 5).

From this, it can be said that the NRPS gene functions as a blue pigment indigoidine synthesis gene, and this NRPS gene was named bpsA (blue-pigment synthetase A) and the protein encoded by this gene was named BPSA.

The above S. lavendulae ATCC11924 strain is non-indigoidin-producing, but is a substance similar to actinomycete hormone, and with the addition of γ- or δ-nonalactone, an aroma component used as a food additive, Idin production is induced. As shown in Examples described later, transcription of the bpsA gene and two genes downstream thereof are induced by the addition of γ-nonalactone (Example 4).

In addition, when this blue pigment indigoidine derived from actinomycetes is produced in Escherichia coli, in addition to the bpsA gene, BPSA is converted to 4'-phosphopantetheinyl, for example, derived from S. verticillus ATCC15003 Co-expression of a 4′-phosphopantetheinyl transferase gene, such as the 4′-phosphopantetheinyl transferase (PPTase) gene (named svp ), was essential (Example 6). That is, BPSA produced in Escherichia coli functions as an enzyme that produces indigoidine only after 4'-phosphopantetheinylation by post-translational modification.

Furthermore, the substrate of BPSA is L-glutamine (L-Gln), and when a protein (named Svp) made by BPSA and svp gene is added to L-Gln in vitro ( in vitro ), Indigoidine was synthesized.

Hereinafter, the protein and gene of the present invention will be described.
In the present specification, the term “gene” is intended to include not only double-stranded DNA but also single-stranded DNAs constituting the sense strand and the antisense strand. Therefore, unless otherwise specified, the gene of the present invention includes single-stranded DNA (sense strand) including double-stranded DNA and cDNA, and single-stranded DNA (antisense strand) having a sequence complementary to the sense strand. Is included.

  The protein of the present invention is specifically shown as a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (BPSA), but is not particularly limited thereto, and mutants of the protein such as alleles, homologs, natural A variant, which is a protein having 90% or more, preferably 95% or more, more preferably 98% or more, even more preferably 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1, The amino acid sequence shown includes a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having the same ability to synthesize indigoidine as BPSA.

  Here, in the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which one or several amino acids are deleted, substituted or added means an amino acid sequence equivalent to the amino acid sequence of SEQ ID NO: 1, This means an amino acid sequence in which 1 to 10, preferably 1 to 10 amino acids have been deleted, substituted or added, and addition includes addition of 1 to several amino acids at both ends.

  The gene of the present invention is specifically shown as DNA consisting of the base sequence shown in SEQ ID NO: 2 that encodes the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, but is not particularly limited to these, and certain modifications described above DNA comprising a base sequence encoding a protein having Further, it is DNA having at least 90% or more, preferably 95% or more, more preferably 98% or more, and further preferably 99% or more identity with the base sequence shown in SEQ ID NO: 2, and the same as BPSA Hybridizes under stringent conditions to DNA encoding a protein having indigoidine synthesis ability or a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2, and has the same indigoidine synthesis ability as BPSA DNA encoding a protein that performs

  Here, “under stringent conditions” includes, for example, conditions in which hybridization is performed at 65 ° C. for 8 to 16 hours with a probe in a hybridization solution of AlkPhos Direct Labeling and detection system manufactured by Amersham Biosciences.

  As an artificial means for the modification, it can be prepared using a known technique such as site-directed mutagenesis. For example, the mutation can be introduced and prepared using a mutation introduction kit (Transformer Site-Directed Mutagenesis Kit manufactured by BD Bioscience) using site-directed mutagenesis.

  The identity of amino acid sequences or nucleotide sequences is measured using known sequence analysis software, for example, the BLAST program of the National Institute of Genetics, National Institute of Genetics, National Institute of Genetics, National Institute of Genetics (registered in the database). Comparison with the amino acid sequence or DNA base sequence).

The gene of the present invention can be easily produced and obtained by a general genetic engineering technique based on the sequence information of the gene of the present invention disclosed in the present specification (Secondary Chemistry Experiment Course “Gene Research Method I”). , II, III ", edited by the Japanese Biochemical Society (1986)).
Specifically, a genomic DNA library is prepared according to a conventional method from a microorganism in which the gene of the present invention is expressed, for example, DCS productivity S. lavendulae ATCC11924, and an appropriate probe specific to the gene of the present invention is prepared from the library. The desired clone can be selected using In the above, separation of total RNA from DCS productivity S. lavendulae , separation and purification of mRNA, acquisition of genomic DNA, and cloning thereof can all be performed according to conventional methods.

  The method for screening the gene of the present invention from a genomic DNA library is not particularly limited, and can be performed according to various usual methods. Specific examples of the method include a plaque hybridization method using a probe that selectively binds to a target nucleic acid sequence, a colony hybridization method, and a combination thereof.

As the probe used in the above method, DNA chemically synthesized based on information on the base sequence of the gene of the present invention can be generally used. In addition, sense primers and antisense primers set based on the base sequence information of the DNA of the present invention can be used as screening probes.
In obtaining the gene of the present invention, a DNA / RNA amplification method by PCR [Science, 230, 1350 (1985)] can be preferably used. As described above, the amplified DNA / RNA fragment can be isolated and purified by a conventional method. For example, it can be performed by gel electrophoresis.
The gene of the present invention obtained according to the above method can be obtained by a conventional method such as the dideoxy method [Proc. Natl. Acad. Sci., USA., 74, 5463 (1977)], the Maxam-Gilbert method [Methods in Enzymology, 65, 499 ( 1980)] and the like, the nucleic acid sequence can be determined. For convenience, the nucleic acid sequence can be determined using a commercially available sequence kit or the like.

