KR101960182B1 - Method for producing caffeic acid phenyl ester or derivatives thereof using p-Coumaroyl Co-A:monolignol transferase - Google Patents

Abstract

The invention p - one Co-A in Kumar: mono league play transferase (p -Coumaroyl Co-A: monolignol transferase; PMT) using recombinant microorganisms overexpressing, cafe acid phenyl ester (caffeic acid phenyl ester; CAPE ) Or a derivative thereof.

Description

[0001] The present invention relates to a method for producing a caffeic acid phenyl ester or a derivative thereof, which comprises using a p-coumaroyl Co-A: monoligonol transferase,

The invention p - one Co-A in Kumar: mono league play transferase (p -Coumaroyl Co-A: monolignol transferase; PMT) for using recombinant microorganisms overexpressing, cafe acid phenyl ester (CAPE) or derivatives thereof And a method for manufacturing the same.

Caffeic acid phenyl ester (CAPE) is a natural flavonoid compound that is known to be a major component of honey propolis. CAPE has a strong antioxidant effect and it can improve the skin moisturizing ability and can be used in various fields such as medicines, cosmetics and health functional foods. In addition, CAPE also exhibits anticancer, anti-inflammatory and neuroprotective effects, and in particular, when CAPE is mixed with an anticancer agent, the anticancer effect is enhanced, and thus the biosafety can be increased in that the natural substance is used There is a bar. In addition, it exhibits physiological activities such as suppression of growth of common eukaryotic fungi and inhibition of acute immune function. In addition to CAPE, its analogous derivatives are expected to have similar effects, and studies on their mass production are required.

CAPE is a polyphenic acid composed of the structure of an ester compound of caffeic acid. Ekfktj is industrially extracted from propolis to use CAPE, or chemically synthesized by linking caffeic acid with 2-phenylethanol via an ester bond. However, the induction of ester linkage by a chemical method has problems such as the production of by-products or the control of the chemical process due to the use of a chemical catalyst, and a method for producing this by using a biological catalyst has been studied.

Lipases are mainly used to catalyze ester bonds using biological catalysts, enzymes. A method of producing CAPE using CALB (lipaseB derived from Candida antartica ) or novozyme 435 (Novozyme 435) in which it is fixed to resin has been known as a representative lipase for commercial use (Non-Patent Document 1, Non-Patent Document 1 Document 2). However, in addition to CAPE, its derivatives are expected to have a variety of utility values, and interest is being focused on the development of enzyme catalysts capable of producing CAPE as well as derivatives thereof through a variety of substrate specificities.

The present inventors have made efforts to develop an enzyme source capable of synthesizing CAPE and its derivatives with various substrate specificities. As a result, it has been found that p -coumaroyl Co-A monoligonol transferase ( p- (Hydroxycinnamic acid) substrate and phenyl alcohol by using recombinant E. coli that overexpresses Coumaroyl Co-A: monolignol transferase (PMT) to produce CAPE and derivatives thereof. Respectively.

 Antonopoulou, Io, et al., &Quot; Enzymatic synthesis of caffeic acid phenethyl ester ", Journal of the Chinese Institute of Chemical Engineers 39 (2008) 413-418.  Eudes, Aymerick, et al., "Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamates and benzoate conjugates in yeast.", Microbial cell factories 15.1 (2016): 198.

The object of the present invention is to provide a use of a protein capable of synthesizing a caproic acid phenyl ester (CAPE) and derivatives thereof by synthesizing hydroxycinnamic acid substrate and phenyl alcohol.

It is another object of the present invention to provide a process for producing CAPE or a derivative thereof using the protein.

In order to achieve the above object, the present invention provides a process for producing CAPE or a derivative thereof, which comprises the following steps i) to iv):

i) p - Kumar In one Co-A: mono league play transferase (p -Coumaroyl Co-A: monolignol transferase; preparing a recombinant microorganism expressing the PMT);

ii) culturing and obtaining the recombinant microorganism produced in step i);

iii) suspending and reacting the recombinant microorganism obtained in step ii) with a reaction solution containing a hydroxycinnamic acid substrate and a phenyl alcohol substrate; And

iv) obtaining a caffeic acid phenyl ester (CAPE) or a derivative thereof from the reaction mixture reacted in step iii).

