KR101578652B1 - 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

TECHNICAL FIELD The present invention relates to a recombinant microorganism producing a stilbene compound and a method for producing the stilbene compound using the recombinant microorganism.

The present invention relates to a recombinant microorganism producing a stilbene compound and a method for producing a stilbene compound using the recombinant microorganism. More particularly, the present invention relates to a recombinant vector comprising a synthetic Sorghum bicolor resveratrol O- methyltransferase 3 ( SbROMT3syn ) A recombinant microorganism having conversion ability to produce a stilbene compound; ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Gene, VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase ) gene or SbROMT3syn gene and RpSTSsyn (synthetic Rheum A recombinant microorganism having the ability to produce a stilbene compound is transformed with a recombinant vector comprising a gene encoding a palmatum stilbene synthase gene; A method of culturing the microorganism to produce a stilbene compound; And ScCCL And a recombinant vector comprising a SbROMT3syn gene and a RpSTSsyn gene as an active ingredient.

Stilbene, which is primarily expressed as a resveratrol compound, is a small class of plant secondary metabolites derived from the common phenylpropanoid pathway, beginning with phenylalanine. Resveratrol (3,4 ', 5- trans- trihydroxystilbene) is a naturally occurring phytoalexin produced in 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 phytoalexins and antioxidants as well as anti-inflammatory, antitumor and anti-aging effects, as well as their important role in plant defense reactions. However, the potential use of trans -resveratrol is limited by its instability in environments that are exposed to light and oxygen or have 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 resveratrol stabilization can be performed by methylation of the resveratrol hydroxyl group to form methylated resveratrol derivatives.

3,5-dimethoxy-4'-hydroxy- trans -stilbene, 3,4 ', 5-trimethoxystilbene, pinostilbene ( pinostilbene; some naturally occurring, including 3,5-dihydroxy-4'-methoxy- trans -stilbene); 3,4'-dihydroxy-5-methoxy- trans -stilbene) and des-oxy reuha Fourteen angiogenin (desoxyrhapotigenin There are derivatives of methylated resveratrol, but their biological activity is largely unknown. Certain methylated resveratrol derivatives have been shown 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 pinostilbene has been reported to exhibit cytotoxicity more than 100-fold over resveratol in cancer cell lines, and this cytotoxic effect is associated with depletion of intracellular polyamine pools Lt; RTI ID = 0.0 > microtubule < / RTI > polymerization. It has also been reported that pinostilbene exerts a more neuroprotective effect on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells than resveratrol (Chao et < RTI ID = 0.0 > al ., J Nutr Biochem 21: 482-489, 2010). In addition, the more hydrophobic pinostilbene can penetrate cell membranes more easily than resveratrol, thus exhibiting more effective intracellular dosage and improved bioavailability. These data suggest that methylation of the hydroxyl group of resveratrol increases its hydrophobicity and enhances the cellular uptake of its derivatives. For this reason, the structure-activity relationship study of resveratrol derivatives is a very interesting field for the development of more stable and robust chemoprotective agents and motivates the production of methylated resveratrol derivatives in recombinant E. coli.

Resveratrol has become an attractive target in metabolic engineering due to its diverse health promoting activity and so far several attempts have been reported to produce resveratrol in recombinant microorganisms (Beekwilder et al ., Appl Environ Microbiol 5670-567, 2006). However, little is known about the production of methylated resveratrol derivatives in microorganisms. Furthermore, although the biosynthetic pathway and essential enzymes of resveratrol have been well characterized, there is a need for further studies on enzymes involved in the formation of methylated resveratrol derivatives.

Korean Patent Publication No. 2003-0067689 discloses 'production of stilbene in a transgenic plant and production method thereof', and Korean Patent No. 1053025 discloses 'production of phenylpropanoid compound in actinomycetes' Method " is disclosed. However, as described in the present invention, a method for producing a recombinant microorganism producing a stilbene compound and a method for producing a stilbene compound using the recombinant microorganism has not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and the present inventors have found that a codon-optimized SbROMT3syn (synthetic Sorghum bicolor Resveratrol O- methyltransferase 3) gene-transformed Escherichia coli and ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) It was confirmed that methylated resveratrol derivatives, pinostilbene and phthalostilbene, were produced in Escherichia coli transformed with the gene, SbROMT3syn or VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase ) gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) Thereby completing the present invention.