  The protein of the present invention can be prepared according to a general gene recombination technique based on the sequence information of the gene provided by the present invention. That is, a recombinant DNA (expression vector) capable of expressing a gene encoding a desired protein in a host cell is prepared, introduced into the host cell, transformed, the transformant is cultured, and then obtained. It can be recovered from the culture.

The host in the above, for example, E. coli (Esherichia coli BL21 (DE3)) generally ones mentioned widely used, such as, preferably prokaryotic cells, preferably in particular using E. coli. When a prokaryotic cell is used as a host, a promoter and SD (Shin and Dalgarno) are used upstream of the gene so that the gene of the present invention can be expressed in the vector. An expression plasmid to which a nucleic acid sequence and an initiation codon necessary for initiation of protein synthesis (for example, ATG) are added can be suitably used. In general, plasmids derived from Escherichia coli, such as pBR322, pBR325, pUC12, and pUC13, are often used as the vector. However, the present invention is not limited to these, and various known vectors can be used. Examples of commercially available vectors used in the expression system using E. coli include pET21, pET28 (Novagen), pGEX-4T (Amersham Pharmacia Biotech), pQE-30, pQE-60 (QIAGEN), etc. Can be illustrated.

  To transform a host using the vector thus obtained, a protoplast method, a competent cell method, an electroporation method, or the like can be used. The obtained transformant may be cultured under suitable conditions using a medium containing an assimilating carbon source, nitrogen source, metal salt, vitamin and the like. Proteins can be collected and purified from the obtained culture broth by a general method.

Next, a method for producing indigoidine using the gene of the present invention will be described.
(1) Production of indigoidine using transformants Generally, NRPS is known to have no activity unless it is phosphopantetheinylated by 4'-phosphopantetheinyl transferase and converted to holo-form. It has been. Also in the protein of the present invention, indigoidine production requires that the protein is phosphopantetheinylated by 4′-phosphopantetheinyl transferase.
Therefore, as a host, in the case of using Escherichia coli (E. Coli BL21 (DE3) ) general bacteria such as the culturing genes and 4'phosphopantetheinyl sulfotransferase gene introduced host of the invention, the culture Indigoidine can be produced by collecting indigoidine from the product.

Here, the 4′-phosphopantetheinyl transferase gene is not particularly limited. For example, S. verticillus- derived 4′-phosphopantetheinyl transferase gene having a clear biochemical function is used. Examples include the svp gene (SEQ ID NO: 4) encoding, the sfp gene derived from Bacillus subtilis, and the like.
As a technique for co-expression, a known genetic engineering technique can be used as described above. For example, an expression vector (for example, pET / bpsA ) into which the gene of the present invention has been inserted and an expression vector (for example, pSTV / svp ) into which a 4′-phosphopantetheinyltransferase gene has been inserted are simultaneously introduced into the host. And a method of selecting co-expressing cells.

Here, the culture means any of culture supernatant, cultured cells or cultured cells, or disrupted cells of cultured cells or cultured cells.
Moreover, the culture method may be performed according to the usual method used for host cell culture as described above.

Indigoidine can be collected and purified, for example, as follows.
That is, after adding γ-nonalactone to a culture solution of S. lavendulae cultured for about 5 hours to a final concentration of about 30 μg / ml, the cells are further cultured for 2 to 3 hours. The blue pigment secreted into the medium and the cells are separated by low-speed centrifugation, and the resulting centrifugal supernatant is further centrifuged at a high speed to obtain a blue pigment precipitate. The precipitate is washed successively with water and methanol and then dried to obtain a crude purified blue pigment. This is dissolved in an organic solvent such as dimethyl sulfoxide, then passed through a 0.2 μm pore membrane filter to remove the remaining cells, and about 5 times the amount of water is added to the resulting filtrate to precipitate blue pigment. . This solution is centrifuged at a high speed to obtain a blue pigment precipitate, washed as described above, and dried to obtain purified indigoidin.

  Moreover, the transformed actinomycetes having the ability to synthesize indigoidine can be obtained by introducing the gene of the present invention into the actinomycetes having no ability to synthesize indigoidine. Indigoidine can also be obtained by liquid culture of the actinomycetes and collecting indigoidine from the culture. In this case, a blue pigment is produced in the medium by culturing at about 28 ° C. for 2-5 days in a YEME medium containing thiostrepton (50 μg / ml).

