JP2007037452A - Gene for producing 2-pyrone-4,6-dicarboxylic acid from gallic acid, transformant transferred with the gene or the like, and method for producing 2-pyrone-4,6-dicarboxylic acid from gallic acid using the transformant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To utilize gallic acid-containing agricultural and forestry wastes including woody wastes such as barks and used tea leaves as new biomass resources by stably and efficiently producing at low cost PDC (2-pyrone-4,6-dicarboxylic acid) of high value as an industrial material from gallic acid found in large quantities as conjugates with tannins and/or catechins in such agricultural and forestry wastes to put PDC on the market. <P>SOLUTION: PDC is ensured to effect mass production stably from gallic acid by the action of transgenic microorganisms through offering a protein having a specific amino acid sequence and having 4-carboxy-2-hydroxymuconate cyclolase enzyme activity and, in association with the protein, a gene encoding the protein and having a specific base sequence. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  In the present invention, a protein having 4-carboxy-2-hydroxymuconate cyclolase enzyme activity involved in the production of 2-pyrone-4,6-dicarboxylic acid from gallic acid, a gene encoding the same, the gene thereof, and the like are introduced. The transformant, and a vector for introducing the gene or the like into the transformant, more specifically, 4-carboxy-2-hydroxymucone produced from gallic acid as a protein having 4-carboxy-2-hydroxymuconate cyclolase enzyme activity. The present invention relates to a production technique for industrially producing 2-pyrone-4,6-dicarboxylic acid stably and efficiently by acting on an acid.

Background of the Invention

2-pyrone-4,6-dicarboxylic acid (hereinafter abbreviated as PDC) is a compound having a carboxyl group and one lactone skeleton in the molecule, but it is a unique compound that is difficult to produce by conventional petrochemical methods and organic synthesis. Has chemical characteristics. Using this compound as a raw material, it is possible to produce various highly functional plastics that require precise physicochemical properties.

As for the production of PDC, high production of PDC using vanillic acid and syringic acid obtained by oxidative degradation of lignin, which is a natural aromatic polymer compound, using transformed microbial cells has been attempted. However, the yields of vanillic acid and syringic acid due to oxidative degradation of lignin are extremely low. Therefore, efficient use of the wood raw material of lignin cannot be realized and there is a problem in cost.

On the other hand, gallic acid (gallic acid, 3,4,5-Trihydroxy-bezoic acid), one of the natural polyphenols, is present in a large amount in various plants as a complex with tannin and catechin. The effective use has not been realized. That is, for example, it exists as a complex with catechins in tea leaves and fermented tea leaves, and the amount is said to occupy about 10% in dry tea leaves.
About 30% of this component is solubilized and drunk when tea is roasted, but the remaining 70% is discarded as it is. However, it is easy to extract gallic acid from the remaining 70%. Therefore, if gallic acid can be converted into a useful substance, a plant-based material that has a large amount of gallic acid and has not been effectively used so far can be used as a new biomass resource. However, so far, no technology has been realized to stably produce PDC that can be an industrial raw material from gallic acid.

The following documents are disclosed in relation to the present invention.
JP 2004-256747 A “Sequencing and expression of PDC hydrolase gene” Toshiyuki Harada (Abstracts of 1999 Graduate School of Biotechnology, Maruyama Laboratory) "Sequencing and Expression of PDC Hydrolase Gene" Shinji Takemiya (Abstracts of 2000 Graduate School of Biotechnology, Maruyama Laboratory) “Cloning of CHMS dehydrogenase gene and its expression in E. coli” Akitaka Ichikawa (Graduation thesis, Maruyama Laboratory, Department of Biotechnology, Gifu University)

A market that stably produces high-efficiency, low-cost PDCs that are highly valuable as industrial raw materials from gallic acid, which is abundant as a complex with tannin and catechin, in agricultural and forestry waste such as bark and tea shells. By using this technology, we will effectively utilize woody waste such as bark and agricultural and forestry waste containing gallic acid such as tea shells as new biomass resources.

