| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | October 8, 2002 |
| PATENT TITLE |
Process for producing coenzyme Q10 |
| PATENT ABSTRACT | A gene of decaprenyl diphosphate synthase, which is the key gene participating in the biosynthesis of coenzyme Q.sub.10 was isolated from a bacterium belonging to the family Rhizobiaceae. By transferring this gene into a microorganism such as Escherichia coli and expressing therein, coenzyme Q.sub.10 can be effectively produced |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | October 10, 2000 |
| PATENT CT FILE DATE | February 3, 2000 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | August 17, 2000 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
H. Matsuda et al., "Molecular colning and mutational analysis of the ddsA gene encoding decaprenyl disphosphate synthase from Gluconobacter suboxydans", Eur. J. Biochem., (1998), vol. 225, No. 1, pp. 52-59. M. Kawamukai et al., "Analysis of the decaprenyl disphosphate synthase (dps) gene in fission yeast suggests a role of ubiquinone as an antioxidant", J. Biochem., (1997), vol. 121, No. 3, pp. 496-505. Kawamukai M. Accession AB006850. Aug. 11, 1998 (Alignment No. 1).* Kawamukai M. Accession AB006850. Aug. 11, 1998 (Alignment No. 2).* Okada et al. Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl disphosphate synthase from Gluconobacter suboxydans. Eur J Biochem Jul. 1, 1998;255(1):52-9. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An isolated polynucleotide which encodes a protein having decaprenyl diphosphate synthase activity, said polynucleotide comprising (a) the nucleotide sequence as set forth in SEQ ID No. 1, (b) a nucleotide sequence which hybridizes under stringent hybridization conditions to the nucleotide sequence as set forth in SEQ ID No. 1, said stringent hybridization conditions comprising hybridizing and washing in 1.times.SSC at about 60.degree. C. or (c) a nucleotide sequence which encodes the amino acid sequence as set forth in SEQ ID No. 2. 2. The isolated polynucleotide according to claim 1 which comprises the nucleotide sequence as set forth in SEQ ID No. 1. 3. The isolated polynucleotide according to claim 1, which encodes a decaprenyl diphosphate synthase comprising the amino acid sequence as set forth in SEQ ID No. 2. 4. A process for producing coenzyme Q.sub.10 comprising transforming a host microorganism with the polynucleotide according to claim 1, culturing the transformed host microorganism, and isolating coenzyme Q.sub.10. 5. The process according to claim 2, wherein the host microorganism is Escherichia coli. 6. The process according to claim 2, wherein the transformed host microorganism is Escherichia coli HB101 pQAD1 (FERM BP-6538). 7. An expression vector comprising the polynucleotide according to claim 1. 8. An expression vector pQAD1, obtained by transferring the polynucleotide according to claim 1 into an expression vector pUCNT. 9. A host microorganism transformed with the polynucleotide according to claim 1. 10. The microorganism according to claim 9, which is an Escherichia coli. 11. The microorganism according to claim 10, wherein the Escherichia coli is Escherichia coli HB 101 pQAD1 (FERM BP-6538). 12. A host microorganism transformed with the expression vector according to claim 7. 13. The microorganism according to claim 12, which is an Escherichia coli. 14. The microorganism according to claim 13, wherein the Escherichia coli is Escherichia coli HB 101 pQAD1 (FERM BP-6538). 15. An isolated protein which is coded by the polynucleotide according to claim 1 and has a decaprenyl diphosphate synthase activity. 16. An isolated protein which comprises the amino acid sequence of SEQ ID No. 2 and has a decaprenyl diphosphate synthase activity. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for producing coenzyme Q.sub.10, which can be used as a pharmaceutical agent and the like. In more detail, the present invention relates to a process for producing coenzyme Q.sub.10 by isolating a gene coding for an enzyme responsible for biosynthesizing coenzyme Q.sub.10 side chain, the key enzyme in the coenzyme Q.sub.10 biosynthetic pathway, i.e. decaprenyl diphosphate synthase, from a bacterium belonging to the family Rhizobiaceae, and transferring said gene into a microorganism to produce coenzyme Q.sub.10. 