In one preferred embodiment of the present invention, the PMT is encoded by the nucleotide sequence of SEQ ID NO: 1, and is a p -coumaroyl Co-A: monolig knock transcription enzyme derived from rice and consisting of the amino acid sequence of SEQ ID NO: have.

In a preferred embodiment of the present invention, the recombinant microorganism of step i) may be E. coli.

In one preferred embodiment of the present invention, the hydroxycinnamic acid substrate of step iii) is selected from the group consisting of caffeic acid having the structure of the following formula (1) or coumaric acid having the structure of the following formula (2) acid may be one of:

[Chemical Formula 1]

; 및

; And

(2)

.

.

In a preferred embodiment of the present invention, the phenyl alcohol substrate of step iii) may be any one selected from the group consisting of:

2-phenylethanol, tyrosol, hydroxy tyrosol, benzyl alcohol, 2-hydroxyphenethyl alcohol, 2- (3-hydroxyphenyl) Hydroxyphenyl alchol, 3-phenyl-1-propanol, 4-phenyl-1-butanol, Phenyl-1-pentanol, 7-phenyl-1-heptanol, 8-phenyl-1-heptanol, 1-nonanol, 2- (3-hydroxyphenyl) alcohol (2-3- (hydroxyphenyl alchol), 2-methanoxypentyl alcohol -methanoxyphenthyl alcohol, 3-methanoxyphenthyl alcohol and 4-methanoxyphenthyl alcohol.

In a preferred embodiment of the present invention, the CAPE or derivatives thereof may be a compound having the structure of the following formula 3:

(3)

(Wherein R < ₁ > is either hydrogen or a hydroxy group,

R ₂ is C ₁ to C ₉ phenylalkyl, methoxyphenethyl, hydroxyphenethyl or dihydroxyphenethyl).

In a preferred embodiment of the present invention, the CAPE or a derivative thereof may be a compound having any one of the following formulas (4) to (24).

Accordingly, the invention is a p separated from the genome of a rice plant genome - one to Kumar Co-A: mono league play transferase (p -Coumaroyl Co-A: monolignol transferase; PMT) provides the use of a. When the recombinant Escherichia coli overexpressing the PMT of the present invention is used, CAPE and derivatives thereof can be synthesized by synthesizing hydroxycinnamic acid substrate and phenyl alcohol.

Brief Description of the Drawings Fig. 1 is a schematic diagram of a reaction scheme for the synthesis of CAPE using caffeic acid and 2-phenylethanol as substrates.
2A shows the results of proton NMR analysis for the structural analysis of CAPE synthesized from caffeic acid and a 2-phenylethanol substrate.
Figure 2B shows HPLC-MS analysis results for quantitative analysis of CAPE synthesized from caffeic acid and 2-phenylethanol substrate.
3 shows HPLC-MS analysis results for the quantitative analysis of CAPE derivatives synthesized from capric acid and tyrosol.
4 is a graph showing the results of a CAPE derivative synthesized from caffeic acid and 7-phenyl-1-heptanol (sample A), 8-phenyl-1-octanol (sample B) The results of HPLC-MS analysis for quantitative analysis are shown.
FIG. 5 shows HPLC-MS analysis results for the quantitative analysis of CAPE derivatives synthesized from capric acid and 2- (3-hydroxyphenyl) alcohol (2-3- (hydroxyphenyl alchol).
6 shows HPLC-MS analysis results for the quantitative analysis of CAPE derivatives synthesized from caffeic acid and 2-methanoxyphenthyl alcohol.
7 shows HPLC-MS analysis results for the quantitative analysis of CAPE derivatives synthesized from caffeic acid and 3-methanoxyphenthyl alcohol.
8 shows HPLC-MS analysis results for the quantitative analysis of CAPE derivatives synthesized from caffeic acid and 4-methanoxyphenthyl alcohol.
9 shows HPLC-MS analysis results for quantitative analysis of CAPE derivatives synthesized from p- coumaric acid and 2-phenylethanol.
10 shows HPLC-MS analysis results for the quantitative analysis of CAPE derivatives synthesized from cumaric acid and tyrosol.
11 shows HPLC-MS analysis results for the quantitative analysis of CAPE derivatives synthesized from cumaric acid and hydroxycrozon.
12 shows HPLC-MS analysis results for quantitative analysis of CAPE derivatives synthesized from cumaric acid and 2-hydroxyphenethyl alcohol.
13 shows HPLC-MS analysis results for quantitative analysis of CAPE derivatives synthesized from cumaric acid and 2- (3-hydroxyphenyl) alcohol (2-3- (hydroxyphenyl alchol).
14 shows HPLC-MS analysis results for quantitative analysis of CAPE derivatives synthesized from cumaric acid and 2-methanoxyphenthyl alcohol.
15 shows HPLC-MS analysis results for quantitative analysis of CAPE derivatives synthesized from cumaric acid and 3-methanoxyphenthyl alcohol.
16 shows HPLC-MS analysis results for quantitative analysis of CAPE derivatives synthesized from cumaric acid and 4-methanoxyphenthyl alcohol.

