KR20030083541A - Recombinant spectinomycin biosynthesis enzyme, gene sequences coding for the same and transformants thereof - Google Patents

Abstract

PURPOSE: Spectinomycin biosynthesis enzyme, the nucleotide sequence thereof and transformants thereof are provided. The spectinomycin biosynthesis enzyme produced from the transformant has improved spectinomycin biosynthesis activity. CONSTITUTION: A gene encoding DTDP-glucose synthase(SpcD) isolated from Streptomyces spectabilis(ATCC 27741) has the nucleotide sequence of SEQ ID NO: 1. A gene encoding DTDP-glucose-4,6-dihydratase(SpcE) isolated from Streptomyces spectabilis(ATCC 27741) has the nucleotide sequence of SEQ ID NO: 3. A gene encoding myo-inositol monophosphatase(SpeA) isolated from Streptomyces spectabilis(ATCC 27741) has the nucleotide sequence of SEQ ID NO: 5. A gene encoding myo-inositol-dehydrogenase(SpcB) isolated from Streptomyces spectabilis(ATCC 27741) has the nucleotide sequence of SEQ ID NO: 3. The transformants of E. coli BL21 are produced by transforming E. coli BL21 with pETSpcD(KACC95003) containing spcD gene, pETSpcE(KACC95004) containing spcE gene, pETSpeA(KACC95001) containing speA gene, and pETSpeB(KACC95002) containing speB gene.

Description

Recombinant spectinomycin biosynthesis enzyme, gene sequences coding for the same and transformants according}

The present invention relates to a recombinant spectinomycin biosynthetic enzyme, a gene sequence encoding the same, and a transformant thereof. More specifically, the present invention relates to a Ditipi-glucose synthase, Ditidipi glucose-4,6-dihydratase from Streptomyces spectabilis ATCC 27741 for biosynthesis of the antibiotic spectinomycin. , A gene sequence and amino acid sequence of myo-inositol monophosphate, myo-inositol dihydrogenase, and a transformant containing the gene are prepared and expressed to express biotinylated spectinomycin. .

Indiscriminate abuse of antibiotics has led to the emergence of bacteria, fungi, and viral cancer cells that are resistant to conventional antibiotics, and actually reported by the US Centers for Disease Control and Prevention (CDC, USA) in 1,300 hospitals in 1992. Patients were reported to have died from bacterial diseases that were highly resistant to antibiotics, which further increased the demand for renal antibiotics to combat them. To date, about 10,000 antibiotics are separated from microorganisms, and only about 100 of them are commercialized. Over time, the success rate and commercialization rate of new functional antibiotics have been significantly reduced. In order to solve this problem, in the case of aminoglycoside antibiotics, a method of neutralizing resistance mechanisms of resistant bacteria by synthesizing and attaching an artificial side chain to an existing antibiotic structure This has been widely done. Therefore, semi-synthetic products such as amikacin developed by nexstar and amplimed, debekacin and arbekacin from meiji Seika, Japan, and ispamycin from Schering-Plough, etc. It has led the market for lycoside antibiotics. However, since these semisynthetic products are targeted only to the target antibiotic, for example kanamycin for amikacin, and gentamicin for ispamicin, It is limited and has a disadvantage in that the unit price is high in the process due to the synthetic product rather than the natural fermentation product. Therefore, in order to overcome the above problems, instead of synthesizing side chains, the focus is on the development of new materials that modify the structure of existing antibiotics by introducing useful genes using genetic engineering techniques and actinomycetes vectors. At some point, antibiotic biosynthesis studies, which underlie these studies, are becoming increasingly important.

An object of the present invention is derived from Streptomyces specabibilis (STreptomyces specabibilis ATCC 27741), which is partly inserted into the cosmid clone into which a population of spectinomycin biosynthetic genes derived from Streptomyces spectabilis (ATCC 27741) A gene encoding SpcD, which is dTDP-glucose synthase, and a gene encoding SpcE, which is dTDP-glucose-4,6-dehydratase , The nucleotide sequence of a gene encoding SpeA, a myo-Inositol monophosphatase, and a gene encoding SpeB, a myo-Inositol dehydrogenase, and an amino acid sequence translated therefrom To provide. Still another object of the present invention is to provide a transformant prepared by connecting a spectinomycin biosynthetic gene derived from Streptomyces spectabilis ATCC 27741 to a plasmid and then introducing into E. coli. Another object of the present invention to provide a recombinant protein SpcD, SpcE, SpeA, SpcB produced by culturing the transformant.

The above object of the present invention is to create a gene library using the genome of S. spectabilis ATCC 27741, a spectinomycin producing strain, amplified by PCR using AG3 and AG4 primers and then PCR Isolates clones containing two cosmids, pHCG121 and pHCG171, that respond positively to the expected size band in the product, and transforms the streptomyces lividans sensitive to spectinomycin with these two cosmids. After confirming the resistance of the transformant to spectinomycin and subcloning all BamHI fragments contained in the two cosmids into the E. coli plasmid pBluescript KS (+), a sequential sequence of about 16 kb was determined by bidirectional sequencing. After confirming that the coding regions of SpcD and SpcE, SpeA and SpeB exist, and comparing the amino acid sequence of this gene, SpcD -Di-glucose synthase and SpcE are ditipid-glucose-4,6-dihydrase, SpeA is myo-inositol-monophosphatase, and SpeA is homologous to myo-inositol dehydrogenase and other microorganisms It was achieved by revealing and confirming its activity.

Figure 1 shows the results of electrophoresis and Southern hybridization of the cosmid pHCG121, pHCG124, pHCG125, pHCG171 into which the spectinomycin biosynthetic gene group was inserted with restriction enzyme Bam HI.

Figure 2 shows 30 kb of spectinomycin biosynthetic gene group and the present invention D. typti-glucose synthase (SpcD), Dtidpi-glucose-4,6- of Streptomyces spectabilis ( S. spectabilis ATCC 27741) Gene maps of dehydratase (SpcE), myo-inositol monophosphatase (SpcA) and myo-inositol dehydrogenase (SpeB) are shown.

FIG. 3 shows the results of homology searches compared to US GenBank data based on the D. tidpi-glucose synthase (SpcD) amino acid sequence derived from S. spectabilis ATCC 27741 of the present invention.

FIG. 4 compares US GenBank data based on the D.T.D.-glucose-4,6-dihydrotase (SpcE) amino acid sequence from S. spectabilis ATCC 27741 of the present invention. FIG. Show homology search results.

FIG. 5 shows the results of homology searches compared to US Genbank data based on the myo-inositol monophosphatase (SpeA) amino acid sequence derived from S. spectabilis ATCC 27741 of the present invention. .

FIG. 6 shows the homology search results compared to US GenBank data based on myo-inositol dehydrogenase (SpeB) amino acid sequence derived from S. spectabilis ATCC 27741 of the present invention. FIG. .

Figure 7 shows the reaction of the enzyme produced by expressing a gene encoding D. typti-glucose synthase (SpcD) derived from S. spectabilis ATCC 27741 of the present invention in E. coli HPLC (High Performance Liquid) Chromatography) shows the results.

Figure 8 is an enzyme produced by expressing a gene encoding E. coli Ditipi-glucose-4,6-dihydratase (SpcE) derived from the present invention Streptomyces spectabilis ( S. spectabilis ATCC 27741) The reaction product is analyzed by HPLC (High Performance Liquid Chromatography).

Figure 9 shows the reaction of the enzyme produced by expressing a gene encoding myo-inositol monophosphatase (SpeA) derived from S. spectabilis ATCC 27741 of the present invention in Escherichia coli HPLC (High Performance) Liquid Chromatography) shows the result.

10 is a HPLC (High Performance Liquid) product of the enzyme produced by expressing a gene encoding myo-inositol dehydrogenase (SpeB) derived from S. spectabilis ATCC27741 of the present invention in Escherichia coli. Chromatography) shows the results.

The present invention comprises the steps of preparing a gene library using the genome of S. spectabilis ATCC 27741, a spectinomycin producing strain; Primer AG3 (5'-CTSGCSGGCGGCWSSGGSACSMGSCTSYACCC-3 ') and AG4 (5) to effectively amplify DTI-DIP-glucose synthase, which is essential for the biosynthesis of the sugar actinectose, the 6-deoxyhexose of spectinomycin. '-RYGTCSGTGATCTCSAGCTCGCCSCG-3') was used to amplify by PCR and isolate the two cosmid clones pHCG121, pHCG171 which showed positive reaction with a band of expected size, and then separated the two cosmid pHCG121, pHCG171 The two cosmids pHCG121 and pHCG171 were specified by transforming Streptomyces lividans sensitive to spectinomycin and confirming that the pHCG171 was resistant to spectinomycin and that the pHCG121 had a common site. Investigating the involvement in thyomycin biosynthesis; After subcloning all BamHI fragments contained in the two cosmids, pHCG121 and pHCG171, into the E. coli plasmid pBluescripKS (+), the nucleotide sequence of about 16 kb fragments was determined by bidirectional sequencing and the SpcD, SpcE, and SpeA of the gene group were determined. And after confirming that the region encoding SpeB exists, comparing the amino acid sequence translated from the determined nucleotide sequence, SpcD is a Dtytipi-glucose synthase, and SpcE is a Dtyti-glucose-4,6-dihydrotase. , SpeA is myo-inositol-mophosphatase and SpeB is a homology of myo-inositol dehydrogenase and Tami organism; Based on the identified homology, the spcD , spcE , speA and speB genes were transformed into Escherichia coli and overexpressed, followed by enzymatic reaction to confirm the product by HPLC.

Hereinafter, the present invention will be described in detail.

The present invention is to cultivate S. spectabilis ATCC 27741, to prepare a genetic library of S. spectabilis ATCC 27741, culturing the chromosome DNA pure, and then purified The separated chromosomal DNA is partially cut with a restriction enzyme and linked to a cosmid vector for producing a gene library to prepare a gene library.

Recombinant cosmid clones containing spectinomycin biosynthetic genes are also selected using PCR and transformed into streptomyces lividans ATCC 1326 to confirm resistance to spectinomycin. At this time, the PCR primer is a primer AG3 (5'-CTSGCSGGCGGCWSSGGSACSMGSCTSYACCC), which targets D.T.-CTSGCSGGCGGCWSSGGSACSMGSCTSYACCC for the biosynthesis of all antibiotics having sugars derived from 6-deoxyhexose of antibiotic structure. -3 ') and AG4 (5'-RYGTCSGTGATCTCSAGCTCGCCSCG-3') are advantageous. As a result of the PCR reaction, the PCR bands of the expected size were identified, plasmids were extracted from the two identified original samples, and PCR was again performed to select two recombinant cosmid clones pHCG121 and pHCG171. The recombinant recombinant cosmid was used to transform streptomyces lividans ATCC 1326, which is not resistant to aminoglycoside antibiotics, and then 20 g / mL thiostrepton, an antibiotic marker of pDW103. Indirectly confirming that the biotin gene was included by selecting transformants having thiostrepton and spectinomycin resistance in a solid medium containing 60 g / mL of spectinomycin, and thus, the pHCG171 recombinant cosmid It can be confirmed that it has the ability to resist.

Gene encoding the present invention Didipid-glucose synthasespcD,Gene encoding Dytidipi-glucose-4,6-dihydratasespcE,Genes encoding myo-inositol monophosphatasespeAAnd Genes encoding myo-inositol dehydrogenasespeBIn order to determine the nucleotide sequence and the amino acid sequence of the two recombinant cosmid restriction enzymeBamHICleaved and subcloned the plasmid into a plasmid and subjected to bidirectional sequencing to obtain Streptomyces spectabilis (S. spectabilisATCC 27741) determine the overall gene sequence of SpcD, SpcE, SpcA and SpcB. The FramePlot 2.3.2 program can be used to search for the portion of the gene encoding the protein and to confirm the presence of an open reading frame. The gene sequence and amino acid sequence are shown in SEQ ID NO: 1 to 8. SpcD is a streptomymias network, dTDP-D-glucose synthase (Streptomyces netropsisSpcK (spectinomycin) and 78% of StreptomycesStreptomyces rishiriensisCouV (coumermycin A1) and 73% (Wang et al., 2000), and Streptomyces spheroidsStreptomyces spheroides)Has 72% homology with NovV (novobiocin) (Steffensky et al., 2000), and SpcE is 77% with SpcJ from Streptomyces necrosis,Streptomyces globisporus) And 69% homologous to SgcA and 65% to Streptomyces spheroids NovT. SpeA is a MT2775 of Mycobacterium tuberculosis CDC1551, a gene encoding inositol monophosphatase, 34%, and mesohibium rotiMesorhizobium loti37% of Mlr4874) and 50% homology with SpcA of Streptomyces netropsis. SpeB is 74% homologous to myo-inositol dehydrogenase, which is encoded by spcB of the same spectinomycin producing strain Streptomyces neuropsis, and NADH-dependent dehydro, which is encoded by mlr2436 of mesohizobiloti. 30% homology with genase (NADH-dependent dyhydrogenase).