2. Description of the Related Art Industrially, coenzyme Q.sub.10 has been produced by isolating a coenzyme from plants such as tobacco and synthetically altering the side chain. In addition, it has been known that coenzyme Q.sub.10 is produced by a wide variety of organisms including from microorganisms such as bacterium and yeast, to higher plants and animals. One of the most effective processes for producing coenzyme Q.sub.10 is believed to be a process which comprises culturing the microorganism and extracting the compound from the culture. Said process has also been used in industrial production of coenzyme Q.sub.10. However, the above known processes do not provide enough productivity because of their low yield or complicated operation. Though there are some differences between the prokaryotic and eucaryotic pathways for biosynthesis of coenzyme Q.sub.10, both pathways consist of complicated multi-step reactions in which many enzymes are involved. They basically consist of the following three steps; synthesizing decaprenyl diphosphate, which is used for the prenyl side chain of coenzyme Q.sub.10 ; synthesizing parahydroxy benzoic acid, which is used for the quinone ring; and binding those two components and converting the substituents sequentially to give coenzyme Q.sub.10. The reaction, which determines the length of the side chain and is believed to be the rate-limiting step in the biosynthetic pathway, that is, the reaction in which decaprenyl diphosphate synthase is involved, is believed to be the most important reaction. Therefore, in order to produce coenzyme Q.sub.10 effectively, it might be a good idea to isolate the decaprenyl diphosphate synthase gene, the key gene in the biosynthetic pathway, and to use said gene for improving productivity. One of the potential candidates for the gene sources is a bacterium belonging to the family Rhizobiaceae, which produces relatively large amounts of coenzyme Q.sub.10. Until now, decaprenyl phosphoric acid synthase genes had been isolated from several microorganisms including Schizosaccaromyces pombe (Japanese Patent Application Laid Open No. H09-173076) and Gluconobacter suboxydans (Japanese Patent Application Laid Open Open No. H10-57072). However, they do not show enough productivity for coenzyme Q.sub.10 and therefore, the art had not yet achieved effective culture, isolation or purification with those microorganisms. Accordingly, it has been desired to isolate a gene of said enzyme from a microorganism having high coenzyme Q.sub.10 producing ability. SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problem of low productivity. The object of the present invention is to provide a process for producing coenzyme Q.sub.10 effectively by means of a microorganism, by isolating a gene coding for coenzyme Q.sub.10 -side chain synthetic enzyme from a bacterium belonging to the family Rhizobiaceae and using the gene. According to the present invention, decaprenyl diphosphate synthase gene, the key gene in the biosynthetic pathway for coenzyme Q.sub.10, was isolated from a bacterium belonging to the family Rhizobiaceae. An effective coenzyme Q.sub.10 production has been achieved by transferring the gene into a microorganism such as Escherichia-coli and expressing therein. The inventors have tried to isolate the gene of decaprenyl diphosphate synthase from a bacterium belonging to the family Rhizobiaceae which produces relatively large amounts of coenzyme Q.sub.10 and succeeded in isolating said gene. Accordingly, the present invention provide a DNA comprising a DNA sequence of the Seq. ID No. 1, or a sequence having deletion, addition or insertion of one or more base in the sequence and coding for decaprenyl diphosphate synthase. The present invention also provide a protein having an amino acid sequence of the Seq. ID No. 2, or an amino acid sequence having deletion, addition or insertion of one or more amino acid in said sequence and having decaprenyl diphosphate synthase activity; and a DNA encoding said amino acid sequence. The present invention also provide a process for producing coenzyme Q.sub.10 comprising the steps of transferring the above described DNA sequence into a host microorganism and culturing the microorganism. The host microorganism used in the present invention is not limited but preferably is Escherichia coli. Although a normal Escherichia coli produces coenzyme Q.sub.8, according to the present invention, Escherichia coli can be modified to produce coenzyme Q.sub.10. In addition, the present invention provides an expression vector comprising the above-described DNA sequence. The expression vector of the present invention may be constructed by using any of known vector systems. For example, pQAD-1, which is constructed by transferring the gene of the Seq. ID No. 1 into the known expression vector system pUCNT, is provided. According to the invention, a host microorganism transformed with the above-described DNA sequence is also provided. For the host microorganism in the present invention, Escherichia coli is preferably used. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a restriction map of plasmid pQAD1 which contains decaprenyl diphosphate synthase gene. FIG. 2 shows a chart of high-speed liquid chromatography detecting coenzyme Q.sub.10 produced by recombinant E-coli comprising decaprenyl diphosphate synthase gene. DESCRIPTION OF THE PREFERRED EMBODIMENTS The inventors studied intensively to isolate the desired gene from the bacterium belonging to the family Rhizobiaceae, which produces relatively high amounts of coenzyme Q.sub.10, and succeeded in obtaining a fragment of said gene by means of PCR technique. The inventors compared known decaprenyl diphosphate synthase genes and polyprenyl diphosphate synthase genes, analogous enzymes of the former that participate in the biosynthesis of longer prenyl chains to provide coenzyme Qs having different length side chains; and based on the homologous regions between those sequences, several PCR primers were designed. Various combinations of the obtained primers were tested to determine PCR condition. It was found that a 400 bp gene fragment of the desired enzyme was amplified from chromosomal gene of Agrobacterium sp. KNK712 (FERM BP-1900) by using DPS-1 (5'-AAGGATCCTNYTNCAYGAYGAYGT-3' (SEQ ID No. 3)) and DPS-2 (5'-AAGGATCCTCRTCNACNARYTGRAA-3' (SEQ ID No. 4)) (wherein R represents A or G, Y represents C or T, and N represents G, A, T or C) as PCR primers, carrying out the PCR process at 94.degree. C. for 1 minute than 25 cycles of thermal cycling at 94.degree. C. 1 min..fwdarw.50.degree. C. 1 min..fwdarw.75.degree. C. 1 min. It was confirmed by sequencing the obtained gene. In order to obtain full length gene of said enzyme, in the next step, Agrobacterium sp. KNK 712 (FERM BP-1900) chromosomal gene was digested with EcoRI restriction enzyme and the obtained fragments were transferred into .lambda. phage vector to provide a recombinant phage library. The plaques were blotted on a nylon filter and the filter was subjected to plaque hybridization with the labeled PCR fragment and then, a clone comprising full length of decaprenyl diphosphate synthase gene could be obtained. Base sequence of the decaprenyl diphosphate synthase gene contained in the obtained clone was determined to give the sequence of Seq. ID No. 1. The amino acid sequence deduced from the base sequence contained regions having sequences characteristic to decaprenyl diphosphate synthase. In order to express the decaprenyl diphosphate synthase gene, it is required to ligate said gene into a vector at a region downstream to an appropriate promoter. For example, an expression vector may be constructed by excising a DNA fragment containing desired gene by means of restriction enzyme or by amplifying the fragment coding for the enzyme by means of PCR, then transferring the fragment into a vector having a promoter. For example, an expression vector system pUCNT (described in WO94/03613) may be transfected with said gene to provide an expression vector pQAD1 for decaprenyl diphosphate synthase gene. Then, an appropriate microorganism may be used for producing coenzyme Q.sub.10 by transforming the same with said expression vector for the enzyme. For example, a Escherichia coli, which produces originally coenzyme Q.sub.8, can be transformed by pQAD1, the expression vector for decaprenyl diphosphate synthase gene, to produces significantly higher amount coenzyme Q.sub.10, which is not originally produced, than the amount of coenzyme Q.sub.8 production. The transformed Escherichia coli, Escherichia coli HB101 pQAD1 was deposited in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry with the accession number of FERM BP-6538. The gene provided by the present invention may be utilized alone or may also be co-transferred into a microorganism with another biosynthetic gene and expressed therein to give a better effect. According to the process of the present invention for producing coenzyme Q.sub.10, the host microorganism transformed with the gene may be cultured to produce coenzyme Q.sub.10. The condition for the culture is not limited and may be determined depending on the selected host microorganism. Conditions for culturing various host microorganisms are well known in the art. After the culture is completed, the host microorganisms may be harvested and coenzyme Q.sub.10 may be isolated and purified by means of an appropriate procedure. The method for isolating coenzyme Qs from the host microorganism is well known in the art. |
| PATENT EXAMPLES | available on request |
| PATENT PHOTOCOPY | available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |