JP2005278549A - Gene for producing 2-pyrone-4,6-dicarboxylic acid by fermentation, plasmid containing gene, transformant containing plasmid and method for producing 2-pyrone-4,6-dicarboxylic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new fermentation production technology for converting a low molecular plant-originated low molecular mixture containing vanillin, syringaldehyde, vanillic acid, syringic acid and protocatechuic acid produced by a low molecule-forming technology such as a chemical decomposition method such as hydrolysis, oxidative decomposition, solvolysis, etc., or physicochemical decomposition utilizing supercritical water and supercritical organic solvent of a plant aromatic component having various chemical structures and complex high molecular structures, through the multiple stages of enzymatic reactions to a single intermediate substance, 2-pyrone-4,6-dicarboxylic acid (PDC) in a high yield by designing and constructing with a recombinant DNA technology. <P>SOLUTION: The gene sequences encoding 4 kinds of enzymes (benzaldehyde dehydrogenase, demethylase, protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase), and a new double strand DNA molecule obtained by joining gene domains associated with the regulation of their expressions are provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is for fermentative production of 2-pyrone-4,6-dicarboxylic acid from plant components or petroleum components or chemically synthesized vanillin, syringaldehyde, vanillic acid, syringic acid, or protocatechuic acid. Gene sequences encoding four types of enzymes (benzaldehyde dehydrogenase, dimethylase, protocatechuate 4,5-dioxygenase, 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase) constituting the multistage reaction process, the multistage The present invention relates to a reaction process control gene, a recombinant vector containing the gene, a transformed cell having the gene, and a production method using the same.

Lignin, a major plant component, is a biomass resource that is universally contained in plant cell walls as an aromatic polymer, accounting for 30% of trees and 15% of rice and corn straw. In addition, plants contain various aromatic compounds as constituents. However, the plant-derived aromatic component mainly composed of lignin has not been developed as an effective utilization technique because it has a variety of chemical structures and a complex polymer structure. Examples of conventional technologies include the practical use of separating and producing vanillin, a fragrance raw material, from low-molecular-weight aromatic degradation products produced by chemical decomposition such as alkaline decomposition of plant-derived aromatic components mainly composed of lignin. Technology. However, there is currently no effective method for utilizing a large amount of low-molecular aromatic substances other than vanillin produced by chemical decomposition. For this reason, lignin produced in large quantities in the papermaking process is burned as a substitute for heavy oil without being effectively used.
Supporting the development of the petrochemical industry today is the conversion of crude oil, which is a mixture of various chemical components, into a single intermediate substance such as benzene and ethylene by catalytic cracking and fractionation. The principle is to manufacture functional plastics. The basic principle that supported the development of the petrochemical industry is a universal principle that can also be applied in the utilization technology of plant aromatic components such as lignin having diverse chemical structures and complex polymer structures. In developing technologies for the use of plant-derived aromatic components based on lignin, processes equivalent to catalytic decomposition of petrochemicals include hydrolysis, oxidative decomposition, and solvent decomposition of plant aromatic components such as lignin. Many low molecular weight technologies such as chemical decomposition methods and physicochemical decomposition methods using supercritical water or supercritical organic solvents have already been studied and developed. However, another intermediate technology, a low-molecular mixture derived from plant components produced by various decomposition methods, can be used as a raw material for various functional plastic materials and chemical products. No conversion / separation technology has been developed. If this technology is developed, the use of plant-derived aromatic components mainly composed of lignin may develop as a technology comparable to petrochemistry.
Under such circumstances, the dimethylase reaction for converting vanillic acid to protocatechuic acid in the presence of NADH has already been studied in microorganisms such as Pseudomonas putida WCS358 and Pseudomonas sp. HR199. A polypeptide having the amino acid sequence shown (VanA), a polypeptide having the amino acid sequence shown in SEQ ID NOs: 6 and 9 (VanB), and a base sequence shown in SEQ ID NOs: 4 and 7 encoding these polypeptides; The gene which has is shown (for example, refer nonpatent literature 1 to 3).

Priefert H., Rabnehorst J., Steinbuchel A., (1997) J. Bacteriol. 179, 2595-2607 Venturi V., Zennaro F., Degrassi G., (1998) Microbiology 144, 965-973 Brunel F., Davison J., (1988) J. Bacteriol. 170, 4924-4930

  The object of the present invention is to provide chemical decomposition methods such as hydrolysis, oxidative decomposition, and solvolysis of plant aromatic components such as lignin having various chemical structures and complex polymer structures, supercritical water and supercritical organic solvents. A single molecule through a multi-stage enzymatic reaction from a low molecular weight mixture derived from plant components including vanillin, syringaldehyde, vanillic acid, syringic acid, and protocatechuic acid produced by low molecular weight technology such as physicochemical decomposition by The design and construction of a new fermentation production technology that converts to the intermediate substance 2-pyrone-4,6-dicarboxylic acid (PDC) in high yield by recombinant DNA technology.

The present inventors have completely decomposed and metabolized vanillin, syringaldehyde, vanillic acid, syringic acid and protocatechuic acid, which are the main components of the low molecular weight mixture, to carbon dioxide and water by the microorganism Sphingomonas paucimobilis SYK-6. Furthermore, it was discovered that these five compounds are degraded in this metabolic pathway via a single intermediate, 2-pyrone-4,6-dicarboxylic acid.
The inventor further provides a multi-step reaction in which these five compounds are converted into a single intermediate 2-pyrone-4,6-dicarboxylic acid, an enzymatic function that catalyzes it, and the genes that encode them. As a result of diligent research on isolation and sequencing, all genes required for the fermentation production process to convert the low-molecular-weight mixture directly into the single intermediate 2-pyrone-4,6-dicarboxylic acid are obtained. succeeded in.
Based on these research results, the present inventor modified various genes involved in the fermentation production process and designed each gene arrangement. From the low-molecular-weight mixture, 2-pyrone-4,6-dicarboxylic acid was obtained by fermentation. A fermentation production process control gene that synchronously and highly expresses the multistage enzyme reaction leading to the production of the present invention has been constructed, and the present invention has been completed.

Accordingly, the present invention provides any of the following DNA molecules (vanAB gene):
(1) a DNA molecule described in SEQ ID NO: 1,
(2) a DNA molecule encoding the amino acid sequence of SEQ ID NO: 2 and / or 3,
(3) a DNA molecule that hybridizes with a DNA molecule comprising SEQ ID NO: 1 or its complementary sequence under stringent conditions and encodes a protein having dimethylase activity against syringic acid, or (4 2) An amino acid sequence described in SEQ ID NO: 2 and / or 3, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added, and having two dimethylase activities against syringic acid A DNA molecule that encodes either or both polypeptides.
The present invention also provides a polypeptide in which a polypeptide that contributes to the stabilization of VanA and does not inhibit the enzyme function of VanA is added to the N-terminus of VanA.

The present invention also provides a recombinant DNA having the following DNA molecule between a promoter and a terminator and operably connected in a suitable host:
A DNA molecule that encodes a polypeptide that contributes to the stabilization of VanA and does not inhibit the enzymatic function of VanA;
vanAB gene;
One of the following DNA molecules (ligA gene):
(5) the DNA molecule set forth in SEQ ID NO: 14,
(6) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 15;
(7) hybridizes under stringent conditions with the DNA molecule of SEQ ID NO: 14 or its complementary sequence, and associates with the polypeptide (ligB) and has protocatechuic acid 4,5-dioxygenase activity A DNA molecule encoding a polypeptide, or (8) an amino acid sequence in which one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 15 are deleted, substituted and / or added, and associated with the polypeptide (ligB) A DNA molecule encoding a polypeptide having protocatechuic acid 4,5-dioxygenase activity,
One of the following DNA molecules (ligB gene);
(9) a DNA molecule described in SEQ ID NO: 16,
(10) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 17,
(11) hybridizes under stringent conditions with the DNA molecule of SEQ ID NO: 16 or a complementary molecule thereof, and associates with the polypeptide (ligA) and has protocatechuic acid 4,5-dioxygenase activity A DNA molecule encoding a polypeptide, or (12) an amino acid sequence in which one or several amino acids of SEQ ID NO: 17 have been deleted, substituted and / or added, and are associated with a polypeptide (ligA) A DNA molecule encoding a polypeptide having protocatechuic acid 4,5-dioxygenase activity,
One of the following DNA molecules (ligC gene):
(13) the DNA molecule set forth in SEQ ID NO: 18,
(14) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 19,
(15) A polypeptide that hybridizes with the DNA molecule of SEQ ID NO: 18 or a complementary DNA sequence thereof under stringent conditions and has 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase activity Or (16) an amino acid sequence in which one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 18 are deleted, substituted and / or added, and 4-carboxy-2-hydroxymuconic acid A DNA molecule encoding a polypeptide having -6-semialdehyde dehydrogenase activity,
Any of the following DNA molecules (ligV gene):
(17) the DNA molecule set forth in SEQ ID NO: 21,
(18) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 22,
(19) a DNA molecule that hybridizes with a DNA molecule comprising SEQ ID NO: 21 or a complementary sequence thereof under stringent conditions and encodes a polypeptide having benzaldehyde dehydrogenase activity, or (20) SEQ ID NO: 22 A DNA molecule comprising an amino acid sequence in which one or several amino acids of the described amino acid sequence are deleted, substituted and / or added, and encoding a protein having benzaldehyde dehydrogenase activity. Here, the polypeptide (ligA) consists of an amino acid sequence represented by SEQ ID NO: 15 or an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 15 are deleted, substituted and / or added, The polypeptide (ligB) is composed of the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 17 are deleted, substituted and / or added.

