Plants have become a promising platform for sustainable bioproduction of an array of natural products and specialty chemicals. Of particular interest are terpenes and the functionalized terpenoids, which represent the largest and most diverse class of natural products. These natural products are commonly used commercially as major constituents of flavorings and fragrances, oils, pigments, and pharmaceuticals, while having many other applications. Given the diversity and structural complexity of many terpenoids, they are often expensive and difficult, if not impossible, to chemically synthesize. Engineering these biosynthetic pathways in plant hosts may provide a sustainable platform to access terpenoids for industrial production. While plants offer a sustainable production platform, metabolic engineering for chemical production has largely focused on microbial hosts, and further development of strategies and tools for plant engineering is needed. In my dissertation, I have taken multi-pronged approaches to further develop sustainable bioproduction of terpenoids in plants. First, I developed strategies to optimize, re-target, and compartmentalize production of squalene, a C30 triterpene, within plant cells to improve yields in plants. Re-targeting the final steps in squalene production, farnesyl diphosphate synthase (FDPS) and squalene synthase (SQS), from the cytosol to plastids enabled compartmentalization of biosynthesis away from competing cytosolic enzymes. I then anchored an optimized FDPS and SQS pair to the surface of cytosolic lipid droplets through fusions to the Nannochloropsis oceanica Lipid Droplet Surface Protein (NoLDSP), where squalene can be sequestered and stored. Scaffolding the pathway to the surface of lipid droplets increased yields to more than twice that of plastidial targeting. Re-targeting this lipid droplet scaffolding to plastids, produced similar squalene yields as the soluble, plastid targeted pathway, and ameliorated some of the negative effects on photosynthesis. Second, I worked to engineer poplar, a bioenergy crop which emits large amounts of the hemiterpene isoprene, with these pathways as a platform for bioproduction and adding value to a bioenergy pipeline. Transformants were successfully created for plastid targeted squalene production, producing up to 0.63 mg/gFW of squalene. The lipid droplet scaffolding strategies appeared toxic during tissue regeneration, suggesting a need for tissue specific engineering of these pathways in future iterations. Third, I developed a pipeline to identify, characterize, and engineer bidirectional promoters (BDPs), which enable divergent expression of two genes and improve gene stacking in plant constructs. As seen above with poplar, plant engineering is often limited by construct size, diverse promoter availability, and expression regulation, and a BDP library enables a range of expression in more compact constructs. I identified 34 BDPs from Populus trichocarpa and Arabidopsis thaliana, characterized their activity via Nicotiana benthamiana transient expression, and engineered select BDPs to further alter activities. Combining these BDPs with previously developed terminator sequences provided further regulation of expression. These genetic tools provide an array of expression activities and enable greater gene stacking options while offering the potential for more fine tuning of expression for multiple genes in a metabolic pathway. The work performed in this dissertation provide strategies to improve production of terpenoids in plants, establish production hosts, and engineer larger, complex pathways.
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