KR101112141B1 - Guanosine tetraphosphate synthase relA and its use for increasing antibiotic expression - Google Patents

Abstract

The present invention relates to a high phosphorylated guanosine nucleotide biosynthetic relA gene, and more specifically to a high phosphorylated guanosine nucleotide biosynthetic relA gene, characterized by encoding a protein having an amino acid sequence of SEQ ID NO: 2. It is about.
According to the present invention, a high phosphorylated guanosine nucleotide biosynthetic relA gene which induces an increase in the expression of natamycin can be obtained from Streptomyces natalensis , and the relA gene is introduced into Streptomyces natalenes. May increase the production of natamycin. Therefore, high production strains of natamycin can be obtained by using the relA gene and the expressed protein of the present invention, and it will be possible to use natamycin antibiotics at a low price. Therefore, the use of the composition for increasing the expression of antibiotics containing the relA gene of the present invention and its expression protein as an active ingredient is expected.

Description

Guanosine tetraphosphate synthase relA and its use for increasing antibiotic expression}

The present invention relates to a high phosphorylated guanosine nucleotide biosynthetic relA gene, and more specifically to a high phosphorylated guanosine nucleotide biosynthetic relA gene, characterized by encoding a protein having an amino acid sequence of SEQ ID NO: 2. It is about.

In food production, safe preservation of food is an important issue above all, which can reduce economic loss as well as consumer health. A major problem in food preservation in general is the decay and toxicity caused by microorganisms. This problem is a serious problem in processed food as well as unprocessed foods such as fruits that cannot be heat-treated and is one of the problems to be solved urgently. One of the solutions to the above problems is to use an antibacterial material that is harmless to the human body and has excellent antibacterial activity.

Currently, natamycin, produced by Actinomycetes Streptomyses natalensis , is a fungicide, a food preservative antibiotic (GRAS) that is not only toxic to humans, but also a fungus on the skin. It is also known to be effective against sexually transmitted diseases. In addition, the natamycin has been reported to play a role in inhibiting a wide range of antifungal activity and a large number of toxins produced by the fungus. In fact, treatment with 10 μg / ml natamycin inhibited the production of aflatoxin B1 by Apergillus flavus by about 62%. Penicilium The penicillic acid produced by patulum was inhibited by 98.8%. These results suggest the superiority of natamycin as an antifungal agent and the possibility of various industrialization of natamycin. Natamycin (purity 50%), which is currently sold by DSM, is sold at a very high price of about 1.5 million won per kilogram, which is one of the major reasons that hamper its use in various applications. . Therefore, in order to utilize the antibiotic in various uses, it is necessary to reduce the selling price, and to overcome this, above all, the productivity improvement by acquiring high production strains of natamycin is urgently required. However, it has been found that there is a limit in productivity improvement in the acquisition of high yielding mutant strains by single cell separation, X-ray and ultraviolet irradiation, and the acquisition of high yielding mutant strains by mutation inducer treatment.

Meanwhile, as a molecular biological study of actinomycetes, Streptomyses coelicolor ) Genetic studies of A3 (2) have been ongoing for a long time, centered on the John Innes Center in the UK. From these studies, not only basic knowledge but also genetic techniques have been accumulated, leading to the production of new bioactive substances by artificial manipulation of polyketide antibiotic biosynthesis genes. But actinomycetes, with their many secondary metabolic productions and complex life histories, have as much control as their features, but only a few have been found in research. Studies reported so far indicate that the control of biosynthesis and morphogenesis of secondary metabolites in Streptomyces actinomycetes is characterized by an autoregulator control system, high phosphorylated guanosine nucleotide (ppGpp) and S-adenosylmethionine. It has been found in part that representative regulatory actions such as S-adenosylmethionine (SAM) and the like are performed independently or organically.

Among these, intracellular accumulation of high phosphorylated guanosine nucleotide (ppGpp) is one of the first reactions to nutrient depletion for growth, and studies by Escherichia coli have shown that ppGpp synthase RelA and ppGpp synthase / hydrolase Functional SpoT synthesizes ppGpp from ATP and GTP, respectively. The RelA protein performs only ppGpp synthesis function with ribosomes under amino acid-limited conditions, while SpoT has ribosome-independent ppGpp synthesis function and magnesium-dependent pyrophosphohydrolase (P2 degradation) function under carbon source restriction conditions. It was found to be a bifunctional enzyme that degrades ppGpp. However, the synthetic activity of ppGpp of SpoT is not generally revealed by the more aggressive degradation activity in vivo.

