Characterization of sesquiterpene lactone biosynthesis in sunflower glandular trichomes
Final Report Abstract
Major objective of project was the characterization of enzymatic steps necessary for the transformation of germacrene A into germacrene A carboxylic acid and the subsequent formation of the lactone ring in α,β-unsaturated γ-lactones of the costunolide type. This was achieved by establishing an EST library representing the transciptome of sunflower glandular trichomes in their biosynthetically active phase and screening for candidate genes. Two enzymes, the germacrene A oxidase (GAO) and the germacrene acid oxigenase (GAAO) were identified, fully sequenced and functionally characterized in vivo using a plasmid-based multigene expression yeast system and in vitro for unambigious confirmation of the in vivo results. GAO and GAAO are both members of the cytochrome P450 protein family and their synthesized products were characterized by GC/MS measurements and 1H NMR. In more detail, GAO was shown to be a multifunctional enzyme transforming germacrene A into gemacrene acid (GAA) in a three step reaction. The hydroxylation of GAA at the C-6 position, necessary for the formation of the 6,7 unsaturated lactone ring in the basic sesquiterpene lactone costunolide was searched for in a similar way. However the identified candidate gene GAAO showed an unexpected regio- and stereo-selectivity for hydroxylating the C-8 instead of C-6 position. This lead to the formation of small amounts of cis-7,8-lactonized STL rather than costunolide. GAAO appears to be essential for a subsequent step in which the side chain esterification at C-8 occurs as it is typical for sunflower STL. Intensive screening of the yet sequenced EST clones did not unravel any other costunolide synthase candidate gene. This major hurdle was overcome by combining GAO with a P450 enzyme from lettuce, showing a very high degree of similarity high homology to sunflower GAAO in the yeast system. In this way, it was not only possible to identify the last missing enzyme on the biosynthetic route from farnesyldiphophate to the basic STL costunolide. It was also possible for the first time to produce mg amounts per liter of de novo synthesized costunolide by heterologeously expressing this biosynthetic pathway using engineered microorganisms. Using one of the heterologously expressed sesquiterpene synthase proteins as antigene, the first antibody proving specificity for sequiterpene synthases was created. This antibody was used as a tool to trace the subcellular localization of germacrene A synthases directly in the cells of sunflower glandular trichomes by means of transmission electrone microscopy (TEM). Immunogold-labelling unravelled the occurrence of the enzyme along the smooth endoplasmatic reticulum in the secretory cells and the upper stalk cells of the biseriate, multicellular trichomes. This suggests a close proximity to the identified membrane anchored P450 enzymes and could be indicative for product channelling in a multienzyme complex.
Publications
- (2007) Genomic, proteomic and metabolomic studies on terpenoid and flavonoid biosynthesis in sunflower glandular trichomes. Botanikertagung, Hamburg, September 2007
Göpfert, J.C. and O. Spring
- "Molekulare und entwicklungsbiologische Charakterisierung von Schlüsselenzymen der Naturstoffbiosynthese in Drüsenhaaren der Sonnenblume". Dissertation, Universität Hohenheim 2008
Jens Göpfert
- (2008). Aspects of flavonoid and sesquiterpene synthesis in sunflower glandular trichomes. PSE Young Scientists` Meeting „Future Trends in Phytochemistry: Compounds - Enzymes - Genes“, Bad Herrenalb, März 2008
Göpfert, J.C. und O. Spring
- (2009). Characterization of a key P450 enzyme at the entry point of sesquiterpene lactone biosynthesis in Asteraceae. Terpnet 2009, Tokio, Japan, Mai 2009
Ro, D.-K., Nguyen, D., Göpfert, J.C. und Ikezawa, N.
- (2009). Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes. BMC Plant Biology 9: 86
Göpfert, J.C., MacNevin, G., Ro, D.-K. and Spring, O.
(See online at https://doi.org/10.1186/1471-2229-9-86) - (2009). Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes. Terpnet 2009, Tokio, Japan, Mai 2009
Göpfert, J.C., Ro, D.-K. and O. Spring
- (2009). Metabolomic studies on glandular trichomes, the biofactory of Asteracean sesquiterpenoids. Proc. 2nd Brazilian Conference on Natural Products, Sao Pedro, Brazil
Spring, O., and J.C. Göpfert
- (2010). Biochemical conservation and evolution of germacrene A oxidase in Asteraceae. ASPB Meeting "Plant Biology 2010", Montreal, Minisymposium 19
Nguyen, D.T., Göpfert J.C., Ikezawa, N., Spring, O. and D.-K. Ro
- (2010). Biochemical conservation and evolution of germacrene A oxidase in Asteraceae. Journal of Biological Chemistry 285 (22): 16588 - 16598
Nguyen, D.T., Göpfert, J.C., Ikezawa, N., MacNevin, G., Kathiresan, M., Conrad, J., Spring, O., Ro, D.- K.
- (2010). Identification and Functional Characterization of a new Sunflower Germacrene A Synthase (HaGAS3). National Product Communication 5 (5): 709 - 715
Göpfert, J.C., Bülow, A.-K., Spring, O.
- (2011). Chemical diversity and evolution written in the genomes of sunflower family. Terpnet 2011, Proc. of the 10th Intern. Meeting, Kalmar, Schweden; p. 19
Ro, D.-K., Nguyen, D.T., Ikezawa, N., Göpfert, J.C. and Spring, O.
- (2011). Cytological and immunological studies on STL- secreting glandular trichomes of sunflower. Terpnet 2011, Proc. of the 10th Intern. Meeting, Kalmar, Schweden; p. 103
Spring, O., Meyfarth E., Göpfert, J. and A. Heller
- (2011). Lettuce costunolide synthase (CYP71BL2) and its homolog (CYP71BL1) from sunflower catalyze distinct regio- and stereoselective hydroxylations in sesquiterpene lactone metabolism. Journal of Biological Chemistry, 286 (24): 21601 - 21611
Ikezawa, N., Göpfert, J.C., Nguyen, D.T., Kim, S.-U., O'Maille, P.E., Spring, O., Ro, D.K.
- (2011). The first molecular identification of cytochrome P450 responsible for the synthesis of the simplest sesquiterpene lactone, costunolide. Terpnet 2011, Proc. of the 10th Intern. Meeting, Kalmar, Schweden; p. 105
Ikezawa, N., Göpfert, C., Nguyen, D.T. and D.-K. Ro
