KR101163542B1 - Methods of preparing for Biotransformed Vanillin from Isoeugenol - Google Patents

Abstract

The present invention relates to a method for producing biotransformed vanillin from isoeugenol. More specifically, the present invention is the production of bioconverted vanillin by bioconverting isoeugenol contained in the culture medium to vanillin by host cells transformed with isoeugenol monooxygenase ( iem ) and iem regulatory gene expression vector It is about a method. The present invention does not require expensive gene expression inducer IPTG (Isopropyl β-D-1-thiogalactopyranoside) or ferulic acid, which is used for expression of external genes in E. coli, and thus economically low cost. There is an advantage that can produce a large amount of natural vanillin. In addition, the present invention can be applied in fields such as pharmaceuticals and foods for mass production of proteins using microorganisms by providing expression constructs capable of mass production of desired specific proteins.

Description

Method of preparing for transforming vanillin from isoeugenol from Isoeugenol

The present invention relates to a process for producing vanillin bioconverted from isoeugenol. More specifically, the present invention relates to a method for bioconverting isoeugenol contained in the culture medium to vanillin by host cells transformed with isoeugenol monooxygenase ( iem ) and iem regulatory gene expression vectors.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a major component of vanilla fruit and is widely used as a flavoring agent in food, drinks and medicine. Currently, most vanillins are chemically synthesized from guayacol ³⁸ . Due to the low yields of plant-derived vanillin produced only in the tropics, synthetic vanillin is traded at lower prices than natural vanillin from vanilla fruit ^24,38 . Natural vanillin production by biobiotechnology, which uses inexpensive natural organic materials as synthons, has recently attracted attention ²⁴ . Plant-derived phenylpropanoid eugenol and isoeugenol are natural lignin precursors that have been suggested as useful precursors for natural vanillin production ^{1,9,11,21,31,34,37,39,43, 44} .

Corynebacterium sp. Was the first microorganism to convert eugenol to vanillin via ferulic acid ³⁴ . Eugenol is metabolized to vanillin through the formation of coniferyl alcohol, coniferyl aldehyde and ferrol acid intermediates ^26,34,35,37 . Pseudomonas sp. In the HR 199 strain, the genes involved in the conversion of eugenol to vanillin are well characterized and the ^pathways used for the synthesis of ^{2,18-20,23,25} (Fig. 1), protocatechuic acid. Is part of.

In this bacterium, the bioconversion of eugenol to coniferyl alcohol is catalyzed by the ehyA and ehyB genes encoded by the flavoytochrome c class enzyme eugenol hydroxyase ²³ . Coney perylene alcohol is converted to vanillin through an -CoA in Coney perylene di-alcohol dehydrogenase hard as by enzyme calA, calB, fcs, ech gene, and by each of the encrypted Coney perylene aldehyde, ferulic acid and metallic ferrule ^{2, 18-20} . Further conversion from vanillin to the protocatechoic eczema is by the vdh and vanAB genes encoding vanillin dehydrogenase and vanillate - O -dimethylase, respectively ²⁵ .

Compared with the relatively complex biosynthetic pathway, isoeugenol is converted to vanillin using fewer steps, including isoeugenol-epoxide and isoeugenol-diol intermediates ^11,43 (FIG. 2). Biotransformation of isoeugenol to vanillin was first reported in the Aspergillus niger ATCC 9142 fungal strain, resulting in low yield (less than 10%) due to the further degradation of vanillin to nanyl alcohol and vanic acid ¹ . However, Bacillus sp. Recently converted from isoeugenol to vanilling. Microorganisms such as B2 ³¹ , Pseudomonas putida I58 ⁹ , Bacillus fusiformis CGMCC1347 ⁴⁴ , Bacillus pumilus S-1 ¹¹ , Pseudomonas chlororaphis CDAE5 ¹³ , Pseudomonas putida IE27 ³⁹ , Bacillus subtilis HS8 ⁴³ and Pseudomonas nitroreducens Jin1 ³⁷ . However, the details of the mechanism for vanillin formation in several bacterial strains are not well known, and due to the polymerization of isoeugenol, dihydrodiisoeugenol is detected in the media ^11,37,43 . Lignostilbene-α, β-dioxygenase produced from Pseudomonas paucimobilis TMY 1009 strain was the first enzyme to bioconvert isoeugenol to vanillin ⁴² . Recently, Iso (isoeugenol monooxygenase) was purified from Pseudomonas putida IE27 ⁴⁰ . Of Iso activity it was only observed only in the presence of oxygen without the co-factors NADH ^40. However, so far only a few genes involved in the bioconversion from isoeugenol to vanillin have been identified ⁴⁰ .

However, no method has yet been reported for the production of bioconverted vanillin from isoeugenol using regulatory genes that regulate the expression of the isoeugenol monooxygenase gene.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have tried to develop a method capable of producing natural vanillin efficiently and economically using microorganisms. As a result, microorganisms were transformed with a gene capable of regulating isoeugenol monooxygenase expression and a vector expressing isoeugenol monooxygenase gene, and then the isoyugenol isoeugenol monooxygena added to the culture medium. The present invention has been accomplished by inducing an aze expression control gene to produce bioconverted vanillin from isoeugenol.

Accordingly, it is an object of the present invention to provide a method for producing vanillin bioconverted from isoeugenol.

Another object of the present invention is to provide an isoeugenol inducible expression vector.

It is another object of the present invention to provide a method for isoeugenol inducible protein expression.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for preparing biotransformed vanillin from isoeugenol, comprising the following steps: (a) the nucleotide sequence of SEQ ID NO: 2 and the sequence Transforming the host cell with a vector having a double stranded nucleic acid molecule consisting of a nucleotide sequence complementary to the host cell; (b) culturing the transformed host cell in a culture medium containing isoeugenol; And (c) obtaining vanillin bioconverted from isoeugenol from the medium of step (b).

The present inventors have tried to develop a method capable of producing natural vanillin efficiently and economically using microorganisms. After transforming the microorganism with the expressing vector, the isoeugenol added to the culture induced an isoeugenol monooxygenase expression control gene to produce bioconverted vanillin from isoeugenol.

The term “isoeugenol” in the present invention is an organic compound having a molecular formula of C ₁₀ H ₁₂ O ₂ , and vanillin is represented by the following Chemical Formula 1.

Formula 1

The term “vanillin” in the present invention is an organic compound having a molecular formula of C ₈ H ₈ O ₃ , wherein vanillin is represented by the following Chemical Formula 2.

Formula 2

As used herein, the term “isoeugenol monooxygenase” refers to an oxygenase enzyme involved in the oxidation of isoeugenol. Preferably the isoeugenol monooxygenase in the present invention catalyzes the bioconversion from isoeugenol to vanillin.

