Production of Coenzyme $Q_{10}$ by Recombinant E. coli Harboring the Decaprenyl Diphosphate Synthase Gene from Sinorhizobium meliloti

  • Seo Myung-Ji (Division of Bio-New Drug Development, Central Research Institute, Chem Tech Research Incorporation (C-TRI)) ;
  • Im Eun-Mi (Division of Bio-New Drug Development, Central Research Institute, Chem Tech Research Incorporation (C-TRI)) ;
  • Hur Jin-Haeng (Division of Bio-New Drug Development, Central Research Institute, Chem Tech Research Incorporation (C-TRI)) ;
  • Nam Jung-Yeon (Department of Biotechnology, Yonsei University) ;
  • Hyun Chang-Gu (Division of Bio-New Drug Development, Central Research Institute, Chem Tech Research Incorporation (C-TRI)) ;
  • Pyun Yu-Ryang (Department of Biotechnology, Yonsei University) ;
  • Kim Soon-Ok (Division of Bio-New Drug Development, Central Research Institute, Chem Tech Research Incorporation (C-TRI))
  • Published : 2006.06.01

Abstract

Decaprenyl diphosphate synthase (DPS) is the key enzyme for the production of coenzyme $Q_{10}$ ($CoQ_{10}$). A dps gene from Sinorhizobium meliioti KCCM 11232 (IFO 14782) was isolated by PCR and then cloned in Escherichia coli. DNA sequencing analysis revealed an open reading frame of 1,017 bp encoding a 338-amino-acid protein. The protein was identical at the 98% level to the putative octaprenyl diphosphate synthase (IspB) of S. meliloti 1021. The deduced amino acid sequence included the DDxxD domains conserved in the majority of the prenyl diphosphate synthases. Heterologous expression in E. coli BL21 (DE3) was carried out, and the $CoQ_{10}$ produced was then analyzed by HPLC. E. coli BL21 (DE3) harboring the dps gene from S. melioti produced CoQ$_{10}$ in addition to endogenous coenzyme Q$_8$ (CoQ$_8$), whereas wild-type E. coli BL21 (DE3) host did not have the ability of producing CoQ$_{10}$. The results suggest that the putative dps from S. meliloti KCTC 2353 encoded the DPS.

References

  1. Alleva, R., M. Tomasetti, L. Andera, N. Gellert, B. Borghi, C. Weber, M. P. Murphy, and J. Neuzil. 2001. Coenzyme Q blocks biochemical but not receptor-mediated apoptosis by increasing mitochondrial antioxidant protection. FEBS Lett. 503: 46-50 https://doi.org/10.1016/S0014-5793(01)02694-1
  2. Asai, K., S. Fujisaki, Y. Nishimura, T. Nishino, K. Okada, T. Nakagawa, M. Kawamukai, and H. Matsuda. 1994. The identification of Escherichia coli ispB (cel) gene encoding the octaprenyl diphosphate synthase. Biochem. Biophys. Res. Commun. 202: 340-345 https://doi.org/10.1006/bbrc.1994.1933
  3. Chen, A., P. A. Kroon, and C. D. Poulter. 1994. Isoprenyl diphosphate synthases: Protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure. Protein Sci. 3: 600-607 https://doi.org/10.1002/pro.5560030408
  4. Ernster, L. and G. Dallner. 1995. Biochemical, physiological and medical aspects of ubiquinone function. Biochim. Biophys. Acta 1271: 195-204 https://doi.org/10.1016/0925-4439(95)00028-3
  5. Joly, A. and P. A. Edwards. 1993. Effect of site-directed mutagenesis of conserved aspartate and arginine residues upon farnesyl diphosphate synthase activity. J. Biol. Chem. 268: 26983-26989
  6. Kawamukai, M. 2002. Biosynthesis, bioproduction and novel roles of ubiquinone. J. Biosci. Bioeng. 94: 511-517 https://doi.org/10.1016/S1389-1723(02)80188-8
  7. Koyama, T., S. Obata, M. Osabe, A. Takeshita, K. Yokoyama, M. Uchida, T. Nishino, and K. Ogura. 1993. Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: Molecular cloning, sequence determination, overproduction, and purification. J. Biochem. (Tokyo) 113: 355-363 https://doi.org/10.1093/oxfordjournals.jbchem.a124051
  8. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  9. Lee, J. K., G. Her, S. Y. Kim, and J. H. Seo. 2004. Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens. Biotechnol. Prog. 20: 51-56 https://doi.org/10.1021/bp034213e
  10. Meganathan, R. 2001. Ubiquinone biosynthesis in microorganisms. FEMS Microbiol. Lett. 203: 131-139 https://doi.org/10.1111/j.1574-6968.2001.tb10831.x
  11. Melzer, M. and L. Heide. 1994. Characterization of polyprenyldiphosphate: 4-Hydroxybenzoate polyprenyltransferase from Escherichia coli. Biochim. Biophys. Acta 1212: 93-102 https://doi.org/10.1016/0005-2760(94)90193-7
  12. Ohnuma, S., K. Narita, T. Nakazawa, C. Ishida, Y. Takeuchi, C. Ohto, and T. Nishino. 1996. A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of farnesyl diphosphate synthase on determination of the final product. J. Biol. Chem. 271: 30748-30754 https://doi.org/10.1074/jbc.271.48.30748
  13. Okada, K., K. Suzuki, Y. Kamiya, X. Zhu, S. Fujisaki, Y. Nishimura, T. Nishino, T. Nakagawa, M. Kawamukai, and H. Matsuda. 1996. Polyprenyl diphosphate synthase essentially defines the length of the side chain of ubiquinone. Biochim. Biophys. Acta 1302: 217-223 https://doi.org/10.1016/0005-2760(96)00064-1
  14. Okada, K., T. Kainou, K. Tanaka, T. Nakagawa, H. Matsuda, and M. Kawamukai. 1998. Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans. Eur. J. Biochem. 255: 52-59 https://doi.org/10.1046/j.1432-1327.1998.2550052.x
  15. Saiki, R., A. Nagata, N. Uchida, T. Kainow, H. Matsuda, and M. Kawamukai. 2003. Fission yeast decaprenyl diphosphate synthase consists of Dps1 and the newly characterized Dlp1 protein in a novel heterotetrameric structure. Eur. J. Biochem. 270: 4113-4121 https://doi.org/10.1046/j.1432-1033.2003.03804.x
  16. Sarter, B. 2002. Coenzyme $Q_{10}$ and cardiovascular disease: A review. J. Cardiovasc. Nurs. 16: 9-20
  17. Song, L. and C. D. Poulter. 1994. Yeast farnesyl-diphosphate synthase: Site-directed mutagenesis of residues in highly conserved prenyltransferase domains I and II. Proc. Natl. Acad. Sci. USA 91: 3044-3048
  18. Suzuki, K., K. Okada, Y. Kamiya, X. F. Zhu, T. Nakagawa, M. Kawamukai, and H. Matsuda. 1997. Analysis of the decaprenyl diphosphate synthase (dps) gene in fission yeast suggests a role of ubiquinone as an antioxidant. J. Biochem. 121: 496-505 https://doi.org/10.1093/oxfordjournals.jbchem.a021614
  19. Takada, M., T. Yuzuriha, K. Katayama, K. Iwamoto, and J. Sunamoto. 1984. Increased lung uptake of liposomes coated with polysaccharides. Biochim. Biophys. Acta 802: 237-244 https://doi.org/10.1016/0304-4165(84)90167-3
  20. Takahashi, S., T. Nishino, and T. Koyam. 2003. Isolation and expression of Paracoccus denitrificans decaprenyl diphosphate synthase gene for production of ubiquinone-10 in Escherichia coli. Biochem. Eng. J. 16: 183-190 https://doi.org/10.1016/S1369-703X(03)00035-4
  21. Tarshis, L. C., M. Yan, C. D. Poulter, and J. C. Sacchettini. 1994. Crystal structure of recombinant farnesyl diphosphate synthase at 2.6-A resolution. Biochemistry 3: 10871-10877
  22. Tarshis, L. C., P. J. Proteau, B. A. Kellogg, J. C. Sacchettini, and C. D. Poulter. 1996. Regulation of product chain length by isoprenyl diphosphate synthases. Proc. Natl. Acad. Sci. USA 93: 15018-15023
  23. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680 https://doi.org/10.1093/nar/22.22.4673