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Fusion of Ginseng Farnesyl Diphosphate Synthase and Centella asciatica Squalene Synthase Involved in Triterpenoid Biosynthesis


Affiliations
1 Department of Herbal Crop Research, National Institute of Horticultural and Crop Science, RDA, Eumseong 27709, Korea, Democratic People's Republic of
2 Department of Botany, University of British Columbia, 6270 University Blvd., Vancouver V6T1Z4, Canada
 

Farnesyl diphosphate synthase (FPS) is a key enzyme in isoprenoid biosynthesis, generating farnesyl diphosphate as the central precursor for the broad classes of sesquiterpenoids and triterpenoids. On the one hand, cyclization of farnesyl diphosphate catalysed by various sesquiterpene synthases leads to structurally diverse sesquiterpenoids, while on the other, dimerization catalysed by squalene synthase (SQS) yields squalene as the first intermediate in the production of triterpenoids. To optimize triterpenoid production, the activities of an FPS generating farnesyl diphosphate and an SQS converting it should be coupled. Here, we constructed a fusion protein combining a ginseng FPS and a Centella asiatica SQS via a short peptide (Gly-Ser-Gly) linker. Heterologous expression in Escherichia coli resulted in a soluble fusion protein detected by SDS-PAGE. The fusion protein had both FPS and SQS activities, at approximately 94% and 71% of the single enzyme levels respectively. This novel fusion protein will serve as a valuable tool for genetic engineering of triterpenoid compounds, including saponins.

Keywords

Farnesyl Diphosphate Synthase, Fusion Protein, Squalene Synthase, Triterpenoids.
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  • Gaffe, J., Bru, J. P., Causse, M., Vidal, A., Stamitti-Bert, L., Carde, J. P. and Gallusci, P., LEFPS1, a tomato farnesyl pyrophosphate gene highly expressed during early fruit development. Plant Physiol., 2000, 123(4), 1351–1362.

  • Kroymann, J., Natural diversity and adaptation in plant secondary metabolism. Curr. Opin. Plant Biol., 2011, 14(3), 246–251.

  • Suzuki, M. and Muranaka, T., Molecular genetics of plant sterol backbone synthesis. Lipids, 2007, 42(1), 47–54.

  • Augustin, J. M., Kuzina, V., Andersen, S. B. and Bak, S., Molecular activities, biosynthesis and evolution of triterpenoid sapoinins. Phytochemistry, 2011, 72(6), 435–457.

  • Grunler, J., Ericsson, J. and Dallner, G., Branch-point reactions in the biosynthesis of cholesterol, dolichol, ubquinone and prenylated proteins. Biochim. Biophys. Acta, 1994, 1212(3), 259–277.

  • Cunillera, N., Boronat, A. and Ferrer, A., The Arabidopsis thaliana FPS1 gene generates a novel mRNA that encodes a mitocondrial farnesyl-diphosphate synthase isoform. J. Biol. Chem., 1997, 27(24), 15381−15388.

  • Oliver, L. M. and Krisans, S. K., Peroxisomal protein targeting and identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes. Biochim. Biophys. Acta, 2000, 1529(1–3), 89−102.

  • Sanmiya, K., Ueno, O., Matsuoka, M. and Yamamoto, N., Localization of farnesyl diphosphate synthase in chloroplasts. Plant Cell Physiol., 1999, 40(3), 348–354.

  • Nakashima, T., Inoue, T., Oka, A., Nishino, T., Osumi, T. and Hata, S., Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase. Proc. Natl. Acad. Sci. USA, 1995, 92(6), 2328–2332.

  • Daudonnet, S., Karst, F. and Tourte, Y., Expression of the farnesyl diphosphate synthase gene of Saccharomyces cerevisiae in tobacco. Mol. Breed., 1997, 3, 137–145.

  • Lee, M. H. et al., Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol., 2004, 45(8), 976–984.

  • Seo, J. W. et al., Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation. Phytochemistry, 2005, 66(8), 869–877.

  • Deng, Y., Sun, M., Xu, S. and Zhou, J., Enhanced (S)-linalool production by fusion expression of farnesyl diphosphate synthase and linalool synthase in Saccharomyces cerevisiae. J. Appl. Microbiol., 2016, 121(1), 187–195.

  • Brodelius, M., Lundgren, A., Mercke, P. and Brodelius, P. E., Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum. Eur. J. Biochem., 2002, 269(14), 3570–3577.

  • Wang, C., Yoon, S. H., Jang, H. J., Chung, Y. R., Kim, J. Y., Choi, E. S. and Kim, S. W., Metabolic engineering of Escherichia coli for -farnesene production. Metab. Eng., 2011, 13(6), 648–655.

  • Kim, O. T., Seong, N. S., Kim, M. Y. and Hwang, B., Isolation and characterization of squalene synthase cDNA from Centella asiatica (L.) Urban. J. Plant Biol., 2005, 48(3), 263–269.

  • Bradford, M. M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72, 248–254.

  • Mekkriengkrai, D., Sando, T., Hirooka, K., Sakdapipanich, J., Tanaka, Y., Fukusaki, E. and Kobayashi, A., Cloning and characterization of farnesyl diphosphate synthase from the rubberproducing mushroom Lactarius chrysorrheus. Biosci. Biotechnol. Biochem., 2004, 68(11), 2360–2368.

  • Kim, O. T., Bang, K. H., Jung, S. J., Kim, Y. C., Hyun, D. Y., Kim, S. H. and Cha, S. W., Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate. Biol. Plant., 2010, 54(1), 47–53.

  • Tansey, T. R. and Shechter, I., Structure and regulation of mammalian squalene synthase. Biochim. Biophys. Acta, 2000, 1529(1–3), 49–62.

  • Carlsson, H., Ljungcrantz, P., Bulow, L. and Mosach, K., Engineering of lactose metabolism in E. coli by introducing β-galactosidase/galactokinase fusion enzymes. Biotechnol. Lett., 1992, 14(6), 439–444.

  • Carlsson, H., Ljung, S. and Bülow, L., Physical and kinetic effects on induction of various linker regions in beta-galactosidase/galactose dehydrogenase fusion enzymes. Biochim. Biophys. Acta, 1996, 1293(1), 154–160.

  • Ohnuma, S., Hirooka, K., Ohto, C. and Nishino, T., Conversion from archael geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase. J. Biol. Chem., 1997, 272(8), 5192–5198.


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  • Fusion of Ginseng Farnesyl Diphosphate Synthase and Centella asciatica Squalene Synthase Involved in Triterpenoid Biosynthesis

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Authors

Su Jin Jung
Department of Herbal Crop Research, National Institute of Horticultural and Crop Science, RDA, Eumseong 27709, Korea, Democratic People's Republic of
Young Chang Kim
Department of Herbal Crop Research, National Institute of Horticultural and Crop Science, RDA, Eumseong 27709, Korea, Democratic People's Republic of
Mei Lan Jin
Department of Herbal Crop Research, National Institute of Horticultural and Crop Science, RDA, Eumseong 27709, Korea, Democratic People's Republic of
Reinhard Jetter
Department of Botany, University of British Columbia, 6270 University Blvd., Vancouver V6T1Z4, Canada
Ok Tae Kim
Department of Herbal Crop Research, National Institute of Horticultural and Crop Science, RDA, Eumseong 27709, Korea, Democratic People's Republic of

Abstract


Farnesyl diphosphate synthase (FPS) is a key enzyme in isoprenoid biosynthesis, generating farnesyl diphosphate as the central precursor for the broad classes of sesquiterpenoids and triterpenoids. On the one hand, cyclization of farnesyl diphosphate catalysed by various sesquiterpene synthases leads to structurally diverse sesquiterpenoids, while on the other, dimerization catalysed by squalene synthase (SQS) yields squalene as the first intermediate in the production of triterpenoids. To optimize triterpenoid production, the activities of an FPS generating farnesyl diphosphate and an SQS converting it should be coupled. Here, we constructed a fusion protein combining a ginseng FPS and a Centella asiatica SQS via a short peptide (Gly-Ser-Gly) linker. Heterologous expression in Escherichia coli resulted in a soluble fusion protein detected by SDS-PAGE. The fusion protein had both FPS and SQS activities, at approximately 94% and 71% of the single enzyme levels respectively. This novel fusion protein will serve as a valuable tool for genetic engineering of triterpenoid compounds, including saponins.

Keywords


Farnesyl Diphosphate Synthase, Fusion Protein, Squalene Synthase, Triterpenoids.

References





DOI: https://doi.org/10.18520/cs%2Fv113%2Fi04%2F785-790