The present invention also provides the recombinant DNA that is a plasmid retained by Escherichia coli XL1-Blue.
The present invention also provides a host organism containing the recombinant DNA.
The present invention also provides a method for producing 2-pyrone-4,6-dicarboxylic acid, comprising culturing the host organism.

  The present invention provides a new fermentation production technology that converts plant component-derived low-molecular-weight mixtures containing vanillin, syringaldehyde, vanillic acid, syringic acid, and protocatechuic acid into 2-pyrone-4,6-dicarboxylic acid in high yield. The use of plant-derived aromatic components mainly composed of lignin is expected to develop as a technology comparable to petrochemistry.

  The fermentative production process control gene of 2-pyrone-4,6-dicarboxylic acid constructed in the present invention is a dimethylase produced by the association of two kinds of polypeptides (vanA and vanB) having the amino acid sequences shown in SEQ ID NOs: 2 and 3. It has a DNA molecule (vanAB gene) described in SEQ ID NO: 1 partially containing a region encoding two polypeptides having activity. Transformed cells carrying a recombinant vector containing the vanAB gene (Figure 1) are converted to protocatechuic acid and 3-methylgallic acid by a demethylase reaction that cleaves the methyl ether bond of vanillic acid and syringic acid in the presence of NADH. To do. Here, the vanAB gene replaces the DNA molecule described in SEQ ID NO: 1, a DNA molecule encoding the amino acid sequence described in SEQ ID NO: 2 and / or 3, a DNA molecule consisting of the DNA molecule described in SEQ ID NO: 1 or a complementary sequence thereof A DNA molecule that encodes two polypeptides that hybridize under stringent conditions and has dimethylase activity against syringic acid, or some amino acids of the amino acid sequence set forth in SEQ ID NOs: 2 and / or 3 May be a DNA molecule encoding one or both of two polypeptides having a deletion, substitution and / or addition, and having dimethylase activity against syringic acid.

The present inventors used the genomic DNA of Pseudomonas putida PpY101 strain as a template, and used primer AS (5′-GGAGAGCCTCATGTACCCGAGAAACACTTCC-3 ′: SEQ ID NO: 10) and primer BO (5′-AATGCCACCTTGCGTTATGC-3 ′: SEQ ID NO: 11). As a result of analyzing the DNA fragment amplified by the PCR method, the base shown by SEQ ID NO: 1 is different from the known dimethylase gene shown by SEQ ID NO: 4 and 7 that cleaves the methyl ether bond of vanillic acid in the presence of NADH. Isolated as a new dimethylase gene with sequence. This gene is an enzyme gene encoding a dimethylase that cleaves the methyl ether bond of vanillic acid and syringic acid in the presence of NADH.
A DNA fragment having the base sequence represented by SEQ ID NO: 1 was ligated to a restriction enzyme Hinc II cleavage site present at the multicloning site of E. coli plasmid pUC119 to prepare a hybrid plasmid pvanAB (FIG. 2). A transformant in which the hybrid plasmid pvanAB was introduced into Escherichia coli XL1-Blue strain (purchased from STRATEGENE, CA, USA) showed a strong demethylase activity against vanillic acid. Furthermore, the transformant showed a strong demethylase activity against syringic acid that was not shown by the known demethylases of Pseudomonas putida WCS358 and Pseudomonas sp. HR199.
Next, a hybrid plasmid pvnnAB was prepared by ligating a DNA fragment having the base sequence represented by SEQ ID NO: 1 to the restriction enzyme SmaI cleavage site present at the multicloning site of E. coli plasmid pUC18 (FIG. 3). The transformant in which the hybrid plasmid pvnnAB was introduced into E. coli XL1-Blue strain (purchased from STRATEGENE, CA, USA) did not show any demethylase activity against vanillic acid and syringic acid.

The hybrid plasmid pvanAB is the nucleotide sequence of SEQ ID NO: 1 at the site cleaved by the restriction enzyme HincII in the multicloning site existing in the gene encoding the LacZ α fragment existing downstream of the LacZ promoter of the E. coli plasmid pUC119. Created by ligating DNA fragments with In the transformant of E. coli XL1-Blue strain introduced with the hybrid plasmid pvanAB, it is not produced as the original dimethylase enzyme protein having the amino acid sequence shown by SEQ ID NO: 2 under the control of the LacZ promoter, but derived from the LacZ α fragment This is produced as a fusion protein in which an oligopeptide consisting of 16 amino acids is added to the N-terminus of the subunit of the dimethylase enzyme shown in SEQ ID NO: 2 (FIG. 4).
On the other hand, in the transformant of E. coli XL1-Blue strain into which the hybrid plasmid pvnnAB has been introduced, it is produced as the original dimethylase enzyme protein having the amino acid sequence shown by SEQ ID NO: 2 under the control of the LacZ promoter (FIG. 4).
The transformant of E. coli XL1-Blue strain introduced with the hybrid plasmid pvanAB shows a strong and stable dimethylase activity, whereas the transformant of E. coli XL1-Blue strain introduced with the hybrid plasmid pvnnAB does not show any demethylase activity. The fact is that the original dimethylase enzyme protein having the amino acid sequence shown in SEQ ID NO: 2 that is thought to be produced in the two transformants is produced as an extremely unstable enzyme protein and rapidly degraded, while the hybrid plasmid pvanAB The dimethylase enzyme protein with an N-terminal oligopeptide consisting of 16 amino acids derived from the LacZ α fragment produced by the transformant of Escherichia coli XL1-Blue strain into which N is introduced is derived from the LacZ α fragment By adding an oligopeptide consisting of 16 amino acids, degradation is avoided and dimethylase activity is reduced. It is thought that it is produced as a stable fusion protein.

To further confirm this possibility, a DNA fragment containing SEQ ID NO: 1 obtained by cutting with the restriction enzymes HindIII and SmaI from the hybrid plasmids pvanAB and pvnnAB is commercially available for the production of proteins to which 6 amino acids of histidine have been added. The hybrid plasmid pQvAB was prepared by ligating to the cleavage site of the restriction enzyme SmaI on the existing expression vector pQE32 by blunting to match the reading frames (FIG. 5). The transformant of E. coli introduced with this hybrid plasmid pQvAB has 16 amino acids derived from the α fragment of LacZ as in the case of pvanAB at the N-terminus of the polypeptide represented by SEQ ID NO: 1 under the control of the LacZ promoter. A fusion protein is produced in which an oligopeptide consisting of 30 amino acids including 6 amino acids of histidine, added to the N-terminus of the polypeptide represented by SEQ ID NO.
Transformed cells prepared by introducing the newly prepared hybrid plasmid pQvAB into E. coli XL1-Blue strain produced a strong and stable enzyme activity using vanillic acid and syringic acid as substrates.
In addition, in the transformant that is introduced into Escherichia coli XL1-Blue strain and exhibits a strong dimethylase activity, a large amount of fusion protein having an increased molecular weight compared to the polypeptide shown in SEQ ID NO: 2 is produced. In the transformant of Escherichia coli XL1-Blue strain into which the plasmid pvnnAB was introduced, accumulation of the original dimethylase enzyme protein calculated from the amino acid sequence represented by sequence A was not observed (FIG. 8).

  Vanillic acid demethylase (VanA and VanB) is produced as an unstable polypeptide with an essentially short half-life in microbial cells. This is due to the fact that in many previous studies using crude enzyme solutions obtained by disrupting wild microbial cells that produce vanillic acid demethylase, it has been detected only as extremely weak enzyme activity, and many documents (Priefert H., Rabnehorst J., Steinbuchel A., (1997) J. Bacteriol. 179, 2595-2607, Venturi V., Zennaro F., Degrassi G., (1998) Microbiology 144, 965-973) In a transformant prepared by introducing a hybrid plasmid having a demethylase gene into E. coli, only a very weak dimethylase activity of 1/3000 of the enzyme activity achieved in the present invention was detected. The importance of the present invention will be explained well. Accordingly, the present invention provides a new and stable realization of the function of a dimethylase that converts vanillic acid possessed by microorganisms such as Pseudomonas to protocatechuic acid in various host cells including E. coli using genetic recombination technology. It is about the method. For example, an oligopeptide consisting of 16 amino acids contained in the α fragment of LacZ or an oligopeptide consisting of 30 amino acids containing 6 histidines, which contributes to the stabilization of VanA and does not interfere with the enzymatic function of VanA. Vanillate demethylase can be stabilized by adding a peptide to the N-terminus of VanA. The structure of the fusion peptide added to the N-terminus does not have to be limited to the amino acid sequence used in the present invention, and any structure that contributes to the stabilization of vanillate demethylase (VanA and VanB) may be used.

