KR102076214B1 - Piperonal synthase and method for producing piperonal using it - Google Patents

Abstract

The present invention relates to a piperonal synthase capable of producing piperonal by biological methods, and to 3,4-(methylenedioxy)cinnamic acid (3,4-MDCA) hydratase-lyase PnMCHL having substrate specificity towards 3,4-MDCA.

Description

Piperonal synthase and method for producing piperonal using it

The present invention provides a method for the discovery of piperonal synthase and the production of piperonal using the same, wherein the side chain C7-C8 double bond of 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid] is cleaved. The present invention relates to a biological piperonal synthesis method using-(methylenedioxy) cinnamic acid hydratase-lyase.

Pepper is one of the world's most important spices, called the "King of Spices" since Roman times. Pepper is a very valuable commodity from ancient times to the Middle Ages and has been used as a currency and is now one of the most commonly used spices in the world. In 1819 Hans Christian Ψrsted purely isolated the hot flavors of pepper and named it piperine. Piperin itself has no significant biological activity, but recently, attention has been shown to increase the bioavailability of the drug by inhibiting cytochrome P450 3A4 (CYP3A4) present in the liver of the human body.

Hydrolysis of pipelin under basic conditions produces piperic acid and piperidine, a cyclic amine. Although the mechanism of piperidine biosynthesis in plants is well established, there is no clear explanation about the mechanism of piperic acid biosynthesis, and only a few hypotheses exist.

Modern textbooks speculate that piperic acid (C ₆ C ₅ ) is produced from 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid], a typical phenylpropanoid (C ₆ C ₃ ). That is, the description that if a Type III polyketide synthase (PKS III) present in the pepper plant, the addition of one 2-carbon (C ₂₎ 3,4-units to MDCA precursor to form a C ₆ C ₅ P FER acid skeleton (See FIG. 10 formula).

However, another piper acid biosynthesis mechanism hypothesis not found in the textbook is that PKS III adds two 2-carbon (C ₂ ) units to a C ₆ C ₁ compound, such as benzoic acid, to produce the piperic acid backbone. It is.

The inventors have conducted studies that specifically reveal the biosynthetic mechanism of piperic acid, which is still hypothesized. Among the possibilities of the two chain extensions, the precursor of the first hypothesis is a typical phenylpropanoid, as described above. It is assumed that 3,4-MDCA derived from ferulic acid will be used, and the precursor of the second hypothesis will be vanillic acid or piperonylic acid. It was predicted that the benzoic acid homologs could be derived from oxidation of benzaldehyde analogs such as vanillin or piperonal (piperonal or 3,4- (methylenedioxy) benzaldehyde).

The piperonal is also known as 3,4- (methylenedioxy) benzaldehyde or heliotropin (heliotropin) is a major pepper constituent usually present in about 1% of the pepper essential oil, and also in vanilla (dilla) and dill (dill) exist. Piperonal has been widely used in foods, cosmetics and fragrances because of its aromatic cherry or floral scent, and has been used as a synthetic precursor for various medicines such as Clarium (Cialis ^™ ) and anti-Pakison disease.

The current process of piperonal synthesis in plants is not known at all, and further studies are needed on how to increase the synthesis of piperonal by biological methods. The present inventors have uncovered a new piperonal synthetase capable of synthesizing piperonal, which is expected to be a precursor of piperic acid in plants, to elucidate the biosynthetic mechanism of piperic acid, which is still hypothetical. The present invention has been completed by conducting research to identify a method for producing piperonal in a plant by biological methods using raw synthase.

Specifically, the vanillin synthase gene ( VpVAN ), which synthesizes an enzyme that produces vanillin from ferulic acid, has been reported to be isolated from vanilla plants. The enzyme is known to be a member of the plant cysteine proteinase superfamily, which maintains the primary structure of amino acids. Although the inventors have found no vanillin in pepper, piperonal, which forms a 3,4- (methylenedioxy) ring in vanillin, accounts for a few percent of pepper essential oil. It was judged that piperonyl acid, an oxide derived from piperonal, was likely to be involved in the formation. In other words, the present inventors completed the present invention by finding and identifying piperonal synthase, which is thought to be involved in the biosynthesis process of piperic acid in pepper.

An object of the present invention is to provide a piperonal synthase that produces piperonal by a biological method, and a piperonal synthesis method using the same.

Hereinafter, the present invention will be described in more detail.

The present inventors obtained next-generation sequencing (NGS) data of transcripts from immature peppercorn, and obtained genetic information of VpVAN isoforms from them , using piperonal polymerase chain reaction (PCR). The present invention was completed by separating the synthase gene and identifying biochemical properties such as substrate selectivity of the enzyme.

The inventors found that the similarity between the structure of piperonal and vanillin, namely that piperonal has a 3,4-methylenedioxy ring in the benzaldehyde matrix and vanillin has an open 3-methoxy-4-hydroxy group Based on the similar structure of the two compounds by day, it was assumed that piperonal would be synthesized directly from 3,4-MDCA, similar to the vanillin biosynthetic pathway (Gallage et al., Nature Communication 2014, 5: 4037). . Therefore, homologous gene contig was investigated in the transcript database using the amino acid sequence of VpVAN (Genbank accession No. AKG47593), a vanillin synthase, and pipero in pepper through a series of selection procedures. A new piperonal synthase was identified, which was briefly named piperonal synthase or more suitably piper nigrum 3,4- (methylenedioxy) cinnamic acid hydratase-lyase (PnMCHL).

One embodiment of the present invention provides a piperonal synthase (PnMCHL) having an amino acid sequence that is at least 70% homologous to the amino acid sequence of SEQ ID NO.

The enzyme may preferably have an amino acid sequence of SEQ ID NO: 1, but the activity of generating 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid] as piperonal is maintained. However, the present invention is not limited thereto and includes all of the amino acids of SEQ ID NO: 1 when substituted, inserted or deleted. Preferably, the amino acid sequence having at least 70%, more preferably at least 80%, and even more preferably at least 90% homology with the amino acid sequence of SEQ ID NO: 1. This comparison of homology can be performed by calculating the homology between two or more sequences as a percentage using a computer program.