To express the spcE, spcD, speA and speB genes and confirm their activity, spcD and spcE were isolated from the recombinant cosmid pHCG121 and linked to pETB21a + to construct pETSpcD and pETSpcE , and speA and speB were isolated from pHCG171 and to pETB21a +. After connecting to construct pETSpeA and pETSpeB, each is inserted into E. coli BL21 and transformed. The recombinant vector was deposited on April 12, 2002 by the Institute of Agricultural Biotechnology, pETSpcD as KACC95003, pETSpcE as KACC95004, pETSpeA as KACC95001, pETSpeB as KACC95002. E. coli transformants are cultured to induce expression. After harvesting and grinding the cells, the cell extracts are separated and purified. The enzyme activity of each of these SpcD, SpcE, SpcA, SpcB can be identified through HPLC analysis. It can be seen that the peak of dTTP used as the substrate for the SpcD reaction decreased significantly after 30 minutes and a new peak of dTDP-D-glucose was formed. It can be seen that it has the function of biosynthesizing di-glucose. In the SpcE reaction, the peak of NAD decreases and the peak of NADH increases as the reaction time elapses, and the new estimated Dtidipi-4-keto-6-deoxy-di-glucose at the retention time of 7 minutes. It can be seen that the peak is generated. In the absorbance experiment after the enzyme reaction using the cell extract, the function of SpcE was also confirmed. When compared with E. coli, which contains only the vector without the gene, NADH, the product of DtiDypi-di-glucose-4,6-dihydratase. The amount of was about three times higher. Therefore, these experiments confirmed that SpcE performs the function of D Tiffy-Di-glucose-4,6-dihydratase. In addition, SpeB analysis showed that the transformants containing only the control vector did not produce any new peaks even after the reaction time. However, the SpeB enzyme reactant produced the enzyme reaction product of myo-inositol dehydrogenase, a commercial enzyme. It can be seen that a new peak is formed at the retention time of 10 minutes. Therefore, it can be seen that this SpeB performs the function of myo-inositol dihydrogenase. As a result of the analysis of SpeA, it can be seen that as the reaction time elapses, a new peak is generated at the same retention time as the standard reagent myo-inositol (3.1 min), so SpeA converts myo-inositol-1-phosphate to myo-inositol It can be seen that it performs the function of myo-inositol monophosphate.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1: Streptomyces spectabilis ( Streptomyces spectabilis Gene library of ATCC 27741)

Streptomyces spectabilis ATCC 27741 was incubated in R2YE medium, the chromosomal DNA was purified according to the method of Hopwood, and the chromosomal DNA was partially cut with restriction enzyme Sau3A, A 30-40 kb fragment of chromosomal DNA doubled with BamHI and HpaI to cosmid vectors for replication in E. coli and actinomycetes. The Streptomyces spectabilis gene library was prepared using a lambda packaging kit.

Example 2: Cloning of Spectinomycin Biosynthesis Genes and Screening for Resistance in Spectinomycin in Streptomyces Lividans

