Metabolic Engineering of Nicotiana benthamiana to Produce Cannabinoid Precursors and Their Analogues

Abstract

In recent years, the perspective towards the use of cannabis has slowly shifted from being an illicit drug to a medicinal plant. The pathway and enzymes involved in the production of cannabinoids are known; however, studies evaluating the production of cannabinoids in heterologous plants and cell cultures are still limited. In this study, we assessed the potential use of N. benthamiana (Nicotiana benthamiana) plants as a heterologous host for producing natural and novel cannabinoids. Transgenic N. benthamiana plants expressing genes encoding cannabis acyl-activating enzyme and olivetol synthase were generated, which were then used for transiently expressing other downstream pathway genes. Production of olivetolic acid and divarinic acid, the universal precursors for major and minor cannabinoids, respectively, was observed in transgenic N. benthamiana plants. To produce novel cannabinoid precursors with different side chains, various fatty acids were infiltrated into the transgenic N. benthamiana plants and the production of novel derivatives was observed. Although we were not able to derive the core intermediate, cannabigerolic acid, from our transgenic plants, possibly due to the low production levels of the precursors, our transgenics plants still serve as a high-potential platform for further development and exploring the N. benthamiana chemical space for generating novel cannabinoids.