(2) Production of indigoidine in vitro Indigoidine can be produced by allowing L-glutamine to act on the protein of the present invention and 4′-phosphopantetheinyltransferase.
Examples of 4′-phosphopantetheinyl transferase include 4′-phosphopantetheinyl transferase (SEQ ID NO: 3) derived from S. verticillus .
This reaction may be carried out at a pH commensurate with the protein of the present invention and 4′-phosphopantetheinyl transferase, and this pH is generally in the range of 6.0 to 8.0, preferably 7.0 to 7.8.
As the reaction solvent, water or a water-organic solvent mixture can be generally used.
The reaction is generally 25-30 ° C., desirably 28-30 ° C., and 2 to 5 minutes, preferably 2-3 minutes after adding L-glutamine as a substrate.
Indigoidine is collected and purified, for example, by centrifugation from the reaction solution at about 15,000 g for about 10 minutes, and the precipitate obtained by removing the supernatant is washed with water and methanol as necessary. And it can carry out by repeating said centrifugation operation.

Materials and methods for implementation (1) Materials (strains and plasmids used and culture media)
As DCS-producing bacteria, S. lavendulae ATCC11924, S. lavendulae ATCC25233, and S. lavendulae FRI-5 were used. S. coelicolor A3 (2), S. lividans 66, S. avermitilis K139, and S. lavendulae JCM4055 were used as DCS non-producing strains.
When preparing protoplasts of Streptomyces genus actinomycetes, they were cultured at 28 ° C. using a YEME medium containing 0.5% glycine. B medium was used when making blue pigments or when preparing total RNA from the bacteria.
As a cloning vector in actinomycetes, pIJ702 (having a melanin pigment production gene and a thiostrepton resistance gene as selection markers, and the target gene is usually inserted into a restriction enzyme site in the melanin pigment synthesis gene. As a result, By making the melanin pigment not produced, colonies whose selection marker is not blackened can be easily selected by inserting the target gene).
Genomic DNA and plasmid DNA were purified according to a conventional method. For expression of the target protein, pET-21a (+) and pET-28a (+) were used as vectors, and Escherichia coli BL21 (DE3) was used as the host at that time. The pSTV28 vector with the pACYC184 origin of replication was used for co-expression. For the purpose of high protein expression, E. coli was cultured in LB medium at 18 ° C. or 23 ° C., and for cloning purposes, it was cultured at 37 ° C. As needed, kanamycin 30 μg / ml, ampicillin 100 μg / ml, and chloramphenicol 34 μg / ml were added to the LB medium.

(2) Southern hybridization A high bond N + membrane was used, and Alphos Direct Labeling and its detection system were used for gene labeling.

Example 1 Cloning of DCS production strain specific gene
In order to clone the biosynthetic genes of secondary metabolites characteristic of DCS producing strain S. lavendulae ATCC11924, first of all, in the DCS producing strain using the genome subtraction method using the non-DCS producing strain S. lavendulae JCM4055 as the control strain Only DNA fragments containing genes that are specifically present were obtained.
That is, PCR-select bacterial genome subtraction kit (Clontech: manufactured by Clontech) was used according to the protocol. However, since the GC content of the actinomycetes gene was high, the hybridization temperature was 75 ° C. The tester strain-specific DNA fragment was subcloned into a pGEM-T vector (Promega) and introduced into E. coli DH5α cells. Of the obtained colonies, about 120 candidate colonies were randomly selected. Each colony was liquid-cultured and a plasmid was extracted from each cell. A plasmid containing a tester-specific DNA fragment was selected using a probe obtained by digesting genomic DNA from a tester and driver strain with the restriction enzyme AluI. The base sequence of the obtained DNA fragment was determined, and the information was analyzed by BLAST and Frame Plot.

In order to clone a 6-kb DNA fragment containing a gene specifically present in S. lavendulae ATCC11924, one of the four clones was selected, and by Southern analysis using this as a probe, S. lavendulae was digested with BamHI. The derived 6-kb DNA fragment was obtained. It was subjected to agarose electrophoresis, extracted, inserted into pUC19 digested with BamHI, and the chimeric plasmid was introduced into E. coli DH5α. The clone obtained by colony hybridization was confirmed to have the gene derived from S. lavendulae ATCC11924 by Southern analysis, and was named A49, and its nucleotide sequence was determined. The DNA base sequence was decoded with an automatic sequencer of ABI Prism 310 and 377, and genetic information was analyzed with GENETYX. Frame analysis was analyzed by Frame Plot, homology was analyzed by FASTX, one of DDBJ programs, and domain structure was analyzed by Domain Database.
As a result, there were four Open Reading Flames (ORF) (Fig. 1). From the homology search, the gene product of orfB was predicted to be NRPS and showed 57% identity with IndC, which was identified in Erwinia chrysanthemi as essential for the synthesis of the blue pigment indigoidine. Furthermore, as a result of Domain Database search, it was predicted that the NRPS encoded by orfB does not have a condensation domain and is a single module type-NRPS with an oxidation domain. This DNA fragment containing orfB was ligated to actinomycetes vector pIJ702 and introduced into S. lividans 66. The transformants from that produced blue dye, named this gene bpsA (b lue p igment s ynthetase -encoding gene A).

Example 2 Expression of bspA in S. lividans 66
By double digesting the A49 clone with BamHI and KpnI, a 4.8-kb fragment containing bspA was obtained and inserted into pIJ702 that was double digested with BglII and KpnI, and the resulting chimeric plasmid was named pIJA49 / bspA did. On the other hand, A49 double digested with BamHI and SphI was a 2.6-kb fragment containing orfC , which was ligated to pIJ702 double digested with BglII and SphI and named the chimeric plasmid pIJA49 / orfC . Each chimeric plasmid was introduced into a protoplast of S. lividanns 66 and regenerated in R5 medium. The obtained colonies were inoculated into YEME or FB medium and cultured at 28 ° C. The results are shown in FIG.

