| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | August 15, 2000 |
| PATENT TITLE |
Method of forming ubiquinone-10 |
| PATENT ABSTRACT | The present invention relates to production of ubiquinone-10 in E. coli by isolating and sequencing a structural gene dds1 of decaprenyl diphosphate synthase derived from photosynthetic bacteria such as Rhodobacter capsulatus., and expressing it in E. coli |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | January 21, 1998 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Abstracts of Lectures of General Congress 1997 of Japan Society for Bioscience, Biotechnology, and Agrochemistry; Extra Edition, vol. 71, Mar. 1997. K. Okada et al; Journal of Bacteriololgy; The ispB Gene Encoding Octaprenyl Diphosphate Synthase Is Essential for Growth of Esherichia coli; vol. 179, No. 9, pp. 3058-3060, May 1997. K. Okada et al; Journal of Bacteriology; Cloning of the sdsA gene Encoding Solanesyl Diphosphate Synthase from Rhodobacter capsulatus and Its Functional Expression in Escherichia coli and Saccharomyces cerevisiae; vol. 179, No. 19, pp. 5992-5998; Oct. 1997 |
| PATENT CLAIMS |
What is claimed is: 1. An isolated DNA comprising a gene coding for decaprenyl diphosphate synthase (DDS) from Rhodobacter capsulatus. 2. The isolated DNA according to claim 1, wherein said gene codes for a decaprenyl diphosphate synthase (DDS) of SEQ ID NO:2. 3. The isolated DNA according to claim 2, wherein said gene coding for DDS, is the dds1 gene comprising the coding sequence of nucleotides 205 to 1182 of SEQ ID NO:1. 4. A plasmid vector comprising the isolated DNA according to claim 1. 5. A plasmid vector, comprising the DNA according to claim 2. 6. Expression plasmid pRC10 comprising the DNA described in claim 2 inserted into vector pUC18. 7. Expression plasmid pRC10 comprising DNA described in claim 3 inserted into vector pUC18. 8. A microorganism transformed with the plasmid vector according to claim 4. 9. The transformed microorganism according to claim 8, which is Escherichia coli. 10. A microorganism transformed with a plasmid described in claim 5. 11. A transformed microorganism according to claim 8 wherein the transformed microorganism is E. coli DH10B/pRC10 (FERM BP-6061), E. coli KO229/pRC10 (FERM BP-6062) or E. coli JM109/pRC10. 12. A microorganism transformed with a plasmid described in claim 7. 13. A microorganism transformed with a plasmid described in claim 6. 14. A transformed microorganism according to claim 12 wherein the transformed microorganism is E. coli DH10B/pRC10 (FERM BP-6061), E. coli KO229/pRC10 (FERM BP-6062) or E. coli JM109/pRC10. 15. A transformed microorganism according to claim 7 wherein the transformed microorganism is E. coli DH10B/pRC10 (FERM BP-6061), E. coli KO229/pRC10 (FERM BP-6062) or E. coli JM109/pRC10. 16. A microorganism transformed with a gene coding for decaprenyl diphosphate synthase (DDS) derived from Rhodobacter capsulatus. 17. A transformed microorganism according to claim 16 wherein a gene coding for octaprenyl disphophate synthase (OPS) has been destroyed. 18. A method of producing ubiquinone-10, which comprises: culturing the transformed microorganism of claim 10 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. 19. A method of producing ubiquinone-10, which comprises: culturing the transformed microorganism of claim 11 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. 20. A method of producing ubiquinone-10, which comprises: culturing the transformed microorganism of claim 13 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. 21. A method of producing ubiquinone-10, which comprises: culturing the transformed microorganism of claim 15 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. 22. A method of producing ubiquinone-10, which comprises: culturing the transformed microorganism of claim 12 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. 23. A method of producing ubiquinone-10, which comprises: culturing the transformed microorganism of claim 14 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. 24. A method of producing ubiquinone-10, which comprises: culturing the transformed microorganism of claim 16 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. 25. A method of producing ubiquinone-10, which comprises: culturing the transformed microorganism of claim 17 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. 26. A method of producing high-purity ubiquinone-10, which comprises: culturing Escherichia coli DH10B/pRC10, Escherichia coli KO229/prC10 or Escherichia coli JM109/pRC10 to produce ubiquinone-10; and isolating ubiquinone-10 produced by the transformed microorganism. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
TECHNICAL FIELD TO WHICH THE INVENTION BELONGS The present invention relates to formation of ubiquinone, particularly ubiquinone-10. The present invention relates in particular to a novel system for forming ubiquinone-10 by isolating and expressing a gene coding for decaprenyl diphosphate synthase (also referred to DDS) derived from photosynthetic bacteria. PRIOR ART Isoprenoid is a natural organic compound of which the fundamental unit is C5 isopentenyl diphosphate (IPP) and occurs widely in the nature. Examples are pigment carotinoid, natural rubber, and isoprenoid quinones acting in the respiratory chain electron transport system. The isoprenoid quinones include a few members such as ubiquinone (UQ), menaquinone and plastoquinone, which are distinguished depending on the difference in the quinone skeleton, and each member has a large number of homologues depending on a difference in the length of its isoprenoid side chain. Ubiquinone is a compound having isoprenoid added to the quinone skeleton, and it is a biological component also called coenzyme (CoQ) having the structure of 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone playing an important role. Ubiquinone occurs as a component in animal and plant tissues and as a microbial component in microorganisms and it is a vitamin-like component also known as anti-oxidant and plays a biologically and biochemically important role as an essential component in the electron transport system. Its analogues ranging mainly from ubiquinone-1 to ubiquione-12 depending on the number of isoprenoid units in the side chain occur in nature. Ubiquinone is studied for pharmacological and clinical effects as well and is considered to have an effect on congestive cardiac insufficiency, muscular dystrophy, anemia etc. Accordingly, it is a substance of high values as a drug for treatment of cardiac diseases and as other pharmaceutical preparations. However, only ubiquione-10 was confirmed to have effect as a pharmaceutical preparation. At present, ubiquinone-10 has been extracted and purified from tobacco leaves, yeast or other microorganisms, but its details are not revealed. In addition, its production does not catch up with its demand, so it is required to develop a further efficient process as a substitute for the conventional process. Isoprenoid, which forms a side chain of this ubiquinone, is a natural organic compound with the fundamental unit of 5C isopentenyl diphosphate (IPP) and occurs widely in nature. Besides its role as a side chain of ubiquinone, its features as carotinoid, natural rubber and prenylated protein are also known. In in vivo biosynthesis, new IPPs are linked to fundamental IPP and FPP (farnesyl diphosphate) thus elongating its chain to form long-chain isoprenoid. An enzyme catalyzing synthesis of isoprenoid in this manner is called prenyl transferase. The presence of many kinds of prenyl transferase has been confirmed in mainly bacteria. The presence of three enzymes FPS, OPS and UPS has been confirmed in E. coli, and the structural gene ispA of FPS and the structural gene ispB of octaprenyl diphosphate synthase (OPS) have been isolated as genes. By the action of this prenyl transferase, octaprenyl diphosphate with an 8-carbon side chain is synthesized in E. coli, and hexaprenyl diphosphate with a 6-carbon side chain is synthesized in budded yeast. Meanwhile, these isoprenoids are added to a benzoquinone skeleton derived from PHB (p-hydroxybenzoate) by an ubiA product in E. coli and a COQ2 product in yeast to complete ubiquinone-8 and ubiquinone-6, respectively. Accordingly, the point catalyzed by the ispB product, ubiA product, etc., is considered important for synthesis of ubiquinone. Ubiquinone-10 draws attention