KR20090097388A - Recombinant vector containing 4cl gene and sht gene, microorganism transformed thereof and method for producing phenylpropanoid amide-based compounds using the same - Google Patents

Abstract

A transformed bacteria by a recombinant vector having a 4CL gene and SHT gene is provided to massively produce phenylpropanoid amide-based compound and reduce production cost. A recombinant vector comprises a serotonin N-hydroxycinnamoyl transferase (SHT) gene, 4-coumarate-CoA ligase (4CL) gene, and a promoter. A method for producing a phenylpropanoid amide-based compound comprises: a step of injecting 0.5mM~5mM of each phenol substrate and amine substrate in a transformed bacteria and culturing at 28~40°C for two to four hours; and a step of isolating and purifying the produced phenylpropanoid amide-based compound from the bacteria.

Description

Recombinant vector containing 4CL gene and SHT gene, microorganism transformed girls and method for producing phenylpropanoid amide- based compounds using the same}

The present invention relates to a method of transforming bacteria by using a recombinant vector comprising a 4CL gene and an SHT gene, and producing a phenylpropanoid amide compound from the transformed bacterium. More specifically, the present invention is serotonin-hydroxy cinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase; SHT) gene and 4- Kumar rate-coenzyme A ligase (4-Coumarate-CoA ligase; 4CL) by introducing a gene sequence, a promoter and The present invention relates to a recombinant vector and a bacterium transformed with the recombinant vector, wherein the recombinant bacterium is operably linked to the recombinant vector. The present invention relates to a method for separating and purifying propaneoid amide compounds to produce phenylpropaneoid amide compounds.

Phenylpropaneoid amide compounds have been reported to have various pharmacological effects. Specifically, Kumaroyl serotonin and Peruroyl serotonin, which are a kind of phenylpropanoid amide compound extracted from safflower seed oil, have antioxidant activity (Kawashima et al., J. Interferron & Cytokine Res. 18, 423-428, 1998), It is reported to have anticancer activity (Zhang et al., Chemical & Pharma. Bull. 44, 874-876, 1996), and is known to be involved in promoting bone formation (Patent No. 10-0354791). It has recently been reported to have an excellent whitening effect (Roh et al., Biol. Pharm. Bull. 27, 1976-1978, 2004). On the other hand, Peruroyl tyramine and caffeoyl tyramine compounds, which are a kind of other phenylpropanoid amide compounds, are also known to have anticancer activity (Park et al., Cancer Let. 202, 161-171, 2003). Loyltyramine has also been reported to have whitening activity (Efdi et al., Biol. Pharm. Bull. 30, 1972-1974, 2007).

The phenyl propaneoid amide compound is a polymer of phenolic compounds and amines compounds, typically peruyl serotonin, coumaroylserotonin, caffeoylserotonin, caffeoylserotonin, Sinapoylserotonin, peruloyltyramine, peruloyloctopamine, coumaroyltyramine and coumaroyloctopamine.

On the other hand, the amines used for biosynthesis of the phenylpropanoid amide compound are tyramine, serotonin, dopamine, phenethylamine, noradrenaline and octopamine. Phenolic compounds include feruloyl-CoA, cinnamoyl-CoA, 4-coumaroyl-CoA, sinapoyl-CoA and Caffeoyl-CoA.

Phenylpropaneoid amide compounds having excellent pharmacological effects as described above are present only in trace amounts in plants. For example, safflower seeds contain about 20 μg of Peruroyl serotonin per gram seed (Baek et al., J. Korean. Soc. Agric. Chem. Biotechnol. 42, 366-368, 1999) and bamboo infected with pathogens It is known that eggplants contain 3-10 μg of peruroyl serotonin and coumaroyl serotonin per gram weight (Tanaka et al., Phytochemistry, 64, 965-969, 2003). It has also been reported that 7 μg of Kumaroyl serotonin, 95 μg of Kumaroyl tyramine, and 21 μg of Peruroyl tyramine are present in the pepper flower (Kang et al., Sci. Hortic . 26, 2009- 2015, 2007). Trace amounts of phenylpropanoid amide compounds have also been found in potatoes and corn (Schmidt et al., J. Biol. Chem . 274, 4273-4280, 1999; Ishihara et al., Biosci. Biotechnol. Biochem. 64 , 1025-1031, 2000). However, as described above, since the phenylpropaneoid amide compound is present only in a small amount in the plant, the method of extracting and producing the phenylpropaneoid amide compound from the plant is not economical, and thus, in a method of artificially mass producing it The situation is urgently needed.