As used herein, the term “complementary” means having complementarity enough to selectively hybridize to the nucleotide sequence described in each sequence in the sequence listing under certain specific hybridization or annealing conditions. Thus, the term “complementary” has a meaning different from that of the term perfectly complementary, and is one or more mismatches so long as it can selectively hybridize to the nucleotide sequence described in each sequence in the sequence listing of the present invention. (mismatch) can have a nucleotide sequence.

Step (a) in the present invention includes the step of transforming the host cell using a vector having a double-stranded nucleic acid molecule complementary to the sequence 2nd sequence and the sequence 2nd sequence, more preferably And transforming the host cell using a vector comprising a double-stranded nucleic acid molecule complementary to the sequence listing first sequence and the sequence listing first sequence.

According to the present invention, the isoeugenol monooxygenase modulator protein induced by external isoeugenol can be produced very effectively by converting isoeugenol monooxenase gene into vanillin which is bioconverted from isoeugenol. Conventional expensive gene expression inducer IPTG (Isopropyl β-D-1-thiogalactopyranoside) has the advantage that can be economically mass-produced than vanillin produced by.

According to a preferred embodiment of the present invention, the first sequence and the second sequence of the sequence listing in the present invention are derived from a microorganism. More preferably, the first and second sequences of the sequence listing are derived from Pseudomonas genus microorganisms, and most preferably from Pseudomonas nitroreducence Jin1 (KCTC 11023BP).

As used herein, the term “nucleotide” is a deoxyribonucleotide or ribonucleotide that exists in single- or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990).

The vector system used in the present invention may be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001). This document is incorporated herein by reference.

In the specification of the present invention, the vector may have a selectable marker for selection. Preferably the selection marker is a polynucleotide encoding a gene that exhibits antibiotic resistance. Genes that exhibit antibiotic resistance are, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

Vectors that can be used in the present invention are plasmids (e.g. pDG1661, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series) And pUC19, etc.), phages (eg, λgt4? ΛB, λ-Charon, λΔz1 and M13, etc.) or viruses (eg, SV40, etc.), most preferably by manipulation of plasmids.

On the other hand, the method of introducing the above-mentioned vector into the cell can be carried out through various methods known in the art, for example, micro-injection method (Capecchi, MR, Cell , 22: 479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094-1099 (1985)), calcium phosphate precipitation (Graham, FL et al., Virology , 52: 456 (1973); and Chen and Okayama, Mol. Cell. Biol. 7: 2745 -2752 (1987)), electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982); and Tur-Kaspa et al., Mol. Cell Biol. , 6: 716-718 ( 1986)), liposome-mediated transfection (Wong, TK et al., Gene , 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta , 721: 185-190 (1982); and Nicolau et al , Methods Enzymol. , 149: 157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol. , 5: 1188-1190 (1985)), and gene bombardment (Yang et al. , Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990)) can be introduced into the cells.

The host cell may use any known host cell capable of stably and continuously expressing the vector.

According to the present embodiment invention is desired, the host cell in step (a) in the present invention is a Gram-negative bacteria, more preferably Escherichia in bacteria and, more preferably, Escherichia genus bacteria is the genus Escherichia coli (Escherichia coli), Escherichia Al Vietnam Tea (Escherichia albertii), Escherichia Blige state (Escherichia blattae), Escherichia Pere old Sony (Escherichia fergusonii), Escherichia Manny Hernandez (Escherichia hermannii) Or Escherichia fire neriseu to (Escherichia vulneris), and most preferably Escherichia coli.

According to another aspect of the invention, the invention is (i) isoeugenol inducible promoter I consisting of the nucleotide sequence of SEQ ID NO: 4, (ii) is operably linked to the isoeugenol inducible promoter I ( isoeugenol inducible comprising an isoeugenol inducible promoter II consisting of a transcriptional regulator-coding nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 3, and (iii) a nucleotide sequence of the SEQ ID NO: 5 It provides an inducible expression vector.

The expression “target protein-coding nucleotide” in the context of the present invention means CDS (coding sequence) nucleotide of the protein of interest.

The desired protein-coding nucleotide to be expressed in the present invention includes any nucleotide sequence. Preferably, the target protein-coding nucleotide is a hormone, a hormone analog, an enzyme, an inhibitor, a signaling protein or a portion thereof, an antibody or a portion thereof, a short chain antibody, a binding protein or a binding domain, an antigen, an adhesion protein, a structural protein. And nucleotide sequences encoding regulatory proteins, toxin proteins, cytokines, transcription factors, blood clotting factors, receptors or portions thereof, transport proteins, vaccine proteins or streptoavidin.

The target protein is, for example, known methods such as error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, Ly It may be a mutant protein in which a natural-occurring protein is mutated by a combination of ligation reassembly, GSSM and a combination of these methods.

Vectors of the invention can typically be constructed as a vector for expression. In addition, in the present invention, the vector may be constructed using Gram-negative bacteria or Gram-positive bacteria as a host cell, and preferably Gram-negative bacteria as a host. More preferably the bacteria Escherichia genus bacteria, and more preferably, Escherichia genus bacteria Escherichia coli (Escherichia coli), Escherichia Al Vietnam Tea (Escherichia albertii), Escherichia Blige state (Escherichia blattae), Escherichia Pere old Sony (Escherichia fergusonii), Escherichia Manny Hernandez (Escherichia hermannii) Or Escherichia fire neriseu to (Escherichia vulneris), and most preferably Escherichia coli.

The present invention can be produced more efficiently and in large quantities by introducing an isoeugenol inducible expression vector comprising isoeugenol inducible promoter II consisting of the nucleotide sequence of SEQ ID NO: 5 into the cell.

According to a preferred embodiment of the present invention, the sequence of (i)-(iii) is located in one expression vector or (iii) and (ii) is located in the first vector and (iii) is located in the second vector. Induction of expression of the protein-coding nucleotide of interest is possible.

More preferably, the nucleotide sequence of the promoter I is SEQ ID NO: 6, the nucleotide sequence of the promoter II is SEQ ID NO: 7.

According to a preferred embodiment of the invention, the isoeugenol inducible expression vector further comprises a protein-coding nucleotide sequence of interest to be expressed and the sequence is operably linked to the isoeugenol inducible promoter II.

Since the isoeugenol-inducible protein expression vector in the specification of the present invention includes a method for expressing a protein by the same mechanism as the method for producing vanillin bioconverted from the isoeugenol, the common content between the two is excessive in this specification. In order to avoid complexity, common elements between the two are omitted.

According to another aspect of the present invention, the present invention provides a method for isoeugenol-induced protein expression, comprising the following steps: (a) (i) an isoeugenol inducible promoter consisting of the nucleotide sequence of SEQ ID NO: 4 I, (ii) a transcriptional regulator-coding nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 2, which is operably linked to the isoeugenol inducible promoter I, (iii) the nucleotide sequence of SEQ ID NO: 5 Transforming the bacterium with a vector comprising isoeugenol inducible promoter II and (iii) a protein-coding nucleotide sequence operably linked to the isoeugenol inducible promoter II; And (b) treating the transformed bacteria with isoeugenol to induce expression of the protein of interest.