The enzyme having an amino acid sequence having at least 70% homology with the amino acid sequence of SEQ ID NO: 1 may have a molecular weight of 35 to 45 kDa, preferably 37 to 40 kDa, more preferably 38 to 39 kDa.

The optimum temperature for the enzyme activity is 10 ℃ to 50 ℃, preferably 20 ℃ to 40 ℃, more preferably 25 ℃ to 35 ℃, for example 30 ℃, the optimum pH is 5.0 to 10.0, Preferably pH 5.5 to pH 8.5, more preferably pH 6.0 to pH 8.0, most preferably pH 6.5 to pH 7.5 conditions.

designation order order
number PnMCHL amino acid sequence masrltliplfvvilaaaaagslsdeenpirlvtdkareaesaihrtlgaahhvmafarfarrfgkqyssvdeirkrfdifvenlelihstnkrglsyklginkfadlsweefkahhlgaaqncsatrgthkltqailpetkdwreegivspvknqghcgscwtfsttgaleaaytqatgksislseqqlvdcasgfnnfgcngglpsqafeyikynggldteesypyagvngicgykienigvkvaesvnitegaedelkhavalvrpvsiafqvvhdfrsykggvytsqecgsapmdvnhavlavgygvengvpywlvknswgndwgvdgyfkielgknmcgvatcasypilsl One PnMCHL Base Sequence atggcgtctcgcctcactctcatcccactcttcgtcgtcatcctcgccgccgccgccgctgggtccctatcagatgaggagaatccgatccggcttgtcaccgacaaggcgcgggaagccgagtcggccattcaccggaccctcggcgccgcccaccatgtcatggcctttgcccggttcgcccgaaggttcggcaagcaatacagctctgtggacgaaatcaggaagaggttcgacatctttgtggagaacttggagctcattcactccaccaacaagcgaggcctctcttataagcttggcatcaacaaatttgcggatttgagctgggaggagttcaaggctcatcacttgggcgccgcacaaaattgttcagctacgaggggcacccacaagctcacacaagctattcttcctgagacgaaagactggagggaagaaggtatagtaagtcctgtcaaaaaccaaggccattgtggatcttgctggacctttagtaccactggcgcgttggaagcagcttacactcaagcaacgggaaagagcatctccctttcggagcagcagcttgtggactgcgccagtggatttaacaattttggctgcaatggaggtcttccttcccaggcattcgagtacatcaaatacaacggtggtctcgacacagaggagtcctatccatacgccggtgtcaatggcatctgcggatacaagatagaaaacattggtgtcaaggtcgctgagtccgtgaatatcacagagggcgccgaagatgaattgaaacatgcagtcgctttggtccgtcccgtcagtattgcgttccaggttgtgcacgacttccgctcatacaaaggaggggtttacacaagtcaagagtgtggtagcgctcccatggatgtaaaccatgctgttctggctgtcggttatggtgtggagaatggcgtgccatattggctggtcaagaattcatggggaaatgattggggggttgatggttatt ttaagatcgagctcggaaagaacatgtgtggtgttgctacttgtgcatcttatcctattctctctctctgtaa 2

According to one embodiment of the present invention, the piperonal synthase having an amino acid sequence having at least 70% homology with the amino acid sequence of SEQ ID NO: 1 is 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid] substrate specificity It may be to have.

As confirmed in Experimental Example 2 below, the piperonal synthase of the present invention is a phenylpropenoic acid (phenylpropene acid) homologue, cinnamic acid, coumaric acid, caffeic acid, ferulic acid, and ferulic acid. (ferulic acid) and piperic acid as substrates did not produce the corresponding hydration-decomposable substance and had 3,4-MDCA substrate specificity.

According to one embodiment of the present invention, the piperonal synthetase having an amino acid sequence having at least 70% homology with the amino acid sequence of SEQ ID NO: 1 is 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid] Side chain C7-C8 The double bond may be cleaved to synthesize piperonal. C7-C8 The decomposition of the double bond may be catalyzed by a hydration-retro-aldol reaction.

The piperonal synthetase of the present invention may be a protein belonging to a cysteine protease group, and it is preferable to have a hydrase and a lyase function functionally.

According to one embodiment of the invention, the piperonal synthase of the present invention can synthesize piperonal by the mechanism shown in FIG. Specifically, the piperonal synthase of the present invention is a hydratase function, hydrated by adding water (H ₂ O) to the 3,4-MDCA substrate, and then undergoes a reverse aldol reaction as a degrading enzyme function, resulting in piperonal and acetic acid. (acetic acid) can be produced.

According to one embodiment of the present invention, the piperonal synthetase may be a piperonal synthetase comprising the amino acid sequence of SEQ ID NO: 1, preferably a piperonal synthetase consisting of the amino acid sequence of SEQ ID NO: 1 Can be.

According to another embodiment, the piperonal synthetase may include an amino acid sequence encoded by the nucleotide sequence of Table 1 SEQ ID NO: 2.

One example of the present invention provides a recombinant expression vector comprising a base sequence encoding the piperonal synthase of the present invention described above.

The piperonal synthetase of the present invention may be an enzyme including a piperonal synthetase having an amino acid sequence having at least 70% homology with the amino acid sequence of SEQ ID NO: 1, preferably an amino acid sequence of SEQ ID NO: 1 .

The base sequence encoding the piperonal synthase may be preferably the base sequence of SEQ ID NO: 2, but is also interpreted to include a sequence showing substantial identity to the base sequence. The substantial identity indicates 70% or more homology when aligning the nucleotide sequence of the present invention with any other sequence to the maximum correspondence, and analyzing the aligned sequence using algorithms commonly used in the art. Nucleotide sequence means a nucleotide sequence that exhibits at least 80% homology, more preferably at least 90% homology.