Hereinafter, the present invention will be described in detail.

The present invention provides a process for the preparation of CAPE or derivatives thereof, which comprises the following steps i) to iv):

i) p - Kumar In one Co-A: mono league play transferase (p -Coumaroyl Co-A: monolignol transferase; preparing a recombinant microorganism expressing the PMT);

ii) culturing and obtaining the recombinant microorganism produced in step i);

iii) suspending and reacting the recombinant microorganism obtained in step ii) with a reaction solution containing a hydroxycinnamic acid substrate and a phenyl alcohol substrate; And

iv) obtaining a caffeic acid phenyl ester (CAPE) or a derivative thereof from the reaction mixture reacted in step iii).

In the method for producing CAPE or a derivative thereof according to the present invention, the PMT is preferably a p -coumaroyl Co-A monoligolnol transferase derived from rice, and more specifically, a protein synthesized from the nucleotide sequence of SEQ ID NO: 1 Most preferably a protein consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

In the method for producing CAPE or derivatives thereof of the present invention, the recombinant microorganism overexpressing PMT can be produced using any method that is used in the art to induce over-expression of a target protein using a recombinant microorganism. More specifically, the recombinant microorganism can be used without limitation as long as it is used for producing a transformant. Specific examples of the recombinant microorganism include E. coli , Saccharomyces cerevisiae , or Corynebacterium , It is preferable to use E. coli. More specifically, E. coli is most preferably used, but not limited thereto.

In the method for producing CAPE or a derivative thereof according to the present invention, the hydroxycinnamic acid substrate of step iii) may be a caffeic acid having a structure of the following formula (1) or a coumaric acid having a structure of the following formula May be any of coumaric acid:

[Chemical Formula 1]

; 및

; And

(2)

.

.

In a preferred embodiment of the present invention, the phenyl alcohol substrate of step iii) may be any one selected from the group consisting of:

2-phenylethanol, tyrosol, hydroxy tyrosol, benzyl alcohol, 2-hydroxyphenethyl alcohol, 2- (3-hydroxyphenyl) Hydroxyphenyl alchol, 3-phenyl-1-propanol, 4-phenyl-1-butanol, Phenyl-1-pentanol, 7-phenyl-1-heptanol, 8-phenyl-1-heptanol, 1-nonanol, 2- (3-hydroxyphenyl) alcohol (2-3- (hydroxyphenyl alchol), 2-methanoxypentyl alcohol -methanoxyphenthyl alcohol, 3-methanoxyphenthyl alcohol and 4-methanoxyphenthyl alcohol.

In a preferred embodiment of the present invention, the CAPE or derivatives thereof may be a compound having the structure of the following formula 3:

(3)

(Wherein R < ₁ > is either hydrogen or a hydroxy group,

R ₂ is C ₁ to C ₉ phenylalkyl, methoxyphenethyl, hydroxyphenethyl or dihydroxyphenethyl).

In a preferred embodiment of the present invention, the CAPE or a derivative thereof may be a compound having any one of the following formulas (4) to (24)

[Chemical Formula 4]

,

,

[Chemical Formula 5]

,

,

[Chemical Formula 6]

,

,

(7)

,

,

[Chemical Formula 8]

,

,

[Chemical Formula 9]

,

,

[Chemical formula 10]

,

,

(11)

,

,

[Chemical Formula 12]

,

,

[Chemical Formula 13]

,

,

[Chemical Formula 14]

,

,

[Chemical Formula 15]

,

,

[Chemical Formula 16]

,

,

[Chemical Formula 17]

,

,

[Chemical Formula 18]

,

,

[Chemical Formula 19]

,

,

[Chemical Formula 20]

,

,

[Chemical Formula 21]

,

,

[Chemical Formula 22]

,

,

(23)

, 및

, And

≪ EMI ID =

.