On the other hand, with the development of genetic engineering technology, a lot of research on mass production of target proteins and compounds using bacteria and various plants and animals is being conducted. To date, Escherichia coli is the most widely used host cell for mass production of many useful proteins and compounds, and the research on this is the most widely conducted (Hodgson, Bio / Technology , 11: 887, 1993; Lee, Trends Biotechnol. , 14 : 98, 1996). However, the mass production method of the target protein and compound using the E. coli should design a specific production method of implementing a recombinant vector and transforming the same according to the type of target protein and compound, and above all, even if the production method as described above Even if it is designed, whether the actual target protein or compound can be efficiently produced, there is a problem that can be easily predicted by those skilled in the art. In particular, a person skilled in the art can actually produce a target protein or compound due to various causes, such as when the target protein is not produced directly from the transformed bacterium or the protein produced from the transformed bacterium fails to react with a separately added substrate. It cannot be easily predicted whether it can be obtained. The various causes may be, for example, the protein is degraded by various proteases present in the cytoplasm, so that the production yield may be significantly low, and the protein may not be fully folded and inactivated. The case where an insoluble aggregate body is formed is mentioned. Alternatively, even when the substrate is treated with the transformed bacterium, the substrate may be degraded by the bacterium, or the processed substrate may not enter the bacterium effectively, thus making it impossible to produce the compound due to the enzymatic reaction between the compound and the protein. Because there is.

The inventors have produced a superior phenyl profile by cannabinoid amide compound serotonin-hydroxy-cinnamate to produce in a plant together transferase (Serotonin N -hydroxycinnamoyltransferase) transformed with the genes in rice, phenyl profile cannabinoid amide compound pharmacological effect . The transformed rice produced 77 μg of coumaroyl serotonin and 96 μg of Peruroyl serotonin per gram of live weight (Jang et al., Plant Physiol. 135, 346-356, 2004; US Pat. No. 7084322). However, in order to purify the phenylpropanoid amide compound in the transformed rice, plant crushing, column purification and high-performance liquid chromatography (HPLC) purification process have to be performed, and the yield is also low and the economic efficiency is not easy. Later, while investigating economic and simple biosynthesis methods, the 4-Coumarate-CoA ligase gene and serotonin hydroxycinnamoyl transferase (Serotonin N- ) went through a lot of trial and error. By designing a recombinant vector capable of expressing the hydroxycinnamoyltransferase gene most efficiently, transforming bacteria using the same, and then administering an amine substrate and a phenol substrate, a large amount of phenylpropanoid amide compound is produced. Was completed.

Accordingly, an object of the present invention is a recombinant vector and a vector transformed with 4-Coumarate-CoA ligase and Serotonin N -hydroxycinnamoyltransferase gene. To provide bacteria.

Still another object of the present invention is to provide a method for producing a phenylpropanoid amide compound using the transformed bacterium of the present invention.

In order to solve the above problems, the present invention is a serotonin hydroxycinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) gene and 4-coumarate-Coenzyme A ligase (4-Coumarate-CoA ligase) gene is sequentially linked It provides a recombinant vector, which is operably linked to a promoter.

The present invention also provides a bacterium transformed with the recombinant vector.

In another aspect, the present invention (a) administering a phenol substrate and an amine substrate to the transformed bacteria to cultivate the bacteria and (b) a phenylpropanoid amide compound produced from the cultured bacteria of step (a) It provides a method for producing a phenylpropaneoid amide compound comprising the step of separating and purifying.

Hereinafter, the present invention will be described in detail.

As used herein, a "recombinant vector" refers to a gene construct that is capable of expressing a protein of interest in a suitable host cell, and includes a gene construct comprising essential regulatory elements operably linked to express the gene insert.

As used herein, the term “operably linked” refers to the polynucleotide of the present invention that is associated with an expression control sequence such that its function or expression is affected by the expression control sequence. That is, maintaining the correct frame of translation such that the sequence of polynucleotides is expressed under the control of expression control sequences to form the desired polypeptide. Such operably linked polynucleotides and expression control sequences may be included in a vector containing a promoter, transcriptional termination, selection marker, and replication origin.

As used herein, the term "promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, the promoter is located at the 5 'non-coding site of the gene, adjacent to the transcriptional initiation site of the structural gene.

The recombinant vector of the present invention is serotonin-hydroxy cinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) gene and 4- Kumar rate-and coenzyme A ligase (4-Coumarate CoA-ligase) gene are connected in sequence, enabling promoter and Operation It is characterized by being connected. Although not limited thereto, preferably, the recombinant vector may be a vector having a cleavage map described in FIG. 2.

