Abstract:
There is provided a process for industrial production of simple 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone from low molecular mixtures derived from plant components such as vanillin, vanillic acid and protocatechuic acid, via a multistage enzyme reaction. A recombinant plasmid containing a vanillate demethylase gene (vanAB genes), benzaldehyde dehydrogenase gene (ligV gene) and protocatechuate 3,4-dioxygenase gene (pcaHG genes); transformants incorporating the plasmid; and a process for production of 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone characterized by culturing the transformants in the presence of vanillin, vanillic acid, protocatechuic acid or a mixture of two or more thereof.

Description:
This application is a 371 of PCT/JP2007/065989 filed Aug. 10, 2007 which claims priority to Japanese allocation No. 2006-218524 filed Aug. 10, 2006. 
     FIELD OF THE INVENTION 
     The present invention relates to a recombinant plasmid comprising genes coding for enzymes participating in a multistage reaction process for fermentative production of 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone from vanillin, vanillic acid, protocatechuic acid or combinations thereof, which are found in low molecularized mixtures of plant aromatic components, to transformants incorporating the recombinant plasmid, and to a process for industrial production of 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone using the same. 
     BACKGROUND OF THE INVENTION 
     Lignin, a major component of plants, is a biomass resource that is ubiquitously present as an aromatic high molecular compound in plant cell walls. However, since plant-derived aromatic components composed mainly of lignin are composed of components with various chemical structures and have complex macromolecular structures, no effective technology has been developed for their use. The known utilization technologies involve separation and production of vanillin as a material for perfumes, from low molecular aromatic decomposition products of alkaline decomposition or other chemical decomposition of aromatic components. Currently, however, no method is known for effective use of large amounts of low molecular aromatic substances, other than vanillin, that are produced by chemical decomposition. Consequently, lignin produced in mass by paper-making processes has been burned as a substitute for heavy oil, without being effectively utilized. 
     The present inventors have found that plant aromatic components such as lignin are converted to low molecular mixtures containing vanillin, syringaldehyde, vanillic acid, syringic acid, protocatechuic acid or the like by chemical decomposition methods such as hydrolysis, oxidative decomposition or solvent decomposition, or physicochemical decomposition methods with supercritical water or supercritical organic solvents, and that these five compounds are converted to the single intermediate substance 2-pyrone-4,6-dicarboxylic acid, which can serve as starting materials for functional plastics or chemical products. 
     The present inventors have also reported a method of producing 2-pyrone-4,6-dicarboxylic acid from vanillin, syringaldehyde, vanillic acid, syringic acid or protocatechuic acid using transformants carrying genes coding for 4 different enzymes (benzaldehyde dehydrogenase, demethylase, protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxy-6-semialdehyde muconate dehydrogenase) that participate in a multistage process for fermentative production of 2-pyrone-4,6-dicarboxylic acid (see Japanese Unexamined Patent Publication No. 2005-278549, for example). 
     However, while numerous intermediates in addition to 2-pyrone-4,6-dicarboxylic acid are known to be obtained by fermentative production from vanillin, syringaldehyde, vanillic acid, syringic acid and protocatechuic acid, their fermentative production processes have not been reported. 
     SUMMARY OF THE INVENTION 
     It is therefore an object of the present invention to provide a process for industrial-scale fermentative production of 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone, as plant-derived components, from low molecular mixtures derived from plant components such as vanillin, vanillic acid and protocatechuic acid, via a multistage enzyme reaction. 
     As a result of much diligent research in light of the current state of the art, the present inventors have completed this invention upon finding that when transformants carrying a recombinant plasmid containing a demethylase gene (vanAB gene) and benzaldehyde dehydrogenase gene (ligV gene), as well as a gene (pcaHG gene) coding for protocatechuate 3,4-dioxygenase which cleaves the protocatechuic acid ring, are cultured in the presence of vanillin or the like, the corresponding 3-carboxy-cis,cis-muconic acid molecules are produced, and further that treatment of the 3-carboxy-cis or cis-muconic acid with an acid can produce 3-carboxymuconolactone at high yield and low cost. 
     Specifically, the invention provides the following. 
     (1) A recombinant plasmid containing a vanillate demethylase gene (vanAB genes), benzaldehyde dehydrogenase gene (ligV gene) and protocatechuate 3,4-dioxygenase gene (pcaHG gene). 
     (2) The recombinant plasmid according to (1) above, wherein the vanAB genes are the DNA molecule set forth in SEQ ID NO: 7. 
     (3) The recombinant plasmid according to (1) or (2) above, wherein the ligV gene is the DNA molecule set forth in SEQ ID NO: 8. 
     (4) The recombinant plasmid according to any one of (1)-(3) above, wherein the pcaH gene is the DNA molecule set forth in SEQ ID NO: 1 and the pcaG gene is the DNA molecule set forth in SEQ ID NO: 3. 
     (5) A transformant incorporating a recombinant plasmid according to any one of (1)-(4) above. 
     (6) The transformant according to (5), wherein the recombinant plasmid according to any one of (1)-(4) is introduced into  Pseudomonas putida  PpY1100. 
     (7) A process for production of 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone, characterized by culturing a transformant according to (5) or (6) above in the presence of vanillin, vanillic acid, protocatechuic acid or a mixture of two or more thereof. 
     According to the invention it is possible to accomplish high-yield and inexpensive fermentative production of simple 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone from vanillin, vanillic acid or protocatechuic acid. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a drawing illustrating the method of constructing recombinant plasmid pULVHG. 
         FIG. 2  is a drawing illustrating the method of constructing recombinant plasmid pKTVDHG of the invention. 
         FIG. 3  is a graph showing an OD increase curve (increase in cell count) during the course of production of 3-carboxy-cis,cis-muconic acid by culturing of  Pseudomonas putida  PpY1100. 
         FIG. 4  is a TLC showing the course of conversion reaction from vanillin, vanillic acid or protocatechuic acid to 3-carboxy-cis,cis-muconic acid.  FIG. 4  shows the conversion (12 hours) from (a): vanillin, (b): vanillic acid, (c): protocatechuic acid, (d): 3-carboxymuconolactone and (e): vanillin, the conversion (12 hours) from (f): vanillic acid and the conversion (12 hours) from (g): protocatechuic acid. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Recombinant plasmid pKTVDHG of the invention is a plasmid obtained by inserting a benzaldehyde dehydrogenase gene (ligV gene) at the ligABC gene site of the known recombinant plasmid pKTVLABC (FIG. 15 of Japanese Unexamined Patent Publication (kokai) No. 2005-278549) which contains genes coding for enzymes (vanA, vanB, ligA, ligB and ligC) that catalyze a multistage process for production of 2-pyrone-4,6-dicarboxylic acid from vanillin or the like, and further downstream from it, by inserting a gene (pcaHG genes) coding for protocatechuate 3,4-dehydrogenase which cleaves protocatechuic acid rings. 
     Recombinant plasmid pKTVDHG of the invention has a wide host range including  Pseudomonas  bacteria, and transformants incorporating the recombinant plasmid are capable of coordinated expression of the ligV gene, vanAB genes and pcaHG genes to produce 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone from either plant- or petroleum-derived or chemically synthesized vanillin, vanillic acid, protocatechuic acid or a mixture thereof. 
     That is, due to the presence of the pcaHG genes, protocatechuic acid is not converted to 2-pyrone-4,6-dicarboxylic acid but rather the protocatechuic acid ring is opened to yield 3-carboxy-cis,cis-muconic acid, as a precursor of 3-carboxymuconolactone. 
