KR101578651B1 - Recombinant microorganism producing stilbene compound and method for producing stilbene compound using the same - Google Patents

Abstract

The present invention relates to a recombinant microorganism producing a stilbene compound and a method for producing a stilbene compound using the recombinant microorganism, wherein a codon-optimized SbROMT3syn (synthetic Sorghum bicolor Recombinant microorganisms transformed with the resveratrol O- methyltransferase 3) gene and ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Using recombinant microorganisms transformed with the gene, SbROMT3syn or VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase ) gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene, It is expected to be able to mass-produce methylated resveratrol derivatives, pinostilbene and phthalostilbene, which exhibit anticancer, antiviral, anti-inflammatory, anti-aging and antioxidant effects.

Description

Recombinant microorganism producing stilbene compound and method for producing stilbene compound using the same}

The present invention relates to a recombinant microorganism producing a stilbene compound and a method for producing a stilbene compound using the same, and more specifically, a recombinant vector containing a sorghum-derived SbROMT3syn (synthetic Sorghum bicolor resveratrol O -methyltransferase 3) gene. A recombinant microorganism having the ability to produce a stilbene compound by being converted; ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Gene, VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase ) gene or SbROMT3syn gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) a recombinant microorganism transformed with a recombinant vector containing the gene to produce a stilbene compound; A method for producing a stilbene compound by culturing the microorganism; And ScCCL It relates to a composition for producing stilbene of a microorganism containing a recombinant vector including a gene and a recombinant vector including the SbROMT3syn gene and the RpSTSsyn gene as an active ingredient.

Stilbenes, mainly expressed as resveratrol compounds, are a small class of plant secondary metabolites derived from the common phenylpropanoid pathway starting with phenylalanine. Resveratrol (3,4',5- trans- trihydroxystilbene) is a naturally occurring phytoalexin produced by some plants such as grapes, peanuts and berries in response to environmental stresses such as UV irradiation or fungal infections. )to be. Resveratrol and its derivatives exhibit a variety of beneficial properties, including anti-inflammatory, anti-tumor and anti-aging effects, as well as an important role as phytoalexin and antioxidant in plant defense responses. However, the potential use of trans -resveratrol is limited due to its instability in environments exposed to light and oxygen or with strong pH conditions. These conditions are cis to cause biological availability (bioavailability) and the biological reduction of the activity (bioactivity) of the compound can cause the isomerization (cis -isomerization) or oxidation. For this reason, it is important to develop resveratrol derivatives with improved stability. An effective method for stabilizing resveratrol can be carried out by methylation of resveratrol hydroxyl groups to form methylated resveratrol derivatives.

Pterostilbene (3,5-dimethoxy-4'-hydroxy- trans- stilbene), 3,4',5-trimethoxystilbene (3,4',5-trimethoxystilbene), pinostilbene ( pinostilbene; 3,4'-dihydroxy-5-methoxy- trans- stilbene) and desoxyrhapotigenin (3,5-dihydroxy-4'- methoxy-trans- stilbene). There are derivatives of methylated resveratrol, but their biological activity is largely unknown. Certain methylated resveratrol derivatives have been demonstrated to be equivalent or more effective than resveratrol for their chemoprotective and anti-tumor activity (Chao et al ., J Nutr Biochem 21:482-489, 2010). For example, trimethylated resveratrol or pinostilben has been reported to exhibit more than 100-fold cytotoxicity than resveratol in cancer cell lines, and this cytotoxic effect can be attributed to depletion of intracellular polyamine pools and Mediated by alteration of microtubule polymerization. In addition, it has been reported that pinostilben exerts a stronger neuroprotective effect than resveratrol on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells (Chao et al. al ., J Nutr Biochem 21:482-489, 2010). In addition, pinostilbene, which is more hydrophobic, can penetrate cell membranes more easily than resveratrol, thus exhibiting a more effective intracellular dose and improved bioavailability. These data suggest that methylation of the hydroxyl group of resveratrol increases its hydrophobicity and enhances cellular uptake of its derivatives. For this reason, the study of the structure-activity relationship of resveratrol derivatives is a very interesting field for the development of more stable and potent chemoprotective agents, and motivates the production of methylated resveratrol derivatives in recombinant E. coli.

Resveratrol has become an attractive target in metabolic engineering because of its diverse health-promoting activity, and several attempts to produce resveratrol in recombinant microorganisms have been reported to date (Beekwilder et al. al ., Appl Environ Microbiol 5670-567, 2006). However, little is known about the production of methylated resveratrol derivatives in microorganisms. In addition, the biosynthetic pathway and essential enzymes of resveratrol are well elucidated, but the enzymes involved in the formation of methylated resveratrol derivatives are still in need of further research.

On the other hand, Korean Patent Application Publication No. 2003-0067689 discloses'production of stilbene in transgenic plants and its production method', and Korean Patent No. 1053025 discloses'production of phenylpropanoid compounds in actinomycetes. The method' is disclosed. However, as in the present invention, a recombinant microorganism for producing a stilbene compound and a method for producing a stilbene compound using the same have not been disclosed.

The present invention was derived from the above requirements, and the inventors of the present invention are codon-optimized SbROMT3syn (synthetic Sorghum bicolor E. coli and ScCCL transformed with resveratrol O -methyltransferase 3) gene ( Streptomyces coelicolor carboxyl CoA ligase) Gene, SbROMT3syn or VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase) gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene were confirmed to be produced in E. coli, methylated resveratrol derivatives, pinostilben and pterostilbene. By doing this, the present invention has been completed.

In order to solve the above problems, the present invention provides a recombinant microorganism having the ability to produce a stilbene compound by transforming with a recombinant vector containing the SbROMT3syn (synthetic Sorghum bicolor resveratrol O-methyltransferase 3) gene derived from sorghum.

In addition, the present invention is ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Gene, VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase ) gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) is transformed with a recombinant vector containing a gene to provide a recombinant microorganism having the ability to produce a stilbene compound.

In addition, the present invention is ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Gene, SbROMT3syn (synthetic Sorghum bicolor resveratrol O- methyltransferase 3) gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) is transformed with a recombinant vector containing a gene to provide a recombinant microorganism having the ability to produce a stilbene compound.

In addition, the present invention provides a method for producing a stilbene compound comprising culturing the microorganism in a medium containing a substrate.

In addition, the present invention is ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) It provides a composition for producing stilbene of microorganisms containing a recombinant vector containing a gene and a recombinant vector containing a SbROMT3syn (synthetic Sorghum bicolor resveratrol O- methyltransferase 3) gene and a RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene as an active ingredient. .

Recombinant microorganism transformed with the codon-optimized SbROMT3syn (synthetic Sorghum bicolor resveratrol O- methyltransferase 3) gene of the present invention and ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) When a recombinant microorganism in which the gene, SbROMT3syn or VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase ) gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene are simultaneously transformed is used, it has improved stability in an environment exposed to light and oxygen, and has excellent stability. The methylated resveratrol derivatives pinostilben and pterostilbene, which exhibit anticancer, antiviral, anti-inflammatory, anti-aging and antioxidant effects, can be produced in large quantities.