PpGpp has also been shown to play an important role in secondary metabolism and morphogenesis in actinomycetes. When the relA gene is destroyed in S. coelicolor A3 (2), the pigment-based antibiotics actinorhodin and undecylprodigiosin produced by Streptomyces cericol Production was suppressed. These findings provide evidence that ppGpp plays a central role in the initiation of antibiotic biosynthesis, and further studies suggest that this is the route by which ppGpp induces the synthesis of actinolodine and undecylprodigiosin. Of actII-ORF4 and redD , genes of path- specific activator It has been found that this is achieved by promoting expression.

In addition, in the case of S. antibioticus , which produces actinomycin, relA gene removal also slowed the growth rate of the cells and produced the ability of actinomycin. It is reported that it is lost.

However, as we have seen, the research on Streptomyces actinomycetes has been actively conducted, while the relA gene study on Streptomyces natalensis, which produces an industrially important antibiotic, natamycin, has not been studied yet. It is a state.

Accordingly, the present inventors have diligently researched to overcome the problems of the prior arts. As a result, when the relA gene, a high phosphorylated guanosine nucleotide (ppGpp) synthase, is introduced into Streptomyces natalensis , the expression of natamycin is increased. The present invention was confirmed to be possible.

Accordingly, a primary object of the present invention is to provide a high phosphorylated guanosine nucleotide biosynthetic relA gene that induces increased expression of natamycin.

Another object of the present invention to provide a recombinant vector for increasing the expression of antibiotics containing the high phosphorylated guanosine nucleotide biosynthetic relA gene and its expression protein as an active ingredient.

According to one aspect of the invention, the invention provides a high phosphorylated guanosine nucleotide (ppGpp) biosynthetic relA gene, characterized by encoding a protein having the amino acid sequence of SEQ ID NO: 2.

The ppGpp is importantly involved in the production and morphogenesis of secondary metabolites of the bacterium, and is biosynthesized by the relA gene. This was confirmed by inhibiting the production of pigment-based antibiotics when the relA gene is destroyed in the bacterium. Therefore, increasing the expression of the relA gene increases the biosynthesis of ppGpp, it will be able to optimize the production of antibiotics.

In the relA gene of the present invention, the gene may have any nucleotide sequence encoding the relA protein, but preferably has a nucleotide sequence of SEQ ID NO: 1.

In a specific embodiment of the present invention, a complete ORF (protein code portion, SEQ ID NO: 1) of 2586 bp size was obtained from Streptomyces natalensis , and the obtained protein had high homology with relA protein derived from actinomycetes. It turns out that it has. Specifically, Streptomyces ( C. Streptomyces) clavuligerus) the homology of 93% in the relA 94% homology, and Streptomyces Scar ratio A (Streptomyces scabiei) of the relA of homology of 95% in the relA, Streptomyces Abbe reumi subtilis (Streptomyces avermitilis) Was observed. Thus, the ORF encodes relA, which encodes a highly phosphorylated guanosine nucleotide synthase in Streptomyces natalensis. It is thought to be a gene.

According to another aspect of the present invention, the present invention provides a high phosphorylated guanosine nucleotide biosynthetic relA protein, which has the amino acid sequence of SEQ ID NO: 2.

The protein of the present invention is characterized by promoting the biosynthesis of high phosphorylated guanosine nucleotides to increase the production of actinorhodin and natamycin.

In a specific embodiment of the present invention, when the relA gene derived from Streptomyces natalissis of the present invention is introduced into an expression vector to measure the production of actinolodine and natamycin, the expression vector (pSET152ET) is not introduced. It can be seen that the production of actinolodine and natamycin is significantly increased compared to). Thus, the 2.6 kb ORF cloned according to the present invention is the relA gene, which biosynthesizes high phosphorylated guanosine nucleotides in Streptomyces natalenes, indicating the actinolodine and Streptomyces natalylenes of Streptomyces Libidans TK24. It can be seen that it plays an important role in promoting the production of mycin.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the high phosphorylated guanosine nucleotidep biosynthetic relA gene.

In the present invention, the relA As a recombinant vector expressing a gene, any expression vector capable of expressing the gene may be used, such as pIJ459, pIJ4090, and preferably, pSET152ET that is expressible in actinomycetes.

The pSET152ET is inserted into the genome of actinomycetes and is stably maintained, and is an expression vector having a promoter of an erythromycin resistance gene whose expression in actinomycetes is confirmed.

In a specific embodiment of the present invention, after inserting the relA gene derived from Streptomyces natalensis obtained through the present invention into the promoter of the expression vector to produce a recombinant vector, streptomyces libby using conjugation transfer method Introduced in Dans TK24 or Streptomyces natalenes.

The recombinant vector of the present invention is characterized in that the pSET152ET_ relA having a cleavage map of FIG.

According to another aspect of the present invention, there is provided a host cell transformed with the recombinant vector of the present invention. The host cell of the present invention is specifically characterized in that the actinomycetes ( Streptomyces ). However, the host cell subjected to the transformation of the present invention does not require special limitation, for example, Streptomyces coelicolor , Streptomyces ambofaciens , Streptomyces You can use virginiae . In the present invention, Streptomyces lividans TK24 or Streptomyces natalansis of the genus Streptomyces is preferably used.

In the present invention, the host cell transformation method may be a protoplast method, an electroporation method, but preferably a conjugation transfer method.

According to an aspect of the present invention, the present invention provides a high phosphorylated guanosine nucleotide biosynthetic relA protein comprising culturing the host cell and obtaining a high phosphorylated guanosine nucleotide biosynthetic relA protein. It provides a method of manufacturing.

According to one aspect of the invention, the present invention provides a recombinant vector for increasing the expression of antibiotics containing the high phosphorylated guanosine nucleotide biosynthetic relA gene.

In the recombinant vector of the present invention, the gene is characterized in that it is inserted into the expression vector.

Figure 1 shows the results of PCR of the relA gene from Streptomyces Natalensis .
2 shows a recombinant vector into which the relA gene is inserted from Streptomyces natalensis .
3 shows the result of measuring the production amount of actinolodine.
Figure 4 shows the production measurement results of natamycin.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Strains and Expression Vectors

The strain used in the present invention is a natamycin-producing strain, the strain strain Streptomyces natalensis ( Streptomyses). natalensis ) pUC19 vector for ATCC 27448 and genome library construction and cloning, vector pSET152ET for relA gene expression and E. coli ( Escherichia coli ) XL-10Gold (Stratagene, UK) host was used from Niihira Professor of Osaka University, Japan. The detailed manufacturing process of the pSET152ET, the vector for relA gene expression is as follows.

First, the vector pSET152 produced by Bierman et al. For use as a vector for conjugation transfer for transformation of an antibacterial bacterium was used as a parent [Bierman M, Logan R, O'Brien K, Seno ET, Rao RN, Schoner BE. (1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene. 116 (1): 43-49]. In order to use the promoter of the erythromycin resistance gene as a promoter for efficient expression of genes cloned from Professor Nihira's team to the pSET152 vector, general expression vector pIJ487 was used [Ward JM, Janssen GR, Kieser T, Bibb MJ, Buttner MJ, Bibb MJ. (1986) Construction and characterization of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. 203 (3): 468-78. To isolate a promoter from the pIJ487 vector, vector pIJ487 was digested with restriction enzymes Eco RI and Bam HI to recover 260 bp DNA fragments. The Eco RI- Bam HI promoter thus obtained was prepared and finally an efficient expression vector pSET152ET (6.0 kb) was inserted into Eco RI- Bam HI of pSET152 [Lee YJ, Kitani S, Kinoshita H, Nihira T. (2010) Null mutation analysis of an afsA -family gene, barX , that is involved in biosynthesis of the γ-butyrolactone autoregulator in Streptomyces virginiae . Microbiology, 156: 206-210.

In addition, spores of the Streptomyces natalenes strain were used as receptors for conjugation transfer, and E. coli ET12567 / pUZ8002 (dam-13 :: Tn9, dcm-6, hsdM, hsdS) lacking methylation was used for conjugation transfer. As a DNA donor strain, pSET152ET (6 kb) into which a promoter of an erythromycin resistance gene was inserted was used as a vector for intracellular expression of the cloned gene.

MS medium (mannitol 20 g / L, soy flour 20 g / L, agar 20 g / L) was used for spore production and conjugation transmission of Streptomyces natalissis strain, and actinol using Streptomyces lividans R5 medium was used as the medium for rodin (actinorhodin) production, ISP2 liquid medium was used as the medium for chromosomal DNA extraction and natamycin production, and LB (for culture and transformation of E. coli and donor strains was used. Luria Bertani) medium was used. Reagents used in the experiment were purchased from Sigma Chemical Co., LTD, USA or Junsei co., LTD, Japan, and the enzyme was used as Takara shuzo co., LTD. Japan).

Example  2. Conjugation transfer to strain

First, donor strain E.coli ET12567 / pUZ8002 containing pSET152ET was transferred to 50 μg / ml of apramycin (Apr) for conjugation of the plasmid from E. coli to Streptomyses natalensis . , 25 μg / ml chloroamphenicol (Cm), 50 μg / ml of kanamycin (Km) was inoculated into the LB medium and then incubated at 600 nm until the absorbance was 0.45. When the culture was completed, washed twice with LB medium, and then resuspended with 0.1 times LB medium was used as a donor strain for conjugation transfer.

After unsustained Streptomyces natalis spores were suspended in 0.5 ml of 2 × YT medium, the donor strain E. coli ET12567 / pUZ8002 containing the expression vector was mixed well with 0.5 ml of the suspension to prepare MS containing 10 mM MgCl ₂ . It was plated on a solid medium and incubated at 28 ° C. for 16-20 hours. After completion of the culture, 0.5 mg of nalidixic acid and 1 mg of apramycin were superimposed on the medium to select a conjugate (transformer), and the selected conjugate was passaged on a selection medium containing antibiotics. Chromosomal DNA was extracted from the cultured cells. Then, the presence or absence of the transformation using PCR.

Example  3. PCR Using Streptomyces From Natalensis relA  Exploration of genes

Streptomyses In order to search for the relA gene in the genome of natalensis ), primers were designed and PCR were performed to design specific primers for the conserved regions obtained by comparing homology of amino acid sequences from relA derived from Streptomyces actinomycetes. PCR performance results are shown in FIG. 1.

As shown in FIG. 1, as a result of PCR, a PCR product of about 832 bp, which matches the expected size, was obtained.

The PCR product was purified, treated with restriction enzyme Eco RI, and then subjected to electrophoresis to recover the PCR product from agarose gel. The recovered product was cloned by inserting the restriction enzyme Eco RI position of pUC19, a cloning vector for E. coli. Thereafter, sequencing of the cloned PCR product was performed.

As a result of the sequencing analysis, relA derived from Streptomyces sp. It was confirmed that the homology was very high above 90% at the amino acid level. These results indicate that the PCR product obtained in the present invention is part of the relA code gene present in Streptomyces natales , and that relA exists in Streptomyces natales . Therefore, using the PCR product as a probe, the entire ORF encoding relA was cloned.

Example 4 From Streptomyces Natalensis relA  Cloning of genes

For cloning of the relA gene, the chromosomal DNA of Streptomyses natalensis was treated with various restriction enzymes, and Southern blots were prepared using the PCR product obtained in Example 3 as a probe. Was performed. For the Southern blot, a detection kit (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche Applied Science, Tokyo, Japan) was used as the probe.

About 7.5 kb of restriction enzyme Kpn I cleaved DNA fragment, which is expected to be able to clone the complete relA gene from the result of Southern blot, was selected for cloning, followed by colony blot. First, on the basis of the results of the Southern blot, a large amount of Streptomyces natalensis genomic DNA was treated with Kpn I enzyme followed by electrophoresis, and a DNA fragment of about 7.5 kb was recovered from an agarose gel. Thereafter, the DNA fragment was linked with the vector pUC19, which had already been cleaved with the restriction enzyme Kpn I, to transform the E. coli to prepare a DNA library. To clone from the prepared DNA library, colony blots were performed using the same probes used in the Southern blots.

As a result of sequencing of genomic DNA fragments obtained through the colony blot, it was confirmed that a complete ORF (SEQ ID NO: 1) having a size of 2586 bp including a PCR product was present. In addition, as a result of analyzing the homology of the obtained ORF using a database, not only the homology with the relA protein derived from actinomycetes at the amino acid level, but also the size of the ORF. Therefore, it was assumed that this ORF was the relA gene encoding high phosphorylated guanosine nucleotide synthase in Streptomyces natalensis . The homology analysis results are shown in Table 1 below.

Table 1. BLASTX search results of protein database using nucleotide query translated from ORF]

The relA gene cloned from the resulting Streptomyces natalenes was unpublished and registered under the accession number AB524881 in DDBJ / EMBL / GeneBank.

Example  5. Preparation of Recombinant Vectors

RelA obtained in Example 4 above For expression in actinomycetes, the pSET152ET vector [Lee YJ, Kitani S, Kinoshita H] has a promoter of the erythromycin resistance gene inserted into the genome of the actinomycetes and maintained stably and confirmed in actinomycetes. , Nihira T. (2010) Microbiology, 156: 206-210. 2.6 kb relA of Streptomyces natalenes obtained in Example 4 to the promoter of the expression vector The recombinant vector was constructed after inserting the gene into the restriction enzyme region Xba I of the expression vector pSET152ET in accordance with the transcription direction. The production process of the recombinant vector will be described in more detail as follows. First, in order to obtain an ORF of relA from a 7.5 kb DNA fragment inserted into the restriction enzyme Kpn I site of pUC19 vector, it was digested with restriction enzyme Xba I. The Xba I- Xba I DNA fragment containing the ORF of 2.6 kb relA obtained therefrom after confirming the transfer direction by inserting a promoter lower restriction sites Xba I of the Streptomyces expression vectors for pSET152ET, a recombinant vector such as pSET152ET_ relA 2 Prepared.

Example 6 Derivation of Streptomyces Natalensis Through High Expression of Genes relA Functional analysis of

It has been reported that relA genes from Streptomyces actinomycetes play a central role in initiating antibiotic biosynthesis by inducing the expression of genes encoding pathway-specific promoters that promote the synthesis of antibiotics. It turned out. In some cases, the growth of cells was also affected by the relA gene. Accordingly, the expression-inducing action of the gene encoding a pathway-specific promoter was confirmed for characterization and function analysis of the Streptomyses natalensis- derived relA gene obtained through the present invention. To this end, it was confirmed using Streptomyces lividans, which is generally used for molecular biology research of the bacterium. Streptomyces lividans are genetically very similar to Streptomyces cericol and normally contain a group of biosynthetic genes capable of producing the pigment-based antibiotics actinolodine and undecylprodigiosin, but induce the onset of antibiotic production. Due to the lack of factors, actinomycetes cannot produce actinolodine and undecylprodigiosin under normal conditions, unlike streptomyces cericol.

First, the recombinant vector for relA expression produced by connecting the 2.6 kb relA gene to the promoter of the pSET152ET vector in Example 5 was introduced into Streptomyces lividans TK24 using conjugation transfer, followed by actinol from the recombinant vector. The yield of rodin was confirmed. The method for measuring the actinolodine production is as follows.

After culture of the Streptomyces lividans TK24 strain into which the recombinant vector for relA expression was introduced, 1 ml of the culture was taken, centrifuged at 4 ° C. and 15,000 × g for 10 minutes, and the supernatant was collected. The collected supernatants were measured using a spectrophotometer at absorbance 600 nm. The measurement results are shown in FIG. 3.

As shown in Figure 3, the wild type strain (W) of Streptomyces lividans TK24, mutant strain (P) introduced only pSET152ET (vector without the relA gene, pSET152ET) into the wild type is very low production of actinolodine On the other hand, the mutant strain (C) into which the recombinant vector into which the relA gene was inserted was confirmed, significantly increased the production of actinolodine. These results suggest that the expression of the relA gene is responsible for the increased expression of pathway-specific promoters that induce the biosynthesis of antibiotics and promote biosynthesis.

