Diverged Plant Terpene Synthases Reroute the Carbocation Cyclization Path towards the Formation of Unprecedented 6/11/5 and 6/6/7/5 Sesterterpene Scaffolds

Abstract

Sesterterpenoids are a relatively rare class of plant terpenes. Sesterterpene synthase (STS)‐mediated cyclization of the linear C25 isoprenoid precursor geranylfarnesyl diphosphate (GFPP) defines sesterterpene scaffolds. So far only a very limited number of STSs have been characterized. The discovery of three new plant STSs is reported that produce a suite of sesterterpenes with unprecedented 6/11/5 and 6/6/7/5 fused ring systems when transiently co‐expressed with a GFPP synthase in Nicotiana benthamiana. Structural elucidation, feeding experiments, and quantum chemical calculations suggest that these STSs catalyze an unusual cyclization path involving reprotonation, intramolecular 1,6 proton transfer, and concerted but asynchronous bicyclization events. The cyclization is diverted from those catalyzed by the characterized plant STSs by forming unified 15/5 bicyclic sesterterpene intermediates. Mutagenesis further revealed a conserved amino acid residue implicated in reprotonation.

Engineering terpenoid production through transient expression in Nicotiana benthamiana

“…Furthermore, the yields of the end products are still in the microgram per litre range in many cases, thus highlighting the need to continue to explore other host systems. The numbers of studies demonstrating the purification of compounds derived from transient expression in plants have increased in recent years, reflecting the increasing popularity of this system (Andersen-Ranberg et al 2016 ; Dickschat et al 2017 ; Geisler et al 2013 ; Huang et al 2017a , b ; Reed et al 2017 ). As described above, transient expression can be achieved at industrial scale, and is currently used for production of pharmaceutical proteins by various companies (Sack et al 2015 ).…”

“…It is clear that N. benthamiana shows a great deal of promise for the production of terpenes and there are a growing number of examples, where this host has been used to obtain isolated yields of the target compounds in milligram to gram quantities (Tables 1 , 2 ) (Andersen-Ranberg et al 2016 ; Huang et al 2017a , b ; Reed et al 2017 ). At the same time, one of the drawbacks to achieving this level of production is the matter of scale.…”

“…1 a/b) is sufficient for most analytical work, translating this approach to infiltrate the volume of plants required for milligram-to-gram quantities of the target compound is laborious. To address this challenge, we and others have utilised vacuum infiltration of N. benthamiana (Andersen-Ranberg et al 2016 ; Huang et al 2017a , b ; Reed et al 2017 ). This process involves inverting the aerial parts of N. benthamiana plants into a bath of A. tumefaciens and using a vacuum to draw the air from the interstitial spaces between the leaf cells (Reed et al 2017 ).…”

Plant terpenes that mediate below‐ground interactions: prospects for bioengineering terpenoids for plant protection

“…94 We recently demonstrated the capacity of engineered N. benthamiana for producing gram-scale amounts of terpenoids. [94][95][96][97] By engineering upstream terpene pathway enzymes (e.g. a feedback-insensitive HMG CoA reductase) and TPSs into a hyper-translation expression vector (pEAQ) together with a custom-designed large-scale vacuum infiltration device, rapid high-level production of various terpenoids was achieved.…”

A Clade II‐D Fungal Chimeric Diterpene Synthase from Colletotrichum gloeosporioides Produces Dolasta‐1(15),8‐diene

“…Neutral biosynthetic intermediates in terpene cyclisations have frequently been described, for example, recently for several plant sesterterpene synthases, but do not always leak from the active site. They can sometimes be obtained from enzyme variants with exchange of a single amino acid residue involved in the reprotonation step .…”