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

The invention also ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Gene, VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase ) gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene to provide a recombinant microorganism having a stilbene compound-producing ability.

The invention also ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Gene, SbROMT3syn (synthetic Sorghum bicolor resveratrol O- methyltransferase 3) gene and RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene to provide a recombinant microorganism having a stilbene compound-producing ability.

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

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

Recombinant microorganisms 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) 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 possible to produce a large amount of methylated resveratrol derivatives, pinostilbene and phthalostilbene, which exhibit anticancer, antiviral, antiinflammatory, anti-aging and antioxidative effects.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a schematic diagram of a construct comprising a biosynthetic pathway of a stilbene compound and a ROMT gene. FIG. (A) A biosynthetic pathway for the production of stilbene compounds from phenylalanine. (B) A recombinant plasmid construct having the synthesized plant ROMT gene. The sequential action of PAL, C4H, 4CL, STS and ROMT results in the conversion of phenylalanine to stilbene compounds, resveratrol and its methylated derivatives. 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).
FIG. 2 shows the expression of HeR-tagged VrROMTsyn and SbROMT3syn recombinant proteins in E. coli BL21-Codon Plus (DE3) -RIPL cells. E. coli cells containing pETDuet-VrROMTsyn or pETDuet-SbROMT3syn constructs were grown in modified M9 medium containing 2% glycerol for 2 hours at a temperature of 25 DEG C and protein expression was induced for 4 hours by the addition of 0.2 mM IPTG. Proteins were separated by 13% SDS-PAGE and stained with CBB (Coomassie brilliant blue). Expression of the His-tag fusion protein was confirmed by Western blot analysis using an anti-heath-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 pinostilbene and < RTI ID = 0.0 > pterostilbene < / RTI > in recombinant E. coli BL21-Codon Plus. E. coli cells containing the pETDuet-VrROMTsyn or pETDuet-SbROMT3syn plasmids were cultured in a modified M9 medium containing 2% glycerol, and 1 mM resveratrol was added to the pre-induced cells for protein expression, Lt; / RTI > Escherichia 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 contents of the pinostilbenes and the phthalostilbenes were measured in comparison with standard curves generated from the known concentrations of pinostilbene and phthalostilbene. (A) Content of pinostilbene. (B) Content of phthalostilbene.
Figure 4 shows HPLC analysis of pinostilbene and ferrostylbenzene produced in recombinant E. coli BL21-Codon Plus. E. coli cells containing pETDuet-VrROMTsyn or pETDuet-SbROMT3syn plasmids were incubated 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 the binary solvent system. Chromatogram STD represents an authentic standard of resveratrol, pinostilbene and phthalostilbene 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 degree of insertion shows an enlarged chromatogram corresponding to the phthalostilbene.
Figure 5 shows the LC-MS analysis of the pinostilbene and the ferrostilbene produced by the recombinant E. coli BL21-Codon plus. The molecular weights of the pinostilbenes and the phthalostilbenes 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 the 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 ferrostilben, respectively.
Fig. 6 shows a recombinant plasmid having ScCCL, RpSTSsyn, VrROMTsyn and SbROMT3syn genes related to 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.
FIG. 7 shows the expression of STS recombinant protein bound to HE-tagged CCL, VrROMTsyn, and SbROMT3syn and S-tag 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 :: S constructs were grown in modified M9 medium containing 2% glycerol for 2 hours at a temperature of 25 ° C 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 He-tag or S-tag fusion protein was confirmed by Western blot analysis using anti-heath-tag antibody or anti-S-tag antibody. (B) pCOLADuet-CCL and pETDuet-STS-VrROMT vectors, and (C) pCOLADuet-CCL and pETDuet-STS-SbROMT3 vectors, Western blot for SDS-PAGE, S-tag and Heath-tag of the transformed microorganism-derived product. M, protein molecular marker (kDa); S, soluble fraction; P, insoluble pellet fraction. E. coli cells containing pCOLADuet-1 and pETDuet-1 were used as a control (Con). The S-tagged STS, the heath-tagged CCL, and the ROMT recombinant protein recognized by the anti-S-tag antibody and the anti-heath tag antibody are shown in the form of an asterisk, an arrow and an arrowhead, respectively.
Figure 8 shows the hourly production of resveratrol and pinostilbene 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) CCL and pETDuet-H :: SbROMT3-STS :: S (CCL + STS-SbROMT3) E. coli cells containing the construct were cultured in a modified M9 medium containing 2% glycerol, before the induced cell 1 mM p - After adding cumaric acid, it was harvested at the indicated time points. Escherichia coli cells containing pColADuet-1 and pETDuet-1 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 resveratrol and the amount of pinostilbene (mg / L) at the indicated time points was compared to a standard curve generated from known concentrations of resveratrol and pinostilbene. (A) The content of resveratrol. (B) the content of the pinostilbene.
FIG. 9 shows HPLC analysis results of resveratrol and pinostilbene 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) E. coli cells containing CCL and pETDuet-H :: SbROMT3-STS :: S (CCL + STS-SbROMT3) constructs were incubated for 48 hours before extraction. HPLC analysis was performed on an Agilent 1200 system using a C18 reversed phase column with a linear gradient of the binary solvent system. Chromatogram STD represents an authentic standard of resveratrol (Res), pinostilbene (Pino) and pterostilbene (Ptero) with retention times of 9.423, 19.327 and 32.009 minutes, respectively. Chromatogram control, CCL + STS, CCL + STS-VrROMT and CCL + STS-SbROMT3 indicate that the sample is produced from p -coumaric acid by recombinant E. coli. The insert shows an expanded chromatogram to show a small amount of the ferro-stilbene production. The arrow indicates the peak corresponding to the heterostilbene.

In order to accomplish the object of the present invention, the present invention provides a recombinant vector comprising SbROMT3syn (synthetic Sorghum bicolor resveratrol O- methyltransferase 3) gene to provide a recombinant microorganism having a stilbene compound-producing ability.

The SbROMT3syn gene preferably comprises the nucleotide sequence shown in SEQ ID NO: 4, but is not limited thereto. The SbROMT3syn gene consisting of the nucleotide sequence of SEQ ID NO: 4 is preferably a nucleotide sequence of the resteratrol O- methyltransferase 3 ( ROMT3 ) gene derived from silkworm comprising the nucleotide sequence of SEQ ID NO: 3, which is redesigned considering the microbial heterologous host codon compatibility And is an artificial gene synthesized after codon optimization for E. coli. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. The homologue is a base sequence having a functional characteristic similar to that of SEQ ID NO: 4, although the base sequence is changed. Specifically, the SbROMT3syn gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 4 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In addition, the present invention relates to a nucleic acid encoding a ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Gene, a VrROMTsyn (synthetic Vitis riparia resveratrol O- methyltransferase) gene and the nucleotide sequence of SEQ ID NO: 7, RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene to provide a recombinant microorganism having the ability to produce a stilbene compound. Preferably, the recombinant microorganism is a ScCCL And a recombinant vector comprising a VrROMTsyn gene consisting of the nucleotide sequence of SEQ ID NO: 2 and a RpSTSsyn gene consisting of the nucleotide sequence of SEQ ID NO: 7 are simultaneously transformed to provide a recombinant microorganism having a stilbene compound- producing ability .

In addition, the present invention relates to a nucleic acid encoding a ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) Gene, a SbROMT3syn (synthetic Sorghum bicolor resveratrol O- methyltransferase 3) gene and the nucleotide sequence of SEQ ID NO: 7, RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) gene to provide a recombinant microorganism having the ability to produce a stilbene compound. Preferably, the recombinant microorganism is a ScCCL And a recombinant vector comprising 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 stilbene compounds.

