KR100997044B1 - Sigma factor r and a method for increasing production of antibiotics using sigma factor r - Google Patents

Abstract

The present invention is Streptomyces (Streptomyces) in the by using the sigma factor R (sigR) gene isolated from Streptomyces method for increasing the production of medically useful substances in Streptomyces genus Streptomyces, the expression vector therefor, and the expression vector It relates to actinomycetes of the genus Streptomyces transformed into. According to the present invention, actinomycetes in Streptomyces can be provided in which the productivity of a medically useful substance is improved while being balanced.

Description

SIGMA FACTOR R AND A METHOD FOR INCREASING PRODUCTION OF ANTIBIOTICS USING SIGMA FACTOR R}

The present invention is Streptomyces (Streptomyces) in the by using the sigma factor R (sigR) gene isolated from Streptomyces method for increasing the production of medically useful substances in Streptomyces genus Streptomyces, the expression vector therefor, and the expression vector It relates to actinomycetes of the genus Streptomyces transformed into.

Actinomycetes in Streptomyces are known to be capable of producing a variety of secondary metabolites, which can be used as medically useful substances such as antibiotics, antifungal agents, and anticancer drugs (Chater et al., Trends Genet., 5: 372-377, 1990; Hopwood et al., Proc. R. Soc.Lond.Series, 235: 2257-2269, 1987; Nakamura et al., Biocechno.Bioproc.Eng., 8: 37- 40, 2004). Already, a large number of medically useful substances have been isolated from actinomycetes in Streptomyces, for example aminoglycosides, anthracyclines, glycopeptides, β-lactams, macrolides, nucleic acid systems, peptide systems, polyenes, etc. It is known that substances with various chemical structures of are derived from actinomycetes in Streptomyces.

In a strain capable of producing a medically useful substance on its own, such as actinomycetes of Streptomyces, the strain can grow in a balanced manner while also improving the productivity of this useful substance and further maintaining this improved productivity. To create the ultimate goal is for researchers in this field of technology.

Accordingly, many efforts have been made to improve productivity of various strains, particularly actinomycetes of Streptomyces sp. In the past, researches on randomly generated mutants or feeding precursors of useful substances to be produced into strains have been mainly conducted, but recently, the latest biology such as recombinant technology of genes and proteins, genome sequencing, bioinformatics, proteomics, etc. More extensive attempts are being made with the development of the technique (Bentley et al., Nature, 417: 141, 2002; Lee et al., J. Mircobiol Biotechnol. 16: 331-348, 2006; Paradkar et al., Crit Rev. Microbiol. 23: 1, 2003). These methods include the use of recombinant DNA technology to directly introduce genes that produce medically useful substances, introduce partial regulatory factors that regulate the expression of these genes, and remove unwanted biosynthetic genes. Methods include metabolic engineering, which eliminates limitations in the rate limiting step, and increased productivity of antibiotics through regulation of antibiotics (Rohlin et al. , Curr. Opin.Microbiol. 4: 330, 2001; Butler et al. Appl.Environ.Microbiol. 66: 3960, 2000; Fujii et al., J. Bacteriol 178: 3402, 1996; Stutzman-engwall et al., J. Bacteriol 174: 144, 1992).

Korean Patent Laid-Open Publication No. 2006-0018190 improves the productivity of gamma-actinolodine that can function as an antibiotic by introducing an afsR (A-factor synthesis R) gene that functions as a transcriptional activator to Streptomyces spp. Has been reported.

On the other hand, beyond the focus on the direct control method of biosynthetic genes in recent years, research has been progressed toward controlling the overall metabolism of actinomycetes in Streptomyces. It is not simply the gene or cluster containing the gene that plays a role in the production of certain metabolites in actinomycetes in Streptomyces, but the overall balance, regulation, and control of primary and secondary metabolism. It is due to the fact that we started to recognize that In particular, efforts have been made to study the external stresses caused by actinomycetes in Streptomyces to produce medically useful secondary metabolites, and to develop factors that can significantly increase resistance and improve the productivity of secondary metabolites. These efforts have led to studies of sigma factors or chaperones that increase expression in actinomycetes in Streptomyces in response to external stress (Mark et al. 17: 5776, 1998; Perry Chou, Appa. Microbiol. Biotechnol. 76: 521, 2007).

Sigma factor is one component of prokaryotic RNA polymerase. The prokaryotic RNA polymerase is composed of several components, consisting of α2, β, and β ', called core enzymes, and the binding of the sigma factor σ to the core enzymes is called a holoenzyme. It is called. Core enzymes have the ability to stretch RNA, but not the ability to selectively recognize promoters, while proenzymes have the ability to selectively recognize promoters. The difference between the core enzyme and the preenzyme is due to the sigma factor, that is, the sigma factor plays an important role in the selective gene expression of the microorganism according to the external situation by participating in the selectivity of the promoter. Prokaryotes, such as actinomycetes in Streptomyces, possess various types of sigma factors necessary to cope with various external situations, and recognize the external situation and increase the production of appropriate sigma factors, thereby eventually leading to Collective expression becomes possible.

Sigma factor R (SigR) is involved in oxidative stress, and the expression of the sigR gene has previously been reported to activate thioredoxin against the stress of an oxidant called diamide (Mark et al., EMBO J 19: 5776, 1998). However, there have been no reports suggesting that the introduction and overexpression of the sigR gene not only improves the growth capacity of actinomycetes in Streptomyces, but also improves the productivity of medically useful substances such as antibiotics, antifungal agents and anticancer agents.

The present invention provides a method for increasing the production of medically useful substances in actinomycetes in Streptomyces using the sigR gene identified in Actinomycetes in Streptomyces, an expression vector for this, and a strain in Streptomyces transformed with the expression vector. It aims to provide actinomycetes.

The inventors of the present invention, after a lot of research, by introducing and overexpressing the DNA of the sigR gene derived from actinomycetes of Streptomyces genus to actinomycetes of Streptomyces, while improving the growth ability than wild-type actinomycetes, such as antibiotics, antifungal agents, anticancer drugs It has been found that the actinomycetes of the transformed Streptomyces genus can improve the productivity of useful substances.

That is, the present invention,

(1) A group consisting of antibiotics, antifungal agents, and anticancer agents by transforming actinomycetes of Streptomyces, comprising DNA fragments consisting of DNA sequences consisting of the sequencing of the sigma factor R (sigR) gene derived from actinomycetes of Streptomyces . Expression vectors for enhancing the productivity of medically useful substances selected from.

