Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Cloning of Geranylgeranyl Pyrophosphate Synthase (CrtE) Gene from Kocuria gwangalliensis and Its Functional Co-expression in Escherichia coli

코쿠리아 광안리엔시스의 제라닐제라닐 피로인산염 합성 효소의 클로닝과 대장균에서 공발현을 통한 효소 활성에 관한 연구

  • Seo, Yong-Bae (Department of Microbiology, College of Natural Sciences, Pukyong National University) ;
  • Kim, Gun-Do (Department of Microbiology, College of Natural Sciences, Pukyong National University) ;
  • Lee, Jae-Hyung (Basic Science Research Institute, Pukyong National University)
  • Received : 2012.07.12
  • Accepted : 2012.08.07
  • Published : 2012.08.30
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Abstract

A gene encoding a novel geranylgeranyl pyrophosphate (GGPP) synthase from Kocuria gwangalliensis has been cloned and expressed in Escherichia coli. The deduced amino acid sequence showed 59.6% identity with a putative GGPP synthase (CrtE) from K. rhizophila. An expression plasmid containing the crtE gene was constructed, and E. coli cells containing this plasmid produced a recombinant protein with a theoretical molecular mass of 41 kDa, corresponding to the molecular weight of GGPP synthase. Due to the lack of crtE, crtB, and crtI in E. coli, the biosynthesis of lycopene was only obtained when the plasmid pCcrtE was co-transformed into E. coli expressing the pRScrtBI-carrying carotenoid biosynthesis crtB and crtI genes, which were sub-cloned from Paracoccus haeundaensis. The biochemical studies on the expressed proteins were performed via HPLC. The results obtained from this study will provide a wider base of knowledge regarding the primary structure of CrtE cloned from K. gwangalliensis at the molecular level.

Kocuria gwangalliensis로부터 카로티노이드 생합성 경로의 첫 번째 단계 기질인 geranylgeranyl pyrophosphate (GGPP)를 생합성하는 GGPP synthase (CrtE)를 암호화하고 있는 crtE를 클로닝 하여 이를 KgGGPP로 명명하였다. 기존 세균에서 밝혀진 GGPP synthase의 아미노산 서열을 NCBI에서 검색하여 KgGGPP synthase의 아미노산 서열과 비교한 결과 Kocuria rhizophila와 59.6%의 상동성을 가지는 것을 확인하였다. crtE 유전자를 대장균에서 발현 시키기 위하여 pCcrtE 재조합 DNA를 구축하였고, 이를 대장균에서 발현시킨 결과 약 41 kDa의 재조합 단백질이 과발현 됨을 확인 할 수 있었으며, 이 단백질은 기존 세균에서 밝혀진 GGPP synthase와 유사한 분자량을 가지고 있다는 것을 알 수 있었다. CrtE 재조합 단백질의 활성을 분석하기 위하여 대장균 내에서 라이코펜의 생합성을 유도 하였다. 대장균의 경우 메발론산 경로를 통하여 FPP와 IPP를 생합성 하지만 crtE, crtB, crtI 유전자가 없기 때문에 라이코펜을 생합성 하지는 못한다. 대장균 내에서 라이코펜의 생합성을 위해서는 crtE, crtB, crtI 유전자의 발현이 필수적으로 요구되기 때문에 crtB, crtI 유전자의 경우는 P. haeundaensis에서 유래한 유전자를 이용하여 pRScrtBI 재조합 DNA를 구축하여 그 발현을 유도하였다. 상기 두 재조합 DNA를 대장균에서 공발현 시켰으며, HPLC 분석법을 이용하여 대장균 내에서 라이코펜의 생산 유무에 따른 KgGGPP synthase의 활성을 분석하였다.