as a physiologically active substance with high pharmaceutically activity as described above, and the present inventors have the new point of view that use of a gene of decaprenyl diphosphate synthase for synthesis of side chains, the ubiA product, and the yeast COQ2 product, can leads to efficient production of ubiquinone-10, and they focused their attention on the necessity for cloning of the decaprenyl diphosphate synthase gene. PROBLEM TO BE SOLVED BY THE INVENTION Under the technical circumstances described above, the present invention was made for the purpose of developing the techniques of producing UQ-10 in microorganisms by use of a gene involved in synthesis of decaprenoid as a side chain of UQ-10. MEANS TO SOLVE THE PROBLEM The present invention was made to achieve the above object, and as a result of extensive examination, the present inventors atempted to produce UQ-10 by isolating a gene necessary for synthesis of UQ-10, that is, a gene for decaprenyl diphosphate synthase necessary for synthesizing its side chain, from an organism producing physiologically active UQ-10 and then expressing the gene in E. coli. To solve this novel technical problem, the present inventors synthesized a mixture of primers designed on the basis of amino acid sequences (amino acid sequences of domains 2 and 6) located in common in various polyprenyl diphosphate synthases (PDSs), and these primers were used to conduct PCR where the genomic DNA from photosynthetic bacterium Rhodobacter capsulatus biosynthesizing UQ-10 was used as the template. Sequencing of the amplification fragment obtained by the PCR suggested that it was a DNA fragment containing the target gene, so genome walking by PCR using cassette and cassette primer at its upstream and downstream sides was conducted to give a gene fragment considered to contain its full-length sequence. The nucleotide sequence of this gene fragment was then determined. As a result, the presence of an ORF consisting of 978 bp (325 amino acids) was confirmed, and as a result of further homology examination, the amino acids deduced from this nucleotide sequence showed high homology with the amino acids of Dds1 (decaprenyl diphosphate synthase) from acetic acid bacterium Gluconobacter suboxydans and IspB (octaprenyl diphosphate synthase) from E. coli, respectively. From the foregoing, the gene cloned in this manner was identified as a gene coding for decaprenyl diphosphate synthase, and it was designated dds1 gene. This gene was cloned in an E. coli expression vector. As a result of expression using a promoter for E. coli lacZ gene, the formation of UQ-9 and UQ-10 was confirmed in addition to UQ-8 originally possessed by the host, and Dds activity was also present. In addition, E. coli from which an octaprenyl diphosphate synthase gene (ispB gene) necessary for synthesis of UQ-8 had been deleted was prepared, and separately, the dds1 gene was cloned in an E. coli expression vector in the same manner as described above, and it was confirmed that UQ-10 could be produced as the major product by expressing the gene in the the ispB-destroyed strain prepared above. As a result, the present inventors succeeded for the first time in production of UQ-10 where in the former case, UQ-10 could be produced along with UQ-8 and UQ9, while in the latter case, UQ-10 can be produced mainly in the ispB destroyed strain to enable efficient production of high-purity UQ-10, and on the basis of these findings, the present inventors arrived at the present invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a restriction enzyme map of the constructed plasmid. FIG. 2 shows the design of specific primers S1 and S2 for PCR. The nucleotide and amino acid sequence shown in the upper part of FIG. 2 correspond to nucleotides 454-847 of SEQ ID NO:1 and residues 85-215 of SEQ ID NO:2, respectively. Primer S1 for upstream amplification is SEQ ID NO:7. Primer S2 for upstream amplification is SEQ ID NO:8. Primer S1 for downstream amplification is nucleotides 460-481 of SEQ ID NO:1. Primer S2 for downstream amplification is nucleotides 488-509 of SEQ ID NO:1. FIG. 3 shows genome walking by PCR by use of cassettes and cassette primers. FIG. 4 shows a nucleotide sequence (SEQ ID NO:1) containing a gene (dds1) coding for Dds1 derived from R. capsulatus, and the amino acid sequence (SEQ ID NO:2) deduced from it. The termination codon is shown by the asterisk. FIG. 5 shows construction of plasmid pRC10 for expression of dds1 gene Primer RcapN as shown is SEQ ID NO:5. Primer RcapC as shown is SEQ ID NO:6. FIG. 6 shows a HPLC chromatogram of UQ extracted from a culture of E. coli DH10B/pRC10. FIG. 7 shows a thin layer chromatogram of a product of E. coli JM109/pRC10. FIG. 8 shows a HPLC chromatogram of UQ extracted from a culture of E. coli JM109/pRC10. FIG. 9 shows a HPLC chromatogram of UQ from the pKA3-deficient strain. FIG. 10 shows a HPLC chromatogram of UQ extracted from a culture of E. coli KO229/pRC10. EMBODIMENT FOR CARRYING OUT THE INVENTION (1) Cloning of dds1 Gene Coding for Decaprenyl Diphosphate Synthase (DDS) from R. capsulatus a) Preparation of primers and PCR Sense primer A and antisense primer B were prepared on the basis of the amino acid sequences of domains II and VI particularly conserved among highly conserved 7 regions located in an enzyme (prenyl diphosphate synthase, PDS) synthesizing isoprenoid. Primer A: 5'-YTNYTNCAYGAYGAYGT-3' (SEQ ID NO:3) Primer B: 5'-RTCNTCNAGYTGRTT-3' (SEQ ID NO:3) where Y is T or C; N is A, C, T or G; and R is A or G. Primers A and B were used to conduct PCR where the genomic DNA of R. capsulatus was used as a template. About 0.5 .mu.g of the genome was thermally denatured at 94.degree. C. for 5 minutes. The reaction involved 25 cycles of PCR, each cycle consisting of thermal denaturation at 94.degree. C. for 30 seconds, annealing to the primers at 55.degree. C. for 2 minutes and elongation reaction at 72.degree. C. for 2 minutes. Then, a part of the reaction solution was subjected to agarose gel electrophoresis, and an amplification product of about 400 bp or thereabout could be confirmed. b) Cloning of the PCR amplification fragment Because a restriction enzyme BamHI recognition site had been added to the 5'-terminal of each of primers A and B, this site was utilized for cloning. Vector pUC18 and the amplification fragment were digested with BamHI, and the vector was subjected to treatment with BAP. DNA ligation Kit Ver. 1 (Takara Shuzo Co., Ltd.) was used for ligation, and a part of the ligation product was introduced into E. coli DH10B by the Hanahan method, and the plasmid was extracted from a clone considered to have the target gene and then confirmed by restriction enzyme reaction. The constructed plasmid was designated pRC3 (FIG. 1). c) Sequencing of the PCR amplification fragment Sequencing was carried out in ABI PRISMTM 377 DNA Sequencer. pRC3 as a template was sequenced starting at both ends by use of universal and reverse primers. When its coding amino acid sequence was examined for homology with other prenyl diphosphate synthase, it was found to have high homology with a long-chain synthases, that is, octaprenyl diphosphate synthase (48.8% homology) encoded by ispB in E. coli and polyprenyl diphosphate synthase encoded by ispB in H. Influenzae (47.7% homology). The photosynthetic bacterium R. capsulatus biosyntheses UQ-10 and is thus considered to have decaprenyl diphosphate synthase activity, so the about 400 bp fragment thus cloned was estimated to be a part of the gene dds1 coding for decaprenyl diphosphate synthase. (2) Cloning of the Full-length dds1 Gene by Use of Cassettes and Cassette Primers a) PCR using cassettes and cassette primers Screening after construction of the plasmid library did not result in cloning of the full length dds1 gene, but the presence of the dds1 gene was confirmed in PCR to be present in the constructed library. Therefore, the present inventors thought that if the probe (400 bp or less) used in screening is larger, false-positiveness may be reduced, so the strategy of elongating a probe or cloning its full length by genome walking in PCR using Takara's cassettes and cassette primers. As shown in FIG. 2, primers S1 and S2, which were complementary to the sequence of a part (about 400 bp) already cloned of the dds1 gene, were synthesized as primers for amplifying its upstream and downstream regions. Because commercially available cassettes