1 shows a schematic diagram of a construct including a biosynthetic pathway of a stilbene compound and a ROMT gene. (A) Biosynthetic pathway of production of stilbene compounds from phenylalanine. (B) Recombinant plasmid construct with synthesized plant ROMT gene. The sequential action of PAL, C4H, 4CL, STS and ROMT results in the conversion of phenylalanine to the stilbene compound, resveratrol and methylated derivatives thereof. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate:coenzyme A ligase; STS, stilbene synthase; ROMT, resveratrol O-methyl transferase dehydratase (O -methyltransferase resveratrol); Pro T7, T7 RNA polymerase promoter; H, Heath-tag (His-tag).
Figure 2 shows the expression of hist-tagged VrROMTsyn and SbROMT3syn recombinant proteins in E. coli BL21-Codon Plus (DE3)-RIPL cells. E. coli cells containing the pETDuet-VrROMTsyn or pETDuet-SbROMT3syn construct were grown for 2 hours at a temperature of 25° C. in a modified M9 medium containing 2% glycerol, and 0.2 mM IPTG was added to induce protein expression for 4 hours. Proteins were separated by 13% SDS-PAGE and stained with CBB (Coomassie brilliant blue). Expression of the hist-tag fusion protein was confirmed by Western blot analysis using an anti-His-tag antibody. (A) SDS-PAGE and Western blot results of VrROMTsyn protein. (B) SDS-PAGE and Western blot results of SbROMT3syn protein. M, protein molecular marker (kDa); S, soluble fraction; P, insoluble pellet fraction.
Figure 3 shows the hourly production of pinostilben and pterostilbene in recombinant E. coli BL21-Codon Plus. E. coli cells containing pETDuet-VrROMTsyn or pETDuet-SbROMT3syn plasmid were cultured in a modified M9 medium containing 2% glycerol, and indicated time points after adding 1 mM resveratrol to pre-induced protein expression cells. Were harvested. E. coli cells containing pETDuet-1 as an empty vector were used as a control. Samples were extracted with ethyl acetate and analyzed by HPLC. E. coli cells were induced by adding 0.2 mM IPTG to express the recombinant protein. The content of pinostilbene and pterostilbene was determined by comparison with a standard curve produced from pinostilbene and pterostilbene at known concentrations. (A) Content of pinostilbene. (B) Content of pterostilbene.
Figure 4 shows the HPLC analysis of pinostilbene and pterostilbene produced in recombinant E. coli BL21-Codon Plus. E. coli cells containing pETDuet-VrROMTsyn or pETDuet-SbROMT3syn plasmid were cultured for 60 hours before extraction. HPLC analysis was performed on an Agilent 1200 system using a C18 reverse phase column with a linear gradient of a binary solvent system. The chromatogram STD represents the authentic standard of resveratrol, pinostilben and pterostilbene with retention times of 9.423, 19.327 and 32.009 minutes, respectively. The chromatogram controls, VrROMT and SbROMT3, indicate that the sample is produced from resveratrol by recombinant E. coli. The inset shows an enlarged chromatogram corresponding to pterostilbene.
Figure 5 shows the LC-MS analysis of pinostilben and pterostilbene produced by recombinant E. coli BL21-Codon plus. The molecular weights of pinostilbene and pterostilbene were analyzed by linear ion trap quadrupole LC-MS in the positive mode of standard atmospheric pressure chemical ionization. Samples were obtained from cells cultured for 60 hours after addition of resveratrol, and the extracted compounds were analyzed by LC-MS, and the injection volume was 10 μl. Chromatograms (A) and (B) show LC-MS spectra showing peaks at m/z 243.2 and 257.2 for pinostilbene and pterostilbene, respectively.
6 shows a recombinant plasmid having ScCCL, RpSTSsyn, VrROMTsyn and SbROMT3syn genes involved in stilbene biosynthesis. CCL, carboxyl CoA ligase; STS, stilbene synthase; ROMT, resveratrol O - methyl transferase dehydratase (resveratrol O -methyltransferase); T7 Pro, T7 RNA polymerase promoter; H, His-tag; S, S-tag. ScCCL was cloned into pCOLADuet-1, and RpSTSsyn , VrROMTsyn and SbROMT3syn were cloned into pETDuet-1.
7 shows the expression of hist-tagged CCL, VrROMTsyn and SbROMT3syn and S-tagged STS recombinant protein in E. coli BL21-Codon Plus (DE3)-RIPL cells. pCOLADuet-H::CCL and pETDuet-STS::S, pCOLADuet-H::CCL and pETDuet-H::VrROMT-STS::S or pCOLADuet-H::CCL and pETDuet-H::SbROMT3-STS:: E. coli cells containing the S construct were grown for 2 hours at a temperature of 25° C. in a modified M9 medium containing 2% glycerol, and 0.2 mM IPTG was added to induce protein expression for 4 hours. Proteins were separated by 13% SDS-PAGE and stained with CBB (Coomassie brilliant blue). Expression of the hist-tag or S-tag fusion protein was confirmed by Western blot analysis using an anti-his-tag antibody or an anti-S-tag antibody. (A) a microorganism transformed with pCOLADuet-CCL and pETDuet-STS vectors, (B) a microorganism transformed with pCOLADuet-CCL and pETDuet-STS-VrROMT vector, and (C) pCOLADuet-CCL and pETDuet-STS-SbROMT3 vectors Western blot for SDS-PAGE, S-tag and hist-tag of transformed microorganism-derived products. M, protein molecular marker (kDa); S, soluble fraction; P, insoluble pellet fraction. E. coli cells containing pCOLADuet-1 and pETDuet-1 were used as controls (Con). The S-tag binding STS, hist-tag binding CCL and ROMT recombinant proteins recognized by the anti-S-tag antibody and the anti-His-tag antibody are indicated by an asterisk, an arrow, and an arrowhead, respectively.
Figure 8 shows the hourly production of resveratrol and pinostilben in recombinant E. coli BL21-Codon Plus. pCOLADuet-H::CCL and pETDuet-STS::S (CCL+STS), pCOLADuet-H::CCL and pETDuet-H::VrROMT-STS::S (CCL+STS-VrROMT) or pCOLADuet-H:: E. coli cells containing CCL and pETDuet-H::SbROMT3-STS::S (CCL+STS-SbROMT3) constructs were cultured in modified M9 medium containing 2% glycerol, and 1 mM p − Harvest at the indicated time points after addition of comaric acid. E. coli cells containing pColADuet-1 and pETDuet-1 were used as controls. Samples were extracted with ethyl acetate and analyzed by HPLC. E. coli cells were induced by adding 0.2 mM IPTG to express the recombinant protein. The contents of resveratrol and pinostilbene at the indicated time points (mg/L) were determined by comparison with a standard curve produced from known concentrations of resveratrol and pinostilbene. (A) Content of resveratrol. (B) Content of pinostilbene.
9 shows the results of HPLC analysis of resveratrol and pinostilben produced by recombinant E. coli BL21-Codon Plus. pCOLADuet-H::CCL and pETDuet-STS::S (CCL+STS), pCOLADuet-H::CCL and pETDuet-H::VrROMT-STS::S (CCL+STS-VrROMT) or pCOLADuet-H:: E. coli cells containing the CCL and pETDuet-H::SbROMT3-STS::S (CCL+STS-SbROMT3) constructs were cultured for 48 hours prior to extraction. HPLC analysis was performed on an Agilent 1200 system using a C18 reverse phase column with a linear gradient of a binary solvent system. The chromatogram STD represents the authentic standard of resveratrol (Res), pinostilbene (Pino) and pterostilbene (Ptero) with retention times of 9.423, 19.327 and 32.009 minutes, respectively. The chromatogram controls, CCL+STS, CCL+STS-VrROMT and CCL+STS-SbROMT3 indicate that the samples are produced from p -coumaric acid by recombinant E. coli. The inset shows an enlarged chromatogram to show the small amount of pterostilbene production. Arrows indicate peaks corresponding to pterostilbene.