The ScCCL gene preferably comprises the nucleotide sequence shown in SEQ ID NO: 5, but is not limited thereto. In addition, the VrROMTsyn gene preferably comprises the nucleotide sequence shown in SEQ ID NO: 2, but is not limited thereto. The VrROMTsyn gene consisting of the nucleotide sequence of SEQ ID NO: 2 preferably comprises a nucleotide sequence of a resveratrol O- methyltransferase ( ROMT ) gene derived from grape ( Vitis riparia ) consisting of the nucleotide sequence of SEQ ID NO: 1, And is an artificial gene synthesized after codon optimization for E. coli. In addition, the SbROMT3syn gene preferably comprises the nucleotide sequence shown in SEQ ID NO: 4, but is not limited thereto. SbROMT3syn gene consisting of the nucleotide sequence of SEQ ID NO: 4, preferably redesigned base sequence of ROMT3 gene of cane origin consisting of the nucleotide sequence of SEQ ID NO: 3 in consideration of a microorganism two kinds of host codon fitness and codon optimized for E. coli And then synthesized artificial genes. In addition, the RpSTSsyn gene may preferably consist of the nucleotide sequence shown in SEQ ID NO: 7, but is not limited thereto. The RPSTSsyn gene consisting of the nucleotide sequence of SEQ ID NO: 7 is preferably a nucleotide sequence of a STS (stilbene synthase) gene derived from Rheum Palmatum consisting of the nucleotide sequence of SEQ ID NO: 6 in consideration of microbial heterologous host codon compatibility It is an artificial gene synthesized after codon optimization for E. coli. Homologues of each of the above base sequences are included within the scope of the present invention. A homologue is a base sequence that changes in its base sequence but has functional properties similar to that of the base sequence.

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 StCCBL gene, the SbROMT3syn gene, and the RpSTSsyn gene are normally expressed and the stilbene compound is produced even when the vector is transformed with the recombinant vector containing the gene, there is no limit to the selectivity of the vector in the construction form or the transformation sequence.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element. Enhancers, ribosome binding sites, termination signals, polyadenylation signals, and promoters, which are available for use in prokaryotic or eukaryotic cells, are well known in the art. Expression vectors containing appropriate transcription / translation control signals can be constructed by methods well known in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

In the recombinant vector of the present invention, in the case of an expression vector having a prokaryotic cell as a host, a strong promoter capable of promoting transcription (for example, pL? Promoter, trp promoter, lac promoter, T7 promoter, tac promoter etc.) Lt; RTI ID = 0.0 > transcription / translation < / RTI > termination sequence. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway, and the left promoter of the phage lambda (pL 貫 promoter) can be used as the regulatory region. The vectors that can be used in the present invention include plasmids (e.g., pETDuet, pCOLADuet, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series, (e.g.,? B,? -charon,?? z1 and M13), a virus (e.g., SV40 or the like) or a fosmid. Preferably, pETDuet or pCOLADuet vectors are used.

The recombinant vector of the present invention may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. The recombinant vector of the present invention can be used as a selectable marker, which is an antibiotic resistant gene commonly used in the art, such as amphicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline resistance gene But are not limited thereto.

As a host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, any host cell known in the art may be used, 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 strains, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

In one embodiment of the invention, are transformed with a recombinant vector of the present invention may be a recombinant microorganism having a steel Ben compound producing ability is preferably Escherichia coli (E. coli), and more preferably be in E. coli BL21 But is not limited thereto.

The recombinant vector of the present invention can be produced by the CaCl ₂ method, Hanahan, D., J. Mol. Biol., 166: 557-580 (1983) And can be delivered into cells.

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

The present invention also provides a method for producing a stilbene compound, comprising culturing a recombinant microorganism transformed with a recombinant vector comprising 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, but not limited to, pinostilbene or phthalostilbene.

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 And simultaneously 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, pinostilbene, or phthalostilbene, but is not limited thereto.