The 4-coumarate-Coenzyme A ligase (4-Coumarate-CoA ligase) gene is characterized in that having a nucleotide sequence of SEQ ID NO: 5, preferably 4-coumarate-Coenzyme A ligase 1 (4-Coumarate -CoA ligase 1), 4-Coumarate-CoA ligase 2, 4-Coumarate-CoA ligase 3 and 4-Coumarate-CoA ligase 3, and 4-Coumarate-Coenzyme A It is characterized in that the gene is selected from the group consisting of ligase 4 (4-Coumarate-CoA ligase 4), most preferably 4-Coumarate-CoA ligase 2 derived from Arabidopsis (4-Coumarate-CoA ligase 2) It is characterized by that. The 4-coumarate-Coenzyme A ligase (4-Coumarate-CoA ligase) has the activity of synthesizing a phenol substrate compound of the active form by attaching a coay group to various phenol substrates.

The serotonin hydroxycinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) gene is characterized in that it has a nucleotide sequence of SEQ ID NO: 6, but is not limited thereto, preferably characterized in that derived from red pepper. The serotonin hydroxycinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) has the activity of biosynthesizing a phenylpropanoid amide compound using an phenolic compound and an amine substrate in an active form in which a koei is bonded to the phenolic substrate. .

Therefore, as described above, the recombinant vector of the present invention is a serotonin hydroxycinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) gene and 4-coumarate-Coenzyme A ligase (4-Coumarate-CoA ligase) gene is sequentially linked, promoter It is operatively linked with and can be used to biosynthesize the phenylpropanoid amide compounds of the present invention.

In one embodiment of the present invention, after performing the RT-PCR of the 4-Coumarate-CoA ligase 2 gene using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, the product is The pETDuet-4CL2 vector was constructed by cleavage with restriction enzymes Nde I and Xho I and insertion into the same restriction enzyme site in pETDuet-1. In addition, the serotonin hydroxycinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) gene derived from red pepper was subjected to PCR using primers of SEQ ID NO: 3 and SEQ ID NO: 4, and then the product was digested with restriction enzymes Nco I and NotI, It was inserted into the prepared pETDuet-4CL2 vector to prepare a pETDuet-SHT + 4CL2 vector (see <Example 1-1>).

The transformed bacteria of the present invention is characterized in that transformed with the recombinant vector. The bacteria, but not limited to Escherichia coli (Escherichia coli), Bacillus sub subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis) and Staphylococcus (Staphylococcus It may be a bacteria selected from the group consisting of and most preferably may be E. coli.

Known methods for introducing the recombinant vector of the present invention into the bacteria include, but are not limited to, CaCl ₂ and heat shock methods, particle gun bombardment, silicon carbide whiskers carbide whiskers), sonication, electroporation, and precipitation by polyethylene glycol (PEG). In an embodiment of the present invention, a method of introducing a recombinant vector into E. coli as a heat shock method was used (see <Example 1-2>).

The transformed bacteria of the present invention prepared by the above-described method can be cultured in large quantities by conventional culture methods. As the culture medium, a medium consisting of a carbon source, nitrogen source, vitamins and minerals may be used. For example, a conventional YEP medium containing ampicillin may be used. Cultivation of bacteria is possible under conventional bacterial culture conditions and may be incubated for 1 hour to 40 hours, for example, in the temperature range of 15 ° C to 45 ° C. Centrifugation or filtration may be performed to remove the culture medium in the culture and to separate only the concentrated cells, which steps may be performed by those skilled in the art as needed. The concentrated cells can be frozen or lyophilized according to a conventional method so as not to lose their activity.

The present inventors named the recombinant E. coli prepared above as 'pETDuet-SHT + 4CL2 (BL21 (DE3))', and deposited it on the Korean Collection for Type culture on February 4, 2008 (deposited). Number: KCTC 11272BP). In addition, the present inventors confirmed that the SHT protein is stably expressed in the recombinant E. coli of the present invention, and considering the activity of the SHT protein, it was found that the phenylpropanoid amide compound could be produced from the recombinant E. coli. (See <Example 2-1>).

Method for producing a phenylpropaneoid amide compound of the present invention comprises the steps of (a) administering a phenol substrate and an amine substrate to the transformed bacteria to culture the bacteria and (b) from the cultured bacteria of step (a) And separating and purifying the produced phenylpropaneoid amide compound.

The phenolic substrate of step (a) is not limited to this, but preferably coumaric acid (coumaric acid), caffeic acid (caffeic acid), ferulic acid (ferulic acid), cinnamic acid (cinnamic acid) and cinamic acid (sinapic acid) The amine substrate of step (a) is tyramine, tryptamine, serotonin, dopamine, doradamine, noradrenaline and octopamine. ) Is selected from the group consisting of.

In addition, in addition to the phenol substrate and the amine substrate, the expression-inducing factor may be additionally administered to the bacteria transformed in step (a). The expression inducing factor is a substance capable of inducing the expression of the gene, but is not limited thereto. Preferably, IPTG (isopropyl-β-thiogalactoside) may be used.

In addition, in the step (a), the phenol substrate and the amine substrate are not limited thereto, but preferably 0.5mM to 5mM, and most preferably, 0.5mM may be administered . In addition, in the case of administering the expression inducer, but not limited to each, preferably 0.01mM to 1mM, and most preferably, 0.1mM can be administered respectively .

In addition, the conditions for culturing the bacteria in the step (a), but is not limited to this, preferably incubated for 2 to 4 hours at the conditions of 28 ~ 40 ℃.

In step (a), the 4-coumarate-CoA ligase enzyme produced by the transformed bacterium attaches a coay group to the phenol substrate to synthesize an active phenolic compound, Serotonin hydroxycinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) enzyme can biosynthesize a phenylpropanoid amide compound using the phenolic compound and amine substrate of the active form.

In one embodiment of the present invention, the transformed bacteria were treated with an expression inducer (IPTG) of 0.1 mM, coumaric acid, 1 mM and serotonin 1 mM, and cultured for 36 hours at 28 ° C. (<Example 2-1>). Reference). In addition, after administering 0.1 mM of the expression-inducing factor (IPTG) to the transformed bacteria, and then treated with coumaric acid (coumaric acid) and serotonin (serotonin), respectively, 0.5, 1, 1.5, 2 or 5mM at 28 ℃ As a result of incubation for 3 hours, it was found that the production of coumaroyl serotonin reached saturation at 2 mM substrate concentration (see <Example 2-2>). Meanwhile, as a result of administering various phenol substrates and amine substrates to the transformed bacteria, it was found that various phenylpropanoid amide compounds can be mass produced (see <Example 3>).

On the other hand, the phenyl propaneoid amide compound of step (b) is a group consisting of coumaroyl serotonin, peruroyl serotonin, caffeoyl serotonin, cinafoyl serotonin, cinamoyl serotonin, coumaroyl octopamine and coumaroyl tyramine. It is characterized in that selected from.

Separating the phenylpropanoid amide compound produced from the cultured bacteria in step (b) is not limited thereto, and can be easily separated by administering an organic solvent to the bacterial culture. The organic solvent may be selected from the group consisting of ether, chloroform, and ethyl acetate, but is not limited thereto. Preferably, ethyl acetate may be used. In addition, if necessary, a phenyl propaneoid amide compound may be purified from the extract by the solvent using a commonly used purification method such as chromatography.

In one embodiment of the present invention, the cellulose propane obtained by centrifugation of the cultured bacteria or the culture solution was administered with ethyl acetate to separate the phenyl propanoid amide compound, and then subjected to high performance liquid chromatography (HPLC) to phenyl Propaneoid amide compounds were quantified (see <Example 2-2>). In particular, more phenylpropanoid amide compounds were isolated and produced in the culture medium than the cell precipitate (see FIG. 4).

The method for producing the phenylpropaneoid amide compound of the present invention is a method for mass production of phenylpropanoid amide compound very simply by administering a phenol substrate and an amine substrate to the transformed bacterium of the present invention. Since the transformed bacteria of the present invention secrete the phenylpropanoid amide-based compound into the culture medium, the transformant does not have to be crushed, so it can be recycled or continuously cultured, which is very economical. In addition, the phenylpropanoid amide compound prepared according to the present invention can be easily separated and purified by large distribution in the culture medium of bacteria, the production method of the present invention is very advantageous in terms of cost and process.

As described above, when the phenol substrate and the amine substrate are administered to the transformed bacteria of the present invention prepared by the recombinant vector of the present invention, it is possible to mass-produce the phenylpropanoid amide compound. Therefore, the method for producing the phenylpropanoid amide compound of the present invention using the transformed bacterium is a very simple method for mass production of the phenylpropanoid amide compound, and in particular, the transformed bacterium of the present invention Since the phenyl propanoid amide compound is secreted into the culture medium, the transformant does not have to be crushed, so it can be recycled or continuously cultured, which is very economical. In addition, the phenylpropanoid amide compound prepared according to the present invention can be easily separated and purified by large distribution in the culture medium of bacteria, the production method of the present invention is very advantageous in terms of cost and process.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Preparation of Recombinant E. Coli for Expression of 4CL2 and SHT Genes

<1-1> Preparation of Expression Vectors for Cloning and Transforming 4CL2 and SHT Genes

The 4CL (4-Coumarate-CoA ligase) gene is obtained by using the nucleotide sequence of the 4CL2 (Gene Bank No. AF106086) ((Ehlting et al., Plant J. 19, 9-20, 1999) gene derived from a known Arabidopsis. It was cloned through reverse transcription-polymerase chain reaction (RT-PCR).

Specifically, total RNA was extracted from the Arabidopsis foliar leaves 12 hours after the wounding using TRI reagent (Molecular Research Center, Inc., USA), and reverse transcription polymerase chain reaction was performed using the entire RNA as a template. First, reverse transcriptase SuperScript II (Invitrogen Inc., USA) was used to synthesize cDNA, and oligo dT was used as a primer. The reverse transcriptase reaction product was used as a template, and the forward primer 5 '-(ata catatg acgacacaagat) -3' (SEQ ID NO: 1) and the Xho I restriction enzyme site (underlined) having an Nde I restriction enzyme site (underlined) PCR reactions were performed using reverse primer 5 ′-(aga ctcgag ctagttcattaatcc) -3 ′ (SEQ ID NO: 2). The 4CL2 gene product (SEQ ID NO: 5) by the PCR reaction was digested with restriction enzymes Nde I and Xho I, gel purified, and inserted into the same restriction enzyme site in the vector pETDuet-1 (Novagen, USA) to pETDuet- 4CL2 vector was constructed.

Also pepper SHT (Serotonin -hydroxycinnamoyltransferase N) full-length cDNA of the gene (gene bank number AF329463) to (Back et al., Plant Cell Physiol. 42, 475-481, 2001, SEQ ID NO: 6) of the mold, Nco I restriction site Forward primer 5 ′-(cata ccatgg cttctgctcct) -3 ′ with (underlined) and reverse primer 5 ′-(tat gcggccgc ctaacagcttcctgcacc) -3 ′ with not I restriction enzyme site (underlined) (SEQ ID NO: 4) was used to perform the PCR reaction. The product by the PCR reaction was digested with restriction enzymes Nco I and Not I, gel purified, and inserted into the same restriction enzyme site in the prepared pETDuet-4CL2 vector to prepare a pETDuet-SHT + 4CL2 vector (FIG. 1). And 2)

<1-2> Preparation of Recombinant E. Coli for Expression of 4CL2 and SHT Genes

1 μg DNA of the pETDuet-SHT + 4CL2 vector prepared in Example 1-1 was mixed with 15 μl of a commercially available strain of E. coli BL21 (DE3) (Novagen, USA) and left on ice for 30 minutes. Heat shock was applied at 42 ° C. for 40 seconds, and after cooling on ice for 1 minute, 60 μl of SOC medium (20 g of Bacto-trypton, 5 g of Bacto-yeast extracts, 0.5 g of NaCl, and 2.5 ml / L of 1M KCl) was added. After incubation for 1 hour at 37 ℃. After culturing, the strain was cultured in YEP solid medium containing ampicillin to prepare recombinant E. coli for 4CL2 and SHT gene expression. The recombinant E. coli prepared above was named 'pETDuet-SHT + 4CL2 (BL21 (DE3))', and on February 4, 2008, the Korean Gene Bank (Korean Collection for Type culture, 52, Eeun-dong, Yuseong-gu, Daejeon) (Accession number: KCTC 11272BP).

<Example 2>

Production of coumaroylserotonin using recombinant E. coli

<2-1> Expression of SHT Protein in Recombinant Escherichia Coli

After incubating the recombinant Escherichia coli 4CL2 and SHT gene expression prepared in <Example 1-2> at 37 ℃ until the absorbance (OD600) of the culture reached 1.5 in conventional ampicillin (ampicillin) containing YEP medium Treatment of the expression inducer (IPTG) with 0.1mM, 1mM of kumaric acid and serotonin at 1mM concentration, and 36 hours of incubation at 28 ° C, the E. coli culture was centrifuged to separate the cell precipitate and the culture medium. .

