KR101091156B1 - The production method of microorganism for overexpression of antibiotics using wblA gene - Google Patents

Abstract

The present invention identifies a gene that functions as a global repressor of the biosynthetic biosynthetic pathway of Streptomyces sp. Microorganisms, and provides a method for producing an antibiotic-strain strain using the gene and a screening method for increasing the production of antibiotics. and, more specifically, the present inventors have found that Streptomyces Percy Precious wild type strain and a gene expression profile for Streptomyces nose elementary color difference between industrial strains (S. peucetius) (S. coelicolor) whole genome microarray to produce doxorubicin Analyzes using a chip, selects genes with specific expression differences, overexpresses the selected genes on the antibiotic-producing strain, and examines the effects of the genes on the production of antibiotics. As a Common Inhibitor in the Material Biosynthetic Pathway The gene for wblA Gene was identified. The invention disclosed above The wblA gene functions as a common inhibitor of the antibiotic biosynthetic pathway in antibiotic-producing microorganisms among Streptomyces microorganisms, thus producing the high-antibiotic strains or producing industrially useful antibiotics produced in Streptomyces. It can be usefully used to screen for compounds that increase or promote this.

Antibiotics, Streptomyces spp., Doxorubicin, high production

Description

The production method of microorganism for overexpression of antibiotics using wblA gene}

1 is a result of measuring a doxorubicin produced on the wild type strain and the industrial strain of S. Percy Precious (S. peucetius):

  A: plate photo;

Upper left corner of the plate: NDYE 6 days in liquid culture medium S. Percy Precious Wild type strains;

Bottom left of the plate: NDYE 6 days in liquid culture medium S. Percy Precious Industrial strains;

Plate right: S. Percy Precious NDYE on plate Wild-type Strains and S. Persistance Industrial strain culture;

Left side of the plate: S. Persistent Wild type strains; And

Right of Rate: S. Persistent Industrial strains.

B: S. Persistent Wild-type Strains and S. Persistance Volumetric productivity of doxorubicin over time of industrial strains;

C: S. Persistent Growth curves of wild-type strains and total RNA harvest time; And

D: S. Persistent Growth curves of industrial strains and time zones for obtaining total RNA.

2 S. Persistent Wild-type and S. Persistent The results of microarray analysis using S. percitus doxorubicin metabolic pathway chip for gene search showing differences in expression patterns among industrial strains.

3 is a result showing the gene expression differences in a gene compared with the S. Ko Eli collar (S. coelicolor) whole genome microarrays:

  A: upregulated genes;

  B: down regulated gene;

  Blue asterisk: SCO5147; And

  Red star: SCO3579.

4 is a pSE34 vector map.

5 is a gel photograph showing the expression level of the antibiotic biosynthesis gene of the S. coelicolor transforming strain overexpressing the selection gene:

  -No blue dye;

  +: Similar to wild type strain;

  ++: produces slightly more than wild type strains;

  +++: significantly more than wild type strains; And

  #: Two different phenotypes seen by some transformants.

Figure 6 is a result of a plate measuring about on the antibiotic production and S. S. Ko elementary color Percy Precious that overexpress SCO5147 and SCO3579 gene of the selection gene:

A: S. coelicolor ;

B: S. Persistance ;

  White asterisk: empty vector transformation;

  Red asterisk: SCO3579 gene transformation; And

  Blue asterisk: SCO5147 gene transformation.

7 is a result of measuring the antibiotic production on S. Ko Eli collar which overexpress SCO3579 gene:

  A: graph of UND and ACT production measurement results; And

  B: CDA production measurement results.

8 is a gel photograph examined by RT-PCR for the expression of antibiotic metabolic pathway related regulatory genes on S. coelicolor :

  M: 100 bp size marker;

Lanes 1-2: actII - ORF4 ;

Lanes 3-4: redD ;

Lanes 5-6: redZ ;

Lanes 7-8: cdaR .;

Lanes 9-10: wlbA ;

  Lanes 11-12: rDNA gene;

Odd number lanes and white star: S. nose Ellie Callahan transformed pSE34 vector; And

Even-number-lane and a red asterisk: a SCO5147 total RNA of the transformed S. gwabyeol nose Eli color Prefecture RT-PCR.

