KR100834257B1 - Actinomadura hibisca mutant with distrupted o- methyltransferase gene of pradimicin and preparing of demethylpradimicin using the same - Google Patents

Abstract

A mutant of Actinomadura hibisca is provided to produce only demethylpradimicin as a new antifungal antibiotic of pradimicin having a new structure by ruining an O-methyltransferase gene of pradimicin associated with methylation. A method for producing an Actinomadura hibisca mutant BC1(KACC 95057P) producing demethylpradimicin represented by the formula(2) comprises the steps of: (1) preparing a genomic DNA library of Actinomadura hibisca by using a cosmid vector, and obtaining a cosmid clone containing a pradimicin biosynthesis gene group; (2) inserting an antibiotic-resistant gene into an O-methyltransferase gene DNA fragment in the pradimicin A biosynthesis gene group to prepare a knock-out cassette; (3) integrating the knock-out cassette into the DNA fragment containing the pradimicin biosynthesis gene group to prepare a knock-out vector; and (4) introducing the knock-out vector into Actinomadura hibisca through conjugation, and transforming Actinomadura hibisca through homologous recombination to knock-out the O-methyltransferase gene.

Description

Actinomadura hibisca mutant with distrupted O-methyltransferase gene of pradimicin and preparing of demethylpradimicin using the gene of O-methyltransferase, a biosynthetic enzyme of pradimycin same}

FIG. 1 shows genes involved in glycation of a variety of tailinging genes involved in the production of a structure of pradimycin and glycosylation of the pradimycin aglycon in a cosmid clone including a pradimycin biosynthesis gene. It is a figure which shows the cleavage map of the DNA fragment containing,

FIG. 2 is a diagram showing a DNA sequence of O -methyltransferase represented by SEQ ID NO: 1, one of the pradimycin biosynthetic genes, and a portion of the nucleotide sequence used to construct a knockout cassette,

Figure 3 is a schematic diagram showing a process of removing the gene of O -methyltransferase in the pradimycin biosynthetic gene using the DNA fragment of Figure 1,

FIG. 4 is a graph showing the results of analyzing the pradimycin compound in the culture extract of the actinomadura hibiscus mutant strain BC1 by high performance liquid chromatography (hereinafter abbreviated as HPLC).

5 is a photograph showing the results of assaying the antimicrobial activity of dimethyl pramycin produced by actinomydura hibisca mutant strain BC1 of the present invention on phytopathogenic fungi in a thin-film chromatography plate, on the left side of fungal spores The picture shows the low support of antimicrobial activity by covering and the right side shows the development of thin-film chromatogram before covering the spores of the fungus.

The present invention relates to an Actinomadura hibisca mutant strain in which the Pradimicin methyltransferases gene in the biosynthetic genes of pradimycin is destroyed and its dimethylpradimycin. More specifically, in the structure of pradimycin, an excellent antifungal active substance, the gene of O-methyltransferase, an enzyme involved in methylation, is destroyed to produce demethylated pradimycin. It relates to Dura hibisca mutant strain and dimethylpradimycin which is a product having antibacterial activity.

Pradimicin is a new antifungal antibiotic of the dihydrobenzo [a] naphthacenequinone family produced by the family belonging to the genus Actinomadura (Tsunakawa). et al., 1989. J. Org. Chem. 54: 2532-2536; Oki et al., 1988. J. Antibiotics. 41 (11) 1701-1704).

This antibiotic (Formula 1) is a benzonaphthacinquinone compound containing D-alanine and disaccharide in the molecule. This antibiotic has an excellent therapeutic effect on systemic infection of the fungus in rats, and T-cells prevent infection by human immunodeficiency virus and inhibit the formation of syncytium by the virus (Hoshino). , et al., 1989. J. Antibiotics 42: 344-346.

화학식 1

Formula 1

Pradimycin is also used in vivo and in against Candida , Cryptococcus , Aspergillus , and many other fungal and phytopathogenic fungi. in vitro It is a compound exhibiting excellent activity in a state.

Until now, only 10 genes involved in biosynthesis of the polyketide moiety are known (Dairi et al, 1997, Biosci. Biotech. Biochem. 61 (9): 1445-1453), and the remaining biosynthesis genes. Genes, particularly those involved in the tailoring phase and those involved in glycosylation, are unknown.

On the other hand, a variety of pramycin has been developed and published, Pradimycin A, B, and C are described in European Patent Application No. 277,621, Pradimycin D, E and their respective deskysilyl derivatives 6 1988 US 203,776, filed May 7, is filed. There is a continuing need for the development of new derivatives of pradimycin with excellent antimicrobial activity.