In order to determine whether SHT protein is expressed in the recombinant Escherichia coli prepared in Example <1-2> during the incubation period, 5 µl of the total culture solution of the recombinant Escherichia coli was separated by electrophoresis (SDS-PAGE), and nitro Blot on nitrocellulose membrane and immunoreact with SHT antibody (Back et al., Plant Cell Physiol . 42, 475-481, 2001). Western blotting kit (Boehringer Manheim, Germany) was used to detect the band due to the reaction and the experimental results are shown in FIG. 3.

As shown in FIG. 3, it was found that SHT protein was stably expressed in recombinant E. coli during the culture period, and the SHT protein biosynthesized a phenylpropanoid amide compound using an amine substrate and a phenolic compound. Considering the enzyme, it can be seen that a representative phenylpropanoid amide compound can be produced from the recombinant E. coli.

<2-2> Production of Coumaroyl Serotonin Using Recombinant Escherichia Coli

In order to quantify the comoylserotonin contained in the cell precipitate or culture medium prepared in <Example 2-1>, the cell precipitate was dissolved in 1 ml of 0.1 M Tris-HCl buffer per gram weight or After diluting 1 mL of the culture solution with an equivalent amount of ethyl acetate, the ethyl acetate fraction was placed in a vacuum concentrator at 40 ° C. to completely remove the solvent, dissolved in 100 μl of methanol, and quantified by high performance liquid chromatography (HPLC). The column used in the high performance liquid chromatography was Wakosil II 3C18HG (4.6 × 150 mm), 35% methanol containing 0.5% TFA as the developing solvent, and the separated material was measured at 320 nm (Jang et al., Plant Physiol. 135, 346-356, 2004), and the results are described in FIG.

As shown in FIG. 4, about 200 mg / l of the culture medium and about 30 mg / l of coumaroyl serotonin were produced in the cell precipitate and a total of 230 mg / L of coumaroyl serotonin was treated for 3 hours after substrate treatment. It was produced in, it was confirmed that the production is stable for 36 hours. In particular, as a result of further subdividing the incubation time after the substrate treatment, it was found that about 30 mg / L of coumaroyl serotonin was produced within 30 minutes, and reached a maximum value of 230-240 mg / L in 2 hours.

Therefore, the recombinant Escherichia coli secretes most of the phenylpropanoid amide compound into the culture medium within a short time of about 2 hours. After extracting the compound from the culture medium, the culture medium is mixed with the E. coli cells and recycled or continuously cultured. It can be seen that the compound can be continuously extracted about 12 times within 24 hours, so that the compound can be obtained in large quantities easily.

<2-3> Comparison of Production of Kumaroyl Serotonin by Substrate Concentration Using Recombinant Escherichia Coli

After administering 0.1 mM of the expression inducer (IPTG) to the recombinant E. coli (OD 600 = 1.5) cultured in Example 2-1, coumaric acid and serotonin 0.5 and 1, respectively. , 1.5, 2 or 5mM and then incubated for 3 hours at 28 ℃ and centrifuged. After centrifugation, the amount of coumaroyl serotonin contained in the culture was quantified in the same manner as in <Example 2-2>, and the results are shown in FIG. 5.

As shown in FIG. 5, when 0.5 or 1 mM of cumaric acid and serotonin were treated, 200 mg / L of coumaroyl serotonin was produced, and when 2 or 5 mM was treated, 250 mg / L of coumaroyl serotonin was produced. Confirmed. From the above results, the higher the concentrations of cumaric acid and serotonin, the higher the yield of coumaroyl serotonin, but the yield of coumaroyl serotonin does not continuously increase in proportion to the substrate concentration. It can be seen that the production reaches saturation. In particular, when treating 0.5 mM and 1 mM coumaric acid and serotonin, all the substrates are used for the production of coumaroyl serotonin, so that the remaining substrate is no longer present in the culture. Therefore, even without the column purification or high-performance liquid chromatography (HPLC) purification process can be obtained easily purified Kumaroyl serotonin of 98-100% purity only by extracting the ethyl acetate of the culture.

< Example  3>

Production of Various Phenylpropanoid Amide Compounds Using Recombinant Escherichia Coli

After administering 0.1 mM of expression inducer (IPTG) to the recombinant Escherichia coli cultured in <Example 2-1>, and then treated with various phenol substrates and amine substrates at a concentration of 1 mM, phenylpropanoid amide The compound was quantified in the same manner as in <Example 2-2>, and the results are shown in FIG. 6.

As shown in FIG. 6, 200 mg / l of coumaroyl serotonin was produced when treated with coumaric acid and serotonin and about 90 mg / l when treated with ferulic acid and serotonin. Confirmed to be produced. In addition, it was confirmed that when Kumaric acid and octopamine were treated, Kumaroyl Octopamine was produced about 200 mg / l, and when Kumaric Acid and tyramine were treated, Kumaroyl Tyramine was produced 180 mg / l.

From the above results, it was found that by expressing the SHT gene and 4CL2 gene in Escherichia coli, various kinds of phenylpropanoid amide compounds can be produced.

1 is a schematic diagram of recombinant E. coli expression vectors for 4CL and SHT gene expression (SHT: serotonin hydroxycinnamoyl transferase, 4CL: 4-coumarate coenzyme A ligase).

2 is a cleavage map of recombinant E. coli expression vectors for 4CL2 and SHT gene expression (SHT: serotonin hydroxycinnamoyl transferase, 4CL2: 4-coumarate coenzyme A ligase, T7 / lac: T7 / lac promoter, rbs: Ribosomal binding site, T7 ter: T7 termination site, f1 origin: origin of replication, Amp ^R : ampicillin resistance gene).

3 is a photograph showing the results of confirming the expression of SHT protein in recombinant E. coli over time (SHT: serotonin hydroxycinnamoyl transferase).

Figure 4 is a graph showing the results of confirming the production of cumuloyl serotonin over time using recombinant E. coli (CS: cumaroyl serotonin).

Figure 5 is a graph comparing the production of the substrate concentration of Kumaroyl serotonin using recombinant E. coli.

6 is a graph showing the yield of various phenylpropanoid amide compounds using recombinant E. coli.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Recombinant vector containing 4CL gene and SHT gene,          microorganism transformed lili and method for preparation          phenylpropanoid amide-based compounds using the same <130> NP08-0013 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 4CL-F <400> 1 atacatatga cgacacaaga t 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 4CL-R <400> 2 agactcgagc tagttcatta atcc 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SHT-F <400> 3 cataccatgg cttctgctcc t 21 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> SHT-R <400> 4 tatgcggccg cctaacagct tcctgcacc 29 <210> 5 <211> 1671 <212> DNA <213> Arabidopsis thaliana <400> 5 atgacgacac aagatgtgat agtcaatgat cagaatgatc agaaacagtg tagtaatgac 60 gtcattttcc gatcgagatt gcctgatata tacatcccta accacctccc actccacgac 120 tacatcttcg aaaatatctc agagttcgcc gctaagccat gcttgatcaa cggtcccacc 180 ggcgaagtat acacctacgc cgatgtccac gtaacatctc ggaaactcgc cgccggtctt 240 cataacctcg gcgtgaagca acacgacgtt gtaatgatcc tcctcccgaa ctctcctgaa 300 gtagtcctca ctttccttgc cgcctccttc atcggcgcaa tcaccacctc cgcgaacccg 360 ttcttcactc cggcggagat ttctaaacaa gccaaagcct ccgcggcgaa actcatcgtc 420 actcaatccc gttacgtcga taaaatcaag aacctccaaa acgacggcgt tttgatcgtc 480 accaccgact ccgacgccat ccccgaaaac tgcctccgtt tctccgagtt aactcagtcc 540 gaagaaccac gagtggactc aataccggag aagatttcgc cagaagacgt cgtggcgctt 600 cctttctcat ccggcacgac gggtctcccc aaaggagtga tgctaacaca caaaggtcta 660 gtcacgagcg tggcgcagca agtcgacggc gagaatccga atctttactt caacagagac 720 gacgtgatcc tctgtgtctg gcctatgttc catatatacg ctctcaactc catcatgctc 780 tgtagtctca gaattggtgc cacgatcttg ataatgccta agttcgaaat cactctcttg 840 ttagagcaga tacaaaggtg taaagtcacg gtggctatgg tcgtgccacc gatcgtttta 900 gctatcgcga agtcgccgga gacggagaag tatgatctga gctcggttag gatggttaag 960 tctggagcag ctcctcttgg taaggagctt gaagatgcta ttagtgctaa gtttcctaac 1020 gccaagcttg gtcagggcta tgggatgaca gaagcaggtc cggtgctagc aatgtcgtta 1080 gggtttgcta aagagccgtt tccagtgaag tcaggagcat gtggtacggt ggtgaggaac 1140 gccgagatga agatacttga tccagacaca ggagattctt tgcctaggaa caaacccggc 1200 gaaatatgca tccgtggcaa ccaaatcatg aaaggctatc tcaatgaccc cttggccacg 1260 gcatcgacga tcgataaaga tggttggctt cacactggag acgtcggatt tatcgatgat 1320 gacgacgagc ttttcattgt ggatagattg aaagaactca tcaagtacaa aggatttcaa 1380 gtggctccag ctgagctaga gtctctcctc ataggtcatc cagaaatcaa tgatgttgct 1440 gtcgtcgcca tgaaggaaga agatgctggt gaggttcctg ttgcgtttgt ggtgagatcg 1500 aaagattcaa atatatccga agatgaaatc aagcaattcg tgtcaaaaca ggttgtgttt 1560 tataagagaa tcaacaaagt gttcttcact gactctattc ctaaagctcc atcagggaag 1620 atattgagga aggatctaag agcaagacta gcaaatggat taatgaacta g 1671 <210> 6 <211> 753 <212> DNA <213> Capsicum annuum <400> 6 atggcttctg ctcctcaacc accaactcta tctgaaaaaa ccactaatct ctcaccggaa 60 aacgacaatg ttacgatcac cggaaagata tatacaagag tccgccttgc tacaaaatct 120 gatctgcacc atgcatacca gttgttttac caaatccacg cataccataa ccaatttcat 180 ttattcaaag caacagagtc ctccttatcg gacttgttct ttaaagaaaa tcctcttccc 240 cttttctacg gaccaaccct acttctactc gaagtctccc cgaccgcttt tactgaaccc 300 Aaaaaataaca aggacgaagg gttcaaaccc gtctttacag cgctcgacct taaatttcct 360 gtcgtggaag gacaagttga ggagtttcgg tccaaatatg atgatggaac cgataaacgt 420 gatgtgttca tcgcgggata tgcttacttt tttgctagtt attcactctt cgggaacgac 480 aaaccgggga tccatttcga cagtctttac ttcagggaaa gctatagaaa attgggaatg 540 ggaaaattgt tgtttggaac tgttgcgtct attgctgcga acaatgggtt tgctgcggtg 600 gagggaattg tagcagtttg gaataaaaag tcatatgatt tttatgtgag tatgggtgtt 660 gaaatgcatg atgactttag gtttggcaaa ttggacggtg aaaatcttca aaagtacgct 720 gataaggaga aaaatggtgc aggaagctgt tag 753  

