KR101726288B1 - Method for producing Phenylacetyl homoserine lactone derivatives - Google Patents

Abstract

The present invention relates to a method for mass production of phenylacetyl homoserine lactone. The production method of phenylacetyl homoserine lactone according to the present invention is characterized in that the yield of phenylacetyl homoserine lactone derivative is increased by using an E. coli expression system and an in vitro enzyme reaction and biotransformation system which have an advantage of being able to rapidly grow at a high concentration in a low- Which is useful for the mass production of phenylacetyl homoserine lactone derivatives.

Description

[0001] The present invention relates to a method for producing phenylacetyl homoserine lactone derivatives,

The present invention relates to an expression vector for biosynthesis of phenylacetyl homoserine lactone, a transformant and a method for producing phenylacetyl homoserine lactone using the same.

Homoserine lactone is a signaling substance between microorganisms. It is a substance used for mutual quorum-sensing signals between microorganisms. It is used as a biofilm between microorganisms and as a signal to suppress propagation. For example, a typical homoserine lactone, acyl-homoserine lactone (AHL), is synthesized as a signal transduction material by an acyl homoserine lactone synthase protein. The synthesized acyl homoserine lactone is freely diffused through the cell membrane during the growth of bacteria and accumulated in the environment outside the cell. When the concentration of the signaling substance accumulated in the outside of the cell reaches a certain concentration due to the increase of the density of the bacteria, the signaling substance enters the cell again and binds to the regulatory protein to promote the expression of the specific gene. And the like. It has also been reported that homoserine lactone is used not only in microorganisms but also in the roots of nearby plants, in the intestines of animals or in the skin and in signal transmission to the host.

These homoserine lactones have different structures depending on the strains, such as partial substituents, and generally have a lactone ring structure bonded with various long fatty acids. Recently, a new type of photosynthetic bacterium, Rhodopseudomonas palustris, a nitrogen-fixing soil bacterium Bradyrhizobium sp. Or Silicibacter pomeroyi, and a specific phenolic acid and lactone ring are combined. Homoserine lactone is reported to be produced.

However, in general strains, production of these compounds is extremely low due to the control of biosynthesis suitable for survival, and production and use of related compounds are lacking in the related research and development. That is, homoserine lactone is a substance that is highly studied and utilized not only as a mutual regulator of microorganisms-microorganisms but also in host plants and animals, but it is difficult to produce materials at a commercial level in general microorganisms.

Under these circumstances, it is necessary to research and develop commercial production of homoserine lactone derivatives conjugated with phenolic acid and lactone rings using E. coli, which has the advantage of rapidly growing at a high concentration in a relatively inexpensive medium.

Korean Patent Laid-Open No. 10-2012-0041115 (disclosed on April 30, 2012)

The present inventors have studied a method for mass production of a phenylacetyl homoserine lactone derivative, which is a useful substance, by using an inexpensive medium, and have found that coenzyme Alaase enzyme, acyl homoserine lactone biosynthesis enzyme, 4-coumarinic acid 3- , And caffeic acid O-methyl transferase can be used to mass-produce phenylacetyl homoserine lactone derivatives through an artificial biosynthetic pathway. Thus, the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing a phenylacetyl homoserine lactone derivative by preparing an expression vector for producing phenylacetyl homoserine and a transformant using the gene encoding the enzyme, and using the same to produce a phenylacetyl homoserine lactone derivative .

In order to solve the above-mentioned necessity, the present invention relates to a method for producing phenylacetyl homoserine lactone (hereinafter, referred to as " expression vector ") for producing phenylacetyl homoserine lactone, which comprises a gene (4CL2nt) encoding a coenzyme A ligation enzyme and a gene (opRpaI) encoding an acyl homoserine lactone biosynthesis enzyme Lt; / RTI >

The present invention also provides an expression vector for producing phenylacetyl homoserine lactone, which further comprises a gene (opTAL) encoding the tyrosine ammonia lyase in the expression vector.

The present invention further provides an expression vector for producing phenylacetyl homoserine lactone, which further comprises a 4-coumarinic acid 3-hydroxylase Sam5 coding gene (sam5) in the above expression vector.

The present invention also provides an expression vector for producing phenylacetyl homoserine lactone further comprising a com gene encoding caffeic acid O-methyltransferase (COMT).

The present invention also provides a transformant for the production of phenylacetyl homoserine lactone wherein the expression vector is introduced.

The present invention also provides a transformant characterized in that the transformant is a mutant strain of tyrosine production.

The present invention also provides a method for producing a transformant, comprising: 1) culturing the transformant in a culture medium; And 2) obtaining phenylacetyl homoserine lactone from the culture broth of step 1); And a method for producing phenylacetyl homoserine lactone.

The present invention also provides a method for producing phenylacetyl homoserine lactone, which comprises adding phenolic acid to the culture medium of step 1).

The present invention also provides a method for producing phenylacetyl homoserine lactone, wherein methionine or S-adenosyl methionine (SAM) is added to the culture medium of step 1).

The present invention also relates to a method for producing (1) a process for treating a coenzyme A ligation enzyme and an acyl homoserine lactone biosynthesis enzyme to a phenolic acid; (2) culturing the treatment liquid of step (1); And (3) obtaining a phenylacetyl homoserine lactone derivative in the culture broth of step (2).

The production method of the phenylacetyl homoserine lactone derivative of the present invention is advantageous in that the yield of the phenylacetyl homoserine lactone derivative can be increased by using an E. coli expression system having an advantage that it can be rapidly cultured at a high concentration in an inexpensive medium, And thus can be usefully used for the mass production of phenylacetyl homoserine lactone derivatives.

FIG. 1 is a graph showing the effect of the 4R2nt expression vector (pET-opRpaI) inserted with the opRpaI gene at the NdeI / XhoI site of the pET28a (+) vector and the 4CL2nt gene inserted at the NdeI / XhoI site of the pET28a (+ his4CL2nt. < / RTI >
Figure 2 shows the results of SDS-PAGE analysis of purified His-tagged opRpaI enzyme (26.7 kDa) and purified His-tagged 4CL2nt enzyme (61.5 kDa).
FIG. 3 shows the results of HPLC and LC / MS analysis of 4-coumarinic acid substrate and enzyme reaction results of the separated opRpaI enzyme and coenzyme A ligation enzyme (4CL2nt).
FIG. 4 shows the results of analysis of caffeic acid substrate and enzyme reaction results of the separated opRpaI enzyme and coenzyme A ligation enzyme (4CL2nt) by HPLC and LC / MS.
FIG. 5 shows the results of analysis of ferulic acid substrate and enzyme reaction results of the separated opRpaI enzyme and coenzyme A ligation enzyme (4CL2nt) by HPLC and LC / MS.
FIG. 6 shows the results of analysis of the results of the reninamic acid substrate and enzyme reaction of the separated opRpaI enzyme and coenzyme A ligation enzyme (4CL2nt) by HPLC and LC / MS.
7 shows a schematic diagram of a production vector (pET-4R) for producing phenylacetyl homoserine lactone containing the coenzyme A ligation enzyme (4CL2nt) and the opRpaI gene.
FIG. 8 shows the results of HPLC analysis of the production yield of each phenylacetyl homoserine lactone by the addition of phenolic acid in E. coli containing the vector (pET-4R) for the production of phenylacetyl homoserine lactone.
A) Cinnamic acid standard (a) and cinnamic acid-added culture (b); B) 4-coumarinic acid standard product (a) and 4-coumaric acid addition culture solution (b); C) Standard product of caffeic acid (a) and culture solution of caffeic acid (b); D) ferulic acid standard (a) and ferulic acid supplemented culture (b);
Peak 1, cinnamic acid; peak 2, p-coumaric acid; peak 3, caffeic acid; peak 4, ferulic acid; peak 5, cinnamoyl-homoserine lactone; peak 6, p-coumaroyl-HSL; peak 7, caffeine-homoserine lactone; peak 8, peruroyl-homoserine lactone.
9 is a graph showing the results of an artificial metabolic pathway vector (pET-1) for producing 4-coumararyol-HSL containing tyrosine ammonia lyase (opTAL), coenzyme A ligation enzyme (4CL2nt) opT4R).
10 shows the results of HPLC analysis of 4-coumararyol-HSL produced in E. coli containing an artificial metabolic pathway vector (pET-opT4R) (*: coumaroyl-homoserine Lactone).
Fig. 11 shows an artificial metabolic pathway vector (pET-opT54R) containing tyrosine ammonia lyase (opTAL), 4-coumarinic acid 3-hydroxylase Sam5 coding gene (co5), coenzyme A ligation enzyme (4CL2nt) Fig.
12 shows the results of HPLC analysis of caffeoyl-HSL production results in E. coli containing the vector pET-opT54R (*: caffeoyl homoserine lactone).
FIG. 13 is a graph showing the activity of tyrosine ammonia lyase (opTAL), 4-coumarinic acid 3-hydroxylase Sam5 coding gene (co5), coenzyme Alaease enzyme (4CL2nt), caffeic acid O- methyltransferase (PET-opT54MR) containing the com gene and the opRpaI gene which encode the COMT gene.
Fig. 14 shows the results of HPLC analysis of feruloyl-HSL production results in E. coli containing the vector pET-opT54MR (*: peroyl homoserine lactone).
FIG. 15 shows the results of confirming the production yields of phenylacetyl homoserine lactone derivatives according to the incubation time of E. coli including pET-opT4R, pET-opT54R and pET-opT54MR.
Figure 16 shows the production of 4-coumaroyl-homoserine lactone in a general E. coli strain (pET-opT4R / C41) or tyrosine high producing strain (pET-opT4R /? COS1) containing the vector pET- Fig.
FIG. 17 is a graph showing the results of culturing a general Escherichia coli strain (pET-opT4R / C41) or a tyrosine high producing strain (pET-opT4R /? COS1) containing pET-opT4R or Met (methionine) (pET-opT4R /? COS1 (PET-opT4R /? COS1 + SAM) and SAM (S-adenosylmethionine) (pET-opT4R /? COS1 + SAM) were added and the amount of p-coumaryol-HSL .
18 shows the results of culturing a general E. coli strain (pET-opT54R / C41) containing pET-opT54R or tyrosine-producing strain (pET-opT54R /? COS1) or Met (methionine) (pET-opT54R / + Met) or SAM (S-Adenosyl methionine) (pET-opT54R / DELTA COS1 + SAM) and then cultured, and then the yield of caffeoyl-HSL was determined.
Fig. 19 shows the results of culturing a general E. coli strain (pET-opT54MR / C41) or tyrosine-producing strain (pET-opT54MR /? COS1) containing pET-opT54MR or Met (methionine) (pET-opT54MR / -Met) or SAM (S-Adenosyl methionine) (pET-opT54MR /? COS1 + SAM) and then cultured.

The present invention relates to a method for producing phenylacetyl homoserine lactone comprising a gene (4CL2nt) encoding a Coenzyme A ligase enzyme and a gene (opRpaI) encoding an acyl-homoserine lactone biosynthesis enzyme Lt; / RTI > expression vector.

The expression vector for the production of phenylacetyl homoserine lactone of the present invention can produce a large amount of phenylacetyl homoserine lactone by performing a series of metabolic processes through an artificial metabolic pathway by bioconversion and gene recombination.

The phenylacetyl homoserine lactone of the present invention means a compound of formula 1 wherein Ra is hydrogen, methoxy or hydroxy, and Rb is hydrogen, methoxy or hydroxy.