A new demethylase gene derived from the Pseudomonas putida PpY101 strain, which is retained on the hybrid plasmid pvanAB and produces the strong demethylase activity in Escherichia coli XL1-Blue strain, isolated in the present invention and represented by SEQ ID NO: 1, is Pseudomonas putida The dimethylase-deficient mutant strain (PpY1100 strain) is more strongly expressed than the E. coli XL1-Blue strain and is expected to maintain stable enzyme activity.
The broad host range plasmid pKT230Mc (FIG. 7) that can be replicated and maintained in Pseudomonas putida is digested with the restriction enzyme EcoRI and then blunted into a DNA fragment that is stable and powerful in vanillic acid demethylase (VanA and VanB) is a broad plasmid ligated with a DNA fragment containing the lacZ promoter region obtained by cleavage with the restriction enzyme PvuII, the α fragment region of LacZ, and the region encoding the demethylase shown in SEQ ID NO: 1. A host range hybrid plasmid pDVZ1 was constructed (FIG. 8). The dimethylase gene on the hybrid plasmid pDVZ1 exists downstream of the E. coli-derived β-lactamase gene promoter of the broad host range plasmid pKT230Mc (FIG. 7), and the β-lactamase gene promoter includes not only E. coli but also the genus Pseudomonas. Due to its function in various microorganisms, Escherichia coli XL1-Blue strain and Pseudomonas putida demethylase-deficient mutant (PpY1100 strain) transformed with the hybrid plasmid pDVZ1 strongly express the dimethylase gene and are stable enzymes. It can be expected to generate activity.
Both transformants of Escherichia coli XL1-Blue strain and Pseudomonas putida demethylase-deficient mutant strain (PpY1100 strain) into which the hybrid plasmid pDVZ1 was introduced showed strong demethylase activity against vanillic acid and syringic acid.

In the transformant of Escherichia coli XL1-Blue, vanillic acid demethylase (VanA and VanB) shown in SEQ ID NOs: 2 and 3 is originally produced as an unstable polypeptide with a short half-life and is stable at the N-terminus of VanA polypeptide. It was judged that the addition of a polypeptide that contributes to the formation of the polypeptide reduced the instability of the polypeptide chain and produced a stable demethylase activity. The possibility that this phenomenon is a universal property observed not only in E. coli but also in Pseudomonas putida transformants was investigated.
A DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 1 obtained by cleaving the E. coli hybrid plasmid pvanAB with restriction enzymes EcoRI and HindIII is ligated to the cleavage site of the broad host range plasmid pKT230MC (FIG. 7) with the restriction enzyme EcoRI. A hybrid plasmid pDV01 was prepared (FIG. 9). The demethylase gene on the hybrid plasmid pDV01 exists downstream of the E. coli-derived β-lactamase gene promoter of pKT230MC, and a demethylase-deficient mutant of E. coli XL1-Blue strain or Pseudomonas putida introduced with the hybrid plasmid pDV01 ( The PpY1100 strain) transformant strongly expresses the dimethylase gene and can produce the original dimethylase enzyme protein having the amino acid sequences shown in SEQ ID NOs: 2 and 3. As expected, transformants of Escherichia coli XL1-Blue strain and Pseudomonas putida PpY1100 strain into which the hybrid plasmid pDV01 was introduced did not show dimethylase activity.
The reason why dimethylase is not produced in the transformants of Escherichia coli XL1-Blue strain and Pseudomonas putida PpY1100 strain into which the hybrid plasmid pDV01 has been introduced is that Shine Dalgano necessary for translation in the upstream region of the dimethylase gene shown in SEQ ID NO: 1. It is conceivable that there is no sequence (SD sequence).
In order to add two types of Shine-Dalgarno sequences (SD sequences) that have been found to function effectively in Pseudomonas bacteria to SEQ ID NO: 1, two types of PCR primer sequences (AS-LIGA (sequences) No. 12) and AS-GAP (SEQ ID NO: 13)) are newly prepared, PCR is performed using primer BO (5′-AATGCCACCTTGCGTTATGC-3 ′: SEQ ID NO: 11) as a downstream primer and Escherichia coli hybrid plasmid pvanAB as a template. Amplified by the method. A DNA fragment newly introduced with the SD sequence on the 5 ′ side of SEQ ID NO: 1 was ligated to a site cleaved by the restriction enzyme SmaI of the multicloning site of the E. coli plasmid pUC18, thereby obtaining a new E. coli hybrid plasmid pvLAB (PCR primer sequence ( AS-LIGA)) and hybrid plasmid pVGAB (containing PCR primer sequence (AS-GAP)) were prepared (FIG. 12). These transformants of E. coli XL1-Blue strain introduced with hybrid plasmid pvLAB or pVGAB did not produce dimethylase as expected.
In addition, a DNA fragment having a dimethylase gene added with an SD sequence obtained by cleaving with the restriction enzymes EcoRI and HindIII from the hybrid plasmids pvLAB and pVGAB of E. coli was obtained using the restriction enzyme EcoRI of the broad host range plasmid pKT230MC (FIG. 7). Ligated plasmids pDVL1 and pDVG1 were prepared by ligation to the cleavage site (FIG. 11). The transformant of Pseudomonas putida PpY1100 strain into which these hybrid plasmids were introduced did not show any dimethylase activity.
The hybrid plasmid pDVZ1 is a transformant of Escherichia coli and Pseudomonas putida that holds the oligopeptide consisting of 16 amino acids derived from the α fragment of LacZ under the control of the LacZ promoter, VanA of the demethylase enzyme shown in SEQ ID NO: 2. It can be produced as a fusion protein added to the N-terminus of subunits to produce strong and stable enzyme activity. Therefore, dimethylase was originally produced as an unstable polypeptide with a short half-life in Pseudomonas putida PpY101 strain, and a polypeptide contributing to stabilization was added to the N-terminus of the polypeptide shown in SEQ ID NO: 2. Thus, stabilization of the dimethylase polypeptide chain represented by the amino acid sequences of SEQ ID NOs: 2 and 3 and a strong dimethylase activity can be obtained.

  Furthermore, the fermentation production process control gene of 2-pyrone-4,6-dicarboxylic acid constructed in the present invention is obtained by association of two types of polypeptides (ligA and ligB) having the amino acid sequences shown in SEQ ID NOs: 15 and 17. And a base sequence (SEQ ID NO: 14 and 16, respectively) encoding two types of polypeptides having protocatechuic acid 4,5-dioxygenase activity, and the amino acid represented by SEQ ID NO: 19 in the downstream region A base represented by SEQ ID NO: 20 which partially contains a base sequence region (SEQ ID NO: 18) encoding a polypeptide (ligC) having 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase activity having a sequence Has an array. The base sequence represented by SEQ ID NO: 20 contains an inverted complementary sequence (inverted repeat) about 24 bases downstream from the stop codon sequence of the region encoding 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase. ) And a ρ-independent transcription terminator structure in which 6 thymines are continuously located (positions 2688 to 2706), and this ρ-independent transcription terminator structure is 2-pyrone-4, By placing the 6-dicarboxylic acid fermentation production process control gene at the most downstream position, efficient transcription of the entire 2-pyrone-4,6-dicarboxylic acid fermentation production process control gene composed of various enzyme genes is possible. become.

  The nucleotide sequence represented by SEQ ID NO: 20 is an unstable intermediate product 4-carboxyl produced from protocatechuic acid by the enzymatic activity of protocatechuic acid 4,5-dioxygenase encoded by the nucleotide sequences represented by SEQ ID NOs: 14 and 16. In order to quickly convert 2-hydroxymuconic acid-6-semialdehyde into stable 2-pyrone-4,6-dicarboxylic acid, two kinds of polypeptides encoded by the nucleotide sequences shown in SEQ ID NOs: 14 and 16 are used. Among these, the gene region encoding the downstream small subunit has a structure in which the promoter sequence of the 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase gene shown in SEQ ID NO: 18 is duplicated. Thus, a hybrid plasmid prepared by ligating a DNA fragment containing the gene downstream of the E. coli-derived β-lactamase gene promoter of the broad host range plasmid pKT230MC (FIG. 7) having E. coli plasmid or Pseudomonas as a host, Since the β-lactamase gene promoter functions not only in E. coli but also in various microorganisms including the genus Pseudomonas, the transformant introduced with the hybrid plasmid is protocatechuic acid 4-carboxy-2-hydroxymuconic acid-6-semialdehyde Converting enzymes protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde to 2-pyrone-4,6-dicarboxylic acid, 4-carboxy-2- Synthetic production of two enzymes, hydroxymuconic acid-6-semialdehyde dehydrogenase, to produce protocatechuic acid Therefore, the stable fermentation process of 2-pyrone-4,6-dicarboxylic acid can be achieved.

  From the genomic DNA of the microorganism Sphingomonas paucimobilis SYK-6 having the genes encoding protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase represented by SEQ ID NO: 20, the restriction enzyme XbaI The DNA fragment (FIG. 12) having the SEQ ID NO: 20 obtained by cleavage is ligated to the cleavage site by the restriction enzyme XbaI present at the multicloning site of the E. coli plasmid pUC119 to prepare a hybrid plasmid pULABC (FIG. 13). The transformant of Escherichia coli XL1-Blue strain into which this hybrid plasmid has been introduced can convert protocatechuic acid to 2-pyrone-4,6-dicarboxylic acid in high yield.

