KR20100024687A - Preparation of the streptomyces strains producing alpha-glucosidase inhibitors transformed by the recombinant vectors harboring the salbostatin biosynthesis gene cluster, and mass-production method of these inhibitors - Google Patents

Abstract

PURPOSE: A strain producing an alpha-glucosidase inhibitor using a salbostatin biosynthesis gene cluster is provided to massively produce valienamine or valiolamine and reduce the production cost of a therapeutic agent for diabetes. CONSTITUTION: A DNA fragment for producing alpha-glucosidase isolated from a salbostatin biosynthesis gene cluster contains a gene of sequence numbers 2 to 14. The DNA fragment further contains the gene of sequence number 1. The alpha-glucosidase inhibitor is valiolamine or Valienamine. A recombinant vector(pWHU or Pwiu) contains a DNA fragment for producing an alpha-glucosidase inhibitor. A method for producing valiolamine or valienamine comprises: a step of inserting the DNA fragment into an expression vector to obtain the recombinant expression vector; a step of transforming Actinomyces with the recombinant expression vector; a step of culturing the transformed Actinomyces in a CST medium to obtain massive amount of valiolamine; and a step of culturing the transformed Actinomyces in a GYM medium.

Description

Preparation of the Streptomyces strains producing alpha-glucosidase inhibitors transformed by the recombinant vectors harboring the salbostatin biosynthesis gene cluster, and mass-production method of these inhibitors}

The present invention relates to the production of a transformed strain producing an alpha-glucosidase inhibitor using a salvostatin (salvostatin) biosynthetic gene group and to a mass production method of an alpha-glucosidase inhibitor. More specifically, the present invention relates to a recombinant vector comprising all or a portion of a gene group designating the biosynthesis of salvostatin from Streptomyces albus KCTC 9015, which is a genus of Streptomyces without alpha-glucosidase activity. The present invention relates to a method for producing a transformant that is introduced into a strain to produce an alpha-glucosidase inhibitor, and a mass production of an alpha-glucosidase inhibitor from the strain.

Salvostatin is produced in Streptomyces albus KCTC 9015 together with salinomycin (FIG. 1). It is a nontoxic antifungal agent that acts as an inhibitor of trehalase, a disaccharide trehalose degrading enzyme, and has recently been developed as a therapeutic agent for diabetes. In addition, valienamine, which is part of the structure of salvostatin, and the valolamine compound derived therefrom, have alpha-glucosidase inhibitory activity, and in this regard, the chemicals of vogliose (voglibose) It can be used as a starting material for synthesis (FIG. 2).

Several compounds have been reported so far, and the compound has a valienamine structure, and acarbose, a diabetic drug having a pharmacological action similar to that of bolibos, and a agricultural antibiotic, Validamycin, are representative compounds (FIG. 1). Cloning of these biosynthetic genes has led to studies of the valenamine structure of these compounds with sedo-heptulose 7-phosphate as a 2-epi-5-epi-valiolone by cyclase enzyme. It is known to convert to (2-epi-5-epi-valiolone), and then go through several steps to finally produce the final product through the sugar binding process (Taifo Mahmud. Nat. Prod. Rep. 2003). , 20, 137). Although the composition of the biosynthetic gene family of salvostatin was expected to be different from that of acarbose or validamycin, the inventors were expected to be essential until the conversion to the first intermediate, 2-epi-5-epi-valiolone, Using the gene of the sedo-heptulose 7-phosphate cycle designating 2-epi-5-epi-valiolon production as a probe, we attempted to isolate the gene group involved in the biosynthesis of salvostatin and successfully completed and reported it. There is a bar. Among them, a patent for 2-epi-5-epi-valiolon synthetase and salQ gene encoding the same is registered (Soon-Kwang Hong etc, Appl. Microbiol. Biotechnol. DOI 10.1007 / s00253-008-1591-2, 2008, Patent Registration 10-0752683.

Boglybose, a diabetic drug, is produced semi-synthetically. First, fermentation process is obtained from Streptomyces hygroscopics KCTC 1717 to obtain the agricultural antibiotic, Validamycin A, and then hydrolyze it to purify the Baliienamine structure. The reaction is carried out to produce a valolinamine, which is then produced through several additional steps of organic synthesis. Therefore, two cyclitols, valienamine and valitolamine, can be used as decisive raw materials for the organic synthesis of Boglybose, and are currently sold at high prices, and the invention can induce low-cost production of these raw materials. Through this, high added value can be created (FIG. 2).

Therefore, the present inventors have made intensive research efforts to overcome the problems of the prior art, the base sequence of SEQ ID NO: 1 to SEQ ID NO: 14, or SEQ ID NO: 1 among the gene group involved in the biosynthesis of salvostatin from Streptomyces albus KCTC 9015 When introducing a recombinant vector comprising a group of genes having a nucleotide sequence of 2 to SEQ ID NO: 14 to the Streptomyces strain that does not have alpha-glucosidase inhibitory activity, the valenceamine and the valenamine which is an alpha-glucosidase inhibitor After confirming that the amine can be mass-produced, the present invention was completed.

Therefore, the main object of the present invention is to produce a recombinant vector containing the nucleotide sequence of some genes from the gene group involved in the biosynthesis of salvostatin cloned from Streptomyces albus KCTC 9015, and this activity The present invention seeks to provide a transformant that is introduced into and expressed in the absence of a streptomyces strain and ultimately has alpha-glucosidase inhibitory activity.

Another object of the present invention to provide a method for mass production of alpha-glucosidase inhibitor using the transformant.

According to one aspect of the invention, the present invention provides a DNA fragment for producing an alpha-glucosidase inhibitor comprising a gene having a nucleotide sequence of SEQ ID NO: 2 to SEQ ID NO: 14 isolated from the salvostatin biosynthetic gene group. The DNA fragment of the present invention is a DNA fragment isolated from the genome of Streptomyces albus KCTC 9015, a salvostatin producing strain, is a DNA fragment containing SalI gene of SEQ ID NO: 2 to SalU gene of SEQ ID NO: 14. The SalI genes of SEQ ID NO: 2 to SalU genes of SEQ ID NO: 14 are SalI genes to SalQ genes, which are part of a gene cluster of SalA to SalQ related to salvostatin biosynthesis, and SalR, sequence of SEQ ID NO: 11 newly discovered by the present inventors. A DNA fragment newly containing SalS of No. 12, SalT of SEQ ID NO: 13, and SalU gene of SEQ ID NO: 14. The DNA fragment of the present invention including all of the SalI genes of SEQ ID NO: 2 to the SalU gene of SEQ ID NO: 14 is a gene encoding all of the enzymes involved in biosynthesis of an alpha-glucosidase inhibitor, Balienamine or Baliolamine. By containing, the alpha-glucosidase inhibitor can be mass-produced in the host cell transfected with the DNA fragment. In the embodiment of the present invention, the present inventors transformed actinomycetes with a recombinant vector in which a DNA fragment comprising the gene group of SEQ ID NO: 2 to SEQ ID NO: 14 was mass-produced an alpha-glucosidase inhibitor baliolamine ( 11 to 13).

In the DNA fragment for producing an alpha-glucosidase inhibitor of the present invention, the DNA fragment preferably further includes a gene having a nucleotide sequence of SEQ ID NO: 1. The DNA fragment of the present invention including all the SalH gene of SEQ ID NO: 1 to SalU gene of SEQ ID NO: 14 includes all of the genes encoding enzymes involved in the synthesis of the valenamine, an alpha-glucosidase inhibitor, Alpha-glucosidase inhibitors can be mass produced in host cells transfected with DNA fragments. In the embodiment of the present invention, the present invention transformed actinomycetes into a recombinant vector in which a DNA fragment comprising the gene group of SEQ ID NO: 1 to SEQ ID NO: 14 was mass-produced an alipha-glucosidase inhibitor, valenamine ( 9, 10).

In the DNA fragment for the production of the alpha-glucosidase inhibitor of the present invention, the alpha-glucosidase inhibitor is preferably valololamine or valienamine. In a preferred embodiment of the present invention, the recombinant vector into which the DNA fragment for alpha-glucosidase inhibitor production containing all the genes having the nucleotide sequence of SEQ ID NO: 2 (SalI) to SEQ ID NO: 14 (SalU) of the present invention is introduced DNA for producing alpha-glucosidase inhibitor, wherein the converted cells produce a valenamine, an alpha-glucosidase inhibitor, and include all genes having a nucleotide sequence of SEQ ID NO: 1 (SalH) to SEQ ID NO: 14 (SalU) It was confirmed that the cells transformed with the recombinant vector into which the fragment was introduced produce a valolamine, an alpha-glucosidase inhibitor.