S. lividans 66 introduced with pIJA49 / bpsA produced blue pigment on FB agar medium (A) and in YEME liquid medium (B). When constructing pIJA49 / bpsA , bpsA is inserted in the opposite direction to the promoter of the tyrosinase gene contained in pIJ702, so it is considered that its own promoter exists upstream of the bpsA gene. On the other hand, S. lividans 66 introduced with pIJ49 / orfC produced actinorhodin on FB agar medium (A). In S. lividans 66, orfC is considered to function as a transcriptional activator, judging from the fact that the actinorhodin biosynthesis gene is normally dormant.

Example 3 Presence / absence of bpsA homologues in other actinomycetes In order to confirm by PCR the presence of bpsA homologues in other DCS producing bacteria, two oligonucleotide primers
bpsA -Ox -F1 (5'-GAGGGCCAGTACCGCTACGAGAAC-3 ') (SEQ ID NO: 5) and
bpsA - Ox -R1 (5'-ACGTCGATCTTGCCGTTGGCGGACA-3 ') (SEQ ID NO: 6) was designed.
By using these primers, a 505 bp gene fragment encoding the Ox domain of BPSA can be specifically amplified. For this reason, the presence or absence of the bpsA homolog in other DCS-producing bacteria can be easily examined. PCR experiments were carried out in a 50 μl reaction containing 5 ng of chromosomal DNA, and heat denaturation (98 ° C) for 1 minute in the first cycle, followed by heat denaturation (96 ° C) for 15 seconds, annealing (68 ° C) The amplification reaction was carried out by repeating a cycle of 15 seconds and elongation (72 ° C.) 30 seconds 28 cycles. The amplified DNA fragment was confirmed by electrophoresis in 0.8% agarose gel. The results are shown in FIG. In each lane, chromosomal DNA of the following microorganism was used as a template.

Lane 1:. S lavendulae ATCC11924 (cycloserine-producing bacteria)
Lane 2: S. lavendulae ATCC25233 (cycloserine producing bacterium)
Lane 3: S. garyphalus (CSH5-12) (cycloserine-producing bacterium)
Lane 4: S. coelicolor A3 (2) (non-cycloserine producing bacteria)
Lane 5: S. lividans 66 (non-cycloserine producing bacteria)
Lane 6: S. avermitilis K139 (non-cycloserine producing bacteria)
Lane 7: S. lavendulae JCM4055 (non-cycloserine producing bacteria)
Lane 8: S. lavendulae FRI-5 (cycloserine-producing bacterium)
Lane 9: positive control

Gene amplification was observed when PCR was performed using chromosomal DNA derived from other cycloserine-producing bacteria as a template, and was not observed when chromosomal DNA derived from non-cycloserine-producing bacteria was used as a template. From this, it is considered that bpsA homolog exists in other cycloserine-producing bacteria.

Example 4 RNase Protection Assay
S. lavendulae ATCC11924 was cultured in medium B at 28 ° C. After seeding, γ-nonalactone (γ-NL) was added 5 hours later, and then total RNA was extracted every hour. Total RNA was extracted according to the manual (FIG. 4).

Riboprobe A containing the region between orfA and bpsA was synthesized as follows. First, a 257 bp DNA fragment using two oligonucleotide primers orfA- sense (5'-TCGAGAACGGATCTGTCACCCGGCTCCTGC-3 ') (SEQ ID NO: 7) and bpsA- antisense (5'-GTAGGCGACCGCGATCGCCTCGGGGTGTTC-3') (SEQ ID NO: 8) Was amplified by PCR and ligated to the pGEM-T vector. Thereafter, in vitro transcription was performed using Ambion MAXIscript in vitro transcription kit and [α- ³² P] UTP. bpsA a riboprobe comprising the region between the orfC B (279 bp), and, riboprobes C (305 bp) including the region between the orfC and orfD were synthesized similarly to the riboprobe A. However, for the synthesis of riboprobe B, oligonucleotide primers bpsA- sense (5'-AGATTGACCGCGTTCTGACATACCTC-3 ') (SEQ ID NO: 9) and orfC- antisense (5'-CTTCTCGCCCCACAGCTCCCGGATGAGG-3') (SEQ ID NO: 10) are used. For the synthesis of Riboprobe C, oligonucleotide primers orfC- sense (5'-AGCGGTTCGTGGTGGGCGCGGCCGTCTGAG-3 ') (SEQ ID NO: 11) and orfD- antisense (5'-CGTGCACCAGACCGGTGGTGATCAGGGTCTC-3') (SEQ ID NO: 12) are used. Using.

