Citronellol is a pleasant-smelling compound produced in rose (Rosa spp.) flowers and in the leaves of many aromatic plants, including pelargoniums (Pelargonium spp.). Although geraniol production has been well studied in several plants, citronellol biosynthesis has been documented only in crab-lipped spider orchid (Caladenia plicata) and its mechanism remains open to question in other species. We therefore profiled 10 pelargonium accessions using RNA-Seq and GC-MS analysis. Three enzymes from the PRISE family were characterized in vitro and subsequently identified as citral reductases (named PhCIRs). Transgenic RNAi lines supported a role for PhCIRs in the biosynthesis of citronellol as well as in the production of mint-scented terpenes. Despite their high amino acid sequence identity, the three enzymes showed contrasting stereoselectivity, either producing mainly (S)-citronellal or a racemate of both (R)- and (S)-citronellal. Using site-directed mutagenesis, we identified a single amino acid substitution as being primarily responsible for the enzyme’s enantioselectivity. Phylogenetic analysis of pelargonium PRISEs revealed three clades and seven groups of orthologs. PRISEs from different groups exhibited differential affinities toward substrates (citral and progesterone) and cofactors (NADH/NADPH), but most were able to reduce both substrates, prompting hypotheses regarding the evolutionary history of PhCIRs. Our results demonstrate that pelargoniums evolved citronellol biosynthesis independently through a three-step pathway involving PRISE homologues and both citral and citronellal as intermediates. In addition, these enzymes control the enantiomeric ratio of citronellol thanks to small alterations of the catalytic site.

Sulfation plays a critical role in the biosynthesis of small molecules, regulatory mechanisms such as hormone signaling, and detoxification processes (phase II enzymes). The sulfation reaction is catalyzed by a broad family of enzymes known as sulfotransferases (SULTs), which have been extensively studied in animals due to their medical importance, but also in plant key processes. Despite the identification of some sulfated metabolites in fungi, the mechanisms underlying fungal sulfation remain largely unknown. To address this knowledge gap, we conducted a comprehensive search of available genomes, resulting in the identification of 174 putative SULT genes in the Ascomycota phylum. Phylogenetic analysis and structural modeling revealed that these SULTs belong to the aryl sulfotransferase family, and they are divided into two potential distinct clusters of PAPS-dependent SULTs within the fungal kingdom. SULT genes from two marine fungi isolated from deep-sea hydrothermal vents, Hortaea werneckii UBOCC-A-208029 (HwSULT) and Aspergillus sydowii UBOCC-A-108050 SULT (AsSULT), were selected as representatives of each cluster. Recombinant proteins were expressed in Escherichia coli and biochemically characterized. HwSULT demonstrated high and versatile activity, while AsSULT appeared more substrate-specific. Here, HwSULT was used to sulfate the mycotoxin zearalenone, enhancing its cytotoxicity toward healthy feline intestinal cells.

Paclitaxel, a natural product produced by yew trees, has become an important drug in the fight against cancer. Although more than 4 decades of research have identified numerous enzymes involved in paclitaxel synthesis, our understanding of this pathway’s architecture remains incomplete, and a few enzymes have yet to be identified. In a new article published in Nature , McClune and colleagues have developed a pipeline of multi-omics approaches to unravel the remaining enzymes performing key steps in the paclitaxel biosynthetic pathway. These findings open up new possibilities for producing this major anticancer drug using innovative biotechnological processes.

Sesquiterpenes are key molecules nurturing the complex and subtle base notes of pelargonium fragrances. Yet, their contribution to the essential oil olfactory profile and their biosynthesis are far from being well understood. We therefore explored sesquiterpene composition in 10 pelargonium accessions and revealed that, although lowly accumulated, their wide diversity participates to the biochemical uniqueness of each fragrance. We further investigated sesquiterpene biosynthesis thanks to a multiomic approach, integrating transcriptomic together with metabolomic data. A phylogenetic analysis of pelargonium TPS-a subfamily first revealed a total of 21 groups of orthology and denoted a strong transcriptional regulation of related enzymes. Seven sesquiterpene synthases were then wisely selected for functional characterization, both in vitro and in planta. Among them, two 6,9-guaidiene synthases were characterized for the first time in plants. Finally, we explored the role of amino acids located in the active site of sesquiterpene synthases in the enzymatic mechanism using site-directed mutagenesis. Altogether, this work highlights the great potential of the multiomic approach to predict TPS functions, but also exposes its limitations, inherent to terpene synthase's plasticity.

Le genre Pelargonium fait partie de la famille des Geraniaceae et réunit plus de 280 espèces, ainsi que de nombreux hybrides et variétés sélectionnés depuis le 18e siècle. Ces accessions regroupent notamment de pélargoniums commercialisés en tant que plantes ornementales (comme les P. x hortorum) mais également des pélargoniums odorants (comme les P. x hybridum cv rosat) qui sont cultivés pour leur huile essentielle (HE). L’HE de P. rosat est stockée dans des structures glandulaires (trichomes glandulaires) présentes sur les feuilles et se compose principalement de mono- et sesqui-terpénoïdes. Ces composés organiques volatils sont à l’origine du parfum « géranium », prisé des parfumeurs pour son profil olfactif complexe et rappelant l’odeur de rose. L’objectif de cette thèse était de mieux comprendre la diversité de terpénoïdes présents dans l’HE de pélargonium, de décrypter les mécanismes sous-jacents à la biosynthèse de ces nombreux composés odorants et plus particulièrement d’analyser les enzymes impliquées dans leur production. Afin de répondre à cet objectif, des études biochimiques et transcriptomiques ont été menées. Celles-ci ont permis de mettre en place une approche multi-omique afin d’étudier le terpénome de dix accessions de pélargoniums odorants, de caractériser structurellement et fonctionnellement des enzymes impliquées dans la biosynthèse de ce terpénome et d’analyser l’effet d’un stress climatique sur celui-ci.