Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves.

Cytosolic lipid droplets are endoplasmic reticulum-derived organelles typically found in seeds as reservoirs for physiological energy and carbon to fuel germination. Here, we report synthetic biology approaches to co-produce high-value sesqui- or diterpenoids together with lipid droplets in plant leaves. The formation of cytosolic lipid droplets is enhanced in the transient Nicotiana benthamiana system through ectopic production of WRINKLED1, a key regulator of plastid fatty acid biosynthesis, and a microalgal lipid droplet surface protein. Engineering of the pathways providing the universal C5-building blocks for terpenoids and installation of terpenoid biosynthetic pathways through direction of the enzymes to native and non-native compartments boost the production of target terpenoids. We show that anchoring of distinct biosynthetic steps onto the surface of lipid droplets leads to efficient production of terpenoid scaffolds and functionalized terpenoids. The co-produced lipid droplets "trap" the terpenoids in the cells.