The extracted RNA sample (5 μg) was hybridized with a radioisotope-labeled riboprobe (4 × 10 ⁴ cpm) overnight at 42 ° C. using an Ambion RPA III RNase protection assay kit. Then treated with a mixture of RNase A and RNase T _1, after precipitation, and electrophoresed in a 5% polyacrylamide containing 8 M urea. As the size marker, φX174 DNA was cleaved with HinfI, dephosphorylated, and labeled with 5 ′ end using T4 polynucleotide kinase (Toyobo) and [γ- ³² P] ATP. Radioactivity in the gel was detected with an imaging plate manufactured by Fuji Film Co., Ltd. and BAS-2000. The results are shown in FIG.
(A): RNA protected by probe A
(B): RNA protected by probe B
(C): RNA protected by probe C
(D): Total RNA used in RNase protection assay detected by 1% formamide agarose gel electrophoresis. This shows that the total RNA amount used is equal.

As a result of the RNase protection assay, it was found that bpsA , orfC and orfD were not expressed in the absence of g-NL addition. On the other hand, these genes were expressed one hour after induction with g-NL and then attenuated. Therefore, bpsA , orfC and orfD were found to be transiently induced by g-NL.

Example 5 Blue Pigment Detection, Purification, and Characterization
Blue pigment production after a predetermined time by S. lavendulae ATCC11924 was detected by adding an equal amount of water to the supernatant obtained by centrifuging the culture solution and then measuring the absorbance at 590 nm. The blue pigment was purified as follows. S. lavendulae ATCC11924 was cultured in medium B (2 L), and γ-NL was added as an inducer 5 hours after the start of the culture. Two hours later, the culture was centrifuged at 4,400 × g for 10 minutes to remove the cells. The obtained supernatant was further centrifuged at 30,000 × g for 30 minutes to obtain a blue pigment precipitate. The precipitate was washed twice with water and methanol and dried in vacuo. Dimethyl sulfoxide was added to the precipitate and dissolved by sonication, and then the fine precipitate was removed using a DISMIC-13JP filter manufactured by Advantech. Thereafter, 5 times the amount of water was added, and the mixture was centrifuged again at 30,000 × g for 30 minutes to obtain a blue pigment precipitate. This precipitate was washed 10 times with water and twice with methanol, and dried in vacuo to obtain a purified blue pigment (11 mg).

For sample preparation for absorbance analysis, the purified blue dye is suspended in various solvents such as dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), tetrahydrofuran (THF), and pyridine and dissolved to the maximum by sonication. I let you. Thereafter, undissolved particles were removed by centrifugation at 15,000 × g for 5 minutes. The visible absorption spectrum was analyzed with a V-550 spectrophotometer manufactured by JASCO Corporation.
The EI mass spectrum was analyzed with a JMS-SX102A spectrometer manufactured by JEOL using a blue dye dissolved in THF. The MALDI-TOF analysis was performed using Voyager ^™ RP-3 manufactured by Applied Biosystems using a blue dye dissolved in dimethyl sulfoxide.

¹ H-NMR analysis was performed with a JEOL JNM-LA500 spectrometer. Tetramethylsilane was used as an internal standard, and deuterated DMSO was used as a solvent.
The IR spectrum was measured by the potassium iodide method, and wave numbers 3443, 3328, 3288, 3196, 3064, 2959, 2924, 2869, 2839, 2364, 1688, 1639, 1596, 1579, 1454, 1371, 1323, 1250, Absorption was observed at frequencies of 1118, 1060, 1037, 956, 885, 824, 779, 752, 718, 689, 661, 634.

The results are summarized in FIG.
In the EI-mass analysis, a peak was present at m / z = 248.0, which agreed with the value calculated from the molecular formula of the structure of the presumed blue pigment (indigoidin).
^{In 1} H-NMR spectrum analysis, signals of 11.30 ppm (s; NH), 8.18 ppm (s; CH), and 6.46 (s; NH ₂ ) were obtained.
From the above, the blue pigment was determined to be indigoidine.

Example 6 Coexpression of svp gene and bpsA gene encoding phosphopantetheinyl transferase derived from S. verticillus in Escherichia coli
The bpsA gene is 5′- CATATG ACTCTTCAGGAGACCAGCGTGCTC-3 ′ (SEQ ID NO: 13) (underlined indicates NdeI site), and 5′- AAGCTT CTCGCCGAGCAGGTAG CGGATGTG-3 ′ (SEQ ID NO: 14) The underlined portion indicates a HindIII site) and was amplified by PCR using KOD-plus polymerase manufactured by Toyobo. The amplified gene was extracted and purified from an agarose gel and ligated to the SmaI site of pUC19 to obtain a chimeric plasmid pUC / bpsA . After confirming the nucleotide sequence of the inserted bpsA gene, pUC / bpsA was double-digested with NdeI and HindIII and incorporated into the same site of pET-28a (+) to obtain a chimeric plasmid pET / bpsA . BPSA expressed from this vector has polyhistidine tags at the N and C terminus.