  A novel protein having a 4-amino-2-hydroxymuconate cyclolase enzyme activity having a specific amino acid sequence is provided to enable stable and high production of PDC from gallic acid to solve the above-mentioned conventional problems It is.

The present invention also provides a gene having a specific base sequence that encodes the protein having the 4-carboxy-2-hydroxymuconate cyclolase enzyme activity described above, and genetically engineered PDC from gallic acid. It aims to solve the above-mentioned conventional problems by enabling high production stably.

Furthermore, the present invention provides a gene for recombination by providing a gene encoding 3,4-dioxygenase that acts on gallic acid to produce 4-carboxy-2-hydroxymuconic acid and linking the gene to the gene recombination technique Facilitates PDC production from gallic acid.

In the above control gene, it may be a gene encoding 4,5-dioxygenase instead of 3,4-dioxygenase.

The present invention also provides a vector containing each of the above genes and contributes to PDC production from gallic acid by DNA recombination.

Furthermore, the present invention provides a transformant obtained by introducing the above vector into a host cell and a transformation method, and enables PDC production from gallic acid in a stable and high yield by an enzyme produced by a recombinant microorganism. Enable.

The present invention also provides a method for producing a protein having 4-carboxy-2-hydroxymuconate cyclolase enzyme activity obtained by culturing the above transformant, which can be stably and efficiently produced from gallic acid in a high yield. Enables PDC production.

In the present invention, 3,4-dioxygenase or 4,5-dioxygenase is allowed to act on gallic acid to produce 4-carboxy-2-hydroxymuconic acid. -Technology for stably producing high yield of 2-pyrone-4,6-dicarboxylic acid by allowing a protein having carboxy-2-hydroxymuconate cyclolase enzyme activity to act, that is, 2-pyrone-4 having the following steps as a gist A 6-dicarboxylic acid production technique is realized to solve the above-mentioned conventional problems.
1: Isolation of 4-carboxy-2-hydroxymuconate cyclolase (chmc) gene 2: Determination of the base sequence of the chmc gene and the amino acid sequence deduced from the sequence 3: chmc gene and 4,5-dioxygenase ( Preparation of ligation plasmid pBchmcligAB of 3,4-dioxygenase) 4: Preparation of broad host range plasmid pKchmcligAB by inserting chmc and ligA, ligB ligation genes into a broad host range plasmid 5: PKchmcligAB plasmid to Pseudomonas spp. Introduction 6: Production of 2-pyrone-4,6-dicarboxylic acid from gallic acid using transformed cells 7: Isolation of produced 2-pyrone-4,6-dicarboxylic acid from fermentation broth

  In the present invention, the following effects can be achieved by the above-described configuration and operation. In other words, from gallic acid that is present in large quantities as a complex with tannin and catechin in agricultural and forestry wastes such as bark and tea husk, PDC, which is highly valuable as an industrial raw material, is stabilized at high cost with low cost by genetic recombination technology. It can be produced and provided to the market and contributes to effective utilization of woody waste such as bark and agricultural and forestry waste containing gallic acid such as tea shells as new biomass resources.

Hereinafter, the present invention will be described in detail. The protein having the enzyme activity according to the present invention comprises 4-carboxy-2-hydroxymuconic acid produced from 4,5-dioxygenase or 3,4-dioxygenase from gallic acid or protocatechuic acid, as 2-pyrone-4 , Which converts to 6-dicarboxylic acid. That is, it is a protein having 4-carboxy-2-hydroxymuconate cyclolase enzyme activity.

In order to obtain the above protein (enzyme), it is necessary to obtain the gene encoded in each claim. Specifically, in order to produce 2-pyrone-4,6-dicarboxylic acid (hereinafter sometimes abbreviated as PDC) from gallic acid in the present invention, 4,5-dioxygenase or 3,4-dioxygenase is encoded. And a gene encoding 4-carboxy-2-hydroxymuconate cyclolase (hereinafter sometimes abbreviated as chmc gene) are required.
Among these, genes encoding 4,5-dioxygenase and 3,4-dioxygenase are known and genes have already been obtained.

