KR100811385B1 - 26- Method for preparing Microorganism expressing Xylene monooxygenase and Method for production of 26-naphthalene dicarboxylic acid using the microorganism - Google Patents

Abstract

The present invention relates to a method for preparing a recombinant microorganism expressing xylene monooxygenase and a method for preparing 2,6-naphthalene dicarboxylic acid using the same, and more particularly, xyl derived from Pseudomonas putida Obtaining a gene encoding Xylene monooxygenase; Inserting the gene into a recombinant expression vector; And obtaining a recombinant microorganism by the method comprising transforming the host cell with the expression vector, and producing 2,6-naphthalene dicarboxylic acid by oxidizing 2,6-dimethylnaphthalene using the microorganism. When the method of the present invention is used, it is possible to efficiently provide 2,6-naphthalene dicarboxylic acid by a biological process.

Xylene monooxygenase, xylM / A gene, 2,6-dimethylnaphthalene, 2,6-naphthalene dicarboxylic acid

Description

Method for preparing microorganism expressing Xylene monooxygenase and Method for production of 2,6-naphthalene dicarboxylic acid using the microorganism}             

1 is a diagram showing the step-by-step oxidation mechanism by the upper TOL metabolic pathway enzyme and the upper TOL operon xyl genes.

2 is a cleavage map of a recombinant expression vector ForexTxylM / A comprising a gene encoding xylene monooxygenase derived from Pseudomonas putida.

3 is a diagram showing an oxidation path converted from 2,6-dimethylnaphthalene to 2,6-naphthalene dicarboxylic acid by xylene monooxygenase.

4 is a high performance liquid chromatography analysis graph capable of confirming 2,6-naphthalene dicarboxylic acid production by recombinant microorganisms in which xylene monooxygenase is inserted.

The present invention relates to a method for preparing a recombinant microorganism expressing xylene monooxygenase and a method for preparing 2,6-naphthalene dicarboxylic acid (2,6-NDA) using the same. In more detail, obtaining a gene encoding Xylene monooxygenase from Pseudomonas putida ( mt-2 (ATCC 33015)); Inserting the gene into a recombinant expression vector; And 2,6-dimethylnaphthalate (2,6-dimethyl) using a method for producing a xylene monooxygenase-expressing microorganism comprising transforming a host cell with the expression vector and a microorganism obtained therefrom. Naphthalate; 2,6-DMN) to a method for producing 2,6-naphthalene dicarboxylic acid.

2,6-naphthalene dicarboxylic acid is a monomer useful in the preparation of high performance polymeric materials such as polyesters and polyamides. Representative high performance polymer materials include polyethylene 2,6-naphthalate (hereinafter referred to as "PEN"), which is now known as 2,6-naphthalene dicarboxylate (NDC) and ethylene glycol. It is synthesized by polymerization. High strength fibers made from PEN can be used to make tire cords, and films made from PEN are useful for making magnetic recording tapes or electronic components. In addition, polyesters prepared from mixtures of terephthalic acid with 2,6-NDA or dimethyl-2,6-naphthalene dicarboxylate have been found to have unique properties such as resistance to gas diffusion. These are useful for making beverage containers or other food containers.

2,6-NDA is most conveniently prepared by using molecular oxygen, in particular air, as the oxygen source for the oxidation reaction and oxidizing 2,6-DMN using a liquid heavy metal catalyst. During this oxidation reaction, the methyl substituents present on the naphthalene ring of 2,6-DMN are oxidized to carboxyl groups. A method for oxidizing 2,6-DMN to 2,6-NDA by such a liquid phase reaction is well known. For example, US Pat. No. 5,183,933 to Harper et al. Discloses a large amount of manganese added to the oxidation reaction mixture. And a method of oxidizing 2,6-DMN to 2,6-NDA using a cobalt oxidation catalyst metal. However, the production of 2,6-NDA by such a chemical method has a disadvantage that the manufacturing process cost is very high due to the environmental pollution, the risk of explosion due to high temperature and high pressure, the size of the equipment used and the excessive consumption of energy.

On the other hand, in general, the production of chemicals using biological processes has the following advantages. The first is that enzymes that catalyze biological reactions have substrate specificities, so that when the reactants are mixtures, they can react only on specific substrates. It is easier to separate intermediate intermediates. In addition, biological processes generally have less concern about environmental pollution than chemical processes. Because of these advantages, the present invention seeks to produce 2,6-naphthalene dicarboxylic acid by oxidizing 2,6-dimethylnaphthalene by a biological method.

Xylene monooxygenase (hereinafter referred to as "XMO") encoded by TOL plasmid pWWO of Pseudomonas putida mt-2 is used for the decomposition of toluene and xylene. It is an enzyme system that plays a key role. XMO belongs to the alkyl hydroxyrase family and selectively hydrolyzes methyl groups of aromatic rings. It is an enzyme used in the early stages of metabolic pathways to form carboxylic acid derivatives that can be converted to substrates of the Krebs cycle via the meta-catabolic pathway. XMO consists of two polypeptide subunits encoded by the XylM and XylA genes. (Acceptor NADH reductase) NADH recipient reductase encoded by the XylA hydratase is the electron transport protein to transport zero-hydroxy-la encoded by the XylM in the reduction equivalent of the film from the NADH. 1 shows stepwise oxidation of toluene to benzyl alcohol, benzaldehyde and benzoic acid by the organization of the upper TOL metabolic pathway enzyme and the xyl genes of the upper TOL operon. XMO stands for xylene monooxygenase, BADH stands for benzyl alcohol dehydrogenase and BZDH stands for benzaldehyde dehydrogenase. P _u indicates the upper TOL operon promoter, and xylW and xylN are genes whose function is unknown. XMO , the enzyme encoded by the xylM and xylA genes, is the first enzyme in the alkane chain catabolic pathway. BADH, the second enzyme of the metabolic pathway, is benzyl alcohol dehydrogenase, a homodimer member of the zinc-containing dehydrogenase family whose substrate is a long chain alcohol. This enzyme is encoded by the xylB gene. BZDH, the third enzyme of the metabolic pathway, is also a homodimer, benzaldehyde dehydrogenase , an enzyme encoded by the xylC gene.

To date, a method of biologically oxidizing methyl groups of aromatic compounds such as toluene and xylene has been reported. A method for preparing aromatic aldehydes and carboxylic acids using XMO has been reported in Korean Patent Application No. 2002-7005344, but only the oxidation of a compound having a mononuclear aromatic ring was demonstrated. Accordingly, the present inventors have attempted to prepare 2,6-NDA by oxidizing 2,6-DMN having a multinuclear aromatic ring using a recombinant microorganism transformed by an electric expression vector and a recombinant expression vector including xylM and xylA genes. As a result of intensive research, the present invention has been reached.

The present invention is to solve the problems of the prior art as described above, to prepare a recombinant microorganism expressing xylene monooxygenase derived from Pseudomonas putida, using 2,6-dimethylnaphthalene in a biological method Oxidation is intended to produce 2,6-naphthalene dicarboxylic acid.

One aspect of the present invention for achieving the above object is to obtain a gene encoding a xylene monooxygenase derived from Pseudomonas putida ( Pseudomonas putida ); Inserting the gene into a recombinant expression vector; And it provides a method for producing a microorganism expressing xylene monooxygenase comprising the step of transforming the host cell with the expression vector.

Another aspect of the present invention for achieving the above object is to provide a microorganism expressing xylene monooxygenase produced by the above method.                         

Another aspect of the present invention for achieving the above object is to provide a method for producing a 2,6-dimethyl naphthalene dicarboxylic acid compound by culturing in a medium containing 2,6-dimethylnaphthalene using the microorganism. will be.

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

Method for producing a recombinant microorganism expressing xylene monooxygenase according to the present invention comprises the steps of obtaining a gene encoding xylene monooxygenase derived from Pseudomonas putida ; Inserting the gene into a recombinant expression vector; And transforming the host cell with the expression vector.

Obtaining a gene encoding the xylene monooxygenase may be performed according to a known method. In the present invention, a gene encoding a xylene monooxygenase, having a size of about 2.3 kb and comprising SEQ ID NO: 1, is a gene derived from Pseudomonas putida mt-2 (hereinafter, referred to as " xylM / A "). Clone .) Preferably, to clone a gene encoding xylM / A derived from Pseudomonas putida mt-2, primer 1 (5'-ATGGACACGCTTCGTTATTACC-) prepared from xylM / A using plasmid DNA (pWWO) as a template DNA. Genes encoding xylene monooxygenase can be obtained by performing polymerase chain reaction (PCR) using 3 ': SEQ ID NO: 2) and primer 2 (5'-CTAGCAAGGAGGTCTATTATAAA-3': SEQ ID NO: 3). have.

The recombinant expression vector into which the gene is inserted can be obtained by linking a gene derived from Pseudomonas putida mt-2 cloned by the above method with a functionally operated promoter to the gene.

Examples of the expression vector may include a known Forex-T vector (TaKaRa), pUC119 (TaKaRa), pBluescript family vector (Stratagene), pET family vector (Merck), and the like. Promoters, T3 promoters, Sp6 promoters and the like can be used.

Preferably, the DNA fragment of about 2.3 kb represented by SEQ ID NO. 1 cloned in the above step is isolated and recombined into a Forex-T vector to prepare the expression vector ForexT- XylM / A shown in FIG. 2.

The process of preparing a recombinant microorganism using the expression vector prepared in the above step may also be carried out through a known method.

Examples of host cells for preparing the recombinant microorganism include MC1061 ( E. coli ), JM109 ( E. coli ), XL1-Blue ( E. coli ), and DH5αF '( E. coli ). As a transformation method, heat shock and electroporation may be used, but the present invention is not limited thereto.

Preferably Recombinant microorganisms expressing xylene monooxygenase can be obtained by introducing the ForexT- XylM / A expression vector prepared above into E. coli MC1061 using conventional transformation methods.

On the other hand, when the recombinant microorganism prepared by the above step is incubated with a substance that can act as a substrate of xylene monooxygenase, the desired product can be obtained by oxidizing the substrate. Culture medium conditions and conditions can be appropriately adjusted depending on the materials desired by those skilled in the art to obtain from known methods.

For example, when the recombinant microorganism of the present invention is cultured in a medium containing 2,6-DMN, metabolic processes as shown in FIG. 3 proceed. In the process of generating 2,6-NDA from 2,6-DMN, both methyl (-CH ₃ ) substituents of 2,6-DMN are substituted with -CH ₂ OH, -COH,-by expression of xylene monooxygenase. An intermediate in the form converted to COOH is produced.

On the other hand, as a method for identifying a recombinant microorganism expressing XylM / A according to the culture of the recombinant microorganism MC1061 (ForexT- XylM / A ), Indigo (Indigo) to the indole (Xdole) by xylene monooxygenase Indigo-generating reactions in which blue colonies form as they are converted can be applied. Preferably, the inoculated recombinant microorganism MC1061 (ForexT- XylM / A ) in an LB liquid medium containing ampicillin (ampicillin, 100mg / L) and cultured with shaking, followed by 1mM Indigo and ampicillin (100mg / L). 100 μl of the culture solution was applied to the LB solid medium containing L) and incubated in a 37 ° C. incubator for 24 hours to finally select blue colonies.

In addition, the recombinant microorganism MC1061 (ForexT- XylM / A ) was inoculated in LB liquid medium containing 1000 ppm DMN and ampicillin (ampicillin, 100mg / L), shaken and cultured at 37 ° C. for a sufficient time, and then concentrated and analyzed by HPLC. It can be seen that through the expression of xylene monooxygenase ( XylM / A) 2,6-DMN is oxidized to produce 2,6-NDA.

Hereinafter, one or more exemplary embodiments will be described in more detail with reference to the following examples, which are intended to illustrate the present invention in more detail, and are not intended to limit the scope of the present invention. In particular, in relation to the xylM / A gene derived from Pseudomonas putida, which is represented by SEQ ID NO: 1 of the present invention, it does not merely include the DNA sequence shown here, but has new properties by introducing mutations through various methods. It includes genes that have been used. In other words, in order to change the properties of the gene or to produce a gene that meets the purpose, the DNA sequence is changed through artificial mutation, chemical mutation, or random mutation introduction by an inducer. Genes having can be prepared. In addition, a gene having a new property can be produced by introducing a mutation by a technique using a polymerase chain reaction.

Example 1 : XylM / A  Cloning of genes

To clone a gene encoding xylM / A from Pseudomonas putida mt-2, plasmid DNA (pWWO) was first isolated (Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Primer 1 (5'-ATGGACACGCTTCGTTATTACC-3 ': SEQ ID NO: 1) and primer 2 (5'-) prepared from xylM / A (GenBank Sequence Database, D63341) using the isolated plasmid DNA (pWWO) as template DNA. The polymerase chain reaction was carried out using CTAGCAAGGAGGTCTATTATAAA-3 ': SEQ ID NO: 2). In the polymerase chain reaction, the first denaturation was performed once at 94 ° C. for 5 minutes, then the second denaturation was performed at 94 ° C. for 1 minute, the annealing at 58 ° C. for 1 minute, and the extension was 2 min at 72 ° C., which was repeated 40 times. DNA fragments of about 2.3 kb represented by SEQ ID NO: 1 were isolated from the fragments obtained by the above method and recombined into a Forex-T vector to prepare an expression vector ForexT- XylM / A as shown in FIG. 2.

Example 2 Cloned Gene Analysis

For sequencing of the cloned recombinant vector (ForexT- XylM / A ), the vector was cleaved using EcoRI, KpnI, BamH1, PstI, and HindIII based on the cleavage maps of the two vectors, and each fragment was digested with M13mp18 and M13mp19. Subcloning was performed, and they were sequenced using an ABI PRISM BigDye primer cycle-sequencing kit (Perkin-Elmer, USA) using AmpliTaq DNA polymerase. At this time, in order to read both directions of both strands of DNA, partially synthesized nucleotides were made, and the sequence was analyzed and reconfirmed.

Example 3 : XylM / A Of recombinant microorganisms secreting

ForexT- XylM / A was introduced into E. coli MC1061 using the calcium chloride method (Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Screened in LB plate medium (yeast extract, 5g / L; tryptone, 10g / L; NaCl, 10g / L) with acillin (ampicillin, 100mg / L) and bacto-agar (15g / L) Recombinant microbial MC1061 (ForexT- XylM / A ) was prepared. The gene sequence of the cloned DNA fragment was analyzed and compared with the base sequence registered in GenBank. As a result, it was confirmed that the gene was xylM / A.

Example 4 : Indigo color reaction xylM / A  Expression confirmation

Was transformed using the ForexT- XylM / A prepared in Example 1 to confirm that the XylM / A expression in accordance with the culture of a recombinant microorganism the recombinant microorganism MC1061 (ForexT- XylM / A), and a recombinant expression vector as a control Wild lines MC1061 not included were cultured under the same conditions to measure the activity of the expressed xylM / A. The expression of xylM / A was confirmed using an indigo color reaction in which Indigo was converted to Indole by monooxygenase to form blue colonies. Two types of E. coli were inoculated in 5 ml LB liquid medium containing ampicillin (100 mg / L), respectively, and then shaken for a sufficient time at a temperature of 25 ° C. to 45 ° C., particularly 37 ° C. 100 L of the culture solution was smeared in an LB solid medium containing 1 mM Indigo and ampicillin (ampicillin, 100 mg / L) and incubated in a 37 ° C. incubator for 24 hours to observe the formation of blue colonies. On the other hand the result is not formed in the control group that does not include the recombinant expression vector is the blue colonies, it is confirmed that the ForexT- XylM / transfected recombinant microorganism that switching the A MC1061 (ForexT- XylM / A) forming a blue colonies It became.

Example 5 : XylM / A NDA Production by Recombinant Recombinant Microorganisms

Reaction experiment of recombinant microorganism MC1061 (ForexT- XylM / A ) and 2,6-DMN was carried out to confirm that 2,6-DMN is oxidized by the expression of XylM / A to produce 2,6-NDA. Recombinant microorganism MC1061 (ForexT- XylM / A ) was inoculated into 5 ml LB liquid medium containing ampicillin (ampicillin, 100 mg / L), and then shaken for a sufficient time at a temperature of 25 ° C. to 45 ° C., particularly 37 ° C. . After inoculating 1% of the culture solution into the medium of the composition shown in Table 1, the culture was shaken for 48 hours in a 37 ℃ incubator. The culture solution was placed in a round-bottom flask, the culture solution was evaporated with a dry evaporator, and the remaining dry powder was dissolved in DMSO, and then analyzed by high performance liquid chromatography (HPLC) under the conditions shown in Table 2 to measure 2,6-NDA production capacity. . As shown in the HPLC analysis results of FIG. 4, it can be seen that 2,6-DMN was converted to 2,6-NDA by the recombinant microorganism MC1061 (ForexT- XylM / A ). In addition, various intermediate compounds produced during the oxidation of 2,6-DMN to 2,6-NDA were also identified by gas chromatography / mass spectrometry. 2-hydroxymethyl-6-naphthaaldehyde, 2-methyl -6-naphthoic acid, and 2,6-dimethylnaphthaaldehyde.

Furtherance content Yeast extract 5 g / L Trypton 10 g / L NaCl 10 g / L 2,6-DMN 1 g / L Empicillin 100 mg / L DMSO 10 mL

HPLC LC 10-AD VP (SHIMADZU) column XTerra ^TM RP18 (4.6 ㅧ 50 mm, Waters) Detector  UV 240 nm Column temperature  40 ℃ Flow rate  1 ml / min. Injection volume  20 μl Mobile phase Time (min) 0.3% phosphoric acid Acetonitrile 0 98 2 5 92 8 28 52 48 30 20 80 35 5 95 36 98 2 49 98 2

The recombinant microorganism expressing the xylM / A gene derived from Pseudomonas putida of the present invention can be easily produced by a biological process of 2,6-naphthalene dicarboxylic acid which is a monomer of polyethylene naphthalate.

Attach sequence list electronic file  

Claims (5)

Obtaining a xylM / A gene represented by SEQ ID NO: 1; Inserting the gene into a recombinant expression vector selected from the group consisting of Forex-T, pUC119, pBluscript and pET; Xyl comprising the step of transforming the host cell selected from the group consisting of MC1061 ( E. coli ), JM109 ( E. coli ), XL1-Blue ( E. coli ) and DH5αF '( E. coli ) with the expression vector. Preparing a microorganism expressing ren monooxygenase; And culturing the microorganism in a medium containing 2,6-dimethylnaphthalene.

Wherein R ₁ and R ₂ are each independently —CH ₂ OH, —COH, or —COOH. delete delete delete delete

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