The present invention has been made to solve the problems of the prior art and the above object, an object of the present invention is to provide a molecular biological method for genes related to the biosynthesis of pradimycin to make a new derivative of pradimycin, an excellent antifungal active substance. And the new Actinomadura hibisca (Acinomadura) produced by destroying the gene of O -methyltransferases involved in methylation in the structure of pradimycin hibisca ) allow the mutant strain to produce a new pradimycin derivative, dimethylpradimycin. An object of the present invention is to provide a new antifungal substance by generating a pradimycin derivative having a new structure having antibacterial activity through the manipulation of such biosynthetic genes.

In order to achieve the above object, the present inventors have prepared a genomic DNA library of the actinomadura hibisca strain of a gene involved in the tailoring (tailoring) of the biosynthetic gene of the pradimycin compound in order to develop a new pradimycin derivative The present invention was completed by developing a mutant strain that produces a pradimycin derivative having a new structure by manipulating a gene involved in methylation among these genes and confirming the antimicrobial activity of the substance.

Thus, the present invention is O which is involved in methylation in the structure of the plastic-di azithromycin - an antibiotic resistance gene in the middle of the methyl transferase gene fragment-gene (SEQ ID NO: 1) and the plastic-di erythromycin A O of the methyl-transferase (Pradimicin methyltransferases) Provide the inserted DNA fragment.

Preferably, the antibiotic resistance gene is an apramycin antibiotic resistance gene.

The present invention also provides a knockout cassette comprising the DNA fragment.

The present invention provides a genetically modified strain wherein the DNA fragment is transformed to destroy the pradimycin A 0 -methyltransferase gene. Preferably, the mutant strain is Actinomadura hibiscus mutant strain BC1 (Microorganism Accession No .: KACC 95057P), and the mutant strain BC1 produces is dimethylpradimycin (Formula 2), which is a novel pradimycin derivative.

화학식 2

Formula 2

Furthermore, the present invention provides a method for producing the genetically modified strain and a method for producing novel dimethylpradimycin from the mutant strain, and a novel antifungal active substance produced therefrom.

Hereinafter, the configuration of the present invention with reference to the accompanying drawings in detail as follows.

The present invention (see SEQ ID NOS. 1, 2) of the O- methyl transferase gene (Pradimicin methyltransferases) which is involved in methylation in the structure of the plastic and the plastic azithromycin di di erythromycin A O - methyl transferase antibiotic in the middle of the gene segments Provided is a DNA fragment into which a resistance gene has been inserted. In the above, a PCR primer including 36 nucleotide sequences of the start codon region and the end codon region of the pradimycin A O -methyltransferase gene was prepared to amplify the apramycin antibiotic resistance gene and the OriT gene simultaneously. DNA fragments comprising part of the gene were isolated (see FIG. 2). The DNA fragment serves as a knockout cassette. A knockout cassette containing the DNA fragment was used to remove the O-methyltransferase and to insert a gene involved in conjugation of the apramycin antibiotic resistance gene with Actinomycetes and Escherichia coli. PCR targeting technology (Gust et al . 2003, Proc. Nat. Acad. Sci. 100 (4): 1541-1546), but various techniques can be applied.

The present invention provides a cosmid vector having a group of pramycin biosynthetic genes. The cosmid vector represented by nucleotide sequence 2 is composed of 33 ORFs (open reading frames) in total of 37.1 kb in total sequence (see FIG. 1) . Within these gene groups, various genes, such as O -methyltransferase, amidotransferase, cyclase, and the like, which are involved in glycosylation, have been found. The cosmid vector is named pBC3-3 ', and the pBC3-3 cosmid clone was strain deposited at the Agricultural Biotechnology Center in the Institute of Agricultural Biotechnology (Microbial Accession No .: KACC 95056P).

In addition, the present invention using the cosmid vector (see Fig. 3) having a group of pradimycin biosynthetic genes transformed with a knockout cassette containing the DNA fragment (see Fig. 3) is the depletion of the Pradimycin A O -methyltransferase gene Provide genetically modified strains. The present inventors transformed the knockout vector prepared above to actinumana hibisca, which is a pradimycin producing strain, to destroy the A O -methyltransferase gene of actinumana hibisca, thereby generating dimethylpradimycin. A strain was prepared and named as Actinumana hibisca BC1 strain, and the strain was deposited in the Agricultural Biological Resources Center of the Institute of Agricultural Biotechnology (Microbial Accession No .: KACC 95057P).

