Compartmentalizing Terpenoid Bioproduction in Plants with Engineered Plastid Pathways and Lipid Droplet Scaffolds

Leveraging synthetic biology approaches, engineered plants offer a sustainable production platform for high-value chemicals and other bioproducts. Squalene, a C30 hydrocarbon, is a biofuel candidate and the precursor to high-value triterpenoids, a diverse class of natural products with applications in the health, cosmetic, and other biotechnological industries. In this work, two strategies have been developed to increase plant production yields of squalene and triterpenoids by hijacking existing cell compartments or building novel compartments to sequester products within cells. Natively, squalene biosynthesis occurs in the cytosol through farnesyl diphosphate synthase (FDPS) and squalene synthase (SQS). The first strategy re-localizes this pathway to plastids, natural intracellular compartments, where biosynthesis can occur separate from native competing enzymes. In plastids, squalene yields were optimized through screening of diverse orthologs and engineered variants of key steps in the pathway. The second strategy re-engineers cytosolic lipid droplets as synthetic storage organelles with biosynthetic enzymes anchored to the surface, synthesizing and storing products in the same location. Fusing FDPS and SQS to the algal Lipid Droplet Surface Protein (LDSP) enables enzyme scaffolding on the surface of lipid droplets, which sequester the hydrophobic products. Preliminary work also demonstrates targeting the SQS-LDSP-FDPS fusion protein to plastids mediates negative effects on photosynthesis, possibly through novel scaffolding and further squalene sequestration. These strategies effectively increased squalene yields using transient expression in N. benthamiana and are being implemented in stable poplar transformants, a target production crop. Effective compartment engineering improves plant production of squalene, while also providing platforms to expand towards higher-value triterpenoids and other bioproducts.