(2) The expression vector according to (1), wherein the nucleotide sequence of the sigR gene is the nucleotide sequence of SEQ ID NO: 1.

(3) The expression vector according to (1), further comprising a DNA fragment consisting of the nucleotide sequence of the afsR gene derived from actinomycetes of Streptomyces spp.

(4) The expression vector according to (3), wherein the nucleotide sequence of the afsR gene is the nucleotide sequence of SEQ ID NO: 3.

(5) Actinomycetes of the genus Streptomyces transformed with the expression vector of any one of (1) to (4).

(6) The method according to (5), wherein the actinomycetes in the transformed Streptomyces genus Streptomyces actinomycetes of the genus Streptomyces, lividans ) or Streptomyces coelicolor .

(7) A medically useful substance selected from the group consisting of antibiotics, antifungal agents, and anticancer agents in actinomycetes in Streptomyces, comprising transforming actinomycetes in Streptomyces in any one of (1) to (4). To improve your productivity.

(8) The method according to (7), wherein the actinomycetes in the Streptomyces genus to be transformed are Streptomyces lividans or Streptomyces coelicolor.

It is about.

The DNA of the sigR gene derived from actinomycetes of the genus Streptomyces used in the present invention has been reported in its sequence in various species. For example, Streptomyces coelicolor, Streptomyces Avermitilis DNA sequences of sigR genes of species such as avermitilis ) are readily available from bioinformatics databases (eg, databases provided by NCBI) that are publicly known in the art. In addition, those skilled in the art with an average knowledge of molecular biology and microbiology can easily find, isolate and analyze the sigR gene in other actinomycetes of Streptomyces through known homology studies based on known sigR genes. Can be found.

The base sequence of the DNA of the sigR gene of Streptomyces coelicolor amplified by the present inventor is shown by SEQ ID NO: 1. The amino acid sequence of the protein encoded by this sigR gene is shown by SEQ ID NO: 2.

The sigR gene used in the present invention may be any object so long as it is derived from any actinomycetes in Streptomyces and can function as the sigR gene in any actinomycetes in Streptomyces. Can be used to achieve the effect. For example, sigR genes such as Streptomyces coelicolor, Streptomyces avermitilis, and the like, which are known in the art, can be used to achieve the desired effect of the present invention.

There is further, a gene having a police Fora (Saccharopolyspora) in the microorganism or salrini spokes La (Salinispora) sequence and similar functions similar to the sigR even in the case of the genus microorganism as saccharide, for other microorganisms, for example, other than the genus Streptomyces can be present, In this case, genes similar to sigR derived from these microorganisms may be utilized in the same manner as the present invention, and may have an effect similar to the desired effect of the present invention.

A "medically useful substance" is any substance that helps humans or animals. Typically, antibiotics, antifungal agents, anticancer agents and the like are medically useful substances that can be produced by actinomycetes in Streptomyces, which are well known in the art (Ikeda H., et al., Nat. Biotechnol. 21: 526-). 531, 2003; Brawner M., et al., Curr Opin Biotechnol 2 (5): 674-81, 1991; Payne G., et al., Appl Microbiol Biotechnol 33 (4): 395-400, 1990; Binnie C , et al., Trends Biotechnol 15 (8): 315-20, 1997). The present invention has the effect of improving the production of such medically useful substances, the specific kinds of antibiotics, antifungal agents, anticancer agents and the like does not have a great effect on this effect. That is, the antibiotics, antifungals, and anticancer agents whose production is improved by the present invention are not particularly limited, and may be any kind that actinomycetes of Streptomyces genus can produce.

Examples of antibiotics in which productivity can be improved by the present invention include actinolodine [streptomyces coelicolor], avermectin [streptomyces avermitilis], streptomycin [streptomyces gliseus ( Streptomyces griseus )], tetracycline [ Streptomyces rimosus )], vancomycin [ Streptomyces orientalis ]], rifamycin [ Streptomyces mediterranei )], chloramphenicol [ Streptomyces venezuelae )], puromycin [ Streptomyces alboniger )], erythromycin [ Streptomyces erythreus ], neomycin [ Streptomyces fradiae ], etc. are mentioned.

Examples of antifungal agents which can be improved in productivity by the present invention include nystatin [ Streptomyces noursei )], amphotericin B [ Streptomyces nodosus )], natamycin [ Streptomyces natalensis )].

An example of an anticancer agent whose productivity can be improved by the present invention is a migrastatin [streptomyces platensis (Streptomyces platensis)], Doxorubicin [streptomyces fusethius (Streptomyces peucetius)], And the like.

A vector refers to a carrier that introduces an external DNA fragment into a host cell or the like and basically includes a replication origin suitable for the host cell. Such vectors are classified into various types according to their use, and their structures vary. Among these, an expression vector refers to a vector for transforming an external DNA fragment related to a specific gene into a target cell and then expressing it in the cell, and the expression vector is usually adjacent to a promoter or promoter together with the origin of replication in the vector. Polycloning site, selection marker. The external DNA fragment is inserted into a polycloning position using a DNA restriction enzyme and a DNA ligase, and after the expression vector is transformed into a host cell, the gene of the external DNA fragment is expressed by a promoter in the expression vector. Various expression vectors are known in the art for each type of target cell.

When an expression vector is introduced into a target cell to express a gene of an external DNA fragment, the expression is regulated by a promoter in the expression vector. Thus, it is possible to obtain a desired form of expression by adjusting and altering the promoter. For example, when a constitutive promoter is used in an expression vector, it causes continuous overexpression of the gene of the foreign DNA fragment in the cell. On the other hand, for example, when a conditional promoter is used, it is possible to control the timing or degree of expression of a gene of an external DNA fragment in a cell, and to cause temporary overexpression.

The expression vector used for transformation in the present invention may have any promoter capable of transforming actinomycetes in Streptomyces and also causing continuous or transient overexpression of the sigR gene in Streptomyces. Any thing as long as the present invention can be used to achieve the desired effect, the kind is not particularly limited. For example, vectors such as pIBR25, pIJ459, pMT3206, pANT1200, pBR322, pKC1139 and the like are expression vectors commonly used in the art for actinomycetes in Streptomyces. Preferably pIBR25 may be used.