The present invention uses a microarray chip to select a gene that affects the antibiotic production pathway, and the antibiotic-producing Streptomyces genus strain produced by the manipulation of the selected gene using a conventional genetic engineering technique and the The present invention relates to a method for screening a substance that increases the production of antibiotics using a transformed gene.

Doxorubicion is a clinically important anticancer agent, and is a highly G + C Gram-positive soil strain, Streptomyces persistia var. Car in siwooseu (. Streptomyces peucetius var caesius) type of biosynthesis by the strain is Ⅱ poly cake containing the structural family of tide compound (Acramone, F. 1984, Med Res Rev. 4:.. 153-188; Nicholls, G. , et al., 1992, J. Pharm. Biomed.Anal. 10: 949-957; Vetrivel, LS. and K. Dharmalingam, 2001, J. Genet. 80: 31-38). Like other Streptomyces secondary metabolites biosynthesis of the species, since the synthesis of doxorubicin is tightly controlled there is a limited production of doxorubicin in wild-type S. Percy Precious (Kalakoutskii, LV et al ., Bacteriol . Rev. 40: 469-524, 1976; Otten, SL, et al ., J. Bacteriol . 177: 1216-1224, 1995; Peggy, AR et al ., J Bacteriol . 137: 891-899, 1979; Stutzman-Engwall, KJ et al ., J. Bacteriol . 174: 144-154, 1992). Although the increased production based on molecular genetics is largely unknown, industrial strains have been improved through random and repetitive mutations, ie, mutation induction using UV or random mutations using chemical therapies such as NTG or HNO _2. Has been used to increase the synthesis of doxorubicin (Acramone, F. Med . Res . Rev. 4: 153-188, 1984; Park, H.-S., et al ., J. Microbiol. Biotechnol . 15: 66-61, 2005; Park, H.-S. et al ., Korean . J. Biotechnol . Bioeng . 18: 289-293, 2003; Rodriguez, E., et al ., J. Int . Microbiol . Biotechnol . 30: 480-488, 2003; Vetrivel, KS, et al ., J. Genet . 80: 31-38, 2001). However, such a random mutation method is expected to consume a lot of time and labor in terms of the probability is low.

Recently, "Omics-derived techniques," including cDNA microarrays, have been used to identify gene expression changes associated with overexpression of secondary metabolites in industrial strains. Erythromycin (erythromycin) produced saccharide as poly seashell erythro Rie (Saccharopolysora erythreae) and to produce tyrosine (tyrosin) Streptomyces furanyl DI Streptomyces nose elementary color transcriptional analysis (Streptomyces fradiae) (Streptomyces coelicolor) cDNA to the Microarrays (Lum, AM, et al., Metabolic Engineering 6: 186-196, 2004) have been reported. In this case, we analyzed the difference between transcripts between wild-type and industrial strains, but we could not experimentally prove genes with significant changes in productivity (Lum, AM, et al., Metabolic Engineering 6: 186-196, 2004). .

The present inventors have analyzed and compared to the wild type strain and expression of the industrial strain of S. Percy Precious producing doxorubicin using S. Ko elementary color full genome microarray, were selected genes differences in expression. After the screening of the gene wblA gene affecting the production of overexpressing a selection gene and S. S. Percy Precious nose antibiotic produced by a strain elementary color, the gene as well as S. S. Percy Precious nose Eli The present invention was completed by confirming that it inhibits the production of antibiotics on the collar, thereby acting as an inhibitor that affects the entire Streptomyces species.

An object of the present invention is to identify a gene that functions as a common inhibitor in the antibiotic biosynthetic pathway of Streptomyces microorganisms, a method for producing high antibiotic production strains using the gene, screening method for increasing antibiotic production compound and the common inhibitor It is to provide a strain of Streptomyces genus that has deleted or introduced the gene.