The gene encoding the piperonal synthase may be used by itself or in the form of a recombinant vector comprising the gene. The recombinant vector refers to a recombinant nucleic acid molecule comprising a target gene sequence and a suitable nucleic acid sequence essential for expressing the gene sequence operably linked in a specific host organism, wherein the appropriate nucleic acid sequence is a transcription and translation terminator. , Transcriptional and translation initiation sequences, and promoters useful for the regulation of expression of specific target nucleic acids. In addition, the gene may be operably linked to, for example, a chemical inducible element and a temperature sensitive element.

The chemical inducible element may be at least one selected from the group consisting of lac operon, T7 promoter, trc promoter, and the like. The T7 promoter is derived from T7 phage which is a virus and includes a T7 terminator with a promoter.

The vector system can be constructed as a vector for cloning or a vector for expression by various methods well known in the art (Franηois Baneyx, Current Opinion in Biotechnology 1999, 10: 411-421).

The vector includes, for example, a plasmid or a virus derived vector. Plasmid refers to a circular double stranded DNA ring to which additional DNA can be linked. Vectors used in the present invention include, for example, plasmid expression vectors, viral expression vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses) and viral vectors capable of performing their equivalent functions, but are not limited to these. It is not limited to. Preferably, pET, pBR, pTrc, pLex, pUC vectors and the like suitable for expression in E. coli may be used, but may be used without particular limitation as long as it can efficiently express the enzyme protein.

According to an example of the present invention, the vector may be a recombinant expression vector including a base sequence encoding the amino acid sequence of SEQ ID NO: 1 and having a cleavage map of FIG. 7 or 8. FIG. 7 is a cleavage map of a recombinant plasmid prepared for expressing PnMCHL in E. coli, and FIG. 8 is a cleavage map of a recombinant plasmid prepared for expressing PnMCHL in yeast. The plasmid shown in Figure 7 includes a T7 promoter, MBP tag, PnMCHL gene, T7 terminator, antibiotic resistance gene, and lacI,

The plasmid shown in FIG. 8 includes antibiotic resistance gene, CYC terminator, PnMCHL gene, GAL1, 10 promoter, FLAG, f1 ori terminator, LEU2 promoter, and LEU2.

Another embodiment of the present invention provides a recombinant cell expressing piperonal synthase, which is transformed with a recombinant expression vector comprising a nucleotide sequence encoding the piperonal synthase as described above.

The piperonal synthetase may be an enzyme including a piperonal synthetase having an amino acid sequence having at least 70% homology with the amino acid sequence of SEQ ID NO: 1, preferably an amino acid sequence of SEQ ID NO: 1.

 In the present invention, "recombination" refers to a molecular chain in which DNA chain fragments or plasmids having genes different from those of the original cells are infiltrated between the cells to bind with the DNA present in the original cell, thereby changing the genotype of the cell. Say the phenomenon.

As a cell to be transformed capable of stably and continuously replicating and / or expressing the vector, any cell known in the art may be used as long as it is capable of overexpressing the active enzyme protein. For example E. a variety of E. coli, such as coli, and the like can be used various yeast, plant cells. Preferably E. coli and yeast.

The transformation method using the vector may be any method known in the art without particular limitation, such as fusion of bacterial protoplasts, electroporation, projectile bombardment, and infection with a viral vector. .

Another example of the present invention includes at least one selected from the group consisting of the piperonal synthase of the present invention described above, a recombinant cell expressing the synthetase, a culture of the recombinant cell, and a lysate of the recombinant cell. To provide a composition for piperonal production.

In one embodiment, the piperonal synthase is an enzyme protein comprising a piperonal synthetase having an amino acid sequence having at least 70% homology with the amino acid sequence of SEQ ID NO: 1, preferably the amino acid sequence of SEQ ID NO: 1 Can be.

The piperonal production composition may be to prepare piperonal using 3,4-MDCA as a substrate. The culture includes an enzyme protein produced from the recombinant cells, and may be in a cell-free form including the recombinant cells or no cells. The lysate refers to a lysate obtained by crushing a recombinant cell or a supernatant obtained by centrifugation of the lysate, and includes an enzyme protein produced from the recombinant cell.

Another embodiment of the present invention is a piperonal synthase of the present invention described above, at least one selected from the group consisting of recombinant cells expressing the synthetase, the culture of the recombinant cells, and the lysate of the recombinant cells Provided is a method for producing piperonal for use.

The piperonal production method includes reacting the enzyme with 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid].

The 3,4-MDCA side chain C7-C8 of 3,4-MDCA through the step of reacting with the composition for piperonal production The double bond may be obtained by cleaving.

Cultivation of the recombinant cells may be carried out under medium and culture conditions readily selected by those skilled in the art according to the characteristics of the cells used. The culture method may be any culture method known in the art, for example, batch, continuous and fed-batch culture methods and the like, but is not limited thereto.

The step of reacting the 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid] with the composition for piperonal production may be preferably performed under optimal activation conditions of the enzyme protein.

As a specific example, it exhibits an enzyme activity of 61% compared to pH 7.0 at pH 5 to pH 8.5 (see FIG. 9), preferably pH 5.5 to pH 8.5, more preferably pH 6.0 to pH 8.0, most preferably The highest enzymatic activity at pH 6.5 to pH 7.5 can maximize the conversion efficiency of 3,4-MDCA to piperonal.

The 3,4-MDCA hydratase-lyase (PnMCHL) enzyme, a novel piperonal synthetase with 3,4-MDCA substrate specificity of the present invention, enables biosynthetic synthesis of piperonal in 3,4-MDCA. do.