In the present invention, a double-stranded nucleic acid molecule comprising a sequence listing first sequence included in a vector and a sequence complementary thereto, a second strand and a double-stranded nucleic acid molecule comprising a sequence complementary thereto, a sequence listing fourth sequence and The double-stranded nucleic acid molecule comprising the complementary sequence and the double-stranded nucleic acid molecule comprising the complementary sequence of the fifth sequence and the complementary sequence include a Shine-Dalgarno sequence (or a shine-dalgarno box). It is included. The shine-dalgarno sequence means a site that binds to ribosomes on prokaryotic mRNA and is generally located upstream of the 16th nucleotide of the initiation codon AUG. In the present invention, SEQ ID NO: 4 sequence includes the shine-dalgarno nucleotide sequence of AGGATT, SEQ ID NO: 5 sequence includes the shine-dalgarno nucleotide sequence of AGGAGA.

Since the method for expressing isoeugenol-induced protein in the specification of the present invention includes a method for expressing a target protein by the same mechanism as the method for producing vanillin bioconverted from the isoeugenol, the contents in common between the two are In order to avoid excessive complexity, common elements between the two omit the description.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a method for producing vanillin biotransformed from isoeugenol, an isoeugenol induced expression vector, and a method for isoeugenol induced protein expression.

(Ii) The present invention does not require expensive gene expression inducer IPTG or ferulic acid, which is used for the expression of external genes in E. coli, thereby producing a large amount of natural vanillin at an economically low cost. There are advantages to it.

(Iii) In addition, the present invention can be applied in fields such as pharmaceuticals and foods that mass-produce proteins using microorganisms by providing expression constructs capable of producing large quantities of desired specific proteins.

1 is a schematic diagram showing a schematic diagram of the plasmid p1500 used in the present invention.
Figure 2 shows genes involved in each step of the metabolic pathway from eugenol and isoeugenol to vanic acid by P. nitroreducens Jin1. EhyAB, calA, calB, fcs, ech, vdh, vdhAB, aat and iem is eugenol hydroxylauryl dehydratase (eugenol hydroxylase), a conical perylene alcohol di hard dehydrogenase kinase (coniferyl alcohol dehydrogenase), Connie perylene aldehyde dehydrogenase (coniferyl aldehyde dehydrogenase), feruloyl-CoA-synthetase, enoyl-CoA-hydratase / aldolase, vanillin dehydrogena dehydratase (vanillin dehydrogenase), banil rate - O - dimethyl la kinase (vanillate- O -demethylase), β- Kane Todi up dehydratase (β-ketothiolase) and iso eugenol mono oxide cyclooxygenase (isoeugenol monooxygenase) encrypts each. Dihydrodiisoeugenol is a dimer of isoeugenol polymerized by P. nitroreducens Jin1.
Figure 3 shows the HPLC extraction profile of vanillic acid produced from isoeugenol by E. coli DH10B (p611A).
4 is a schematic diagram showing the gene region in each strain. Panel (A) shows a 55-kb region in P. nitroreducens Jin1, and panel (B) shows Pseudomonas sp. The 32-kb region in HR199, panel (C), compares the gene sequences of the 6.9 kb region in P. putida IE27, respectively. Pseudomonas sp. Gray bars impinged at the 32-kb DNA terminus of HR199 represent regions showing low nucleotide homology to the corresponding P. nitroreducens Jin1 region. trc1 ^* and 2 ^* are named in this study.
Figure 5 shows the HPLC extraction profile of vanillin produced from isoeugenol by E. coli BL21 (DE3) (pET-IEM) expressing the isoeugenol motooxygenase of P. nitroreducens Jin1. The chromatogram shows (A) authentic vanillin and (B) isoeugenol, and biological vanillin was incubated for (C) 10 minutes and (D) E. coli BL21 (DE3) (pET-IEM). ) From isoeugenol.
Figure 6a-6b shows the nucleotide and predicted amino acid sequence iemR and iem of P. nitroreducens Jin1. a Shine-Dalgarno (S / D) sequence of iemR (bottom strand) and iem (upper strand) was screen box. The translation start sites of the two genes are indicated by underlines and arrows. Predicted promoter sequence and the transcription initiation site is up to the -35 and -10 represents the slit or underscore expressed as TS in iemR or iem. Translation termination sites are indicated by three asterisks. The location of the hairpin-like structures downstream of iem R and iem is indicated by the insertion of an arrow.
7A-7B show homology with other bacterial transcriptional regulators and transcriptional regulators IemR and Iem (isoeugenol monooxygenase) and bacterial oxygenase, respectively, in P. nitroreducens Jin1. Figure 6a is Pseudomonas sp. HR199 (Accession number CAB69486) ORF 3, P. putida IE27 (Accession number BAF62887) Expected transcriptional regulator, Sphingobium herbicidovorans MH (Accession number CAF32814) transcriptional regulator, Acidovorax venae subsp. citrulli AAC00-1 (Accession number YP_968536) Transcription regulators of the AraC / XylS family and Bradyrhizobium sp. HW13 cad gene transcription regulators shows the result of the alignment and P. nitroreducens Jin1 IemR deduced amino acid sequence of the iemR (Accession number BAB78520). 6b shows Pseudomonas sp. LSD (the lignostilbene-dioxygenase) ^{33 of} HR199 (Accession number CAB69485) ³³ , Iso (isoeugenol monooxygenase) ⁴⁰ of P. putida IE27 (Accession number BAF62888), LSD-I (lignostilbene-α) of S. paucimobilis TMY1009 (Accession number AAC60447) , β-dioxygenase isozymes I) ¹² , carotenoid oxygenase (CRTO) ⁷ and Synechocytis sp. of Novosphingobium aromaticivorans DSM 12444 (Accession number YP_496081). It shows the result of the alignment and iso eugenol mono oxide cyclooxygenase of PCC 6803 (Accesion number NP_441748) of ACO (apocarotenoid-15,15'-oxygenase) of P. nitroreducens Jin1 (Jin1_Iem) deduced the amino acid sequence of ¹⁴ to iem . Matching sequences are shown in the last column. A gap (-) was included in the sequence to align in line. White letters on a black background represent homologous amino acid residues. Typical amino acid residues are indicated in white letters on gray background. Black letters on a gray background mean blocks of similar residues. Somewhat similar residues are indicated in black letters on a light gray background.
FIG. 8 shows a dendrogram of P. nitroreducens Jin1 Iem and other bacterial oxygenases. The amino acid sequences were aligned and used as a phylogenetic tree using the Vector NTI9.0 program.
9 is P. nitroreducens of eugenol and iso-eugenol metabolic genes involved in metabolism processes in Jin1 (ehyA, calA, calB, fcs, ech, aat, vdh and iem) real-time RT-PCR (Real- for the transfer of the Time reverse transcription PCR analysis. Grey, white, black and dashed bars represent glucose, eugenol, isoeugenol and vanillin added to the medium as carbon sources of P. nitroreducens Jin1 and error bars represent standard deviations.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Unless stated otherwise, solids / solids are (weight / weight) parts or%, solids / liquids are (weight / volume) parts or%, and liquids / liquids are (volume / volume) parts or%.

