Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Abstract

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ϳ8% 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.

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

“…premnaspirodiene) and eremophilanes (5-epi-aristolochene, epi-eremophilene and (+)-valencene). In the recently published work by Nguyen et al [12], the chicory germacrene A oxidase is shown to selectively oxidise at the C12 position of both germacrene A and amorpha-4,11-diene. Thus it would seem that two categories of sesquiterpene modifying P450 enzymes can be distinguished in the plant kingdom: those that act on the A-ring, such as the H. muticus premnaspirodiene oxygenase, and those that act on the allylic C12 position, such as the chicory germacrene A oxidase and the A. annua amorpha-4,11-diene oxidase.…”

“…Two sequences with high similarity to both genes were identified, one of which encoded the recently described C. intybus germacrene A oxidase [12]. The second sequence encodes a novel protein CYP71AV8 with 50% sequence identity to premnaspirodiene oxygenase, 78% to amorpha-4,11-diene oxidase and 81% to the germacrene A oxidase from C. intybus.…”

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…The authors hypothesized that the biosynthesis of commonly occurring germacrene A acid, a transient intermediate in the routes to highly diverse sesquiterpene lactones, represents the evolutionary origin for the biosynthesis of artemisininc acid. Several members of the CYP71AV family from divergent Asteraceae species have consistently been functionally characterized as germacrene A oxidases, but interestingly also accept the non-natural substrate amorphadiene, whereas Artemisia CYP71AV1 displayed no significant activity towards germacrene A [41]. Hence, CYP71AV1 represents an example of biochemical neofunctionalization.…”

“…Recent work in the Asteraceae family has yielded highly illustrative insights into the evolution of sesquiterpenoid chemical diversity. Fuelled by the finding that Artemisia annua CYP71AV1 catalyses a three-step oxidation of amorphadiene to artemisinic acid [39,40], a detailed analysis of evolutionarily conserved sesquiterpene lactone biosynthetic routes was performed across a range of Asteraceae [41]. The authors hypothesized that the biosynthesis of commonly occurring germacrene A acid, a transient intermediate in the routes to highly diverse sesquiterpene lactones, represents the evolutionary origin for the biosynthesis of artemisininc acid.…”

Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

“…The amplicon was inserted into NotI and SpeI restriction sites of pESC-CPR in-frame with a FLAG-tag sequence yielding pESC-CPR/CFS. A synthetic PsSPS (CYP719A20) gene (GenBank KF481962), codon-optimized for expression in S. cerevisiae and containing a sequence encoding the N-terminal membrane-spanning domain from the Lactuca sativa (lettuce) germacrene A oxidase (43 amino acids) 58 replacing the corresponding native domain (29 amino acids) to increase protein stability 59 , was inserted into the NotI and SpeI restriction sites of pESC-CPR 25 in-frame with a FLAG-tag sequence yielding pESC-CPR/ SPSDN. For heterologous gene expression, S. cerevisiae strain YPH499, harbouring the plasmid of interest, was grown in yeast nitrogen broth, synthetic dropout supplement lacking leucine, 0.2% D-glucose, 1.8% D-galactose and 1% D-raffinose for 48 h at 30°C 25 .…”