The svp gene encoding phosphopantetheinyltransferase (PPTase) derived from S. verticillus was 5′- CATATG ATCGCCGCCCCCCTGCCCTCCTG-3 ′ (SEQ ID NO: 15) (SEQ ID NO: 15) (underlined indicates NdeI site) and 5'- CTCGAG CGGGACGGCGGTCCGGTCGTCCGC-3 '(SEQ ID NO: 16) (underlined indicates the XhoI site) was used as a template DNA and amplified using the S. verticillus chromosome. This amplified fragment was ligated to the SmaI site of pUC19 to obtain a chimeric plasmid pUC / svp . After confirming the nucleotide sequence of the inserted svp gene, pUC / svp was double-digested with NdeI and XhoI and incorporated into the same site of pET-21a (+) to obtain a chimeric plasmid pET / svp . Svp expressed from this vector has a polyhistidine tag at the C-terminus. By cutting out a 2.2 kb DNA fragment containing the vector-derived T7 promoter and the svp gene linked downstream from pET / svp by double digestion with SphI and PvuI, and ligating it to the same site of pSTV28. A chimeric plasmid pSTV / svp was obtained.
pET / bpsA and pSTV / svp were simultaneously introduced into E. coli BL21 (DE3) strain to obtain a BPSA and Svp co-expression strain. Considering the solubility of both proteins when expressed in large quantities, expression experiments were performed at 18 ° C. The results of culturing at 18 ° C. for 48 hours are shown in FIG.

Escherichia coli into which pET / bpsA and pSTV / svp were simultaneously introduced produced blue pigment, whereas Escherichia coli into which only pET / bpsA was introduced did not produce blue pigment. From this, it is considered that the own PPTase possessed by Escherichia coli cannot convert BPSA to a holo type, and Svp converts BPSA to a holo type, and holo type BPSA catalyzes blue pigment synthesis.

Example 7 Large-scale expression and purification of BPSA and BPSAΔTE in E. coli
In order to produce BPSAΔTE (amino acid number 1-1014) lacking the TE-domain, a bpsA ΔTE expression vector pET / bpsA ΔTE was constructed using pET-28a (+) as in the case of full-length bpsA . The PCR primer used was the 5 ′ phosphorylated sense primer used for bpsA amplification and the 5 ′ phosphorylated antisense primer 5′- AAGCTT CTGGGCGACCTCGCGCTCCAG-3 ′ (SEQ ID NO: 17) (underlined indicates HindIII site). BPSAΔTE protein was produced as an N-terminal and C-terminal histidine tagged protein.

E. coli BL21 (DE3) strain carrying pET / bpsA or pET / bpsAΔTE was cultured at 18 ° C. in 2.5 liters of LB medium containing kanamycin. In view of the solubility of the expressed protein, the culture was performed for 30 hours without induction by isopropyl-β-D-galactopyranoside (IPTG). The cells were collected by centrifugation and suspended in binding buffer [20 mM Tris-HCl (pH 7.9), 500 mM sodium chloride, 5 mM imidazole]. All processes after suspension were performed at 4 ° C. Cells were disrupted by ultrasonic waves, and cell debris was removed by centrifugation at 27,000 × g for 20 minutes. The resulting supernatant was added to a His-Bind resin column (1 × 10 cm, Novagen) previously equilibrated with a binding buffer, and the column was washed with Washing Buffer I [20 mM Tris-HCl (pH 7.9). ), 500 mM sodium chloride, 30 mM imidazole]. Elution was performed with a linear imidazole concentration gradient from 30 mM to 500 mM. Fractions containing BPSA or BPSAΔTE were collected and dialyzed against a binding buffer containing 1 mM ethylenediaminetetraacetic acid (twice), followed by dialysis against the binding buffer (twice). The dialyzed solution was again added to the His-Bind resin column, and the column was washed with Wash Buffer II [20 mM Tris-HCl (pH 7.9), 500 mM sodium chloride, 60 mM imidazole]. Elution was performed with a linear imidazole concentration gradient from 60 mM to 1000 mM. At this stage, BPSA and BPSAΔTE were purified as almost a single protein as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (FIG. 7). EpoB with Ox-domain (Chen, HW, O'Conner, S., Cane, DE, and Walsh, CT (2001). Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit. Chem. Biol. 8 , 899-912.), Purified BPSA and BPSAΔTE had a yellow color because they contained flavin mononucleotide. The purified protein was stored at −20 ° C. in the presence of 50% glycerol until use.

Example 8 Large-scale expression and purification of Svp in E. coli
E. coli BL21 (DE3) strain carrying pET / svp was cultured at 23 ° C. in 3 liters of LB medium containing ampicillin. Considering the solubility of Svp, the culture was performed for 24 hours without induction by IPTG. Cells were collected by centrifugation. After suspending the cells in binding buffer, all steps were performed at 4 ° C. Cells were disrupted by ultrasonic waves, and cell debris was removed by centrifugation at 27,000 × g for 20 minutes. The resulting supernatant was added to a His-Bind resin column (1 × 10 cm) previously equilibrated with a binding buffer. After washing the column with wash buffer II, elution was performed with a linear gradient of imidazole from 60 mM to 500 mM. Through these processes, Svp was purified almost as a single protein (FIG. 7). The purified Svp protein was stored at −20 ° C. in the presence of 50% glycerol until use.

Example 9 Synthesis of blue pigment in a test tube
660 nM BPSA, 810 nM Svp, 0.1 mM coenzyme A and 1 mM chloride prepared using 50 mM sodium phosphate buffer (pH 7.8) for 4'-phosphopantetheinylation of BPSA using Svp A solution containing magnesium (1.4 ml) was incubated at 30 ° C. for 10 minutes. Blue dye synthesis was initiated by adding 200 μl of 10 mM adenosine triphosphate solution (final concentration 1 mM) and 400 μl of 5 mM L-amino acid solution (final concentration 1 mM). The synthesis of the blue pigment in the test tube was monitored by measuring the absorption at 590 nm (FIG. 8). The molecular weight of the synthesized blue dye was analyzed by MALDI-TOF as described above.