  A sequence obtained by cleavage of the restriction enzyme XbaI from the hybrid plasmid pULABC shown in FIG. 13 at the site of cleavage of the hybrid plasmid pvanAB (FIG. 2) by the restriction enzyme Xba1, which produces a strong dimethylase activity in the E. coli XL1-Blue strain Escherichia coli hybrid plasmid ligated with DNA fragments encoding protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase having the base sequence shown by No. 20: pUVLABC (FIG. 14) It was created. The transformant of Escherichia coli XL1-Blue strain into which this hybrid plasmid was introduced was composed of three enzymes under the control of the LacZ promoter, dimethylase, protocatechuic acid 4,5-dioxygenase and 4-carboxy-2-hydroxymuconic acid-6. -Synchronously expressing the gene sequence encoding semialdehyde dehydrogenase and derived from plant component, petroleum component, or chemically synthesized vanillic acid, syringic acid, protocatechuic acid, 2-pyrone-4,6-dicarboxylic acid The acid could be produced by fermentation.

A broad host range plasmid pKT230MC (Fig. 7) that can be replicated and maintained in Pseudomonas putida, and a hybrid plasmid pvanAB (Fig. 7) that realized the production of a stable and powerful vanillate demethylase (VanA, B) in E. coli transformants. The broad host range hybrid plasmid pDVZ1 (Fig. 8) ligated with the DNA fragment obtained by cleavage with the restriction enzyme PvuII in 2) is a transformant of E. coli XL1-Blue strain or Pseudomonas putida into which the demethylase gene has been introduced. Strongly expressed and stable enzyme activity production. Protocatechuic acid 4,5 having the base sequence represented by SEQ ID NO: 20 obtained by digestion of the restriction enzyme XbaI from the hybrid plasmid pULABC shown in FIG. 13 at the cleavage site of the hybrid plasmid pDVZ1 (FIG. 8) by the restriction enzyme Xba1. A broad host range hybrid plasmid ligated with DNA fragments encoding -dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase: pKTVLABC (FIG. 15) was prepared. Transformants of Pseudomonas putida PpY1100 strain and E. coli XL1-Blue strain into which this hybrid plasmid has been introduced may contain a mixture of vanillic acid, syringic acid, and protocatechuic acid, or each may be highly enriched in 2-pyrone-4,6-dicarboxylic acid. Yield conversion was possible.
The broad host range recombinant plasmid pKTVLABC (FIG. 15) has a wide host range including Pseudomonas bacteria, and in transformed cells introduced with the recombinant plasmid, three types of enzyme demethylase, protocatechuic acid 4,5 Synthetic expression of a gene sequence encoding -dioxygenase, 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, derived from plant components or petroleum components, or chemically synthesized vanillic acid, syringa 2-pyrone-4,6-dicarboxylic acid can be produced from acid and protocatechuic acid, but for high production of 2-pyrone-4,6-dicarboxylic acid, it is derived from plant components, chemical synthesis or petroleum Degradation of any of vanillic acid, syringic acid, and protocatechuic acid into 2-pyrone-4,6-dicarboxylic acid, and 2-pyrone-4,6-dicarboxylic acid It is necessary to use a transformant using a microorganism that has lost its degrading enzyme function as a host.

  SEQ ID NO: 22 for E. coli hybrid plasmid pUVLABC (FIG. 14) for fermentative production of 2-pyrone-4,6-dicarboxylic acid from vanillic acid, syringic acid, and protocatechuic acid, and broad host range hybrid plasmid: pKTVLABC (FIG. 15) A hybrid plasmid prepared by further linking the base sequence represented by SEQ ID NO: 21, which partially contains a region encoding a polypeptide having a benzaldehyde dehydrogenase activity (ligV) having the amino acid sequence represented by In the presence, vanillin and syringaldehyde are oxidized to vanillic acid and syringic acid, and further benzaldehyde and P-hydroxybenzaldehyde are oxidized to benzoic acid and P-hydroxybenzoic acid. Therefore, a function of oxidizing vanillin and syringaldehyde contained in the plant component-derived low-molecular mixture to convert them into vanillic acid and syringic acid is newly added.

  A hybrid plasmid pULV (FIG. 16) is prepared by ligating the gene encoding benzaldehyde dehydrogenase represented by SEQ ID NO: 21 to a cleavage site by the restriction enzyme SmaI present at the multicloning site of E. coli plasmid pUC119. A transformant of E. coli XL1-Blue strain introduced with this hybrid plasmid can produce benzaldehyde dehydrogener. Further, the gene encoding benzaldehyde dehydrogenase represented by SEQ ID NO: 21 is ligated to the cleavage site of the broad host range plasmid pKT230MC (FIG. 7) that can be replicated and maintained in Pseudomonas putida by the restriction enzyme SmaI to hybridize the plasmid pKTLV. (FIG. 16) is created. A transformant of Pseudomonas putida PpY1100 strain into which this hybrid plasmid has been introduced can produce benzaldehyde dehydrogener.

2-pyrone-4,6-dicarboxylic acid is fermentatively produced from vanillic acid, syringic acid, and protocatechuic acid from a DNA fragment obtained by digestion with restriction enzyme SmaI of Escherichia coli hybrid plasmid pULV (FIG. 16) carrying SEQ ID NO: 21. Sequences encoding three enzymes (dimethylase, protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase) and their expression After partial cleavage of the E. coli hybrid plasmid pUVLABC (FIG. 14) having a gene region involved in regulation with the restriction enzyme XbaI, the DNA fragment obtained by the end treatment is ligated with a known DNA ligase to thereby bind the E. coli recombinant plasmid pUVDLABC (FIG. 17). ).
The prepared E. coli recombinant plasmid pUVDLABC (FIG. 17) was introduced into E. coli and under the control of the LacZ promoter, four types of enzyme demethylase, benzaldehyde dehydrogenase, protocatechuic acid 4,5-dioxygenase and 4-carboxy-2-hydroxymuconone. Synchronously expressing a gene sequence encoding acid-6-semialdehyde dehydrogenase from plant components, petroleum components, or chemically synthesized vanillin, syringaldehyde, vanillic acid, syringic acid, protocatechuic acid 2-pyrone-4,6-dicarboxylic acid can be produced by fermentation.

2-pyrone-4,6-dicarboxylic acid is fermentatively produced from vanillic acid, syringic acid, and protocatechuic acid from a DNA fragment obtained by digestion with restriction enzyme SmaI of Escherichia coli hybrid plasmid pULV (FIG. 16) carrying SEQ ID NO: 21. Sequences encoding three enzymes (dimethylase, protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase) and their expression The broad host range recombinant plasmid pKTVLABC (FIG. 15) having a gene region involved in regulation is partially cleaved with the restriction enzyme XbaI and then joined with a DNA fragment obtained by terminal treatment with a known DNA ligase. pKTVDLABC (FIG. 18) is created.
The prepared broad host range recombinant plasmid pKTVDLABC (FIG. 18) has a wide host range including Pseudomonas bacteria, and in transformed cells into which the recombinant plasmid has been introduced, four types of enzyme demethylase and benzaldehyde dehydrogenase are used. , Synchronously expressing gene sequences encoding protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, derived from plant or petroleum components, or chemically 2-pyrone-4,6-dicarboxylic acid can be produced from synthesized vanillin, syringaldehyde, vanillic acid, syringic acid, and protocatechuic acid. The microorganism used as a host for high production of 2-pyrone-4,6-dicarboxylic acid replicates the broad host range recombinant plasmid pKTVDLABC (FIG. 18) and participates in 2-pyrone-4,6-dicarboxylic acid production. It is not limited as long as it can express the enzyme gene, but any of plant component-derived, chemically synthesized or petroleum-derived vanillin, syringaldehyde, vanillic acid, syringic acid, or protocatechuic acid is used as 2-pyrone-4,6- It is necessary to use a transformant having a microorganism that has lost its function of degrading and metabolizing enzymes to dicarboxylic acids and the function of degrading enzymes to 2-pyrone-4,6-dicarboxylic acid as a host.

(Acquisition of vanillic acid demethylase gene (vanAB gene))
In order to produce a multistage reaction process control gene designed and constructed for fermentation production that converts to high yield of 2-pyrone-4,6-dicarboxylic acid of the present invention, first, vanillic acid and syringic acid in the presence of NADH. It is necessary to isolate the vanillic acid demethylase gene (vanAB gene), which is converted to protocatechuic acid and 3-methylgallic acid by breaking the methyl ether bond. The means for separating this gene is not particularly limited, and for example, a microorganism host vector system can be used. The dimethylase gene (vanAB gene) is produced as follows using, for example, recombinant DNA technology. That is, using a microorganism having the ability to produce a demethylase that cleaves methyl ether of vanillic acid and syringic acid in the presence of NADH as a DNA donor, genomic DNA is extracted from the microorganism, cleaved with a restriction enzyme, etc. And On the other hand, restriction enzyme ends into which genome fragments can be inserted are prepared using restriction enzymes or the like for vector DNA such as phages and plasmids, and restriction enzymes or the like for genomic DNA. 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 transformant cell holding the target recombinant vector, and isolating the target recombinant vector from the transformant cell.
The DNA donor for separating the gene is not particularly limited as long as it is a microorganism capable of producing a dimethylase that cleaves methyl ether of vanillic acid and syringic acid in the presence of NADH. For example, Pseudomonas putida PpY101 strain is preferable.