[Chemical Formula 1]

The gene coding for the acyl homoserine lactone biosynthesis enzyme is a gene encoding the acyl homoserine lactone biosynthetic enzyme expressed by the ratio of tRNA to amino acid sequence in each of the amino acid sequences in E. coli based on the amino acid sequence (SEQ ID NO: 1) of the acyl homoserine lactone biosynthesis enzyme (RpaI) of Rhodopseudomonas palustris (Codon usage). ≪ / RTI > According to one embodiment of the present invention, the codon usage frequency can be the codon usage frequency provided in the kazusa database (http://www.kazusa.or.jp). The gene has a nucleotide sequence optimized for E. coli expression, and preferably has a polynucleotide represented by SEQ ID NO: 2, or a functionally equivalent property, and has a nucleotide sequence of 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the nucleotide sequence.

Accordingly, the present invention provides an expression vector for producing phenylacetyl homoserine lactone, wherein the acyl homoserine lactone biosynthesis enzyme is encoded by the polynucleotide represented by SEQ ID NO: 2.

The gene coding for the coenzyme A ligation enzyme is selected from the amino acid sequence (SEQ ID NO: 3) of 4-coumaroyl coenzyme A (4CL2) of tobacco (Nicotiana tabacum) Lt; RTI ID = 0.0 > codon usage. ≪ / RTI > In order to improve the expression of genes in the E. coli expression system, the codon usage was determined through an optimization system of E. coli expression by Biona Co., According to one embodiment of the present invention, the codon usage frequency can be the codon usage frequency provided in the kazusa database (http://www.kazusa.or.jp). The gene has a nucleotide sequence optimized for E. coli expression, and preferably has a polynucleotide represented by SEQ ID NO: 4 or a functionally equivalent property. The gene has 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the nucleotide sequence.

Thus, the present invention provides an expression vector for producing phenylacetyl homoserine lactone, wherein said coenzyme A ligation enzyme is encoded by a polynucleotide represented by SEQ ID NO: 4.

The present invention also relates to a gene encoding a coenzyme A ligation enzyme (4CL2nt), a gene coding for an acyl homoserine lactone biosynthesis enzyme (opRpaI), and a gene coding for tyrosine ammonia lyase (opTAL) Thereby providing an expression vector for producing phenylacetyl homoserine lactone.

In one embodiment of the present invention, the genes of the expression vector are preferably sequentially linked in the order of opTAL-4CL2nt-opRpaI, and they are referred to as pET-opT4R vectors in the present invention.

The gene encoding the tyrosine ammonia lyase is based on the amino acid sequence of tyrosine ammonia lyase (TAL) identified in the actinomycetes Saccharothrix espanaensis (KCTC9392) which converts tyrosine into 4-coumaric acid, Can be produced using optimal codon usage according to the ratio of tRNA to each amino acid in E. coli. The gene has a sequence optimized for E. coli expression, and preferably has a polynucleotide of SEQ ID NO: 5, or a functionally equivalent property thereof, and is selected from the group consisting of 90%, 91%, 92%, 93 , 94%, 95%, 96%, 97%, 98%, 99% or more of the nucleotide sequence of the polynucleotide sequence.

Accordingly, the present invention provides an expression vector for producing phenylacetyl homoserine lactone, wherein said tyrosine ammonia ligase is encoded by the polynucleotide of SEQ ID NO: 5.

The vector named pET-opT4R vector can effectively produce phenylacetyl homoserine lactone, preferably coumaroyl-homoserine lactone, through an artificial metabolic pathway.

The present invention also relates to a gene encoding a coenzyme A ligation enzyme (4CL2nt), a gene coding for an acyl homoserine lactone biosynthesis enzyme (opRpaI), a gene encoding opioids such as tyrosine ammonia lyase (opTAL) And an expression vector for the production of phenylacetyl homoserine lactone comprising the enzyme Sam5 (4-coumarate 3-hydroxylase Sam5) encoding gene (sam 5).

In one embodiment of the present invention, the genes of the expression vector are preferably sequentially linked in the order of opTAL-Sam5-4CL2nt-opRpaI, and in the present invention, they are named pET-opT54R vector.

The 4-coumarinic acid 3-hydroxylase Sam5 coding gene is a gene that is synthesized from 4-coumarinic acid 3-hydroxylase Sam5 identified in Saccharothrix espanaensis, which converts 4-coumarinic acid into caffeic acid. 15, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96% or more of the polynucleotide of SEQ ID NO: 15 or a functionally equivalent property thereof. , 97%, 98%, 99% or more of the nucleotide sequence of the polynucleotide sequence.

Accordingly, the present invention provides an expression vector for producing phenylacetyl homoserine lactone, wherein the 4-coumarinic acid 3-hydroxylase Sam5 coding gene is a polynucleotide represented by SEQ ID NO: 15.

The vector named pET-opT54R vector can effectively produce phenylacetyl homoserine lactone, preferably caffeoyl-homoserine lactone, through an artificial metabolic pathway.

The present invention also relates to a gene encoding a coenzyme A ligation enzyme (4CL2nt), a gene encoding an acyl homoserine lactone biosynthetic enzyme (opRpaI), a gene encoding a tyrosine ammonia lyase (opTAL) There is provided an expression vector for producing phenylacetyl homoserine lactone comprising a com gene encoding the enzyme Sam5 coding gene (sam 5) and caffeic acid O-methyltransferase (COMT).

In one embodiment of the present invention, the genes of the expression vector are preferably sequentially connected in the order of opTAL-Sam5-4CL2nt-com-opRpaI, and in the present invention, they are named pET-opT54MR vector.

Com, which encodes the caffeic acid O-methyltransferase (COMT), is an O-methyltransferase of caffeic acid identified in Arabidopsis thaliana, which converts caffeic acid to ferulic acid 17, and preferably has a polynucleotide represented by SEQ ID NO: 17, or a polynucleotide having a functionally equivalent property thereto, and has 90% or 91% sequence identity with the nucleotide sequence of SEQ ID NO: 17, , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology.

Accordingly, the present invention provides an expression vector for producing phenylacetyl homoserine lactone, wherein the com gene encoding the caffeic acid O-methyltransferase (COMT) is a polynucleotide represented by SEQ ID NO: 17 .

The vector named pET-opT54MR vector can effectively produce phenylacetyl homoserine lactone, preferably perroyl-homoserine lactone, through an artificial metabolic pathway.

In the present invention, the phenylacetyl homoserine lactone may include any of its analogs without limitation, and preferably is selected from the group consisting of coumaro homoserine lactone represented by any of the following Chemical Formulas 2 to 5, caffeoyl-homoserine lactone , Perroyl-homoserine lactone, and cinnamoyl homoserine lactone.

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

(3) is caffeoyl-homoserine lactone, (4) is peryloyl-homoserine lactone, and (5) is cinnamoyl-homoserine lactone to be.

The expression vector of the present invention can be produced according to a vector recombinant method known in the art. Any expression vector that can be used in the present invention can be used as long as it is preferably used for expressing an exogenous protein in a prokaryotic or eukaryotic cell. Recombinant vectors for prokaryotic cells are preferred, and recombinant vectors for eukaryotic cells can be used for recombinant vectors for yeast insects or mammalian cells, but it is preferable to use recombinant vectors for yeast. Commercially available vectors for prokaryotic cells include but are not limited to pET vectors (Novagen, Inc., USA), pQE vectors (Qiagen, USA) and pGEX (Pharmacia Biotech Inc., USA) PCT1 (Stratagene, USA), pSG5 (Stratagene, USA), pCMNeo (Clontech, USA), pcDNA3 (InVitrogen, USA) ), PBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), EBO-pSV2- pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and lZD35 (ATCC 37565), and may include promoters and terminators so that they can undergo independent expression control.

The present invention also provides a transformant for producing phenylacetyl homoserine lactone, wherein the expression vector for producing phenylacetyl homoserine lactone is introduced.

The transformant of the present invention may be any system capable of expressing an expression vector for producing a phenylacetyl homoserine lactone derivative, and preferably an E. coli expression system is used. In the present invention, any method known in the art may be used for transformation using an expression vector. Preferably, an expression vector is inserted into E. coli using a method of applying heat shock to an activated cell prepared by chemically treating E. coli. .

According to the embodiment of the present invention, the transformant of the present invention is Escherichia coli C41 (DE3), and an artificial biosynthetic pathway of phenylacetyl homoserine lactone can be established by the introduced expression vector, and phenylacetyl homoserine lactone Can be produced in large quantities.

In addition, according to one embodiment of the present invention, the transformant of the present invention may be a tyrosine-producing mutant strain (? COS1).

The above tyrosine high production mutant strains are characterized in that the tyrAfbr of the nucleotide sequence of SEQ ID NO: 27 (feedback-inhibition of the chorismate mutase / prephenate dehydrogenase gene, tyrA) -resistant, fbr) gene; And aroGfbr [3-deoxy-D-arabino-haptenulosonate-7-phosphate (DAHP-3-deoxy-D- arabinose-heptulosonate-7-phosphate synthase gene consisting of the nucleotide sequence shown in SEQ ID NO: ), a feedback-inhibition-resistant (fbr) gene of a promoter, a trypticase, a synthase, aroG) is inserted into the tyrosine DNA-binding transcriptional repressor regulatory gene region.

The tyrosine high production mutant strain thus produced may preferably be pET-opT4R /? COS1, pET-opT54R /? COS1, pET-opT54MR /? COS1 to which pET-opT4R, pET-opT54R or pET- .

When the mutant strains of high tyrosine production are used, a larger amount of phenylacetyl homoserine lactone can be produced as compared with the production ability of introducing a vector for expression of phenylacetyl homoserine lactone into general E. coli, which is industrially useful.

The present invention also provides a method for producing phenylacetyl homoserine lactone comprising the steps of: 1) culturing a transformant for producing phenylacetyl homoserine lactone in a culture medium; And 2) obtaining phenylacetyl homoserine lactone from the culture broth of step 1); And a method for producing phenylacetyl homoserine lactone.

The method for producing homoserine phenylacetyl homoserine lactone is a method for producing phenylacetyl homoserine lactone by adding phenolic acid as a substrate and biotransformation thereof according to the type of transformant to be used and a method for producing artificial metabolism And a method for producing phenylacetyl homoserine lactone through a route.

Accordingly, the present invention provides a method for producing phenylacetyl homoserine lactone, which comprises adding phenolic acid to the culture medium of step 1).

In the present invention, the phenolic acid can be used as a substrate for producing a phenylacetyl homoserine lactone derivative, and preferably 4-coumarinic acid, caffeic acid, ferulic acid, cinnamic acid and cinnamic acid can be used, At least one selected from the group consisting of acetic acid, caffeic acid, ferulic acid and cinnamic acid can be used.

In an embodiment of the present invention, in the case where the production method is a method for producing phenylacetyl homoserine lactone through biotransformation, the transformant of step 1) is preferably a transformant into which a pET-4R vector has been introduced .

Also, in one embodiment of the present invention, when the production method is a method of producing phenylacetyl homoserine lactone through artificial metabolic pathway construction, the transformant of step 1) is selected from pET-opT4R, pET-opT54R, pET- RTI ID = 0.0 > opT54MR < / RTI >

The culture medium for the transformation of the transformant in the step 1) is preferably a medium for growth of the transformant. For example, in LB, YT, M9 medium or the like.

The protein expression of the transformant cultured in step 1) can be induced by each protein induction method according to the type of the expression vector such as IPTG treatment. After the expression of the protein is induced, further cultivation is performed to obtain phenylacetyl homolog Induces the expression of serine lactone. Additional cultures may be carried out in the presence of, for example, 3 g / L KH ₂ PO _{4 ,} 7.3 g / LK ₂ HPO ₄ , 8.4 g / L MOPS, 2 g / L NH ₄ Cl, 0.5 g / L NaCl, 0.1 ml / / L (NH ₄ ) ₂ SO ₄ , 5 g / L MgSO ₄ and 15 g / L glucose.

The phenylacetyl homoserine lactone can be obtained by separating the compound by HPLC or the like or by isolating the compound by a conventional method known in the art in the step 2).

The transformant of step 1) may preferably be E. coli C41 (DE3), more preferably a tyrosine-producing mutant strain (DELTA COS1).

Also, the present invention provides a method for producing phenylacetyl homoserine lactone, wherein methionine or S-adenosyl methionine (SAM) is added to the culture medium of step 1).

By adding the methionine or S-adenosylmethionine to the medium, the transformant can produce a significantly increased amount of phenylacetyl homoserine lactone than that produced in the general E. coli and tyrosine production mutant strains. Therefore, the addition of a simple additive can bring about a remarkable improvement in the production efficiency.

The present invention also provides a method for producing a coenzyme A ligation enzyme comprising the steps of: 1) treating a coenzyme A ligation enzyme and an acyl homoserine lactone biosynthesis enzyme to a phenolic acid; 2) culturing the treatment liquid of step 1); And 3) obtaining phenylacetyl homoserine lactone in the culture broth of step 2).

In step 1), Coenzyme Alaase enzyme is an enzyme that can be identified in tobacco (Nicotiana tabacum) and converts phenolic acid into phenyl coenzyme A. According to an embodiment of the present invention, the enzyme is 4-Coumarate CoA Ligase 2 from Nicotiana tabacum (4CL2nt).

According to an embodiment of the present invention, the acyl homoserine lactone biosynthesis enzyme may be one isolated from Rhodopseudomonas palustris (RpaI).

In the step 2), the reaction is preferably carried out at a temperature of 30 ° C in a solution containing CoA, ATP and SAM (S-adenosyl methionine).

The phenylacetyl homoserine lactone can be obtained by separating the compound by HPLC or the like or by isolating the compound by a conventional method known in the art in the step 3). The isolated phenylacetyl homoserine lactone derivative may preferably be a homoserine lactone derivative bonded with a phenolic acid and a lactone ring. According to an embodiment of the present invention, 4-coumaroyl-homoserine lactone (formula 2) is prepared by using 4-coumarinic acid as a substrate for producing a phenylacetyl homoserine lactone derivative, caffeoyl-homoserine The cinnamoyl-homoserine lactone (Formula 5) can be produced using perroyl-homoserine lactone (Formula 4) and cinnamic acid using lactone (Formula 3) and ferulic acid.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

[ Example  1] in vitro opRpaI  Identification of the homoserine lactone synthesis activity of the gene

Example  1-1. Acyl homoserine  A gene encoding the lactone biosynthesis enzyme ( opRpaI  Gene) and coenzyme A Liege  Enzyme (4 CL2nt  Gene)

Based on the amino acid sequence (SEQ ID NO: 1) of the acyl homoserine lactone biosynthesis enzyme (opRpaI) of Rhodopseudomonas palustris for improving the gene expression in the E. coli expression system based on the frequency of codon usage revealed for E. coli, A new gene (SEQ ID NO: 2) was determined using an optimized program of bioneer using optimal codon usage according to the ratio of tRNA to each of the sequences, and the 4-coumarin of Nicotiana tabacum Based on the amino acid sequence (SEQ ID NO: 3) of 1 coenzyme A (4CL2nt), the optimum gene for the optimal protein expression was determined using the optimal codon usage according to the tRNA ratio to each amino acid sequence in E. coli of the US DNA2.0 SEQ ID NO: 4) was determined.

Example  1-2. 4 CL2nt  And opRpaI  Generation of recombinant vectors containing genes

On the basis of the nucleotide sequence (SEQ ID NO: 2) determined in Example 1-1, opRpaI gene synthesis was performed by Bioneer Co., Ltd. so as to have a restriction enzyme NdeI position and a backward XhoI position in front of the nucleotide sequence. The nucleotide sequence thus prepared is shown in SEQ ID NO: 6.

Based on the nucleotide sequence (SEQ ID NO: 4) determined in Example 1-1 with reference to DNA2.0, the 4CL2nt gene was synthesized so as to have a restriction enzyme NcoI site in front of the nucleotide sequence and a HindIII site in the back thereof. The nucleotide sequence thus prepared is shown in SEQ ID NO: 7.

The opRpaI gene having a restriction enzyme NdeI position and an XhoI position at the back of the nucleotide sequence was digested with restriction enzymes NdeI and XhoI, and pET-28a (+) vector, an E. coli expression vector, The recombinant vector pET-opRpaI was prepared by linking the NdeI and XhoI sites of opRpaI with the NdeI and XhoI sites of the pET-28a (+) vector by digesting with restriction enzymes NdeI and XhoI, and the schematic diagram thereof is shown in FIG. .

In addition, 4TL2nt-F and 4CL2nt-R primers were prepared by using pET-4CL2-Nt vector containing a 4CL2nt gene having a restriction enzyme NcoI site and a HindIII site at the back of the base sequence prepared according to DNA2.0 as a template DNA PCR product was obtained by pairing PCR product. The PCR product was inserted into T-blunt vector using T-Blunt PCR Cloning method provided by Solgent Corporation to construct His4CL2nt TA vector. The pET-28a (+) vector, an Escherichia coli expression vector, was digested with restriction enzymes NdeI and XhoI to obtain NdeI and XhoI digests of 4CL2nt and pET-28a (+) digestion sites of 4CL2nt. ) Vector, the recombinant vector pET-his4CL2nt was constructed, and the schematic diagram thereof is shown in FIG. 1 (b).

Using the prepared vector, the expression of the acyl homoserine lactone biosynthesis gene and the 4-coumaroyl coenzyme A ligation gene was confirmed and separated, and SDS-PAGE was shown in FIG.

FIG. 2 (a) shows the size of the pET-opRpaI vector, M denotes the size marker, 1 denotes the solution passing through the Ni-NTA resins, 2 denotes the solution passing through the washing buffer, SDS-PAGE results of each elution buffer solution are shown.

Fig. 2 (b) shows the result of SDS-PAGE of the elution buffer solution in Ni-NTA resins. Fig. 2 (b) shows the size marker using pET-his4CL2nt vector.

Example  1-3. In vitro opRpaI  Enzymatic Phenylacetyl  Synthesis of homoserine lactone compound

In order to convert phenolic acid into acyl coenzyme A, 4CL2nt, which was expressed and purified in the state of N-terminal His-tagged soluble protein in Example 1-2, was used, and in acyl coenzyme A, homoserine lactone In the same manner as in Example 1-2, opRpaI, an acyl homoserine lactone synthase enzyme which was expressed and purified by N-terminal His-tagged soluble protein was used.

Using 4-coumarinic acid, caffeic acid, ferulic acid, and cinnamic acid purchased from Sigma (USA) as reference substrates, the 4CL2nt and opRpaI enzymes were reacted together at 30 ° C for 1 hour Respectively.

[Table 1]

The same amount of ethyl acetate as that of the reaction solution was added to extract, centrifuged at 12000 rpm for 2 minutes, and the supernatant ethyl acetate layer was concentrated. After the concentrated extract was dissolved in methanol, 20ul of the solution was analyzed by HPLC under the following conditions. All reaction extracts were YMC J'sphere ODS-H80, 150x4.6 mm I.D. The column was used to carry out HPLC analysis of CH3CN-H2O (0.05% TFA) mobile phase at a rate of 1 ml / min from 10% to 100% for 25 minutes.

4-coumarinic acid, caffeic acid, ferulic acid and cinnamic acid were used as substrates and the results are shown in Figs. 3 to 6. Fig. FIG. 3 shows the result of using 4-coumarinic acid as a substrate, FIG. 4 shows the results of using caffeic acid as a substrate, FIG. 5 shows results using ferulic acid as a substrate, and FIG. 6 shows results using cinnamic acid as a substrate. In Figures 3 to 6, * means newly formed phenylacetyl homoserine lactone.

As shown in FIG. 3, peaks having the same UV spectrum at the time (Rt, 8.0 minutes) faster than 4-coumarinic acid were identified in each of the reaction solutions using 4-coumarinic acid as a substrate. LC / (M / z 248.14 [M + H] +). The analysis confirmed that the opRpaI enzyme produced the 4-coumaroyl-homoserine lactone compound represented by the following formula (2).

(2)

4, a peak having the same UV spectrum at a time (Rt, 7.0 minutes) earlier than that of caffeic acid was identified in the reaction solution using caffeic acid as a substrate. LC / MS analysis showed that the peak of the same molecular weight m / z 264.00 [M + H] +). From the analysis results, it was confirmed that the opRpaI enzyme produced the caffeoyl-homoserine lactone compound represented by the following formula (3).

(3)

As shown in Fig. 5, peaks having the same UV spectrum at the time (Rt, 8.2 minutes) earlier than that of ferulic acid were confirmed in the reaction solution using ferulic acid as a substrate. LC / MS analysis showed that the peaks having the same molecular weight m / z 278.19 [M + H] +). From the analysis results, it was confirmed that the opRpaI enzyme produced the peruroyl-homoserine lactone compound represented by the following formula (4).

[Chemical Formula 4]

6, peaks having the same UV spectrum at a time (Rt, 11.0 minutes) earlier than that of cinnamic acid were confirmed in a reaction solution using cinnamic acid as a substrate. LC / MS analysis showed that the peaks having the same molecular weight m / z 232.06 [M + H] +). From the results, it was confirmed that the opRpaI enzyme produced the cinnamoyl-homoserine lactone compound represented by the following formula (5).

[Chemical Formula 5]

From the above analysis results, it can be seen that in vitro, the opRpaI enzyme is selectively degraded from each added substrate cininamic acid, 4-coumarinic acid, caffeic acid, and ferulic acid by 4-coumaroyl homoserine lactone, caffeoyl homoserine lactone, It was confirmed that a phenylacetyl homoserine lactone compound such as homoserine lactone could be produced.

[ Example  2] pET -4R Production of caffeoyl-homoserine lactone and perroyl-homoserine lactone of transformed microorganisms

Example  2-1. 4 CL2nt - opRpaI  A recombinant vector containing the gene ( pET -4R) production

The pET-his4CL2nt TA PCR product of Example 1-2 was digested with restriction enzymes NdeI and SpeI, and the pET-opRpaI TA product was digested with restriction enzymes SpeI and XhoI. In addition, the pET-28a (+) vector was digested with restriction enzymes NdeI and XhoI, and the product of the prepared two genes was ligated to a vector and transformed into E. coli.

At this time, the NdeI site of the pET-his4CL2nt TA PCR product was ligated to the NdeI site of the cleaved pET-28a (+) vector, and the SpeI site of the pET-his4CL2nt TA PCR product was ligated to the SpeI site of the pET-opRpaI TA product. Two genes were synthesized by linking the XhoI site of the pET-opRpaI TA product to the XhoI site of the pET-28a (+) vector and inserted into one expression vector pET-28a (+).

The 4CL2nt and opRpaI genes of pET-4R thus constructed were constructed so as to independently regulate their expression with the respective T7 promoter and T7 terminator, which was named pET-4R. A schematic diagram of the prepared vector is shown in Fig.

Example  2-2. pET Synthesis of Homoserine Lactone Compound of -4R Transforming Microorganism

PET-4R / C41 was prepared by transforming E. coli prepared in Example 2-1 into Escherichia coli. To the medium containing the Escherichia coli was added a standard product of cinnamic acid, 4-coumarinic acid, caffeic acid, and ferulic acid Were each treated with 30 mg / L and cultured.

A portion of the culture was filtered (Sartorius Minisart RC 4, 0.2 um) and 20 ul of the solution was subjected to HPLC analysis. More specifically, HPLC analysis was carried out using a YMC Jsphere ODS-H80, 150 x 4.6 mm ID, (YMC, Japan) column and CH3CN-H2O (JTBaker, USA) (0.05% TFA; Sigma-Aldrich, USA) mobile phase was performed at a rate of 1 ml / min from 10% to 100% for 25 minutes. Cinnamic acid, 4-coumarinic acid, caffeic acid, and ferulic acid were purchased from Sigma (USA) and analyzed, and the results of the analysis are shown in FIG.

As shown in Fig. 8, peaks having the same UV spectrum at a time point earlier than the phenolic acid added in the culture medium of pET-4R-transformed Escherichia coli pET-4R / C41 were identified. As a result of LC / MS analysis, when the cinnamic acid was added, the same molecular weight (m / z 232 [M + H] +) as in Chemical Formula 5 was confirmed, and the transformed strain was found to have a cinnamoyl- Serine lactone compound (Fig. 8A). (M / z 248 [M + H] < + >) was confirmed by adding 4-coumarinic acid to the compound of Formula 2, whereby the transformed strain was found to contain the coumaroyl-homoserine lactone compound (Fig. 8B). When caffeic acid was added, the same molecular weight (m / z 264 [M + H] +) as in Chemical Formula 3 was confirmed, and the transformed strain produced the caffeoyl-homoserine lactone compound represented by Chemical Formula 3 (Fig. 8C). When ferulic acid was added, the same molecular weight (m / z 278 [M + H] +) as in Chemical Formula 4 was confirmed, whereby the transformed strain produced the peruroyl-homoserine lactone compound represented by Chemical Formula 4 (Fig. 8D).

In addition, 30 mg / L of cinnamic acid, 4-coumarinic acid, caffeic acid, and ferulic acid were added as a substrate in the transformed strain and cultured for 36 hours. HPLC was performed to evaluate the conversion by pET-4R / C41 . These results indicate that pET-4R / C41 can be used as a homoserine lactone compound, which is a homocellin lactone compound, which is a homocellin lactone, a catechol-homoserine lactone, a catechol-homoserine lactone, a cinnamoyl-homoserine lactone, And the conversion rate was 34, 47, 72, and 46%, respectively.

[ Example  3] 4- Kumaroto  Production of homoserine lactone biosynthesis artificial metabolic pathway

Example  3-1. optal -4 CL2nt - opRpaI  A recombinant vector containing the gene ( pET -opT4R) production

The enzyme that converts tyrosine into 4-coumaric acid is based on the amino acid sequence of tyrosine ammonia lyase (TAL) identified in the actinomycetes Saccharothrix espanaensis (KCTC9392) The nucleotide sequence (opTAL, SEQ ID NO: 5) was synthesized using the optimal codon usage according to the tRNA ratio. The enzyme that converts 4-coumaric acid into 4-coumaroyl Coenzyme A is a base of nicotiana tabacum cinnamate / 4-coumarate (CoA ligase) (4CL2nt, SEQ ID NO: 4) was synthesized. Also, the above-described nucleotide sequence (opRpaI, SEQ ID NO: 2), which is an enzyme for converting 4-coumaroyl coenzyme A into a 4-coumaroyl homoserine lactone compound, was synthesized and used.

In order to construct a modular artificial pathway for production of 4-coumaroyl homoserine lactone, a vector was constructed by sequentially connecting three genes, opTAL, 4CL2nt and RpaI, which was named pET-opT4R and its schematic diagram is shown in FIG. .

Specifically, a PCR reaction was performed using a pET-optal vector as a template DNA as a pair of opTAL-F and pET-CPac primers. PCR was performed with nPfu-special DNA polymerase for 28 cycles of annealing at 60 ° C for 30 seconds and extension at 72 ° C for 2 minutes and 30 seconds to obtain pET-opTAL TA PCR product.

Also, the pET-his4CL2nt TA PCR product was obtained by performing PCR as described above using the pET-his4CL2nt vector prepared in Example 1-2 as a pair of pET-NPac and pET-CSpe primers as a template DNA.

Based on the pET28a (+) vector, PCR was performed to have a SpeI site in the vicinity of NdeI site by about 400 bp. The prepared vector had T7 promoter and RBS between SpeI and NdeI sites, XhoI site after NdeI, XhoI has a T7 terminator behind the spot. The vector was digested with NdeI and XhoI, and the synthesized pGEM-opRpaI was digested with restriction enzymes NdeI and XhoI, and the NdeI and XhoI digests of the digested vector were ligated to the NdeI and XhoI digests of the digested gene. The truncated gene was ligated with a vector.

The pET-opTAL TA PCR product was digested with restriction enzymes NdeI and PacI, and the pET-his4CL2nt TA PCR product was digested with restriction enzymes PacI and SpeI. The pET-opRpaI TA product was digested with restriction enzymes SpeI and XhoI. The pET-28a (+) vector was digested with restriction enzymes NdeI and XhoI, and the PCR products of the prepared three genes were ligated to the vector.

The NdeI site of the pET-opTAL TA PCR product was ligated to the NdeI site of the cleaved pET-28a (+) vector. The PacI site of the pET-opTAL TA PCR product and the PacI site of the pET-his4CL2nt TA PCR product were ligated, The SpeI site of the pET-his4CL2nt TA PCR product was ligated to the SpeI site of the pET-opRpaI TA product. PET-opT4R was constructed by inserting three genes synthesized by linking the XhoI site of the pET-opRpaI TA product to the XhoI site of the pET-28a (+) vector into one expression vector pET-28a (+) . Specifically, the C41 (DE3) cell was treated with CaCl2 to induce instability in the cell membrane structure to allow the DNA to enter well. A pET-opT4R gene was added to C41 (DE3) Escherichia coli and heat shocked PET-opT4R was transformed into E. coli.

The prepared optal, 4CL2nt and opRpaI genes of pET-opT4R were constructed so as to independently regulate expression with T7 promoter and T7 terminator, respectively. The schematic diagram is shown in FIG. 9, and the nucleotide sequences of the primers used above are shown in Table 2 .

 [Table 2]

Example  3-2. pET - opT4R  The 4- Kumaroto  Homoserine lactone compound synthesis

The pET-opT4R / C41 Escherichia coli prepared in Example 3-1 was inoculated in 50 ml of LB medium (50 μg / l kanamycin) and cultured at 37 ° C. to a DO _{600 of} 0.6. After 10 min of cold shock Protein expression was induced with 1 mM IPTG. Then, the cells were further cultured at 26 DEG C for 6 hours, and cells were transferred to 30 ml of SM medium (modified synthetic medium; 15 g / l; 50 g / l Kan, 1 mM IPTG) Lt; / RTI >

A portion of the culture liquid filtering (Sartorius Minisart RC 4, 0.2um ) to the solution 20ul SunFire ™ C18 column (250 × 4.6 mm, 5 μm; Waters, USA) using a column _{CH 3 CN-H 2 O (} JTBaker , USA) (0.05% TFA; Sigma-Aldrich, USA). The mobile phase was subjected to HPLC analysis at a rate of 1 ml / min from 10% to 60% for 25 minutes. The 4-coumaric acid standard product was purchased and analyzed by Sigma (USA), and the analysis results are shown in FIG.

As shown in Fig. 10, in the culture solution of E. coli strain pET-opT4R / C41 transformed with pET-opT4R, a peak with the same UV spectrum appeared at a time (Rt, 12.2 minutes) faster than 4-coumarinic acid. As a result of LC / MS analysis, it was confirmed that this was a substance having the same molecular weight (m / z 248.11 [M + H] +) as that of the formula 2, whereby the transformed strain was transformed from 4-coumarinic acid - coumaroyl - homoserine lactone compound.

It was additionally confirmed whether the E. coli strain pET-opT4R / C41 transformed with pET-opT4R could overproduce a 4-coumaroyl-homoserine lactone compound. pET-opT4R / C41 was cultured three times under the same culture conditions as above. The amount of the product produced after the incubation time (1, 4, 15, 25, 40 hours) was measured by HPLC, and the result is shown in FIG.

As shown in Fig. 15, it was confirmed that pET-opT4R / C41 produced a 4-coumaroyl homoserine lactone compound at a high level of up to 16 mg / L in a culture medium for 40 hours.

[ Example  4] Production of caffeine-homoserine lactone through biosynthetic artificial metabolism pathway

Example  4-1. Cafe oil - for homoserine lactone production optal - Sam5 -4 CL2nt - RpaI  A recombinant vector containing the gene ( pET - opT54R Production

To produce caffeine oil - homoserine lactone through a biosynthetic artificial metabolic pathway, a modular artificial pathway was constructed. More specifically, a gene encoding 4-coumarate 3-hydroxylase Sam5 (4-coumarate 3-hydroxylase) identified in Saccharothrix espanaensis, which converts 4-coumarinic acid into caffeic acid No. 15) was additionally introduced into the previously prepared vector pET-opT4R. The synthetic Sam5 nucleotide sequence containing the restriction enzyme sequence is shown in SEQ ID NO: 16. Finally, a vector was constructed by sequentially connecting the four genes, opTAL, Sam5, 4CL2nt, and opRpaI.

Specifically, the pET-Sam5 vector was used as a template DNA, and PCR amplification was performed with a pair of pET-NPac and pET-CPac primers to obtain pET-Sam5 TA PCR product. Based on the pET28a (+) vector, it was designed to have a PacI site about 800 bp before the NdeI site. The constructed vector had the T7 promoter and RBS between the sites of PacI and NdeI and the HindIII site after NdeI , And a T7 terminator behind the HindIII digits. The vector was digested with PacI, the pET-opT4R vector of Example 3-1 was digested with restriction enzyme PacI, and the digested gene was ligated with the vector. The vector thus prepared was named pET-opT54R. The pET-opT54R vector consists of opTAL, Sam5, 4CL2nt, and opRpaI genes, each of which has a T7 promoter and a T7 terminator to be independently regulated in expression. The constructed vector is named pET-opT54R and the schematic diagram thereof is shown in Fig.

Example  4-2. pET - opT54R  Synthesis of caffeic oil homoserine lactone compound of transformed microorganism

In order to confirm that pET-opT54R produced in Example 4-1 efficiently produced caffeine homoserine lactone compound from 4-coumarinic acid through the artificial metabolic pathway, Escherichia coli was transformed with the pET-opT54R vector, Respectively.

pET-opT54R was transformed into E. coli to produce pET-opT54R / C41. The E. coli prepared was cultured in the same manner as in Example 3-2. A portion of the culture was filtered (Sartorius Minisart RC 4, 0.2 um) and 20 ul of the solution was subjected to HPLC analysis. HPLC analysis was performed using a column of SunFire ™ C18 column (250 × 4.6 mm, 5 μm; Waters, USA) and CH ₃ CN-H ₂ O (JTBaker, USA) (0.05% TFA; Sigma-Aldrich, USA) The mobile phase was run from 10% to 60% at a rate of 1 ml / min for 25 minutes. The caffeic acid standard product was purchased from Sigma (USA) and analyzed. The results are shown in FIG.

As shown in Fig. 12, peaks having the same UV spectrum at a time (Rt, 9.9 min) earlier than that of caffeic acid were identified in the culture of E. coli strain pET-opT54R / C41 transformed with pET-opT54R. This was analyzed by LC / MS and the same molecular weight (m / z 264.34 [M + H] +) as in Chemical Formula 3 was confirmed. Namely, it was confirmed that the transformed strain can effectively produce the caffeoyl-homoserine lactone compound represented by the formula (3) through the artificial metabolic pathway.

In order to confirm the amount of caffeine homoserine lactone produced by the transformant strain, pET-opT54R / C41 was cultured three times in the same manner as the above culture conditions. The amount of production according to the culture time (1, 4, 15, 25, 40 hours) was measured by HPLC and the results are shown in Fig.

As shown in FIG. 15, it was confirmed that the caffeine homoserine lactone compound was effectively produced at a level of 5 mg / L at the maximum for 15 hours.

[ Example  5] Production of Peruroyl Homoserine Lactone via Biosynthetic Artificial Metabolism Pathway

Example  5-1. Production of peroxylated homoserine lactone was performed using a recombinant vector ( pET - opT54MR Production

A modular artificial pathway was constructed to produce peroxylated homoserine lactone through a biosynthetic artificial metabolic pathway. More specifically, it has been found that caffeic acid O-methyltransferase (TKA), which has been identified in Arabidopsis thaliana, which converts caffeic acid to ferulic acid for the construction of a modular artificial pathway for the production of peroylo-homoserine lactone (SEQ ID NO: 17) coding for Caffeic acid O-methyltransferase (COMT) was used. The com base sequence containing the restriction enzyme site is shown in SEQ ID NO: 18. Finally, five genes consisting of opTAL, Sam5, 4CL2nt, com and RpaI were sequentially linked to construct a vector.

More specifically, a pET-COM TA PCR product was obtained by PCR amplification using pET-NSpe and pET-CSpe primer pairs and pET-COM vector as template DNA. The vector was constructed with a T7 promoter and RBS between SpeI and NdeI sites, HindIII site after NdeI, and a HindIII site after NdeI. It has a T7 terminator behind the HindIII site. The vector was digested with SpeI, the pET-opT54R vector of Example 4-1 was digested with restriction enzyme SpeI, and the digested gene was ligated with the vector. The vector thus constructed was designated as pET-opT54MR, which was constituted by opTAL, Sam5, 4CL2nt, com and opRpaI genes, and was independently regulated through T7 promoter and T7 terminator. The prepared vector was named pET-opT54MR vector, and the schematic diagram thereof is shown in Fig.

Example  5-2. pET - opT54MR  Synthesis of Peruroyl Homoserine Lactone Compound in Transgenic Microorganism

The pET-opT54MR prepared in Example 5-1 was transformed into E. coli to produce pET-opT54MR / C41, and the E. coli thus prepared was cultured in the same manner as in Example 3-2, and the product was analyzed . Ferulic acid standard was purchased from Sigma (USA) and analyzed, and the results of the analysis are shown in Fig.

As shown in Fig. 14, peaks having the same UV spectrum at a time (Rt, 12.7 minutes) earlier than ferulic acid in a culture medium of E. coli strain pET-opT54MR / C41 transformed with pET-opT54MR were confirmed. This was analyzed by LC / MS to confirm the same molecular weight (m / z 278.47 [M + H] +) as in Chemical formula 4. That is, it was confirmed that the transformed strain produced the feruloyl-homoserine lactone compound represented by the formula (4).

Further, pET-opT54MR / C41 was cultured three times in the same manner as the above culture conditions to confirm the yield of peroyl homoserine lactone by pET-opT54MR / C41. The amount of the product produced in accordance with the culture time (1, 4, 15, 25, 40 hours) was measured and analyzed by HPLC. The result is shown in FIG.

As shown in FIG. 15, it was confirmed that pET-opT54MR / C41 produced a feruloyl homoserine lactone compound at a maximum level of 5.6 mg / L in a culture medium for 25 hours.

[ Example  6] tyrosine-producing strain Phenyl  Increased productivity of acetylated homoserine lactone

Example  6-1. Production of Tyrosine High Production Strain

(3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase) and tyrA (chorismate mutase / prephenate dehydrogenase gene) feedback-suppression resistance -inhibition resistance, fbr).

The gene design of aroGfbr and tyrAfbr was followed according to the method described in Lutke-Eversloh (Appl Environ Microbiol 2005, 71: 7224-7228) and Stephanopoulos (Appl Microbiol Biotechnol 2007, 75: 103-110) (Kang SY et al., Microb Cell Fact 2012, 11: 153). AroGfbr and FRT-neo-FRT fragments were used as templates to produce pET-AG (Kang SY et al., Microb Cell Fact 2012, 11: 153) and FRT-neo-FRT fragments (Gene Bridges).

(SEQ ID NO: 19) and IF-C1 primer (SEQ ID NO: 20), both of which contain both the tyrAfbr and aroGfbr gene cassettes in which the RBS and T7 promoters precede each gene. Other fragments containing FRT-neo-FRT were amplified using the IF-FRT1 primer (SEQ ID NO: 21) and the IF-FRT2 primer (SEQ ID NO: 22). The two fragments were ligated between the SpeI sites of pUC19 using an In-fusion kit (Clontech Laboratories, Inc., USA) to construct pUC-AGFRT. A 5.9-kb insertion PCR product was generated using pUC-AGFRT as a template, and a tyrRr primer (SEQ ID NO: 23) and an Inf-tyrRfAG primer (SEQ ID NO: 24) were used. The gene cassette was inserted between tyrR genes for gene insertional inactivation and was performed by conventional methods using RED / ET recombination including Quick & Easy E. coli Gene deletion kit (Gene Bridges) .

In order to confirm that gene insertion was inactivated properly, the cells were transformed with the FLP recombinant expression plasmid 707-FLPe (Gene Bridges) to remove the kanamycin selection marker from the chromosome. The insert-inactivated tyrosine high production mutant strain (hereinafter referred to as "ΔCOS1") constructed through the above process was identified as tyrA-F primer (SEQ ID NO: 25) and aroG-R primer (SEQ ID NO: 26).

The sequences of the primers used in the tyrosine-producing strain are summarized in Table 3 below.

[Table 3]

Example  6-2. Production of homoserine lactone by using high tyrosine production strain

PET-opTT4R /? COS1, pET-opT54R /? COS1, pET-opT54R, pET-opT54R and pET-opT54MR prepared in Example were introduced into the tyrosine-producing E. coli DELTA COS1 prepared in Example 6-1, -OPT54MR /? COS1. These vectors are vectors capable of producing 4-coumaroyl homoserine lactone, caffeine homoserine lactone, and peryloyl homoserine lactone, respectively, and thus it was confirmed whether or not their yields were increased in? COS1 into which they were introduced.

The E. coli thus prepared were cultured and analyzed in the same manner as in Example 3-2, and the area value of each homoserine lactone compound peak was compared with the area value of 4-coumaroyl homoserine lactone standard at a wavelength of 300 nm by HPLC Production was measured.

Production of 4-coumaroyl homoserine lactone

As shown in Fig. 16, in the same time zone (Rt, 12.2 min) as the 4-coumaroyl homoserine lactone peak produced in the culture of pET-opT4R / C41 of Example 3-2 in the culture solution of pET-opT4R / Lt; RTI ID = 0.0 > UV < / RTI > Thus, it was confirmed that pET-opT4R /? COS1 produces a 4-coumaroyl homoserine lactone compound. The yield of the 4-coumaroyl homoserine lactone compound thus produced was compared with the yield of pET-opT4R / C41 E. coli. The results are shown in FIG.

As shown in Fig. 17, in the culture solution of pET-opT4R /? COS1, 4-coumaroyl homoserine lactone compound was produced at a level of 61 mg / L, which is three times higher than that of pET-opT4R / C41. Therefore, it was confirmed that the 4-coumaroyl homoserine lactone compound can be produced in a large amount in the strain producing high tyrosine.

Production of caffeine oil homoserine lactone

Similarly, in the culture solution of pET-opT54R /? COS1, a peak having the same UV spectrum in the same time zone (Rt, 9.9 minutes) as the caffeine homoserine lactone peak produced in the culture of pET-opT54R / C41 of Example 4-2 Respectively. It was confirmed that pET-opT54R / COS1 produced caffeine homoserine lactone compound. The yield of the caffeine homoserine lactone compound thus produced was compared with the yield of pET-opT54R / C41 E. coli. The results are shown in FIG.

As shown in FIG. 18, in the culture solution of pET-opT54R /? COS1, caffeine homoserine lactone compound was produced at a level of 30 mg / L, which is about 6 times higher than that of pET-opT54R / C41. Therefore, it was confirmed that caffeine homoserine lactone compound can be produced in a large amount in the strain producing high tyrosine.

Peroxylated homoserine lactone production

In addition, a peak having the same UV spectrum in the same time zone (Rt, 12.7 minutes) as the peruroyl homoserine lactone peak produced in the culture of pET-opT54MR / C41 of Example 5-2 in the culture solution of pET-opT54MR / Respectively. From this, it was confirmed that pET-opT54MR /? COS1 produces a peruroyl homoserine lactone compound. However, as shown in Fig. 19, it was confirmed that, in the culture solution of pET-opT54MR / DELTA COS1, the feruloyl homoserine lactone compound in a very small amount was specifically produced. However, the production of cumaric acid, which is a metabolic pathway intermediate, is remarkably increased, and it is considered that the production of peroyl homoserine lactone, which is the final product, is less than that of pET-opT54MR / C41 due to the metabolic flow problem.

Example  7. Methionine ( 메티오 로 ) And SAM (S- Adenosyl 메티오 로 )of In addition  Increased homoserine lactone productivity

(SAM) or methionine was added to the culture medium of pET-opT4R /? COS1, pET-opT54R /? COS1, pET-opT54MR /? COS1 prepared in Example 6-2, The productivity of serine lactone was compared.

The pET-opT4R /? COS1, pET-opT54R /? COS1, and pET-opT54MR /? COS1 prepared in Example 6-2 were cultured three times in the same manner as in the above cultivation conditions, and SAM or methionine 1 mM final concentration and incubated for 25 hours each. A portion of each culture was filtered (Sartorius Minisart RC 4, 0.2 μm) and 20 μl of the solution was used for HPLC analysis on a SunFire ™ C18 column (250 × 4.6 mm, 5 μm; Waters, USA) (JTBaker, USA) (0.05% TFA; Sigma-Aldrich, USA) The mobile phase was analyzed from 10% to 60% for 25 minutes at a rate of 1 ml / min. To compare the yield of 4-coumaroyl homoserine lactone, caffeine homoserine lactone, and peryloyl homoserine lactone, the area value of the peak of the 4-coumaroyl homoserine lactone compound shown at a wavelength of 300 nm of the culture solution was Mu] m < / RTI > homoserine lactone.

4-coumaroyl homoserine lactone productivity increase

As shown in Fig. 17, in the culture solution to which SAM was added, 93 mg / L of 4-coumaroyl homoserine lactone was produced. This is about 6.2 times higher than that of pET-opT4R / C41 without SAM. It is also about 2.3 times higher than the culture of pET-opT4R / △ COS1 without SAM. In the culture medium containing methionine (Met), it was confirmed that 143 mg / L of 4-coumaroyl homoserine lactone was produced. This was the highest production of 4 - coumaroyl homoserine lactone, which was 1.5 times higher than that of SAM - supplemented culture.

Increase productivity of caffeine homoserine lactone

As shown in Fig. 18, it was confirmed that caffeoyl homoserine lactone of 51 mg / L was produced in the culture supplemented with SAM. This amount is about 12.8 times higher than that of pET-opT54R / C41 without SAM or methionine, which is about 1.7 times higher than that of pET-opT54R / ΔCOS1 culture. Methionine (Met) supplemented with 50 mg / L of caffeoyl homoserine lactone produced similar increase in SAM production.

Increase productivity of homoserine lactone perroyl

As shown in Example 6, the final yield of peryloyl homoserine lactone was lower in pET-opT54MR /? COS1, a tyrosine-producing strain, than pET-opT54MR / C41. However, as shown in Fig. 19, in the medium supplemented with SAM or methionine (Met), the yield of peroyl homoserine lactone by pET-opT54MR /? COS1 markedly increased. In particular, about 10 mg / L of feruloyl homoserine lactone was produced in the culture medium supplemented with methionine, and the yield of peroyl homoserine lactone was increased to about 1.7 times as compared with the culture solution of pET-opT54MR / C41.

From the above results, it was confirmed that phenylacetyl homoserine lactone can be efficiently produced using a vector including 4CL2nt and opRpaI genes, and optionally opTAL, sam5, and com. It was also confirmed that the production of phenylacetyl homoserine lactone can be significantly increased by introducing the vector into the tyrosine-producing strain? COS1 or by simply adding S-adenosylmethionine or methionine to the culture medium thereof.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for producing Phenylacetyl homoserine lactone derivatives <130> 1-16p-1 <150> KR 10-2014-0091762 <151> 2014-07-21 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> RPA1 amino acid <400> 1 Met Gln Val His Val Ile Arg Arg Glu Asn Arg Ala Leu Tyr Ala Gly   1 5 10 15 Leu Leu Glu Lys Tyr Phe Arg Ile Arg His Gln Ile Tyr Val Val Glu              20 25 30 Arg Gly Trp Lys Glu Leu Asp Arg Pro Asp Gly Arg Glu Ile Asp Gln          35 40 45 Phe Asp Thr Glu Asp Ala Val Tyr Leu Leu Gly Val Asp Asn Asp Asp      50 55 60 Ile Val Ala Gly Met Arg Met Val Pro Thr Thr Ser Pro Thr Leu Leu  65 70 75 80 Ser Asp Val Phe Pro Gln Leu Ala Leu Ala Gly Pro Val Arg Arg Pro                  85 90 95 Asp Ala Tyr Glu Leu Ser Arg Ile Phe Val Val Pro Arg Lys Arg Gly             100 105 110 Glu His Gly Gly Pro Arg Ala Glu Ala Val Ile Gln Ala Ala Ala Met         115 120 125 Glu Tyr Gly Leu Ser Ile Gly Leu Ser Ala Phe Thr Ile Val Leu Glu     130 135 140 Thr Trp Trp Leu Pro Arg Leu Val Asp Gln Gly Trp Lys Ala Lys Pro 145 150 155 160 Leu Gly Leu Pro Gln Asp Ile Asn Gly Phe Ser Thr Thr Ala Val Ile                 165 170 175 Val Asp Val Asp Asp Asp Ala Trp Val Gly Ile Cys Asn Arg Arg Ser             180 185 190 Val Pro Gly Pro Thr Leu Glu Trp Arg Gly Leu Glu Ala Ile Arg Arg         195 200 205 His Ser Leu Pro Glu Phe Gln Val Ser Ser     210 215 <210> 2 <211> 657 <212> DNA <213> Artificial Sequence <220> <223> novel op RPA1 gene <400> 2 atgcaagttc atgttattcg tcgcgagaac cgcgcgctct atgccggttt gctagaaaaa 60 tatttccgta ttcgtcacca aatctacgtc gtagaacgcg gctggaaaga attggatcgg 120 ccggatggac gagaaattga tcagttcgat accgaagacg cggtgtatct tttaggtgtc 180 gacaatgatg atattgtagc tggcatgcgt atggtgccga ccacgtctcc gacacttctg 240 agcgatgttt ttccacaact cgcgctggct ggtccagtaa gaaggcctga tgcctatgag 300 ttatctcgga tatttgtggt tcctcgtaag cgcggagagc atgggggccc acgtgctgag 360 gcagtgatac aggcggccgc aatggaatac ggtttatcga ttggcctgtc agcctttact 420 atcgtactgg aaacttggtg gctgccgcga ctggttgacc agggctggaa agcaaaacct 480 ttaggtctgc ctcaggatat caatggattt tccaccacag cagtcatcgt tgatgttgac 540 gatgatgctt gggtcggtat ttgtaataga cgcagtgttc ccggacccac gttagaatgg 600 agagggttag aagcaatacg gcgtcatagt cttccggaat ttcaggtgat ttcataa 657 <210> 3 <211> 542 <212> PRT <213> Artificial Sequence <220> <222> 4CL2nt amino acid <400> 3 Met Glu Lys Asp Thr Lys Gln Val Asp Ile Ile Phe Arg Ser Ser Lys Leu   1 5 10 15 Pro Asp Ile Tyr Ile Pro Asn His Leu Pro Leu His Ser Tyr Cys Phe              20 25 30 Glu Asn Ile Ser Glu Phe Ser Ser Arg Pro Cys Leu Ile Asn Gly Ala          35 40 45 Asn Lys Gln Ile Tyr Thr Tyr Ala Asp Val Glu Leu Asn Ser Arg Lys      50 55 60 Val Ala Ala Gly Leu His Lys Gln Gly Ile Gln Pro Lys Asp Thr Ile  65 70 75 80 Met Ile Leu Leu Pro Asn Ser Pro Glu Phe Val Phe Ala Phe Ile Gly                  85 90 95 Ala Ser Tyr Leu Gly Ala Ile Ser Thr Met Ala Asn Pro Leu Phe Thr             100 105 110 Pro Ala Glu Val Val Lys Gln Ala Lys Ala Ser Ser Ala Lys Ile Ile         115 120 125 Val Thr Gln Ala Cys His Val Asn Lys Val Lys Asp Tyr Ala Phe Glu     130 135 140 Asn Asp Val Lys Ile Ile Cys Ile Asp Ser Ala Pro Glu Gly Cys Leu 145 150 155 160 His Phe Ser Val Leu Thr Gln Ala Asn Glu His Asp Ile Pro Glu Val                 165 170 175 Glu Ile Gln Pro Asp Val Val Ala Leu Pro Tyr Ser Ser Gly Thr             180 185 190 Thr Gly Leu Pro Lys Gly Val Met Leu Thr His Lys Gly Leu Val Thr         195 200 205 Ser Val Ala Gln Gln Val Asp Gly Glu Asn Pro Asn Leu Tyr Ile His     210 215 220 Ser Glu Asp Val Met Leu Cys Val Leu Pro Leu Phe His Ile Tyr Ser 225 230 235 240 Leu Asn Ser Val Leu Leu Cys Gly Leu Arg Val Gly Ala Ala Ile Leu                 245 250 255 Ile Met Gln Lys Phe Asp Ile Val Ser Phe Leu Glu Leu Ile Gln Arg             260 265 270 Tyr Lys Val Thr Ile Gly Pro Phe Val Pro Ile Val Leu Ala Ile         275 280 285 Ala Lys Ser Pro Met Val Asp Asp Tyr Asp Leu Ser Ser Val Arg Thr     290 295 300 Val Met Ser Gly Ala Ala Pro Leu Gly Lys Glu Leu Glu Asp Thr Val 305 310 315 320 Arg Ala Lys Phe Pro Asn Ala Lys Leu Gly Gln Gly Tyr Gly Met Thr                 325 330 335 Glu Ala Gly Pro Val Leu Ala Met Cys Leu Ala Phe Ala Lys Glu Pro             340 345 350 Phe Glu Ile Lys Ser Gly Ala Cys Gly Thr Val Val Arg Asn Ala Glu         355 360 365 Met Lys Ile Val Asp Pro Lys Thr Gly Asn Ser Leu Pro Arg Asn Gln     370 375 380 Ser Gly Glu Ile Cys Ile Arg Gly Asp Gln Ile Met Lys Gly Tyr Leu 385 390 395 400 Asn Asp Pro Glu Ala Thr Ala Arg Thr Ile Asp Lys Glu Gly Trp Leu                 405 410 415 Tyr Thr Gly Asp Ile Gly Tyr Ile Asp Asp Asp Asp Glu Leu Phe Ile             420 425 430 Val Asp Arg Leu Lys Glu Leu Ile Lys Tyr Lys Gly Phe Gln Val Ala         435 440 445 Pro Ala Glu Leu Glu Ala Leu Leu Leu Asn His Pro Asn Ile Ser Asp     450 455 460 Ala Ala Val Val Pro Met Lys Asp Glu Gln Ala Gly Glu Val Pro Val 465 470 475 480 Ala Phe Val Val Arg Ser Asn Gly Ser Thr Ile Thr Glu Asp Glu Val                 485 490 495 Lys Asp Phe Ile Ser Lys Gln Val Ile Phe Tyr Lys Arg Ile Lys Arg             500 505 510 Val Phe Phe Val Asp Ala Ile Pro Lys Ser Pro Ser Gly Lys Ile Leu         515 520 525 Arg Lys Asp Leu Arg Ala Lys Leu Ala Ala Gly Leu Pro Asn     530 535 540 <210> 4 <211> 1632 <212> DNA <213> Artificial Sequence <220> <223> novel 4CL2nt gene <400> 4 atggagaaag acacgaagca agttgacatc atttttcgct cgaaactgcc ggacatttac 60 attccgaatc atctgccgct gcatagctac tgcttcgaga acatttctga attttctagc 120 cgtccgtgtc tgattaacgg tgccaataaa cagatctata cgtacgcgga cgtcgagttg 180 aacagccgta aggtcgcagc gggtctgcac aagcaaggca tccagcctaa agataccatc 240 atgattctgt tgccaaattc tccggagttt gtgtttgcgt ttatcggcgc aagctacctg 300 ggtgcgatta gcacgatggc aaatccgctg tttaccccgg ctgaggttgt taaacaagca 360 aaagccagca gcgcgaagat catcgtgacc caagcatgcc acgtcaacaa agttaaggac 420 tatgccttcg aaaatgacgt caagatcatt tgcatcgata gcgcgcctga aggttgtctg 480 catttcagcg ttctgacgca ggctaacgaa cacgatattc cggaagttga gattcagccg 540 gacgatgtgg tggccctgcc gtactccagc ggtaccaccg gcctgccgaa aggcgttatg 600 ctgacccaca agggcctggt gacgagcgtc gcccagcagg tcgatggtga aaacccgaac 660 ctgtacatcc acagcgaaga tgttatgctg tgtgttctgc cactgttcca catctattcc 720 ctgaacagcg tcctgctgtg cggcctgcgt gtgggcgctg ccattttgat tatgcagaag 780 tttgacattg tcagcttctt ggaactgatc caacgctaca aggtgacgat cggtccgttc 840 gtcccgccga ttgttttggc cattgcaaaa agcccaatgg tggatgacta tgacctgtcg 900 agcgtgcgta ccgtgatgtc cggtgcagcg ccgctgggca aagagctgga ggataccgtt 960 cgtgcgaagt ttccgaatgc gaaactgggt caaggctacg gtatgactga agcaggtccg 1020 gtgctggcga tgtgcttggc gttcgcgaaa gagccgttcg aaatcaaaag cggtgcgtgc 1080 ggtaccgtgg tgcgtaatgc tgaaatgaaa attgtggatc cgaaaaccgg caacagcctg 1140 ccgcgcaacc agagcggtga gatttgtatt cgcggtgacc agattatgaa gggctacctg 1200 aatgacccgg aggccactgc gcgtacgatc gacaaagagg gttggctgta taccggcgac 1260 atcggttata tcgatgacga cgacgagctg ttcatcgttg atcgcctgaa agagttgatt 1320 aagtacaagg gtttccaagt tgcgcctgcg gaactggagg ctctgctgtt gaatcatccg 1380 aacattagcg atgcagcagt cgttccgatg aaggatgagc aggcgggtga agttccggtc 1440 gcgtttgttg tgcgtagcaa cggcagcacg atcaccgagg atgaggtaaa ggatttcatt 1500 tccaaacaag tcatcttcta taagcgtatc aagcgtgtgt ttttcgtcga tgcaatcccg 1560 aaaagcccgt ccggtaagat cctgcgcaaa gacttgcgtg cgaagctggc ggcaggtctg 1620 ccgaattagt aa 1632 <210> 5 <211> 1533 <212> DNA <213> Artificial Sequence <220> <223> optal gene <400> 5 atgacccagg tggttgaacg ccaggccgat cgcctgagta gtcgtgaata cttagctcgc 60 gtcgttcgta gtgccggctg ggatgcgggc ctaacctctt gtacagatga agaaattgtt 120 cgcatgggcg cgtcagcccg caccatcgag gaatatttaa aaagtgataa accgatttat 180 ggtttaaccc aaggcttcgg cccgctggta ctgtttgatg cggatagcga attagaacag 240 ggtggtagcc tgattagcca tctgggcacc ggtcagggcg cgccgctggc gccggaagtg 300 agtcgtttaa ttctgtggct gcgtattcaa aacatgcgca aaggttatag cgccgttagc 360 ccggttttct ggcaaaaact ggcagaccta tggaataaag gctttacccc ggcaattccg 420 cgtcatggta ccgtttccgc ctcgggtgat ctgcaaccgc tggcgcatgc cgcgctggca 480 ttcaccggcg tgggtgaagc gtggacccgc gatgcagatg gccgctggag caccgttccg 540 gctgttgatg ccctggcagc gctgggtgcc gaaccgtttg attggcctgt ccgcgaagca 600 ctggcgtttg ttaatggcac cggagccagc ctggcggttg ctgttttaaa tcatcgttct 660 gccctgcgcc tggttcgcgc gtgtgcggta ctgagcgcac gcctggcgac cctgctgggc 720 gcaaatccgg aacattatga cgttggtcat ggcgttgccc gcggtcaggt tggccagctg 780 accgcggcgg aatggattcg tcagggcctg ccacgtggta tggtgcgcga tggaagccgt 840 ccgttgcagg aaccttatag ccttcgctgc gctccgcagg ttctaggcgc tgttctggat 900 cgctggacg gtgcgggtga cgtgctggcc cgcgaagttg atggttgcca ggataaccct 960 attacctacg aaggtgaatt gctgcatggc ggtaacttcc atgccatgcc ggttggtttt 1020 gcaagtgatc agattggtct ggcgatgcac atggcggcct acctggctct acgccagctg 1080 ggcctgctgg ttagcccggt aaccaatggt gatttaccac cgatgctgac cccgcgtgcc 1140 ggccgtggtg cgggtcttgc tggcgtccag atttctgcca ccagcttcgt ttctcgtatt 1200 cgccaactgg ttttcccggc gtctctgacc accctgccga ccaacggttg gaatcaagac 1260 catgtaccga tggcactgaa tggcgctaat agcgttttcg aagcactgga actgggttgg 1320 ttaaccgttg gaagcctggc ggtgggcgtt gcacagctgg cggcgatgac cggtcatgcg 1380 gctgaagggg tttgggcaga actggcaggc atttgcccgc cgttagatgc cgaccgtccg 1440 ctgggtgcgg aagttcgcgc agcccgtgat ctgctgagcg cgcacgctga tcagctgttg 1500 gtggacgaag ccgatggtaa agactttggc taa 1533 <210> 6 <211> 666 <212> DNA <213> Artificial Sequence <220> <223> op RPA1 gene comprising restriction enzyme <400> 6 catatgcaag ttcatgttat tcgtcgcgag aaccgcgcgc tctatgccgg tttgctagaa 60 aaatatttcc gtattcgtca ccaaatctac gtcgtagaac gcggctggaa agaattggat 120 cggccggatg gacgagaaat tgatcagttc gataccgaag acgcggtgta tcttttaggt 180 gtcgacaatg atgatattgt agctggcatg cgtatggtgc cgaccacgtc tccgacactt 240 ctgagcgatg tttttccaca actcgcgctg gctggtccag taagaaggcc tgatgcctat 300 gagttatctc ggatatttgt ggttcctcgt aagcgcggag agcatggggg cccacgtgct 360 gaggcagtga tacaggcggc cgcaatggaa tacggtttat cgattggcct gtcagccttt 420 actatcgtac tggaaacttg gtggctgccg cgactggttg accagggctg gaaagcaaaa 480 cctttaggtc tgcctcagga tatcaatgga ttttccacca cagcagtcat cgttgatgtt 540 gacgatgatg cttgggtcgg tatttgtaat agacgcagtg ttcccggacc cacgttagaa 600 tggagagggt tagaagcaat acggcgtcat agtcttccgg aatttcaggt gatttcataa 660 ctcgag 666 <210> 7 <211> 1638 <212> DNA <213> Artificial Sequence <220> <223> 4CL2nt gene comprising restriction enzyme <400> 7 ccatggagaa agacacgaag caagttgaca tcatttttcg ctcgaaactg ccggacattt 60 acattccgaa tcatctgccg ctgcatagct actgcttcga gaacatttct gaattttcta 120 gccgtccgtg tctgattaac ggtgccaata aacagatcta tacgtacgcg gacgtcgagt 180 tgaacagccg taaggtcgca gcgggtctgc acaagcaagg catccagcct aaagatacca 240 tcatgattct gttgccaaat tctccggagt ttgtgtttgc gtttatcggc gcaagctacc 300 tgggtgcgat tagcacgatg gcaaatccgc tgtttacccc ggctgaggtt gttaaacaag 360 caaaagccag cagcgcgaag atcatcgtga cccaagcatg ccacgtcaac aaagttaagg 420 actatgcctt cgaaaatgac gtcaagatca tttgcatcga tagcgcgcct gaaggttgtc 480 tgcatttcag cgttctgacg caggctaacg aacacgatat tccggaagtt gagattcagc 540 cggacgatgt ggtggccctg ccgtactcca gcggtaccac cggcctgccg aaaggcgtta 600 tgctgaccca caagggcctg gtgacgagcg tcgcccagca ggtcgatggt gaaaacccga 660 acctgtacat ccacagcgaa gatgttatgc tgtgtgttct gccactgttc cacatctatt 720 ccctgaacag cgtcctgctg tgcggcctgc gtgtgggcgc tgccattttg attatgcaga 780 agtttgacat tgtcagcttc ttggaactga tccaacgcta caaggtgacg atcggtccgt 840 tcgtcccgcc gattgttttg gccattgcaa aaagcccaat ggtggatgac tatgacctgt 900 cgagcgtgcg taccgtgatg tccggtgcag cgccgctggg caaagagctg gaggataccg 960 ttcgtgcgaa gtttccgaat gcgaaactgg gtcaaggcta cggtatgact gaagcaggtc 1020 cggtgctggc gatgtgcttg gcgttcgcga aagagccgtt cgaaatcaaa agcggtgcgt 1080 gcggtaccgt ggtgcgtaat gctgaaatga aaattgtgga tccgaaaacc ggcaacagcc 1140 tgccgcgcaa ccagagcggt gagatttgta ttcgcggtga ccagattatg aagggctacc 1200 tgaatgaccc ggaggccact gcgcgtacga tcgacaaaga gggttggctg tataccggcg 1260 acatcggtta tatcgatgac gacgacgagc tgttcatcgt tgatcgcctg aaagagttga 1320 ttaagtacaa gggtttccaa gttgcgcctg cggaactgga ggctctgctg ttgaatcatc 1380 cgaacattag cgatgcagca gtcgttccga tgaaggatga gcaggcgggt gaagttccgg 1440 tcgcgtttgt tgtgcgtagc aacggcagca cgatcaccga ggatgaggta aaggatttca 1500 tttccaaaca agtcatcttc tataagcgta tcaagcgtgt gtttttcgtc gatgcaatcc 1560 cgaaaagccc gtccggtaag atcctgcgca aagacttgcg tgcgaagctg gcggcaggtc 1620 tgccgaatta gtaagctt 1638 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> opTAL-F primer <400> 8 catatgaccc aggtggttga acgcc 25 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pET-Cpac primer <400> 9 ttaattaatg cgccgctaca gggcgcgtcc 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pET-Npac primer <400> 10 ttaattaatc gccgcgacaa tttgcgacgg 30 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> pET-Cspe primer <400> 11 actagttcct cctttcagca aaaaacccct c 31 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <222> 4CL2nt-F primer <400> 12 catatggaga aagacacgaa gcaagttgac atc 33 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <222> 4CL2nt-R primer <400> 13 aagcttacta attcggcaga cctgccgcc 29 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pET-Nspe <400> 14 actagtaggt tgaggccgtt gagcaccgcc 30 <210> 15 <211> 1539 <212> DNA <213> Artificial Sequence <220> <223> sam <400> 15 atgaccatca cgtcacctgc gccggcgggc cggctcaaca acgtccgccc gatgacgggc 60 gaggagtacc tggaatcact gcgggacggc cgagaggtct acatctacgg cgagcgggtc 120 gacgacgtca ccacgcacct ggccttccgc aacagcgtgc gctccatcgc gcggctctac 180 gacgtgctgc acgacccggc gtccgaaggt gtgctgcggg tgcccaccga caccggcaac 240 ggcgggttca cccacccgtt cttcaagacc gcccggtcgt cggaggacct ggtcgccgcg 300 cgcgaggcca tcgtcggctg gcagcggctg gtgtacgggt ggatgggccg caccccggac 360 tacaaggcgg cgttcttcgg cacgctcgac gccaacgccg agttctacgg gccgttcgag 420 gccaacgccc gccgctggta ccgcgacgcc caggaacggg tgctgtactt caaccacgcg 480 atcgtgcacc cgccggtcga ccgggaccgg cccgccgacc ggaccgccga catctgcgtg 540 cacgtggagg aggagaccga cagcgggttg atcgtctccg gcgccaaggt ggtcgcgacc 600 ggctccgcga tgaccaacgc gaacctcatc gcgcactacg ggcttccggt gcgggacaag 660 aagttcggcc tggtgttcac ggtcccgatg aactcgcccg gcctcaagct catctgccgc 720 acctcctacg agctgatggt cgcgacgcag ggctcgccct tcgactaccc gctgtcgagc 780 cggctcgacg agaacgactc gatcatgatc ttcgaccggg tgctggtgcc ctgggagaac 840 gtgttcatgt acgacgcggg cgcggccaac tccttcgcca ccgggtcagg cttcctcgaa 900 cgcttcacct tccacggctg cacccgcctc gcggtcaagc tggacttcat cgccggctgc 960 gtcatgaagg cggtggaggt caccggcacc acgcacttcc ggggcgtgca ggcgcaggtc 1020 ggcgaagtgc tcaactggcg cgacgtgttc tggggcctgt ccgacgcgat ggccaagtcg 1080 ccgaactcgt gggtcggcgg ctcggtgcag ccgaacctca actacgggct cgcgtaccgc 1140 accttcatgg gcgtgggcta cccgcgcatc aaggagatca tccagcagac cctcggcagc 1200 gggttgatct acctgaactc ctcggccgcc gactggaaga accccgacgt ccgcccgtac 1260 ctcgaccgct acctgcgcgg ctcgcggggc atccaggcga tcgaccgggt caagctgctg 1320 aagctgctgt gggacgcggt cggcaccgag ttcgccggcc ggcacgagct ctacgagcgc 1380 aactacggcg gcgaccacga gggcatccgg gtgcagaccc tgcaggcgta ccaggcgaac 1440 ggccaagccg ccgcgctcaa gggtttcgcc gagcagtgca tgtccgagta cgacctcgac 1500 ggctggacga ggcccgacct gatcaacccc ggcacctga 1539 <210> 16 <211> 1547 <212> DNA <213> Artificial Sequence <220> 230 <400> 16 catatgacca tcacgtcacc tgcgccggcg ggccggctca acaacgtccg cccgatgacg 60 ggcgaggagt acctggaatc actgcgggac ggccgagagg tctacatcta cggcgagcgg 120 gtcgacgacg tcaccacgca cctggccttc cgcaacagcg tgcgctccat cgcgcggctc 180 tacgacgtgc tgcacgaccc ggcgtccgaa ggtgtgctgc gggtgcccac cgacaccggc 240 aacggcgggt tcacccaccc gttcttcaag accgcccggt cgtcggagga cctggtcgcc 300 gcgcgcgagg ccatcgtcgg ctggcagcgg ctggtgtacg ggtggatggg ccgcaccccg 360 gactacaagg cggcgttctt cggcacgctc gacgccaacg ccgagttcta cgggccgttc 420 gaggccaacg cccgccgctg gtaccgcgac gcccaggaac gggtgctgta cttcaaccac 480 gcgatcgtgc acccgccggt cgaccgggac cggcccgccg accggaccgc cgacatctgc 540 gtgcacgtgg aggaggagac cgacagcggg ttgatcgtct ccggcgccaa ggtggtcgcg 600 accggctccg cgatgaccaa cgcgaacctc atcgcgcact acgggcttcc ggtgcgggac 660 aagaagttcg gcctggtgtt cacggtcccg atgaactcgc ccggcctcaa gctcatctgc 720 cgcacctcct acgagctgat ggtcgcgacg cagggctcgc ccttcgacta cccgctgtcg 780 agccggctcg acgagaacga ctcgatcatg atcttcgacc gggtgctggt gccctgggag 840 aacgtgttca tgtacgacgc gggcgcggcc aactccttcg ccaccgggtc aggcttcctc 900 gaacgcttca ccttccacgg ctgcacccgc ctcgcggtca agctggactt catcgccggc 960 tgcgtcatga aggcggtgga ggtcaccggc accacgcact tccggggcgt gcaggcgcag 1020 gtcggcgaag tgctcaactg gcgcgacgtg ttctggggcc tgtccgacgc gatggccaag 1080 tcgccgaact cgtgggtcgg cggctcggtg cagccgaacc tcaactacgg gctcgcgtac 1140 cgcaccttca tgggcgtggg ctacccgcgc atcaaggaga tcatccagca gaccctcggc 1200 agcgggttga tctacctgaa ctcctcggcc gccgactgga agaaccccga cgtccgcccg 1260 tacctcgacc gctacctgcg cggctcgcgg ggcatccagg cgatcgaccg ggtcaagctg 1320 ctgaagctgc tgtgggacgc ggtcggcacc gagttcgccg gccggcacga gctctacgag 1380 cgcaactacg gcggcgacca cgagggcatc cgggtgcaga ccctgcaggc gtaccaggcg 1440 aacggccaag ccgccgcgct caagggtttc gccgagcagt gcatgtccga gtacgacctc 1500 gacggctgga cgaggcccga cctgatcaac cccggcacct gaagctt 1547 <210> 17 <211> 1092 <212> DNA <213> Artificial Sequence <220> <223> com <400> 17 atgggttcaa cggcagagac acaattaact ccggtgcaag tcaccgacga cgaagctgcc 60 ctcttcgcca tgcaactagc cagtgcttcc gttcttccga tggctttaaa atccgcctta 120 gagcttgacc ttcttgagat tatggccaag aatggttctc ccatgtctcc taccgagatc 180 gcttctaaac ttccgaccaa aaatcctgaa gctccggtca tgctcgaccg tatcctccgt 240 cttcttacgt cttactccgt cttaacctgc tccaaccgta aactttccgg tgatggcgtt 300 gaacggattt acgggcttgg tccggtttgc aagtatttga ccaagaacga agatggtgtt 360 tccattgctg ctctttgtct tatgaaccaa gacaaggttc tcatggaaag ctggtaccat 420 ttgaaggatg caattcttga tggtgggatt ccattcaaca aggcttatgg aatgagcgcg 480 ttcgagtacc acgggactga ccctagattc aacaaggtct ttaacaatgg aatgtctaac 540 cattccacaa tcaccatgaa gaagattctt gagacctata agggttttga aggattgact 600 tctttggttg atgttggtgg tggcattggt gctacactca aaatgattgt ctccaagtac 660 cctaatctta aaggcatcaa ctttgatctc ccacatgtca tcgaagatgc tccttctcat 720 cctggtattg agcatgttgg aggagatatg tttgtaagtg tccctaaagg tgatgccata 780 ttcatgaagt ggatatgtca tgactggagt gacgaacatt gcgtgaaatt cttgaagaac 840 tgctacgagt cacttccaga ggatggaaaa gtgatattag cagagtgtat acttccagag 900 acaccagact caagcctctc aaccaaacaa gtagtccatg tcgattgcat tatgttggct 960 cacaatcccg gaggcaaaga acgaaccgag aaagagtttg aggcattagc caaagcatca 1020 ggcttcaagg gcatcaaagt tgtctgcgac gcttttggtg ttaaccttat tgagttactc 1080 aagaagctct aa 1092 <210> 18 <211> 1099 <212> DNA <213> Artificial Sequence <220> <223> com comprising restriction enzymes seqence <400> 18 catatgggtt caacggcaga gacacaatta actccggtgc aagtcaccga cgacgaagct 60 gccctcttcg ccatgcaact agccagtgct tccgttcttc cgatggcttt aaaatccgcc 120 ttagagcttg accttcttga gattatggcc aagaatggtt ctcccatgtc tcctaccgag 180 atcgcttcta aacttccgac caaaaatcct gaagctccgg tcatgctcga ccgtatcctc 240 cgtcttctta cgtcttactc cgtcttaacc tgctccaacc gtaaactttc cggtgatggc 300 gttgaacgga tttacgggct tggtccggtt tgcaagtatt tgaccaagaa cgaagatggt 360 gtttccattg ctgctctttg tcttatgaac caagacaagg ttctcatgga aagctggtac 420 catttgaagg atgcaattct tgatggtggg attccattca acaaggctta tggaatgagc 480 gcgttcgagt accacgggac tgaccctaga ttcaacaagg tctttaacaa tggaatgtct 540 aaccattcca caatcaccat gaagaagatt cttgagacct ataagggttt tgaaggattg 600 acttctttgg ttgatgttgg tggtggcatt ggtgctacac tcaaaatgat tgtctccaag 660 taccctaatc ttaaaggcat caactttgat ctcccacatg tcatcgaaga tgctccttct 720 catcctggta ttgagcatgt tggaggagat atgtttgtaa gtgtccctaa aggtgatgcc 780 atattcatga agtggatatg tcatgactgg agtgacgaac attgcgtgaa attcttgaag 840 aactgctacg agtcacttcc agaggatgga aaagtgatat tagcagagtg tatacttcca 900 gagacaccag actcaagcct ctcaaccaaa caagtagtcc atgtcgattg cattatgttg 960 gctcacaatc ccggaggcaa agaacgaacc gagaaagagt ttgaggcatt agccaaagca 1020 tcaggcttca agggcatcaa agttgtctgc gacgcttttg gtgttaacct tattgagtta 1080 ctcaagaagc tctaagctt 1099 <210> 19 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> IF-N1 primer <400> 19 cggtacccgg ggatcactag ttgatcggcg cgagatttaa tcgccgcgac aat 53 <210> 20 <211> 48 <212> DNA <213> Artificial Sequence <220> IF-C1 primer <400> 20 gtacca <210> 21 <211> 46 <212> DNA <213> Artificial Sequence <220> IF-FRT1 primer <400> 21 actagtttaa ttaaccctca ctaaaggggg ccgcgaagtt cctatt 46 <210> 22 <211> 54 <212> DNA <213> Artificial Sequence <220> IF-FRT2 primer <400> 22 cgactctaga ggatcactag taatacgact cactataggg ctcgaggaag ttcc 54 <210> 23 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> tyrRr primer <400> 23 atcaggcata ttcgcgctta ctcttcgttc ttcttctgac tcagaccata taatacgact 60 cactataggg ctc 73 <210> 24 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Inf-tyrRfAG primer <400> 24 gtcatatcat catattaatt gttctttttt caggtgaagg ttcccatgcg tactagtcgt 60 tctaccatcg acacc 75 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> tyrA-F primer <400> 25 ccatggttgc tgaattgacc gcattacg 28 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> aroG-R primer <400> 26 aagcttaacc acgacgcgct ttcacagc 28