In order to achieve the object of the present invention, the present invention is SbROMT3syn (synthetic Sorghum) derived from sorghum consisting of the nucleotide sequence of SEQ ID NO: 4 bicolor Resveratrol O -methyltransferase 3) It is transformed with a recombinant vector containing a gene to provide a recombinant microorganism having the ability to produce stilbene compounds.

The SbROMT3syn gene may preferably consist of a nucleotide sequence represented by SEQ ID NO: 4, but is not limited thereto. The SbROMT3syn gene consisting of the nucleotide sequence of SEQ ID NO: 4 is preferably redesigned in consideration of the compatibility of the microbial heterologous host codon with the nucleotide sequence of the sorghum-derived ROMT3 (resveratrol O- methyltransferase 3) gene consisting of the nucleotide sequence of SEQ ID NO: 3 It is an artificial gene synthesized after codon optimization for E. coli. In addition, homologs of the nucleotide sequence are included within the scope of the present invention. The homolog is a nucleotide sequence that has a nucleotide sequence change, but has functional characteristics similar to the nucleotide sequence of SEQ ID NO: 4. Specifically, the SbROMT3syn gene has a nucleotide sequence of 70% or more, more preferably 80% or more, more preferably 90% or more, most preferably 95% or more of the nucleotide sequence of SEQ ID NO: 4, respectively. Can include. The "% of sequence homology" for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

In addition, the present invention is ScCCL consisting of the nucleotide sequence of SEQ ID NO: 5 ( Streptomyces coelicolor carboxyl CoA ligase) VrROMTsyn (synthetic Vitis) consisting of the gene and the nucleotide sequence of SEQ ID NO: 2 riparia RpSTSsyn (synthetic Rheum) consisting of the resveratrol O -methyltransferase) gene and the nucleotide sequence of SEQ ID NO: 7 Palmatum stilbene synthase) is transformed with a recombinant vector containing a gene to provide a recombinant microorganism having the ability to produce a stilbene compound, preferably ScCCL consisting of the nucleotide sequence of SEQ ID NO: 5 A recombinant vector including a gene and a recombinant vector including a VrROMTsyn gene consisting of a nucleotide sequence of SEQ ID NO: 2 and a RpSTSsyn gene consisting of a nucleotide sequence of SEQ ID NO: 7 are simultaneously transformed to provide a recombinant microorganism having the ability to produce a stilbene compound.

In addition, the present invention is ScCCL consisting of the nucleotide sequence of SEQ ID NO: 5 ( Streptomyces coelicolor carboxyl CoA ligase) Gene, SbROMT3syn (synthetic Sorghum) consisting of the nucleotide sequence of SEQ ID NO: 4 bicolor resveratrol O -methyltransferase 3) RpSTSsyn (synthetic Rheum) consisting of the gene and the nucleotide sequence of SEQ ID NO: 7 Palmatum stilbene synthase) is transformed with a recombinant vector containing a gene to provide a recombinant microorganism having the ability to produce a stilbene compound, preferably ScCCL consisting of the nucleotide sequence of SEQ ID NO: 5 A recombinant vector containing a gene and a recombinant vector including the SbROMT3syn gene consisting of the nucleotide sequence of SEQ ID NO: 4 and the RpSTSsyn gene consisting of the nucleotide sequence of SEQ ID NO: 7 are simultaneously transformed to provide a recombinant microorganism having the ability to produce a stilbene compound.

The ScCCL gene may preferably consist of a nucleotide sequence represented by SEQ ID NO: 5, but is not limited thereto. In addition, the VrROMTsyn gene may preferably consist of a nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto. The VrROMTsyn gene consisting of the nucleotide sequence of SEQ ID NO: 2, preferably, the nucleotide sequence of the resveratrol O- methyltransferase (ROMT) gene derived from grape (Vitis riparia ) consisting of the nucleotide sequence of SEQ ID NO: 1, considering the suitability of the codon for the heterogeneous host of microorganisms. It is an artificial gene synthesized after redesigning and optimizing codons for E. coli. In addition, the SbROMT3syn gene may preferably consist of a nucleotide sequence represented by SEQ ID NO: 4, but is not limited thereto. The SbROMT3syn gene consisting of the nucleotide sequence of SEQ ID NO: 4 , preferably, the nucleotide sequence of the sorghum-derived ROMT3 gene consisting of the nucleotide sequence of SEQ ID NO: 3 is redesigned in consideration of the suitability of the codon for the heterogeneous host of microorganisms, and codon optimization for E. coli It is an artificial gene synthesized after doing so. In addition, the RpSTSsyn gene may preferably consist of a nucleotide sequence represented by SEQ ID NO: 7, but is not limited thereto. The RPSTSsyn gene consisting of the nucleotide sequence of SEQ ID NO: 7 is preferably the nucleotide sequence of the STS (stilbene synthase) gene derived from Rheum Palmatum consisting of the nucleotide sequence of SEQ ID NO: 6 in consideration of the suitability of the codon for the heterogeneous host of microorganisms. It is an artificial gene designed and synthesized after codon optimization for E. coli. Homologs of each of the above nucleotide sequences are included within the scope of the present invention. The homolog is a nucleotide sequence that has a functional property similar to that of the corresponding nucleotide sequence, although the nucleotide sequence is changed.

Recombinant microorganism having the above-mentioned stilbene compound producing ability is, for example, in the case of the transformed microorganisms in ScCCL gene, SbROMT3syn gene and RpSTSsyn gene, one of ScCCL gene on one vector, SbROMT3syn gene and RpSTSsyn gene, 2 or 3 If the ScCCL gene, the SbROMT3syn gene, and the RpSTSsyn gene are normally expressed even when transformed with a recombinant vector containing two genes to produce a stilbene compound, the possibility of selection is not limited in terms of the construction form or transformation sequence of the vector.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells may express genes or gene segments that are not found in the natural form of the cell in either a sense or antisense form. In addition, recombinant cells can express genes found in cells in their natural state, but the genes are modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment(s), a nucleic acid molecule, which is delivered into a cell. Vectors replicate DNA and can be reproduced independently in host cells. The term “carrier” is often used interchangeably with “vector”. The term "expression vector" refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a particular host organism. In general, any plasmid and vector can be used as long as they can replicate and stabilize in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element. Translational regulatory elements that can be used in prokaryotic or eukaryotic cells, enhancers, ribosome binding sites, termination signals, polyadenylation signals, and promoters are known in the art. Expression vectors containing appropriate transcription/translational control signals can be constructed by methods well known in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. In addition, the expression vector may include a ribosome binding portion and a transcription terminator as a translation initiation site.

In the recombinant vector of the present invention, in the case of an expression vector using prokaryotic cells as a host, a strong promoter capable of proceeding transcription (eg, pLλ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.), initiation of translation It is common to include a ribosome binding site for and a transcription/translation termination sequence. When E. coli is used as a host cell, a promoter and operator site of the E. coli tryptophan biosynthetic pathway, and a left-handed promoter of phage λ (pLλ promoter) may be used as a regulatory site. Vectors that can be used in the present invention include plasmids used in the art (e.g., pETDuet, pCOLADuet, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.), phage (e.g., λgt4. λB, λ-Charon, λΔz1 and M13, etc.), viruses (e.g., SV40, etc.) or fosmid may be manipulated, and preferably pETDuet or pCOLADuet vector may be used, but is not limited thereto.