In order to achieve the above object, the present invention provides a method for producing antibiotic high strain Streptomyces strain.

The present invention also provides a Streptomyces strain wherein the wblA gene has been engineered by conventional genetic engineering techniques.

The present invention also provides a transgenic Streptomyces strain introduced with the wblA gene.

In addition, the present invention provides a method for screening a compound that increases the production of antibiotics produced by itself in Streptomyces microorganism.

Hereinafter, the present invention will be described in detail.

The inventors cultured wild-type strains and industrial strains of S. percitus (see FIG. 1A), and obtained a sample by measuring the time to become the same OD value between the two strains (see FIGS. 1B-D). Since wild-type strains and industrial strains of S. pertussis using the doxorubicin metabolic pathway chip (Kang, S.-H. et al., Korean J. Biotechnol. Bioeng. 20: 239-246, 2005) Gene expression was examined. The metabolic pathway chip contains S. Persistent Over 40 genes have been identified in the stomach as doxorubicin metabolic pathway genes. As a result, most of the doxorubicin metabolic pathway genes were expressed late in the wild-type strain sample, and the industrial strain was confirmed that the expression of doxorubicin metabolic pathway-related genes increased from the beginning of the exponential growth phase of the strain (see Fig. 2). The inventors then investigated gene expression between wild type strains and industrial strains of S. percitus using the whole genome microarray chip of S. coelicolor . As a result, over 160 genes were selected that showed more than two-fold differences in expression in the T0 time zone. After that, genes that did not show significant difference in expression in two strains of the selected genes and proteins whose functions were not yet known (hypothetical proteins) were excluded first. Later, we looked at S. Percytus wild strains and compared them to genes that significantly down-regulate the genes that were significantly up-regulated, whereas among them S. Genes suspected of differing or related to secondary metabolism were selected (FIG. 3).

After comparing expression patterns between wild-type strains and industrial strains at time zones T0, T1 and T2, 10 genes up-regulated and 10 genes down-regulated were selected (see FIG. 3). . Thereafter, the selected 20 genes were inserted into the pSE34 vector (see FIG. 4). S. Coelicolor And S. Transfection was performed to determine the degree of antibiotic production. The results show that SCO5147 is an estimated positive regulator and SCO3579 is an estimated negative regulator in S. coelicolor. The SCO5147 and SCO3579 genes were then transformed and overexpressed S. Coelicala And Antibiotic production in S. Persistent was confirmed. As a result, in both strains, overexpression of SCO5147 gene showed no significant change in the production of antibiotics, whereas overexpression of SCO3579 gene inhibited the production of antibiotics (see FIG. 6). This allows the SCO3579 gene to be expressed in S. coelicolor. And It was found that it acts as an antibiotic inhibitor in S. percitus. Since the empty vector was transformed by transfection or by introducing the SCO3579 gene, the production of antibiotics (UND, ACT, CDA) produced in S. coelicolor was measured. In this case, it was confirmed that the antibiotics were normally produced in the strain transformed with the empty vector, but in the case of introducing the SCO3579 gene, the production of antibiotics was strongly inhibited. RT-PCR was performed to investigate the effect of SCO3579 gene on the expression of other antibiotic metabolic pathway control genes. As a result, it was confirmed that S. coelicolor transformed with the SCO3579 gene reduces the expression of genes related to metabolic regulation of antibiotics (see FIG. 8). This confirmed that the SCO3579 gene is an inhibitor of the overall biosynthesis of antibiotics of Streptomyces spp. Therefore, the SCO3579 gene identified by the present inventors can be usefully used for screening compounds for producing antibiotic-producing high strains and increasing or promoting antibiotic production.

The present invention provides a method for producing antibiotic high production Streptomyces strain.

