Cargando…

Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus

Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C(20) precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-gui...

Descripción completa

Detalles Bibliográficos
Autores principales: Czechowski, Tomasz, Forestier, Edith, Swamidatta, Sandesh H., Gilday, Alison D., Cording, Amy, Larson, Tony R., Harvey, David, Li, Yi, He, Zhesi, King, Andrew J., Brown, Geoffrey D., Graham, Ian A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9173813/
https://www.ncbi.nlm.nih.gov/pubmed/35584121
http://dx.doi.org/10.1073/pnas.2203890119
Descripción
Sumario:Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C(20) precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-guided transcriptomic approach and a genomics approach that led to the discovery of two casbene-derived diterpenoid gene clusters yielded a total of 68 candidate genes that were transiently expressed in Nicotiana benthamiana for activity toward jolkinol C and other lathyranes. We report two short-chain dehydrogenases/reductases (SDRs), identified by RNA sequencing to be highly expressed in Euphorbia peplus latex. One of these, EpSDR-5, is a C3-ketoreductase, converting jolkinol C to the lathyrane jolkinol E. Gene function of EpSDR-5 was further confirmed by heterologous expression in Saccharomyces cerevisiae. To investigate the in vivo role of EpSDR-5, we established virus-induced gene silencing (VIGS) in E. peplus, resulting in a significant reduction in jatrophanes and a corresponding increase in ingenanes. VIGS of Casbene Synthase results in a major reduction in both jatrophanes and ingenanes, the two most abundant classes of E. peplus diterpenoids. VIGS of CYP71D365 had a similar effect, consistent with the previously determined role of this gene in the pathway to jolkinol C. These results point to jolkinol C being a branch point intermediate in the pathways to ingenanes and jatrophanes with EpSDR-5 responsible for the first step from jolkinol C to jatrophane production.