     A method for preparation of recombinant plasmid pKTVLABC is described in detail in Japanese Unexamined Patent Publication (kokai) No. 2005-278549. The vanAB genes incorporated into plasmid pKTVLABC is (i) the vanillate demethylase gene from  Pseudomonas putida  PpY101 (SEQ ID NO: 1 of said publication) or (ii) a DNA molecule coding for vanillate demethylase (SEQ ID NO: 2 and/or 3 of said publication). Preferred of these vanAB genes is the vanillate demethylase gene from  Pseudomonas putida  PpY101, which is herein set forth in SEQ ID NO: 7. 
     The ligV gene used for the invention may be a DNA molecule listed in Japanese Unexamined Patent Publication (kokai) No. 2005-278549, selected from among (i) the benzaldehyde dehydrogenase gene from  Sphingomonas paucimobilis  SYK-6 (SEQ ID NO: 21 of said publication), (ii) a DNA molecule coding for benzaldehyde dehydrogenase (SEQ ID NO: 22 of said publication), (iii) a DNA molecule that hybridizes under stringent conditions with the DNA molecule set forth in SEQ ID NO: 21 of said publication or a DNA molecule composed of its complementary sequence and that codes for a polypeptide with benzaldehyde dehydrogenase activity, or (iv) a DNA molecule that comprises the amino acid sequence set forth in SEQ ID NO: 22 of said publication with a deletion, substitution and/or addition of one or more amino acids and that codes for a protein with benzaldehyde dehydrogenase. Preferred of these ligV genes is the benzaldehyde dehydrogenase gene from  Sphingomonas paucimobilis  SYK-6, which is herein set forth in SEQ ID NO: 8. There are no particular restrictions on the methods of separation and fragmentation of the ligV gene from the microorganisms, and the same methods described in the aforementioned publications may be used. 
     The pcaHG genes used for the invention may be obtained with reference to J Bacteriol. 1989 November; 171 (11): 5915-21 or the total genome data of  Pseudomonas putida  KT2440 (NCBI accession number: NC — 002947). 
     There are no particular restrictions on the specific method of obtaining the pcaHG genes, and as an example, the genomic DNA may be extracted from strain KT2440 and cleaved with a restriction enzyme or the like to obtain DNA fragments, and the restriction enzyme used to create restriction enzyme ends allowing insertion of the genomic DNA fragments, from vector DNA such as phage or plasmid. The recombinant vector may be constructed from the genomic DNA fragments and vector DNA using known DNA ligase. The recombinant vector may then be introduced into suitable host cells, transformants retaining the recombinant vector of interest selected and the recombinant vector of interest separated from the transformants. 
     The genome extraction may be accomplished by ordinary methods. For example, it is preferably accomplished by collecting cultured cells of a microorganism and, after bacteriolysis with protease K, for example, carrying out a combination of appropriate methods such as deproteinization treatment by phenol extraction, protease treatment, ribonuclease treatment, genomic DNA precipitation with an alcohol and centrifugal separation. 
     The plasmid used is preferably pUC18, pUC19, pUC118, pUC119, pKT230 MC, Bluescript or the like, with  E. coli  as the host. After cleavage with the restriction enzyme, the cleaved ends may be dephosphorylated as appropriate. T4 DNA ligase may be mentioned as an example of a known DNA ligase. 
     The nucleotide sequence of the open reading frame of the PcaH gene obtained from  Pseudomonas putida  KT2440 is set forth in SEQ ID NO: 1, its amino acid sequence is set forth in SEQ ID NO: 2, the nucleotide sequence of the reading frame of the PcaG gene is set forth in SEQ ID NO: 3 and its amino acid sequence is set forth in SEQ ID NO: 4. 
     The recombinant plasmid pKTVDHG of the invention may be constructed in the following manner, for example. 
     (1) First, a known ligase is used to link the ligV gene set forth in SEQ ID NO: 21 in Japanese Unexamined Patent Publication (kokai) No. 2005-278549 at a restriction enzyme XbaI-cleaved site within the multicloning site in a gene coding for the α fragment of LacZ located downstream from the LacZ promoter of a suitable plasmid such as Bluescript, to construct recombinant plasmid pBluescript II SK − /ligV.
 