1 is an experimental result confirming the substrate specificity of PnMCHL enzyme and its function as 3,4-MDCA hydrate-degrading enzyme using gas chromatography-mass spectrometry (GC-MS) method.
2 is In vitro reaction was performed using PnMCHL enzyme expressed and purified in E. coli and the reaction product was analyzed. 2A is the result of adding 3,4-MDCA as a substrate and B in FIG. 2 as a substrate to the reaction.
Figure 3 is a schematic diagram of the expected mechanism of PnMCHL enzyme reaction, showing that the PnMCHL enzyme cleaves the side chain C7-C8 double bond of 3,4-MDCA according to the hydrase-degrading enzyme mechanism to produce piperonal.
4 is Yeast mutant YPH499 Δ PTF production method and mutation identification are shown. 4A shows a strategy of simultaneously inactivating phenylacrylic acid decarboxylase (PAD) and ferulic acid decarboxylase (FDC), and FIG. 4B is a DNA agarose gel image. PAD (P), phenylacrylic acid decarboxylase, FDC (F), ferulic acid decarboxylase, and TRP (T) tryptophan synthase.
5 is Polyacrylamide gel electrophoresis of purified maltose binding protein-PnMCHL (maltose binding protein-PnMCHL, MBP-PnMCHL) is shown.
Figure 6 shows the tissue-specific transcription level of the PnMCHL gene in pepper plants.
7 shows a cleavage map of recombinant plasmids prepared for the expression of PnMCHL in E. coli.
8 shows a cleavage map of the recombinant plasmid prepared for PnMCHL expression in yeast.
Figure 9 shows the results of the enzyme activity of PnMCHL according to the pH.
10 is 3,4-MDCA, cinamic acid (cinamic acid), caffeic acid (caffeic acid), ferulic acid (ferulic acid), piperic acid (piperic acid), piperonal (piperonal), piperonylic acid (piperonylic acid), vanillin and vanillic acid.

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited only to the following examples.

Example 1 Bacteria, Yeast Strains and Culture Medium

(mrr-hsdRMS-mcrBC) Φ80d lacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara, leu)7697 galU galK rpsL λ ^- endA1 nupG]와 단백질 발현을 위해 대장균 Rosetta2(DE3)[F^- ompT hsdS _B (r_B ^-m_B ^-) gal dcm(DE3) pRARE2(Cam^R)] 균주를 사용했다. 효모 PAD1과 FDC1 파괴 돌연변이인 YPH499 ΔPTF(MATa ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1 Δpad1-fdc1::TRP1)(트립토판 선택성)를 개발하였고 이 돌연변이체를 PnMCHL의 생체내 기능 분석 실험에 사용했다.E. coli DH10B [F-mcrA for gene cloning

( mrr-hsdRMS-mcrBC ) Φ80d lacZ Δ M15 Δ lacX74 deoR recA1 araD139 Δ (ara, leu) 7697 galU galK rpsL λ ^- endA1 nupG ] and E. coli Rosetta2 (DE3) [F ^- ompT hsdS _B (r _B ) for protein expression _{^{- m B -) gal dcm (}} DE3) pRARE2 (Cam R)] used a strain. The yeast PAD1 and FDC1 disruption mutants YPH499 Δ PTF (MATa ura3-52 lys2-801 ade2-101 trp1- Δ 63 his3- Δ 200 leu2- Δ 1 Δ pad1-fdc1 :: TRP1 ) (tryptophan selectivity) were developed. Sieve was used for in vivo functional assays of PnMCHL .

E. coli was cultured in Luria-Bertani (LB) medium (MBcell, Korea) and a solid LB medium plate was prepared by adding 1.5% (w / v) Micor-agar (Duchefa, Netherlands). Plasmid-transformed strains were cultured in LB medium added with kanamycin (50 μg / ml).

Wild type yeast (YPH499) was cultured in YPDA medium (Clontech, USA). Mutant yeasts were cultured in a 0.67 g / L tryptophan DO (dropout) supplement and 26.7 g / L Minimal SD Base (Clonetech, Madison, Wis.). Transformants were cultured in the medium under leucine and tryptophan deficient conditions, and 2% of Bacto ^™ Agar (Becton, USA) was used as the solid medium.

Example 2 Enzymes and Chemicals

Restriction enzymes, T4 DNA ligase, Ex Taq polymerase, and Phusion ^® High-Fidelity DNA Polymerase were purchased from TaKaRa (Japan) or NEB (USA). Acrylamide / bis solution (40%) and Coomassie Blue protein quantitative reagent for polyacrylamide gel electrophoresis were purchased from Bio-Rad (USA). The medium used for the cultivation of yeast, including the DO Supplement, was purchased from Clontech (USA). Adenine hemisulfate, ATP, dithiothreitol (DTT), IPTG, NAD ⁺ , MgCl ₂ , CH ₂ Cl ₂ , Tris-HCl and phenylpropenic acid (cinnamic acid, kumaric acid, caffeic acid, ferulic acid, 3,4-MDCA) Piperic acid was purchased from Sigma-Aldrich. Amylose resin for purification of Maltose Binding Protein (MBP) tagged proteins was purchased from (NEB, USA).

Example 3 Oligonucleotides

Primer pairs used in the polymerase chain reaction (PCR) were synthesized by COSMO Genetech (Seoul, Korea), and a list of primer sequences used in the experiment is shown in Table 2 below. The underlined portion is the restriction enzyme site.

Primer Name Sequence (5 '→ 3') SEQ ID NO: PnMCHL F atggcgtctcgcctcactctc 3 PnMCHL noER F atggaggagaatccgatccggctt 4 PnMCHL R ttacagagagagaataggataagatg 5 pESC-Leu2 BamHI noER MCHL F cg ggatcc gatggaggagaatccgatccg 6 pESC-Leu2 SalI PnMCHL R acgc gtcgac cagagagagaataggataagatg 7 pMBP noER NdeI F cgc catatg gaggagaatccgatccggctt 8 pMBP noER XhoI R cccg ctcgag cagagagagaataggata 9 PnMCHL QRT F cagcttacactcaagcaacggg 10 PnMCHL QRT R ggaagacctccattgcagcca 11 PAD F atgctcctatttccaagaagaa 12 FDC R ttatttatatccgtaccttttcca 13 TRP SphI F acat gcatgc accataaacgacattactatatata 14 TRP SpeI R gg actagt aatttcctgatgcggtattttc 15 pET28 MBP NcoI F catg ccatgg atgaaaatcgaagaaggtaaact 16 pET28 MBP NdeI R cgc catatg agtctgcgcgtctttcagg 17

Example 4 Isolation of Piperonal Biosynthesis-Related Genes

Using the amino acid sequence of vanillin synthase VpVAN (Genbank accession No. AKG47593), homologous gene contigs were examined in the transcript database using the Local tBLASTn method. Based on the sequence of the contigs, primer pairs PnMCHL F and PnMCHL R were designed to clone the target ORF from the cDNA library using Takara Ex Taq Polymerase (Takara, Japan) (Table 2). Was cloned.