In the present invention, the production of expression vectors, transformation of actinomycetes in Streptomyces, and the like may be performed using conventional genetic engineering techniques. Samples, processes, and the like used in such conventional genetic engineering techniques are well known in the art, and specific processes or samples may be adjusted according to the type of expression vector, specific species belonging to actinomycetes in Streptomyces, and the like. It's within the scope of creation and common sense. For example, those skilled in the art will be familiar with the literature [J. Sambrook, et al., "Molecular Cloning: A Laboratory Manual"; F.M. Ausubel, et al., "Current Protocols in Molecular Biology"; A.L. Demain, et al., "Manual of Industrial Microbiology and Biotechnology"; M.J. Gait, et al., "Oligonucleotide Synthesis"; Mullis, et al., "PCR: The Polymerase Chain Reaction"] may select appropriate samples and processes according to specific embodiments. As long as an expression vector comprising a DNA fragment of the sigR gene is constructed and the expression vector can be normally introduced and overexpressed into actinomycetes cells of Streptomyces, the type and process of the sample used for this transformation are not particularly limited. Do not.

Actinomycetes in various types of Streptomyces are capable of producing specific antibiotics, antifungal agents, anticancer agents, or the like, or have the ability to produce specific antibiotics, antifungal agents, anticancer agents, and the like. The actinomycetes for transformation used in the present invention can achieve any desired effect of the actinomycetes of any species so long as it belongs to Streptomyces genus and is capable of producing or capable of producing a desired medically useful substance. Can be used for

For example, Streptomyces coelicolor, which produces the antibiotic actinolodine, Streptomyces avermitilis, which produces the antibiotic avermectin, Streptomyces griceus, which produces the antibiotic streptomycin, antibiotic tetracycline Produces Streptomyces rimosus, which produces antibiotics, Streptomyces Orientalis, which produces the antibiotic vancomycin, Streptomyces Mediterranea, which produces the antibiotic rifamycin, and Streptomyces Venezuela, which produces the antibiotic chloramphenicol, and antibiotic puromycin. Streptomyces alboniger, Streptomyces erythrius, which produces the antibiotic erythromycin, Streptomyces pradier, which produces the antibiotic neomycin, Streptomyces norus, which produces the antifungal nystatin, and antifungal amphoteric God B Streptomyces nodosus to produce, Streptomyces natalenes to produce the antifungal agent natamycin, Streptomyces platensis to produce the anti-cancer drug mygrastatin, Streptomyces fusethius to produce the anticancer drug doxorubicin, etc. Can be used in

The DNA of the afsR gene derived from actinomycetes of Streptomyces genus used in the present invention has been reported in its sequence in various species. (Ikeda, H. et al., Nat. Biotechnol. 21 (5), 526-531; Horinouchi, S. et al., Gene 95 (1), 49-56; Palaniappan, N. et al., Chem. Biol. 13 (7), 753-764; Parajuli, N. et al., Res.Microbiol.156 (5-6), 707-712; Sekurova, O. et al, FEMS Microbiol.Lett.177 (2) Umeyama, T. et al., Microbiology (Reading, Engl.) 145. PT 9) 2281-2292; Oliynyk, M. et al., Mol. Microbiol. 49 (5), 1179-1190). In addition, those skilled in the art with an average knowledge of molecular biology and microbiology can easily find the afsR gene in other actinomycetes of Streptomyces genus through homology studies, and isolate and analyze the nucleotide sequence. All contents of Korean Patent Laid-Open Publication No. 2006-0018190 with respect to the afsR gene are incorporated herein.

The base sequence of the DNA of the afsR gene of Streptomyces coelicolor amplified by the present inventor is shown in SEQ ID NO: 3. The amino acid sequence of the protein encoded by this afsR gene is shown by SEQ ID NO: 4.

The afsR gene used in the present invention may be any object as long as it is derived from any actinomycetes in Streptomyces and can function as an afsR gene in any actinomycetes in Streptomyces. Can be used to achieve the effect. For example, the afsR gene of SEQ ID NO: 3 can be used to achieve the desired effect of the present invention.

In one aspect of the invention, the actinomycetes of Streptomyces, including DNA fragments consisting of the nucleotide sequence of the sigR gene derived from actinomycetes of Streptomyces genus, is transformed to be selected from the group consisting of antibiotics, antifungal agents, and anticancer agents Expression vectors are provided for improving the productivity of medically useful substances. This vector has the purpose of transforming actinomycetes in Streptomyces to improve the growth ability of actinomycetes and the productivity of medically useful substances. Expression vectors with this use are novel which are not known in the art.

Preferably, the sigR gene is derived from Streptomyces coelicolor, the nucleotide sequence is the same as the nucleotide sequence of SEQ ID NO: 1.

Also preferably, the expression vector further includes a DNA fragment consisting of a nucleotide sequence of the sigR gene derived from actinomycetes in Streptomyces and a DNA fragment consisting of the nucleotide sequence of the afsR gene derived from Actinomycetes in Streptomyces. . As a result, the effect of the sigR gene disclosed in the present invention is superimposed on the effect of the conventionally known afsR gene, and thus it is possible to exert an excellent effect.

Particularly preferably, the afsR gene is derived from Streptomyces coelicolor, and its nucleotide sequence is the same as that of SEQ ID NO: 3.

In another aspect of the present invention, actinomycetes of the genus Streptomyces transformed with the expression vector are provided. Actinomycetes in the transformed Streptomyces genus will increase the productivity and the productivity of medically useful substances. Actinomycetes of the genus Streptomyces with this effect are novel and are not known in the art.

Preferably, the actinomycetes in the transformed Streptomyces genus is Streptomyces lividans or Streptomyces coelicolor.

In another aspect of the present invention, the productivity of a medically useful substance selected from the group consisting of antibiotics, antifungal agents, and anticancer agents in actinomycetes in Streptomyces, comprising transforming actinomycetes in Streptomyces with the expression vector. Provided are methods for improving this. This is a method that can improve the productivity of the actinomycetes in Streptomyces and medically useful substances. Methods of transforming actinomycetes in Streptomyces with this effect are novel which are not known in the art.

Preferably, the actinomycetes in the transformed Streptomyces genus is Streptomyces lividans or Streptomyces coelicolor.

Sequence Listing

SEQ ID NO: 1: base sequence of DNA of sigR gene of Streptomyces coelicolor

SEQ ID NO: 2 amino acid sequence of a protein encoded by the sigR gene of Streptomyces coelicolor

SEQ ID NO: 3: nucleotide sequence of the DNA of the afsR gene of Streptomyces coelicolor

SEQ ID NO: 4: amino acid sequence of the protein encoded by the afsR gene of Streptomyces coelicolor

SEQ ID NO: 5: Primer-F for amplifying the sigR gene of Streptomyces coelicolor

SEQ ID NO: 6: Primer-R for amplifying the sigR gene of Streptomyces coelicolor

SEQ ID NO: 7: Primer afsR-F for amplifying the afsR gene of Streptomyces coelicolor

SEQ ID NO: 8: primers afsR-R for amplifying the afsR gene of Streptomyces coelicolor

Microbial deposit

Streptomyces coelicolor A3 (2) M145 (BGC12) transformed with the pIBR25 expression vector (pIBR25-sigR-afsR) containing both the DNA of the sigR gene and the DNA of the afsR gene was deposited with the Institute of Agricultural Biotechnology. The accession number is KACC 91361P.

According to the present invention, by introducing and overexpressing the DNA of the sigR gene derived from actinomycetes of Streptomyces genus to actinomycetes of Streptomyces, the growth ability of the actinomycetes of the wild type, while improving the growth ability of antibiotics, antifungal agents, anticancer agents, etc. Actinomycetes of the transformed Streptomyces genus can be provided with improved productivity.

The invention is described in more detail by the following examples which can represent the invention. However, this is for illustrative purposes only and does not in any way limit the scope of the invention.

< Example  1>

Streptomyces  From coelicolor sigR  Gene Identification and Expression Vector Construction

To identify the sigR gene derived from Streptomyces coelicolor, the Streptomyces coelicolor sequencing database (http://stereptomyces.org.kr) was referred to. Based on this database, two primers were devised:

Primer-F: aaa aaa gga tcc tgc caa gac cgc ccc tat (SEQ ID NO: 5)

Primer-R: aaa aaa tct aga tca tga ccc cga gcc ttt (SEQ ID NO: 6)

The genome of Streptomyces coelicolor A3 (2) M145 (available from Accession No. 20364 of Seoul National University Microbiological Resource Center) was extracted by a conventional method, and then PCR amplification reaction using the two primers [Taq of 2.5 U]. DNA polymerase, 10% dimethylsulfoxide, 2.5 pmol primer, 0.2 mM dNTP and 0.1 μg of genomic DNA were dissolved in a titration buffer to 50 μl, denaturated at 94 ° C., annealed at 65 ° C., kidney at 74 ° C. Repeated 30 cycles] was used to amplify the DNA (SEQ ID NO: 1) of the sigR gene from Streptomyces coelicolor A3 (2) M145.

 Using DNA recombination techniques, the DNA of the amplified sigR gene was inserted into the pIBR25 expression vector. The configuration of the pIBR25 expression vector and the configuration of the pIBR25 expression vector (pIBR25-sigR) into which the DNA of the sigR gene is inserted are shown in FIG. 1.

< Example  2>

sigR  Overexpressed genes Streptomyces At Lividans Actinorodin  productivity

A 200 ml flask with 50 ml of R5-media was inoculated with Streptomyces lividans (KCTC 1167) and incubated for 2 days. The culture was centrifuged to remove the supernatant and the cells washed three times with 15 ml of 10.3% sucrose. P-buffer with 20 mg of lysozyme added to the isolated cells [25 ml of sucrose (10.3%), 75 ml of distilled water, 0.2 ml of trace component solution, 1 ml of K ₂ SO ₄ (2.5%), and 9.3 ml of this mixture 10 ml is prepared by mixing 0.2 ml of CaCl ₂ (5M) and 0.5 ml of tris-maleic acid buffer, where the trace component solution is 40 mg of ZnCl ₂ , FeCl ₃ .6H ₂ O 200 mg, CuCl ₂ .2H ₂ O. ₂ ml of 10 mg, MnCl ₂ 4H ₂ O 10 mg, Na ₂ B ₄ O ₇ 10H ₂ O 10 mg, and (NH ₄ ) 6 Mo ₇ O ₂₄ 4H ₂ O 10 mg were added to make 2 ml. 20 ml were added, and incubated at 30 ° C. for 1 hour and 15 minutes, the cell solution was filtered with cotton to obtain a protoplasm present in the filtered cell solution. The protoplasts were centrifuged to remove the supernatant to obtain a high concentration of the protoplasts, and then 10 μl of the solution in which the pIBR25-sigR vector was dissolved was added to the obtained protoplasts. Buffer [103 g of sucrose, 0.25 g of K ₂ SO ₄ , 2.02 g of MgCl ₂ .6H ₂ O, 2 ml of a trace component solution, and an appropriate amount of distilled water are mixed to make an 800 ml solution, and then 80 ml of the solution is KH. _{2 PO 4 (0.5%) 1} ml, CaCl 2 and _{2H 2 O (3.68%) 10} ml, and TES buffer solution (5.73%, pH7.2) prepared a 101 ml mixture of 10 ml, wherein the minor constituent solution is ZnCl ₂ 40 mg, FeCl ₃ 6H ₂ O 200 mg, CuCl ₂ 2H ₂ O 10 mg, MnCl ₂ 4H ₂ O 10 mg, Na ₂ B ₄ O ₇ 10H ₂ O 10 mg, and (NH ₄ ) 2 ml was prepared by adding a suitable amount of distilled water to 10 mg of 6Mo ₇ O ₂₄ 4H ₂ O] and a solution mixed with a 1: 2 ratio and 500 μl of T-buffer, respectively, were added sequentially and mixed. The mixed solution was plated on an R5-solid medium, incubated at 30 ° C. for 24 hours, followed by a screening process using thiostrepton, and transformed Streptomyces lividans (BGL11) over which the sigR gene was introduced. Obtained.

The wild type Streptomyces lividans (BGL00) and sigR genes were introduced and overexpressed, transformed Streptomyces lividans (BGL11) were incubated for 72 hours at a temperature of 30 ° C. on R5-solid medium.

Actinordine is a medically useful substance that can function as an antibiotic, and the actinordine is basically blue, and it is possible to estimate the production and production of actinordinin according to the presence and degree of blue.

The result of detecting actinolodine is shown in FIG. As can be seen in FIG. 2, wild-type Streptomyces lividans (BGL00) produce substantially no activinin, whereas transformed Streptomyces lividans (BGL11), which is overexpressed by the introduction of the sigR gene, A large amount of actinolodine was produced. The results demonstrate that actinomycetes in Streptomyces, overexpressed with the sigR gene, can produce antibiotics that do not produce in the wild-type state.

< Example  3>

sigR  Overexpressed genes Streptomyces Coelicolor  Growth capacity and Actinorodin  productivity

The sigR gene was transformed in the same manner as in Example 2, except that the Gram-positive bacterium Streptomyces coelicolor A3 (2) M145 (available from Accession No. 20364 of the Seoul National University Microbial Resources Center) was used. A transgenic Streptomyces coelicolor (BGC11) was introduced to overexpress.