Materials and methods

Bacterial Strains, Plasmids and Media

The bacterial strains and plasmids used in this study are shown in Tables 1 and 2. Pseudomonas nitroreducens Jin1 was incubated in nutrient broth or basal minimal medium containing a carbon source ³⁷ . Escherichia coli strains were cultured in LB (Luria-Bertani) medium or M9 minimal medium ¹⁹ , and ampicillin and chloramphenicol were added at 50 and 12.5 μg / ml, respectively, if necessary.

Amp stands for ampicillin and Chl stands for chloramphenicol.

Amp stands for ampicillin and Chl stands for chloramphenicol.

BAC Library Fabrication

P. nitroreducens Jin1 was cultured in nutrient broth medium, fixed on 1% agarose plugs, and then the method disclosed in (http://www.hpa.org.uk/web/HPAwebFile/HPAweb_C/1194947324361). Dissolved accordingly. Genomic DNA was partially cut using Hind III and fragments were separated using CHEF-DR II (Bio-Rad, Richmond CA, USA) pulsed field gel electrophoresis (PFGE). After recovering DNA fragments (100-150 kb) from agarose gels, cloned into pIndigoBAC536 ¹⁵ digested with Hind III and transformed E. coli DH10B using an electroporator (Eppendorf, Hamburg, Germany). One hundred hundred chloramphenicol resistant white colonies were collected and stored at -70 ° C.

Library Screening and Gene Sequencing

Each clone obtained from the P. nitroreducens Jin1 BAC library was inoculated into 24-well microplates containing 1 ml LB medium and then incubated overnight at 37 ° C. at 200 rpm. 100 μl of each culture from 8 wells were put in 160-ml serum bottles each containing 20 ml LB medium and 500 isoeugenol and incubated at 30 ° C. for 20 hours. The bacterial cultures were recovered by centrifugation at 6,000 × g for 10 minutes and cells were washed twice with pH 7, 20 mM phosphate buffer. 2 ml MSB medium residual cell suspension was added at OD 600 nm = 10 and supplemented with 50 isoeugenol and incubated at 30 ° C. for 20 hours. Isoeugenol and metabolites were extracted with 1 ml of ethyl acetate and analyzed by gas chromatography. Individual clones showing isoeugenol conversion in the reaction mixture were tested for isoeugenol metabolism. BAC p611A converting isoeugenol was isolated using a Qiagen Large-Construct kit (Qiagen, Valencia, Calif.), And was sequenced by Macrogen (Seoul, Korea).

RNA extraction and cDNA synthesis

P. nitroreducens Jin1 was cultured in nutrient broth without pre-induction. Isoeugenol, eugenol and vanillin were added to the culture medium at a final concentration of 3 mM and then incubated at 27 ° C. at 200 rpm. Negative control cultures contained 7 mM glucose as a single carbon source. Except for the culture containing vanillin with OD 600 nm = 0.25, 500 μl cell culture was mixed with 1 ml of RNAprotect Bacteria Reagent (Qiagen, Valencia, CA) when the optimal cell density reached OD 600 nm = 0.45. . The mixture was incubated for 5 minutes at room temperature for 5 minutes and total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA). DNA contained in the RNA samples was removed using RQ1 RNase-Free DNase (Promega, Madison, Wis.) For 30 minutes at 37 ° C. The same amount of each phenol / chloroform / isoamyl alcohol solution was added to stop the enzymatic reaction, and ethanol was added to recover RNA. RNA concentrations in the samples were measured using a NanoDrop spectrometer (NanoDrop Technologies, Wilmington, DE), and total RNA samples showed a 260/280 ratio between 1.8 and 2.1. 500 ng RNA was reverse transcribed using 50 pmol random hexamer primers (Genotech, Deajeon, Korea) and 200 U CycleScript reverse transcriptase (Bioneer, Deajeon, Korea) to synthesize cDNA. The 30-μl reaction mixture contained 1 mM DTT, 0.25 mM dNTP, and 5X reaction buffer. CDNA was synthesized using Mastercycler personal (Eppendorf, Hamburg, Germany) under the following conditions: reverse transcription primer annealing at 15 ° C. for 30 seconds, cDNA synthesis for 4 minutes at 42 ° C., RNA template secondary structure at 55 ° C. Denaturation and cDNA synthesis step are 12 cycles, inactivating reverse transcriptase for 5 minutes at 90 ℃.

Real-Time reverse transcription (RT) PCR.

Real-time RT-PCR primers were prepared from P. nitroreducens Jin1 based on eight metabolic genes ( ehyA, calA, calB, fcs, ech, vdh, iem and aat ) and predictive regulatory gene ( iemR ) sequences (Table 3). . cDNAs were diluted 2-fold with RNase-free water and 2 μl diluted samples were used for real-time PCR analysis. CDNA was further diluted 10-fold for 16S rRNA amplification. The forward and reverse primers of each gene were used at 0.3 μM concentration. 2X QuantiTech SYBR Green Master Mix (Qiagen, Valencia, Calif.) And RNase-free water were used with 25 μl total reaction volume. The cycle conditions are as follows: 15 minutes 1 cycle at 95 ° C., 30 seconds at 94 ° C., 30 seconds at 53 ° C. and 30 cycles at 72 ° C., 30 seconds. After the final cycle, the melting protocol is maintained for 45 seconds at 53 ° C., heated at 53-95 ° C. and 5 seconds at each 1 ° C. increased temperature during fluorescence monitoring. Real-time PCR was performed using Rotor-Gene 3000 (Corbett Research, Sydney, Australia), and the relative value (RV) of the number of mRNA copies for each gene was calculated using the following equation: RV = ( Number of gene copies) / (number of iemR copies)

Subcloning and Functional Analysis of the Iem (isoeugenol monooxygenase) Gene in E. coli

The isoeugenol motooxygenase gene was PCR amplified using forward primer 5′-AAAACATATGGCGAGACTCAACCGCAAC-3 ′ and reverse primer 5′-AAAACTCGAGTTAAGGTCTGGGTACCCAGC-3 ′. In addition, stop codons (TAA) were added to the reverse primers. PCR products amplified in pGEM-T easy vectors (Promega, Madison, Wis.) were cloned and the plasmids were designated as pG-IEM. In using the electrophoretic purification of the pG-IEM in iem gene fragment, and by connecting the pET21-a was produced pET-IEM, E. coli BL21 ( DE3) - agarose gel was cut with Nde I and Xho I The prepared pET-IEM was transformed. His-tag in pET-IEM did not fuse with the iem gene due to the stop codon preceding the Xho I sequence in the reverse primer. E. coli BL21 (DE3) (pET-IEM) was inoculated in LB medium, incubated with shaking at 100 rpm at 37 ° C. until the OD 600 nm reached 0.1-0.2, and then incubated at 20 ° C. When the OD 600 nm value reached 0.4 to 0.6, 0.1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to the cell medium and further incubated at 20 ° C. at 100 rpm for 12 hours. Cells were harvested by centrifugation at 6,000 × g for 10 minutes, washed twice with pH 7.0, 20 mM phosphate buffer, and bacterial pellets suspended in phosphate buffer until OD 600 nm = 5. After addition of 2 mM isoeugenol to a 30 ° C. 50-ml serum bottle, the 3 ml cell suspension was incubated with shaking at 200 rpm. The mixture was extracted with 6 mL ethyl acetate, and 4 mL ethyl acetate was dried in vacuo . The dry sample was dissolved in 0.5 ml methanol and analyzed using 0.5 ml high performance liquid chromatography (HPLC) as follows.

Upstream Region Analysis of the IEM (isoeugenol monooxygenase) Gene

Using PCR to investigate the 50-1,500 bp gene region of the region upstream of the start iem prepare a forward primer. Reverse primers were designed to amplify the 150 bp downstream region of the translation stop codon of the iem gene. Bgl II recognition sequences were added to all PCR primers, and the amplified PCR products were digested with Bgl II and linked to a pKK232-8 vector without a proboter digested with Bam HI ²² . E. coli BL21 (DE3) ^41, which does not have vanillin metabolism, was used as the host strain in this experiment. Cells were cultured in LB medium containing ampicillin (50 μg / ml) with shaking at 200 rpm for 12 hours at 37 ° C. Cells grown in LB (1%) were seeded in 250-ml flasks containing 50 ml of M9 minimal medium with 0.4% (w / v) glucose. Cells were incubated with shaking at 200 rpm for 12 hours at 30 ° C., harvested by centrifugation, and washed twice with M9 medium. Cells were transferred to 160-ml serum bottles containing 30 ml of M9 medium with 0.4% glucose and incubated with or without 0.5 mM isoeugenol, eugenol and vanillin at OD 600 nm = 0.1. The bacterial cultures were incubated with shaking at 200 rpm for 12 hours at 30 ° C. and recovered by centrifugation. Cells were washed with pH 7, 20 mM phosphate buffer, suspended in the same buffer at OD 600 nm = 1.0 and 1 ml cell suspension was added to a 15-ml serum bottle containing 1 mM glucose and 2 mM isoyugenol. The suspension was incubated with shaking at 200 rpm for 2 hours at 30 ° C. and extracted with 1 ml ethyl acetate to determine the amount of vanillin produced using HPLC according to the method described below. Cells grown in LB medium without substrate were used as negative controls and all experiments were repeated three times.

Analysis method

Substrates and metabolites were detected using a Shimadzu GC-17A gas chromatograph (GC) equipped with a flame ionization detector (Shimadzu, Kyoto, Japan). A 1 μl aliquot of ethyl acetate extract was injected into the GC using a gas-tight syringe (Hamilton, Reno, NV). The GC was equipped with an RTX-5 capillary column (30 m, 0.32 mm and 1.0 μm) and an injector and the detector temperature was 300 ° C. The colum temperature was set at 200 ° C. for 5 minutes, the temperature was raised to 250 ° C. at a rate of 15 ° C./min, then increased to 300 ° C. at a rate of 50 ° C./min, and left for 2 minutes. HPLC was performed using Varian ProStar HPLC equipped with a photodiode array (PDA) analyzer (Varian, Walnut Creek, Calif.) And reversed-phase C18 Spherisorb (Waters, Milford, Mass.) Colum (5 μm particle size, 4.6 mm x 25 cm) Was carried out. The mobile phase consisted of acetonitrile with 0.1% formic acid and water, and the gradient was as follows: 0 to 10% acetonitrile, 5 to 20% acetonitrile, 12 to 40 Stand in% acetonitrile, 90% acetonitrile at 17 minutes, and 90% acetonitrile for 5 minutes.

20 μl aliquots of each sample were injected at a flow rate of 1 ml / min. UV detection was performed at 270 nm.

Nucleotide Sequence Access Number

The nucleotide and amino acid sequence data described in this study were submitted to GenBank under accession number FJ851547.

Results and review

Library screening

The BAC library of total DNA from P. nitroreducens Jin1 contains insert DNA in the range of 50-100 kb, after which 1,100 BAC clones were examined and one colon, E. coli DH10B (p611A), replaced isoeugenol. Converted to nilic acid (FIG. 2). However, clone p611A did not convert eugenol to vanic acid. In addition, no dihydrodiisoeugenol, an isoeugenol dimer, was detected in the supernatants incubated with isoeugenol (FIG. 2). The compound has already been detected in culture medium inoculated with P. nitroreducens Jin1 ³⁷ .

Sequence and annotation of open reading frames (ORFs)

Insert DNA in BAC p611A was sequenced and 57.4 kb Contig 1 and 80.5 kb Contig 2 were constructed. The contigs from about 30 kb of the gene region 2 is reported to be involved in the metabolism of the prototype catheter chyuik acid from eugenol by vanillin intermediates Pseudomonas sp. It showed 98.5% sequence homology with the 30 kb gene region in HR199 (FIG. 4) ³³ . 4 is Pseudomonas sp. A comparison of ORFs detected in regions sequenced from P. nitroreducens Jin1 to annotate ORFs from HR199 (Accession No A92090) and P. putida IE27 (Accession No AB291707). The results in FIG. 4 are exactly the same as those found in P. nitroreducens Jin1 and the gene sequence found in the eugenol metabolic gene cluster of Pseudomonas sp.HR199 and 23 ORFs transcription directions. However, Pseudomonas sp. The 1,898 nucleotide gene region 1,248 bp upstream of the predicted transcription start site of ORF 1 in HR199 showed 45% nucleotide homology with 2,103 nucleotides upstream of the ORF 38 predicted transcription start site in P. nitroreducens Jin1 (FIG. 4). Although P. putida IE27 contains iso genes for isoeugenol metabolism, the array of genes surrounding iso is ORF 27 and lsd of P. nitroreducens Jin1 , and Pseudomonas sp . Each was different from the arrangement of surrounding genes of HR199.