.

In an specific embodiment of the invention, the present inventors from rice genome p - one Co-A in Kumar: mono league play transferase (p -Coumaroyl Co-A: monolignol transferase; PMT) to remove the gene, to overexpress this E. coli transformants were prepared. Using the transformant E. coli thus prepared, caffeic acid or a coumaric acid substrate and a phenyl alcohol substrate were synthesized to confirm that CAPE or a derivative thereof was produced.

Accordingly, the present invention p-one Co-A in Kumar: mono league play transferase (p -Coumaroyl Co-A: monolignol transferase; PMT) provides the use of a. When the recombinant Escherichia coli overexpressing the PMT of the present invention is used, CAPE and derivatives thereof can be synthesized by synthesizing hydroxycinnamic acid substrate and phenyl alcohol.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

p - Coomarocyl Co-A: Monolig knock-in transgene ( p -Coumaroyl Co-A: monolignol transferase; PMT) overexpression of E. coli

For use as a catalyst for the whole-cell biocatalysis of the present invention, a transformed E. coli overexpressing p -coumaroyl Co-A: monoligolnt transferase (PMT) was prepared.

Specifically, the PMT gene (Sequence Listing 1) was isolated from the rice genome genome. Both ends of the separated gene sequence were digested with SmaI restriction enzyme and inserted into the SmaI site of the pBluescript (Stratagene) vector. The inserted pBluescript vector was further digested with EcoRI and Xho I (New England BioLab) and transferred to EcoRI and Xho I sites of a pGEX vector used as an E. coli expression vector to prepare a vector for expression of PMT. The prepared expression vector was transformed into E. coli DH-5? Strain (Invitrogen). The transformed Escherichia coli was selected on LB medium supplemented with ampicillin. Plasmid was isolated from the selected Escherichia coli, and PMT gene was confirmed to be contained therein and selected as a final transformant.

The final selected transformant E. coli was seeded in 2 ml of LB medium and shaken at 37 ° C overnight at 200 rpm. On the next morning, 500 μl of the cultured medium was inoculated in 50 ml of fresh LB medium, and main culture was performed while shaking at 37 ° C at 200 rpm. When the cultured medium reached a bacterial density at an OD ₆₀₀ of 0.6 to 0.8, a protein inducer, IPTG (final concentration: 0.1 mM), was added to the culture. Then, the transformant Escherichia coli was cultured for 4 hours while shaking at 30 DEG C at 200 rpm to induce overexpression of the PMT protein.

Synthesis of Caffeic acid phenethyl ester (CAPE) using PMT overexpressing E. coli

The synthesis of caffeic acid phenyl ester (CAPE) using caffeic acid and 2-phenyl ethanol as substrates was carried out using E. coli transformants (Fig. 1).

Concretely, a culture solution in which the PMT-overexpressing transformant Escherichia coli prepared in [Example 1] was cultured was obtained. Then, the culture was centrifuged at 15,000 rpm for 10 minutes to recover the transformed E. coli. The recovered E. coli is an E. coli concentration in 10 ㎖ M9 medium containing 10% DMSO were prepared again the reaction medium and suspended to a 1.5 OD _600. Then, ethanol containing 10 mg / ml caffeic acid aqueous solution and 10 mg / ml 2-phenylethanol was added to the reaction medium prepared above using a stock solution, and the final concentration of aqueous solution of caffexic acid and 2-phenylethanol Were added to the reaction mixture to give 200 μM and 400 μM, respectively, followed by shaking at 30 ° C. and 200 rpm for 67 hours to carry out the CAPE synthesis reaction. 2-phenylethanol was volatile and lost in the reaction. After 24 hours and 53 hours from the start of the reaction, 400 μM of 2-phenylethanol was further added (the total amount of 2-phenylethanol at the start of the reaction was 1,200 [mu] M). After completion of the reaction, a reaction medium was obtained and extracted twice with ethylacetate. The extract was vacuum-dried, and the obtained sample was dissolved in DMSO to proton NMR analysis to confirm its structure and quantitated by HPLC-MS analysis. CAPE standard samples for quantification were prepared by dissolving CAPE in ethanol and analyzed by HPLC to quantify the CAPE produced based on the quantitative curve.