Wild-type Streptomyces coelicolor (BGC00) and transformed Streptomyces coelicolor (BGC11) overexpressed with the sigR gene were incubated at 230 rpm for 144 hours at a temperature of 30 ° C. in 15 mL of R5- medium. It was. Thereafter, the cell weight and the production of actinolodine were measured at regular intervals.

The measurement of the weight of the cells was carried out by placing 1 ml of the culture solution in an Eppendorf tube, centrifuging to separate the supernatant, and measuring the weight of the wet cells with a balance.

The measurement of the production of actinolodine was performed by adding 50 μl of KOH (1N) to 400 μl of the separated supernatant, mixing the mixture, and measuring absorbance in the 630 nm wavelength region with a UV spectrometer.

The measurement results of the weight of the cells and the production amount of actinolodine are shown in FIG. 3. As can be seen in the graph of FIG. 3, the wild-type Streptomyces coelicolor in the stationary phase at which the transforming Streptomyces coelicolor (BGC11), which is introduced and overexpressed by the sigR gene, produces the antibiotic actinolodine The cell weight was heavier than that of (BGC00) and the amount of actinolodine produced was higher. These results demonstrate that sigR is introduced and overexpressed in actinomycetes in Streptomyces, which not only improves cell growth capacity but also improves the productivity of antibiotics compared to wild type actinomycetes.

< Example  4>

Streptomyces Coelicala  Origin afsR  Gene Identification and Expression Vector Construction

The DNA of the afsR gene (SEQ ID NO: 3) was amplified in the same manner as in Example 1 except that the following two primers were used.

afsR-F: aaa aaa tct aga aag ctg atg gag aac ctg (SEQ ID NO: 7)

afsR-R: aaa aaa aag ctt tca ccg cgc cac act gcg (SEQ ID NO: 8)

Using DNA recombination techniques, the DNA of the amplified afsR gene was inserted into the pIBR25 expression vector. In addition, separately from both the DNA of the sigR gene and the DNA of the afsR gene identified in Example 1 were inserted into the pIBR25 expression vector. The configuration of the pIBR25 expression vector (pIBR25-afsR) into which the DNA of the afsR gene was inserted and the pIBR25 expression vector (pIBR25-sigR-afsR) into which both the DNA of the sigR gene and the DNA of the afsR gene were inserted are shown in FIG. 4.

< Example  5>

sigR  Gene and afsR  Both genes introduced Streptomyces In Coelicolor Actinorodin  productivity

Transformation was carried out in the same manner as in Example 2 except for using pIBR25-afsR and Gram-positive bacteria Streptomyces coelicolor A3 (2) M145 (available from Accession No. 20364 of the Seoul National University Microbial Resources Center). , transgenic Streptomyces coelicolor (BGC01) was introduced to which the afsR gene was introduced and overexpressed. In addition, except that pIBR25-sigR-afsR and Gram-positive bacteria Streptomyces coelicolor A3 (2) M145 (available from Accession No. 20364 of Seoul National University Microbial Resource Center) were used separately from Transformation was performed in the same manner to prepare a transgenic Streptomyces coelicolor (BGC12) in which both the sigR gene and afsR gene were introduced and overexpressed. BGC12 was deposited with the Institute of Agricultural Biotechnology and its accession number is KACC 91361P.

Wild-type Streptomyces coelicolor (BGC00), transgenic Streptomyces coelicolor (BGC11) introduced with sigR gene, transgenic Streptomyces coelicolor (BGC01) overexpressed with afsR gene introduced, And transformed Streptomyces coelicolor (BGC12), in which both the sigR gene and the afsR gene were introduced and overexpressed, were cultured in the same manner as in Example 3, and the weight of the cells and the production of actinolodine were measured.

The measurement results of the weight of the cells and the production amount of actinolodine are shown in FIG. 5. As can be seen in the graph of FIG. 5, the transgenic Streptomyces coelicolor (BGC12), which is overexpressed by introducing both the sigR gene and the afsR gene, has three other types of Streptomyces coelicolor (BGC00, BGC11, And BGC01) more particularly in the production of actinolodine. Of particular note is that transgenic Streptomyces coelicolor (BGC12), in which both the sigR and afsR genes are introduced and overexpressed, is compared to trans Streptomyces coelicolor (BGC01) in which the afsR gene is introduced and overexpressed. The production of actinolodine was quite good. This result indicates that the effect of introducing sigR can be superimposed on the effect of introducing afsR, that is, by introducing and overexpressing sigR and afsR at the same time, the effect of individual genes can be exerted, especially in the production of antibiotics. Prove that there is.

1 shows the construction of a pIBR25 expression vector and the construction of a pIBR25 expression vector (pIBR25-sigR) into which DNA of the sigR gene is inserted.

Figure 2 shows the results of the detection of the antibiotic activinodin after the culture of the transformed Streptomyces Lividans (BGL00) and sigR gene is overexpressed transformed Streptomyces Lividans (BGL11).

Figure 3 shows the results of measuring the weight of the cells and the production of actinorodin after the culture of the transformed Streptomyces coelicolor (BGC11) over-expressed by introducing wild-type Streptomyces coelicolor (BGC00) and sigR gene .

4 shows the construction of a pIBR25 expression vector (pIBR25-afsR) into which the DNA of the afsR gene is inserted and a pIBR25 expression vector (pIBR25-sigR-afsR) into which both the DNA of the sigR gene and the DNA of the afsR gene are inserted.

FIG. 5 shows wild-type Streptomyces coelicolor (BGC00), transgenic Streptomyces coelicolor (BGC11) introduced by overexpression of sigR gene, and transgenic Streptomyces coelicolor (BGC01) overexpressed by introduction of afsR gene. ), And the results of measurement of the weight of cells and the production of actinolodine after the culture of the transformed Streptomyces coelicolor (BGC12), which is introduced and overexpressed by both the sigR gene and the afsR gene.

<110> SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION <120> Sigma Factor R And A Method For Increasing Production Of          Antibiotics Using Sigma Factor R <130> Y07KP-083 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 684 <212> DNA <213> Streptomyces coelicolor <220> <221> gene (222) (1) .. (684) <223> sigR <400> 1 gtgggtccgg tcactgggac cgacgcaggg accgaacacg gccaggcgga gcagcccgag 60 ggccggggta cgggcgcgga gtcgaccgcg gagcgcagcg cgcgcttcga gcgggacgcg 120 ctggagttcc tcgaccagat gtactcggcc gcgctgcgca tgacgcgtaa tccggccgac 180 gccgaggacc tggtgcagga gacctacgcc aaggcgtacg cgtccttcca ccagttccgc 240 gagggcacca acctcaaggc gtggctgtac cgcatcctca ccaacacctt catcaactcg 300 taccgcaaga agcagcgcga accccagcgc agcgcggccg aggagatcga ggactggcag 360 ctcgcccgtg ccgagtcgca catgtcgacg ggtctgcgct ccgcggagtc gcaggcgctc 420 gaccacctgc ccgactcgga cgtgaagcag gcgctccagg cgatccccga ggaattccgt 480 atcgccgtgt atctggcgga cgtcgagggc tttgcctaca aggagatcgc ggacatcatg 540 gggacaccca tcggtacggt gatgtcccgg ctgcaccgtg gacgccgtca gctgcgcggc 600 atgctggagg actacgcccg cgaccgcggt ctggtccccg ccggcgccgg agagtcgaac 660 gaagcgaaag gctcggggtc atga 684 <210> 2 <211> 227 <212> PRT <213> Streptomyces coelicolor <220> <221> PEPTIDE (222) (1) .. (227) <223> sigR <400> 2 Met Gly Pro Val Thr Gly Thr Asp Ala Gly Thr Glu His Gly Gln Ala   1 5 10 15 Glu Gln Pro Glu Gly Arg Gly Thr Gly Ala Glu Ser Thr Ala Glu Arg              20 25 30 Ser Ala Arg Phe Glu Arg Asp Ala Leu Glu Phe Leu Asp Gln Met Tyr          35 40 45 Ser Ala Ala Leu Arg Met Thr Arg Asn Pro Ala Asp Ala Glu Asp Leu      50 55 60 Val Gln Glu Thr Tyr Ala Lys Ala Tyr Ala Ser Phe His Gln Phe Arg  65 70 75 80 Glu Gly Thr Asn Leu Lys Ala Trp Leu Tyr Arg Ile Leu Thr Asn Thr                  85 90 95 Phe Ile Asn Ser Tyr Arg Lys Lys Gln Arg Glu Pro Gln Arg Ser Ala             100 105 110 Ala Glu Glu Ile Glu Asp Trp Gln Leu Ala Arg Ala Glu Ser His Met         115 120 125 Ser Thr Gly Leu Arg Ser Ala Glu Ser Gln Ala Leu Asp His Leu Pro     130 135 140 Asp Ser Asp Val Lys Gln Ala Leu Gln Ala Ile Pro Glu Glu Phe Arg 145 150 155 160 Ile Ala Val Tyr Leu Ala Asp Val Glu Gly Phe Ala Tyr Lys Glu Ile                 165 170 175 Ala Asp Ile Met Gly Thr Pro Ile Gly Thr Val Met Ser Arg Leu His             180 185 190 Arg Gly Arg Arg Gln Leu Arg Gly Met Leu Glu Asp Tyr Ala Arg Asp         195 200 205 Arg Gly Leu Val Pro Ala Gly Ala Gly Glu Ser Asn Glu Ala Lys Gly     210 215 220 Ser Gly Ser 225 <210> 3 <211> 2982 <212> DNA <213> Streptomyces coelicolor <220> <221> gene (222) (1) .. (2982) <223> afsR <400> 3 atggacggtg gaccgcgggt tccggagcag cggcgtcccg gattcccggc ggaagaggag 60 gaatcgggcg cgctgcgctt cggtgtgctc gggccggtgc gcgcctggcg ggacggggag 120 acgctggcca ccggctcccc gcagcagcgc gcgctgctgg ccgccctgct gctgcgcgag 180 ggccgtacgg ccacggccgg cgagctgatc gacgccctgt ggggcgagga accgccgtcg 240 caggcgctgg cggcggtgcg gacgtacgcg tcccggctgc ggaaggtcct cgacccgggg 300 gtgctggtca gcgagtccgg cgggtacgcc gtgcggggcc tcgccgaggg ggcgctggac 360 ctggcgcggg cccaggacct ggcctcggcg gccgagaagg ccaggagcgc cggggacctg 420 tgccacgccc gtgacctgct gcgccgggcg ctggacctgt gggacggcga ggtgctggcc 480 ggcgtgccgg ggccgtacgc gcagacgcag cgggtccgtc tcggcgaatg gcggctccaa 540 ctcctcgaaa cccgcctcga catggacctc gaccagggct gtcacgccga ggcggtctcg 600 gaactgacgg cgctgaccgc ggcgcacccg ctgcgcgaac gcctgcgcga gctgctgatg 660 ctggccctgt accgcagcgg ccgccaggcg gaggcgctcg ccgtctacgc ggacacgcgc 720 cgcctgctcg ccgacgagct gggcgtcgac ccgcgccccg gcctccagga actccagcag 780 cgcatcctcc aggccgaccc ggccctggcc gagctctcgg cggccaccgc ggagaccgcg 840 acggccacgc tgcgcccggc gcaactgccc gcgaccgttt ccgacttcac cggccgcgcg 900 gccttcgtcc gggagctgag cgacgtactg tccgccgcgt ccggggagtc ggcgtcgggc 960 cgcgtgatgg cggtctcggc gctggccggc atcggcggcg tcggcaagac gaccctcgcg 1020 gtgcacgtcg cccaccgggc gcgggccgcc ttcccggacg ggcagctgta cgtcgacctc 1080 cagggcgcgg gcgcgcggcc cgcggagccg gagacggtgc tcggttcctt cctgcgggcc 1140 ctgggcacgg ccgactcggc catcccggac tccctggagg agcgcgccgc cctctaccgc 1200 tccgtcctgg acggccgccg tgtcctggtc ctgctggaca acgcccgcga cgccgcccag 1260 gtgcggcccc tgctgcccgg cacggacggg tgcgcggcgc tggtgacggc acgggtgcgg 1320 atggtggacc tcgccggggc gcacctggtc gacctggacg tgatggcgcc cgaggaggcg 1380 ctggccctct tcacgaagat cgtgggcgag gagcgggtgg cctcggaacg gcaggcggcg 1440 ctcgacgtgg tcggcgcgtg cggtttcctg ccgctggcga tccgcatcgc cgcgtcccgg 1500 ctggccgccc gccgcacctg gacggtctcg gtcctggccg cgaagctggc ggacgaacgg 1560 cgccggctgg acgagctgca ggccggcgac caggcggtcg aggccacctt cgaactgggc 1620 tacggccagc tggaaccggc ccaggcccgc gccttccgcc tgctgggcct ggccgacggc 1680 ccggacatct ccctcgcggc ggcggcggcc gtactggacc tcccggcgca ggacacggag 1740 gacctcctgg agtccctggt cgacacctcg ctgctcgaat cggcggcccc gggccgctac 1800 cgcttccacg acctggtccg cctctacgcg cgtgcctgcg cggaacggac ggaacgggac 1860 gggaacgcgc cgagcgagcg gggggcggcg ctgtcgcggc tgctggactt ctacctggcg 1920 acggcggcgg gggtgtacgc gatcgagcgg ccgggggacc ggctggtgga cggcctggag 1980 ccgacggagt acccggggct gacgttcacc gagggctcgg cggcgctgga ctggctgtac 2040 acggaggccg cgccgctgct ggcgtgcgtg cggcagtccg ccgggacggc caggctgcgg 2100 cgggcggtgg atctgctgtg ggcggccaag gacctgacgg agtccggcgc gaactcacac 2160 cagtacgagg cgacggccag ggcgatgtgc gacgccaccg gttccgctgc ggacacccgc 2220 gccgagggaa gggctcgcac cgttctcagc gatgtgctcc tggtgtccgg tcgcatcgag 2280 catgccgagg aagaggcacg gctggccatg cggctggcgg gctccgccga ggactcggcc 2340 gccgtgagct gggtcgccaa caaccgagga ctcgtctgcc tgcaccagcg ccggtacgcc 2400 gagggcaagg gcctcttcca ccaggccatc gcgggtttcc gggcaacgga caaccgcgcg 2460 ggcgaggcgt ccgcgttgtc gaacctctcc cgtgcccagc tcggcatggg caacgtcgcg 2520 gaggccgtcg acatcgcccg gcaaggactc gcggtgtacg cggagctggg ccgcaccatg 2580 cgcctcgcca acggccactt cgccctggga gtggcactga ccagggcagg ccggcacgaa 2640 gaggcactgg gcgagttcgc cgaggcgctg gacctcttcg gggaccaccg gcagcgcctg 2700 tgggagggcg ccaccaactt ccgcctcgcc gaggtgcacc tggccgccgg acgcccctcc 2760 tcggcggccc agcacgccga gcaggcactc gccctgggat gcatcggggg cgaccggatg 2820 cggggcaacg tcctggctct