Cloning to remove ER targeting peptides used PnMCHL noER F and PnMCHL R primer pairs. The reaction conditions were initially reacted for 5 cycles at 98 ° C. for 5 minutes, 98 ° C. for 15 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 1 minute 6 seconds for 35 cycles, followed by 5 minutes at 72 ° C. Maintained. Next, PCR products were purified using the Inclone ^™ Gel & PCR Purification Kit (Inclone Biotech, Korea) and cloned into pMD19 T vector (Takara, Japan).

Example 5 Yeast PAD1 , FDC1  Destruction mutation

Yeast phenylacrylic acid decarboxylase (PAD1, YDR538W) and ferulic acid decarboxylase (FDC1, YDR539W) are connected back and forth on chromosome IV (Fig. 4). Using the PAD F and FDC R primer pairs (Table 2), DNA fragments containing the PAD1 and FDC1 genes (2.7 kbp) were cloned from the yeast chromosome.

The PCR product was then linked to the pMD19 T vector and sequence confirmed by DNA sequencing. After this step, by using the Sph I and Spe I were each double-cut the PAD1 and FDC1 which the 368 bp and 996 bp downstream, respectively from the ATG, the tryptophan gene synthesis (1.06kbp) obtained from pESC-TRP vector Sph I and Spe I was linked to the double cleavage site (FIG. 4).

Next, gene fragments for knockout experiments were prepared. Fragments were amplified using PAD F and FDC R primer pairs and 5 μg of PCR product was transformed into yeast YPH499 strain using LiAc / SS carrier DNA / PEG method (Gietz and Schiestl 2007). Finally, to select the Δ pad1-fdc1 mutation named YPH499 Δ PTF Transformants were plated in medium excluding tryptophan.

Example 6 Heterologous Expression and Protein Purification

The ORF of the gene was determined by Gibson Assembly ^® Cloning Kit (NEB, USA) using the Nde I and Xho I doubles of the pET28 a (+)-MBP vector with C-terminal 6ХHis tag ( obtaining MBP sequence from pMAL-c2x vector). Cloned to digestion site. Recombinant plasmids containing PnMCHL - pET: MBP-PnMCHL (or pET: MBP-noER-PnMCHL from which the endoplasmic reticulum targeting peptide was deleted) were obtained from Rosetta? 2 (DE3) was transformed and incubated overnight at 37 ° C. in an agar plate containing 50 μg / ml kanamycin and 35 μg / ml chloramphenicol. Next, only selected colonies were taken and inoculated in 5 mL LB medium to which 50 μg / ml of kanamycin and 35 μg / ml chloramphenicol were added and incubated overnight.

For protein expression, 50 ml LB medium was inoculated with 0.5 ml of the culture solution (5 ml LB medium culture solution), and when OD ₆₀₀ reached 0.5, 0.1 mM IPTG was added and the culture temperature was lowered to 20 ° C. and incubated for 16 hours. Cells were then collected by centrifugation at 5000 rpm (Beckman F1202 rotor) for 5 minutes at 4 ° C. The pellet obtained by centrifugation was then resuspended in 10 ml of lysis buffer (20 mM Tris-HCl, pH 7.0, 300 mM NaCl added). The suspension was then sonicated (Sonic Dismembrator 550, Fisher Scientific) for a total of 10 minutes at 3 second run, 1 second stop intervals to disrupt cells.

The lysed protein fractions were centrifuged at 6000 rpm (Beckman F1202 rotor) at 4 ° C for 30 minutes. MBP-tagged target proteins were bound to amylose resin and eluted with lysis buffer to which 10 mM maltose was added in supernatant by affinity chromatography. SDS-PAGE results of purified PnMCHL are shown in FIG. 5. MBP (MBP-PnMCHL fusion protein) fused to the N-terminus of PnMCHL showed a molecular weight of 75 kD (FIG. 5).

Example 7 In Vitro Analysis

The assay was performed with 0.2 mM substrate (ferulic acid and 3,4-MDCA), 2.5 mM DTT, 5 mM MgCl ₂ , and 1 μg purified protein (PnMCHL) in a total volume of 1 ml Tris-HCl buffer (100 mM, pH 7.5). ) Was added and reacted at 30 占 폚 for 30 minutes, and then the reaction mixture was heated at 100 占 폚 for 3 minutes to terminate the reaction. Control experiments were performed in the same manner, except that the heat treated enzyme (100 ° C., 3 minutes) was used.

Example 8 Metabolite Analysis

For product analysis, 500 μl of CH ₂ Cl ₂ was added and the reaction was vigorously vortexed for 1 minute and centrifuged (4000 g) for 5 minutes to separate the CH ₂ Cl ₂ layer. The CH ₂ Cl ₂ extraction process was then repeated twice, and the CH ₂ Cl ₂ layer was concentrated to 500 μl under weak nitrogen gas flow before analysis. Thereafter, the piperonal was analyzed on GC-MS according to the procedure of Example 9.

Example 9 GC-MS Analysis

Reaction products including piperonal were analyzed by gas chromatograph-mass spectrometry (Agilent model 6890, USA). The gas chromatography column was ZB-5MSi capillary (60 m Х 0.25 mm Х 0.25 μm, Zebron) and helium was flowed at 1 ml / min flow rate as carrier gas. For enzyme reaction product analysis, 1 μl of CH ₂ Cl ₂ extract was injected into a gas chromatograph running with the following temperature program. Injection at 220 ° C., initial temperature of 70 ° C., increased to 220 ° C. at a rate of 5 ° C./min, and then to 320 ° C. (maintained for 1 minute) at 25 ° C./min The mass spectrometer was Agilent HP 5973 (USA) with a transfer line temperature of 320 ° C., an ion source of 250 ° C., a quadrupole temperature of 150 ° C., and an ionization potential of 70 eV. The scan range was 50-200 atomic mass units. The product was identified and identified as compared to the piperonal standard using the MS Search Program v.2.0 (NIST Standard Reference Database, USA).