The recombinant vector of the present invention may preferably contain one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, and all genes capable of distinguishing a transformed cell from a non-transgenic cell correspond to this. The recombinant vector of the present invention as a selectable marker, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline resistance genes, which are antibiotic resistance genes commonly used in the art. It may include, but is not limited thereto.

Host cells capable of continuously cloning and expressing the vector of the present invention while being stable in prokaryotic cells may be any host cell known in the art. For example, E. coli BL21, E. coli JM109, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringensis, and other strains of the genus Bacillus, and Salmonella typhimurium, Serratia marsessons, and various Pseudomonas Intestinal bacteria and strains such as species are included.

In one embodiment of the present invention, the recombinant microorganism transformed with the recombinant vector of the present invention and having the ability to produce a stilbene compound may preferably be E. coli , more preferably E. coli BL21. However, it is not limited thereto.

When the host cell of the present invention is a prokaryotic cell, the recombinant vector of the present invention is a host cell by the CaCl ₂ method, one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)), and the electroporation method. It can be transported into cells.

In one embodiment of the present invention, the stilbene compound may be a methylated resveratrol compound, preferably pinostilbene or pterostilbene, but is not limited thereto.

In addition, the present invention provides a method for producing a stilbene compound comprising culturing a recombinant microorganism transformed with a recombinant vector containing the SbROMT3syn gene in a medium containing resveratrol as a substrate.

In one embodiment of the present invention, the stilbene compound may be a methylated resveratrol compound, preferably pinostilbene or pterostilbene, but is not limited thereto.

The present invention is a recombinant vector comprising a recombinant vector and SbROMT3syn gene and RpSTSsyn gene is a recombinant vector comprising a recombinant vector and VrROMTsyn gene and RpSTSsyn genes including ScCCL gene containing the transformed recombinant microorganism or ScCCL genes At the same time, it provides a method for producing a stilbene compound comprising culturing the transformed recombinant microorganism in a medium containing p-coumaric acid as a substrate.

In one embodiment of the present invention, the stilbene compound may be resveratrol, pinostilben, or pterostilbene, but is not limited thereto.

The cultivation of the recombinant microorganism of the present invention can be carried out using conventional conditions used for culturing a microbial host. As the cultivation method, any method used for cultivation of ordinary microorganisms, such as a batch type, a flow batch type, a continuous culture, and a reactor type, can be used. The medium for culturing the transformant obtained using bacteria such as E. coli as a host may be a complete medium or a synthetic medium (LB medium, NB medium, M9 medium, etc.), but is not limited thereto. Carbon sources provided to the medium for the growth of microorganisms include, for example, sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; Lower alcohols such as ethanol, propanol, and butanol; Polyhydric alcohols such as glycerol; Organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid, and gluconic acid; Fatty acids such as propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid can be used, and the nitrogen source is, for example, ammonia, ammonium chloride, ammonium sulfate, In addition to ammonium salts such as ammonium phosphate, those derived from natural products such as peptone, meat juice, yeast extract, malt extract, casein decomposition products, and corn steep liquor can be used. In addition, as the inorganic material, for example, first potassium phosphate, second potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, or the like can be used. In addition, according to the antibiotic resistance of the recombinant microorganism, appropriate antibiotics such as ampicillin, chloramphenicol, kanamycin, and streptomycin can be added and cultured. When culturing a microorganism transformed with an expression vector containing an inducible promoter In this case, an inducer suitable for the type of promoter can be added to the medium and cultured. The inducer may be, for example, isopropyl-D-1-thiogalactopyranoside (IPTG), tetracycline, or IAA (indoleacetic acid).

In addition, the present invention is ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) consisting of the nucleotide sequence of SEQ ID NO: 5 A recombinant vector containing a gene and a recombinant vector including a SbROMT3syn (synthetic Sorghum bicolor resveratrol O -methyltransferase 3) gene consisting of a nucleotide sequence of SEQ ID NO: 4 and a RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene consisting of a nucleotide sequence of SEQ ID NO: 7 It provides a composition for producing stilbene of microorganisms containing as an active ingredient. The composition comprises a recombinant vector comprising a RpSTSsyn gene consisting of the nucleotide of the consisting of the recombinant vector and the nucleotide sequence of SEQ ID NO: 4 comprising a ScCCL gene consisting of the nucleotide sequence of SEQ ID NO: 4 as an active ingredient SbROMT3syn gene and SEQ ID NO: 7, SEQ ID NO: And, by transforming the genes into a microorganism, it is possible to produce a stilbene compound in the microorganism.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1. Biosynthetic pathway of resveratrol and pterostilbene

The stilbene production process of the present invention follows the stilbene biosynthetic pathway shown in FIG. 1. Is created Kumar acid (p -coumaric acid), 4CL-tyrosine, from amino acids such as phenylalanine, p (4-coumarate: CoA ligase ) p - by the one -CoA (p -coumaroyl-CoA) into Kumano is generated. The p -coumaroyl-CoA produced in this way is converted to resveratrol by stilbene synthase (STS), and resveratrol is again methylated by the methylating enzyme ROMT (resveratrol O -methyltransferase) and pinostilbene. ), which is again methylated and converted to pterostilbene. The present inventors used a CCL (carboxyl CoA ligase) protein of actinomycetes, which is known to have the same enzymatic activity as 4CL, in producing p-coumaroyl-CoA.

Example 2. Pterostilbene synthetic transcription factor VrROMT And SbROMT3 Gene cloning

In order to develop a pterostilbene production system, the transcription factor genes VrROMT (1074 bp, SEQ ID NO: 1) and SbROMT3 (1125 bp, SEQ ID NO: 3) related to pterostilbene biosynthesis from grapes (Vitis riparis ) and sorghum ( Sorghum bicolor) ) Was used separately. The gene was isolated by amplification by homology-based PCR using PCR primers obtained from VrROMT (GenBank registration number: FM178870) and SbOMT3 (GenBank registration number: EF189708) genes. The primers used for PCR are as follows: VrROMT-F: 5'-ATGGATTTGGCAAACGGTGTGA-3' (SEQ ID NO: 8), VrROMT-R: 5'-TCAAGGATAAACCTCAATGAGGGA-3' (SEQ ID NO: 9), SbROMT3-F: 5'- ATGGTACTCATCAGCGAGGACAGT-3' (SEQ ID NO: 10), SbROMT-R: 5'-TCATGGATATAGCTCAATGATCGATC-3' (SEQ ID NO: 11). Grape samples were obtained from the Chungcheongbuk-do Institute of Agricultural Technology Grape Research Institute (www.ares.chungbuk.kr), and sorghum samples were obtained from the Agricultural Genetic Institute Information Center (http://www.genebank.go.kr/) of the National Institute of Agricultural Science, Rural Development Administration. I did.

The obtained grape or sorghum plant-derived genes are used for the synthesis of artificial genes VrROMTsyn (SEQ ID NO: 2) and SbROMT3syn (SEQ ID NO: 4) after redesigning the base sequence in consideration of the codon compatibility of the heterogeneous host of microorganisms, optimizing the codon for E. coli. I did. The synthesized artificial gene was used to induce productivity improvement of proteins and functional substances.

Example 3. Resveratrol methylation gene ( ROMT ) Microbial overexpression vector production

In order to find out whether the synthesis of resveratrol and pterostilbene can be increased when the VrROMTsyn and SbROMT3syn genes cloned in Example 2 are overexpressed in a microorganism, the T7 promoter of the microbial expression vector pETDuet having an ampicillin resistance gene A vector was constructed by inserting the VrROMTsyn gene or the SbROMT3syn gene between the and T7 terminators. The constructed overexpression vectors were named pETDuet-His::VrROMTsyn (pETDuet-VrROMTsyn) and pETDuet-His::SbROMT3syn (pETDuet-SbROMT3syn), respectively (Fig. 1B). E. coli DH5α strain was used to construct the vector.

Example 4. Production of recombinant microorganisms producing stillbens

The pETDuet-His::VrROMTsyn (pETDuet-VrROMTsyn) and pETDuet-His::SbROMT3syn (pETDuet-SbROMT3syn) microbial expression vectors prepared in Example 3 are chloramphenicol (chloramphenicol)-resistant protein-expressing Escherichia coli (BL21 Plus RI PL21). DE3)) was transformed by a heat shock method. The prepared recombinant microorganisms producing pinostilben or pterostilbene were selected in LB plate medium to which ampicillin and chloramphenicol were added at 50 µg/ml and 30 µg/ml, respectively.

Example 5. Analysis of expression of stilbene protein in recombinant microorganisms

Western blot analysis was performed to confirm whether the VrROMTsyn gene and the SbROMT3syn gene were stably expressed and translated in each of the recombinant microorganisms selected in Example 4 above. Recombinant microorganisms were pre-cultured in LB liquid medium containing chloramphenicol and ampicillin, passed to 50 ml of liquid medium, and cultured at 37° C. so that the absorbance at 600 nm was 0.6 to 0.7. Then, 0.5 mM IPTG (Isopropyl-D-1-thiogalactopyranoside) was added to induce protein expression, and 10-20 ml of samples were collected over time to isolate the protein. As a result of Western blot analysis using an anti-His-tag antibody, the proteins were identified at positions corresponding to the theoretical molecular weights (41.7 and 42.5 kDa, respectively) of VrROMT and SbROMT3 proteins (FIG. 2). That is, it was confirmed that the VrROMT and SbROMT3 genes, which synthesize pinostilben and pterostilbene by attaching a methyl group to resveratrol, were stably expressed and translated.

Example 6. Analysis of Stilbene Compound Content in Recombinant Microbial Culture Solution

In order to confirm that each recombinant microorganism whose expression of protein was confirmed in Example 5 could catalyze the production of methylated resveratrol derivatives, 16.5 μg/ml chloramphenicol and 50 μg/ml ampicillin were pre-cultured in LB medium. 1% was inoculated into 200 ml of the LB medium and cultured at 37° C. so that the absorbance was 0.6 to 0.7. After that, in order to induce protein production, 0.1 mM IPTG was added and incubated for 3 hours at 25°C. After collecting only the recombinant protein-expressing strain by centrifuging the culture medium, the modified M9 (M9 broth, 0.5% glycerol, 1 mM MgSO ₄ , 0.125% yeast extract) liquid medium suitable for producing pinostilben and pterostilbene Resuspended in. Ampicillin, chloramphenicol and 0.5 mM IPTG were added to the medium, and 1 mM resveratrol to be used as a substrate was added, followed by incubation at 25° C. for 60 hours. During the cultivation, 20 ml of culture solution samples were collected every hour (0, 6, 12, 24, 48 and 60 hours), and the collected sample was adjusted to pH 9.0 with 5 N sodium hydroxide and the same amount of ethyl acetate The desired material was extracted with. After the ethyl acetate was removed by evaporation, the extracted sample was dissolved in 1 ml of HPLC methanol, filtered through a 0.2 µm PTFE filter (hydrophilic, ADVANTEC, Japan), and filtered through HPLC (High-performance liquid chromatography) and LC-MS (liquid chromatograph). -mass spectrometer) was used for analysis. HPLC was analyzed using an Agilent technology 1200 series, and a quaternary pump system and a Zorbax Eclipse XBD-C18 (5 mm, 4.6×150 mm; agilent) column were used. Water (A, 0.05% trifluoroacetic acid) and acetonitrile (B, 0.05% trifluoroacetic acid) were used as the mobile phase, and the extract was separated using gradient elution.

As a result of analyzing the extracts produced by each recombinant microorganism, it was confirmed that pinostilbene and trace amounts of pterostilbene were detected in the culture medium of the recombinant microorganism (FIG. 4), and the extract detection results were summarized by time and graphed (Fig. 3). In the SbROMT3syn clone, pinostilbene levels began to increase from 6 hours after resveratrol addition and continued to accumulate up to 60 hours. Cultured cells containing the pETDuet-SbROMT3syn plasmid produced high levels of methylated derivatives with pinostilbene at a concentration of 34 mg/L and a small amount of pterostilbene at a concentration of 0.16 mg/L as the main products in a 60-hour culture. (Fig. 3). On the other hand, in the cultured cells containing the pETDuet-VrROMTsyn plasmid, very small amounts of pinostilben (about 0.16 mg/L) and pterostilbene (0.04 mg/L) were detected in 48-shiga culture (FIG. 3 ). In cells expressing the empty vector pETDuet-1, pinostilbene and pterostilbene were not produced. In addition, as a result of performing an LC-MS analysis using a sample cultured for 60 hours, it was confirmed again that the newly generated peaks were pinostilbene and pterostilbene (FIG. 5). Therefore, the present inventors confirmed that pterostilbene is produced from the resveratrol substrate, and based on this result, as shown in FIG. 1, p -coumaric acid attempted to produce pterostilbene.

Example 7. p -Coumaroyl-CoA synthetic transcription factor ScCCL Gene cloning

In order to develop a production system for p -coumaroyl- CoA, a transcription factor gene ScCCL (1569 bp) related to p-coumaroyl-CoA biosynthesis was isolated from actinomycetes (Streptomyces coelicolor) and used. For genetic information, reference was made to a paper by Kaneko et al. (J. Bacterial. 185: 20-27, 2003), and in order to increase the productivity of the protein expressed in the microbial production system, the CCL derived from the microorganism rather than the plant-derived 4CL gene shown in FIG. 1 The gene was used. Gene samples were obtained from the Korea Research Institute of Bioscience and Biotechnology Chemical Biology Research Center (www.kribb.re.kr), and the ScCCL gene (SEQ ID NO: 5) was obtained by separating from the pET28-ScCCL vector using Nde I and Hind III.

Example 8. Resveratrol synthetic transcription factor RpSTS Gene cloning

To develop a resveratrol production system, the transcription factor gene RpSTS related to resveratrol biosynthesis from Rheum palmatum (1176 bp, SEQ ID NO: 6) was isolated and used. The gene was isolated by amplification by homology-based PCR using PCR primers obtained from the RpSTS (GenBank registration number: JX673939) gene. The primers used were as follows: RpSTS-F: 5'-CATATGGCACCGGAGGAGT-3' (SEQ ID NO: 12), RpSTS-R: 5'-ACTAGTTCAGGTAATTAGCGGC-3' (SEQ ID NO: 13). Jangyeop rhubarb samples were obtained from the Northern Agricultural Experiment Station (www.ares.gangwon.kr), Gangwon-do Agricultural Technology Institute, Rural Development Administration. The gene derived from the Jangyeop rhubarb plant was used for the synthesis of the artificial gene RpSTSsyn (SEQ ID NO: 7) after redesigning the base sequence in consideration of the codon compatibility of the heterogeneous host of the microorganism, and optimizing the codon for E. The synthesized artificial gene was used to induce productivity improvement of proteins and functional substances.

Example 9. Construction of a vector for microbial overexpression of stilbene biosynthetic gene

The cloned VrROMTsyn , SbROMT3syn , ScCCL and RpSTSsyn In order to confirm whether the synthesis of resveratrol and pterostilbene can be increased when the gene is overexpressed in a microorganism, a vector in which ScCCL is inserted between the T7 promoter and the T7 terminator of the microbial expression vector pCOLADuet having a kanamycin resistance gene was constructed. Also it was produced Amphitheater of between cylinder resistant to having microbial expression vector pETDuet of the T7 promoter and T7 terminator RpSTS gene insertion vector, RpSTS gene and the VrROMT gene is inserted at the same time vector and RpSTS gene and SbROMT3 vector the genes are simultaneously sapin each . The constructed overexpression vectors are pCOLADuet-His::ScCCL (pCOLADuet-CCL), pETDuet-RpSTS::S (pETDuet-STS), pETDuet-His::VrROMT-RpSTS::S (pETDuet-STS-VrROMT) and Named His::SbROMT3-RpSTS::S (pETDuet-STS-SbROMT3), respectively (Fig. 6). E. coli DH5α strain was used to construct the vector.

Example 10. Production of recombinant microorganisms producing stillbens

PCOLADuet-His::ScCCL (pCOLADuet-CCL), pETDuet-RpSTS::S (pETDuet-STS) prepared in Example 9, pETDuet-His::VrROMT-RpSTS::S (pETDuet-STS-VrROMT) and pETDuet-His::SbROMT3-RpSTS::S (pETDuet-STS-SbROMT3) microbial expression vector was transformed into E. coli (BL21 Codon Plus RIPL(DE3)) expressing a chloramphenicol resistant protein by a heat shock method. . First, pCOLADuet-His::ScCCL (pCOLADuet-CCL) and pETDuet-RpSTS::S (pETDuet-STS) vectors were simultaneously transformed to confirm the production of resveratrol, and pCOLADuet for the production of pinostilben and pterostilbene. -His::ScCCL (pCOLADuet-CCL) and pETDuet-His::VrROMT-RpSTS::S (pETDuet-STS-VrROMT) or pCOLADuet-His::ScCCL (pCOLADuet-CCL) and pETDuet-His: ::S (pETDuet-STS-SbROMT3) vector was transformed at the same time. The prepared stilbene-producing microorganisms were selected on an LB plate medium to which ampicillin, kanamycin, and chloramphenicol were added, respectively, at 50 µg/ml, 25 µg/ml, and 30 µg/ml.

Example 11. Analysis of Resveratrol Synthetic Protein Expression of Recombinant Microorganisms

To confirm whether the ScCCL , RpSTS , VrROMT and SbROMT3 genes are stably expressed and translated in each of the selected recombinant microorganisms, Western blot analysis was performed. After inducing protein expression by adding 0.5 mM IPTG (Isopropyl-D-1-thiogalactopyranoside) to LB liquid medium containing chloramphenicol, ampicillin, and kanamycin, the results of analysis on recombinant microorganisms showed resveratrol and pterostilbene. Proteins of molecular weight estimated to be the transcription factors of ScCCL, RpSTS, VrROMT and SbROMT3 were confirmed (FIG. 7).

Example 12. In the culture of recombinant microorganisms Stillben Compound content analysis

Each recombinant microorganism in which stable protein expression was confirmed above was extracted after culturing in the same manner as described in Example 6, and p -coumaric acid was used as a substrate instead of resveratrol. The extracted sample was dissolved in 1 ml of HPLC methanol, filtered through a 0.2 µm PTFE filter (hydrophilic, ADVANTEC, Japan), and used for high-performance liquid chromatography (HPLC) analysis. HPLC was analyzed using an Agilent technology 1200 series, and a quaternary pump system and a Zorbax Eclipse XBD-C18 (5 mm, 4.6×150 mm; agilent) column were used. Water (A, 0.05% trifluoroacetic acid) and acetonitrile (B, 0.05% trifluoroacetic acid) were used as the mobile phase, and the extract was separated using gradient elution. The detection results were summarized by time and graphed (FIG. 8), and as a result of analysis of the extract, resveratrol, pinostilbene and pterostilbene newly produced from p-coumaric acid substrate in the recombinant microorganism culture were confirmed (FIG. 9). . As a result of simultaneous expression of ScCCLsyn and RpSTSsyn proteins using p-coumaric acid as a substrate, 0.51 mg/L of resveratrol was produced in a 48-hour culture medium and 0.50 mg/L of resveratrol in a recombinant microorganism culture that simultaneously expressed ScCCLsyn and RpSTSsyn and VrROMTsyn proteins. , ScCCLsyn and RpSTSsyn and SbROMT3syn proteins were simultaneously expressed in recombinant microorganism culture, resulting in 0.41 mg/L of resveratrol and 0.71 mg/L of pinostilben. As a result, it was confirmed that resveratrol produced by ScCCLsyn and RpSTSsyn was methylated by SbROMT3syn protein to become pinostilbene.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Recombinant microorganism producing stilbene compound and method for producing stilbene compound using the same <130> PN15158 <160> 13 <170> KopatentIn 2.0 <210> 1 <211> 1074 <212> DNA <213> Vitis riparia <400> 1 atggatttgg caaacggtgt gatatcagct gagctgcttc atgctcaagc tcatgtctgg 60 aaccatatat tcaacttcat caagtctatg tcactaaaat gtgctactca actaggcatc 120 ccagacatca tccacaacca tggcaagccc atgactcttc ctgagctggt cgctaagctc 180 ccagtccacc ctaaaaggag tcagtgcgtg taccgtctca tgcgcattct tgttcattct 240 ggcttccttg ctgcgcaaag agttcaacaa ggtgaggaag aagaggggta tgtgcttaca 300 gatgcctcta ggctccttct aatggatgac tccttgagca taaggccctt ggtgcttgcc 360 atgctcgacc caattttaac taaaccatgg cattatctga gtgcttggtt tcaaaatgat 420 gatcccactc cgttccacac tgcttatgag cggccatttt gggattatgc cggccatgaa 480 cctcagctca acaattcctt caatgaagcc atggctagcg atgctcgctt actcaccagc 540 gtgctgatta aggagggcaa gggcgtattt gcggggttga actcattagt tgatgtaggg 600 ggtggcaccg gaaaagtggc caaggccatt gctaacgctt tcccacattt gaactgcacc 660 gtgttagatc tcccccacgt ggttgctggc ttgcaaggga gcaagaactt gaactacttt 720 gcaggtgata tgtttgaggc aattcctcct gcagatgcaa ttttactcaa gtggatactg 780 cacgactgga gcgatgaaga atgcgtgaag atactaaagc gatgcaggga agcaattccg 840 agcaaggaaa acggaggaaa ggtgattatc atagacatga tcatgatgaa gaatcaagga 900 gactacaagt ccatagaaac acagctgttc tttgatatga cgatgacgat tttcgccccg 960 ggtagagaga gggacgagaa cgaatgggag aagctattct tggatgctgg tttcagtcac 1020 tacaagataa ctcccatttt gggtttgagg tccctcattg aggtttatcc ttga 1074 <210> 2 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> VrROMTsyn <400> 2 atggatctgg cgaacggtgt gatcagcgcc gaactgctgc atgcacaggc tcacgtttgg 60 aaccatatct tcaacttcat caaaagcatg tctctgaaat gcgcgaccca actgggtatt 120 ccggatatta tccataatca cggcaaaccg atgacgctgc cggaactggt tgccaaactg 180 ccggtccacc cgaaacgttc ccagtgtgtg tatcgtctga tgcgcatcct ggttcattca 240 ggctttctgg cggcccaacg cgttcagcaa ggtgaagaag aagaaggcta cgtcctgacc 300 gatgcgtctc gtctgctgct gatggatgac agtctgtcca tccgtccgct ggtgctggca 360 atgctggacc cgattctgac gaaaccgtgg cattatctga gtgcttggtt tcagaacgat 420 gacccgaccc cgttccacac ggcgtatgaa cgtccgtttt gggattacgc cggtcatgaa 480 ccgcagctga acaattcatt caacgaagcg atggcctcgg acgcacgtct gctgaccagc 540 gtcctgatca aagaaggtaa aggtgttttt gccggtctga attccctggt cgatgtgggc 600 ggtggcaccg gtaaagtcgc aaaagctatt gcgaacgcct tcccgcacct gaattgcacg 660 gttctggatc tgccgcatgt ggttgcaggt ctgcaaggct ctaaaaacct gaattatttt 720 gcaggcgata tgttcgaagc tattccgccg gcagacgcta tcctgctgaa atggattctg 780 cacgattggt cggacgaaga atgcgtgaaa atcctgaaac gttgtcgcga agccattccg 840 agcaaagaaa acggtggcaa agttatcatc atcgatatga tcatgatgaa aaatcagggt 900 gactacaaaa gtattgaaac ccaactgttt ttcgatatga ccatgacgat ctttgcaccg 960 ggccgtgaac gcgatgaaaa tgaatgggaa aaactgtttc tggacgctgg tttctctcat 1020 tataaaatca ccccgattct gggcctgcgt agtctgattg aagtgtaccc gtaa 1074 <210> 3 <211> 1122 <212> DNA <213> Sorghum bicolor <400> 3 atggtactca tcagcgagga cagtagggag ttgctccaag cccacgtcga gctatggaac 60 cagacctaca gctttatgaa gtcggtggcg ctcgccgttg ctttagacct ccgcatcgct 120 gatgccatcc accgctgcgg tggcgccgcc accctctccc agatactcgg agagattggt 180 gtccgcccat gtaagcttcc cgccctccac cgcctaatgc gtgttctgac cgtctcagga 240 accttcacca tcgtccagcc atcagcggca accatgtcat tggtgtcgga cgggaatgag 300 cttgtctata agctgacaac agcgtcccgc ctcctcgtca gcagcgaaag ctcggcgacg 360 gcgagcttgt ctcctatgct gaaccacgtg cttagcccct tccgtgactc gcccctcagc 420 atggggctca ctgcgtggtt ccggcacgat gaagatgaac aggcgcctgg cccatgcccg 480 ttcaccctga tgtacggcac aaccttgtgg gaggtgtgca gtcgtgacga cgcaatcaac 540 gcgttgttca acaacgccat ggccgcagac agcaacttcc tgatgcagat tgtgttgagg 600 gagttcggca aggtcttcca cgggatagac tcgctggtcg acgtcggcgg tggggttggg 660 ggagccacca tggccattgc cacggcgttc ccgtctttga agtgtaccgt actagacctc 720 cctcacgttg tcgccaaggc tccgtccagt tctattggca acgtgcagtt tgttgggggt 780 gacatgtttg agagcattcc accagcgaat gttgtcttcc tcaagtggat tttgcatgac 840 tggagcaatg acgagtgtat caagatatta aagaactgca agcaagctat cccttctaga 900 gatgcaggag gaaagataat aatcattgat gttgtggttg ggtctgagtc atcagacacc 960 aagcttctgg agacacaagt aatgtatgat ctccatctca tgaaaattgg tggggttgaa 1020 cgagatgagc aagagtggaa gaaaatattc ctcgaagctg gatttaagga ctacaatatt 1080 ataccagttt taggcctccg atcgatcatt gagctatatc ca 1122 <210> 4 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> SbROMT3syn <400> 4 atggtgctga ttagcgaaga tagccgtgaa ctgctgcaag cgcatgtgga actgtggaac 60 cagacctact catttatgaa aagcgtcgct ctggcggttg ccctggatct gcatattgca 120 gacgctattc atcgtcgcgg cggtgcggca accctgtctc agattctggg cgaaatcggt 180 gttcgtccgt gcaaactgcc gggcctgcat cgtattatgc gcgtgctgac cgttagtggt 240 acctttacga tcgtccaacc gtcggcggaa acgatgagct ctgaaagcga tggccgcgaa 300 ccggtttata aactgaccac ggccagttcc ctgctggtct catcggaaag ctctgcgacc 360 gcctctctga gtccgatgct gaaccatgtt ctgagcccgt ttcgtgattc cccgctgtca 420 atgggtctga ccgcatggtt ccgccacgat gaagacgaac aggctccggg catgtgcccg 480 tttacgctga tgtacggtac cacgctgtgg gaagtgtgtc gtcgcgatga cgcgattaac 540 gccctgttta acaatgcaat ggcagctgat agcaatttcc tgatgcagat tctgctgaaa 600 gaattttctg aagtcttcct gggcatcgat agtctggttg acgttgccgg cggtgtgggc 660 ggtgcaacca tggctattgc ggccgcattc ccgtgcctga aatgtacggt gctggatctg 720 ccgcatgtgg ttgcaaaagc tccgagttcc tcaatcggta acgtccagtt tgtgggcggt 780 gatatgttcg aatcgattcc gccggcgaat gtcgtgctgc tgaaatggat tctgcacgat 840 tggtcaaacg acgaatgcat caaaatcctg aaaaactgta aacaagcgat tccgtcccgt 900 gatgccggcg gtaaaattat cattatcgac gttgtcgtgg gctcagattc gagcgacacc 960 aaactgctgg aaacgcaagt gatttatgat ctgcacctga tgaaaatcgg cggtgttgaa 1020 cgcgacgaac aagaatggaa gaaaattttt ctggaagcgg gtttcaaaga ttacaaaatt 1080 atgccgattc tgggtctgcg ctccattatt gaactgtatc cgtgactcga caagcttgcg 1140 gccgcataa 1149 <210> 5 <211> 1569 <212> DNA <213> Streptomyces coelicolor <400> 5 atgttccgca gcgagtacgc agacgtcccg cccgtcgacc tgcccatcca cgacgccgtg 60 ctcggcgggg ccgccgcctt cgggagcacc ccggcgctga tcgacggcac cgacggcacc 120 accctcacct acgagcaggt ggaccggttc caccggcgcg tcgccgccgc cctcgccgag 180 accggcgtgc gcaagggcga cgtcctcgcc ctgcacagcc ccaacaccgt cgccttcccc 240 ctggccttct acgccgccac ccgcgcgggc gcctccgtca ccacggtgca tccgctcgcg 300 acggcggagg agttcgccaa gcagctgaag gacagcgcgg cccgctggat cgtcaccgtc 360 tcaccgctcc tgtccaccgc ccgccgggcc gccgaactcg cgggcggcgt ccaggagatc 420 ctggtctgcg acagcgcgcc cggtcaccgc tccctcgtcg acatgctggc ctcgaccgcg 480 cccgaaccgt ccgtcgccat cgacccggcc gaggacgtcg ccgccctgcc gtactcctcg 540 ggcaccaccg gcacccccaa gggcgtcatg ctcacacacc ggcagatcgc caccaacctc 600 gcccagctcg aaccgtcgat gccgtccgcg cccggcgacc gcgtcctcgc cgtgctgccg 660 ttcttccaca tctacggcct gaccgccctg atgaacgccc cgctccggct cggcgccacc 720 gtcgtggtcc tgccccgctt cgacctggag cagttcctcg ccgccatcca gaaccaccgc 780 atcaccagcc tgtacgtcgc cccgccgatc gtcctggccc tcgccaaaca ccccctggtc 840 gccgactacg acctctcctc gctgaggtac atcgtcagcg ccgccgcccc gctcgacgcg 900 cgtctcgccg ccgcctgctc gcagcggctc ggcctgccgc ccgtcggcca ggcctacggc 960 atgaccgaac tgtccccggg cacccacgtc gtccccctgg acgcgatggc cgacgcgccg 1020 cccggcaccg tcggcaggct catcgcgggc accgagatgc gcatcgtctc cctcaccgac 1080 ccgggcacgg acctccccgc cggagagtcc ggggagatcc tcatccgcgg cccccagatc 1140 atgaagggct acctgggccg ccccgacgcc accgccgcca tgatcgacga ggagggctgg 1200 ctgcacaccg gggacgtcgg acacgtcgac gccgacggct ggctgttcgt cgtcgaccgc 1260 gtcaaggaac tgatcaagta caagggcttc caggtggccc ccgccgaact ggaggcccac 1320 ctgctcaccc accccggcgt cgccgacgcg gccgtcgtcg gcgcctacga cgacgacggc 1380 aacgaggtac cgcacgcctt cgtcgtccgc cagccggccg cacccggcct cgcggagagc 1440 gagatcatga tgtacgtcgc cgaacgcgtc gccccctaca aacgcgtccg ccgggtcacc 1500 ttcgtcgacg ccgtcccccg cgccgcctcc ggcaagatcc tccgccgaca gctcagggag 1560 ccgcgatga 1569 <210> 6 <211> 1176 <212> DNA <213> Rheum Palmatum <400> 6 atggcaccgg aggagtcgaa gcatgctgaa actgctaaca gagccactgc caccgtcctg 60 gccatcggca ctgccaaccc tccaaactgc tactaccagg ccgactttcc cgacttctac 120 ttccgtgtca ccaacagcga ccacctcacg cacctcaaga ataaattcaa gagcatttgt 180 gagaggtcga agattgagaa acgttacctc cacttgacgg aagaaattct caaggagaat 240 ccgaatattg cttcctacga ggcgccatca ttagatgtaa gacaaaacat tcaagtgaaa 300 gaagtggtga agctcgggaa agaggcagct ttgaaggcca tcaatgagtg gggccaaccc 360 aagtcaaaga tcacgcacct cattgtgtgt tgtattgcag gcgttgacat gcccggcgca 420 gactatcaac ttactaaagt tcttggctta caactctctg ttaagcggtt tatgttttac 480 cacctaggat gctatgccgg tggcaccgtc ctttgccttg caaaggacat agcagagaac 540 aacaagggag ctcgtgttct catcgtttgc tctgagatga cgccaatctg tttccgtggg 600 ccatccgaaa cccacataga ctccatggta gggcaagcaa tatttggtga cggtgctgcg 660 gctgtcatag ttggtgcaca tccggaccta tccatcgaaa ggccgatttt cgagttgatt 720 tcaacatccc aaactatcat acctgaatcc gacggtgcga ttgagggaca tttgcttgaa 780 gttggactca gtttccatct ccaccagacc gttccctcat taatctctaa ttctatccaa 840 acttgtcttt caaaggcttt cacacctctt aacattagtg attggaactc gctattctgg 900 attgcacacc ctggtggccg tgctatcctt gacgatattg aggctactgt aggtctcaag 960 aaggagaaac ttatggcaac aagacaagtt ttgaacgatt atgggaacat gtcaagtgct 1020 tgcgtatttt tcatcatgga tgagatgagg aagaagtcga ttgcaaacgg tcaagtaacc 1080 actggagaag gactcaagtg gggtgttctt tttgggttcg gcccaggtgt tactgtggaa 1140 actgtggttc tacacagtgt gccgctaatt acctga 1176 <210> 7 <211> 1188 <212> DNA <213> Artificial Sequence <220> <223> RpSTSsyn <400> 7 atggcagatc ccatggcgcc ggaagaaagt aaacatgctg aaaccgcaaa ccgtgcaacc 60 gcaacggtgc tggccattgg tacggcaaac ccgccgaatt gctattacca ggccgatttt 120 ccggactttt atttccgcgt taccaacagc gatcatctga cgcacctgaa aaacaaattc 180 aaaagtatct gtgaacgttc caaaatcgaa aaacgctatc tgcacctgac cgaagaaatt 240 ctgaaagaaa acccgaatat cgcttcatac gaagcgccgt cgctggatgt tcgtcagaac 300 attcaagtca aagaagtggt taaactgggt aaagaagcgg ccctgaaagc catcaatgaa 360 tggggccagc cgaaaagcaa aattacccat ctgatcgtgt gctgtattgc cggtgttgat 420 atgccgggcg cagactacca gctgacgaaa gtcctgggtc tgcaactgtc tgtgaaacgc 480 tttatgttct atcacctggg ttgctacgcc ggcggtaccg tgctgtgtct ggccaaagat 540 attgcagaaa acaataaagg cgcacgtgtc ctgattgtgt gcagcgaaat gaccccgatt 600 tgctttcgcg gtccgagcga aacgcatatt gattctatgg ttggccaggc catcttcggc 660 gacggtgcag ctgcggttat tgtcggtgca cacccggatc tgtcaatcga acgtccgatt 720 tttgaactga tcagtacctc ccaaacgatt atcccggaat cggacggtgc aatcgaaggc 780 catctgctgg aagttggcct gagttttcat ctgcaccaga ccgtcccgtc cctgatttca 840 aactcgatcc aaacctgcct gtctaaagcg tttacgccgc tgaacattag cgattggaat 900 tctctgttct ggatcgctca cccgggcggt cgtgcgattc tggatgacat cgaagctacc 960 gtgggtctga aaaaagaaaa actgatggcg acgcgccagg tcctgaacga ttatggcaat 1020 atgagctctg cttgtgtgtt tttcatcatg gacgaaatgc gtaaaaaatc tatcgcgaat 1080 ggtcaagtga ccacgggcga aggtctgaaa tggggcgttc tgtttggctt cggtccgggc 1140 gtgaccgttg aaacggtcgt gctgcattcc gtgccgctga ttaccctc 1188 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 atggatttgg caaacggtgt ga 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 tcaaggataa acctcaatga ggga 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 atggtactca tcagcgagga cagt 24 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 tcatggatat agctcaatga tcgatc 26 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 catatggcac cggaggagt 19 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 actagttcag gtaattagcg gc 22

Claims (7)

A recombinant vector comprising a ScCCL (Streptomyces coelicolor carboxyl CoA ligase) gene comprising the nucleotide sequence of SEQ ID NO: 5 and a SbROMT3syn (synthetic Sorghum bicolor resveratrol O-methyltransferase 3) gene comprising the nucleotide sequence of SEQ ID NO: And a recombinant vector comprising RpSTSsyn gene (synthetic rheumatum palmatum stilbene synthase) gene is simultaneously transformed to produce pinostilbene. delete delete delete A method for producing pinostilbene comprising culturing the recombinant E. coli of claim 1 in a medium containing p-coumaric acid as a substrate. delete A recombinant vector comprising a ScCCL (Streptomyces coelicolor carboxyl CoA ligase) gene comprising the nucleotide sequence of SEQ ID NO: 5 and a SbROMT3syn (synthetic Sorghum bicolor resveratrol O-methyltransferase 3) gene comprising the nucleotide sequence of SEQ ID NO: Wherein the recombinant vector comprises RpSTSsyn (synthetic rheumatum palmatum stilbene synthase) gene as an active ingredient.

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