As a result, the over-expression of E. coli using the PMT, as shown in Figure 2, Cafe acid and 2-phenylimidazole was by structural analysis that CAPE is synthesized from ethanol to make the temperament NMR analysis results were as follows (Fig. 2a): ¹ H (2H, t, J = 7.0 Hz), 6.26 (1H, d, J = 16.0 Hz), 6.86 (2H, (1H, d, J = 8.2 Hz), 7.02 (1H, dd, J = 8.2, 2.0 Hz), 7.14 7.52 (1H, d, J = 16.0 Hz). As a result of the quantitative analysis, it was confirmed that 110.9 μM of CAPE was synthesized after completion of the reaction (FIG. 2B). When the remaining amount of caffeic acid added was confirmed, it was confirmed that about 55.5% of caffeic acid was converted into CAPE.

Synthesis of CAPE derivatives using PMT overexpressing E. coli

It was confirmed that the synthesis of caffeic acid phenyl ester (CAPE) could be induced by using PMT overexpressing E. coli, and the synthesis reaction of the CAPE derivative was induced by changing the substrate.

<3-1> Synthesis of CAPE derivatives using caffeic acid as substrate

Specifically, a culture solution in which the PMT-overexpressing transformant Escherichia coli prepared in [Example 1] was cultured was obtained and cultured in the same manner as in Example 2 using the aqueous solution of caffeic acid and the substrate described in the following [Table 1] CAPE derivatives were synthesized as described above.

Substrates for synthesis of CAPE and its derivatives and synthesized CAPE and its derivatives number temperament Product (CAPE derivative) One

2-페닐에탄올(2-phenylethanol)

2-phenylethanol &lt; / RTI &gt;

2

Tyrosol

3

Benzyl alcohol

4

3-phenyl-1-propanol (3-phenyl-1-propanol)

5

4-phenyl-1-butanol &lt; / RTI &gt;

6

5-phenyl-1-pentanol &lt; / RTI &gt;

7

7-phenyl-1-heptanol &lt; / RTI &gt;

8

8-phenyl-1-octanol &lt; / RTI &gt;

9

9-phenyl-1-nonanol (9-phenyl-

10

2- (3-hydroxyphenyl) alcohol
(2-3- (hydroxyphenyl alchol)

11

2-methanoxypentyl alcohol
(2-methanoxyphenthyl alcohol)

12

3-methanoxypentyl alcohol
(3-methanoxyphenthyl alcohol)

13

4-methanoxypentyl alcohol
(4-methanoxyphenthyl alcohol)

As a result, as shown in FIG. 3 to FIG. 8, it was confirmed that various CAPE derivatives could be synthesized as the alcohol substrate reacted with caffeic acid was changed (FIG. 9). On the other hand, synthesis reaction with caffeic acid was induced by using hydroxy tyrosol or 2-hydroxyphenethyl alcohol as a substrate, but the synthesis reaction was not significant, -MS analysis. &Lt; / RTI &gt;

<3-2> Synthesis of CAPE derivatives using coumaric acid as a substrate

Caffeic acid has two hydroxyl groups in the benzene ring, and the coumaric acid has one hydroxyl group and is similar in structure. Thus, the present inventors conducted a reaction to determine whether CAPE derivatives could be synthesized even when the substrate was changed to a coumaric acid in the reaction of synthesizing CAPE and its derivatives using caffeic acid as a substrate.

Specifically, a culture solution in which the PMT-overexpressing transformant Escherichia coli prepared in [Example 1] was cultured was obtained, and Escherichia coli was suspended as described in Example 2 to prepare a reaction medium. Then, an aqueous solution of cumaric acid And the final concentrations of the substrates described in [Table 2] were adjusted to 300 μM, respectively, and shaken at 30 ° C. and 200 rpm for 69 hours to carry out the CAPE derivative synthesis reaction. 24 hours after the initiation of the reaction, 500 [mu] M was further added to each of the substrates described in [Table 2] below to carry out the reaction. After completion of the reaction, the same procedure as in [Example 2] was carried out and quantified by HPLC-MS analysis.