Extraction of genomic DNA from the microorganism is performed by collecting cultured cells of the microorganism, lysing the bacterial body with, for example, protease K, and then removing protein by phenol extraction, protease treatment, ribonuclease treatment, genomic DNA by 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 autonomously growing in a host microorganism. As the plasmid, for example, pUC18, pUC19, pUC118, pUC119, Bluescript, pKT230MC (FIG. 7) and the like using E. 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 the foreign gene trait is stably expressed. Or Pseudomonas putida can be used. As a method for transferring the recombinant vector into the host microorganism, any method such as a conjugation method, an electroporation method, or a competent cell method can be used.
Selection of transformants can be based on the selection marker of the vector used, for example, drug resistance acquired by DNA recombination of the transformant. 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 the probe label, for example, any of radioisotopes, digoxigenin, enzymes, and the like can be used.

In order to extract a recombinant vector from the transformant cells thus selected, extraction may be performed by a conventional method, for example, using an alkaline lysis method (Cold Spring Harbor Laboratory Press, Molecular Cloning Second Edition (1989)). be able to. The extracted recombinant vector can be recombined again using DNA technology as necessary. The vanillic acid demethylase gene (vanAB gene) that is converted from the resulting recombinant vector into protocatechuic acid and 3-methylgallic acid by cleaving the methyl ether bond of vanillic acid and syringic acid in the presence of NADH contained in the gene of the present invention ) Can be cut out using a restriction enzyme or the like.
The base sequence of the vanillic acid demethylase gene contained in the gene of the present invention thus obtained can be decoded and determined by, for example, the dideoxy method. SEQ ID NO: 1 shows the base sequence of the region containing the vanillate demethylase gene contained in the gene of the present invention, and SEQ ID NOs: 2 and 3 show the amino acid sequences deduced from the base sequence.

(Acquisition of protocatechuic acid 4,5-dioxygenase gene (ligAB gene))
To create a multistage reaction process control gene designed and constructed for the fermentative production of 2-pyrone-4,6-dicarboxylic acid according to the present invention, an aromatic ring of protocatechuic acid and 3-methylgallic acid is used as the second gene. Isolation of the enzyme protocatechuate 4,5-dioxygenase gene (ligAB gene) that opens the ring is necessary. The means for separating this gene is not particularly limited, and for example, a microorganism host vector system can be used. The protocatechuic acid 4,5-dioxygenase gene is produced as follows using, for example, recombinant DNA technology. That is, using a microorganism capable of producing protocatechuic acid 4,5-dioxygenase as a DNA donor, genomic DNA is extracted from the microorganism and cleaved with a restriction enzyme or the like to obtain a DNA fragment. On the other hand, restriction enzyme ends into which genome fragments can be inserted are prepared using restriction enzymes or the like for vector DNA such as phages and plasmids, and restriction enzymes or the like for genomic DNA. 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 transformant cell holding the target recombinant vector, and isolating the target recombinant vector from the transformant cell.
The DNA donor for separating the gene is not particularly limited as long as it is a microorganism capable of producing protocatechuic acid 4,5-dioxygenase that opens the aromatic ring of protocatechuic acid and 3-methylgallic acid. For example, Sphingomonas paucimobilis SYK-6 strain is preferable.

Extraction of genomic DNA from the microorganism, fragmentation of the genomic DNA, preparation of vectors and assembly vectors, host microorganism for introducing the obtained recombinant vector, its introduction method, and transformant containing the target recombinant vector And the method for extracting a recombinant vector from the selected transformant cell is the same as described above (obtain vanillic acid demethylase gene (vanAB gene)). In order to excise the protocatechuic acid 4,5-dioxygenase gene (ligAB gene) 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 protocatechuic acid 4,5-dioxygenase gene (ligAB gene) contained in the gene of the present invention thus obtained can be determined by decoding, for example, by the dideoxy method. SEQ ID NOs: 14 and 16 (including SEQ ID NO: 20) include the bases of the region containing the protocatechuate 4,5-dioxygenase gene (ligAB gene) isolated from the genomic DNA of the microorganism for producing the gene of the present invention. The amino acid sequences deduced from the nucleotide sequences are shown in SEQ ID NOs: 15 and 17, respectively.

(4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase gene (ligC gene))
In the production of the multistage reaction process control gene designed and constructed for the fermentative production of 2-pyrone-4,6-dicarboxylic acid according to the present invention, 4-carboxy-2-hydroxymuconic acid- It is necessary to isolate the 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase gene (ligC gene), which converts 6-semialdehyde to 2-pyrone-4,6-dicarboxylic acid. The means for separating this gene is not particularly limited, and for example, a microorganism host vector system can be used. The 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase gene (ligC gene) is produced as follows using, for example, recombinant DNA technology. That is, using a microorganism capable of producing 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase (ligC) as a DNA donor, genomic DNA is extracted from the microorganism, cleaved with a restriction enzyme or the like, and then a DNA fragment And On the other hand, restriction enzyme ends into which genome fragments can be inserted are prepared using restriction enzymes or the like for vector DNA such as phages and plasmids, and restriction enzymes or the like for genomic DNA. 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 transformant cell holding the target recombinant vector, and isolating the target recombinant vector from the transformant cell.
As a DNA donor for the separation of the gene, 4-carboxy-2-hydroxymuconic acid-6 which converts 4-carboxy-2-hydroxymuconic acid-6-semialdehyde to 2-pyrone-4,6-dicarboxylic acid -Any microorganism that has the ability to produce semialdehyde dehydrogenase (ligC) is not particularly limited. For example, Sphingomonas paucimobilis SYK-6 strain is preferable.

Extraction of genomic DNA from the microorganism, fragmentation of the genomic DNA, preparation of vectors and assembly vectors, host microorganism for introducing the obtained recombinant vector, its introduction method, and transformant containing the target recombinant vector And the method for extracting a recombinant vector from the selected transformant cell is the same as described above (obtain vanillic acid demethylase gene (vanAB gene)). In order to excise the 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase gene (ligC gene) 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 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase gene (ligC gene) contained in the gene of the present invention thus obtained can be determined by decoding, for example, by the dideoxy method. SEQ ID NO: 20 contains a 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase gene (ligC gene) isolated from the genomic DNA of the microorganism Sphingomonas paucimobilis SYK-6 strain for the production of the gene of the present invention The nucleotide sequence of the region is shown in SEQ ID NO: 18, the nucleotide sequence of the 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase gene (ligC gene) region, and SEQ ID NO: 19 of the enzyme protein deduced from the nucleotide sequence. Amino acid sequence is shown.

(Arrangement design of each gene and construction of metabolic gene)
The three enzymes thus obtained (vanillate demethylase (VanA and VanB), protocatechuate 4,5-dioxygenase (ligA and ligB) and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC) )) And a DNA fragment containing the gene region involved in their expression control, excised from each recombinant plasmid with restriction enzymes and ligated with DNA ligase based on the designed gene arrangement, and the three enzymes Restriction-cleavage DNA fragments containing genes are ligated together. A novel double-stranded DNA molecule of a control gene for a multistage reaction process that rapidly fermentatively produces 2-pyrone-4,6-dicarboxylic acid in large quantities from vanillic acid, syringic acid, or protocatechuic acid can be prepared.

(Arrangement design of each gene and construction of metabolic gene 1: Preparation of Escherichia coli expression plasmid pvanAB of dimethylase)
In the present invention, a DNA fragment having the base sequence of SEQ ID NO: 1 excised by the restriction enzyme HincII from a transformation plasmid holding a DNA fragment having the gene sequence shown in SEQ ID NO: 1 as a part thereof is encoded by the E. coli plasmid pUC119. A new E. coli expression plasmid pvanAB is prepared by binding to a DNA fragment prepared by cleaving at the cleavage site of the restriction enzyme HincII present in the LacZα fragment gene (FIG. 2). When the E. coli expression plasmid pvanAB is introduced into E. coli or the like using the electroporation method or the competent cell method, the α fragment polypeptide existing downstream of the Lac promoter in the transformant cells and SEQ ID NOs: 2 and 3 are shown. Produced as a fusion polypeptide with a dimethylase polypeptide.
SEQ ID NO: 1 is linked to a LacZα fragment gene encoded by E. coli plasmid pUC119, and an oligopeptide that contributes to the stabilization of the dimethylase enzyme derived from the α fragment gene at the N-terminus of the polypeptide shown in SEQ ID NOs: 2 and 3. By producing the added fusion peptide in a transformant cell such as Escherichia coli, a stable and high demethylase fermentation activity can be obtained. However, as in the E. coli expression plasmid pvnnAB shown in FIG. 2-2, the dimethylase gene of SEQ ID NO: 1 is directly linked downstream of the promoter sequence to produce a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 and 3. In this case, dimethylase activity cannot be obtained because it is produced as an unstable polypeptide in the transformant cells. In addition, oligopeptides contributing to stabilization added to SEQ ID NOs: 2 and 3 for stable production of dimethylase include not only oligopeptides derived from LacZα fragments but also 6 amino acids of histidine, such as pQvAB in FIG. An oligopeptide composed of 30 amino acids may be used as long as it contributes to stabilization of SEQ ID NOs: 2 and 3.

(Arrangement design of each gene and construction of metabolic system 2: Construction of a wide host range plasmid pDVZ1 (Fig. 8) of dimethylase)
From the E. coli recombinant plasmid pvanAB (FIG. 2), a Lac promoter sequence excised by the restriction enzyme PuvII, a gene encoding 16 amino acids of the α fragment existing downstream thereof, and a DNA fragment comprising SEQ ID NO: 1 fused thereto were obtained as Pseudomonas By binding with a known DNA ligase to a DNA molecule obtained by blunting both ends of a DNA fragment obtained by digestion with a restriction enzyme EcoRI of a broad host range plasmid pKT230MC (FIG. 7) that can be replicated and maintained in putida A new broad host range plasmid pDVZ1 (FIG. 8) is generated. When the broad host range plasmid pDVZ1 (FIG. 8) is introduced into Pseudomonas putida or the like by electroporation or competent cell method, the α fragment polypeptide and the sequence number present downstream of the Lac promoter in the transformed cell. It is produced as a fusion polypeptide with the polypeptide of dimethylase represented by 2 and 3, and a powerful and stable dimethylase can be produced.