are EcoRI, HindIII, PstI, SalI, XbaI and Sau3AI Cassettes, the corresponding restriction enzymes were used to digest genomic DNA from R. capsulatus, and the resulting fragment was ligated to each cassette by use of Ligation high (Toyobo). A part of the ligation product was subjected, as the template, to first PCR using cassette primer C1 l and primer S1 complementary to the known region. Then, 1 .mu.l of the first PCR reaction solution was used to carry out second PCR, and the product was subjected to electrophoresis. As a result, about downstream 1.4 kb and upstream 2.2 kb specific amplification products were observed which resulted from amplification using the ligated PstI cassette and the ligated HindIII cassette respectively. However, PCR using other cassettes resulted in a plurality of unspecific amplification fragments, so their cloning was difficult (FIG. 3). b) Cloning of the upstream and downstream amplification fragments First, the upstream amplification fragment, 1.4 kb, and the downstream amplification fragment, 2.2 kb, were confirmed to be parts of the dds1 gene by Southern hybridization, and its cloning was then carried out. "A" is added to the terminal of the PCR amplification fragment because of the character of LA Taq DNA polymerase used in PCR. Hence, the amplification fragment was ligated into vector pT7 Blue T-Vector (Novagen) with one nucleotide (T) protruded at the 3'-side, and a part of the reaction solution was introduced into E. coli JM109 by electroporation. Plasmids were extracted to obtain plasmids pHCA and pPCB having the target upstream amplification fragment 1.4 kb and downstream amplification fragment 2.2 kb inserted into them respectively (FIG. 1). c) Determination of the nucleotide sequences (Sequencing of the upstream region) Plasmid pHCA having the amplification fragment, about 1.4 kb, inserted therein was subjected to sequencing reaction using universal primer and reverse primer and then it was sequenced by ABI PRISMT M377 DNA Sequencer. However, the size of the insert DNA was too large to determine the whole region, so its subcloning was conducted. As shown in FIG. 1, it was digested at the SmaI site in the vector and SamI site in the insert DNA and then the resulting fragment was cloned into the EcoRV site of pUC18 to construct plasmid pSMI. It was sequenced starting at both ends whereby the nucleotide sequence of the upstream amplification fragment, 1.4 kb, was determined. (Sequencing of the downstream region) Plasmid pPCB having the downstream amplification fragment about 2.2 kb inserted therein was also sequenced using the universal primer and reverse primer in the same manner as for pHCA, and as a result, the universal primer side was found to be located at the N-terminal. To sequence the region whose nucleotide sequence could not be determined by this sequencing step, a deletion plasmid was constructed as follows: After the absence of KpnI and BamHI sites in the insert DNA was confirmed, pPCB was completely digested with KpnI and BamHI and then with Exonuclease III and Mung bean nuclease, to give plasmid pPCB .increment.300 from which about 400 bp of the upstream region had been deleted. After ligation, the product was introduced into DH10B, and the target gene was isolated and sequenced. As a result, the presence of an ORF consisting of 978 kb (325 amino acids) was confirmed. Its nucleotide sequence and the amino acid sequence deduced from it were shown in FIG. 4. (d) Examination of Homology The amino acid sequence deduced from the nucleotide sequence of the ORF (open reading frame) i.e. the region whose genetic information is transcribed into mRNA and then translated into a protein has high homology with the amino acid sequences of other prenyl diphosphate synthases, that is, 43.6% homology with IspB from E. coli and 41.6% homology with Dds1 from acetic bacterium Gluconobacter suboxydans. From this, the presently cloned gene is considered to be a structural gene of polyprenyl diphosphate synthase. The photosynthetic bacterium R. capsulatus biosyntheses UQ-10 and thus this gene was expected to code for a protein having decaprenyl diphosphate synthase activity, so this gene was designated gene dds1 coding for decaprenyl diphosphate synthase. The nucleotide sequence thereof, along with the amino acid sequence of DDS, were shown in SEQ ID NOs:1 (corresponding to nucleotides 205 to 1188) and 2, respectively. (3) Expression of the dds1 Gene in E. coli a) Construction of a plasmid for expression of the dds1 gene As shown in FIG. 5, PCR was carried out using primers RcapN and RcapC (to which restriction enzyme BamHI and HindIII sites had been added) capable of amplifying the ORF in the dds1 gene. Then, the amplification fragment was cloned into the BamHI-HindIII site in pUC118 to result in constructing plasmid pRC10 in which said fragment was inserted so as to be adjusted to the translational frame of lacZ gene. b) Expression in E. coli (1) Plasmid pRC10 thus constructed was introduced into E. coli DH10B and cultured where IPTG (isopropyl-4-thio-.beta.-D-galactoside) was added for induction at a concentration of 2 mM to the microorganism just before logarithmic growth phase. UQ was extracted from the E. coli, then developed by TLC and analyzed by HPLC, and a peak of UQ-10 was thus confirmed in addition to a peak of originally biosynthesized UQ-8 (FIG. 6). Because the peak of UQ-10 disappeared upon addition of the reducing agent NaBH.sub.4, it was confirmed that the E. coli biosynthesised UQ-10. As is evident from the foregoing, E. coli DH10B (Escherichia coli DH10B/pRC10), that is, a microorganism transformed with dds1 derived from R. capsulatus was confirmed to express dds1 and to produce UQ-10. This transformant was designated E. coli DH10B/pRC10 and deposited as FERM BP-6061 on Aug. 15, 1997 under the Budapest Treaty with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan. UQ extraction, TLC development, and HPLC analysis were carried out respectively as follows: (UQ extraction) 50 ml culture (yeast cells) and 20 ml culture (E. coli) were centrifuged and 3 ml of acetone/methanol (7:2) was added to the pellet which was then sonicated for 30 seconds and placed in ice-cold water for 30 seconds. The above step was repeated 6 times. After centrifugation, the supernatant was dried under vacuum. 400 .mu.l of chloroform/methanol (1:1) and an equal volume of 0.7% NaCl were added to the pellet and then stirred. After centrifugation, the lower layer was extracted, dried, and suspended in 100 .mu.l of chloroform/methanol (2:1). (TLC development) The sample was spotted onto a TLC plate. It was developed with benzene/acetone (93:7) for about 1 hour, and UQ was confirmed by irradiation of the plate with UV ray. A part considered to contain UQ was scratched. 400 .mu.l Chloroform/methanol (1:1) was added to it and stirred, and after centrifugation, the supernatant was dried under vacuum. It was suspended in 100 .mu.l of 100% ethanol, and 20 .mu.l of the resulting solution was used as analysis sample. (HPLC analysis) Analysis was carried out under the following conditions. Column: Shim-pack CLC-ODS. Eluent: 100% ethanol. Flow rate: 1.0 ml/min. Wavelength: 275 nm. An example of formation of UQ-10 accompanied by UQ-8 by use of E. coli DH10B (E. coli DH10B/pRC10) (FERM BP-6061) transformed with plasmid pRC10 has been described, and another example is hereinafter described. c) Expression in E. coli (2) E. coli JM109 (Escherichia coli JM109/pRC10) carrying pRC10, that is, a microorganism transformed in the same manner as above with dds1 derived from R. capsulatus, was cultured, and measurement of the activity of the dds1 gene product and identification of the product were carried out as follows: .sup.14 C-labelled IPP (isopentenyl diphosphate: Amersham Life Science Co., Ltd.) and FPP (farnesyl diphosphate) were used as substrate, and a crude enzyme (0.05 mg protein) from E. coli where the gene was expressed was allowed to react with the substrate at 30.degree. C. for 2 hours, and then polyprenyl diphosphate extracted with butanol was hydrolyzed and developed in the form of polyprenol by TLC. As a result, as shown in FIG. 7, decaprenol was detected in JM109 carrying pRC10 (lane 2). From the results, it was made evident that the dds1 gene obtained by the present inventors is a gene involved in synthesis of side chain decaprenyl diphosphate necessary for synthesis of UQ-10, and it was proved that formation of UQ-10 is made feasible by expression of the dds1 gene in E. coli. As is evident from the drawings, lane 1 is JM109 as the control, where octaprenol is detected. On the other hand, in lane 2 (gene expression lane), solanesol and decaprenol are confirmed to be formed in addition to octaprenol, and from these results it was made evident that dds1 is a structural gene for solanesyl, decaprenyl diphosphate synthase. Then, UQ was extracted from JM109 expressing dds1 and analyzed by HPLC, and peaks of UQ-9 and UQ-10 were confirmed in addition to a peak of originally biosynthesized UQ-8 (FIG. 8). On the other hand, JM109 as the control formed UQ-8 only, and from these results, the production of UQ-10 by E. coli JM109 (Escherichia coli JM109/pRC10) transformed with plasmid pRC10 was confirmed. In other words, dds1 is the structural gene of DDS and further its expression in the heterologous microorganism was confirmed. (4) Test of Complementation with the Mutation in E. coli ispB-Deficient Strain KO 229 (.increment.ispB::CAT) by Dds1 Because E. coli KO229 is a strain in which ispB gene coding for octaprenyl diphosphate synthase in the genome has been destroyed by insertion of a gene coding for CAT (chloramphenicol acetyl transferase), it cannot biosynthesize UQ-8, and it cannot grow as such, so the mutation is complemented by introducing plasmid pKA3 carrying the ispB gene. Generally, a plasmid not necessary for growth will not be segregated into daughter cells at the time of cell division so that the plasmid is deleted from it. However, because plasmid pKA3 (having spectinomycin resistant gene) possessed by KO229 complements a mutation and is thus essential for growth, pKA3 cannot be deleted even by several sub-cultures in a non-selective medium not containing spectinomycin. Hence, a plasmid deletion experiment was carried out considering that if the function of IspB in E. coli can be replaced in vivo by that of Dds1 protein, pKA3 having the ispB gene inserted into it can be deleted. That is, after pRC10 was introduced into KO229, the microorganism was subjected to several sub-cultures in a non-selective medium (Cm+Amp). Colonies were formed in a non-selective plate not containing spectinomycin and replicated in a non-selective plate and a selective plate (Cm+Spc), and some strains showing sensitivity to spectinomycin as the selective marker of pKA3, that is, strains considered to delete the plasmid, could be obtained. UQ was extracted from the strains showing sensitivity to spectinomycin and analyzed by HPLC, and as a result, peaks of UQ-9 and UQ-10 having isoprenoid chain with a length considered to be supplied by Dds1 protein were observed as shown in FIG. 9. This indicates that plasmid pKA3 could be successfully deleted by complementing the mutation with Dds1 protein in place of IspB protein. In view of its usefulness, the strain whose complementation was thus confirmed was designated E. coli KO229/pRC10 and deposited as FERM BP-6062 on Aug. 15, 1997 under the Budapest Treaty with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology. UQ was extracted in the manner as above from this strain (FERM BP-6062), and the result is shown in FIG. 10. As is evident from this result, formation of UQ-10 was confirmed. EFFECT OF THE INVENTION According to the present invention, a structural gene for decaprenyl diphosphate synthase derived from a wide variety of photosynthetic bacteria such as Rhodobacter capsulatus was not only successfully isolated and sequenced but could also be expressed in E. coli for the first time. As a result, UQ-10 in addition to UQ-8 and UQ-9 can be made feasible using E. coli, and by use of the ispB gene-deficient strain, UQ-10 can be selectively produced in high purity and in a large amount while inhibiting production of UQ-8. The microorganisms R. capsulatus, E. coli DH10B, E. coli JM109, E. coli KO229 etc. used in the present invention are described in literatures, are commercially available, and are obtainable from Dr. Matuda, Shimane University, Japan. __________________________________________________________________________ # SEQUENCE LISTING |
| PATENT PHOTOCOPY | available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |