Characterization of the Eucalyptus grandis Terpenome Using Engineered Yeast Strains

Sequenced in 2014, Eucalyptus grandis has more terpene synthases than any other plant studied to date. The essential oil of E. grandis is highly complex, making it difficult to purify and study the terpene constituents that may be useful as fuels, chemicals, and pharmaceuticals. To simplify the search for novel terpenes and to connect them with their corresponding synthases in E. grandis, we have engineered S. cerevisiae to be used as a chassis for terpene synthase expression. Each strain contains an engineered mevalonate pathway, including the expression of heterologous genes to circumvent the native feedback regulation at HMG1 and overexpression of the precursor prenyl diphosphate synthase. The remainder of the ergosterol pathway is downregulated by the introduction of a copper repressible promoter, diverting flux towards terpene production. In collaboration with the Joint Genome Institute, the 2014 E. grandis genome was scanned with stringent filters for terpene synthases, finding 73 terpene synthases and 25 of the triterpene-producing oxidosqualene cyclases. In total 98 enzymes were synthesized and are being expressed and characterized in the yeast strains engineered for high terpene flux.