The recombinant microorganism culture of the present invention can be carried out using conventional conditions used for culturing microbial hosts. As the culture method, any method used for culturing ordinary microorganisms such as a batch method, a flow batch method, a continuous culture method, and a reactor type can be used. The culture medium for culturing the transformant obtained by using a bacterium such as Escherichia 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 to be supplied to the medium for the proliferation of microorganisms include 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; There may be used 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, and the nitrogen source may be exemplified by ammonia, ammonium chloride, ammonium sulfate, In addition to ammonium salts such as ammonium phosphate, those derived from natural materials such as peptone, meat juice, yeast extract, malt extract, casein decomposition product, and corn steep liquor can be used. The inorganic substance may be, for example, potassium primary phosphate, potassium secondary phosphate, magnesium phosphate, magnesium sulfate, sodium chloride or the like. In addition, it can be cultured by adding appropriate antibiotics such as amphicillin, chloramphenicol, kanamycin, streptomycin and the like according to the antibiotic resistance of the recombinant microorganism. In the case of culturing a microorganism transformed with an expression vector containing inducible promoter An inducer suitable for the kind of promoter can be added to the medium and cultured. For example, IPTG (isopropyl-D-1-thiogalactopyranoside), tetracycline, indoleacetic acid (IAA)

The present invention also relates to a ScCCL ( Streptomyces coelicolor carboxyl CoA ligase) comprising the nucleotide sequence of SEQ ID NO: A recombinant vector comprising a SbROMT3syn (synthetic Sorghum bicolor resveratrol O -methyltransferase 3) RpSTSsyn (synthetic Rheum Palmatum stilbene synthase) consisting of the base sequence of the gene and the gene SEQ ID NO: 7 consisting of the nucleotide sequence of the recombinant vector containing the gene, and SEQ ID NO: 4 As an active ingredient, a composition for producing stilbenes of microorganisms. 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 the stilbene compound can be produced from the microorganism by transforming the genes into microorganisms.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1. Biosynthesis pathway of resveratrol and pterostilbene

The stilbene production process of the present invention follows the stilbene biosynthetic pathway shown in Fig. 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 resulting p - coumaroyl - CoA is converted to resveratrol by stilbene synthase (STS), and resveratrol is methylated again by methylating enzyme ROMT (resveratrol O - methyltransferase) to form pinostilbene ), Which is again methylated and converted to the thermostilbene. The present inventor used CCL (carboxyl CoA ligase) protein of actinomycetes known to have an enzyme activity such as 4CL in producing p -coumaroyl-CoA.

Example 2: Synthesis of a thermostilbene synthesis transcription factor VrROMT  And SbROMT3  Cloning of genes

In order to develop a ferrostilbene production system, VrROMT (1074 bp, SEQ ID NO: 1) and SbROMT3 (1125 bp, SEQ ID NO: 3) related to the thermostilbene biosynthesis- related genes from grape ( Vitis riparis and sorghum bicolor ) ) Were separately used. Was isolated and amplified by based PCR (homology-based PCR) -: (GenBank Registration No. EF189708) homology with the PCR primers obtained from the gene: The gene has VrROMT (GenBank Registration No. FM178870) and SbOMT3. The primers used in the PCR were as follows: VrROMT-F: 5'-ATGGATTTGGCAAACGGTGTGA-3 '(SEQ ID NO: 8), VrROMT-R: 5'-TCAAGGATAAACCTCAATGAGGGA- ATGGTACTCATCAGCGAGGACAGT-3 '(SEQ ID NO: 10), SbROMT-R: 5'-TCATGGATATAGCTCAATGATCGATC-3' (SEQ ID NO: 11). Grape samples were obtained from the Chungcheongbuk-do Agricultural Research Institute, Grape Research Institute (www.ares.chungbuk.kr), and the samples were obtained from the Rural Development Administration, National Institute of Agricultural Science and Technology (http://www.genebank.go.kr/) Respectively.

The gene derived from the grape or sperm plant thus obtained was redesigned in consideration of the codon compatibility of the microorganism Ricinus persica , and codon optimized for E. coli was used for the synthesis of artificial genes VrROMTsyn (SEQ ID NO: 2) and SbROMT3syn (SEQ ID NO: 4) Respectively. The synthesized artificial gene was used to induce the productivity improvement of protein and functional substance.