Example 10 Bioinformatics Analysis

Local tBLASTn searches for each gene were performed using BioEdit (version 7.2.0). The threshold for tBLASTn search was set at 25% or higher for protein identity and 5 × 10 ⁻²⁰ or lower for e-value. Alignment of nucleotide and amino acid sequences was performed using Vector NTI Advance ^® software (Thermo Fisher Scientific, USA). Gene sequence translation, reverse translation, protein molecular weight prediction and restriction enzyme site prediction were performed at www.bioinformatics.org/sms2/.

Example 11 Quantitative Real-Time PCR or qRT-PCR

Quantitative PCR reaction conditions were prepared with 20 μl total reaction volume with 10 μl of 2Х QuantiMix SYBR Kit PCR system (PhileKorea, Korea), 1 μl of diluted cDNA, 0.25 μM of primer and double-distilled water, respectively. Quantitative PCR instrument conditions were performed using a Rotor-Gene 2000 Real Time Cycler (Corbett Research, Australia) with a temperature program of 40 cycles of 5 minutes at 95 ° C, 15 seconds at 95 ° C, 20 seconds at 58 ° C and 20 seconds at 72 ° C. Was performed. Standard curves were generated by quantifying pMD19-PnMCHL plasmids ranging from 10 ² to 10 ⁷ copy / μl, and cDNA copy numbers were calculated by methods described by Yin et al., Immunology and Cell Biology (2001).

Experimental Example 1 Pipelinen Biosynthesis-Related Genes in-silico  Search

The method of Example 4 was used to find a gene candidate for an enzyme synthesizing piperonal or vanillin from 3,4-MDCA or ferulic acid, and before the function was confirmed, it was tentatively identified as P. nigrum cutting enzyme. , PnCuE) and presented in Table 3. The ORF sequence of PnCuE is shown in Table 1 SEQ ID NO: 1. This finding of VpVAN homologs from less ripe peppercorn through next-generation sequencing technology (NGS) indicates that vanillin or piperonal is biosynthesized from pepper fruit.

First, the similarity was evaluated by comparing the base and amino acid sequences of candidate genes with VpVAN, and the sequences were verified by in-silico , and then the function verification of the enzyme encoded by the PnCuE gene, which is assumed to be a hydrase, was performed.

Table 3 below shows a comparison of PnCuE, which is presumed to be a piperonal or vanillin biosynthesis gene, and VpVAN used in its tBLASTn search query. The PnCuE protein, believed to be phenylpropenic acid side chain cleavage enzyme, consists of 356 amino acid residues and is expected to have a molecular weight of 38.7 kDa, showing a high similarity of 72% to the cysteine protease group belonging to VpVAN.

designation ORF (bp) function Protein molecular weight (kDa) VpVAN 1,071 Vanillin synthase (ferulic acid side chain C ₇ -C ₈ double bond cleavage) 39.0 PnCuE 1,071 Estimated by Side Chain C ₇ -C ₈ Cleavage of Phenylpropene Acid 38.7

One complete ORF sequence (SEQ ID NO: 1) with about 72% amino acid sequence homology to the VpVAN was found in-silico and the gene was cloned using the method of Example 4.

Experimental Example 2 Substrate Specificity and Enzyme Activity Test of Enzyme

2-1. PAD1  And FDC1  Gene Production of Yeast Mutants Deficient

Hydration-degrading enzyme activity of PnCuE (PnMCHL) enzymes was conveniently screened, and in vivo experiments with yeast were performed to confirm substrate specificity of PnCuE.

Since wild type yeast YPH499 has several sets of ferulic acid decarboxylase, when ferulic acid is provided as a substrate, it is impossible to confirm the PnMCHL enzymatic reaction product by making the decarboxyl reaction product as a by-product. Therefore, a variant that inactivates ferulic acid decarboxylase is required. Do. 4A illustrates a mutant manufacturing method, and illustrates a strategy of simultaneously inactivating phenylacrylic acid decarboxylase (PAD) and ferulic acid decarboxylase (FDC1). The mutant prepared as in FIG. 4A was named as YPH499 Δ PTF , and Example 5 shows a method for preparing PAD1 and FDC1 disrupting mutant mutant yeast.

4B shows a DNA agarose gel image and could identify gene fragments of expected size in the mutants. PAD (P) represents phenylacrylic acid decarboxylase, FDC (F) represents ferulic acid decarboxylase, and TRP (T) represents tryptophan synthase.

2-2. Confirmation of Substrate Specificity of PnCuE (PnMCHL) Enzyme

After transforming and incubating the PnCuE gene cloned in the YPH499 Δ PTF mutant yeast, five phenylpropene homologues (cinnamic acid, kumaric acid, kumaric acid, ferulic acid, 3,4-MDCA) and piperic acid ( piperic acid) was added as a substrate and reacted, and then C7-C8 binding cleavage was assayed by gas chromatography-mass spectrometry (GC-MS) shown in Example 9, and the results are shown in FIG.

As shown in FIG. 1, only 3,4-MDCA of the acid provided as a substrate produced piperonal as an enzymatic reaction product, and the remaining cinnamic acid, coumaric acid, caffeic acid, ferulic acid, and piperic acid were used as substrates. In that case no hydration-decomposition material was produced. In the ferulic acid reaction, only 2-methoxy-4-vinyl phenol was detected instead of vanillin, which is caused by ferulic acid decarboxylase remaining in yeast as a decarboxyl product derived from ferulic acid. The specific ion chromatogram (EIC) was followed at pipernal molecular weight m / z 150.