(Arrangement design of each gene and construction of metabolic gene 3: Construction of E. coli plasmid pULABC from protocatechuic acid to PDC (Fig. 13))
Cleavage of the restriction enzyme XbaI present in the LacZα fragment gene encoded by the Escherichia coli plasmid pUC119 is a DNA fragment having the nucleotide sequence of SEQ ID NO: 20 excised from the E. coli plasmid containing the nucleotide sequence represented by SEQ ID NO: 20 by the restriction enzyme XbaI. A new E. coli plasmid pULABC (FIG. 13) is prepared by binding to a DNA fragment prepared by cleaving at the site by a known DNA ligase. When E. coli plasmid pULABC (FIG. 13) is introduced into E. coli or the like using electroporation method or competent cell method, protocatechuate 4,5-dioxygenase enzyme gene (ligAB gene) and 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase enzyme gene (ligC gene) is expressed synchronously and can produce two types of enzymes.

(Arrangement design of each gene and construction of metabolic gene 4: Recombinant plasmid pUVLABC from vanillic acid and syringic acid to PDC (Fig. 14))
The DNA fragment obtained by cleaving restriction enzyme XbaI of E. coli plasmid pvanAB shown in FIG. 2 is bound to the DNA fragment containing SEQ ID NO: 20 obtained by cleaving restriction enzyme XbaI of E. coli plasmid pULABC shown in FIG. 13 by a known DNA ligase. 3 enzymes constituting a multi-stage reaction process for fermentative production of 2-pyrone-4,6-dicarboxylic acid from vanillic acid, syringic acid and protocatechuic acid (vanillic acid demethylase, protocatechuic acid 4, 2-pyrone containing a novel double-stranded DNA molecule linking a gene sequence encoding 5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde) and a gene region involved in their expression control A -4,6-dicarboxylic acid fermentation-producing Escherichia coli recombinant plasmid pUVLABC (FIG. 14) is prepared. When the E. coli recombinant plasmid pUVLABC (FIG. 14) is introduced into E. coli or the like using electroporation or competent cell methods, the transformed cells are derived from plant components, petroleum components, or chemically synthesized. 2-pyrone-4,6-dicarboxylic acid can be produced by fermentation from the prepared vanillic acid, syringic acid, and protocatechuic acid.

(Arrangement design of each gene and construction of metabolic gene 5: Wide host range recombinant plasmid pKTVLABC producing PDC from vanillic acid and syringic acid (FIG. 15))
A DNA fragment having SEQ ID NO: 20 obtained by cleavage of the restriction enzyme XbaI of the E. coli recombinant plasmid pULABC (FIG. 13) is obtained by cleavage of the restriction enzyme XbaI of the broad host range plasmid pDVZ1 (FIG. 8) capable of producing dimethylase. By ligating the obtained DNA fragment with a known DNA ligase, 2-pyrone-4,6-dicarboxylic acid fermentation production wide host range recombinant plasmid pKTVLABC (FIG. 15) having various host ranges can be produced. Recombinant plasmid pKTVLABC (Fig. 15) for 2-pyrone-4,6-dicarboxylic acid fermentation production with various host ranges contains 2-pyrone-4,6-dicarboxylic acid from vanillic acid, syringic acid, and protocatechuic acid. A gene sequence encoding three types of enzymes (dimethylase, protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase) that constitute a multi-stage reaction process for fermentative production; New double-stranded DNA molecules that link gene regions involved in their expression control are included. When the broad host range recombinant plasmid pKTVLABC (FIG. 15) is introduced into the host microbial cells using electroporation or competent cell methods, the transformed cells are derived from plant components or petroleum components. Alternatively, 2-pyrone-4,6-dicarboxylic acid can be produced by fermentation from chemically synthesized vanillic acid, syringic acid, and protocatechuic acid.
Recombinant plasmid pKTVLABC (FIG. 15) having 2-pyrone-4,6-dicarboxylic acid fermentation production with various host ranges is obtained from vanillic acid, syringic acid, and protocatechuic acid downstream of β-lactamase gene promoter. It contains a gene sequence that encodes three types of enzymes that constitute a multistage reaction process for the fermentation production of 6-dicarboxylic acid, has a wide host range, including Pseudomonas bacteria, and uses electroporation and competent cell methods. In the transformed cells into which the recombinant plasmid was introduced, 3 types of enzyme demethylase, protocatechuic acid 4,5-dioxygenase, and 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase were encoded. Synthetic expression of the gene sequence, derived from plant or petroleum components, or chemically synthesized vanillin Can produce 2-pyrone-4,6-dicarboxylic acid from syringic acid and protocatechuic acid, but for high production of 2-pyrone-4,6-dicarboxylic acid, it is derived from plant components, chemical synthesis Or, it has the function of degrading and metabolizing enzymes from any of petroleum-derived vanillic acid, syringic acid, and protocatechuic acid to 2-pyrone-4,6-dicarboxylic acid and degrading enzyme function to 2-pyrone-4,6-dicarboxylic acid It is necessary to use a transformant with the lost microorganism as the host.

(Benzaldehyde dehydrogenase gene (ligV gene))
To fermentatively produce 2-pyrone-4,6-dicarboxylic acid from plant components or petroleum components or chemically synthesized vanillin and syringaldehyde via vanillic acid, syringic acid, and protocatechuic acid Sequences encoding four types of enzymes (benzaldehyde dehydrogenase, dimethylase, protocatechuate 4,5-dioxygenase, 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase) that constitute the multistage reaction process A new double-stranded DNA molecule is created by linking and arranging gene regions involved in the regulation of gene expression by genetic engineering methods, and four types of enzyme genes are synchronously increased in the transformed cells that hold them. Vanillin, syringaldehyde, vanillic acid expressed and derived from plant components or petroleum components, or chemically synthesized In order to construct a control gene for a multistage reaction process that rapidly fermentatively produces 2-pyrone-4,6-dicarboxylic acid from syringic acid and protocatechuic acid in large quantities, oxidation and conversion of vanillin to vanillic acid were newly performed. It is necessary to isolate a benzaldehyde dehydrogenase gene (ligV gene) having an oxidative activity of lingaldehyde to syringic acid. The means for separating this gene is not particularly limited, and for example, a microorganism host vector system can be used. The benzaldehyde dehydrogenase gene (ligV gene) is produced as follows using, for example, recombinant DNA technology. That is, using a microorganism having the ability to produce benzaldehyde dehydrogenase capable of oxidizing vanillin and syringaldehyde as a DNA donor, genomic DNA is extracted from the microorganism and cleaved with a restriction enzyme or the like to obtain a DNA fragment. On the other hand, restriction enzyme ends into which genome fragments can be inserted are prepared using restriction enzymes or the like for vector DNA such as phages and plasmids, and restriction enzymes or the like for genomic DNA. 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 transformant cell holding the target recombinant vector, and isolating the target recombinant vector from the transformant cell.
The DNA donor for separating the gene is not particularly limited as long as it is a microorganism capable of producing benzaldehyde dehydrogenase capable of oxidizing vanillin and syringaldehyde. For example, Sphingomonas paucimobilis SYK-6 is preferable.

Extraction of genomic DNA from the microorganism, fragmentation of the genomic DNA, preparation of vectors and assembly vectors, host microorganism for introducing the obtained recombinant vector, its introduction method, and transformant containing the target recombinant vector And the method for extracting a recombinant vector from the selected transformant cell is the same as described above (obtain vanillic acid demethylase gene (vanAB gene)). In order to excise the benzaldehyde dehydrogenase gene (ligV gene) having the activity of oxidizing vanillin contained in the gene of the present invention into vanillic acid and oxidizing syringaldehyde into syringic acid from the obtained recombinant vector, a restriction enzyme or the like is used. be able to.
The base sequence of the benzaldehyde dehydrogenase gene (ligV gene) contained in the gene of the present invention thus obtained can be determined by decoding, for example, by the dideoxy method. SEQ ID NO: 21 shows the base sequence of the region containing the benzaldehyde dehydrogenase gene (ligV gene) contained in the gene of the present invention, and SEQ ID NO: 22 shows the amino acid sequence deduced from the base sequence.