Example 3. Resveratrol methylation gene ( ROMT ) For microbial overexpression

To confirm whether the VrROMTsyn and SbROMT3syn genes cloned in Example 2 could be increased in the microbial over-synthesis of resveratrol and pterostilbene, the T7 promoter of the microorganism expression vector pETDuet with the ampicillin resistance gene And the T7 terminator was constructed by inserting the VrROMTsyn gene or the SbROMT3syn gene. The prepared over-expression vector was designated pETDuet-His :: VrROMTsyn (pETDuet-VrROMTsyn) and pETDuet-His :: SbROMT3syn (pETDuet-SbROMT3syn), respectively (FIG. Escherichia coli DH5? Strain was used for the above vector production.

Example 4. Production of recombinant microorganisms producing stilbene

The pETDuet-His :: VrROMTsyn (pETDuet-VrROMTsyn) and pETDuet-His :: SbROMT3syn (pETDuet-SbROMT3syn) microbial expression vectors prepared in Example 3 were transformed with BL21 Codon Plus RIPL (chloramphenicol) DE3) were transformed by heat shock method. The recombinant microorganism producing pinostilbene or phthalostilbene produced was selected on LB plate medium supplemented with 50 μg / ml and 30 μg / ml of ampicillin and chloramphenicol, respectively.

Example 5. Analysis of stilbene protein expression of recombinant microorganisms

Western blot analysis was performed to confirm that 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, and then cultured in 50 ml of a liquid medium at 37 ° C so as to have an absorbance of 0.6 to 0.7 at 600 nm. Then, 0.5 mM IPTG (Isopropyl-D-1-thiogalactopyranoside) was added to induce protein expression, and 10 to 20 ml of the sample was taken over time to separate the proteins. Western blot analysis using an anti-His-tag antibody confirmed the protein at positions corresponding to the theoretical molecular weights of VrROMT and SbROMT3 proteins (41.7 and 42.5 kDa, respectively) (FIG. 2). That is, it was confirmed that the VrROMT and SbROMT3 genes synthesizing pinostilbene and phthalostilbene were stably expressed and translated by attaching a methyl group to resveratrol.

Example 6. Analysis of content of stilbene compounds in recombinant microorganism culture

In order to confirm that the recombinant microorganism having expression of the protein in Example 5 was able to catalyze the production of the methylated resveratrol derivative, the recombinant microorganism was pre-cultured in LB medium, and then 16.5 / / ml of chloramphenicol and 50 / / And incubated at 37 [deg.] C with an absorbance of 0.6 to 0.7. Then, 0.1 mM IPTG was added to induce protein production and further cultured at 25 ° C for 3 hours. After centrifuging the culture solution to collect only the recombinant protein expression strain Pinot stilbene and program for interrogating a suitably modified to produce a stilbene M9 (M9 broth, 0.5% glycerol, 1 mM MgSO _4, 0.125% yeast extract) liquid medium . Ampicillin, chloramphenicol and 0.5 mM IPTG were added to the medium, and 1 mM resveratrol to be used as a substrate was added and cultured at 25 DEG C for 60 hours. During the incubation, 20 ml of the culture broth was collected at time intervals (0, 6, 12, 24, 48 and 60 hours), and the collected samples were adjusted to pH 9.0 with 5 N sodium hydroxide and extracted with an equal volume of ethyl acetate To extract the desired substance. After the ethyl acetate was evaporated off, the extracted sample was dissolved in 1 ml of methanol-use methanol and filtered through a 0.2 μm PTFE filter (hydrophilic, ADVANTEC, Japan), followed by HPLC (high performance liquid chromatography) -mass spectrometer) analysis. HPLC was performed using Agilent Technologies 1200 series, using a quaternary pump system and a Zorbax Eclipse XBD-C18 (5 mm, 4.6 x 150 mm; agilent) column. The mobile phase was extracted with water (A, 0.05% trifluoroacetic acid) and acetonitrile (B, 0.05% trifluoroacetic acid) using gradient elution.

