Identification of early quassinoid biosynthesis in the invasive tree of heaven (Ailanthus altissima) confirms evolutionary origin from protolimonoids

Abstract

The tree of heaven, Ailanthus altissima (MILL.) SWINGLE, is a globally invasive plant known to secrete allelopathic metabolites called quassinoids. Quassinoids are highly modified triterpenoids. So far, nothing has been known about the biochemical basis of quassinoid biosynthesis. Here, based on transcriptome and metabolome data of Ailanthus altissima, we present the first three steps of quassinoid biosynthesis, which are catalysed by an oxidosqualene cyclase and two cytochrome P450 monooxygenases, resulting in the formation of the protolimonoid melianol. Strikingly, these steps are identical to the first steps of the biosynthesis of limonoids, structurally different triterpenoids from sister plant families within the same order Sapindales. Our results are therefore not only important to fully understand the biosynthesis of complex triterpenoids in plants, but also confirm the long-standing hypothesis that quassinoids and limonoids share an evolutionary origin. In addition, our transcriptome data for Ailanthus altissima will be beneficial to other researchers investigating the physiology and ecology of this invasive tree.

Post-Cyclase Skeletal Rearrangements in Plant Triterpenoid Biosynthesis by a Pair of Branchpoint Isomerases

“…Many recent examples demonstrate that co-expression analysis is a helpful tool to discover novel biosynthetic genes. [24][25][26][27] During our recent discovery of the genes required for melianol (8) biosynthesis in A. altissima based on de novo transcriptome sequencing, [23] we observed that these genes were highly co-expressed. We therefore expected that further, yet unknown genes involved in triterpenoid biosynthesis may share a similar expression profile.…”

“…We therefore expected that further, yet unknown genes involved in triterpenoid biosynthesis may share a similar expression profile. We therefore searched our A. altissima expression data from previous work [23] for gene candidates co-expressed with the first three genes in the pathway. To facilitate visual analysis of the underlying multidimensional dataset, we employed self-organising map (SOM) analysis (Figure 2), which arranges large numbers of transcripts into clusters based on their expression profile.…”

“…To facilitate visual analysis of the underlying multidimensional dataset, we employed self-organising map (SOM) analysis (Figure 2), which arranges large numbers of transcripts into clusters based on their expression profile. [28] This process successfully grouped the two previously identified cytochrome P450 monooxygenase (CYP450) genes ( AaCYP71CD4 and AaCYP71BQ17 ) [23] into a single cluster with predominant average expression in roots. This cluster was of high quality, i.e.…”

“…[21,22] Protolimonoid biosynthesis requires three steps starting from the common triterpenoid precursor 2,3-oxidosqualene (5). [21,23] First, 2,3oxidosqualene ( 5) is converted by an oxidosqualene synthase (OSC) into tirucalla-7,24-dien-3β-ol (6), followed by multiple oxidations carried out by two cytochrome P450 monooxygenases (CYP450s) which sequentially oxidise 6 to dihydroniloticin (7) and then to the protolimonoid melianol (8), which is considered to be a key intermediate in these metabolic pathways (Figure 1). [21,23] Remarkably, the carbon skeletons of protolimonoids exhibit distinct differences to other Sapindales triterpenoid classes, namely the positioning of a methyl group at either C-14 or C-8, and the presence or absence of a cyclopropane ring.…”

Cytochrome P450 monooxygenase-mediated tailoring of triterpenoids and steroids in plants

“…Limonoids are highly oxidised, modified and truncated triterpenoids; one of the most well-known limonoids is the eponymous compound limonin (18), which contributes to the bitter taste of citrus products (Figure 6C) [104]. The first steps of limonoid biosynthesis were recently explored by functional characterisation in heterologous hosts [29,[105][106][107]. There, CYP enzymes MaCYP71CD2 and MaCYP71BQ5 from Melia azedarach initiate the ring formation on the side chain of the triterpene precursor tirucalla-7,24-dien-3β-ol (19) in a sequential manner.…”