(Arrangement design of each gene and construction of metabolic gene 6: Recombinant plasmid pUVDLABC of E. coli from vanillin and syringaldehyde to PDC (Fig. 17))
From the Escherichia coli recombinant plasmid pULV (FIG. 16) containing the benzaldehyde dehydrogenase gene (ligV gene) thus obtained, a DNA fragment containing the gene sequence encoding the benzaldehyde dehydrogenase gene (ligV gene) was excised by the restriction enzyme SmaI and already constructed. DNA fragment obtained by partial digestion with restriction enzyme XbaI of Escherichia coli plasmid pUVLABC (Fig. 14), which rapidly fermentatively produces 2-pyrone-4,6-dicarboxylic acid from vanillic acid, syringic acid and protocatechuic acid in large quantities A multi-stage reaction process that contains four types of enzyme genes linked by ligase, and rapidly fermentatively produces 2-pyrone-4,6-dicarboxylic acid in large quantities from vanillin, syringaldehyde, vanillic acid, syringic acid, and protocatechuic acid Novel double-stranded DN that encodes and expresses two enzyme genes that catalyze An E. coli recombinant plasmid pUVDLABC (FIG. 17) with an A molecule can be generated.
When the E. coli recombinant plasmid pUVDLABC (FIG. 17) is introduced into E. coli or the like using electroporation or competent cell methods, the transformant cells are derived from plant components, petroleum components, or chemically. Four enzymes that comprise a multistage reaction process for the fermentative production of 2-pyrone-4,6-dicarboxylic acid from synthesized vanillin and syringaldehyde via vanillic acid, syringate and protocatechuic acid ( Benzaldehyde dehydrogenase, vanillic acid demethylase, protocatechuic acid 4,5-dioxygenase, and 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase) genes are expressed synchronously and 2-pyrone-4,6- Dicarboxylic acid can be produced by fermentation.

(Arrangement design of each gene and construction of metabolic system gene 7: Wide host range recombination plasmid pKTVDLABC from vanillin and syringaldehyde to PDC (FIG. 18))
A DNA fragment containing the gene sequence encoding the benzaldehyde dehydrogenase gene (ligV gene) was cut out from the E. coli recombinant plasmid pULV (Fig. 16) containing the benzaldehyde dehydrogenase gene (ligV gene) by the restriction enzyme SmaI. The DNA fragment obtained by partial cleavage with the restriction enzyme XbaI of the broad host range plasmid pKTVLABC (FIG. 15), which rapidly fermentatively produces 2-pyrone-4,6-dicarboxylic acid from acid and protocatechuic acid in large quantities, is obtained using DNA ligase. Concatenates four enzyme genes into a multi-stage reaction process that rapidly fermentatively produces 2-pyrone-4,6-dicarboxylic acid in large quantities from vanillin, syringaldehyde, vanillic acid, syringate, and protocatechuic acid. Has a novel double-stranded DNA molecule that encodes and expresses four types of enzyme genes that catalyze It is possible to create a wide host range recombinant plasmid PKTVDLABC (Figure 18)).
The broad host range recombinant plasmid pKTVDLABC (FIG. 18) has a broad host range including Pseudomonas bacteria, and in transformed cells into which the recombinant plasmid has been introduced using the electroporation method or competent cell method, Synchronously express gene sequences encoding four enzymes, benzaldehyde dehydrogenase, vanillate demethylase, protocatechuate 4,5-dioxygenase, and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase 2-pyrone-4,6-dicarboxylic acid can be produced from plant components or petroleum components or chemically synthesized vanillin, syringaldehyde, vanillic acid, syringic acid, and protocatechuic acid, For high production of 2-pyrone-4,6-dicarboxylic acid, it is derived from plant components, chemical synthesis or Decomposing metabolic enzyme function from any of petroleum-derived vanillin, syringaldehyde, vanillic acid, syringate, and protocatechuic acid to 2-pyrone-4,6-dicarboxylic acid, and 2-pyrone-4,6-dicarboxylic acid It is necessary to use a transformant having a microorganism that has lost its function of degrading the enzyme as a host.

  In addition, the present invention includes a plant component-derived, chemically synthesized or petroleum-derived vanillin, syringaldehyde, vanillic acid, syringic acid, protocatechuic acid, or any number thereof, including culturing a host organism containing the above-described plasmid. Or a method for producing 2-pyrone-4,6-dicarboxylic acid by fermentation from a mixture containing all of them.

(Example 1)
2-pyrone-4,6-dicarboxylic acid from vanillic acid and syringic acid by a transformed cell in which a plasmid pKTVLABC (Fig. 15) for fermentation of 2-pyrone-4,6-dicarboxylic acid was introduced into Pseudomonas bacteria (Pseudomonas putida PpY1100) Example of acid production (1) Recombinant plasmid pKTVLABC (FIG. 15) containing an enzyme gene of a multistage reaction process for fermentation production of 2-pyrone-4,6-dicarboxylic acid was transformed into Escherichia coli HB101, 25 mg The LB medium (100 ml) containing / L ampicillin was cultured with shaking at 37 ° C. for 18 hours, and the recombinant plasmid pKTVLABC (FIG. 15) was extracted from the grown cultured cells.
(2) Decomposing metabolic enzyme function from plant component-derived, chemically synthesized or petroleum-derived vanillin, syringaldehyde, vanillic acid, syringic acid, or protocatechuic acid to 2-pyrone-4,6-dicarboxylic acid, and 2 -Pseudomonas bacterium (Pseudomonas putida PpY1100), a microorganism that has lost its function of degrading enzyme for pyrone-4,6-dicarboxylic acid, was cultured in LB liquid medium 500 ml at 28 ° C. for 23 hours and cooled in ice for 30 minutes. The cells were collected by centrifugation at 10000 rpm at 4 ° C. for 10 minutes, washed gently with 500 ml of 0 ° C. distilled water, and then collected again by centrifugation. Subsequently, the cells were gently washed with 250 ml of 0 ° C. distilled water and then collected by centrifugation. Further, the cells were gently washed with 125 ml of 0 ° C. distilled water and collected by centrifugation. The collected microbial cells were suspended in distilled water containing 10% glycerol and kept at 0 ° C.
(3) Place 4 μl of distilled water containing about 0.05 μg of plasmid pKTVLABC (FIG. 15) in (1) into a 0.2 cm cuvette and add 40 μl of cell suspension suspended in distilled water containing 10% glycerol from (2). , (25 μF, 2500 V, 12 msec).
(4) The total amount of the treated cells was inoculated into 10 ml of LB liquid medium and cultured at 28 ° C. for 6 hours. After culturing, the cells were collected by centrifugation, spread on an LB plate containing 25 mg / L kanamycin, and cultured at 28 ° C. for 48 hours to obtain a kanamycin-resistant transformant carrying the plasmid pKTVLABC (FIG. 15). This bacterium was named Pseudomonas putida PpY1100 (pKTVLABC) strain.
(5) Pseudomonas putida PpY1100 (pKTVLABC) 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. Pre-culture cell suspension of Pseudomonas putida PpY1100 (pKTVLABC) strain prepared using 8L LB liquid medium and 3ml antifoam (Antiform A) using 10L jar fermenter (fermentor) 200 ml was mixed and cultured at 28 ° C. under aeration and stirring at 500 rpm / min until OD660 = 13 to 14 (10 hours to 12 hours). The growth curve accompanying culture is shown in FIG.
(6) When OD660 = 13 to 14 was reached in culture using a 10 L jar fermenter (fermentor), 500 ml of the culture solution was extracted from the fermentor into an Erlenmeyer flask and stored on ice.
(7) Perista pump with 500 ml of 0.1N NAOH solution (adjusted to pH 8.5) containing vanillic acid (40 g) and syringic acid (40 g) as substrates in the culture medium of the fermenter that reached OD660 = 13-14 Over 5-7 hours. Due to the production of 2-pyrone-4,6-dicarboxylic acid as the reaction proceeds, the pH of the culture solution decreases. To prevent this, a 0.1N NAOH solution was added with a peristaltic pump connected to a pH sensor to maintain the pH of the culture solution.
The progress of the reaction was confirmed by Tin Layer Chromatography (TLC). As shown in FIGS. 20 to 22, after 16 hours when the vanillic acid and syringic acid added were almost disappeared by TLC analysis, 500 ml of the ice-cold cell suspension prepared in (5) was added to the culture medium of the fermenter. In addition, the culture was continued for 12 hours.
(8) 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 pH3. 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 low temperature to give 2-pyrone-4,6 -The dicarboxylic acid was isolated as a precipitate. The produced 2-pyrone-4,6-dicarboxylic acid reached about 65 g and was produced in a yield of about 90% of the added substrate. FIGS. 23 to 25 show NRM spectra and MS spectra of purified products obtained by further purifying the produced 2-pyrone-4,6-dicarboxylic acid by treatment with activated carbon or the like.
(Example 2)
A broad host range plasmid with the gene function to produce 2-pyrone-4,6-dicarboxylic acid from vanillin and syringaldehyde via vanillic acid, syringic acid and protocatechuic acid to Pseudomonas genus bacteria (Pseudomonas putida PpY1100) Example (9) Production of 2-pyrone-4,6-dicarboxylic acid from vanillin and syringaldehyde by transformed cells into which pKTVDLABC (FIG. 18) has been introduced (9) 2-pyrone-4,6- produced by the present invention Recombinant plasmid pKTVDLABC (FIG. 18) containing the enzyme gene of the multistage reaction process for fermentative production of dicarboxylic acid was transformed into Escherichia coli HB101 strain, and LB medium (100 ml) containing 25 mg / L ampicillin was used at 37 ° C. for 18 hours. Recombinant plasmid pKTVDLABC (FIG. 18) was extracted from cultured cells grown by shaking over time.
(10) Decomposing metabolic enzyme function from plant component-derived, chemically synthesized or petroleum-derived vanillin, syringaldehyde, vanillic acid, syringic acid, or protocatechuic acid to 2-pyrone-4,6-dicarboxylic acid, and 2 -Pseudomonas bacterium (Pseudomonas putida PpY1100), a microorganism that has lost its function of degrading enzymes for pyrone-4,6-dicarboxylic acid, was cultured in LB liquid medium at 28 ° C for 23 hours and cooled in ice for 30 minutes . The cells were collected by centrifugation at 10000 rpm at 4 ° C. for 10 minutes, washed gently with 500 ml of 0 ° C. distilled water, and then collected again by centrifugation. Subsequently, the cells were gently washed with 250 ml of 0 ° C. distilled water and then collected by centrifugation. Further, the cells were gently washed with 125 ml of 0 ° C. distilled water and collected by centrifugation. The collected microbial cells were suspended in distilled water containing 10% glycerol and kept at 0 ° C.
(11) Plasmid pKTVDLABC of (9) (FIG. 18) 4 μl of distilled water containing about 0.05 μg of DNA is placed in a 0.2 cm cuvette, and 40 μl of cell suspension suspended in distilled water containing 10% glycerol of (2) is added. , (25 μF, 2500 V, 12 msec).
(12) The total amount of the treated cells was inoculated into 10 ml of LB liquid medium and cultured at 28 ° C. for 6 hours. After culturing, the cells were collected by centrifugation and developed on an LB plate containing 25 mg / L kanamycin and cultured at 28 ° C. for 48 hours to obtain a transformant exhibiting kanamycin resistance carrying the plasmid pKTVDLABC (FIG. 18). This bacterium was named Pseudomonas putida PpY1100 (pKTVDLABC) strain.
(13) Pseudomonas putida PpY1100 (pKTVDLABC) 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. Pre-cultured cell suspension of Pseudomonas putida PpY1100 (pKTVDLABC) strain prepared using 8L LB liquid medium and 3ml antifoam (Antiform A) using 10L jar fermenter (fermentor) 200 ml was mixed and cultured at 28 ° C. under aeration and stirring at 500 rpm / min until OD660 = 13 to 14 (10 hours to 12 hours).
(14) When OD660 = 13-14 was reached in culture using a 10 L jar fermenter (fermentor), 500 ml of the culture solution was extracted from the fermentor into an Erlenmeyer flask and stored on ice.
(15) Vanillin (40 g) and syringaldehyde (40 g) were added as substrates to the culture medium of the fermenter that reached OD660 = 13 to 14, and the culture was continued as in (7). The progress of the reaction was confirmed by Tin Layer Chromatography (TLC). Twenty hours after the disappearance of the vanillin and syringaldehyde added was confirmed by TLC analysis, 500 ml of the ice-cold cell suspension prepared in (14) was added to the culture medium in the fermentor and the cultivation was continued for 12 hours. Thereafter, as shown in (8), 2-pyrone-4,6-dicarboxylic acid was separated as a precipitate. The produced 2-pyrone-4,6-dicarboxylic acid was produced with a yield of about 90% of the added substrate.