  When BPSA 4'-phosphopantetheinylated with Svp was used, blue pigment synthesis was observed only when L-glutamine was used as a substrate. In addition, when BPSA that was not 4′-phosphopantetheinylated with Svp was used, synthesis of blue pigment was not observed even when L-glutamine was used as a substrate. This suggests that 4'-phosphopantetheinylated holo-type BPSA synthesizes a blue pigment using L-glutamine as the only substrate.

Example 10 Adenosine Triphosphate / Pyrophosphate-Exchange Assay The activity of the adenylation domain of BPSA was measured using an adenosine triphosphate / pyrophosphate exchange reaction.
The amino acid-dependent adenosine triphosphate / pyrophosphate-exchange assay is basically a previously described method (Du, L., Chen, M., Zhang, Y., and Shen, B. (2003). BimIII and BlmIV nonribosomal peptide synthetase-catalyzed biosynthesis of the bleomycin bithiazole moiety involving both in cis and in trans aminoacylation. Biochemistry 42, 9731-9740.) The reaction solution (100 μl) consists of 300 nM histidine-tagged protein, 5 mM adenosine triphosphate, 1.72 μM [ ³² P] pyrophosphate (1 μCi, 60 Ci / mmol; PerkinElmer Life Siences), 1 mM pyrophosphate, 1 Contains mM magnesium chloride, 0.1 mM ethylenediaminetetraacetic acid, 0.5 mM amino acid and 20 mM Tris-HCl (pH 7.8). After incubating the solution at 30 ° C. for 20 minutes, the reaction was stopped by adding 0.9 ml of a solution containing 0.25 M perchloric acid, 1.6% activated carbon and 0.1 M tetrasodium pyrophosphate. Activated carbon was collected by centrifugation, washed twice with 1 ml of water, and suspended in 0.5 ml of water. After liquid scintillation (ACSII, Amersham Biosciences) was added, the radioactivity bound to the activated carbon was measured with a liquid scintillation counter. A reaction solution containing no amino acid was used as a control. When determining the kinetic parameters of BPSA and BPSAΔTE, the reaction was run for 6 minutes with varying concentrations of L-glutamine. Using the specific activity of [ ³² P] pyrophosphate, the radioactivity bound to the activated carbon was converted to a reaction rate. The results are shown in FIG. The figure shows the activity against L-glutamine as 100% and the activity against other amino acids as its relative activity.
From FIG. 9, it is considered that the substrate of BPSA is L-glutamine.

Example 11 Pyrophosphate Release Assay
The activity of the BPSA adenylation domain against L-glutamine was measured by a pyrophosphate release assay.
The rate of pyrophosphate release by the A-domain was measured by a continuous spectroscopic method using MesG, which is a guanosine analog, using the EnzCheck Pyrophosphatase Assay Kit (Molecular Probe). The reaction solution is 20 mM Tris-HCl (pH 7.8), 1 mM magnesium chloride, 0.1 mM ethylenediaminetetraacetic acid, 0.2 mM MesG, 0.2 U purine nucleoside phosphorylase, 0.2 U inorganic pyrophosphatase, 5 mM adenosine triphosphate, 3 mM Contains L-glutamine, 100 nM Svp, 0.5 mM coenzyme A and 100 nM BPSA. When converting BPSA from apo form to holo form, 100 nM BPSA was previously incubated with 100 nM Svp at 30 ° C. for 30 minutes in a solution not containing L-glutamine and adenosine triphosphate. In this case, the reaction was started by adding 3 mM L-glutamine and 5 mM adenosine triphosphate, and then absorption at 360 nm was measured every 10 seconds for 6 minutes. The slope from 0 to 100 seconds in the resulting spectrum correlated with a standard curve generated using standard pyrophosphate (FIG. 10).
In FIG. 10, diamonds indicate data obtained using a holoenzyme, and squares indicate data obtained using an apoenzyme. By using the gradient from 0 to 100 seconds, the rotation speed of holo BPSA was calculated to be 107 / min, and the rotation speed of apo BPSA was calculated to be 76 / min.

  The rotational speed increased slightly by converting the apo type into the holo type. This is considered to be because L-glutamine activated by the A domain quickly migrates to the T domain in holo-type BPSA.

Example 12 Aminoacylation assay of [ ¹⁴ C] L-glutamine to BPSAΔTE T-domain Holo BPSA and holo BPSAΔTE in which T-domain is phosphopantetheinylated by purified Svp, or each apoenzyme not activated by Svp Was used to examine the binding of ¹⁴ C-labeled L-glutamine to the T-domain.
[ ¹⁴ C] L-glutamine binding to the T-domain of BPSAΔTE was examined by autoradiography. 100 μl of the reaction solution consisted of 20 mM Tris-HCl (pH 7.8), 1 mM magnesium chloride, 0.1 mM ethylenediaminetetraacetic acid, 3 mM adenosine triphosphate, 10 mM [ ¹⁴ C] L-glutamine (0.2 μCi, 210 mCi / mmol; Moravek Biochemicals), 300 nM Svp, 0.5 mM coenzyme A and 300 nM BPSA or BPSAΔTE but not [ ¹⁴ C] L-glutamine in advance for 30 minutes at 30 ° C As a result, the T-domains of these proteins were phosphopantetheinylated before [ ¹⁴ C] L-glutamine was added to initiate the reaction. After adding [ ¹⁴ C] L-glutamine, after various times, the reaction was stopped by adding 0.8 ml of 10% ice-cold trichloroacetic acid containing 2% bovine serum albumin. The precipitated protein was collected, washed with 10% ice-cold trichloroacetic acid and acetone, and then its radioactivity was measured. When autoradiography was performed, the reaction was stopped 15 minutes after adding [ ¹⁴ C] L-glutamine, and the protein precipitate was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the dried gel was exposed to an imaging plate and visualized using a BAS-2000 apparatus (FIG. 11).

  When full-length BPSA was used, L-glutamine was not bound on BPSA, regardless of the presence or absence of 4'-phosphopantethenylation by Svp. On the other hand, when BPSAΔTE was used, binding of L-glutamine was observed only to BPSAΔTE 4′-phosphopantetheinylated with Svp. This suggests that the TE domain at the C-terminus of full-length BPSA catalyzes the detachment of L-glutamine (or its derivatives) bound to the T domain, and L-glutamine activated by the A domain. Is thought to bind to the 4'-phosphopantetheinylated T domain.

FIG. 2 is a schematic diagram showing the structure of orf present in the cloned DNA fragment of about 6-kb and the domain structure of BPSA. Open arrow: incomplete gene, black arrow: complete gene It is the photograph which showed the expression of bspA in S. lividans 66. (A) Each transformant that grows on FB agar medium containing thiostrepton (50 μg / ml), (B) Each transformant that grows on YEME liquid medium containing thiostrepton (50 μg / ml) body It is the figure which showed the agarose electrophoresis pattern of each gene amplified by PCR. Lane 1: S. lavendulae ATCC11924, Lane 2: S. lavendulae ATCC25233, Lane 3: S. garyphalus (CSH5-12), Lane 4: S. coelicolor A3 (2), Lane 5: S. lividans 66, Lane 6: S. avermitilis K139, lane 7: S. lavendulae JCM4055, lane 8: S. lavendulae FRI-5, lane 9: positive control It is a figure which shows the result of an RNase protection assay. (A): RNA protected by probe A, (B): RNA protected by probe B, (C): RNA protected by probe C, (D): Total RNA used in RNase protection assay 1 Detected by% formamide agarose gel electrophoresis. NL (-): γ-NL not added, NL (+): γ-NL added It is the figure which showed the chemical property and structure of the blue pigment synthesize | combined from S. lavendulae . (A): Structure. (B) EI-mass analysis, (C) ¹ H-NMR spectrum A photograph showing the production of blue pigment in E. coli . (1) E. coli BL21 (DE3) strain coexpressed with bpsA and svp , (2) E. coli BL21 (DE3) strain expressing only bpsA , (3) E. coli BL21 carrying the expression vector pET-28a (+) ( DE3) shares It is the figure which showed the expression and refinement | purification protein sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern. Lane 1: purified BPSA, lane 2: purified BPSAΔTE, lane 3: Svp, M: standard protein, left number: molecular weight. Is a graph showing the production of blue pigment in an In vitro. Solid line: When BPSA and Svp are used, Broken line: When only BPSA is used It is the figure which showed the activity of the adenylation domain of BPSA. It is the figure which showed the activity with respect to L-glutamine of a BPSA adenylation domain. Diamond: holoenzyme, square: apoenzyme FIG. 6 shows the ability of ¹⁴ C-labeled L-glutamine to bind to the T-domain. Left: After dodecyl sulfate-polyacrylamide gel electrophoresis, stained with Coomassie, Right: After electrophoresis, ¹⁴ C-labeled L-glutamine detected by autoradiography

Claims (10)

The following protein (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1,
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a), and having the ability to synthesize indigoidine.   A gene encoding the protein according to claim 1. A gene comprising the following DNA (a) or (b):
(A) DNA comprising the base sequence shown in SEQ ID NO: 2,
(B) A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) and encodes a protein having the ability to synthesize indigoidine.   The gene according to claim 2 or 3, which is a single-module non-ribosomal peptide synthase gene.   A recombinant vector containing the gene according to claim 2 or 3.   A transformant comprising the recombinant vector according to claim 5.   The method for producing a protein according to claim 1, wherein the transformant according to claim 6 is cultured, and the protein according to claim 1 is collected from the culture.   A method for producing indigoidine characterized by culturing actinomycetes into which the gene according to claim 2 or 3 has been introduced and collecting indigoidine from the culture.   A method for producing indigoidine, comprising culturing a host into which the gene according to claim 2 or 3 and a 4′-phosphopantetheinyltransferase gene have been introduced, and collecting indigoidine from the culture.   A method for producing indigoidine, which comprises reacting the protein according to claim 1 and 4'-phosphopantetheinyl transferase with L-glutamine.