Metabolic Engineering of Tobacco for the Production of the Anti-Cancer Drug Etoposide

Etoposide is a potent topoisomerase inhibitor used clinically to treat a variety of cancers including lung cancer, testicular cancer, and lymphoma. Currently etoposide is produced semi-synthetically from podophyllotoxin, a natural product that must be extracted from the rhizome of the endangered Himalayan mayapple plant (Sinopodophyllum hexandrum). However, this process is expensive, unsustainable, and yields very low quantities of the drug, resulting in the global supply of etoposide being both unreliable and insufficient to satisfy demand. In previously reported work, we utilized Agrobacterium tumefaciens-mediated T-DNA transfer to transiently express the minimal set of mayapple enzymes necessary to produce the etoposide aglycone—a more direct precursor to etoposide than podophyllotoxin—in tobacco (Nicotiana benthamiana). Here, we first identify flux of coniferyl alcohol, a metabolite found naturally in all vascular plants, as a limiting factor for generating high yields of the desired product through this tobacco system. We then demonstrate that by increasing the amount of available coniferyl alcohol via expression of an additional set of eight enzymes involved in phenylpropanoid and monolignol biosynthesis, we can achieve a 250-fold increase in yield compared to expression of the minimal set alone. This engineered system is thus a promising solution to the problem of etoposide supply reliability, and we also hope our work will facilitate advancements in the use of tobacco as a platform organism for metabolic engineering in the future.