FIG. 3 is a structural diagram of vanA and vanB genes derived from P. putida PpY101. It is a structural diagram of pvanAB. It is a structural diagram of pvnnAB. It is the amino acid sequence of the protein produced from pvanAB and pvnnAB. It is a structural diagram of pQvAB. Protein analysis in each recombinant is shown. It is a structural diagram of pKT230MC. The construction of pDVZ1 is shown. Construction of the inactive broad host range plasmid pDV01. The introduction of SD sequences derived from ligA derived from S. paucimobilis SYK-6 and P. aeruginosa GAPDH is shown. The construction of a broad host plasmid containing the ligA from S. paucimobilis SYK-6 and the SD sequence from P. aeruginosa GAPDH is shown. FIG. 2 is a structural diagram of LigA, LigB and LigC genes derived from S. paucimobilis SYK-6. The construction of pULABC is shown. The construction of pUVLABC is shown. The construction of pKTVLABC is shown. The creation of pULV and pKTLV is shown. The creation of pUVDLABC is shown. Shows the creation of pKTVDLABC. It is a graph which shows the growth curve of P. putida PpY1100 (pKTVLABC). It is a figure which shows a Tin Layer Chromatography (TLC) result. It is a figure which shows a Tin Layer Chromatography (TLC) result. It is a figure which shows a Tin Layer Chromatography (TLC) result. ¹ is a ¹ H-NRM spectrum of 2-pyrone-4,6-dicarboxylic acid. It is a ¹³ C-NRM spectrum of 2-pyrone-4,6-dicarboxylic acid. 2 is an MS spectrum of 2-pyrone-4,6-dicarboxylic acid. The vanAB gene derived from Pseudomonas putida PpY101 is shown (SEQ ID NO: 1). This shows the vanAB gene derived from Pseudomonas putida. WCS358 (SEQ ID NO: 4). The vanAB gene derived from Pseudomonas sp. HR199 is shown (SEQ ID NO: 7). The ligABC gene derived from S. paucimobilis SYK-6 is shown (SEQ ID NO: 20). This shows the ligV gene derived from S. paucimobilis SYK-6 (SEQ ID NO: 21).

Claims (11)

Any of the following DNA molecules:
(1) a DNA molecule described in SEQ ID NO: 1;
(2) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 2 and / or 3;
(3) A DNA molecule that hybridizes with a DNA molecule comprising SEQ ID NO: 1 or a complementary sequence thereof under stringent conditions and encodes two polypeptides having dimethylase activity against syringic acid Or (4) the amino acid sequence of SEQ ID NO: 2 and / or 3, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added, and having a demethylase activity against syringic acid; A DNA molecule that encodes one or both of the two polypeptides.   A polypeptide in which a polypeptide that contributes to the stabilization of VanA and does not inhibit the enzymatic function of VanA is added to the N-terminus of VanA. A promoter,
A DNA molecule according to claim 1;
Recombinant DNA to which terminators are operably connected in this order in an appropriate host.   The recombinant DNA according to claim 3, further comprising a DNA molecule encoding a polypeptide that contributes to the stabilization of VanA and does not inhibit the enzymatic function of VanA between the promoter and the DNA molecule. The recombinant DNA according to claim 3 or 4, further comprising the following DNA molecule group between a promoter and a terminator:
Here, the DNA molecule group is selected from the group consisting of DNA molecules (ligA gene), DNA molecules (ligB gene), DNA molecules (ligC gene) and combinations thereof,
A DNA molecule (ligA gene) is one of the following DNA molecules:
(5) the DNA molecule set forth in SEQ ID NO: 14;
(6) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 15;
(7) hybridizes under stringent conditions with the DNA molecule of SEQ ID NO: 14 or its complementary sequence, and associates with the polypeptide (ligB) and has protocatechuic acid 4,5-dioxygenase activity A DNA molecule encoding a polypeptide; or (8) consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence of SEQ ID NO: 15 have been deleted, substituted and / or added, and associated with the polypeptide (ligB) A DNA molecule encoding a polypeptide having protocatechuic acid 4,5-dioxygenase activity;
A DNA molecule (ligB gene) is one of the following DNA molecules:
(9) the DNA molecule set forth in SEQ ID NO: 16;
(10) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 17;
(11) hybridizes under stringent conditions with the DNA molecule of SEQ ID NO: 16 or a complementary molecule thereof, and associates with the polypeptide (ligA) and has protocatechuic acid 4,5-dioxygenase activity A DNA molecule encoding the polypeptide; or (12) consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 17 have been deleted, substituted and / or added, and associated with the polypeptide (ligA) A DNA molecule encoding a polypeptide having protocatechuic acid 4,5-dioxygenase activity;
A DNA molecule (ligC gene) is one of the following DNA molecules:
(13) the DNA molecule set forth in SEQ ID NO: 18;
(14) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 19;
(15) A polypeptide that hybridizes with the DNA molecule of SEQ ID NO: 18 or a complementary DNA sequence thereof under stringent conditions and has 4-carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase activity Or (16) consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 18 are deleted, substituted and / or added, and 4-carboxy-2-hydroxymuconic acid A DNA molecule encoding a polypeptide having -6-semialdehyde dehydrogenase activity;
The polypeptide (ligA) consists of the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 15 are deleted, substituted and / or added,
The polypeptide (ligB) is composed of the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 17 are deleted, substituted and / or added. Any of the following DNA molecules:
(17) the DNA molecule set forth in SEQ ID NO: 21;
(18) a DNA molecule encoding the amino acid sequence set forth in SEQ ID NO: 22;
(19) a DNA molecule that hybridizes with a DNA molecule comprising SEQ ID NO: 21 or a complementary molecule thereof under stringent conditions and encodes a polypeptide having benzaldehyde dehydrogenase activity; or (20) SEQ ID NO: 22 A DNA molecule comprising an amino acid sequence in which one or several amino acids of the described amino acid sequence are deleted, substituted and / or added, and encoding a protein having benzaldehyde dehydrogenase activity. A promoter,
A DNA molecule according to claim 6;
Recombinant DNA to which terminators are operably connected in this order in an appropriate host.   The recombinant DNA according to any one of claims 3 to 5, further comprising the DNA molecule according to claim 6 between a promoter and a terminator.   The recombinant DNA according to any one of claims 3 to 5, 7 or 8, wherein the recombinant DNA is a plasmid retained by Escherichia coli XL1-Blue.   A host organism comprising the recombinant DNA according to claim 9.   A method for producing 2-pyrone-4,6-dicarboxylic acid, comprising culturing the host organism according to claim 10.

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