1000 colonies of the Streptomyces spectabilis gene library prepared in Example 1 were inoculated using a yellow tip into a 1.5 mL Eppendorf tube containing 500 μL of LBA liquid medium and incubated in an incubator for one day. Samples were transferred to a new Eppendorf tube in 30 μL cultures from 50 samples. Eventually 50 samples were transferred to one Eppendorf tube and the number of samples transferred to the new tube was reduced from the first 1000 to 20. Plasmids were extracted from the collected 20 samples, and PCR reactions were performed using the DNA of 20 samples. The PCR primer used was TDP-glucose synthase (TDP-) involved in the initial synthesis of sugars for biosynthesis of all antibiotics with sugars derived from 6-deoxyhexose such as spectinomycin. AG3 (5'-CTSGCSGGCGGCWSSGGSACSMGSCTSYACCC-3 ') and AG4 (5'-RYGTCSGTGATCTCSAGCTCGCCSCG-3') primers developed for glucose synthase) were used. As a result of the PCR reaction, PCR bands of two sizes among 20 samples were identified, plasmids were extracted from 100 samples (470 μL) used to prepare two samples, and PCR was performed again. Recombinant cosmid clones pHCG171, pHCG121 were selected. Two isolated cosmids were transformed into streptomyces lividans ATCC 1326, which is not resistant to aminoglycoside antibiotics, using the method of Hopwood, followed by antibiotics of pDW103. It was incubated in a solid medium containing 20 g / mL of the archer thiostrepton and 60 g / mL of spectinomycin. By selecting the transformants having the resistance to thiostrepton and spectinomycin, the indirect confirmation of the inclusion of biosynthetic genes was confirmed that the pHCG171 recombinant cosmid was resistant to the two antibiotics. Figure 1 shows the result of electrophoresis of the pHCG171 resistance and the cosmid pHCG121 clone that shows positive in PCR reaction using AG3, AG4 primers but not resistant to the restriction enzyme BamHI, pHCG121 is spectinomycin resistance Is not visible, but the two clones overlapped each other.

Example 3: Invention spcD Wow spcE Genetic and Amino Acid Sequence Determination and Similarity Search

Since enzymes involved in antibiotic biosynthesis generally exist in a certain region of the genome, aminoglycosides also exist in the insertion fragments of 30-40 kb of two cosmid clones pHCG171 and pHCG121 where spectinomycin biosynthetic gene populations exist. Since there will be a dTDP-glucose synthase gene used for antibiotic production, the two cosmid clones were digested with restriction enzyme BamHI, and then all fragments were subcloned into E. coli plasmid pBluescript KS (+). Bidirectional sequence analysis was performed at 800 bp to determine the overall sequence of SpcD and SpcE from pHCG171 and SpeA and SpeB from pHCG121.

The base sequences of the SpcD, SpcE, SpeA and SpeB genes and the amino acid sequences translated from the base sequences are shown in SEQ ID NOs: 1-8. Using the FramePlot 2.3.2 program (ishikawa, J. and Hotta, K. FEMS Microbiol. Lett. 174: 251-253, 1999), the portion encoding the protein was searched based on the primary DNA sequence results. Experimental results show that there is an open reading frame encoding proteins in this region.

The protein named SpcD consists of 291 amino acids starting with start codon ATG and ending with stop codon TGA, SpcE consists of 330 amino acids starting with ATG and ending with stop coton TGA, and SpeA starts with ATG and stop codon It consists of 265 amino acids ending in TGA and SpeB consists of 374 amino acids starting with GTG and ending with stop codon TAA. In addition, as a result of amino acid homology search through the database, SpcD was 78% of SpcK (spectinomycin) of Streptomyces netropsis , a streptomyces netropsis, It has a homology of 72% with CouV (coumermycin A1) of Streptomyces rishiriensis and 73% (Wang et al., 2000) and NovV (novobiocin) of Streptomyces spheroides (Steffensky et al. , 2000), SpcE showed 77% homology with SpcJ of Streptomyces necrosis, 69% with SgcA of Streptomyces globisporus , and 65% with NovT of Streptomyces spheroids. At least 60% homology with other genes encoding D. tidypi-glucose-4,6-dihydratase. SpeA is MT2775 and 34% of Mycobacterium tuberculosis CDC1551, a gene encoding inositol monophosphatase, Mlr4874 and 37% of Mesorhizobium loti , streptomycin High homology with SpcA of Streptomyces netropsis was 50%. SpeB obtained 74% homology with myo-inositol dihydrogenase, which is encoded by spc B of the same spectinomycin-producing strain Streptomyces neuropsis, and NADH-D, which is encoded by mlr2436 of mesozozium loti. A homology of 30% was observed with NADH-dependent dyhydrogenase.