gctgggacgc gccctgtcgg ccctgggcca ggcggaccgc 2880 gcgcgagcgt gctggcgcga ggcgctcagc ctctacgagc agcacgacgc ccaggaggtc 2940 ggcgaggtaa gggcgttgct ggctcgcagt gtggcgcggt ga 2982 <210> 4 <211> 993 <212> PRT <213> Streptomyces coelicolor <220> <221> PEPTIDE (222) (1) .. (993) <223> afsR <400> 4 Met Asp Gly Gly Pro Arg Val Pro Glu Gln Arg Arg Pro Gly Phe Pro   1 5 10 15 Ala Glu Glu Glu Glu Ser Gly Ala Leu Arg Phe Gly Val Leu Gly Pro              20 25 30 Val Arg Ala Trp Arg Asp Gly Glu Thr Leu Ala Thr Gly Ser Pro Gln          35 40 45 Gln Arg Ala Leu Leu Ala Ala Leu Leu Leu Arg Glu Gly Arg Thr Ala      50 55 60 Thr Ala Gly Glu Leu Ile Asp Ala Leu Trp Gly Glu Glu Pro Pro Ser  65 70 75 80 Gln Ala Leu Ala Ala Val Arg Thr Tyr Ala Ser Arg Leu Arg Lys Val                  85 90 95 Leu Asp Pro Gly Val Leu Val Ser Glu Ser Gly Gly Tyr Ala Val Arg             100 105 110 Gly Leu Ala Glu Gly Ala Leu Asp Leu Ala Arg Ala Gln Asp Leu Ala         115 120 125 Ser Ala Ala Glu Lys Ala Arg Ser Ala Gly Asp Leu Cys His Ala Arg     130 135 140 Asp Leu Leu Arg Arg Ala Leu Asp Leu Trp Asp Gly Glu Val Leu Ala 145 150 155 160 Gly Val Pro Gly Pro Tyr Ala Gln Thr Gln Arg Val Arg Leu Gly Glu                 165 170 175 Trp Arg Leu Gln Leu Leu Glu Thr Arg Leu Asp Met Asp Leu Asp Gln             180 185 190 Gly Cys His Ala Glu Ala Val Ser Glu Leu Thr Ala Leu Thr Ala Ala         195 200 205 His Pro Leu Arg Glu Arg Leu Arg Glu Leu Leu Met Leu Ala Leu Tyr     210 215 220 Arg Ser Gly Arg Gln Ala Glu Ala Leu Ala Val Tyr Ala Asp Thr Arg 225 230 235 240 Arg Leu Leu Ala Asp Glu Leu Gly Val Asp Pro Arg Pro Gly Leu Gln                 245 250 255 Glu Leu Gln Gln Arg Ile Leu Gln Ala Asp Pro Ala Leu Ala Glu Leu             260 265 270 Ser Ala Ala Thr Ala Glu Thr Ala Thr Ala Thr Leu Arg Pro Ala Gln         275 280 285 Leu Pro Ala Thr Val Ser Asp Phe Thr Gly Arg Ala Ala Phe Val Arg     290 295 300 Glu Leu Ser Asp Val Leu Ser Ala Ala Ser Gly Glu Ser Ala Ser Gly 305 310 315 320 Arg Val Met Ala Val Ser Ala Leu Ala Gly Ile Gly Gly Val Gly Lys                 325 330 335 Thr Thr Leu Ala Val His Val Ala His Arg Ala Arg Ala Ala Phe Pro             340 345 350 Asp Gly Gln Leu Tyr Val Asp Leu Gln Gly Ala Gly Ala Arg Pro Ala         355 360 365 Glu Pro Glu Thr Val Leu Gly Ser Phe Leu Arg Ala Leu Gly Thr Ala     370 375 380 Asp Ser Ala Ile Pro Asp Ser Leu Glu Glu Arg Ala Ala Leu Tyr Arg 385 390 395 400 Ser Val Leu Asp Gly Arg Arg Val Leu Val Leu Leu Asp Asn Ala Arg                 405 410 415 Asp Ala Ala Gln Val Arg Pro Leu Leu Pro Gly Thr Asp Gly Cys Ala             420 425 430 Ala Leu Val Thr Ala Arg Val Arg Met Val Asp Leu Ala Gly Ala His         435 440 445 Leu Val Asp Leu Asp Val Met Ala Pro Glu Glu Ala Leu Ala Leu Phe     450 455 460 Thr Lys Ile Val Gly Glu Glu Arg Val Ala Ser Glu Arg Gln Ala Ala 465 470 475 480 Leu Asp Val Val Gly Ala Cys Gly Phe Leu Pro Leu Ala Ile Arg Ile                 485 490 495 Ala Ala Ser Arg Leu Ala Ala Arg Arg Thr Trp Thr Val Ser Val Leu             500 505 510 Ala Ala Lys Leu Ala Asp Glu Arg Arg Arg Leu Asp Glu Leu Gln Ala         515 520 525 Gly Asp Gln Ala Val Glu Ala Thr Phe Glu Leu Gly Tyr Gly Gln Leu     530 535 540 Glu Pro Ala Gln Ala Arg Ala Phe Arg Leu Leu Gly Leu Ala Asp Gly 545 550 555 560 Pro Asp Ile Ser Leu Ala Ala Ala Ala Ala Val Leu Asp Leu Pro Ala                 565 570 575 Gln Asp Thr Glu Asp Leu Leu Glu Ser Leu Val Asp Thr Ser Leu Leu             580 585 590 Glu Ser Ala Ala Pro Gly Arg Tyr Arg Phe His Asp Leu Val Arg Leu         595 600 605 Tyr Ala Arg Ala Cys Ala Glu Arg Thr Glu Arg Asp Gly Asn Ala Pro     610 615 620 Ser Glu Arg Gly Ala Ala Leu Ser Arg Leu Leu Asp Phe Tyr Leu Ala 625 630 635 640 Thr Ala Ala Gly Val Tyr Ala Ile Glu Arg Pro Gly Asp Arg Leu Val                 645 650 655 Asp Gly Leu Glu Pro Thr Glu Tyr Pro Gly Leu Thr Phe Thr Glu Gly             660 665 670 Ser Ala Ala Leu Asp Trp Leu Tyr Thr Glu Ala Ala Pro Leu Leu Ala         675 680 685 Cys Val Arg Gln Ser Ala Gly Thr Ala Arg Leu Arg Arg Ala Val Asp     690 695 700 Leu Leu Trp Ala Ala Lys Asp Leu Thr Glu Ser Gly Ala Asn Ser His 705 710 715 720 Gln Tyr Glu Ala Thr Ala Arg Ala Met Cys Asp Ala Thr Gly Ser Ala                 725 730 735 Ala Asp Thr Arg Ala Glu Gly Arg Ala Arg Thr Val Leu Ser Asp Val             740 745 750 Leu Leu Val Ser Gly Arg Ile Glu His Ala Glu Glu Glu Ala Arg Leu         755 760 765 Ala Met Arg Leu Ala Gly Ser Ala Glu Asp Ser Ala Ala Val Ser Trp     770 775 780 Val Ala Asn Asn Arg Gly Leu Val Cys Leu His Gln Arg Arg Tyr Ala 785 790 795 800 Glu Gly Lys Gly Leu Phe His Gln Ala Ile Ala Gly Phe Arg Ala Thr                 805 810 815 Asp Asn Arg Ala Gly Glu Ala Ser Ala Leu Ser Asn Leu Ser Arg Ala             820 825 830 Gln Leu Gly Met Gly Asn Val Ala Glu Ala Val Asp Ile Ala Arg Gln         835 840 845 Gly Leu Ala Val Tyr Ala Glu Leu Gly Arg Thr Met Arg Leu Ala Asn     850 855 860 Gly His Phe Ala Leu Gly Val Ala Leu Thr Arg Ala Gly Arg His Glu 865 870 875 880 Glu Ala Leu Gly Glu Phe Ala Glu Ala Leu Asp Leu Phe Gly Asp His                 885 890 895 Arg Gln Arg Leu Trp Glu Gly Ala Thr Asn Phe Arg Leu Ala Glu Val             900 905 910 His Leu Ala Ala Gly Arg Pro Ser Ser Ala Ala Gln His Ala Glu Gln         915 920 925 Ala Leu Ala Leu Gly Cys Ile Gly Gly Asp Arg Met Arg Gly Asn Val     930 935 940 Leu Ala Leu Leu Gly Arg Ala Leu Ser Ala Leu Gly Gln Ala Asp Arg 945 950 955 960 Ala Arg Ala Cys Trp Arg Glu Ala Leu Ser Leu Tyr Glu Gln His Asp                 965 970 975 Ala Gln Glu Val Gly Glu Val Arg Ala Leu Leu Ala Arg Ser Val Ala             980 985 990 Arg     <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aaaaaaggat cctgccaaga ccgcccctat 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aaaaaatcta gatcatgacc ccgagccttt 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aaaaaatcta gaaagctgat ggagaacctg 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aaaaaaaagc tttcaccgcg ccacactgcg 30  

