Total Biosynthesis for Milligram-Scale Production of Etoposide Inter-mediates in a Plant Chassis.

Etoposide is a plant-derived drug used clinically to treat several forms of cancer. Recent shortages of etoposide demonstrate the need for a more depend-able production method to replace the semi-synthetic method currently in place, which relies on extraction of a precursor natural product from Himalayan mayapple. Here we report mg-scale production of (-)-deoxypodophyllotoxin, a late stage biosynthetic precursor of the etoposide agly-cone, using an engineered biosynthetic pathway in tobacco. Our strategy relies on engineering the supply of coniferyl alcohol, an endogenous tobac-co metabolite and monolignol precursor to the etoposide aglycone. We show that transient expres-sion of 16 genes, encoding both coniferyl alcohol and main etoposide aglycone pathway enzymes from mayapple, in tobacco leaves results in the ac-cumulation of up to 4.3 mg/g dry plant weight (-)-deoxypodophyllotoxin, and enables isolation of high-purity (-)-deoxypodophyllotoxin after chro-matography at levels up to 0.71 mg/g dry plant weight. Our work reveals that long (>10 step) pathways can be efficiently transferred from diffi-cult-to-cultivate medicinal plants to a tobacco plant production chassis, and demonstrates mg-scale total biosynthesis for access to valuable precursors of the chemotherapeutic etoposide.