The present invention provides a method for producing an antibiotic high producing strain consisting of the following steps: 1) obtaining total RNA from the antibiotic high producing strain of Streptomyces genus and its wild-type strain, respectively; 2) comparing the degree of gene expression between the high production strain of Streptomyces antibiotic and its wild type strain using the obtained total RNA of step 1); 3) selecting genes showing differences in expression in the compared gene expression results of step 2); 4) overexpressing the selected gene of step 3) into an antibiotic producing strain of Streptomyces; 5) measuring the amount of antibiotics produced in the overexpressed strain by introducing the selected gene of step 4); 6) selecting the genes related to the antibiotic production pathway by comparing the antibiotic production age of step 5) with the antibiotic production in the antibiotic producing strain of Streptomyces before the selected gene introduction; 7) preparing a mutant strain by manipulating the selected gene in a Streptomyces genus antibiotic producing strain; And 8) selecting an antibiotic high producing strain from the mutant strain of step 7).

In the above method, the genus Streptomyces (Streptomyces) microorganisms of step 1) is Streptomyces Percy Precious (Streptomyces peucetius ), Streptomyces coelicolor), Venezuela Streptomyces (Streptomyces venezuela ) , Streptomyces hygroscopius ( S. hygroscopicus ) is preferably any one selected from the group consisting of. The antibiotic high producing strain is preferably an industrial Streptomyces strain prepared by repeated mutant methods for mass production of doxorubicin, and more preferably, S. pertussis industrial mutant strain.

In the method, the antibiotic of step 1) is any one selected from the group consisting of doxorubicin, actinorhodin, picromycin, geldanamycin The present invention is not limited thereto, and all antibiotics produced by Streptomyces microorganism may be included in the present invention.

In the above method, the method for measuring gene expression in step 2) is preferably a microarray chip, but is not limited thereto. Any method for measuring gene expression known to those skilled in the art may be used in the present invention. In the present invention, the S. coelicolor whole genomic microarray chip is used, but is not limited thereto. Any microarray chip in which all genes of the strain of the genome of which Streptomyces genus has been found is integrated can be used in the present invention. .

In the above method, in step 3), it is preferable to select a gene whose expression increases or decreases by more than two times the difference in gene expression.

In the method, the selected gene of step 6) is wblA ( whiB -like putative transcription factor gene: SCO3579), and the gene has a nucleotide sequence as set forth in SEQ ID NO: 1, but is not limited thereto, and a gene having a sequence showing 60 to 99% homology from the sequence of the gene is It can be used in the present invention.

In the above method, the genetic engineering method of step 7) is preferably a method of inserting, substituting or deleting one or more nucleotides of a selected gene, and the method is applicable to all conventional genetic manipulation techniques, but is not limited thereto. It doesn't happen.

In addition, the present invention is wblA Provided are Streptomyces strains whose genes have been engineered by conventional genetic engineering techniques.

The selection gene wblA (SCO3579) is represented by SEQ ID NO: 1, but is not limited thereto. A gene having a sequence showing 70 to 99% homology with the sequence may be used in the present invention. In addition, the antibiotic is characterized in that the doxorubicin or actinolodine, but is not limited thereto. The strain is It is characterized by, but not limited to, Streptomyces Persischeus or Streptomyces coelicolor. The deletion method is preferably using a general method known to those skilled in the art, and more preferably using a gene deletion method through homologous recombination based on a PCR tergeting system.

The strain of the present invention is a strain lacking wblA , a gene that acts as a common inhibitor of the antibiotic biosynthesis pathway of Streptomyces sp. Microorganisms producing antibiotics, and is simpler than an industrial strain prepared by a conventional repeated mutation method. It can be made practically.

The conventional genetic engineering technique is preferably, but is not limited to, a method of inserting, replacing, or deleting one or more nucleotides of the wblA gene.

In addition, the present invention is wblA Provided are Streptomyces strains into which the genes have been introduced.

The selection gene wblA (SCO3579) has a nucleotide sequence set forth in SEQ ID NO: 1, but is not limited thereto, a gene having a sequence showing 70 to 99% homology with the sequence is available in the present invention. In addition, the antibiotic is characterized in that the doxorubicin or actinolodine, but is not limited thereto. The strain is It is characterized by, but not limited to, Streptomyces Persischeus or Streptomyces coelicolor.

In addition, the present invention provides methods for screening compounds that increase the production of antibiotics.

The present invention provides a screening method consisting of the following steps: 1) wblA Introducing the gene and overexpressing it in the Streptomyces strain; 2) treating the test compound to the strain of step 1); 3) measuring antibiotic production of Streptomyces strain treated with the test compound of step 2); 4) selecting a test compound having an effect of increasing antibiotic production by comparing the measurement result of step 3) with the antibiotic production measurement of the strain of Streptomyces before the test compound treatment.

In the above method, the wblA gene of step 1) has a nucleotide sequence set forth in SEQ ID NO: 1, but is not limited thereto, and any gene having a sequence showing 70 to 99% homology with the sequence is useful in the method of the present invention. Can be used.

Streptomyces sp in the step 1) in the above process is Streptomyces Percysis or Streptomyces It is characterized by being coelicolor, but is not limited thereto.

In the above method, the test compound of step 2) is an artificial compound or a natural compound or a derivative thereof.

In the method, the antibiotic of step 3) is characterized in that the doxorubicin or ectinorodin.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following Examples illustrate the present invention in detail, and the content of the present invention is not limited by the Examples.

< Example  1> Industrial S. Persistent  Strain and wild type S. Persistent  Strain culture

Repeatedly mutated doxorubicin overexpressed S. Persistance industrial mutant strains (provided by Boryeong Pharmaceuticals, South Korea) and wild type S. persistia strains [ S. peucetius subsp. caesius ATCC27952, purchased from the American Type Cell Collection, was cultured via shake flask culture using NDYE medium (Hopwood, DA, et. al ., Practical Streptomyces Genetics, The John Innes Foundation, Norwich, UK, 2000). Supernatant and cell pellet samples were obtained while incubated for 10 days, and HPLC analysis was performed for concentration measurements of doxorubicin titer and cell growth (Kang, S.-H., et al ., Korean J. Biotechnol . Bioeng. 20: 239-246, 2005; Park, H.-S., et al., J. Microbiol . Biotechnol. 15: 66-71, 2005). Doxorubicin was used as a C-18 reverse phase (250 × 4.6 mm) column (Waters, Ireland), and the extraction was performed by taking a certain amount of the culture solution and extracting 1 vol. Of extract (Iso-propyl alcohol: 30% HCl = 50: 1). ) Was mixed and stirred for 30 minutes, followed by centrifugation at 15,000 rpm for 10 minutes to filter the supernatant. The mobil phase for HPLC analysis was mixed with acetonitrile / water (1: 1 v / v) mixture with SDS (1.327 g / l) and phosphoric acid (0.68 ml / l). The flow rate was 1 ml / min and the column temperature was maintained at room temperature to quantify the production of doxorubicin at 254 nm with a UV detector. Wild-type strains and mutant S. Percysis strains were determined by incubating at similar rates.

As a result, the industrial mutant strain produced approximately 20-30 mg / L doxorubicin, but no doxorubicin was detected in the wild type strain (FIG. 1A).

In order to identify possible transcriptional differences between wild type and mutant strains, samples were taken during incubation at five different growth phases to isolate total RNA (FIGS. 1B-D). Total RNA was extracted using the RNeasy mini kit (Ambion Inc., USA).

< Example  2> Microarray  Industrial S through analysis. Persistent  Strain and wild type S. Persistent  Gene Expression Changes of Strains

It is possible to obtain the genetic sequence of the S. Percy Precious gene is released, S. Koh the elementary color cDNA microarrays and S. Percy Precious of doxorubicin pathway mini cDNA chips containing the gene of the gene 40 S. Percy Precious former was used in the evaluation of the transfer member (huang, J. et al, Genes Dev 15:...... 3183-3192, 2001; Kang, S. -H, et al, Joran J. Biotechnol Bioeng 20: 239 -246, 2005; Lum, AM et al., Metabolic Engineering 6: 186-196, 2004).