Using a knockout vector of the present invention liquid Martino wife when transformed Hibi Scar knockout vector set within the O-methyl transferase some nucleotide sequence and chromosome I O of the gene - the same methyl transferase gene sequences to between sequences Homologous recombination is induced. The homologous recombination produces a mutant strain in which the O -methyltransferase gene is destroyed.

The inventors confirmed that the mutant strain produced dimethylpradimycin using HPLC (see FIG. 4), and also confirmed the antibacterial activity of the dimethylpradimycin using thin layer chromatography (see FIG. 5).

The method for producing a genetically modified strain wherein the pradimycin A O -methyltransferase gene is destroyed according to the present invention and a method for producing a new dimethylpradimycin from the modified strain,

1) preparing a genomic DNA library of Actinu hibiscus hibisca using a cosmid vector to obtain a cosmid clone containing a group of pradimycin biosynthetic genes (step 1);

2) preparing an knock-out cassette by inserting an antibiotic resistance gene into a DNA fragment with an O -methyltransferase gene in a group of pradimycin A biosynthesis genes (step 2);

3) integrating the knockout cassette to a DNA fragment comprising the pradimycin biosynthetic gene group (step 3);

4) The transformed DNA fragments were introduced into the Actinomadura hibisca through conjugation, and then transformed into the Actinomadura hibisca strain through homologous recombination, thereby producing O -methyl. Removing the transferase gene (step 4);

5) selecting a variant strain in which the O -methyltransferase gene is destroyed using an antibiotic (step 5);

6) culturing the selected strains O -methyltransferase is cultivated in ISP 2 liquid medium and is separated and purified from the culture filtrate to produce dimethylpradimycin (step 6).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

< Example  1> Pradimycin  Detection and Identification of Biosynthetic Genes

Referring to step 1 specifically, a chromosomal library (Chromosomal DNA library) was prepared from the actinmadura hibiscus ATCC 53557, a pradimycin producing strain, and this library was constructed using a cosmid vector pCC2FOS, and then Clones containing polyketide synthase (PKS) genes using oligonucleotides containing ketosynthase (Dairi et al., Biosci. Biotech. Biochem. 1997, 61 (9): 1445 ~ 1453) Was selected using the PCR technique.

In order to confirm whether the selected cosmid clone contains a group of pradimycin biosynthetic genes, the ketocinase gene was knocked out through the method of Examples 2 and 3 below. As a result, it was confirmed by HPLC that the red pradimycin produced in Actinomadura hibisca is no longer produced.

The cosmid vector containing the pradimycin biosynthetic gene group was named 'pBC3-3'. The clone was analyzed by Genotech Co., Ltd. using modified shotgun whole genome sequencing (SEQ ID NO: 2).

Next, the entire base sequence of 37.1 kb was subdivided into a total of 33 open reading frames (ORFs) using the BLAST function of the National Center for Biotechnology Information (NCBI) (FIG. 1) . Within these gene groups, various genes, such as O -methyltransferase, amidotransferase, cyclase and the like, and genes involved in glycosylation have been found. The pBC3-3 cosmid clone was deposited in the Agricultural Biological Resource Center in the Agricultural Biotechnology Research Institute (Accession No .: KACC 95056P).

< Example  2> Pradimycin  One of the biosynthetic genes O Methyltransferase ( O -methyltransferase) gene Knock-out cassette  Production and In E. coli  Sangyo Recombination ( homologous recombination  Removal method of the biosynthetic gene by

An antibiotic resistance gene cassette was prepared in order to remove only O -methyltransferase from the cosmid clone (pBC3-3) obtained in Example 1. PCR targeting technology using OriT gene, a gene involved in the conjugation of actinomycetes and Escherichia coli, to knock out the O -methyltransferase gene (FIG. 2) (Gust et al . 2003, Proc. Nat. Acad. Sci. 100 (4): 1541-1546) was used.

To this end, the sequences of the start codon and the end codon of the O -methyltransferase gene, respectively, having the apramycin antibiotic resistance gene and the OriT gene in the outer 5 'and 3' ends of the gene, respectively A knock-out cassette containing 36 nucleotides was prepared and used.

To remove O -methyltransferase present in the group of pradimycin biosynthetic genes encoded in the selected cosmid clones (pBC3-3), the pBC3-3 vector was first subjected to electroporation to strain BW25113. Transformants were selected by culturing in LB (Luria-Bertani) agar medium containing 50 μg / ml of ampicillin and 50 μg / ml of chloramphenicol.