Claims (8)

Streptomyces Libby thiooxidans (Streptomyces lividans) or Streptomyces nose Eli collar (Streptomyces coelicolor) (Streptomyces lividans) , Streptomyces Libby thiooxidans comprising a DNA fragment consisting of a nucleotide sequence of the sigma factor R (sigR) gene derived from Or an expression vector for transforming Streptomyces coelicolor to improve the productivity of a medically useful substance selected from the group consisting of antibiotics, antifungal agents, and anticancer agents. The expression vector according to claim 1, wherein the nucleotide sequence of the sigR gene is the nucleotide sequence of SEQ ID NO: 1. According to claim 1, consisting of the nucleotide sequence of Streptomyces Libby thiooxidans (Streptomyces lividans) or Streptomyces nose Eli collar (Streptomyces coelicolor) a afsR (A- factor synthesis modulators, A-factor synthesis regulator) gene derived from An expression vector further comprising a DNA fragment. The expression vector according to claim 3, wherein the nucleotide sequence of the afsR (A-factor synthesis regulator) gene is the nucleotide sequence of SEQ ID NO: 3. Streptomyces lividans or Streptomyces coelicolor transformed with the expression vector of any one of claims 1 to 4. delete Any one of claims 1 to 4 to any one of the expression vectors Streptomyces Libby thiooxidans (Streptomyces lividans) or Streptomyces nose Eli collar (Streptomyces coelicolor) transformants, Streptomyces Libby thiooxidans (Streptomyces, comprising the step of conversion of wherein lividans ) or Streptomyces coelicolor for improving the productivity of a medically useful substance selected from the group consisting of antibiotics, antifungal agents, and anticancer agents. delete

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