It was confirmed that PnCuE has substrate specificity to 3,4-MDCA and functions as a 3,4-MDCA hydratase (PnMCHL or piperonal synthase). The tentatively named PnCuE was identified as P. nigrum 3,4- (methylenedioxy) cinnamic acid hydratase-lygase (PnMCHL) or piperonal synthase.

Experimental Example 3 Confirmation of the Characteristics and Substrate Specificity of PnMCHL Produced from Transgenic Escherichia Coli

To determine whether the production of piperonal by C7-C8 binding cleavage of 3,4-MDCA is an artificial product occurring in E. coli in vivo, and to confirm the similarity of this enzyme with the HCHL bacterial enzyme, PnMCHL enzyme expressed in E. coli was purified using the method of Example 6.

Figure 5 shows the results of polyacrylamide gel electrophoresis of maltose binding protein-PnMCHL (MBP-PnMCHL). Purified protein showed nearly 100% purity.

Next, the purified PnMCHL enzyme was reacted in vitro with the 3,4-MDCA substrate, which was previously confirmed in vivo, using the method of Example 7, and the metabolite was analyzed using the methods of Examples 8 and 9. .

The analysis results are shown in A of FIG. 2. FIG. 2A shows the result after reaction with 3,4-MDCA as the substrate and B in FIG. 2 with the ferulic acid as the substrate. As shown in A of FIG. 2 and B of FIG. 2, denatured PnMCHL (heated MCHL) was inactive, and undenatured PnMCHL protein did not react with ferulic acid, but only with 3,4-MDCA as a substrate. It was confirmed to produce a ronal. Thus, the substrate specificity for 3,4-MDCA of the PnMCHL enzyme could once again be established. In addition, unlike P. fluorescens HCHL, PnMCHL was confirmed to decompose 3,4-MDCA into piperonal even without NAD ⁺ and ATP (Fig. 2, Fig. 3).

Specifically, PnMCHL enzymatic reaction is the side chain C7-C8 of 3,4-MDCA, as shown in Figure 3 It was confirmed that the double bond was cleaved by PnMCHL hydrase and produced piperonal. From this, it was again confirmed that the enzyme belonged to the plant cysteine protease group.

Experimental Example 4 In Pepper Plant Tissues PnMCHL  Check transcription level of gene

In order to confirm the level of transcription in the pepper plant tissues (roots, stems, leaves and fruits) of the PnMCHL piperonal biosynthetic gene found through the previous experiments, the primers PnMCHL QRT F (SEQ ID NO: 10) and PnMCHL QRT R ( Transcription levels were determined by qRT-PCR using SEQ ID NO: 11) pairs. Four experimental replicates and five plant tissue specimens were repeated and the results are shown in FIG. 6.

As shown in Figure 6, PnMCHL is expressed in the whole pepper plants, it was confirmed that the highest level in the leaves, and high levels in the fruit and stem and low levels in the roots.

Experimental Example 5 piperonal synthase activity according to pH change

In order to confirm the optimum pH condition of the activity of the PnMHL piperonal synthase purified in Experimental Example 3-1 to the maximum, the enzyme was treated with substrate 3,4-MDCA and pH 6.0 using the method of Example 7. Three replicate experiments were reacted at pH conditions of pH 7.0, pH 8.0, pH 9.0, and pH 10.0. In the method of Example 9, the area corresponding to the piperonal peak was calculated by GC-MS analysis. Enzyme activity was 100% in pH 7.0 buffer conditions with the largest piperonal area, and enzyme activity was expressed as a percentage (%) compared to pH 7.0 under other pH conditions.

As a result, as shown in Figure 9, it showed 61% of the enzyme activity compared to pH 7.0 at pH 6.0 to pH 8.0 conditions, the highest at pH 7.0, that is, the enzyme activity of 100%. The above experiment confirmed that the pH 7.0 condition is a pH condition maximizing the conversion efficiency of 3,4-MDCA to piperonal.

<110> Seoul National University R & DB Foundation <120> Piperonal synthase and method for producing piperonal using it <130> DPP20177471KR <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 356 <212> PRT <213> Artificial Sequence <220> PnMCHL Amino acid sequence <400> 1 Met Ala Ser Arg Leu Thr Leu Ile Pro Leu Phe Val Val Ile Leu Ala   1 5 10 15 Ala Ala Ala Ala Gly Ser Leu Ser Asp Glu Glu Asn Pro Ile Arg Leu              20 25 30 Val Thr Asp Lys Ala Arg Glu Ala Glu Ser Ala Ile His Arg Thr Leu          35 40 45 Gly Ala Ala His His Val Met Ala Phe Ala Arg Phe Ala Arg Arg Phe      50 55 60 Gly Lys Gln Tyr Ser Ser Val Asp Glu Ile Arg Lys Arg Phe Asp Ile  65 70 75 80 Phe Val Glu Asn Leu Glu Leu Ile His Ser Thr Asn Lys Arg Gly Leu                  85 90 95 Ser Tyr Lys Leu Gly Ile Asn Lys Phe Ala Asp Leu Ser Trp Glu Glu             100 105 110 Phe Lys Ala His His Leu Gly Ala Ala Gln Asn Cys Ser Ala Thr Arg         115 120 125 Gly Thr His Lys Leu Thr Gln Ala Ile Leu Pro Glu Thr Lys Asp Trp     130 135 140 Arg Glu Glu Gly Ile Val Ser Pro Val Lys Asn Gln Gly His Cys Gly 145 150 155 160 Ser Cys Trp Thr Phe Ser Thr Thr Gly Ala Leu Glu Ala Ala Tyr Thr                 165 170 175 Gln Ala Thr Gly Lys Ser Ile Ser Leu Ser Glu Gln Gln Leu Val Asp             180 185 190 Cys Ala Ser Gly Phe Asn Asn Phe Gly Cys Asn Gly Gly Leu Pro Ser         195 200 205 Gln Ala Phe Glu Tyr Ile Lys Tyr Asn Gly Gly Leu Asp Thr Glu Glu     210 215 220 Ser Tyr Pro Tyr Ala Gly Val Asn Gly Ile Cys Gly Tyr Lys Ile Glu 225 230 235 240 Asn Ile Gly Val Lys Val Ala Glu Ser Val Asn Ile Thr Glu Gly Ala                 245 250 255 Glu Asp Glu Leu Lys His Ala Val Ala Leu Val Arg Pro Val Ser Ile             260 265 270 Ala Phe Gln Val Val His Asp Phe Arg Ser Tyr Lys Gly Gly Val Tyr         275 280 285 Thr Ser Gln Glu Cys Gly Ser Ala Pro Met Asp Val Asn His Ala Val     290 295 300 Leu Ala Val Gly Tyr Gly Val Glu Asn Gly Val Pro Tyr Trp Leu Val 305 310 315 320 Lys Asn Ser Trp Gly Asn Asp Trp Gly Val Asp Gly Tyr Phe Lys Ile                 325 330 335 Glu Leu Gly Lys Asn Met Cys Gly Val Ala Thr Cys Ala Ser Tyr Pro             340 345 350 Ile Leu Ser Leu         355 <210> 2 <211> 1071 <212> DNA <213> Artificial Sequence <220> PnMCHL DNA sequence <400> 2 atggcgtctc gcctcactct catcccactc ttcgtcgtca tcctcgccgc cgccgccgct 60 gggtccctat cagatgagga gaatccgatc cggcttgtca ccgacaaggc gcgggaagcc 120 gagtcggcca ttcaccggac cctcggcgcc gcccaccatg tcatggcctt tgcccggttc 180 gcccgaaggt tcggcaagca atacagctct gtggacgaaa tcaggaagag gttcgacatc 240 tttgtggaga acttggagct cattcactcc accaacaagc gaggcctctc ttataagctt 300 ggcatcaaca aatttgcgga tttgagctgg gaggagttca aggctcatca cttgggcgcc 360 gcacaaaatt gttcagctac gaggggcacc cacaagctca cacaagctat tcttcctgag 420 acgaaagact ggagggaaga aggtatagta agtcctgtca aaaaccaagg ccattgtgga 480 tcttgctgga cctttagtac cactggcgcg ttggaagcag cttacactca agcaacggga 540 aagagcatct ccctttcgga gcagcagctt gtggactgcg ccagtggatt taacaatttt 600 ggctgcaatg gaggtcttcc ttcccaggca ttcgagtaca tcaaatacaa cggtggtctc 660 gacacagagg agtcctatcc atacgccggt gtcaatggca tctgcggata caagatagaa 720 aacattggtg tcaaggtcgc tgagtccgtg aatatcacag agggcgccga agatgaattg 780 aaacatgcag tcgctttggt ccgtcccgtc agtattgcgt tccaggttgt gcacgacttc 840 cgctcataca aaggaggggt ttacacaagt caagagtgtg gtagcgctcc catggatgta 900 aaccatgctg ttctggctgt cggttatggt gtggagaatg gcgtgccata ttggctggtc 960 aagaattcat ggggaaatga ttggggggtt gatggttatt ttaagatcga gctcggaaag 1020 aacatgtgtg gtgttgctac ttgtgcatct tatcctattc tctctctgta a 1071 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PnMCHL forward primer <400> 3 atggcgtctc gcctcactct c 21 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PnMCHL noER forward primer <400> 4 atggaggaga atccgatccg gctt 24 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PnMCHL reverse primer <400> 5 ttacagagag agaataggat aagatg 26 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pESC-Leu2 BamHI noER MCHL forward primer <400> 6 cgggatccga tggaggagaa tccgatccg 29 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> pESC-Leu2 SalI PnMCHL reverse primer <400> 7 acgcgtcgac cagagagaga ataggataag atg 33 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pMBP noER NdeI forward primer <400> 8 cgccatatgg aggagaatcc gatccggctt 30 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> pMBP noER XhoI reverse primer <400> 9 cccgctcgag cagagagaga ataggata 28 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PnMCHL QRT forward primer <400> 10 cagcttacac tcaagcaacg gg 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PnMCHL QRT reverse primer <400> 11 ggaagacctc cattgcagcc a 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PAD forward primer <400> 12 atgctcctat ttccaagaag aa 22 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FDC reverse primer <400> 13 ttatttatat ccgtaccttt tcca 24 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> TRP SphI forward primer <400> 14 acatgcatgc accataaacg acattactat atata 35 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TRP SpeI reverse primer <400> 15 ggactagtaa tttcctgatg cggtattttc 30 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> pET28 MBP NcoI forward primer <400> 16 catgccatgg atgaaaatcg aagaaggtaa act 33 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> pET28 MBP NdeI reverse primer <400> 17 cgccatatga gtctgcgcgt ctttcagg 28

Claims (13)

Piperonal synthetase, consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 1, wherein the piperonal synthase is C7-C8 located in the side chain of 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid] Piperonal synthase, which is to synthesize a piperonal by cutting a double bond. The piperonal synthetase of claim 1, wherein the piperonal synthase has 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid] substrate specificity. delete delete The piperonal synthetase of claim 1, wherein the piperonal synthetase is encoded by the nucleotide sequence of SEQ ID NO: 2. 6. An expression vector comprising a nucleotide sequence encoding the piperonal synthase according to claim 1. The recombinant expression vector of claim 7, wherein the expression vector has a cleavage map of FIG. 7 or 8. The recombinant expression vector of claim 7, wherein the nucleotide sequence encoding the piperonal synthase consists of a nucleotide sequence of SEQ ID NO: 2. 9.  A recombinant cell expressing piperonal synthase, transformed with the expression vector according to claim 7. The recombinant cell of claim 10, wherein the cell is a yeast or E. coli cell. The piperonal synthase according to any one of claims 1 to 3 and 6, the recombinant cell according to claim 10 or 11, the culture of the recombinant cell, and the lysate of the recombinant cell. Comprising one or more selected from the group consisting of, piperonal production composition. A method for producing piperonal, comprising the step of reacting the composition for producing piperonal according to claim 12 with 3,4-MDCA [3,4- (methylenedioxy) cinnamic acid].

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