According to another aspect of the invention, the invention has a DNA fragment for producing an alpha-glucosidase inhibitor comprising a gene having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 14 or a base sequence of SEQ ID NO: 2 to SEQ ID NO: 14 It provides a recombinant expression vector inserted with a DNA fragment for producing an alpha-glucosidase inhibitor containing a gene (Figs. 4 and 5). The expression vector of the present invention refers to a plasmid, virus or other medium into which a DNA fragment for producing an alpha-glucosidase inhibitor can be inserted or introduced.

DNA fragments according to the present invention each comprises a gene group of SEQ ID NO: 2 to SEQ ID NO: 14, or a gene group of SEQ ID NO: 1 to SEQ ID NO: 14, respectively, so that all of these respective genes can be expressed in SEQ ID NO: 1 To genes of SEQ ID NO: 14 may be operably linked to an expression control sequence, wherein the operably linked gene sequence and expression control sequence are to be included in an expression vector including a selection marker and a replication origin. Can be. “Operably linked” can be genes and expression control sequences linked in such a way as to enable gene expression when the appropriate molecule is bound to the expression control sequences. The "expression control sequence" refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Examples of such plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b +), Bacillus subtilis-derived plasmids (pUB110 and pTP5), actinomycetes-derived plasmids (pIJ101, pIJ702, pUWL201pw and pWHM3) and yeast-derived plasmids (YEp13, YEp24 and YCp50) and the like can be used. A vector suitable for introducing a polynucleotide according to the present invention into a host cell can be used, and preferably a vector designed to facilitate protein expression induction and isolation of the expressed protein. In a specific embodiment of the present invention, recombinant plasmids (pWIU and pWHU) for the production of alpha-glucosidase inhibitors were prepared by inserting into expression vector pWHM3 vectors (JESUS VARA et al., Journal of Bacteriology, 1989, 171, 5872-5881). 4, 5). In the present invention, when the pWHM3 vector is used to introduce the DNA fragment for producing alpha-glucosidase inhibitor of the present invention into the vector, the pWHM3 vector is E. coli-actinomycete shuttle vector, so it is easy in E. coli instead of genetic manipulation in difficult actinomycetes. When introduced into actinomycetes, it has the advantage of maintaining a high gene copy number (100-200 copies / chromosome DNA).

In the recombinant expression vector of the present invention, the recombinant expression vector is a recombinant vector (pWHU) having a cleavage map of FIG. 4 or a sequence in which a DNA fragment comprising a gene group of SEQ ID NO: 1 to SEQ ID NO: 14 is introduced into an expression vector pWHM3. It is preferable that the recombinant vector (pWIU) having the cleavage map of FIG.

According to another aspect of the present invention, the present invention provides a cell transformed with a recombinant expression vector inserted with a DNA fragment for producing alpha-glucosidase inhibitor. The cells of the present invention can be used in any cell capable of producing an alpha-glucosidase inhibitor by being transduced with a recombinant expression vector into which a DNA fragment for alpha-glucosidase inhibitor production is inserted, E. coli, Bacillus, It is preferable to use any one cell selected from the group consisting of lactic acid bacteria, yeast, and actinomycetes, and it is more preferable to use actinomycetes cells because the gene expression is easy and stable using its own promoter. The method of transforming a recombinant vector into the cell includes all conventional methods of transforming a vector including a foreign gene.

According to another aspect of the present invention, the present invention inserts a DNA fragment for producing an alpha-glucosidase inhibitor comprising a gene having a nucleotide sequence of SEQ ID NO: 2 to SEQ ID NO: 14 isolated from the salvostatin biosynthetic gene group into an expression vector Preparing a recombinant expression vector; Transforming actinomycetes with the recombinant expression vector; And culturing the transformed actinomycetes in CST medium to mass-produce a valolinamine, thereby providing a method for producing a valolinamine, which is an alpha-glucosidase inhibitor.

According to another aspect of the invention, the present invention is inserted into a vector DNA fragment for the production of alpha-glucosidase inhibitor comprising a gene having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 14 isolated from the salvostatin biosynthetic gene group Preparing a recombinant expression vector; Transforming actinomycetes with the recombinant expression vector; And culturing the transformed actinomycetes in a GYM medium, thereby providing a mass production method of a valenamine, an alpha-glucosidase inhibitor, comprising mass production of a valenamine.

Hereinafter, the DNA fragment for producing the alpha-glucosidase inhibitor of the present invention, the recombinant vector into which the DNA fragment is introduced, the preparation of the transformed strain with the recombinant vector and the production of the alpha-glucosidase inhibitor are more specifically step by step. Explain.

The present invention is expected to direct alpha-glucosidase inhibitors valienamine or baliolamine biosynthesis in a group of 37-kb salvostatin biosynthetic gene cloned from the Streptomyces albus KCTC 9015 genome, a salvostatin producing strain. Subcloning the gene fragment to the Escherichia coli-actinomycete shuttle vector, introducing the recombinant vectors pWHU and pWIU prepared by subcloning into the actinomycete, and the actinomycetes transformant Streptomyces lividans into which the recombinant vector is introduced ( Streptomyces lividans TK24) and Streptomyces albus G153 transgenic strains. The present invention is derived from Streptomyces albus KCTC 9015, 14 genes from salH to salU or from salI to salU among genes indicating the sequencing and amino acid sequences from salA to salU registered in GenBank as a salvostatin biosynthetic gene group Recombinant vectors containing 13 genes are provided.

The reasoning function of the enzymes designated by 14 genes from salA to salU is glycotransferase (SEQ ID NO: 1: SalH, sucrose-phosphate synthase; SEQ ID NO: 2: SalI, N-acetyl glucosaminyl transferase / Trehalose phosphorylase), epimerase (SEQ ID NO: 3: salJ, NDP-epimerase), Transporter (SEQ ID NO: 4: SalK, MFS Transporter), 2-Epi-5Epi-Valonone 7-Kinase (SEQ ID NO: 5: SalL, 2-epi-5 -epi-valiolone 7-kinase), dehydrogenase (SEQ ID NO: 6: SalM, Cyclitol dehydrogenase), oxidoreductase (SEQ ID NO: 7: SalN, Cyclitol oxidoreductase), ballyon-7-phosphate epimerase (SEQ ID NO: 8: SalO, Valiolone-7-phosphate 2-epimerase, Hexose Mutase (SEQ ID NO: 9: SalP, phosphohexomutase / phosphatase), 2-Epi-5-Epivaliolone Synthetase (SEQ ID NO: 10: SalQ, 2-epi- 5-epi-valiolone synthase), enti-diphosphatase (SEQ ID NO: 11: SalR, NTP pyrophosphohydrolases), dehydrogenase (SEQ ID NO: 12: SalS, galaci tol-1-phosphate dehydrogenase), regulatory proteins (SEQ ID NO: 13: TetR-family repressor), CoA-ligase (SEQ ID NO: 14: SalU, long-chain-fatty-acid-CoA ligase) genes.

According to the present invention, the recombinant vectors pWHU (including SalH to SalU, FIG. 4) and pWIU (including SalI to SalU, FIG. 5) comprising some nucleotide sequences of the salvostatin biosynthetic gene group described in FIG. It was confirmed that mass-producing Balinamine or Baliolamine by introducing into and culturing the transformant obtained therefrom.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate the invention, and therefore the scope of the invention is not to be construed as limited by these examples.

Example  One. Salvostatin  Acquisition and interpretation of biosynthetic gene population

Salvostatin producing strain Streptomyces albus KCTC 9015 was cultured in R2YE medium (Kieser et al., Practical Streptomyces genetics, A laboratory manual, 2000; John Innes Foundation, Norwich, United Kingdom) The chromosomal DNA was isolated purely according to the method (Kieser et al., Practical Streptomyces genetics, A laboratory manual, 2000), and then the cleaved chromosomal DNA was partially digested with restriction enzyme Sau3A. The partially cut chromosome DNA of about 37Kb was double-cut into BamHI and HpaI, and the T4 DNA ligase was connected to a cosmid vector for gene library production that can be replicated in actinomycetes, followed by Lambda Packaging Kit (Stratagene). A S. albus KCTC9015 gene library was prepared using a packaging kit.

1,000 colonies of the Streptomyces albus gene library prepared above were inoculated in a 1.5 ml Eppendorf tube containing 0.5 ml of LBA liquid medium using a yellow tip, and then cultured in an incubator for one day. The culture solution was transferred to a new Eppendorf tube by 30ul, and the plasmids were extracted, respectively, and PCR reactions were performed. The PCR primers used in this study are expected to have similar biochemical mechanisms to acbC (2-epi-5-epi-valiolone synthase) and acbC gene among acarbose biosynthesis genes having a valenamine structure in salvostatin compound structure. Are Val-F (5'-GGSGGCGTCCTCATGGACGTCGCCGG-3 ') and Val-R (5'-GCCATGTCCACGCACACCGCCTCCCCGTG-) developed for comparative analysis with the 3-dehydroquinate synthase gene. 3 ') primers were used. As a result of the PCR reaction, the final four recombinant cosmid clones were selected (pSH2101, pSH2102, pSH2103 and pSH2104).

In this embodiment, since enzymes involved in biosynthesis of bioactive substances are generally present in a certain region of the genome, four cosmid clone 30-40 kb fragments containing salvostatin biosynthesis gene groups are involved in salvostatin biosynthesis. It is expected that most of the enzymatic genes will be present. Among the 4 cosmids, the pSH2104 clone was partially cut into 1 ~ 2 kb and subcloned into plasmid pBluescript KS (+) to make a shotgun library and then 400 libraries were obtained. The base sequence was determined using T7 and T3 primers to determine the base sequence of about 20 kb. After identifying the nucleotide sequence of 17 enzyme genes from SalA to SalQ and the amino acid sequence translated from this nucleotide sequence, the bases of the 21 enzyme genes from SalA to SalU are linked again by connecting salR to salU through PCR-gapping. Sequence and amino acid sequence were revealed. 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 gene family consists of 39 open reading frames (ORFs), of which 21 are expected to be directly involved in salvostatin production and are named SalA to SalU in alphabetical order, starting with the ORF located at 5 prime of the gene population (Fig. 3). Here, the function of each gene investigated on each database is shown in FIG. 3 and the GenBank registration number of each gene is as follows [SalA; EU822205, SalB; EF394361, SalC; EF394362, SalD; EF394363, SalE; EF394364, SalF; EF394365, SalG; EU822206, SalH; EU822207, SalI; EU822208, SalJ; EU822209, SalK; EU822210, SalL; EU822214, SalM; EU822215, SalN; EU822216, SalO; EU822217, SalP; EU822211, SalQ; EU141958, SalR; EU822212, SalS; EU822213, SalT; EU822218, Sal U; EU822219 / SalR to SalU sequences are unpublished until application of the present invention. The newly discovered SalR gene is required for extracellular transport through dephosphorylation of the produced compound, or to remove phosphate groups from activated sugars, and the SalS gene is a reduction process during biosynthesis of alpha-glucosidase inhibitors. The SalT gene is expected to act as an expression regulator gene of the SalS gene.

Example  2. Alpha Glucosidase  Inhibitor Biosynthesis Gene Cassette Construction

About 17-kb fragment containing salH (SEQ ID NO: 1) to salU (SEQ ID NO: 14) of the salvostatin biosynthetic gene group cloned to produce a gene cassette capable of directing alpha glucosidase inhibitor biosynthesis in a heterologous strain; About 15-kb fragments containing the salI (SEQ ID NO: 2) to salU (SEQ ID NO: 14) genes were cut with restriction enzymes and the actinomycete-E. coli shuttle vector pWHM3 (JESUS VARA et al., Journal of Bacteriology, 1989, 171, 5872). Recombinant vectors pWHU and pWIU obtained after introduction into -5881p) were added to Actinomycetes Streptomyces lividans and Streptomyces albus G153 (Kieser et al., Practical Streptomyces genetics, A laboratory manual, 2000, John Innes Foundation, Norwich, United Kingdom). ) Transformants were obtained (Fig. 4, Fig. 5).

The pWHU recombinant vector is a 16,564-bp fragment obtained by cutting the restriction enzymes NdeI and EcoRI from the cosmid (pSH2104) prepared in Example 1 into which the salvostatin biosynthetic gene group is inserted, and the pUWL201PW vector (M. Doumith et al., Molecular and General Genetics). , 2000, 264, 477-485), and then cloned into pWHM3 (JESUS VARA et al., Journal of Bacteriology, 1989, 171, 5872-5881) using restriction enzymes XbaI and EcoRI. The pWIU recombinant vector was prepared by cloning the cosmid (pSH2104 cosmid) DNA prepared in Example 1 with restriction enzymes SphI and EcoRI, and then cloned into pWHM3 by treating the 14,862-bp fragment with the same enzyme.

Example  3. Preparation of Actinomycetes Transformant with Recombinant Vector

To transform the recombinant plasmid DNA into actinomycetes, strains of actinomycetes Streptomyces albus G153 (Kieser et al., Practical Streptomyces genetics, A laboratory manual, 2000, John Innes Foundation, Norwich, United Kingdom) were transferred to YEME medium (Kieser et al. , Practical Streptomyces genetics, A laboratory manual, 2000, John Innes Foundation, Norwich, United Kingdom) and R2YE medium (Kieser et al., Practical Streptomyces genetics, A laboratory manual, 2000, John Innes Foundation, Norwich, United Kingdom) 100 ml Incubated for 2-3 days. After incubation, the cells were immersed in 2,800 rpm for 5 minutes, washed with phosphate buffer (Kieser et al., Practical Streptomyces genetics, A laboratory manual, 2000, John Innes Foundation, Norwich, United Kingdom), and then again at 2,800 ppm. Cells were obtained by centrifugation for 5 minutes. A solution of 20 mg of lysozyme (Sigma-Aldrich) dissolved in 10 ml of phosphate buffer was filtered through a 0.22 μm filter, and the cells were suspended and reacted at 30 ° C. for 20 minutes. After 10 minutes, the tube was shaken three to four times to ensure good lysozyme treatment. The solution was collected through a cotton filter and centrifuged at 2,800 alpha, washed once more with phosphate buffer to remove lysozyme, and centrifuged again at 2,800 alpha to dissolve the cells obtained in 10 ml of phosphate buffer. Actinomycetes strains were sterilized by dispensing 50 ~ 100μl in a sterile tube and used at -70 ℃. After dissolving the protoplasts stored at -70 ° C, 200 µl of a polyethylene glycol (molecular weight 1000) solution and the pWHU recombinant vector and pWIU recombinant vector prepared in Example 2 were allowed to stand for 5 minutes, and then R2YE prepared in advance. Dispense 5 ~ 6 plate medium and slightly smeared and incubated at 30 ℃. After 12 to 15 hours, antibiotics and 1 ml of distilled water were mixed and overlayed. After drying, the mixture was further incubated at 30 ° C. for 3 to 4 days. The transformants obtained were transferred to R2YE plate medium containing antibiotics and identified. The antibiotic thiostrepton (Sigma-Aldrich) was used at a concentration of 50 μg / μl. The solution and medium composition used for the transformation of actinomycetes were followed by Keiser et al. [Kieser et al. (2000) Practical Streptomyces genetics. A laboratory manual. John Innes Foundation, Norwich, United Kingdom].

Example  4. Streptomyces Lividans  Transformant Analysis

The transformants were transformed into Streptomyces lividans TK24 by transforming the recombinant vectors pWHU and pWIU prepared in Example 2 in the same manner as in Example 3 to identify metabolites of the transformants. Inoculated in R2YE medium and cultured for 12 days, thin layer chromatography (TLC) analysis and alpha-glucosidase inhibitory activity were measured using the culture medium.

For thin layer chromatography analysis, silica 60 (Merck) was used, and the developing solution was used in combination with 1-propanol, acetic acid, and water (4: 1: 1). The color development after development was phosphomolybdic acid (H ₂ O, 25g), sulfate (cer (IV) -sulfate.4H ₂ O, 10g), concentrated sulfuric acid (60ml), and distilled water (940ml) and 0.2g. % Ninhydrin reagent was used together.

In addition, alpha-glucosidase inhibitory activity was shown to be 67 mM (pH 6.8) potassium phosphate buffer and alpha-glucosidase enzyme solution (0.15 to 0.3 unit / ml), and inhibitors (valienamine, Baliolamine or culture medium) was mixed and reacted at 37 ° C. for 20 minutes, and then a 10 mM paranitrophenyl alpha diglucoside (PNP-Gluc) solution was added to the substrate, followed by 20 at 37 ° C. After the reaction, the reaction was stopped by adding 100 mM sodium carbonate solution (NaCarb). The degree of reaction was measured for absorbance at 400nm using a spectrophotometer.

As a result, in both experiments for the two recombinant expression vectors pWHU or pWIU transformed strain prepared in Example 2, no significant difference was found between the control group and the vector-introduced Streptomyces Lividans trait. It was judged that no special substance was made in the converter.

Example  5. Streptomyces Albus  Transformants ( pWHU ) Analysis

Transformants obtained by transforming the recombinant vectors pWHU and pWIU into Streptomyces albus G153, which do not produce salvostatin, respectively, were prepared using CST medium [1% glucose, 5% starch, 3% tryptone, 3% corn glutenmeal]. , 0.5% NaCl, 1% CaCO ₃ ], and then cultured for 14 days, TLC analysis and alpha-glucosidase inhibitory activity was measured in the same manner as in Example 4 using the cell culture. In particular, when measuring alpha-glucosidase inhibitory activity using a culture medium, 77.2% of the pWHU transformants and 80.15 of the pWIU transformants were compared to the alpha-glucosidase inhibitory activity (65.38%) of the control group into which the vector only was introduced. Alpha-glucosidase inhibitory activity corresponding to% was found, indicating that the transformant produced a substance having alpha-glucosidase inhibitory activity.

In addition, TLC analysis of the transformant culture of the pWHU, it was confirmed that a substance having an R.f value consistent with the ballienamine (R.f = 0.41) used as a standard from the cell culture. As a result of the two analyzes, it was concluded that the valenamine is produced in the transformant of pWHU, and was analyzed by purification using a chromatography method in the following examples.

Example  6. Streptomyces  Recombinant vector stability analysis of transformants

As the results of the two strains used for transformation were different, the stability of the recombinant vector of each transformant over the culture period was investigated to analyze the cause. For this purpose, primers (salQ-F; 5-CAG CAT ATG ACC GGT ACG AGC CTG AC-3, salQ-R; 5-CGG GGA TCC GCG) can be used to extract DNA from each transformant and to detect salQ and salI genes. Polymerase chain reaction using AGG GTG ATT CTC-3, salI-F; 5-GGC AGC CCC ATA TGG CCT GCC GCC GG-3, salI-R; 5-GTG ACG AAT TCG ACG CTC ATG CGA GGG-3) (PCR) was performed. As a result, PCR products of the salQ gene were observed in the Streptomyces lividans transformant, but PCR products of the salI gene could not be confirmed. These results indicated that the high copy number plasmids with large insertion fragments were unstable in Streptomyces lividans TK24. On the other hand, it was confirmed that genes were stably replicated in the Streptomyces albus transformant (FIG. 6).

Example  7. Streptomyces Albus  Transformants ( pWHU Product analysis

After pretreatment of 250 ml of the cell culture medium (GYM medium, 30 degrees, 10 days culture) of the Streptomyces albus transformant (pWHU) prepared in Example 5, the strong cation exchange resin [Dowex 50W resin (NH4 ⁺ -form)] was used for the first purification. The solvent used for purification at this time is 0.5-1N NH ₄ OH. The purified sample was concentrated and subjected to TLC analysis. As a result, the spot having the same Rf value as the standard valenamine was identified, and concentrated under reduced pressure to remove ammonia water and dissolved in distilled water to investigate the effect of alpha glucosidase. As a result, it was confirmed that there is a significant alpha glucosidase inhibitory effect similar to the standard ballienamine (Fig. 7). In order to confirm the presence of valenamine in the prepared sample, mass (ESI-Mass) analysis was performed. The analysis confirmed a peak of 174.9, similar to the expected molecular weight of 175.18 of valenamine (FIG. 8). Therefore, it was confirmed that the valenamine was synthesized in the Streptomyces albus transformant (pWHU).

Example  8. Balienamine  Medium Optimization of Recombinant Strains for Mass Production

Five mediums were selected for the production of the Baliienamine of the recombinant strain, and the transformants were cultured and the Baliienamine productivity of the culture was compared. The composition of each medium was as follows (Medium 1 (M1; Starch 4g, Yeast extract 4g, Malt extract 10g, CaCO ₃ 5g), Medium 2 (M2; Maltose 4g, Yeast extract 4g, Malt extract 10g, pH 7.2), Medium 3 (M3; Starch 25 g, Corn steep liquor 10 g, (NH ₄ ) ₂ SO ₄ 5 g, NaCl 5 g, CaCO ₃ 5 g), Medium 4 (M4; Soymeal 30 g, NH ₄ Cl 4 g, CaCO ₃ 5 g, Glycerol 40 ml, pH 7.5), Medium 5 (M5; 10 g Glucose, 50 g Starch, 15 g Tryptone, 30 g Corn gluten meal, 5 g NaCl, 10 g CaCO ₃ )].

The transforming actinomycetes strain prepared in Example 3 transformed with the recombinant vectors pWHU and pWIU, respectively, were cultured in the above five media for 9 days, and then subjected to ion exchange chromatography, followed by chromatography (TLC and HPLC). Enamines were analyzed, but no valenamines could be detected. These results indicate that the type of medium affects the production of valenamine.

In order to find an optimized medium composition for producing alpha-glucosidase in transgenic actinomycetes strains, we found CST medium (1% Glucose, 5% Soluble starch, 3% Tryptone, 3% Corn gluten meal, 0.5% NaCl, 1% CaCO ₃ ), GYM medium (glucose 0.4%, Yeast extract 0.4%, Malt extract 1%, CaCO ₃ 0.2%), FM II medium (Soluble starch 60g / L, Sucrose 30g / L, Beef extract 35g / L, Experiments were carried out by reselecting three media of NaCl 0.75 g / L, MgSO ₄ 2 g / L, and CaCO ₃ 8 g / L).

As a result, it was confirmed that 5 ppm of valenamine was produced by HPLC analysis of the actinomycetes strain transformed with the pWHU recombinant vector in a culture medium (7-day culture) in GYM medium. The Balinenamine analysis was used to derivatize the culture with para-nitrofluorebenzene (p-nitrofluorebenzene) and analyzed by HPLC (Jorunal of chromatography B, 8. 24. 2005, p341-347). To this end, the culture medium is lyophilized, concentrated five times, and then 775 μl of D, N, dimethylformamide, 150 μl of triethylamine, and 75 μl of para-nitrofluorobenzene. After mixing, the reaction was carried out at 90 degrees for 60 minutes. 20 µl of the derivatized sample was analyzed using HPLC. The column was a Luna C18 RP column, the solvent was acetonitrile and distilled water at a ratio of 12:88, and the elution rate was 1 ml / min and the column temperature was 40 degrees. And detection at 396 nm. As a result of HPLC analysis, peaks consistent with the Balienamine standard were observed only in the culture medium of the transformant vector of the recombinant vector pWHU (FIG. 9). As a result of performing LC-MS analysis under the same conditions, the peak expected was en-para-nitrophenyl volley. The expected molecular weight 295 (the expected molecular weight in the case of M-1 negative mode 296.28-1 = 295.28) of the enamine (Np-nitrophenyl valienamine) was consistent with FIG. 10. The valenamine productivity calculated from the HPLC peaks was 5 ug / ml.

Example  9. Streptomyces Albus  Transformants ( pWIU Product analysis

Streptomyces albus transformant (pWIU) was incubated for 12 days in the CST medium of Example 8, and then TLC analysis and alpha-glucosidase inhibitory activity were measured using cell culture. As a result, the alpha-glucosidase inhibitory activity was strongly detected in the transformants transformed with the recombinant vector pWIU vector (80.15% inhibition) than the control group (transformants transformed with only the parent vector pWHM3, 65.38% inhibition). It became. The metabolite was separated by TLC for detailed analysis, scraped off the portion corresponding to the spot visible only at the transformant, extracted with methanol, and then filtered using a 0.2 μm polytetrafluoroethylene (PTFE, PolyTetraFluoroEthylene, Whatman) filter. Filtered. Some of the filtered samples were concentrated under reduced pressure and dissolved in distilled water to measure alpha-glucosidase inhibitory activity. The inhibitory activity was confirmed to be stronger than that of the standard ballienamine, resulting in a special alpha-glucosidase inhibitor in the transformant. Was estimated (Fig. 11). To verify these results, mass spectrometry (ESI-MS) of the sample extracted with methanol and filtered was carried out, and it was confirmed that a substance corresponding to [M +] 194.8 was present, which was a valeolinol (molecular weight 193.20). It was confirmed that the match (Fig. 12). To verify this, gas chromatography and mass spectrometry (GC / MS analysis) were performed on the sample extracted with methanol and filtered. To this end, after drying the methanol of the sample to be measured, a few drops of SIGMA-SIL-A (Sigma) without distilled water were methylated to the hydroxyl group (-OH) of the compound and extracted with hexane (n-Hexane) and analyzed. As a result, a substance having a molecular weight of 263 corresponding to pentamethylvaliolamine was detected at a peak of 8.8 minutes in gas chromatography (FIG. 13). Therefore, it was confirmed that the potent alpha-glucosidase inhibitor produced by the Streptomyces albus transformant (pWIU) is valenamine.