For growth-dependent microarray hybridization, the “zero” sample, the initial time point of doxorubicin production with OD ₆₀₀ of 1, was labeled with a Cy3 fluorescent die (green), and all subsequent time point samples were labeled with a Cy5 fluorescent die (red). And hybridized with “zero” samples of the same strain (FIG. 2 first and second column). Wild type strain cDNA samples labeled with Cy3 and industrially used mutant samples labeled with Cy5 were separated at the same time and compared directly (FIG. 2 third column).

Analysis using a mini cDNA chip containing about 40 genes of doxorubicin metabolic pathway genes revealed that most of the doxorubicin metabolic pathway genes were induced late in wild-type strain samples. High expression of doxorubicin metabolic pathway-related genes was observed from the beginning of the growth phase (FIG. 2).

Since for detecting the overall change in the large amount of mRNA associated with doxorubicin over-expression on the S. Percy Precious, S. Percy Precious wild type strain and the mutant strain on the comparative analysis of transcripts containing a known sequence S. nose knife elementary micro-la cDNA Arrays (Huang, J. et al ., Genes Dev . 15: 2183-3192, 2001).

As a result, approximately 160 S. coelicolor potential candidate genes were identified as genes that showed at least a 2-fold difference in transcription between wild-type and mutant strains at the initial T0 time point. An analysis of the growth phase transcription profile of the potential candidate genes was then performed, with over 20 genes selected to show large transcriptional changes between the two strains (FIGS. 3A, B). As a result, S. Percy Precious industrial mutant expressed in the strain of S. SCO5147 Percy Precious body was increased four times in the wild type strain S. Percy Precious T3 time point (Fig. 3A), the SC03579 S. Percy Precious homologues Was inhibited by about 50% in industrial mutant strains (FIG. 3B). Real-time RT-PCR targeting the two possible regulatory genes on S. percitus confirmed the transcriptional changes observed by microarray analysis (data not shown).

< Example  3> Investigation of changes in antibiotic production through selection gene transformation

The genes pSE34 Streptomyces expression vectors of the strong ermE (Fig. 4) was overexpressed in control under the nose S. elementary color of the promoter.

Each gene was prepared with primers based on the already known Streptomyces whole genome sequence (http://streptomyces.org.uk/), the ribosomal binding site at the front of the gene and the enzyme loci at both ends. (enzyme site) is included. The PCR product was then cloned into pMD18 T-vector (Takara, Japan), cloned into pSE34 using the corresponding restriction enzyme and obtained by transformation into E. coli . Then, the transformed S. Ko Eli collar R2YE (Kieser et al . 2000. Practical Streptomyces Genetics. A laboratory manual Norwich, UK: John Innes Foundation) cultured in a medium plate.

As a result, the gene that mostly increases the production of the double blue pigment antibiotic actinorhodin was induced by putative positive regulator SCO5147. A large decrease in activinin expression was observed in the expressing cells of SCO3579 (FIG. 5).

The presumptive positive modulator as the selection gene of the SCO5147 gene is classified as a 50 ECF- subfamily sigma factor (sigma factor) gene, present in the genome of the S. Ko elementary color. Although the biological rules of the gene have not been revealed, it can be seen that deletion in S. coelicolor has an important function for bacterial survival by showing inactivity. Adjusting the estimated speech design SCO3579 is w (wblA in S. Ko elementary color hi B - l ike putative transcription factor gene ) (http://streptomyces.org.uk./; Soliveri, JA et al ., Microbiology 146: 333-343, 2000).

After the selection gene and the SCO5147 SCO3579 gene cloned into the expression vector pSE34, S. S. Ko Percy Precious and was transformed with each of the elementary color, the impact on the production of antibiotics of the transformed strain, respectively Was investigated.

The transformed S. Ko elementary color strains were cultured on R2YE medium plate, the transformed S. Persistent strains were cultured on NDYE medium plates.

As a result, the transformant of S. Ko elementary color conversion strains inhibited the generation of the exciter Martino Rodin in SCO3579 gene-overexpressed strain (left a red asterisk), S. Percy Precious In the strain, it was also observed that the production of doxorubicin was effectively inhibited in the strain overexpressed the SCO3579 gene (right red star) (FIG. 6).

Subsequently, the S. coelicolor transformed by introducing the selection gene. Antibiotics [ACT (Actinorhodin), UND (Undecylprodigiosin-undecylprodigiosin) and CDA (calcium dependent antibiotics) produced in the above phase were measured and compared by Hopwood et al. (Hopwood, DA et al. al ., Practical Streptomyces Genetics. The Jone Innes Foundation, Norwichm UK, 2000). At this time, the transformation medium was cultured in YEME medium, ACT and UND production was measured by absorbance (Fig. 7A), and for analysis of CDA production, the control strain, the same amount of spores of the transformed strain into which the SCO3579 gene was introduced, were fed agar medium. (nutrient agar medium) was incubated for 3 days. 15 mM Ca (NO ₃₎ in the culture medium two sheets ₂ are made of soft nutrient agar medium in the presence or absence, Bacillus Mai of log phase state Koh des (Bacillus mycoides ) cells were mixed to 0.5 ml / ml, dried over 5 ml of this medium and incubated overnight.

As a result, it was confirmed that the ACT, UND and CDA production of the transformed strain introduced with the SCO3579 gene was significantly inhibited compared to the transformed strain without introducing the SCO3579 gene (Fig. 7A, B).

< Example  4> WblA Study on the effects of genes on antibiotics

Due to overexpression of genes WblA Eli S. nose color In order to investigate the effect on the expression of genes associated with antibiotic production in the strain, S. Ko was inserted into the SCO3579 gene of elementary color to pSE34 vectors overexpressing SCO3579 by transformation S. Ko Eli collar Strains were prepared in the same manner as in Example 3. After that, the expression of antibiotics-related genes was investigated by RT-PCR method. The sequence of the primer pair used at this time is as follows: rDNA (Forward primer: 5'-GAC TCC TAC GGG AGG CAG CA-3 ': SEQ ID NO: 2; Reverse primer: 5'-CGC CCA ATA ATT CCG GAC AA-3 ': SEQ ID NO: 3), actII -ORF4 (forward primer: 5'-TCC CTG GTA ATT TCG CAT CC-3': SEQ ID NO: 4; reverse primer: 5'-CCA TGT GCA TAC GCT GGA TT-3 ': sequence Number 5), redD (forward primer: 5'-CCC TGG AGG ATC TCA TCA GC-3 ': SEQ ID NO: 6; reverse primer: 5'-GTA CGA CTC CAG GGC GTC TC-3': SEQ ID NO: 7), redZ (Forward primer: 5'-ACG TCG GTC GAA GAA CTG GT-3 ': SEQ ID NO: 8; reverse primer: 5'-GAG GAG GAC TTC CGT TTC CC-3': SEQ ID NO: 9), and cdaR (forward primer: 5'-CCA TCG AAG AGA TCG GTC TTG-3 ': SEQ ID NO: 10; Reverse primer: 5'-GCT ACG CCC GAT GAA GTA GG-3': SEQ ID NO: 11). Total RNA was extracted from S. coelicolor comprising SCO3579 and S. coelicolor strain containing the empty vector pSE34 incubated in R2YE plates for 3 days. DNA samples treated with DNA were used to synthesize cDNA containing AMV reverse transcriptase (Takara, Japan), random hexamer, and dNTP at the same concentration. RT-PCR was performed using the synthesized cDNA as a template, and the conditions were repeated 35 times at 95 ° C for 1 minute, 50 ° C for 1 minute, and 68 ° C for 1 minute.