Gene encoding 4-carboxy-2-hydroxymuconate cyclolase (chmc gene)
Can be obtained, for example, using recombinant DNA technology as follows.
That is, using a microorganism capable of producing 4-carboxy-2-hydroxymuconate cyclolase as a DNA donor, genomic DNA is extracted from the microorganism and cleaved with a restriction enzyme or the like to prepare a DNA fragment.
On the other hand, restriction enzyme ends into which genome fragments can be inserted are prepared from vector DNA such as phages and plasmids using restriction enzymes. These genomic DNA fragments and linearized vector DNA are combined using DNA ligase to obtain a recombinant vector.
The recombinant vector is produced by transferring the recombinant vector into a host cell, selecting a transformed cell carrying the target recombinant vector, and isolating the target recombinant vector from the transformed cell.

In addition to the above method, if the entire sequence or a partial sequence of the chmc gene has already been clarified, it can also be obtained by a known PCR method.
As a DNA donor for separation of the gene, it has the ability to produce 4-carboxy-2-hydroxymuconic acid cyclolase that converts 4-carboxy-2-hydroxymuconic acid into 2-pyrone-4,6-dicarboxylic acid If it is microorganisms, it will not restrict | limit in particular.

Extraction of genomic DNA from the microorganism involves collecting cultured cells of the microorganism, lysing the cells with protease K, for example, deproteinization treatment with phenol extraction, protease treatment, ribonuclease treatment, genomic DNA with alcohol It is preferable to appropriately combine methods such as precipitation and centrifugation. For fragmenting the separated genomic DNA, for example, digestion with a restriction enzyme of the genomic DNA is performed.

As the vector, it is preferable to use a vector constructed for the purpose of a recombinant vector from a phage or plasmid capable of growing independently in a host microorganism. As the plasmid, for example, pUC18, pUC19, pUC118, pUC119, Bluescript, pKT230MC and the like using Escherichia coli as a host are preferable. These vectors are used after, for example, creating restriction enzyme ends into which DNA fragments can be inserted using restriction enzymes and dephosphorylating the ends as necessary. The binding of the genomic DNA fragment and the vector DNA fragment is preferably performed using a known DNA ligase such as T4 DNA ligase.

The host microorganism into which the obtained recombinant vector is introduced may be any microorganism as long as the recombinant vector can be stably and autonomously replicated and can stably express the properties of a foreign gene. For example, Escherichia coli and Pseudomonas
You can use putida. As a method for transferring a recombinant vector into a host microorganism, any method such as a conjugation method, an electroporation method, or a competent cell method can be used.

The selection of transformants can be based on, for example, drug resistance acquired by DNA recombination of the transformants. Among these transformants, selection of a transformant containing the target recombinant vector is preferably performed, for example, by a colony hybridization method using a partial DNA fragment of a gene as a probe. As a label for this probe, for example, any of radioisotopes, digoxigenin, enzymes and the like can be used.

In order to extract the recombinant vector from the transformed cells selected in this manner, extraction may be performed by a conventional method, for example, alkaline lysis (Cold Spring Harbor
Laboratory Press, Molecular Cloning Second Edition (1989)) can be used.
The extracted recombinant vector can be recombined using DNA technology as necessary. In order to excise the gene (chmc gene) encoding 4-carboxy-2-hydroxymuconate cyclolase contained in the gene of the present invention from the obtained recombinant vector, a restriction enzyme or the like can be used.

The base sequence of the gene encoding 4-carboxy-2-hydroxymuconate cyclolase contained in the gene of the present invention thus obtained can be determined by decoding, for example, by the dideoxy method.
The nucleotide sequence of the gene encoding 4-carboxy-2-hydroxymuconate cyclolase is shown in SEQ ID NO: 1 in the sequence listing, and 4-carboxy-2-hydroxymuconate cyclolase enzyme activity is shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence of the protein is shown.