Through the results of the above example, transformed with a recombinant vector into which a DNA fragment for alpha-glucosidase inhibitor production containing all genes having a nucleotide sequence of SEQ ID NO: 2 (SalI) to SEQ ID NO: 14 (SalU) of the present invention was introduced. Cells produce an alpha-glucosidase inhibitor, and a fragment of DNA for producing an alpha-glucosidase inhibitor comprising all genes having a nucleotide sequence of SEQ ID NO: 1 (SalH) to SEQ ID NO: 14 (SalU). It was confirmed that the cells transformed with the introduced recombinant vector produced a valolamine, an alpha-glucosidase inhibitor.

As described above, according to the present invention, 14 enzymes are derived from Streptomyces albus KCTC 9015, a salvostatin producing strain having trehalase inhibitory activity, and are salH of SEQ ID NO: 1 to salU of SEQ ID NO: 14 Or a recombinant vector containing an alpha-glucosidase inhibitory DNA fragment containing a gene group encoding 13 enzymes from salI of SEQ ID NO: 2 or salU of SEQ ID NO: 14 to salU of SEQ ID NO: 14 is stably maintained in heterologous actinomycetes Can be expressed.

In addition, the present invention is to produce an alpha-glucosidase inhibitor in a heterologous strain transformed with a recombinant vector into which the alpha-glucosidase inhibitory DNA fragment is introduced, and from the transformant, a valenamine or a valenol. The amines can be mass produced.

Therefore, the salvostatin biosynthetic gene group of the present invention is not only utilized as a gene source for the development of a salvostatin derivative having a novel nontoxic trehalase inhibitory activity, but also with a Balienamine which is a synthetic starting material of a semisynthetic diabetic agent such as Boglybose. It is a very useful invention for the biological and pharmaceutical industries because it can be bioavailable in mass production of valolinamine and thus can be very usefully applied to the development of various diabetes treatments.

BRIEF DESCRIPTION OF THE DRAWINGS The chemical structures of the valenamine containing compound salvostatin, validamycin, acarbose are shown.

FIG. 2 is a diagram illustrating an alpha-glucosidase production process including boglibosses, and compares the process advantages of producing a valenamine or a valenamine directly from fermentation.

3 is a map of the salvostatin biosynthetic gene group cloned from Streptomyces albus, SalH (sucrose-phosphatesynthase, glycosyltransferase), SalI (N-acetyl glucosaminyl transferase / Trehalose phosphorylase), SalJ (dehydrogenase / NDP-) used in the present invention. epimerase) SalK (MFS Transporter), SalL (2-epi-5-epi-valiolone7-kinase), SalM (Cyclitol dehydrogenase), SalN (Cyclitol oxidoreductase), SalO (Valiolone-7-phosphate2-epimerase), SalP (phosphohexomutase) / phosphatase (glycosidichydrolase), SalQ (2-epi-5-epi-valiolone synthase), SalR (NTP pyrophosphohydrolases), SalS (galacitol-1-phosphate dehadrogenase), SalT (TetR-family repressor), SalU (long-chain-) fatty-acid--CoA ligase) gene.

4 shows a cleavage map of the recombinant vector pWHU.

5 shows a cleavage map of the recombinant vector pWIU.

Figure 6 shows the stability of the recombinant plasmid DNA in long-term culture in S. albus host (S. albus).

7 shows the activity of thin-layer chromatography (TLC) analysis (left) of the pWHU transformant culture recovered from Dowex 50W and alpha-glucosidase inhibitory activity of the material recovered from thin-layer chromatography ( Right).

FIG. 8 shows that pWHU transformant cultures recovered from Dowex 50W are producing valenamine by ESI-Mass analysis.

9 is a result of chromatographic analysis (HPLC) of the fermentation broth of S. albus transformed with the recombinant expression vector pWHU to confirm that the derivatized valenamine peak is present in the culture. .

FIG. 10 shows a fermentation broth of S. albus transformed with the recombinant expression vector pWHU by mass spectrometry (LC-MS). The analysis results also show agreement with the derivatized valenamine mass.

FIG. 11 shows chromatographic (TLC) analysis (left) of fermentation broth of S. albus transformed with recombinant expression vector pWIU (left) and alpha-glucosidase inhibitory effect of the material recovered from chromatography (right). ).

12 is a mass analysis of the fermentation broth of S. albus transformed with the recombinant expression vector pWIU [Total ESI Mass (M + mode)] analysis, there is a substance corresponding to the valerian amine in the culture Indicates.

Figure 13 is a result of confirming the presence of the valerian amine as a result of mass spectrometry (GC-MS) of the fermentation broth of S. albus transformed with the recombinant expression vector pWIU (S. albus).

