The isoprenoids or terpenoids are a family of compounds comprising no less than 50,000 diverse structures discoveredso far. They are mostly produced by plants and are involved in generalized and specialized metabolism. Terpenoidshave diverse biological roles and have wide applications within the food, pharma and chemical industries. Sourcing ofuseful terpenoids is primarily derived from extraction from natural sources or chemical synthesis. Biosyntheticproduction is gaining significant focus today, as this method reduces environmental impact and pursues more efficientproduction than chemical synthesis can provide. The complex terpenoids are difficult to produce by chemical synthesis;in particular the sesquiterpene lactone thapsigargin produced by the plant Thapsia garganica. Thapsigargin has beenshown to have apoptotic and anti-cancer activity. The opening chapter of this thesis provides an introduction toT.garganica, thapsigargin’s biological activity and usefulness as the bioactive ingredient in the anticancer drugMipsagargin that is currently undergoing clinical trials. Then the terpenes are described with their roles in nature,usefulness, and biosynthesis with a focus on the sesquiterpenes and sesquiterpene lactones.Chapter 2 provides a review of factors that are important for the sesquiterpene synthases that are responsible for the firstbiosynthetic step, including the critical cofactors, substrate preference and peptide motifs that bind cofactors. Studies ofthe active site and residues that are important for the catalytic activity and product specificity are also reviewed.Specificity is determined by specific amino-acid residues in the lining of the active site and more distant residues, and isa result of the interactions of spatial constraints and amino-acid residues in and around the active site of the terpenesynthases. Based on these studies our group devised a strategy to engineer the sesquiterpene synthase TgTPS2, theenzyme responsible for the first step of thapsigargin biosynthesis, toward a less promiscuous product profile thanwildtype, which may potentially find application in a future biosynthetic production platform for thapsigargin.Chapter 3 describes past and recent developments in the pursuit of a sustainable production platform for thapsigargin,including chemical synthesis, the discovery of TgTPS2 as the first biosynthetic step (Publication 2: Pickel et al., 2012),the discovery of the second step performed by a cytochrome P450 that incorporates the lactone ring in the corestructure, and finally a novel method that enables in vitro cultivation of T.garganica plantlets.Lastly, the development of a heterologous, yeast based expression platform for sesquiterpene synthases for the purposeof biochemical characterization is described in Chapter 4. A radioisotopic method was developed for characterizingthese enzymes and has potential for higher throughput, reduction in reagent use and waste production (Publication 3:Manczak and Simonsen, 2016). The method was useful both for purifying high levels of enzyme using yeast and fordetermining enzymatic parameters. An additional enzyme, CaTPS, discovered in C.acutatum, a fungus pathogenic toagricultural crops was also characterized by this method (Publication 4: Amby et al., 2016). The enzymatic parametersobtained were in accordance with previous studies of sesquiterpene synthases, however, there are some improvementsto the method that should be implemented to strengthen the validity. Nevertheless, this is, to our knowledge, the firstreported attempts to determine the enzymatic parameters of a plant sesquiterpene synthase in vitro using yeast asexpression platform.