Terpenoids are a structurally diverse family of natural compounds with over 50,000 known members that are produced in all lineages of life. Many of these compounds are known to play essential roles maintaining cell physiology, such as respiration (ubiquinone) and photosynthesis (chlorophylls and carotenoids), in addition to important functions as carbohydrate carriers (dolichol), hormones (steroids and plant hormones), pheromones, and phytoalexins. Because of these diverse functions, it is not surprising that terpenoids have found many applications in human health as medicines and neutraceuticals as well as other uses as pesticides, fragrances, and flavors. For example, terpenoids include the WHO-recommended anti-malarial agent, artemisinin and the potent anticancer drug, taxol. However, since these compounds are naturally produced in small quantities as secondary metabolites, isolation from biological sources can be costly and inefficient. Moreover, because of the structural complexity of these molecules, the chemical syntheses of terpenoids are also difficult to scale for industrial drug production. Thus, we have taken an alternative approach, using a synthetic bacterial system to prepare large quantities of these molecules at lower cost.

Terpenoids are built from the C₅ isoprene units, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (Figure 1). These universal precursors can be synthesized from two distinct pathways, the mevalonate pathway and the more recently discovered non-mevalonate pathway. In the next step, IPPs are then condensed sequentially to the DMAPP primer, yielding linear the diphosphate intermediates geranyl diphosphate (C10, GPP, monoterpenoid precursor), farnesyl diphosphate (C15, FPP, sesquiterpenoid and steroid precursor), and geranylgeranyl diphosphate (C20, GGPP, diterpenois and carotenoid precursor). This consecutive condensation can further continues to form longer prenyl chains to that vary in size from triterpenoids to natural rubber. Each of these prenyl diphosphate precursors is then cyclized by different classes of terpene synthases to yield diverse terpene olefin backbones.

Currently, the use of bacterial system for mass-production of terpene olefin backbones is limited by the availability and the quality of enzymes capable of catalyzing desired chemical reactions. However, the number of known terpene synthases to date falls short of covering all possible cyclization reactions. In addition, many terpene synthases derived from plants are poorly expressed in E.coli. Thus, redesigning functions of existing enzymes using protein engineering strategies is becoming increasingly more attractive solutions. Redesigning reaction selectivity of FPP synthase and several sesquiterpene synthases will be discussed in the following chapters.