Claims (12)

Serotonin hydroxycinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) gene and 4-Coumarate-CoA ligase (4-Coumarate-CoA ligase) gene is characterized in that the sequential connection and operably linked to the promoter Recombinant vector. The recombinant vector of claim 1, wherein the 4-Coumarate-CoA ligase gene has a nucleotide sequence of SEQ ID NO: 5. 8. According to claim 1, wherein the serotonin hydroxycinnamoyl transferase (Serotonin N -hydroxycinnamoyltransferase) gene is a recombinant vector, characterized in that having a nucleotide sequence of SEQ ID NO: 6. The recombinant vector according to claim 1, wherein said recombinant vector has a cleavage map as described in FIG. A bacterium transformed with the recombinant vector of claim 1. The bacterium of claim 5, wherein the transformed bacterium is Escherichia coli. The bacterium according to claim 6, wherein the E. coli is Accession No. KCTC 11272BP. (a) culturing the bacteria of claim 5 by administering a phenol substrate and an amine substrate; And (b) isolating and purifying the phenylpropanoid amide compound produced from the cultured bacterium of step (a). The phenolic substrate of step (a) is coumaric acid (coumaric acid), caffeic acid (caffeic acid), perulic acid (ferulic acid), cinnamic acid (cinnamic acid) and sinapic acid (sinapic acid) Process for producing a phenylpropanoid amide compound, characterized in that selected from the group consisting of. According to claim 8, wherein the amine substrate of step (a) is a group consisting of tyramine, tryptamine, serotonin, dopamine, noradrenaline and octopamine Process for producing a phenyl propaneoid amide compound, characterized in that selected from. According to claim 8, wherein the phenyl propaneoid amide compound of step (b) is cumaroyl serotonin, peruroyl serotonin, caffeoyl serotonin, cinapoyl serotonin, cinamoyl serotonin, cumaroyl octopamine and coumaro Process for producing a phenylpropanoid amide compound, characterized in that selected from the group consisting of yltyramine. The method of claim 8, wherein incubating the phenol substrate and the amine substrate to the bacteria of step 5 of step (a) by administering 0.5 mM to 5 mM of the phenol substrate and the amine substrate, respectively, under the conditions of 28-40 ° C. A method for producing a phenylpropanoid amide compound, which is incubated for 4 hours.

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