(2) The pcaHG gene is then linked at a restriction enzyme XbaI-cleavage site in the multicloning site of a suitable plasmid to construct recombinant plasmid pBluescript II SK − /pcaHG.
 
(3) Next, a DNA fragment of a plasmid containing the LacZ promoter region, obtained by cutting of the recombinant plasmid pBluescriptII SK − /pcaHG with restriction enzymes PvuII and BamHI followed by end treatment, and a DNA fragment obtained by cutting of the recombinant plasmid pBluescriptII SK − /ligV with restriction enzyme FbaI followed by end treatment, are linked with a known ligase to construct recombinant plasmid pBluescriptII SK − /pcaHG-LigV. Also, a DNA fragment obtained by cutting of pBluescriptII SK − /pcaHG-LigV with XbaI and a DNA fragment obtained by cutting of the known recombinant plasmid pKTVLABC with restriction enzyme XbaI followed by end treatment were linked with a known ligase to construct recombinant plasmid pKTVDHG.
 
     The microorganisms that may be used as hosts for high-yield production of 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone are not particularly restricted so long as they allow replication of the recombinant plasmid of the invention and can express enzyme genes that participate in production of 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone, but it is necessary to use transformants whose host is a microorganism that is permeable to plant-derived, chemically synthesized or petroleum-derived vanillin, vanillic acid or protocatechuic acid, and has lost the catabolic enzyme function from any of these to 2-pyrone-4,6-dicarboxylic acid, as well as catabolic enzyme function for 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone. Preferred microorganisms of this type are  Pseudomonas  bacteria, and especially  Pseudomonas putida  PpY1100. 
     A known method such as a protoplast method, competent cell method or electroporation method may be used to transform the host microorganism using the recombinant plasmid. 
     Selection of transformants may be accomplished based on a selective marker for the plasmid used, such as drug resistance acquired by DNA recombination in the transformants. The transformants containing the recombinant plasmid of interest are preferably selected from among the transformants by colony hybridization using a partial DNA fragment of the gene as the probe. Labeling of the probe may be carried out using a radioactive isotope, digoxigenin, an enzyme or the like. 
     The obtained transformants may be cultured under appropriate conditions using medium containing a carbon source, a nitrogen source, metal salts, minerals, vitamins and the like. The pH of the medium may be a pH in a range that allows growth of the transformants, and the pH is preferably adjusted to about 6.0 to 8.0. The culturing conditions may be shake culturing or submerged culturing for 2 to 7 days at between 15 and 40° C. and preferably between 28 and 37° C. 
     The culture solution containing the 3-carboxy-cis,cis-3-muconic acid obtained by the culturing may be subjected to acid treatment for efficient conversion to 3-carboxymuconolactone. The acid used is preferably hydrochloric acid at about pH 1 to 2. 
     The 3-carboxy-cis,cis-muconic acid and/or 3-carboxymuconolactone obtained by the production process of the invention, as a plastic material, chemical product material or the like, can exhibit functions different from 2-pyrone-4,6-dicarboxylic acid or higher functions thereof, and therefore can serve as a useful plastic material. 
     EXAMPLES 
     The present invention will now be described in greater detail by examples, with the understanding that the invention is not limited to these examples. 
     Example 1 
     Construction of Recombinant Plasmid pKTVDHG 
     (1) Construction of Recombinant Plasmid pKTVLABC 
     Recombinant plasmid pKTVLABC was constructed by the method described in Japanese Unexamined Patent Publication (kokai) No. 2005-278549. 
     (2) Construction of Recombinant Plasmid pULV 
     Recombinant plasmid pULV was constructed by the method described in Japanese Unexamined Patent Publication (kokai) No. 2005-278549. 
     (3) Construction of Recombinant Plasmid pBluescriptII SK − /pcaHG 
     Using the universal primer: 5′-GGTGTCAGGCAAAGGTGTTAAGAC-3′ (SEQ ID NO: 5) and reverse primer: 5′-AGTGGGGTTCTGCTGGTTCGGC-3′ (SEQ ID NO: 6) as PCR primers, pcaHG was amplified from the genome of strain KT2440 and linked to a DNA fragment obtained by cleaving pBluescriptII SK with XbaI, so that it was in-frame with Lac. 
     (4) Construction of Recombinant Plasmid pULVHG 
     A DNA fragment of a plasmid containing the LacZ promoter region, obtained by cutting of the recombinant plasmid pBluescriptII SK − /pcaHG with restriction enzymes PvuII and BamHI followed by end treatment, and a DNA fragment obtained by cutting of the recombinant plasmid pULV with restriction enzyme BamHI followed by end treatment, were linked with a known ligase to construct recombinant plasmid pULVHG. 
     (5) Construction of Recombinant Plasmid pKTVDHG 
     A DNA fragment obtained by cutting of pULVHG with XbaI and SacI followed by end treatment and a DNA fragment obtained by cutting of the known recombinant plasmid pKTVLABC with restriction enzyme XbaI followed by end treatment were linked with a known ligase to construct recombinant plasmid pKTVDHG. 
     Example 2 
     Production of 3-Carboxymuconolactone from Vanillin 
     (1) Conversion from Vanillin to 3-carboxy-cis,cis-muconic Acid 
     (1-1) Recombinant plasmid pKTVDHG constructed in Example 1 was used to transform  E. coli  HB101, and the transformants were shake cultured at 37° C. for 18 hours in LB medium (100 ml) containing 25 mg/L ampicillin, after which the recombinant plasmid pKTVDHG was extracted from the proliferated cultured cells.
 