Example 4 Gene of the Invention spc D and spc E, spe A, spe Preparation, Expression, and Confirmation of Enzyme Activity Expressed with B

The spcD and spcE genes were separated from pHCG121 by PCR, and then digested with EcoRI-XhoI and connected to pET21a + digested with the same enzymes to construct pETSpcD and pETSpcE.SpE and speB genes were separated from pHCG171 by PCR. Then, it was digested with EcoRI-XhoI and connected to pET21a + digested with the same type of enzyme to construct pETSpeA and pETSpeB. PETSpcD as KACC95003, pETSpcE as KACC95004, pETSpeA as KACC95001 and pETSpeB as KACC95002 dated April 12, 2002. pETSpcD, pETSpcE, pETSpeA and pETSpeB were transformed into E. coli BL21 and incubated for 14 hours in 3 mL LB medium containing 50 g / mL ampicillin. Each cultured E. coli transformant was inoculated 1% in 250 mL LB medium (50 g / mL ampicillin), incubated at 600 nm to OD 0.5, and then added to a concentration of 1 mM IPTG to induce expression. Cultured cells were collected by growing until cell growth became OD 1 at 600 nm and centrifuging for 15 minutes at 5,000 rpm. The collected cells were pulverized by ultrasonication and the cell free extracts were separated by centrifugation, and the proteins of SpcD, SpcE, and SpeA and SpeB were purified through his-resin colunm. Enzymatic reactions to confirm the function of SpcD were carried out with purified SpcD containing 50 mM Tris-HCl (pH 8.0), 12 mM MgCl ₂ , 24 mM di-glucose-1-phosphate, 6 mM dTTP, 1.8 U inorganic pyrophosphatase. The total volume of the reaction mixture was adjusted to 300 L for 0 minutes, 30 minutes, and 60 minutes at 37 ° C. 1 mL of 50 mM phosphate buffer solution (pH 3.0) was added to terminate the reaction. The reaction mixture was stored at -20 ° C until analysis. . Enzyme reaction to confirm the function of SpcE was carried out by adding 160 mM Tris-HCl (pH7.9), 8 mM NAD +, 1.6 mM Didepi-glucose and SpcE cell extract to 300 μL of the total reaction mixture and mixing well at 37 ° C. After reacting for 30 minutes, 60 minutes, immediately stored at −20 ° C. until analysis. Enzyme reaction to confirm the function of SpeA was performed by mixing 20 mM Tris-HCl (pH 7.5), 3 mM MgCl ₂ , 0.7 mM myo-inositol-1-phosphate and SpeA cell extract in 300 μL of the whole reaction mixture. After reacting for 30 minutes and 60 minutes, immediately stored at -20 ° C until analysis. Enzyme reaction to confirm the function of SpeB was performed by mixing 200 mM myo-inositol, 4 mM NAD +, 50 mM Tris-HCl (pH 9.5) and SpeB cell extract in 300μL of the whole reaction mixture. After reacting for a minute, immediately stored at −20 ° C. until analysis, and commercial enzyme (Sigma I0255) was also reacted under the same conditions. Enzyme reaction products of SpcD, SpcE, SpeA, and SpeB were identified through HPLC. Analytical conditions for SpcD were anion exchange resin N (CH3) 2-1101-N column (0.46 x 10 cm), solvent at flow rate 1.5 mL / min and mobile phase 50 mM potassium phosphate (pH). 4.0) (solution A) and 600 mM potassium phosphate (pH 4.0) (solution B) were run in a concentration gradient for 20 minutes. Concentration gradient was carried out so that only A solvent was flowed for the first 1 minute, and then B concentration was 100% for 15 minutes, and again, concentration gradient was made to be 100% A solvent for 5 minutes, followed by 1 minute solvent A only. More flow to stabilize the column. The detector was a UV detector and the analysis of the enzyme reaction product was carried out at 254 nm ultraviolet wavelength. Dititipi and Ditipi-Glucose (Sigma) were used as standard reagents. As a result, it can be seen that the peak of Dititi used as a substrate for the SpcD reaction was significantly reduced after 30 minutes and a new Ditipi-di-glucose peak was formed, which was the standard reagent Dtidipi-di- purchased from Sigma. Because of the same retention time as glucose, SpcD was found to have the function of biosynthesis of Dtyti-di-glucose. The analysis conditions of SpcE and SpeB are the same as SpcD, but the flow rate was 1.0 mL / min, and there are slight differences in concentration gradient conditions as follows. In the first 2 minutes, only A solvent was flowed, and the concentration gradient was adjusted so that B solvent became 50% for 10 minutes. Then, the concentration gradient was adjusted so that A solvent became 100% for 5 minutes. The column was stabilized. As a result, it was confirmed that the peak of NAD decreases and the peak of NADH increases as the reaction time elapses in SpcE reaction, and it is estimated to be Dinipi-4-keto-6-deoxy-D-glucose at the retention time of 7 minutes. It was confirmed that a new peak was generated. In the absorbance experiment after the enzyme reaction using the cell extract, the function of SpcE was also confirmed. When compared with E. coli, which contains only the vector without the gene, NADH, the product of DtiDypi-di-glucose-4,6-dihydratase. The amount of was about three times higher. Therefore, these experiments confirmed that SpcE performs the function of D Tiffy-Di-glucose-4,6-dihydratase. In addition, SpeB analysis showed that the transformants containing only the control vector did not produce any new peaks even after the reaction time. However, the SpeB enzyme reactant produced a commercial enzyme, myo-inositol dehydrogenase. It can be seen that a new peak is formed at the retention time of 10 minutes. Therefore, this SpeB was found to perform the function of myo-inositol dehydrogenase. The SpeA analysis was performed using the reverse phase Bondapak TMC18 (3.9 × 300 mm, Waters) column and 20% acetonitrile as the mobile phase at 0.8 mL / min at 254 nm. Analyzed. A standard reagent of myo-inositol was used (myo -inositol) (Sigma). As a result, it was found that as the reaction time elapsed, a new peak was generated at the same retention time as the standard reagent myo-inositol (3.1 min). Therefore, SpeA converts myo-inositol-1-phosphate to myo-inositol. It can be seen that there is a function of myo-inositol monophosphatase.

