| Numerous plant-derived terpenoid compounds have been found to be of commercial, pharmaceutical and agricultural value, and several of these important biochemical pathways have been elucidated and many of the enzymatic reactions have been fully characterized. The demand for bioactive terpenoid compounds, such as the potent diterpenoid chemotherapeutic agent paclitaxel, several monoterpene flavor and fragrance compounds and diterpenoid resin acid synthesis intermediates, has led to the investigation of alternate means of economically producing these often complex molecules in larger quantities than are naturally available. With the current knowledge of terpenoid biochemistry, and through application of molecular tools, we can now look towards bioengineering plant metabolism for the improved production of these compounds in increased yields via enzyme modification and genetic engineering of plant and microbial hosts.; It is now feasible to engineer terpenoid biosynthetic catalyst by applying several techniques, including X-ray structural studies, molecular modeling, site-directed mutagenesis and directed molecular evolution. The bifunctional diterpene cyclase, abietadiene synthase, which catalyzes two distinct reactions in two separate active sites, was used as a model for defining structure-function relationships of a terpenoid synthase that conducts two fundamental cyclization types. In these studies in which the enzyme was split into two separate functional domains, we demonstrated that catalysis at both sites is dependent upon interdomain contacts. In another approach, the redirection of pathway precursor flux toward monoterpene biosynthesis in a heterologous microbial host was investigated. For a model system, the (−)-carvone biosynthetic pathway from spearmint (Mentha spicata) was installed in Escherichia coli because this pathway represents a simple, yet representative four-step metabolic sequence in which all of the major reactions of terpenoid biosynthesis occur, i.e. prenyltransfer, cyclization, hydroxylation and oxidation. This work established the basic parameters for the functional assemblage of a multi-step pathway, precursor supply, transport processes and the biochemical and physiological limitations of microbial hosts for terpenoid production.; The future goals of bioengineering natural products formation include the successful manipulation of terpenoid metabolism through designing more efficient catalysts and by gaining a better understanding of the limitations of foreign hosts as production platforms. The present studies address some limitations in the redesign of critical terpenoid cyclization enzymes and in the transplantation of terpenoid biosynthetic pathways into alternate host, and present some examples of approaches which can be taken to address these challenges. |