(1-2)  Pseudomonas putida  PpY1100, a microorganism that has lost the catabolic enzyme function from plant-derived, chemically synthesized or petroleum-derived vanillin, syringaldehyde, vanillic acid, syringic acid, protocatechuic acid, p-hydroxybenzaldehyde or p-hydroxybenzoic acid to 2-pyrone-4,6-dicarboxylic acid, as well as catabolic enzyme function for 3-carboxymuconolactone, 3-carboxy-cis,cis-muconic acid, was cultured in 500 ml of LB liquid medium at 28° C. for 23 hours, and then cooled in ice for 30 minutes. The cells were collected by 10,000 rpm centrifugation at 4° C. for 10 minutes, and after mild rinsing with 500 ml of 0° C. distilled water, they were re-centrifuged. This was followed by additional mild rinsing with 250 ml of 0° C. distilled water and re-centrifugation. This was followed by still further mild rinsing with 125 ml of 0° C. distilled water and re-centrifugation. The collected microorganic cells were suspended in distilled water containing 10% glycerol and stored at 0° C.
 
(1-3) After placing 4 μl of distilled water containing about 0.05 μg of plasmid pKTVDHG DNA in a 0.2 cm cuvette, 40 μl of the cell solution suspended in distilled water containing 10% glycerol obtained in (1-2) above was added, and the mixture was subjected to electroporation under conditions of 25 μF, 2500 V, 12 msec.
 
(1-4) The total amount of cells obtained in (1-3) above was seeded in 10 ml of LB liquid medium and cultured at 28° C. for 6 hours. The cells were recovered by centrifugation after culturing, and then spread on an LB plate containing 25 mg/L kanamycin and cultured at 28° C. for 48 hours, and transformants carrying plasmid pKTVDHG and exhibiting kanamycin resistance were obtained. This cell line was named  Pseudomonas putida  PpY1100(pKTVDHG).
 