Absorbance Assay of BlmE Enzyme Experiment Gene structure Product of Didipid-di-glucose-4,6-dihydratase SpcE NADH (nmol / min / mg of protein) IPTG + IPTG pET21a + (vector) 9.63 ± 1.92 10.25 ± 0.642 pETBlmE (vector with spc E inserted) 11.54 ± 1.25 32.1 ± 1.283

As described above, SpcD, SpcE, SpeA, and SpeB prepared by the present invention have an excellent effect as an important enzyme commonly involved in not only spectinomycin antibiotics but also other aminoglycoside antibiotic biosynthesis, and its product, Diff-glucose and myo-inositol dehydrogenase are not only excellent for their use as substrates and enzymes important for antibiotic biosynthesis research, but also because of their economic value, such as being sold as expensive reagents by pharmaceutical companies. It is a very useful invention.

Claims (12)

The gene base sequence set forth in SEQ ID NO: 1 encoding Dytipid-glucose synthase (SpcD) from Streptomyces spectabilis ATCC 27741. The gene base sequence set forth in SEQ ID NO: 3 encoding Dytipid-glucose-4,6-dihydratase (SpcE) from Streptomyces spectabilis ATCC 27741. The gene base sequence set forth in SEQ ID NO: 5 encoding myo-inositol monophosphatase (SpeA) from Streptomyces spectabilis ATCC 27741. The gene base set forth in SEQ ID NO: 3 encoding myo-inositol-dehydrogenase (SpcB) from Streptomyces spectabilis ATCC 27741. Transformation prepared by introducing pETSpcD (KACC95003), which was linked to pET21a +, by integrating the gene spcD encoding StiD-glucose synthase (SpcD) from Streptomyces spectabilis ATCC 27741 into E. coli BL21 sieve. E. coli BL21 was linked to pETSpcE (KACC95004), which was linked to pET21a + by the gene spcE , which encodes Dtipid -glucose-4,6-dihydratase (SpcE) from Streptomyces spectabilis ATCC 27741. Transformants prepared by introduction into. Transformants prepared by introducing pETSpeA (KACC95001) in which the gene speA encoding myo-inositol monophosphatase (SpeA) derived from Streptomyces spectabilis ATCC 27741 to pET21a + was introduced into E. coli BL21. . PETSpeB (KACC95002) linking gene speB encoding myo-inositol-dihydrogenase (SpcB) derived from Streptomyces spectabilis ATCC 27741 to pET21a + was introduced into E. coli BL21. Transformants. Recombinant protein Dtyti-glucose synthase (SpcD) produced by culturing the transformed Escherichia coli according to claim 5. Recombinant protein Dtyti-glucose-4,6-dihydratase (SpcE) produced by culturing the transformed Escherichia coli according to claim 6. Recombinant protein myo-inositol monophosphatase (SpeA) produced by culturing the transformed Escherichia coli according to claim 7. Recombinant protein myo-inositol-dehydrogenase (SpcB) produced by culturing the transformed Escherichia coli according to claim 8.