<110> Myongji University Industry and Academia Cooperation <120> Preparation of the Streptomyces strains producing          alpha-glucosidase inhibitors transformed by the recombinant          vectors harboring the salbostatin biosynthesis gene cluster, and          mass-production method of these inhibitors <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 1269 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 1 gtgaatgggg aacttcttcc cggtgtcctg gttgtcaaca gcaattattt tccgagatcg 60 cgcggggcgg ccgcccgagt gggtgccacc tccttcgcgc tcgaggcggc cgcctgcctc 120 gaggaagccg gggtcttctc cggcgtcatc ctgtacaaaa ggcaggaggg tcagcggaca 180 ccgacgctgc acccggtcac caggaacggc atgccctgcg tggaaatgcg cttcaacttc 240 tccatgccca ccggggagtt gtgccgcgct ctccgcagag cggccgaaga gctgacccac 300 cgggcggcgg ccaccgccac gggcccgccc atgctctact accagaccga tacgctgctg 360 aggtatcacc cccgcgactt acccgcctgc gtcacccatc acggcccctt cttcgacgac 420 ttcgcggacc ggttctccac tcaggacacc ttccaggcat tcggcagcgc ggagaaggcc 480 ctgcacctca tgcgccagca ggaacgggga ctcctcgaac tcgtcgccgc acgccatatc 540 ttcgtgatgc agcactccag gatgcagcgc gactacctgg tcggacaggg aatggacccg 600 gcgcgcatgc gcgaggtcag cccgcccatc cgcccggaga aggtcccgag cgtccgcccg 660 ccgaccggca tcctgcgttt catcgaatcg gcgccgcttc tcctgtgcac cgcggtggcc 720 cgactcgact acttcaagaa cctcgacctg ctgatcagcg cggccacgga actgatggag 780 cggggacttc ccgtgaaggt cctgctgatc ggcggcgacg aatgcgacga cgtgctgcgc 840 aaatcgctcg tcagagaagt cccggcacac cacgccgaca gattcctgct gaccccgaga 900 ctcacgaagg gcgaactcta cgcggtgttc cggacggccc ggcggacctc ggtattcgtc 960 tgcacctcgc ggtacgagac cctgggcatc accccgctcg aagcagcgct gagcggcctc 1020 tccaccgtcg tccccgactc ccgccccgtc gaggccgcca gattctttcc cgccgacgcc 1080 cgatacgagc ccaccgcgaa cggcctggtg aaactgatcg aacggatcgc ctccgtggac 1140 ctgttccggc gcggcgcgga actgtgcagg ctgctagaag ggcagatctc cgtcgcatgg 1200 ttccgcagag atctgatgcg ggcctggcgc agtttttccg agatgggcag gagcgccctc 1260 cagcaatga 1269 <210> 2 <211> 1329 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 2 atggcctgcc gccggcaatc gacgcagagc gaaggagagc acatgccgac accggcatgg 60 gaggtcgaac ccaccggcga gcgccggagg ctcgtacacg tcaacgcgac ggcccgcggc 120 ggcggagtcg cggagctgct gcacggcctg gtgccctgcc agcaggccgc cgggctgaac 180 gccggctggg ccgtgatcgg cggcgacgag gacttctacg cggtgaccaa gctcttgcat 240 cacctgctgc acggcacggc cgatccggac cggctgcgag ccgagtcgct gcggacctac 300 cgcgagcggc tggccgcgca ggcctcctgg ttcaccgagc ggctcacccg cgacgacgtg 360 gtggtcctgc acgatccgca gaccctcgga ctcgcaccgc tgctgagcgc caccggcgcc 420 cgtgtcgtct ggcactgcca tgtcggggcg gacgtccccc cggaccgggg accgggtgcc 480 gtctggcgcg ccttcgcggc cgaactgtcc gcggtcgacg cggtgatcac caccctgccg 540 gagttcgcgc ccccctccgt acccctggcc aggaggttcg tcgccgcccc ggcgatcgac 600 ccctcggccc cgaagaaccg gcacctggag ccgagggaag tcgccgcgct gctggacagg 660 gccggactca ccgcggaccg cacggccccg gacgtcaggc tcgtccagca gacgacgctg 720 ccggccgacg cccgtgtcgt cctgcaggtc tcccgctggg atccgctgaa ggacatgccc 780 ggcgtggtgc gctgcctcgc gggcctcgcc ccggatgtgc acctggtgct cgcgggcacc 840 gaccccaccg agataccgga cgacccggaa ggcctggccg tcctcgccga cgtacagcag 900 acgctcgccg gactggcccc ggcggaccgg gcgcgagtcc atctggtcaa tgtctccatg 960 cgcgacccgc tgggcaacgc gctgctcgtg aacgcgctgc agcgccgcgc ggacgtggtg 1020 ctgcagaaga gcctggagga gggcttcggc ctgaccgtga ccgaggcgat ggtcaagggc 1080 cgggcggtgg tggcctcttc ggtgggcgga ctgcggcagc aggtgatcca cgagcgcaac 1140 gggctcctgg tcgagccgac cgacctcgcc ggggtgcggg ccgcgctcac ccggcttctc 1200 gacgaccccg ccctgcgccg ccgactcggc gagcaggccc gcaacgacac cctggagcgc 1260 tacacgatga cacggctcgt cacggactac ggagccatcg ccggctcggc agagggggcc 1320 ctcgcatga 1329 <210> 3 <211> 759 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 3 atgagcgtcg ccgtcgtcac cgccgccagc ggcggaatcg gtcgtgccgt ggtgcacacc 60 ctgctcgcgg agaaggtcgt ctcccgggtg atcgccgtcg acgtggcgga cccgccggcc 120 tccctgcccg acggcgcgga agcgctgcgc tgcgacctgc gcaccgagga ggggcttgcc 180 gccctcgccg aggccgtgcc cgaggagatc accgtcctcg tcaatgtcct cggcggggag 240 cggcagccgc cgctggagcc catcgaggac gtggcctggc cgccgccgga ggtgtgggac 300 gacatcgtcg acctcaacct ctccggcgtc taccgggtca cccggctgct cgccggacgc 360 ctcctgccgg gcggcgcgat ctgccaggtc agctccatcg ccgccaccat gccctgggtc 420 gtctcgcccg cgtacggggc cgccaaggcc gccctggagc actggaacga ctcgcttgcc 480 gtgctcctgg ccgaccgcgg gatcagggcc aacctcgtcc gtcccgggtt cgtctggagc 540 cgccagtggc agctggtcga ccgggccgag ttcgaggagg tggtacggga ccgggtgccg 600 ctccggcagg tcaccggcac gacgccgacc gaccgggagc agaccgcgga cgacgtcgcc 660 caggccgtgt cgttcctctg ctcccccgcg gcggcgcacc tcaccggtca ggccatcaac 720 gtggacggcg gcgcggccct cgtccgcgcg gcccgctga 759 <210> 4 <211> 1182 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 4 atggaaagca ccgctctgcc cccggcggcc aagctgcgcg gcagcaccgt cttcctgggc 60 ttcggcgcct tcggactgct gtggggcctg tacgccgcgg cgctgccgga gatcaaggac 120 aacaccggtg tctcggacgg tgaactcggc accgcgctcg cctgcgtggc cctcgcggcg 180 gcgcccgcga tgttcctgac cggccgcctc ctcgaccgct acggaaggcc ggtggccatc 240 ggctcgctgg tcctgttcgc cgcggtcgcc tccctgccga ccctcgccac gtccacgagc 300 acactggtcg tgacgcttct gctcttcgga ttcggctcgg gctgctgcga tgtggtgatc 360 aacagcctgg cggcgacggt cgaggccgag acgggcggac gggtgctcaa ccgggcccac 420 gccctgttca gcgtgggact gctcgtcggc gccgtcggca ccagcatcac ccacgccctc 480 ggggcgagcg tcacctggcc gctcgccgta ctggcggcct gcaccgtcgt gggcgcgtgg 540 gcgctccgca ccagggtgcc gccgcgcctg ggccgcgccc cccagccgga ccggccgacc 600 gggcgccgca aggtcgacaa ggtcgtgctc ggcttcggcc tgctcgccgc gctcgccatg 660 ctggtggaga gcggcgtcca gcagtggagc gccgtcttcc tggcggacgg ggtcggcgcc 720 gcggacggtc tgtccggcct cgcaccgggg gtgttcgccg gttcgatggc gctgggcagg 780 ctcgccggac actggctgtc cacccggtgg tccgaccgtg tcgtactgct gctctcgggg 840 ctggtgtccg ccttcggtgt gctggtcgtc gcctggtcgc agcggccgct gctcgcgctg 900 gccggattcg ccgtcaccgg tctggccatc tcggcggcgg cgccgaccgt gtacagcgtg 960 gccggacgca acgccccggc ggagcgccgc ggggccgtca tcggctccac cgccgccatc 1020 ggctacgtgg gactcctgct cggccccgtg gtcgtcggcc aggtcgcgga cttcacccaa 1080 ctccgcaccg ccatcggctc cttggtcgtg gtgtccctgg cactgagcgc cgcggccctc 1140 ctgctgccga cgcccggggc agaagaccgt acggcccagt ag 1182 <210> 5 <211> 942 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 5 gtgcggcgca gaccgtcgcc gagccggacc agcctgccgg acgccgctgt cgacgaactg 60 cagtcggcgc tggtgcgggc catggccggg ccggttccgc ccgggggtgt cgccggggtc 120 tccttcggcg cggcactcga ccaccgcacc gggaccgtgt acgcctccgc tccgctgtgg 180 ggcgcgcaca cccggccctt cgacctgctc ggggccctgc gctcggcccg gcccgacgtg 240 cggtggcatg tcgtcaacga cgtcaccgcg gccctgctgc acgtggcgga ctcaccgacc 300 gcgcacgacc ggggcaagat cctgctcgcc accatcagca ccggcatcgc ctgccgcacc 360 atggaccggc gtaccggtgg gatcccggtc gacggctgcg gtctgcaagg cgagatcggt 420 catctgcccg ccttcgtcgc cctcgacggc cttccggtcg aactgcgctg cgactgcggc 480 gagccgggcc atctggcggc gttctcctcc ggtccgggca tccggcggct cggcgaggtg 540 gtgcgggacc gcgcacccga ccggtgggag gcctcccggc tcggcgccgg gctcgcggcc 600 ggagcgacct tcgagacggc gctcgcgaaa gcgctcgccg aaggcgatcc ggtggcgaac 660 cggctgctcg acgcggcgac cggcccggtg gccgatgtcg tacgcaccgc gctgtgcctc 720 gacccgggga tcgacctggt cgccttcacc ggtggggtcg cgaccggtct cggcgaccac 780 taccgcgagg cgctcctgag gcacctggac cgggccggtc tctatctgac cagcgagcgg 840 gagcccggct gggtgcgcga gcgcatcctc gtctgcggac cgggcgaggc ggacggcctc 900 gtcggcgccg gactcgccgc gctggccggg gaggcggcat ga 942 <210> 6 <211> 1098 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 6 atgaccgagc accgcgcgat catccgcacg agcaccaccg tgtccgtcgg cccgcggccc 60 accacggcgc ccgggcccgg ggagttgagc atcgcgacgc tgtacgcggg cctgtgcggc 120 accgacatcc agatgctgcg cggcctgcgc gacgacccct cgccggtgat cggccacgag 180 gggatcgccc gggtggtgac cgccggtgcg ggcgcgcccg aatggtgcgc cccgggcacc 240 ctggtggcgg tcaacccgac gcatccgacg gacccgggct tcctgctggg ccacaacgtc 300 gacggactgc tgcaggaacg cacgctgctg cccgcctccg cgctcaccgg cggcatggtc 360 ctgccgctcc ccgcgaccac ggacgtcggc ctggcccccc tcctggagcc gctcgcggtg 420 gtccgctacg cgctgagcga gctgcgcgcc ttcgcccccc gcaccctgct cgtcatcgga 480 gacggcacga tcgggcacct cgccctccgt gccgcccccc gctggctcgg cgaggacgta 540 cgggtcgcgc tggtgcacca cacccccgag ggccgcgcct tcagccaggc gcggccgcac 600 cgggccgaac tgctgctcgg gatcgaggag ttggccgccc ggcggtggga cgccccggtc 660 gcggccctgc tggccacgcc ccggaacgcg accctcgccg ccctggaggc cgtgctcgcc 720 gccgcggggc cggacctcgc cgtggacatc gtgggcgggc tgcccccggg cgcgaccacc 780 ccctcgctgc ccggtctcga cctcggtgcc gtccgcgcgg ccaactgcgg gggcatcccc 840 gacccggcgc tcgtcaccac caccgcaccg ggcgtgcgcc tgctcgggca ccggggcgtg 900 ggcaaccggc acctgctcga cgccgccgcg gaactcgccc gcgcccccga gcagtaccgg 960 gacctcatca cccacgagtc ggacctcgcc ggtgcggccc gcctcatgcg gagcctggcg 1020 ggctcagggc accgcctctt cgacggccga cgcctcgtca agctcgccgt gcgcgtcaac 1080 gacagggagc agacatga 1098 <210> 7 <211> 762 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 7 atgacgacag acaccccgct cgccgaccac cgcgccctcg tcgtcggcgg ctccacgggc 60 atcggccgcg gcatcgccga cgcctgggcc gccgcgggcg ccgaggtggt cgtctgcagc 120 cgcagccgcc ccaccggccc cggcgccgaa gcgctgcgct gggaggcact ggacctcacc 180 cggcccgagc aggcacaccc gcggctgtac gagctcgcct ccgggcccct caaggccgtg 240 tgcttcgcct ccgtccacta cggcgccggg cgcgccccct tcagcgaggt cgccgaacag 300 gagtggctgg accaactggc cgtcaacacc acgggcctgt ggcacaccct ggcggcgagc 360 ctgccgtcgc tgcgtaccgc cgcgcccggg ctcttcctgg gtgtctcctc cgaggtcgcc 420 ttcaacgccg gtccgggcag gtccggttac gccgcgacga aggcggcgtc caagaccctc 480 ctcgactcgg tggcccagga ggaggacagc gcggccgtac gcatcgtgca ggtgctgccc 540 gccgggatgg tggacagccc gggcatccgc aaccggcgac ccgaggactt cgacttcagc 600 tcgtacatga agccctccga cttcggcgcg ctggcccggg agttggccgt gacctccggg 660 gagaagtacc acggcgactc cctcgtggtc ggcggcgacg gccagtggtg gtcggcctac 720 ggctcggcgc cggtctccca gacccggtcg gtgcgctcat ga 762 <210> 8 <211> 804 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 8 atgagctgcc gattggccct cgccgaatgg cggctgcccg cctcggggcc cgaggcgctg 60 cggctggccc ggacggtcgg agccgacggt ctgcaactgg acctcggcgg gcccggccgc 120 ggcgagtggc tcgacgggcc cggccggatc gacgcggtgc gcgccgaggc ggagtcgacc 180 ggagtgcggc tgctcgcggt cgccgggaac cacctcaacg atgtcggcct gatgtcaccg 240 gccgcgcgcc cggtcctgga acgcctcctg gacaccgccg ccgcgctcgc tgtgccgctg 300 gccttcgtgc ccagcttccg ccgcagcgcg atcgaggcac cggccgacct ggaacgcacc 360 gccgaggtgc tggcctgggc cgccggagag gccggggccc gcggtctcct cctggcgagc 420 gagaacgtgc tgaccggcga gcaggctcgc gcgctggtcc ggcgggtcgg ctcccccgcc 480 ttccgcgtgg tgctcgacac gttcaacccg gtcgcggccg ggctgtcccc cgaggtgctg 540 gtcgccgaac tgcacgacgt cctggccgac caggtccacc tcaaggacgg cccgccgacc 600 accggcgcga ccccgcccct gggcagcggc accggacggc tcgacgacac cctccgtgcg 660 ctgcgcgcgc accaggtgcc ggtgcgggcc gcggtcctgg agaacgacta ccgcgacggc 720 gatcgggccc ggctgctcgc cgatctgcgc tgggcgcggc agcgcaccgc ctcactcgcc 780 acgacctcgg ggaaggaaga atga 804 <210> 9 <211> 675 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 9 atgagcactc cgtcactgcc agtcgccgtc gtcacctcgg ccctgctctt cgacatggac 60 ggcacgctgg tcgactccac agcggtggtc gagcggacct ggcgccgctt cgcccgcaga 120 cacggcgtgc gcgccgaaga gatcctcgcc gtctcgcacg gccgccgtac ggaggagacc 180 gtcgcgcggt tcgcccccgc ggacgtcgac gccgcagcgg aagcacggcg tgtgatcgcc 240 gaggaggtcg aggacacccg ggggatcacc gccatccccg gtgccgccga actcctcgcc 300 tcgctgccgg aggccggctg ggccctggtg acctccgcgg gccgccggct ggccgaggcg 360 aggatgcgcg ccgccgggct tcccctgccg cccgtactcg tcagcgcgga cgacgtcgcc 420 cagggcaagc cgagcccgga ggggtatctg caggcggccc gtcggctggg gcgctccccc 480 gagtcgctcg tcgtcttcga ggacgccgag gcggggatcc tcgcggcccg ggcctccggc 540 gccagaaccg tcgtggtcgg cccgtcccgc tgcgaggccg ccgaggggct cgaccaggtg 600 accgacctgc gggacgtccg ggtcgacatc gacacctcga ccggccggct gcgggtgtcc 660 ctgcgagccg actga 675 <210> 10 <211> 1233 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 10 atgaccggta cgagcctgac cgacacctcc tccggcctct acttcaggga ccactcccag 60 gggtggctgc tgcgcgccca gaagcagatc agctacgagg tccggctacg ggacggaatc 120 ttccgccccg agtgcaccga ccttctggag cagggggcgg gaactcccgg gcgatcacgc 180 cgcttcgtcg tggtggacag caatgtcgac ttaatgtacg gaaatcgcat ccggtcctat 240 ttcgactacc acggtgtcga ctgctcgatc atggtggtcg aggcgaacga gacgctcaag 300 aacctggaga cggcgacccg catcgtcgac gagatcgacg ccttcggcat agcccgccgc 360 aaggagccgc tgatcgtgat cggcggcggc gtactgatgg acatcgtcgg cctggtcgcc 420 agcctgtacc gccggggtgc gccgttcgta cgggttccca ccaccctcat cggcctggtg 480 gacgccgggg tcggcgtcaa gaccggcgtg aacttcaacg gccacaagaa ccggctcggc 540 acctacacgc ccgcggacct cacgctcctg gaccgccagt tcctggccac cctggaccgg 600 cggcacatcg gcaacggcct cgccgagatc ctgaagatcg ccctgatcaa ggacctcagc 660 ctgttcgcgg cactggagga gcacggcccc accctgctcg acgagaagtt ccagggcagt 720 acggcggcgg gcgaccgggc cgcgcggtcg gtgctgcact ccgcgatcca cggaatgctc 780 gacgaactgc agcccaacct ctgggaggcc gagctcgaac gttgcgtcga ctacgggcac 840 accttcagcc ccaccgtgga gatgcgcgcc ctgcccgagc tgctgcacgg cgaggccgtc 900 tgcgtggaca tggcgctcac caccgtcatc gcctggcgcc gcggcctgct caccgaggcc 960 caacgcgacc ggatcttcgc ggtgatggcc gcactggagc taccgagctg gcaccccatc 1020 ctcgacccgg acgtcctggt gaacgccctc caggacaccg tgcgccaccg ggacggactg 1080 cagcggctgc cgctgccggt cggaatcggc ggcgtcacct tcgtcaacga cgtcacaccc 1140 cgggaactgg aggccgcggt caccctgcag caggaactgg gggacgcgcg gacaccgaag 1200 acgagcgggg accgcggcgg caggaacctt tag 1233 <210> 11 <211> 546 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 11 gtgatcgaac cgctgcgctc ccgcgaggtc taccgcaacg cgtggatgac cgtacgggag 60 gacgacgtcc ggcacagcaa cggccaccag ggcatctacg gagtggtaga caagccggac 120 tacgccctgg tcatcccccg ccagggcgac cggctccacc tggtgcagca gtaccgctac 180 ccggccggcg gccggttctg ggagttcccc cagggctcct ggccgggcgg gcgctccccc 240 acggacccga gcgaactggc ccgcaccgaa ctgcgcgagg agaccgggct gcgggcaggc 300 cggatgaccg gcctcgggcg gctccatgtc gcgtacggat acgccagcca gggctgccat 360 gtgttcctgg ccgaggacct ggaagcaggc gagcccgagc gtgaggcgac cgagtccgac 420 atgcggcagc gctgggtcga cccggacgag tggtgggccc tgatccgggc gggcgagatc 480 accgacgcgg ccaccatcgc cgcgttcgcc ctgctcagcc gaccggaatc ggccccgggc 540 acctga 546 <210> 12 <211> 960 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 12 atgaaggcag tgtcgatcaa ggaacccggc ggtcccgagg tcctggagtg gaccgaggtc 60 cccgacccct cgcccgccgc cggtgaagtg gtggtcgacg tggtggccgg cgcgctgaac 120 cgggccgatg tcatgcagcg gatgggcctc tacccggtgc ccgcgggcgc ttcgccgtac 180 ccgggtcttg aggtctccgg gcggatcagc gccgtcggca ccggagtgac cggctggaag 240 gtcggcgacg aggtctgcgc gctgctcacc gggggcggct atgcgcagaa ggtcgcggtc 300 cccgcggggc agttgctcac cgtgccccag ggcatcggcc tggtggaggc ggcgggcctg 360 ccggaggcgg tcgccacggt gtggtccaac atcgtcatga ccgcgggcct gaaggagggc 420 gagaccttcc tcgtgcacgg ggggaccggt ggcgtcggca ccgcggcgat ccagatcgcc 480 aaggcgatcg gcgcccgcgt cgtcaccacc gtcggcagcc ccgaaaaggc cgagcgcgcc 540 cgcgagttgg gcgccgacct ggccatcgac caccgcaccc aggacttcac cgagcacggc 600 ccctacgacg tgatcctcga cgtggtcggc ggctcgtacc tggcaggcaa tgtccggtcc 660 ctggccgcgg acggccgcct ggtcgtcatc ggcctccagg acgggctgga gggccgactc 720 aacctcgccg acatggtgtt caagcgcctc tccgtgcacg gcaccaccct gcgcacccgg 780 tccgcggcgc agaaggccgc catcgtggcc gaggtgcagg agaaggtctg gccgctgatc 840 gagaacggca ccgtgaagct ggtcgtcgac cggaccgtgc ccatggccga ggccgccgag 900 gcccaccggc tcatggacac cggtcggcat atcggcaaga tcctgctggt caacgactga 960                                                                          960 <210> 13 <211> 612 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 13 gtgaccaagg ccgacgctgg gacgacctgg aggacgtacg cgggcagtgg gctgccgccg 60 atcctggaga ccgccctggc ctgcttcatg gagcacggct accacgggac gaccatccgg 120 acggtggcct cccgcgccgg gctctccgtg ccggggctgt actaccacta cccctcgaag 180 caggccctgc tggtcgccgt cgtctcctac gccatggacg acctccggga gcgcagcgaa 240 gccgccctgg aggaggccgg gtcggatgtg cagcggcgcc tcgacctgct cgtggagtgc 300 ctcgtgctct tccacgcgta ccggcgggac ctcgccttca tcgcgtacag cgagatccgc 360 agcctcgtcg gggacgcccg cgccacctac atcggcgccc gcgaccgcca acagcgtctg 420 atggacggcg tgttggcgga cggggtcgcg cggaaggtct tcaccacccc ctacccgcgc 480 gaggtcagcc gggcgatcgt caccatgtgc accggtgtcg cccagtggta ccgcgcggag 540 ggcgcgctca cgccgcacga gctggcggag cggtaccgcg ccatgacgcg gatgacggtc 600 ggcgcgccgt ga 612 <210> 14 <211> 1680 <212> DNA <213> Streptomyces albus KCTC 9015 <400> 14 atgaccgtgc ccgtggagcc gccgtggggc gcgccgctcc cgaccggaac cggggtgagg 60 atcggccagg gctccctggt ggacgcctgg cgggcgcggg tggcgaggaa tccgggcggt 120 atcgcgctgc gctacttcga cggcgccatg tcggcgcggg aggcggacgc cgcatccgac 180 gccctcgcgg cggcgttcca ggcccggggc accggacgcg gcgaccgcgt cggcgtctgt 240 ctgcagaaca tcccccagta cgcgctggtg ctgctcgccc tgtggaagct gggcgcgacc 300 gccctgggga tcaacccgat gtaccggcgg caggagctgc gccgcctcgt cgacgactcc 360 ggcgcgaccg gcctggtctg cgcggacacc gaggccgagc agacccgcga caccctcgcc 420 gggagcacgg tccgctggct gctcagcacc tcggcgctgg accaccagga gcgcgacgac 480 ccgcgggtct tcccgacgag gcagcgcccc gcgcccgcgc cggacggcga tctccgggcg 540 ctgatcgagg agttcgcggg cgcccgtccg gagccggtgc ggccgaccac cgacgaggtc 600 gcgctcctga cctacacctc cggcaccacc gggccgccga agggtgccat gaacacccac 660 ggcaacatcc tgcacgtggt gcggacctac gcggcctgga ccggtctcgc cgagggggac 720 gtggtgctcg ccctcgcccc gctgttccac atcaccggcg cggtggtcaa cgcgagcctc 780 tcgctgctca ccgacaccac cctcgtcctc gcgggccggt tccgtcccga ggtcgccctc 840 gacgccatcg ccgagcacgg ggtcacctcc accatcggct ccatcacggc gtaccacgcg 900 ctctacgagg tgccgggcgc ggggccggag cacttcgcct cggtgaaggc cctctactcc 960 gggggcgccc cgatcccgcc cgcgaccgtg gagaggttcc aggagcggtt cggggtctac 1020 ctgcacaacg gctacgggat gaccgagacg agttccgcgg tgatcgcggt gcccccgggg 1080 cggcgggccc cggtgcaccg gccgagcggc accctctcca tcggcctgcc cctgccggga 1140 ctcaccgcgc gggtcgtgga tcccagcggc gatccggtgc ccggcgggca gcagggcgaa 1200 ctcgaactga gcggcccgca ggtggtgccc ggctactggc agcagcccgc ggccacccag 1260 gaggccatgc cccagggccg gttgcgtacc ggcgacggcg cgatcgtcga cgaggagggc 1320 tgggtctacc tggtggaccg gctcaaggac cagatcaatg tctccggtta caaggtctgg 1380 ccgcgcgagg tcgaggacgc cctgtacgag cacccggcgg tgcacgaggc cgccgtggtg 1440 ggagtgccgg acgactaccg cggcgagacg gtcgtcgccc atgtctcgct gaaggcgggc 1500 caccgggcca ccgccgagga gctgatcgcc ttctcgcgcg cacgcctcgc ggcctacaag 1560 tgcccgcgcg aggtccacct ccgcaccgag ctgcccaaga cccagaccgg caagatccgc 1620 cgcgccgaac tccgcgaggc cggtggcggc ggacccgcct cccccgccgc ggagggctga 1680                                                                         1680  