(1-5)  Pseudomonas putida  PpY1100(pKTVDHG) was seeded in 200 ml of LB liquid medium (containing 25 mg/L kanamycin) and cultured at 28° C. for 16 hours to obtain a preliminary cultured cell suspension. Next, 5 L of LB liquid medium and 3 ml of an antifoaming agent (Antiform A) were prepared using a 10 L-volume jar fermenter, and 200 ml of the preliminary cultured cell suspension of  Pseudomonas putida  PpY1100(pKTVLABC) was mixed therewith and cultured to OD660 13-14 with aerated stirring at 28° C., 500 rpm/min (10 hours-12 hours).  FIG. 3  shows the OD increase curve (increase in cell volume) (—black triangle—) for production of 3-carboxy-cis,cis-muconic acid. In  FIG. 3 , the—black square—symbols represent oxygen concentration (81 ppm/min flow rate), and the—black diamond—symbols represent preparation to pH 6.5 with aqueous hydrochloric acid and sodium hydroxide.
 
(1-6) When the OD660 reached 13-14 with culturing using a 10 L-volume jar fermenter, 500 ml of culture solution was removed from the fermenter into an Erlenmeyer flask and stored on ice.
 
(1-7) Next, 500 ml of a 0.1 N NaOH solution (adjusted to pH 8.5) containing 50 g of vanillin as substrate was added to the culture solution in the fermenter that had reached OD660 13-14, using a peristaltic pump over a period of 5 to 7 hours. In order to prevent reduction in pH of the culture solution with production of 3-carboxy-cis,cis-muconic acid as the reaction proceeded, a 0.1 N NaOH solution was added with a peristaltic pump connected to a pH sensor to maintain the pH of the culture solution.
 
     After 16 hours, when virtually all of the added vanillin was confirmed to have disappeared by TLC analysis, 500 ml of the ice-cooled cell suspension prepared in (1-6) above was added to the culture solution in the fermenter and culturing was continued for 12 hours. Progression of the reaction was confirmed by thin-layer chromatography (TLC). 
     (2) Conversion from 3-carboxy-cis,cis-muconic Acid to 3-carboxymuconolactone 
     Upon completion of the reaction, the medium in the fermenter was transferred to a plastic container (bucket). The cell component was precipitated and removed from the culture solution by centrifugal separation (6000 rpm, 20° C.), hydrochloric acid was added to the obtained supernatant to lower the pH to below 1.0, and the mixture was stored at low temperature for conversion of the 3-carboxy-cis,cis-muconic acid to 3-carboxymuconolactone. Complete conversion to 3-carboxymuconolactone was confirmed by TLC, HPLC and GC-MS.  FIG. 4(   e ) shows TLC with spotting of the solution extracted with ethyl acetate after hydrochloric acid treatment. After confirming complete conversion to 3-carboxymuconolactone, an organic solvent was used for extraction of the 3-carboxymuconolactone. Approximately 1.9 g of 3-carboxymuconolactone was extracted and dried from 200 ml of culture solution, which was a yield of about 88.5% as the ratio of added substrate based on the total culture solution volume of 5.7 L. The obtained 3-carboxymuconolactone was further purified by active carbon treatment or the like and the structure was confirmed by its NMR spectrum. 
       1 H-NMR (400 MHz, DMSOd 6 ) δ(ppm): 2.67, 3.10, 5.55, 6.81, 12.5-13.0 
       13 C-NMR (100 MHz, DMSOd 6 ) δ(ppm): 36.5, 78.5, 125.9, 157.9, 162.1, 170.4, 170.8 
     Ms m/z: 402 (M + ) (as TMS (trimethylsilyl) form of 3-carboxymuconolactone) 
     Example 3 
     Production of 3-Carboxymuconolactone from Vanillic Acid 
     Following the same procedure as in Example 2, except for using vanillic acid as the substrate, 3-carboxymuconolactone was obtained at a yield of about 88.5% in terms of the ratio of added substrate. 
     Example 4 
     Production of 3-Carboxymuconolactone from Protocatechuic Acid 
     Following the same procedure as in Example 2, except for using protocatechuic acid as the substrate, 3-carboxymuconolactone was obtained at a yield of about 88.5% in terms of the ratio of added substrate.