A list of substrates and CAPE derivatives used with coumaric acid to synthesize derivatives of CAPE number temperament Product (CAPE derivative) One

2-페닐에탄올(2-phenylethanol)

2-phenylethanol &lt; / RTI &gt;

2

Tyrosol

3

Hydroxy tyrosol &lt; RTI ID = 0.0 &gt;

4

2-hydroxyphenethyl alcohol &lt; RTI ID = 0.0 &gt;

5

2- (3-hydroxyphenyl) alcohol (2-3- (hydroxyphenyl alchol)

6

2-methanoxyphenthyl alcohol

7

3-methanoxyphenthyl alcohol

8

4-methanoxyphenthyl alcohol

As a result, as shown in FIG. 9 to FIG. 16, it was confirmed that various CAPE derivatives could be synthesized by changing the alcohol substrate reacting with the coumaric acid. In particular, the Kumar acid, about 98.2% of the p-substrate was used the product when reacted with a tee sol-tyrosine is converted into Kumano In one brush (p -coumaroyl tyrosol), and finally of 758.7 μM p-one to Kumar It was confirmed that thyrosol was synthesized.

<110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Method for producing caffeic acid phenyl ester or derivatives          its using p-Coumaroyl Co-A: monolignol transferase <130> 1062693 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1320 <212> DNA <213> Homo sapiens <400> 1 atggggttcg cggtggtgag gacgaaccgg gagttcgtgc ggccgagcgc ggcgacgccg 60 ccgtcgtccg gcgagctgct ggagctgtcc atcatcgacc gcgtggtggg gctccgccac 120 ctggtgcggt cgctgcacat cttctccgcc gccgccccga gcggcggcga cgccaagccg 180 tcgccggcgc gggtgatcaa ggaggcgctg gggaaggcgc tggtggacta ctacccgttc 240 gcggggaggt tcgtggacgg cggcggcggg ccggggagcg cccgcgtgga gtgcaccggc 300 gagggcgcct ggttcgtgga ggccgccgcc ggctgcagcc tcgacgacgt gaacggcctc 360 gaccacccgc tcatgatccc cgaggacgac ctcctccccg acgccgcccc cggtgtccac 420 cccctcgacc tccccctcat gatgcaggtg acggagttca gttgcggagg gttcgtggtg 480 ggcctgatct cggtgcacac gatggcggac gggctagggg ccgggcagtt catcaacgcg 540 gtgggcgact acgcccgcgg gctggacagg ccgagggtga gcccggtctg ggcccgcgag 600 gccatcccga gcccgccgaa gctgcccccg ggcccgccgc cggagctgaa gatgttccag 660 ctccgccacg tcaccgccga cctgagcctg gacagcatca acaaggccaa gtccgcctac 720 ttcgccgcca ccggccaccg ctgctccacc ttcgacgtcg ccatcgccaa gacgtggcag 780 gcgcgcaccc gcgcgctccg cctcccggaa cccacctccc gcgtcaacct ctgcttcttc 840 gccaacaccc gccacctcat ggccggcgcc gccgcctggc ccgcacccgc cgccggcggc 900 ggcggagagc 960 ggggcggtgg aggcggcgga cgtggccggg gtggtgggga tgatacggga ggcgaaggcg 1020 aggctgccgg cggacttcgc gcggtgggcg gtggccgact tcagggagga tccgtacgag 1080 ctgagcttca cgtacgattc cctgttcgtc tccgactgga cgcggctggg gttcctggag 1140 gcggactacg ggtgggggcc gccgtcgcac gtcataccct tcgcgtacta cccgttcatg 1200 gccgtcgcca tcatcggcgc gccgccggtg cccaagaccg gcgcccggat catgacgcag 1260 tgcgtcgagg acgaccacct gccggcgttc aaggaggaga tcaaggcctt cgacaagtaa 1320                                                                         1320 <210> 2 <211> 439 <212> PRT <213> Homo sapiens <400> 2 Met Gly Phe Ala Val Val Arg Thr Asn Arg Glu Phe Val Arg Pro Ser   1 5 10 15 Ala Ala Thr Pro Ser Ser Gly Glu Leu Glu Leu Ser Ile Ile              20 25 30 Asp Arg Val Val Gly Leu Arg His Leu Val Arg Ser Leu His Ile Phe          35 40 45 Ser Ala Ala Pro Ser Gly Gly Asp Ala Lys Pro