A gene designed by cutting out the gene (chmc) encoding 4-carboxy-2-hydroxymuconate cyclolase obtained in this way and the 4,5-dioxygenase (ligA and ligB) already obtained using restriction enzymes. Based on the arrangement, the DNA fragments are ligated with DNA ligase to ligate restriction enzyme-cleaved DNA fragments containing three types of enzyme genes. This makes it possible to create a control gene for a multi-step reaction process that rapidly fermentatively produces 2-pyrone-4,6-dicarboxylic acid from gallic acid in large quantities.

In the present invention, DNA fragments retaining the ligA and ligB gene sequences obtained by the XbaI treatment of pUCligAB to the XbaI site of the transformation plasmid pBchmc that retains the DNA fragment having the gene sequence shown in SEQ ID NO: 1 as a part thereof are publicly known. E. coli expression plasmid pBchmcligAB inserted using DNA ligase is prepared (see Fig. 1).

A DNA fragment containing the Lac promoter sequence, chmc, ligA and ligB obtained by digesting the restriction enzyme ApaLI digested fragment from the E. coli recombinant plasmid pBchmcligAB shown in FIG. The Pseudomonas
After digestion with the restriction enzyme EcoRI of the broad host range plasmid pKT230MC that can be maintained with putida, the digested ends are blunted with a known Klenow fragment, etc., and further ligated to a DNA fragment obtained by digestion with the restriction enzyme KpnI with a known DNA ligase. As a result, a new broad host range plasmid pKchmcligAB as shown in FIG. 2 is prepared.

When introduced into Pseudomonas putida, etc. using the electroporation method, competent cell method, conjugation transfer method, etc., the broad-host-range plasmid pKchmcligAB is transformed into 4-carboxy-2 -Hydroxymuconate cyclolase and 4,5-dioxygenase are produced strongly and stably.

Recombinant plasmid pKchmcligAB (Fig. 2), which produces 2-pyrone-4,6-dicarboxylic acid from gallic acid with various host ranges, is downstream of β-lactamase gene promoter and Lac promoter from 2-pyrone-4,6-dicarboxylic acid from gallic acid. It contains a gene sequence that encodes an enzyme that constitutes a multistage reaction process for fermentative production of acid, and has a wide host range including Pseudomonas bacteria. Synchronize 4-carboxy-2-hydroxymuconate cyclolase and 4,5-dioxygenase in transformed cells into which the recombinant plasmid has been introduced using the electroporation method, competent cell method, conjugation transfer method, etc. It can be expressed to produce 2-pyrone-4,6-dicarboxylic acid from gallic acid, but for high production of 2-pyrone-4,6-dicarboxylic acid, it is necessary to incorporate gallic acid into cells. It is necessary to use a transformant that uses a microorganism that can digest gallic acid but cannot decompose gallic acid. As a microorganism capable of taking gallic acid into cells but not degrading gallic acid, Pseudomonas as shown in Examples below.
putida PpY1100 can be used.

2-pyrone-4,6-dicarboxylic acid from gallic acid using transformed cells with 4-carboxy-2-hydroxymuconate cyclolase and 4,5-dioxygenase genes (hereinafter sometimes referred to as PDC producing cells) For production, for example, a jar fermenter system is preferably used. PDC-producing cells are cultured in a medium containing the components necessary for growth, and when fully grown, the addition of gallic acid is started, and a culture solution is produced by producing 2-pyrone-4,6-dicarboxylic acid from gallic acid. Acidification of aqueous alkaline solution eg 5M
By adding NaOH solution etc., fermentative production of 2-pyrone-4,6-dicarboxylic acid is performed while neutralizing. In this case, the culture solution components are not limited as long as they are optimal for the growth of microorganisms. For example, it is preferable to use a known LB medium or the like.

After fermentation is complete, acidify the fermentation broth by adding, for example, hydrochloric acid, stop the reaction, adjust the pH to about 1.0 by adding hydrochloric acid, etc., and then concentrate under reduced pressure or store at a low temperature. 4,6-dicarboxylic acid can be recovered as a precipitate. At this time, since various nutrient components and the like present in the culture solution do not precipitate, it can be recovered as very high purity 2-pyrone-4,6-dicarboxylic acid.

An embodiment according to the present invention will be described with reference to the drawings.
First, a gene chmc encoding 4-carboxy-2-hydroxymuconate cyclolase was obtained as follows.
That is, the gene chmc encoding 4-carboxy-2-hydroxymuconate cyclolase was isolated from the genomic library of Sphingomonas paucimobilis SYK-6 strain. The DNA sequence is shown in SEQ ID NO: 1 in the sequence listing. Then, 4-carboxy-2-hydroxymuconic acid cyclolase produced from this gene quickly converts 4-carboxy-2-hydroxymuconic acid into 2-pyrone-4,6-dicarboxylic acid.

Construction of gene structure to produce PDC from gallic acid
PBchmc was prepared by linking chms isolated from the genomic library of Sphingomonas paucimobilis SYK-6 strain to the multiple cloning site of pBluescript (see FIG. 1). LigA encoding 4,5-dioxygenase enzyme,
The ligA-ligB fragment obtained by digestion with the restriction enzyme XbaI from the plasmid pUCligAB carrying ligB on the multicloning site was transformed into the XbaI of pBchmc.
Inserted into the site, pBchmcligAB was created (see Figure 1).

Next, as shown in FIG. 2, pBchmcligAB was digested with restriction enzyme ApaLI, the cleavage site was smoothed with Klenow Fragment (TAKARA co.LTD), and further digested with restriction enzyme KpnI to obtain Lac.
A chmc-ligA-ligB fragment containing promotor was obtained.
Furthermore, after digesting the high-range host vector pKT230MC with the restriction enzyme EcoRI, smoothing the cleavage site with Klenow Fragment, the gene fragment and Lac cleaved with the restriction enzyme KpnI
The chmc-ligA-ligB fragment containing promotor was ligated with ligase (TOYOBO) to obtain pKchmcligAB (Fig. 2). The obtained pKchmcligAB was introduced into Escherichia coli DH5α strain, and pKchmcligAB was further obtained by the triple parental junction transfer method.
Was introduced into Pseudomonas putida PpY1100 strain. Pseudomonas putida PpY1100
(pKchmcligAB) strain.

PDC production from gallic acid using Pseudomonas putida PpY1100 (pKchmcligAB)
Pseudomonas putida PpY1100 (pKchmcligAB) strain was inoculated into 200 ml of LB liquid medium (containing 25 mg / L kanamycin) and cultured at 28 ° C. for 16 hours to obtain a precultured cell suspension.
Pseudomonas prepared using 8L LB liquid medium and 3ml antifoam (Antiform A) using 10L jar fermenter (fermentor)
200 ml of a precultured cell suspension of putida PpY1100 (pKchmcligAB) strain was mixed and cultured at 28 ° C. under aeration and agitation at 500 rpm / min until OD660 = 13 to 14 (10 hours to 12 hours). In addition, the growth curve accompanying this culture was shown in FIG.

When OD660 = 13-14 was reached in culture using a 10L jar fermenter (fermentor), 500 ml of the culture broth was extracted from the fermentor into an Erlenmeyer flask and stored on ice (ice-cold cell suspension). Suspension). The fermentation broth that reached OD660 = 13-14 contains 80 g of gallic acid as a substrate.
500 ml of 0.1N NaOH solution (pH 8-9) was added over 5 hours using a peristaltic pump. Due to the formation of 2-pyrone-4,6-dicarboxylic acid as the reaction proceeds, the pH of the culture broth
Decreases. To prevent this, the pH of the culture solution was maintained by adding a 0.1N NaOH solution with a peristaltic pump connected to a pH sensor.

The progress of the reaction was confirmed by thin layer chromatography (TLC). 24 hours after the start of the addition of gallic acid, 500 ml of the ice-cold cell suspension prepared above was added to the culture solution in the fermentor and the culture was continued for 12 hours. After completion of the reaction, the culture medium in the fermenter was transferred to a plastic container (bucket) and hydrochloric acid was added to stop the reaction at pH 3.
Next, the bacterial cell components are precipitated and removed from the culture by centrifugation (6000 rpm, 20 ° C.), and hydrochloric acid is added to the resulting supernatant to lower the pH to 1.0 and stored at a low temperature to obtain 2-pyrone-4. 6-Dicarboxylic acid was isolated as a precipitate. The produced 2-pyrone-4,6-dicarboxylic acid reached about 65 g, which means that it was produced in a yield of about 90% of the added substrate.

The purified product obtained by further purifying the produced 2-pyrone-4,6-dicarboxylic acid by activated carbon treatment was analyzed. That is, 2-pyrone-4,6-dicarboxylic acid (PDC) produced in the above example was analyzed by ¹ H-NMR and ¹³ C-NMR. As a result, it was clarified that the substance produced as shown in FIG. 4 was indeed 2-pyrone-4,6-dicarboxylic acid (PDC) and very high purity.

It is a schematic diagram of the production process of E. coli recombinant plasmid pBchmcligAB. It is a schematic diagram which shows the vector preparation process containing the control gene for functioning a 4, 5- dioxygenase (ligAB) gene and a chmc gene. It is a graph which shows the growth condition of Pseudomonas putida PpY1100 (pKchmcligAB) strain | stump | stock in the culture | cultivation which concerns on an Example. The NMR spectrum of the refinement | purification which further raised the purity of 2-pyrone-4,6-dicarboxylic acid produced with the manufacturing method which concerns on an Example by activated carbon treatment etc. is shown.

Claims (14)

4-carboxy-2-hydroxymucone having an amino acid sequence described in SEQ ID NO: 2 in the sequence listing, or an amino acid sequence deleted or substituted or added from the amino acid sequence described in SEQ ID NO: 2 A protein having acid cyclolase enzyme activity. A gene encoding a protein having 4-carboxy-2-hydroxymuconate cyclolase enzyme activity according to claim 1. The gene of Claim 2 which has the base sequence of sequence number 1 of a sequence table. The control gene which links the gene which codes 3, 4- dioxygenase and the gene in any one of Claim 2 or 3. A control gene that links a gene encoding 4,5-dioxygenase and the gene according to claim 2 or 3. A vector comprising the gene according to claim 2 or 3. The vector according to claim 6, further comprising a gene encoding 3,4-dioxygenase and a regulatory gene according to claim 4. The vector according to claim 6, further comprising a gene encoding 4,5-dioxygenase and a regulatory gene according to claim 5. A transformant obtained by introducing the vector according to claim 7 or 8 into a host cell. A transformation method comprising introducing a gene comprising the gene according to claim 2 or 3 and the gene according to claim 4 or 5 into a host cell. A method for producing a protein, comprising culturing the transformant according to claim 9 and collecting a protein having 4-carboxy-2-hydroxymuconate cyclolase enzyme activity from the obtained culture. The benzene ring of gallic acid is cleaved by 3,4-dioxygenase or 4,5-dioxygenase to convert to 4-carboxy-2-hydroxymuconic acid, and the 4-carboxy-2-hydroxymuconic acid is converted to 4-carboxy-2-hydroxymuconic acid. A process for producing 2-pyrone-4,6-dicarboxylic acid, which comprises converting a protein having 4-carboxy-2-hydroxymuconate cyclolase enzyme activity to 2-pyrone-4,6-dicarboxylic acid. A gallic acid is converted into 4-carboxy-2-hydroxymuconic acid by a culture obtained by culturing the transformed microorganism according to claim 10 in a fermenter, and the 4-carboxy-2-hydroxymuconic acid is converted. A process for producing 2-pyrone-4,6-dicarboxylic acid, wherein muconic acid is further converted into 2-pyrone-4,6-dicarboxylic acid. In Claim 13, 2-pyrone-4,6-dicarboxylic acid is obtained by precipitating and removing bacterial cell components from the culture solution in the fermenter after completion of the production process, and then setting the pH to a predetermined value and keeping it at a low temperature. A method for producing 2-pyrone-4,6-dicarboxylic acid, wherein the precipitate is separated by precipitation.

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