Next, Escherichia coli strain BW25113 containing pBC3-3 was grown into a competent cell, and the knockout cassette was put into Escherichia coli strain BW25113 using electroporation to be a lambda red recombination vector existing therein. Recombination with pIJ790 led to integration with pBC3-3.

Recombined transformants were then selected by culturing in LB (Luria-Bertani) agar medium containing 50 μg / ml of ampicillin and 50 μg / ml of apramycin antibiotic (Bertolt G). et al ., John Innes Centre, England).

In order to confirm the removal of O -methyltransferase from the selected E. coli strain (pBC3-3M / BW25113), a nucleotide sequence about 100bp away from both primers used for knockout was prepared as a primer and selected colonies after recombination ( PCR with colony) confirmed that the knockout cassette of about 1.6 kb was integrated instead of O -methyltransferase. Therefore, it was confirmed that the cosmid vector (pBC3-3M) of the E. coli strain had a biosynthetic gene DNA fragment in which the O -methyltransferase gene was removed from the pradimycin biosynthesis gene.

For the introduction of the cosmid vector pBC3-3M knocked out of the O -methyltransferase gene into actinomycetes, pBC3-3M is used to avoid the strong methyl-specific restriction system of actinomycetes. Was isolated from E. coli strain BW25113, introduced into the methylation-deficient E. coli ET12567 / pUZ8002 by electroporation, and 50 μg / ml of apramycin and 50 μg / ml. Transformed cells were selected from LB agar medium containing kanamycin.

< Example  3> Pradimycin O - Methyltransferase  Gene destroyed Actinomadu la Hibisca  Preparation of Mutant Strains

Pradimycin biosynthetic gene group knocked out O -methyltransferase made in Example 2 to obtain a mutant strain in which the O -methyltransferase gene is destroyed among the actinmadura hibisca's pradimacin biosynthesis gene of the present invention A cosmid vector (pBC3-3M) containing was introduced from the E. coli strain ET12567 into the Actinomadura hibisca strain through conjugation.

For this purpose, the ET12567 / pBC3-3M strain was used in liquid medium LB containing 50 µg / ml of apramycin and 50 µg / ml of kanamycin. After inoculating 1% of the preculture to 20 ml of LB medium containing amicycin (apramycin) and 50 µg / ml kanamycin, the cells were incubated at 37 ° C. for 4 hours until the OD ₆₀₀ value was 0.6. The culture is washed twice with the same amount of liquid medium LB.

At the same time, aerial hypha of Actinomadura hibiscar grown at 28 ° C. for 21 days on ISP 2 agar medium (4 g yeast extract, 10 g malt extract, 4 g glucose) was looped. Scrape well and suspend in 500 μl of 2 × YT liquid medium (Tryptone 16g, Yeast extract 10g, Sodium chloride 5g). The suspension is washed once in the same 500 μl 2 × YT liquid medium. Next, the suspension is subjected to heat-shock at 50 ° C. for 10 minutes and cooled at room temperature for 5 minutes.

Next, 500 μl of E. coli strain ET12567 / pBC3-3M and Actinomyce dura Hibiscar were mixed, centrifuged at 13,000 rpm for 30 seconds, and then discarded most of the supernatant, and the remaining suspension contained 10 mM MgCl _2. Smear in MS agar medium [20g Mannitol, 20g Soybean meal], incubate at 28 ℃ for 20 hours, and 25mg / ml concentration of 0.5mg nalidixic acid and 50µg / ml concentration. 1 ml of sterile water containing 1.25 mg of apramycin was covered.

After incubation at 28 ° C. for 5 days, when the flora was formed, it was transferred to MS agar medium containing 25 μg / ml of nalidixic acid and 50 μg / ml of apramycin. . The mutant strain formed was confirmed by PCR and southern hybridization that the O -methyltransferase had a Pradimycin biosynthetic gene group (see FIG. 3) having a knockout cassette introduced therein. The mutant strain from which the O -methyltransprase was removed was named Actinomadura hibisca BC1 strain and deposited in the Agricultural Biological Resources Center of the Institute of Agricultural Biotechnology (Accession Number: KACC 95057P).

< Example  4> Pradimycin O - Methyltransferase  Gene destroyed Mutant strain  BC1 From  Produced Dimethylpramycin  Purifying and Antimicrobial Activity

After knockout experiments, the mutant strain BC1 was incubated with rotation shaking at 170 rpm at 28 ° C. for 7 days in 100 ml ISP 2 liquid medium in a 1 L flask.

ISP  2 Badge Composition

Yeast extract 4g

Malt Extract 10g

Glucose (Dextrose) 4g

The pH of the culture solution obtained above was adjusted to 2.0, and it was possible to extract with butanol and methanol using fractional fermentation, and extracted from actinomycetes cells with the same amount of butanol and 1% methanol.

The extract was centrifuged at 8,000 rpm for 30 minutes to separate culture filtrate and mycelium.

The separated culture filtrate was finally dissolved in 1 mL of methanol using a reduced pressure evaporator and stored.

The methanol was purified by fractionation by performing HPLC (High-Performance Liquid Chromatography) under conditions using a flow rate of 2 mL / min, UV detection 210 nm, 20% -95% methanol including 0.1% solvent formic acid (FIG. 4).

Hydrogen nuclear magnetic resonance was used to confirm the structure of purified dimethylpradimycin, which removed methoxy protons at 3.96 ppm of the chemical shift found in the structure of pradimycin produced by Actinumazura hibisca strain. By confirming that, the substance produced by the above strain was confirmed that the dimethylpradimycin of the formula (2) in which the methyl group was removed from the methoxy group of the pradimycin structure.

화학식 2

Formula 2

In order to assay the antimicrobial activity of purified dimethylpradimycin, thin layer chromatography was carried out by attaching a sample to thin film silica and using a developing solvent in which butanol, acetic acid, and distilled water were mixed at a ratio of 3: 0.5: 1. (TLC) was performed. The silica gel plate thus developed was subjected to a biological assay (bioassay) to investigate the antimicrobial activity. Biological Assay, Fusarium oxysporum ) showed mycelial growth inhibitory effect (see FIG. 5).

As described above, the present invention secures a gene group involved in biosynthesis of pradimycin from the actinomadura hibisca, which produces the antifungal antibiotic pradimycin, and removes O -methyltransferase from these biosynthetic genes. By preparing the strain, dimethylpradimycin (demethylpradimicin), a novel pradimycin derivative, is not known. The novel pradimycin derivative is a Fusarium of Fusarium of phytopathogenic fungi. oxysporum ) was confirmed to have antimicrobial activity. Therefore, the present invention is a useful invention that produced a novel antimicrobial active material through the manipulation of the biosynthetic gene of antibiotics.

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Claims (15)

delete Methyl transferase intermediate antibiotic resistance gene has been inserted the DNA fragment of the gene segments of (Pradimicin A -methyltransferases O) - O Prado de-azithromycin A in the structure of the plastic-di azithromycin shown in SEQ ID NO: 1, which is involved in methylation. The DNA fragment of claim 2, wherein the antibiotic resistance gene is an apramycin antibiotic resistance gene. The DNA fragment of claim 2, wherein the pradimycin A 0 -methyltransferase gene segment is part of a nucleotide sequence of a start codon site and a stop codon site. A knockout cassette comprising said DNA fragment according to any one of claims 2 to 4. delete A knockout vector comprising a cosmid clone pBC3-3 (microbial accession number; KACC 95056P) having a DNA fragment of claim 2 and a group of pradimycin biosynthetic genes represented by SEQ ID NO: 2. Claim is transformed with the knockout vector described in section 7 PRA di erythromycin A O - methyl transferase gene is disrupted gene mutant. According to claim 8, wherein the variant strain Actinomadura hibiscus mutant strain BC1 (Microorganism Accession No .: KACC 95057P). The mutant strain BC1 according to claim 9, wherein the mutant strain BC1 produces dimethylpradimycin which is a pradimycin derivative. 1) preparing a genomic DNA library of Actinu hibiscus hibisca using a cosmid vector to obtain a cosmid clone containing a group of pradimycin biosynthetic genes (step 1); 2) preparing a knock-out cassette by inserting an antibiotic resistance gene into the O -methyltransferase gene DNA fragment in the pradimycin A biosynthesis gene group (step 2); 3) preparing a knockout vector by integrating the knockout cassette into a DNA fragment containing the pradimycin biosynthetic gene group (step 3); 4) O -methyltransferase gene by introducing the knockout vector into the Actinomadura hibisca through conjugation and transforming the actinomadura hibisca strain through homologous recombination. A method for producing a genetically modified strain, wherein the pradimycin A O -methyltransferase gene is removed, comprising the step (step 4). delete delete delete In the method for producing dimethylpradimycin having an antimicrobial activity represented by the formula (2),

Formula 2 Dimethylpradimycin is a dimethyl pradi consisting of culturing the actinomadura hibisca mutant strain BC1 (Microbial Accession No .: KACC 95057P) in ISP 2 liquid medium and separating and purifying from the culture filtrate. Method for preparing mycin.

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