Engineered Biosynthesis Of High Value Therapeutics In A Plant Chassis PDF Download

Plants are known to produce a wide array of chemicals, and many plant natural products display medicinal properties beneficial to human health. These compounds have served as an inspiration to modern medicine, and recent technological advances have accelerated discovery and engineering of plant biosynthetic pathways. With the improved understanding of plant chemistry, we engineered biosynthesis of natural products and their analogs for therapeutic development. To demonstrate the value of engineered biosynthesis, we sought to improve production of etoposide aglycone, the complete biosynthetic pathway of which has been fully elucidated and reconstituted in a plant chassis, Nicotiana benthamiana. We first demonstrate milligram-scale production of a late-stage intermediate to etoposide aglycone and its desmethoxy analog through overexpression of primary metabolic enzymes and engineering of secondary metabolism. We then aimed to exploit the native transcriptional regulation for metabolic engineering and show that a single transcription factor can not only improve the pathway flux for etoposide aglycone biosynthesis but also suppress the competing metabolism. Additionally, we proposed to improve etoposide through development of its analogs with an altered activity profile. Previously, a hepatic metabolite of the chemotherapeutic etoposide has been implicated with treatment-related leukemogenesis. To address this issue, we hypothesized that etoposide analogs resistant to hepatic metabolism could be biosynthetically prepared. The first step of the pathway, coupling of coniferyl alcohol, is stereoselectivity-guided by a plant dirigent protein. We show that the plant dirigent protein demonstrates expanded substrate versatility, promoting asymmetric heterocoupling of coniferyl alcohol and its analog. Furthermore, we show that the heterocoupled (+)-pinoresinol analogs can be further processed by the rest of the pathway enzymes and display reduced propensity for oxidation. Taken together, we showcase flexibility of heterologous biosynthetic pathways in a plant chassis for improved biosynthetic yields of high-value therapeutic compounds and expanded chemical space of therapeutic analogs previously inaccessible by traditional synthetic approaches.