Claims (23)

(4CL2nt) encoding the coenzyme A ligation enzyme represented by SEQ ID NO: 4, and a gene (opRpaI) encoding the acyl homoserine lactone biosynthesis enzyme represented by SEQ ID NO: 2, the phenylacetyl homoserine lactone Expression vector for production:
[Chemical Formula 1]

Wherein Ra is hydrogen, methoxy or hydroxy, and Rb is hydrogen, methoxy or hydroxy.
delete delete The expression vector for producing phenylacetyl homoserine lactone according to claim 1, further comprising a gene (opTAL) encoding the tyrosine ammonia lyase represented by SEQ ID NO: 5.
5. The vector according to claim 4, wherein the expression vector is a gene in which the gene is linked in the order of opTAL-4CL2nt-opRpaI.
delete 5. The expression vector for producing phenylacetyl homoserine lactone according to claim 4, further comprising the 4-coumaric acid 3-hydroxylase Sam5 coding gene (sam 5) represented by SEQ ID NO:
8. The vector according to claim 7, wherein the gene is linked in the order of opTAL-Sam5-4CL2nt-opRpaI.
delete The expression vector for producing phenylacetyl homoserine lactone according to claim 7, further comprising a com gene coding for caffeic acid O-methyltransferase (COMT) represented by SEQ ID NO: 17.
delete 11. The vector according to claim 10, wherein the genes are linked in the order of opTAL-Sam5-4CL2nt-com-opRpaI.
12. The pharmaceutical composition according to any one of claims 1, 4, 5, 7, 8, 10, and 12, wherein the phenylacetyl homoserine lactone is represented by the formula (2) An expression vector for producing phenylacetyl homoserine lactone, which is at least one selected from the group consisting of homoserine lactone, caffeoyl-homoserine lactone, peryloyl-homoserine lactone and cinnamoyl homoserine lactone;
(2)

,
(3)

,
[Chemical Formula 4]

,
[Chemical Formula 5]

.
A transformant for producing phenylacetyl homoserine lactone, wherein the expression vector of any one of claims 1, 4, 5, 7, 8, 10, and 12 is introduced.
15. The transformant according to claim 14, wherein the transformant is a mutant of tyrosine production.
The tyrosine production mutant strain according to claim 15, wherein the tyrosine production mutant strain comprises a tyrAfbr comprising the nucleotide sequence of SEQ ID NO: 27 (feedback inhibition of chorismate mutase / prephenate dehydrogenase gene, tyrA) Feedback-inhibition-resistant (fbr) gene]; And aroGfbr [3-deoxy-D-arabino-haptenulosonate-7-phosphate (DAHP-3-deoxy-D- arabinose-heptulosonate-7-phosphate synthase gene consisting of the nucleotide sequence shown in SEQ ID NO: wherein the feedback-inhibition-resistant (fbr) gene of the promoter of the promoter is inserted into the tyrosine DNA-binding transcriptional repressor regulatory gene region.
1) culturing the transformant of claim 14 in a culture medium; And
2) obtaining phenylacetyl homoserine lactone from the culture broth of step 1); &Lt; / RTI &gt;
18. The method for producing phenylacetyl homoserine lactone according to claim 17, wherein phenol acid is added to the culture medium of step 1).
The method for producing phenylacetyl homoserine lactone according to claim 18, wherein the phenolic acid is at least one selected from the group consisting of 4-coumarinic acid, caffeic acid, ferulic acid and cinnamic acid.
18. The method according to claim 17, wherein the transformant of step 1) is a mutant strain of tyrosine production.
18. The method for producing phenylacetyl homoserine lactone according to claim 17, wherein methionine or S-adenosyl methionine (SAM) is added to the culture medium of step 1).
18. The pharmaceutical composition according to claim 17, wherein the phenylacetyl homoserine lactone is at least one selected from the group consisting of 4-coumaroyl homoserine lactone, caffeoyl-homoserine lactone, and peryloyl-homoserine lactone, Production method.
1) treating a coenzyme A lasease enzyme and an acyl homoserine lactone biosynthesis enzyme to a phenolic acid;
2) culturing the treatment liquid of step 1); And
3) A method of producing phenylacetyl homoserine lactone represented by the following Formula 1, which comprises the step of obtaining phenylacetyl homoserine lactone in the culture medium of Step 2)
[Chemical Formula 1]

Wherein Ra is hydrogen, methoxy or hydroxy, and Rb is hydrogen, methoxy or hydroxy.

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