In addition, in order to investigate the effect of relA on natamycin production in Streptomyces natalensis, the recombinant vector containing the relA gene constructed in the Streptomyces Libidans study was inserted in the same manner as above. After introduction into cis, the production amount of natamycin from the recombinant vector was confirmed. The method of measuring natamycin production is as follows.

After culture of the Streptomyces natalensis strain into which the recombinant vector for relA expression was introduced, 0.1 ml of the culture solution was mixed with 0.9 ml of methanol, and centrifuged at 4 ° C. and 15,000 × g for 10 minutes. The supernatant was collected. With the collected supernatant, the production of natamycin was measured using the following two methods. The first method uses a disk agar diffusion method using Saccharomyces cerevisiae as an indicator strain, and the second method uses 60% (v / v) methanol / water on a C18 reversed-phase column using HPLC. The solvent was measured using a method that flowed at a flow rate of 0.8 ml / min and measured by a UV detector at 305 nm. The measurement results are shown in FIG. 4.

As shown in Figure 4, at 36 hours of liquid culture, the wild type strain (W), strain (P) introduced only pSET152ET (vector without the relA gene inserted) into the wild type increased production but recombinant vector inserted with the relA gene The introduced strain (C) is started production in 24 hours, it can be confirmed that the production time is advanced by rel A. In addition, the production of natamycin in the wild type strain (W), the mutant strain (C) into which the recombinant vector into which the relA gene was inserted was introduced, compared to the mutant strain (P) into which only the pSET152ET (vector without the relA gene inserted) was introduced into the wild type. It was confirmed that about 2.5 times increased.

Therefore, the 2.6 kb ORF cloned according to the present invention is the relA gene for biosynthesis of high phosphorylated guanosine nucleotides (ppGpp) in Streptomyces natalenes, the production of Actinolodine of Streptomyces Libidans and Streptomyces natal It can be seen that it plays an important role in promoting the production of natamycin in lensis.

As described above, according to the present invention, a high phosphorylated guanosine nucleotide biosynthetic relA gene which induces an increase in the expression of natamycin can be obtained from Streptomyces natalensis , and the relA gene is obtained from Streptomyces natal When introduced into lanceis, it is possible to increase the production of natamycin.

Therefore, high production strains of natamycin can be obtained by using the relA gene and the expressed protein of the present invention, and it will be possible to use natamycin antibiotics at a low price. Therefore, the use of recombinant vectors for increasing the expression of antibiotics containing the relA gene of the present invention as an active ingredient is expected.

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Claims (11)

A high phosphorylated guanosine nucleotide biosynthetic relA gene, characterized by encoding a protein having an amino acid sequence of SEQ ID NO: 2.
The high phosphorylated guanosine nucleotide biosynthetic relA gene according to claim 1, wherein the gene has a nucleotide sequence of SEQ ID NO: 1.
A high phosphorylated guanosine nucleotide biosynthetic relA protein, having the amino acid sequence of SEQ ID NO: 2.
The biosynthesis of guanosine nucleotide biosynthesis according to claim 3, wherein the protein promotes biosynthesis of high phosphorylated guanosine nucleotides to increase the production of actinorhodin and natamycin. relA protein.
A recombinant vector comprising the high phosphorylated guanosine nucleotide biosynthetic relA gene according to claim 1.
The method of claim 5, wherein the recombinant vector is a recombinant vector, characterized in that pSET152ET_ relA having a cleavage map of FIG.
A host cell transformed with the recombinant vector of claim 5.
8. The transformed host cell of claim 7, wherein the host cell is actinomycetes.
Of claim 7 wherein the step of culturing a host cell according to the deceased oxidizing guanosine nucleotide biosynthesis Highly oxidized guanosine nucleotide comprising the step of obtaining a relA protein (guanosine nucleotide) biosynthesis relA Method of producing protein.
Recombinant vector for increasing the expression of antibiotics containing the high phosphorylated guanosine nucleotide biosynthesis relA gene according to claim 1 as an active ingredient. delete

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