As a result, antibiotic-specific metabolic pathway control genes of S. coelicolor , including act II- ORF4 , redD / Z and cdaR , were reduced in the expression of wblA overexpressing strains (FIG. 8). This suggests that wblA is a global inhibitor of antibiotic biosynthesis among Streptomyces spp.

The present invention uses a microarray chip in which the genome cDNAs of other wild-type strains are integrated into genes showing differences in expression patterns in the industrial mutant Streptomyces strain and its wild-type strains produced by repeated mutation techniques for mass production of antibiotics. The search was completed by identifying genes that act as common inhibitors in antibiotic-producing microorganisms of Streptomyces microorganisms. The method for producing an antibiotic mass production strain by manipulating the identified gene by a conventional genetic engineering technique, and the strain produced by this is simpler and more efficient than the conventional repeated mutation technique and mass production of antibiotics at low cost. In addition, it is expected that the use of the transformed strain into which the genes identified above can be introduced will allow for simple screening of novel substances that promote or increase the production of antibiotics produced in Streptomyces microorganisms.

<110> INHA UNIVERSITY INDUSTRY PARTHNERSHIP INSTITUTE <120> The production method of microorganism for overexpression of          antibiotics using wblA gene <130> 7p-03-06 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> wblA <400> 1 atgggctggg taaccgactg gagtgcgcag gccgcctgcc gcactaccga tccggacgaa 60 ctgttcgttc agggagcagc gcagaacagg gccaaggcgg tgtgcaccgg atgtccggtg 120 cggaccgagt gcctggccga cgcgctcgac aaccgtgtcg agttcggcgt gtggggaggc 180 atgacggagc gggaacgccg cgcgctgctg cgcaggcggc ccaccgtgac ctcctggcgc 240 cggctcctcg agacggcgcg gacggagtac gaacgagggg tcggcatcgt ccccctcgac 300 gacgacgagg tgtacgagaa ctacgcggcc gtgggctga 339 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rDNA forward primer <400> 2 gactcctacg ggaggcagca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rRNA reverse primer <400> 3 cgcccaataa ttccggacaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> actII-ORF4 forward primer <400> 4 tccctggtaa tttcgcatcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> actII-ORF4 reverse primer <400> 5 ccatgtgcat acgctggatt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> redD forward primer <400> 6 ccctggagga tctcatcagc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> redD reverse primer <400> 7 gtacgactcc agggcgtctc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> redZ froward primer <400> 8 acgtcggtcg aagaactggt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> redZ reverse primer <400> 9 gaggaggact tccgtttccc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> cdaR forward primer <400> 10 ccatcgaaga gatcggtctt g 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cdaR reverse primer <400> 11 gctacgcccg atgaagtagg 20  

Claims (23)

delete delete delete delete delete delete An antibiotic low-producing Streptomyces strain transduced with a whibl-like putative transcription factor gene (SCO3579) gene as set forth in SEQ ID NO: 1. delete One) Treating the test compound with the strain of Streptomyces spp. 2) measuring the antibiotic production of the strain Streptomyces strain treated with the test compound of step 1); And 3) comparing the measured value of step 2) with the antibiotic production of the Streptomyces strain before the test compound treatment of step 1), and selecting the test compound having increased antibiotic production. Method for screening a compound to increase. The screening method according to claim 9, wherein the test compound of step 1) is an artificial compound or a natural compound or a derivative thereof. The method of claim 9, wherein the Streptomyces strain of step 1) is Streptomyces persis ( S. peucetius ), Streptomyces coelicolor ( S. coelicolor ), Streptomyces Venezuela ( S. venezuela ) and Streptococcus Screening method, characterized in that any one selected from the group consisting of Mises hygroscopic picus ( S. hygroscopicus ). The method of claim 9, wherein the antibiotic is any one selected from the group consisting of doxorubicin, actinorhodin, picromycin and geldanamycin. delete delete delete delete delete delete delete delete delete delete delete

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