Abstract
Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes approximately 8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.
Highlights
Council of Canada Discovery Grant, the Canada Research Chair program, and the Canada Foundation for Innovation
The chemical diversification of sesquiterpene lactone (STL) is interlocked with plant speciation in Asteraceae, and the structural features of STLs have been routinely used as chemosystematic markers in Asteraceae (10 –12)
Isolation of germacrene A oxidase (GAO) and Pathway Reconstitution in Engineered Yeast—Lettuce (L. sativa) accumulates STLs inside its laticiferous cells in stems and leaves [30], and we first aimed at functionally identifying a gene encoding the enzyme for germacrene A oxidation from lettuce
Summary
Fractions showing blue spots on TLC were further monitored by HPLC (Waters 279 Separation Module; Waters Nova-Pak C18 column 4 m, 3.9 ϫ 150 mm; Waters 2996 Photodiode Array Detector with UV wavelength at 194 nm) to distinguish germacrene A acid (13.3 min retention time) from minor amounts of ilicic acid (9.3 min) and costic acids (14.5 min) This separation was achieved with a solvent gradient of 95:5 (A:B) to 20:80 (A:B) over 20 min at 1 ml minϪ1 and 40 °C column temperature (A: H2O with 1% acetic acid; B: 100% acetonitrile). LC-MS Analyses of Sesquiterpenoids—Metabolite mass profiles were generated by an Agilent 1200 Rapid Resolution LC system coupled with an Agilent 6410 MS using 10-l injections of samples onto a reverse phase C18 column (2.1 ϫ 50 mm, 1.8 m, Eclipse plus C18 Zorbax) with a solvent gradient of 80:20 (A:B) to 20:80 (A:B) over 12 min at 0.4 ml minϪ1 at 40 °C column temperature (A: H2O with 1% acetic acid; B: 100% acetonitrile). Sequence Deposition—The DNA sequences for the GAO genes have been deposited in the GenBankTM data library under the following accession numbers: GU198171 (L. sativa), GU256644 (C. intybus), GU256645 (S. lappa), GU256646 (Helianthus annuus), and GU256647 (B. spinosa)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