Claims (11)

DNA fragment for producing an alpha-glucosidase inhibitor comprising a gene having a nucleotide sequence of SEQ ID NO: 2 to SEQ ID NO: 14 isolated from the salvostatin biosynthetic gene group. The DNA fragment of claim 1, wherein the DNA fragment further comprises a gene having a nucleotide sequence of SEQ ID NO: 1. According to claim 1, wherein the alpha-glucosidase inhibitor is a DNA fragment for the production of alpha-glucosidase inhibitors, characterized in that the valolol (valiolamine). According to claim 2, wherein the alpha-glucosidase inhibitor is a DNA fragment for producing an alpha-glucosidase inhibitor, characterized in that the Balienamine (Valienamine). A recombinant expression vector into which a DNA fragment for producing an alpha-glucosidase inhibitor of claim 1 or 2 is inserted. The recombinant expression vector according to claim 5, wherein the recombinant expression vector is a recombinant vector having a cleavage map of FIG. 4 (pWHU) or a recombinant vector having a cleavage map of FIG. 5 (pWIU). A cell transformed with the recombinant expression vector according to claim 5. 8. The transformed cell of claim 7, wherein said cell is an actinomycete cell. 8. The transformed cell of claim 7, wherein said cell is Streptomyces albus G153. Preparing a recombinant expression vector by inserting the DNA fragment according to claim 1 into an expression vector; Transforming actinomycetes with the recombinant expression vector; And Method for producing a valolamine alpha-glucosidase inhibitor comprising culturing the transformed actinomycetes in CST medium mass production. Preparing a recombinant expression vector by inserting the DNA fragment of claim 2 into a vector; Transforming actinomycetes with the recombinant expression vector; And A method for mass production of a valenamine, an alpha-glucosidase inhibitor, comprising culturing the transformed actinomycetes in a GYM medium to mass-produce a Balienamine.

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