Engineering S. Cerevisiae for Production of Euphorbiaceae Diterpenes

The Euphorbiaceae family of flowering plants produces a class of unique diterpenoids with interesting core structures. Among these compounds are molecules with anticancer properties and activity against latent HIV reservoirs. While the biosynthesis of these diterpenoids has yet to be elucidated, they are believed to be derived from a precursor, casbene, which is common to many species within the Euphorbiaceae. In order to identify novel genes involved in the biosynthesis of these diterpenoids, we assembled a de novo transcriptome for Euphorbia esula with representation from various core tissues. By studying gene expression under various conditions, co-expression analysis can be used to identify genes that are coordinately expressed with casbene synthase, and likely a part of the biosynthetic pathway. We also sought to engineer S. cerevisiae for high casbene production in order to aid in the functional validation of genes responsible for downstream transformations in plants. This was accomplished by overexpressing genes in the mevalonate pathway in S. cerevisiae, and incorporating heterologous enzymes for increasing cellular GGPP pools. In addition, a combinatorial strain- crossing approach was used to optimize pathway balance.