Characterization of Terpene Synthase Family Members

Wednesday, March 09, 2022: 1:35 PM - 2:10 PM

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Description

Cannabis Sativa, a diecious angiosperm, has long history of domestication by humans tracing back to Neolithic times (Ren2021, Tihelka 2021). The plant has been highly sought for its medicinal metabolites and is most well-known for its bioaccumulation of specialized compounds called cannabinoids and terpenoids. Monoterpenes such as Terpinolene, alpha-pinene, and beta myrcene undergo enzymatic synthesis from the precursor Geranyl pyrophosphate (GPP), while sesquiterpenes such as beta caryophyllene, beta farnescene, and alpha bisabolol are synthesized from the precursor farnesyl pyrophosphate (FPP)(Bohlmann 2011). To assess the activity of specific terpene synthase genes among Cannabis varieties in our germplasm, we utilized molecular cloning and functional gene characterization techniques to clone and characterize dominantly expressed terpene synthase genes TPS37 and TPS20 genes from Front Range Biosciences varieties using a bacterial expression system. We performed downstream enzymatic characterization after supplementing the enzyme with FPP/GPP substrate in vitro. Synthesized products were analyzed by selective ion monitoring (SIM) on a Shimadzu TQ8040 to characterize the enzymatic activity. We found that TPS37 produced terpinolene from GPP as a dominant product with several terpenes (alpha-cedrene, alpha-phellandrene, alpha-pinene, alpha-terpinene, alpha-terpineol, beta-myrcene, beta-pinene, E-beta-ocimene, and gamma-terpinene) present in lower quantities. Many of the lower abundance terpenes synthesized by TPS37 may not have been detected without selective ion monitoring, highlighting the need for more sensitive detection methodologies in characterizing terpene synthases. We also found that TPS20 produced alpha/beta farnescene and valencene in similar quantities from FPP.

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Track(s)

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  Cannabis/Hemp

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Additional Info

Category: Please choose one (1):
Cannabis/Hemp

Keywords: Please select up to 4 keywords ONLY:
Enzyme Assays,Terpenes,Agriculture