Analysis of the extracts produced from each recombinant microorganism revealed that pinostilbene and a trace amount of heterostilbene were detected in the recombinant microorganism culture (FIG. 4), and the detection results of the extracts were summarized and plotted over time 3). In the SbROMT3syn clone, the level of pinostilbene began to increase from 6 hours after resveratrol addition and continued to accumulate until 60 hours. The cultured cells containing the pETDuet-SbROMT3syn plasmid produced a high level of methylated derivatives with a concentration of 34 mg / L of pinostilbene and 0.16 mg / L of small amounts of phthlostylbenzene as main products in a 60-hour culture (Fig. 3). On the other hand, very small amounts of pinostilbene (about 0.16 mg / L) and pterostilbene (0.04 mg / L) were detected in culture at 48 hours in cultured cells containing the pETDuet-VrROMTsyn plasmid (FIG. In the cells expressing pETDuet-1 as the empty vector, no pinostilbene and no pterostilbene were produced. LC-MS analysis using a 60-hour cultured sample again confirmed that the newly generated peak was pinostilbene and phthalostilbene (FIG. 5). Therefore, the present inventors have confirmed that the fermentation product of the resveratrol substrate results in the formation of the phthalostilbene. Based on these results, it has been attempted to produce the phthalostilbene from p -coumaric acid as shown in FIG.

Example 7. p - coumaroyl-CoA synthetic transcription factor ScCCL  Cloning of genes

In order to develop the p - coumaroyl - CoA production system, the transcription factor gene ScCCL (1569 bp) related to p - coumaroyl - CoA biosynthesis was isolated from Streptomyces coelicolor . Genetic information was obtained from Kaneko et al. (J. Bacterial. 185: 20-27, 2003). In order to increase the productivity of the protein expressed in the microbial production system, the microorganism-derived CCL Genes were used. The gene samples were obtained from the Center for Chemical Biology Research, Korea Research Institute of Bioscience and Biotechnology (www.kribb.re.kr), and the ScCCL gene (SEQ ID NO: 5) was isolated from the pET28-ScCCL vector using Nde I and Hind III.

Example 8. Resveratrol < RTI ID = 0.0 > RpSTS  Cloning of genes

To develop production systems resveratrol, resveratrol jangyeop rhubarb biosynthesis-related transcription factor gene from (Rheum palmatum) RpSTS (1176 bp, SEQ ID NO: 6) was isolated and used. The gene was amplified by homology-based PCR using a PCR primer obtained from RpSTS (GenBank registration number: JX673939) gene. The primers used were: RpSTS-F: 5'-CATATGGCACCGGAGGAGT-3 '(SEQ ID NO: 12), RpSTS-R: 5'-ACTAGTTCAGGTAATTAGCGGC-3' (SEQ ID NO: 13). The samples of Jangbyeon Rahuang were obtained from the Northern Agricultural Experiment Station (www.ares.gangwon.kr) of Gangwon-do Agricultural Research and Extension Service of Rural Development Administration. The gene derived from the Changdeobu rhubarb plant was redesigned in consideration of the codon compatibility of the microorganism Jongsoo Sook and used for the synthesis of the artificial gene RpSTSsyn (SEQ ID NO: 7) after codon optimization for E. coli. The synthesized artificial gene was used to induce the productivity improvement of protein and functional substance.

Example 9: Construction of vector for overexpression of microorganism of stilbene biosynthesis gene

The cloned VrROMTsyn , SbROMT3syn , ScCCL and RpSTSsyn In order to confirm that the synthesis of resveratrol and pterostilbene could be increased when the gene was overexpressed in the microorganism, a vector in which ScCCL was inserted between the T7 promoter and the T7 terminator of the microorganism expression vector pCOLADuet having the 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 . PETDuet-His :: VrROMT-RpSTS :: S (pETDuet-STS-VrROMT) and pETDuet-His :: ScCCL (pCOLADuet-CCL) His :: SbROMT3-RpSTS :: S (pETDuet-STS-SbROMT3) (Fig. 6). Escherichia coli DH5? Strain was used for the above vector production.