Keywords

References

  1. Amar, Y., Kiron, V., Satoh, S. and Watanabe, T. 2004. Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products. Fish Shellfish Immunol. 16, 527-537. https://doi.org/10.1016/j.fsi.2003.09.004
  2. Breitenbach, J., Visser, H., Verdoes, J. C., van Ooyen, A. J. and Sandmann, G. 2011. Engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in Xanthophyllomyces dendrorhous. Biotechnol. Lett. 33, 755-761. https://doi.org/10.1007/s10529-010-0495-2
  3. Chemler, J. A., Yan, Y. and Koffas, M. A. 2006. Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae. Microb. Cell Fact. 23, 5-20.
  4. Christianson, D. W. 2008. Unearthing the roots of the terpenome. Curr. Opin. Chem. Biol. 12, 141-150. https://doi.org/10.1016/j.cbpa.2007.12.008
  5. Engprasert, S., Taura, F., Kawamukai, M. and Shoyama, Y. 2004. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq. BMC Plant Biol. 4, 18 https://doi.org/10.1186/1471-2229-4-18
  6. Fernandez-Garayzabal, J. F., Dominguez, L., Pascual, C., Jones, D. and Collins, M. D. 1995. Phenotypic and phylogenetic characterization of some unknown coryneform bacteria isolated from bovine blood and milk: description of Sanguibacter gen. nov. Lett. Appl. Microbiol. 20, 69-75. https://doi.org/10.1111/j.1472-765X.1995.tb01289.x
  7. Hemmi, H., Noike, M., Nakayama, T. and Nishino, T. 2003. An alternative mechanism of product chain-length determination in type III geranylgeranyl diphosphate synthase. Biochem. 270, 2186-2194.
  8. Hunter, W. N. 2007 The non-mevalonate pathway of isoprenoid precursor biosynthesis. J. Biol. Chem. 282, 21573-21577. https://doi.org/10.1074/jbc.R700005200
  9. Irlinger, F., Bimet, F., Delettre, J., Lefevre, M. and Grimont, P. A. D. 2005. Arthrobacter bergerei sp. nov. and Arthrobacter arilaitensis sp. nov. novel coryneform species isolated from the surfaces of cheeses. Int. J. Syst. Evol. Microbiol. 55, 457-462. https://doi.org/10.1099/ijs.0.63125-0
  10. Lin, J., Jin, Y. J., Zhou, X.,and Wang, J. Y. 2010. Molecular cloning and functional analysis of the gene encoding geranylgeranyl diphosphate synthase from Jatropha curcas. Biotechnol. 9, 3342-3351.
  11. Lee, J. H. and Kim, Y. T. 2006. Cloning and characterization of the astaxanthin biosynthesis gene cluster from the marine bacterium Paracoccus haeundaensis. Gene 370, 86-95. https://doi.org/10.1016/j.gene.2005.11.007
  12. Lee, J. H., Kim, Y. S., Choi, T. J., Lee, W. J. and Kim, Y. T. 2004. Paracoccus haeundaensis sp. nov., a Gram-negative, halophilic, astaxanthin-producing bacterium. Int. J. Syst. Evol. Microbiol. 54, 1699-1702. https://doi.org/10.1099/ijs.0.63146-0
  13. Morran, C. P. Jr., Lang, N., Le Grice, F. J., Lee, G., Stephens, M., Sonenshein, A. L., Pero, J. and Losick, R. 1982. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol. Gen. Genet. 186, 339-346. https://doi.org/10.1007/BF00729452
  14. Saitou, N. and Nei, M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425.
  15. Sambrook, J. and Russell, D. W. 2001. Molecular cloning: a laboratroy manual. pp. 4.7-4.10. 3th eds. Cold Spring Harbor Laboratroy Press, Cold Spring Harbor, New York, USA.
  16. Seo, Y. B., Kim, D. E., Kim, G. D., Kim, H. W., Nam, S. W., Kim, Y. T. and Lee, J. H. 2009. Kocuria gwangalliensis sp. nov., an actinobacterium isolated from seawater. Int. J. Syst. Evol. Microbiol. 59, 2769-2772. https://doi.org/10.1099/ijs.0.008482-0
  17. Seo, Y. B., Choi, S. S., Nam, S. W., Lee, J. H. and Kim, Y. T. 2009. Cloning and characterization of the zeaxanthin glucosyltransferase Gene (crtX) from the astaxanthin-producing marine bacterium, Paracoccus haeundaensis. J. Microbiol. Biotechnol. 19, 1542-1546. https://doi.org/10.4014/jmb.0904.04033
  18. Seo, Y. B., Hwang H. H. and Kim, Y. T. 2011. Molecular characterization and tissue expression of carboxypeptidase H (CPH) gene in flounder (Paralichthys olivaceus). Biotechnol. Bioprocess Eng. 16, 374-382. https://doi.org/10.1007/s12257-010-0278-6
  19. Sitthithaworn, W., Kojima, N., Viroonchatapan, E., Suh, D. Y., Iwanami, N., Hayashi, T., Noji, M. Saito, K., Niwa, Y. and Sankawa, U. 2001. Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis. Chem. Pharm. Bull. 49, 197-202. https://doi.org/10.1248/cpb.49.197
  20. Takarada, H., Sekine, M., Kosugi, H., Matsuo, Y., Fujisawa, T., Omata, S., Kishi, E., Shimizu, A., Tsukatani, N., Tanikawa, S., Fujita, N. and Harayama, S. 2008. Complete genome sequence of the soil actinomycete Kocuria rhizophila. J. Bacteriol. 190, 4139-4146. https://doi.org/10.1128/JB.01853-07
  21. Wang, K. and Ohnuma, S. 1999. Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution. Trends Biochem. Sci. 24, 445-451. https://doi.org/10.1016/S0968-0004(99)01464-4
  22. Young, J. M., Saddler, G. S., Takikawa, Y., De Boer, S. H., Vauterin, L., Gardan, L., Gvozdyak, R. I. and Stead, D. E. 1996. Names of plant pathogenic bacteria 1864-1995. Rev. Plant Pathol. 75, 721-763.
  23. Young, M., Artsatbanov, V., Beller, H. R., Chandra, G., Chater, K. F., Dover, L. G., Goh, E. B., Kahan, T., Kaprelyants, A. S., Kyrpides, N., Lapidus, A., Lowry, S. R., Lykidis, A., Mahillon, J., Markowitz, V., Mavrommatis, K., Mukamolova, G. V., Oren, A., Rokem, J. S., Smith, M. C., Young, D. I. and Greenblatt, C. L. 2010. Genome sequence of the Fleming strain of Micrococcus luteus, a simple free-living actinobacterium. J. Bacteriol. 192, 841-860. https://doi.org/10.1128/JB.01254-09
  24. Xu, Z., Tian, B. Sun, Z., Lin, J., and Hua, Y. 2007. Identification and functional analysis of a phytoene desaturase gene from the extremely radioresistant bacterium Deinococcus radiodurans. Microbiology 153, 1642-1652. https://doi.org/10.1099/mic.0.2006/002857-0