Using Agrobacterium tumefaciens to Assemble Multi-step Metabolic Pathways in Nicotiana benthamiana

Within the realm of the natural world, plants emerge as prolific producers of diverse bioactive compounds with pharmaceutical, nutritional, and industrial applications. However, many of these compounds are scarce with low concentrations and specific distributions among species, prompting the exploration of methods for producing them in plant biofactories. Typically, pathways comprising several enzymatic steps need to be engineered in plant hosts to produce the desired product of interest from available metabolic precursors. Transient expression systems, specifically agroinfiltration of Nicotiana benthamiana leaves with Agrobacterium tumefaciens, is a potent and cost-effective method for testing synthetic gene combinations. Here, we present a protocol to produce metabolites through a multi-step pathway, exemplifying the assembly of a carotenoid synthesis pathway within the plant cell cytosol. The approach showcases the efficiency and simplicity of agroinfiltration-mediated transient expression systems in reconstructing metabolic pathways, offering a valuable and sustainable alternative to stably transformed lines.