P. putida IE27 (orf3) of vanillin in ⁴⁰ dehydrogenase amino acid sequence is shown, but the vanillin dehydrogenase (ORF 24) and 35.5% amino acid homology in the P. nitroreducens Jin1, P. nitroreducens Jin1 38 ORFs of And P. putida IE27 orf2 showed no similarity with amino acid sequence. ORFs 16 to 38 found in the -30 kb gene region of P. nitroreducens Jin1 in Contig 2 in BlastP assay were identified as Pseudomonas sp. It had very high amino acid sequence homology (83.2-100%) with the amino acid sequence for 23 ORFs found in the HR199 Eugenol metabolic gene cluster. Similarity values for the proteins translated from each ORF are shown in Table S2. Ten of 23 ORFs from ORF 16 to ORF 38 of P. nitroreducens Jin1 were identified as Pseudomonas sp. It may be a metabolic gene homologous to the genes used for eugenol metabolism by HR199. These P. nitroreducens Jin1 genes are encoded coniferyl alcohol aldehydes ( calA ), coniferyl aldehyde dehydrogenase ( calB ), aat (β-ketothiolase) represented by ORFs 16, 19, 22, 23, 24, 25, 30, 32, 35, and 36. ), fcs (feruloyl-CoA-synthetase), vdh (vanillin dehydreogenase), ech (enoyl-CoA-hydratase / aldolase), ehyB (eugenol hydroxylase flavoprotein subunit), ehyA (eugenol hydroxylase cytochrome C subunit), vanB (vanillate- O -demethylase oxidoreductase reductase-subunit) and vanA (vanillate- O -demethylase oxidoreductase a-subunit) (FIG. 4). ORF 27 has been annotated with isoeugenol monooxygenase and is predicted to be involved in isoeugenol metabolism. In addition, these gene regions included ORF 10, 17, 18, 20, 26 and 37, six predictive transcriptional regulators. ORF 37 of P. nitroreducens Jin1 was expressed in P. putida WCS358 (Accession number CAB64602) and Pseudomonas syringae pv. tomato str. Amino acid homology of 67.8% and 66.4% with VanR protein in DC3000 (Accession number NP_792704), respectively. The ORF was cleaved and transcribed from P. nitroreducens Jin1 vanAB and predicted as a transcriptional regulator for vanAB , which encodes vanylate - O -dimethylase. We found that genes involved in the metabolism of eugenol and isoeugenol were closely arranged to share metabolites in two different metabolic pathways of eugenol and isoeugenol, finally by vanillin and P. nitroreducens Jin1. Estimated ³⁷ . ORFs (ORFs 26 and 27), thought to be involved in the metabolism of isoeugenol to vanillin, were located in the eugenol metabolic gene cluster of P. nitroreducens Jin1.

Contig 2 also contained two integrases (encoded in ORFs 2 and 4) and was located downstream of the cluster involved in eugenol metabolism by P. nitroreducens Jin1. ORF 2 amino acids are P. putida GB-1 (Accession number YP_001671010), Pseudomonas syringae pv. tomato str. DC3000 (Accession number NP — 794482) and Marinomonas sp. Homologous phage integrase of MWYL1 (Accession number YP_001343052) showed 38.8%, 37.7% and 35.0% homology, respectively. Similarly, the ORF 4 amino acid is P. putida GB-1 (Accession number YP_001671012), P. syringae pv. tomato str. DC3000 (Accession number NP — 794484) and Marinomonas sp. The homology of 33.3%, 33.0% and 37.3% was shown for phage integrase of MWYL1 (Accession number YP_001343050), respectively. There was no significant amino acid sequence homology between ORF 2 and ORF 4. The 30 kb eugenol metabolic gene regions found in P. nitroreducens Jin1 and HR199 showed very high nucleotide sequence homology (98.5%), while the 1.9 kb region from the HR 199 32 kb region showed low homology (45%). And located in genomic DNA ²⁵ . According to Method ³⁴ described in the prior art, Pseudomonas sp. Partial 16S rDNA gene of HR199 was amplified and sequenced. Based on PHYDIT program analysis ⁶ , the nucleotide sequence of partial 16S rDNA from HR199 strain was best matched with Pseudomonas nitroreducens (ATCC 33634) strain with 99.23% (10/1307) homology, followed by P. nitroreducens Jin1 ( Accession Number EF534986) and 98.92% homology (15/1392). These results indicate that bacteria isolated geographically from different regions contain almost similar DNA clusters in the metabolism of the plant-derived phenylpropanoid compounds eugenol and isoeugenol. However, P. nitroreducens Jin1 and Pseudomonas sp. A very homologous and large genetic region in HR199 suggests that the genetic region has been obtained by lateral gene transfer of these microorganisms isolated from different geographical regions. Gene horizontal transfer between microbial species has been shown to play a pivotal role in the evolution and adaptation of prokaryotes ^17,32 . This phenomenon has been shown to occur through mobile genetic elements (MGE) including plasmids, integral and conjugative elements (ICE), transposons, insertion sequences (IS) and homologous recombination ^17,32 . Recently, Gaillard ¹⁰ et al. Described Pseudomonas sp. On> 100 kb DNA fragments containing homologous 18 bp sequence ²⁸ of tRNA ^Gly at genomic islands (GEI) and intB13 (integrase genes) and at the left and right ends ( attL and attR ). The clc component of the B13 strain was compared with the clc component of several bacteria capable of metabolizing 3- and 4- chlorocatechol . Surprisingly, Pseudomonas sp. The B13 strain clc component showed 100% homology over the total length of chromosomal sites found in Burkholderia xenovorans LB400 except for two separate regions ¹⁰ . In this study, two integrases encoded by ORFs 2 and 4 were identified as Pseudomonas sp. It was not similar to the integrase gene sequence found in the B13 strain and was not flanked by attL and attR . Therefore, while the eugenol metabolic gene regions found in P. nitroreducens Jin1 and Pseudomonas HR199 are almost homologous, it is not yet known how these migrate and connect in different microorganisms. Understanding these mechanisms requires further study.

P. nitroreducens Jin1 ORFs 26 and 27.

ORF 27 of P. nitroreducens Jin1 was identified as Pseudomonas sp. HR199 ³³ amino acid sequence of lignostilbene dioxygenase and P. putida IE27 (Accession number BAF62888) ⁴⁰ Iso, respectively The homology was 97.9% and 81.4%. E. coli DH10B with BAC p611A was analyzed to convert isoeugenol to vanillin. These results meant that the ORF 27 iem (isoeugenol monooxygenase). The activity of iem was further confirmed through heterologous expression of ORF 27 by regulating the T7 promoter in E. coli BL21 (DE3), producing vanillin from isoeugenol (FIG. 5). iemR and iem genes consists of 951 and 1437 nucleotides, respectively, iem gene was present in a single copy in P. nitroreducens Jin1. and the iemR iem The protein sizes predicted from the amino acids were 34.5 kDa and 54.4 kDa, respectively.

iemR isopropyl emulsion of P. nitroreducens Jin1 containing play mono oxide cyclooxygenase gene upstream region is to determine whether the gene expression involved in iem, -1500, -502, -200, -100 and -50 bp upstream of the translation initiation codon Five plasmids containing the structural regions of the and iem genes were prepared using a promoter-free pKK232-8 plasmid. The p1500 plasmid included all coding regions of iemR (Table 4). The highest Iem activity producing 1 mM vanillin from isoeugenol was observed in the remaining cells containing the p1500 plasmid incubated with glucose and isoeugenol (Table 4). iem and Other plasmids containing fragments of the upstrip region did not show Iem activity. Cells cultured in minimal medium containing glucose and eugenol or glocose and vanillin, or LB medium, exhibited about 14%, 14% and 4% activity of cells grown in minimal medium containing glucose and isoeugenol (Table 4). The IemR the results of the present study was found to induce transcription atom iem gene.

^a plasmid thought illustrates the number of nucleotides of Plasmid name indicates number of nucleotides of upstream region of iemgene inserted into a region upstream of the gene iem sapin the plasmid pKK232-8.

^{The b} value represents the mean ± standard deviation of three experiments.

^c The amount of vanillin not detectable.

iemR and iem are each estimated transfer Adjuster (ORF 26) and iso eugenol mono oxide cyclooxygenase (ORF 27) refers to the gene.

iem's transcription modulator

3.1 kb nucleotide sequences from 1500 bp upstream to 163 bp downstream of iem are shown in FIGS. 6A-6B. 951 bp which is located upstream of iemR iem is transferred is derived from iem. The transcription initiation codon (ATG) of iemR is advanced at the -175 nucleotide position by the Shine-Dalgarno sequence (AGGATT) at six nucleotides distance and 99 bp upstream of the predicted transcription initiation site of the transcription initiation codon. The predicted promoter sequence (TTGCCG-N ₇ -GTTTATGTT) was located 10 nucleotides upstream of the transcription initiation site (FIG. 6A). The iem transcription initiation codon (ATG) is located at +1 and is advanced by the Shine-Dalgarno sequence (AGGAGA) at 8 nucleotides distance and the transcription initiation site located 48 bp upstream of the predicted transcription initiation site of the transcription initiation codon. . The iem promoter sequence (ATGAAA-N ₁₄ -AACATAAAC) was located 8 nucleotides upstream of the transcription initiation site (FIG. 6A). a transcription termination sequence for iemR iem and is represented by the inserted repeat sequence will be positioned at 15 and 26 bp down stream of the termination codon of each gene (Figure 6a-6b).

Alignment of the amino acid sequence of IemR and five other bacterial transcriptional regulators revealed by BlastP analysis is shown in FIG. 7A. The amino acid sequence of the iemR gene is Pseudomonas sp. HR199 (Accession number CAB69486) ORF 3, P. putida IE27 (Accession number BAF62887) Expected transcriptional regulator, Sphingobium herbicidovorans MH (Accession number CAF32814) transcriptional regulator, Acidovorax venae subsp. citrulli AAC00-1 (Accession number YP_968536) Transcription regulators of the AraC / XylS family and Bradyrhizobium sp. The amino acid sequence of the HW13 cad gene transcription regulator (Accession number BAB78520) was 97.5%, 46.6%, 26.5%, 26.3% and 22.4% homology, respectively. The transcriptional regulator AraC / XylS family generally acts as an activator in the presence of chemical effectors. The N-terminal portion of the family of transcription factors is changeable and binds to a variety of inducers, while the C-terminal portion contains helix-turn helix motifs that mediate DNA-binding ^16,29,36 . The helix-turn helix prediction tool ^{10 indicated} that IemR and other modulators contained helix-turn-helix motifs from 236 to 257 of the aligned amino acid sequence (FIG. 7B). In addition, the C-terminal sequence of P. nitroreducens Jin1 IemR (229-313 nucleotides) is described in P. putida IE27, S. herbicidovorans MH, A. venae subsp. citrulli AAC00-1 and Bradyrhizobium sp. The amino acid sequence of the HW13 transcriptional regulator showed 54.7%, 43.5%, 34.1% and 41.4% homology, respectively.

We report that P. nitroreducens Jin1 strains preincubated with eugenol, isoeugenol and vanillin grew faster in minimal media containing isoeugenol as a carbon and energy source than cells grown without preincubation. ³⁷ . In this study, Iem was well expressed by E. coli containing iemR and iem, iem expression was induced by eugenol, iso-eugenol and vanillin. In contrast, vanillin-induced activity was not induced by eugenol and vanillin in P. putida IE27 ³⁷ . These results indicate that IemR in P. nitroreducens Jin1 and orf 1 in IE27 strains responded to vanillin and eugenol inducers differentially.

Relationship between P. nitroreducens Jin1 Iem and other oxygenases

P. nitroreducens Jin1 the nucleotide sequence of iem was compared with the nucleotide sequence of various bacteria oxide cyclooxygenase. In BlastN results in Figure 3, iem the Pseudomonas sp. Lsd of lsd (lignostilbene dioxygenase) gene, P. putida IE27 iso (isoeugenol monooxygenase ) gene and the S. paucimobilis TMY1009 (Accession number S65040) of (Accession number AB291707) of HR199 (Accession number A92110) (lignostilbene -α, β-dioxygenase isozymes I) gene and 98.6, 70.6 and 53.5 nucleotide homology, respectively.

The amino acid sequence of five bacterial oxygenases and the Iem amino acid sequence of P. nitroreducens Jin1 were compared (FIG. 7B). Iem amino acid sequence is Pseudomonas sp. LSD (the lignostilbene-dioxygenase) ^{33 of} HR199 (Accession number CAB69485) ³³ , Iso (isoeugenol monooxygenase) ⁴⁰ of P. putida IE27 (Accession number BAF62888), LSD-I (lignostilbene-α) of S. paucimobilis TMY1009 (Accession number AAC60447) , β-dioxygenase isozymes I) ¹² , carotenoid oxygenase (CRTO) ⁷ and Synechocytis sp. of Novosphingobium aromaticivorans DSM 12444 (Accession number YP_496081). The amino acid sequence of the acocarotenoid-15,15'-oxygenase (ACO) ¹⁴ amino acid sequence of PCC 6803 (Accesion number NP_441748) was 97.9, 81.4, 41.8 and 39.2, respectively. Iso of P. putida IE27 does not require NADH or metal ions for its activity ⁴⁰ . Similarly, Iem purified from P. nitroreducens Jin1 did not require NADH or externally added metals to exhibit the activity of converting isoeugenol to vanillin under aerobic conditions. In contrast, lsd for dioxygen activity And ACO have been shown to require iron metal ions ^12,14 . Recently, four histidine residues at positions 183, 238, 304 and 484 in the crystal structure of ACO have been known to bind four ligands of Fe ²⁺ . Interestingly, these four histidine residues are conserved in lined bacterial oxygenase (indicated by the arrow in FIG. 7B). In addition, the phylogenetic tree of the six bacterial oxygenases is divided into two groups (FIG. 8). Future structural studies of Iem will find it useful to determine how oxygen atoms bind to isoeugenol without the help of NADH and metal cofactors.

Induction of P. nitroreducens Jin1 genes in cells grown under different carbon sources

6 genes (ehyA, calA, calB, fcs , ech and vdh) and two genes related to the metabolism of the vanillin from the iso eugenol (iemR and iem) and ferrule associated with the metabolic processes of the bar nilrik acid from eugenol Real-time reverse transcription PCR (RT-PCR) was used to investigate the effect of carbon sources on the pattern of induction of single genes ( aat ) associated with secondary metabolism from Rick to vanillyl-CoA. RT-PCR values were normalized to copy number of iemR gene. In FIG. 9, the relative values of the seven genes induced by eugenol were the highest except for iem among the tested samples. In P. nitroreducens Jin1, ehyA, calA, calB, fcs, ech, vdh and aat genes were more strongly induced by eugenol than isoeugenol, vanillin or glucose. Ech coding for the enzymes that convert an -CoA (feruloyl-CoA) to vanillin by a ferrule has been induced more than the other gene. Further, the eugenol has led to the expression of iem by inducing expression of ehyA, calA, calB, fcs, aat and vdh. And, iso eugenol was induced (which is involved in the process of metabolism of alcohol in Coney perylene Coney perylene aldehyde) iem, and ech calA expression. However, unlike eugenol , isoeugenol did not induce ehyA, calB, fcs, aat and vdh gene expression. In addition, vanillin induced low levels of transcription of all genes, and the induction pattern was similar to that found for eugenol inducers. Glucose did not induce most of the gene expression tested except for ech and iemR . The mRNA copy numbers of regulatory iemRs when glucose, isoeugenol, eugenol and vanillin were included were 156, 158, 116, and 375, respectively, but were relatively uninduced compared to the induction of other genes. These results are transferred to the structural iemR low level in P. nitroreducens Jin1 means is increased under the transcription of iem iso eugenol present. This pattern is similar to nodD gene induction in Bradyrhizobium japonicum USDA 110 ³ .

Vanillin dehydrogenase encoded by vdh was identified as Pseudomonas sp. It is known that HR199 is involved in the bioconversion of vanillin to vanic acid ²⁰ . In P. nitroreducens Jin1, expression of vdh was induced about 39-fold higher during growth with eugenol than when cells were grown with vanillin. In the previous study, ^26,37 , vanillin, an intermediate product during the bioconversion from ferulic acid to vanillic acid, was synthesized on Pseudomonas sp. No HR199 and P. nitroreducens Jin1 strains were detected during growth. However, vanillin was observed in culture media containing P. nitroreducens Jin1 residual cells and isoeugenol ³⁷ . This result is not surprising considering the transcription pattern of P. nitroreducens Jin1 gene induced by isoeugenol.

In summary, we have identified a gene region containing isoeugenol monooxygenase that can convert isoeugenol to vanillin in a single step reaction. So far, this study is the first report on the two genes, ie functions of iem and iemR that are related to the metabolism of vanillin from eugenol isobutane. Isoeugenol monooxygenase is a single component enzyme and has been found to provide a biocatalytic route for the microbial production of natural product vanillin. This is in contrast to previously reported complexes, multicomponents, oxygenases such as toluene deoxygenase and styrene monooxygenase ^4,45 . In addition, the present researchers have presented evidence that a bacterial strain containing and iemR iem gene provides a useful and cost-effective means of producing vanillin from natural products play isopropyl emulsion. Such compounds may play a dual functioning role as substrates and gene inducers required for vanillin biosynthesis.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Attach an electronic file to a sequence list

Claims (11)

Biotransformed vanillin preparation from isoeugenol comprising the following steps:
(a) isooperatively coupled to the isoeugenol inducible promoter I consisting of the nucleotide sequence of SEQ ID NO: 4 in the 5 'to 3' direction, and (ii) the isoeugenol inducible promoter I (operatively linked) a vector comprising a transcriptional regulator-coding nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 3 and (i) an isoeugenol inducible promoter II consisting of the nucleotide sequence of SEQ ID NO: 5 in the host cell Converting;
(b) culturing the transformed host cell in a culture medium containing isoeugenol; And
(c) obtaining vanillin bioconverted from isoeugenol from the medium of step (b).
The method according to claim 1, wherein the third to fifth sequences of the sequence listing are sequences derived from Pseudomonas nitroreducens Jin1 strain (KCTC 11023BP).
The method of claim 1, wherein the host cell is a Gram-negative bacterium.
4. The method of claim 3, wherein the Gram-negative bacterium is a bacterium of the genus Escherichia .
The method of claim 4, wherein the Escherichia genus bacterium is Escherichia coli (Escherichia coli), Escherichia al Corbett T (Escherichia albertii), Escherichia Blossom state (Escherichia blattae), Escherichia FER obtain Sony ( Escherichia fergusonii ) , Escherichia hermannii Or wherein the Escherichia fire in neriseu (Escherichia vulneris).
(I) isoeugenol inducible promoter I consisting of the nucleotide sequence of SEQ ID NO: 4 in the 5 'to 3' direction, and (ii) a operatively linked sequence to the isoeugenol inducible promoter I Isoeugenol inducible expression comprising a transcriptional regulator-coding nucleotide sequence consisting of the nucleotide sequence of list 3, and (iii) an isoeugenol inducible promoter II consisting of the nucleotide sequence of sequence 5 vector.
The method of claim 6, wherein the sequence of (i)-(iii) is located in one expression vector, or (iii) and (ii) is located in a first vector, and (iii) is located in a second vector. Expression vector.
7. The expression vector according to claim 6, wherein the isoeugenol inducible expression vector further comprises a protein-coding nucleotide sequence of interest to be expressed, and the sequence is operably linked to the isoeugenol inducible promoter II. vector.
Isoeugenol inducible protein expression method comprising the following steps:
(a) (i) isoeugenol inducible promoter I consisting of the nucleotide sequence of SEQ ID NO: 4, (ii) the nucleotide sequence of SEQ ID NO: 3 operably linked to the isoeugenol inducible promoter I A target protein operably linked to the isoeugenol inducible promoter II consisting of a transcriptional regulator-coding nucleotide sequence consisting of (i) the nucleotide sequence of SEQ ID NO: 5, and (iii) the isoeugenol inducible promoter II Transforming the bacteria with a vector comprising the coding nucleotide sequence; And
(b) treating the transformed bacteria with isoeugenol to induce expression of the protein of interest.
10. The method of claim 9, wherein the bacteria are Gram-negative bacteria or Gram-positive bacteria.
The method of claim 10 wherein the bacteria is characterized in that the Escherichia coli (Escherichia coli).

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