Ser Pro Ala Arg      50 55 60 Val Ile Lys Glu Ala Leu Gly Lys Ala Leu Val Asp Tyr Tyr Pro Phe  65 70 75 80 Ala Gly Arg Phe Val Asp Gly Gly Gly Gly Pro Gly Ser Ala Arg Val                  85 90 95 Glu Cys Thr Gly Glu Gly Ala Trp Phe Val Glu Ala Ala Ala Gly Cys             100 105 110 Ser Leu Asp Asp Val Asn Gly Leu Asp His Pro Leu Met Ile Pro Glu         115 120 125 Asp Asp Leu Leu Pro Asp Ala Ala Pro Gly Val His Pro Leu Asp Leu     130 135 140 Pro Leu Met Met Gln Val Thr Glu Phe Ser Cys Gly Gly Phe Val Val 145 150 155 160 Gly Leu Ile Ser Val His Thr Met Ala Asp Gly Leu Gly Ala Gly Gln                 165 170 175 Phe Ile Asn Ala Val Gly Asp Tyr Ala Arg Gly Leu Asp Arg Pro Arg             180 185 190 Val Ser Pro Val Trp Ala Arg Glu Ala Ile Pro Ser Pro Pro Lys Leu         195 200 205 Pro Pro Gly Pro Pro Pro Glu Leu Lys Met Phe Gln Leu Arg His Val     210 215 220 Thr Ale Asp Leu Ser Leu Asp Ser Ile Asn Lys Ala Lys Ser Ala Tyr 225 230 235 240 Phe Ala Ala Thr Gly His Arg Cys Ser Thr Phe Asp Val Ala Ile Ala                 245 250 255 Lys Thr Trp Gln Ala Arg Thr Arg Ala Leu Arg Leu Pro Glu Pro Thr             260 265 270 Ser Arg Val Asn Leu Cys Phe Phe Ala Asn Thr Arg His Leu Met Ala         275 280 285 Gly Ala Ala Ala Trp Pro Ala Pro Ala Ala Gly Gly Asn Gly Gly Asn     290 295 300 Gly Phe Tyr Gly Asn Cys Phe Tyr Pro Val Ser Val Val Ala Glu Ser 305 310 315 320 Gly Ala Val Glu Ala Ala Asp Val Ala Gly Val Val Gly Met Ile Arg                 325 330 335 Glu Ala Lys Ala Arg Leu Pro Ala Asp Phe Ala Arg Trp Ala Val Ala             340 345 350 Asp Phe Arg Glu Asp Pro Tyr Glu Leu Ser Phe Thr Tyr Asp Ser Leu         355 360 365 Phe Val Ser Asp Trp Thr Arg Leu Gly Phe Leu Glu Ala Asp Tyr Gly     370 375 380 Trp Gly Pro Pro Ser Ser Val Valle Pro Phe Ala Tyr Tyr Pro Phe Met 385 390 395 400 Ala Val Ala Ile Ile Gly Ala Pro Pro Val I Lys Thr Gly Ala Arg                 405 410 415 Ile Met Thr Gln Cys Val Glu Asp Asp His Leu Pro Ala Phe Lys Glu             420 425 430 Glu Ile Lys Ala Phe Asp Lys         435

Claims (7)

(a), p rice origin of the amino acid sequence of SEQ ID NO: 2-1 in Kumar Co-A: mono league play transferase (p -Coumaroyl Co-A: monolignol transferase; PMT) for producing a recombinant microorganism expressing the step;
(b) culturing and obtaining the recombinant microorganism produced in the step (a);
(c) contacting the recombinant microorganism obtained in the step (b)
i) coumaric acid having the structure of the following formula (2);
(2)

ii) suspending and reacting in a reaction solution containing tyrosol; And
to obtain the one tee brush (p -coumaroyl tyrosol) into Kumano - (d) p made of a structure of the following [formula 18] from the reaction solution in the reaction step (c);
, P containing -yl tee brush in Kumar (p -coumaroyl tyrosol) produced by:
[Chemical Formula 18]

.
delete The method of claim 1 wherein the (a) step of the recombinant microorganism is Escherichia coli (E.coli), p, characterized in that - one way tee brush in Kumar (p -coumaroyl tyrosol) production. delete delete delete delete

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