Abstract
The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Here we report the first discovery of an epistatic network of residues that controls the onset of terpene cyclization in Artemisia annua. We begin with amorpha-4,11-diene synthase (ADS) and (E)-β-farnesene synthase (BFS), a pair of terpene synthases that produce cyclic or linear terpenes, respectively. A library of ~27,000 enzymes is generated by breeding combinations of natural amino-acid substitutions from the cyclic into the linear producer. We discover one dominant mutation is sufficient to activate cyclization, and together with two additional residues comprise a network of strongly epistatic interactions that activate, suppress or reactivate cyclization. Remarkably, this epistatic network of equivalent residues also controls cyclization in a BFS homologue from Citrus junos. Fitness landscape analysis of mutational trajectories provides quantitative insights into a major epoch in specialized metabolism.
Highlights
The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored
Cyclization is the major gateway to chemical diversity in isoprenoid specialized metabolism; terpene synthases (TPSs) convert a few universal substrates into hundreds of often stereochemically complex mono- and polycyclic hydrocarbons[7], which seed the biosynthesis of thousands of derivatives through downstream metabolic pathways
By contrast, (E)-b-farnesene synthase (BFS) catalyses one of the simplest TPS reactions where the linear carbocation is quenched before cyclization can happen; formally, this involves release of pyrophosphate followed by proton elimination from either a transoid or cisoid farnesyl carbocation to produce the linear hydrocarbon (E)-b-farnesene, an aphid alarm pheromone[13]
Summary
The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Cyclic terpenes mediate essential interactions between organisms, enabling plants to attract pollinators[2] and natural enemies of herbivores[3], repel microbial pathogens[4], and to conduct symbiotic relations[5] They provide a rich source of bioactive compounds for human uses ranging from flavours and fragrances to medicinal compounds such as artemisinin, a naturally occurring terpenoid extracted from Artemisia annua, the most effective treatment for malaria[6]. We calculate the fitness landscapes of A. annua and C. junos to directly measure how the protein background shapes the functional roles and pattern of epistatic interactions among residues in the network. These results provide unique insights into the emergence of cyclization across distinct plant lineages that underlie the evolutionary expansion of specialized metabolism
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