Example 10. Preparation of recombinant microorganisms producing stilbene

The pCOLADuet-His :: ScCCL (pCOLADuet-CCL), pETDuet-RpSTS :: S (pETDuet-STS), pETDuet-His :: VrROMT-RpSTS :: S (pETDuet-STS-VrROMT) The pETDuet-His :: SbROMT3-RpSTS :: S (pETDuet-STS-SbROMT3) microbial expression vector was transformed by heat shock method to a protein expressing E. coli (BL21 Codon Plus RIPL (DE3)) resistant to chloramphenicol . First, pCOLADuet-His :: ScCCL (pCOLADuet-CCL) and pETDuet-RpSTS :: S (pETDuet-STS) vectors were simultaneously transformed to confirm resveratrol production, and pCOLADuet -His :: ScCCL (pCOLADuet-CCL) and pETDuet-His :: VrROMT-RpSTS :: S (pETDuet-STS-VrROMT) or pCOLADuet-His :: ScCCL (pCOLADuet-CCL) and pETDuet-His :: SbROMT3-RpSTS :: S (pETDuet-STS-SbROMT3) vector were simultaneously transformed. The manufactured stilbene production microorganisms were selected on LB plate medium supplemented with 50 쨉 g / ml, 25 쨉 g / ml and 30 쨉 g / ml of ampicillin, kanamycin and chloramphenicol, respectively.

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

Western blot analysis was performed in order to confirm that the ScCCL , RpSTS , VrROMT and SbROMT3 genes were stably expressed and translated in each of the selected recombinant microorganisms. The expression of the protein was induced by adding 0.5 mM IPTG (Isopropyl-D-1-thiogalactopyranoside) to the LB liquid medium containing chloramphenicol, ampicillin and kanamycin. The recombinant microorganisms were analyzed for resveratrol and pterostilbene (Fig. 7), which are presumed to be transcription factors of ScCCL, RpSTS, VrROMT and SbROMT3.

Example  12. In the recombinant microbial culture solution Stilben  Compound content analysis

Each of the recombinant microorganisms in which stable protein expression was confirmed was cultured and extracted in the same manner as described in Example 6, and p -coumaric acid was used instead of resveratrol as a substrate. The extracted sample was dissolved in 1 ml of methanol for HPLC and filtered through a 0.2 μm PTFE filter (hydrophilic, ADVANTEC, Japan) and used for high-performance liquid chromatography (HPLC) analysis. HPLC was performed using Agilent Technologies 1200 series, using a quaternary pump system and a Zorbax Eclipse XBD-C18 (5 mm, 4.6 x 150 mm; agilent) column. The mobile phase was extracted with water (A, 0.05% trifluoroacetic acid) and acetonitrile (B, 0.05% trifluoroacetic acid) using gradient elution. As a result of analysis of the extracts, resveratrol, pinostilbene and phthlostilbene newly produced from the p -coumaric acid substrate in the culture medium of the recombinant microorganism was 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 culture solution for 48 hours, and 0.50 mg / L of resveratrol was produced in a recombinant microorganism culture in which ScCCLsyn and RpSTSsyn and VrROMTsyn proteins were simultaneously expressed , A recombinant microorganism culture in which ScCCLsyn, RpSTSsyn, and SbROMT3syn proteins were co-expressed resulted in 0.41 mg / L resveratrol and 0.71 mg / L pinostilbene. As a result, resveratrol produced by ScCCLsyn and RpSTSsyn was methylated by SbROMT3syn protein to be 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> PN13264 <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 gcccgaccc 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 cgcctgccc cgcctcgcc 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 cccgaccgt 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 gt; 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 ggatcgctc 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 gtaccgttg 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 (12)

A recombinant Escherichia coli transformed with a recombinant vector comprising a recombinant vector comprising SbROMT3syn (synthetic Sorghum bicolor resveratrol O-methyltransferase 3) gene derived from a silkworm consisting of the nucleotide sequence of SEQ ID NO: 4 and having the ability to produce pinostilbene. delete delete delete delete delete delete A method for producing pinostilbene comprising culturing the recombinant E. coli of claim 1 in a medium containing resveratrol as a substrate. delete delete delete A synthetic Sorghum bicolor resveratrol O-methyltransferase 3 (SbROMT3syn) gene derived from a silkworm comprising the nucleotide sequence of SEQ ID NO: 4 as an active ingredient.

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