Rapid Combinatorial Coexpression of Biosynthetic Genes by Transient Expression in the Plant Host Nicotiana benthamiana

Metabolic engineering of heterologous hosts requires coexpression of multiple genes, often more than ten for a single pathway. Traditional approaches to create genetic constructs for this purpose are highly inflexible and suffer from very low throughput. In this book chapter, we describe a powerful method to overcome this bottleneck, namely, combinatorial co-expression in the Australian tobacco plant Nicotiana benthamiana. This system is based on Agrobacterium tumefaciens-mediated transient gene expression, often called agroinfiltration. Herein, instead of creating complex multigenic constructs, coexpression is achieved by simply mixing different plasmid-bearing Agrobacterium strains without the need for different selection markers. We present a practical guide for coexpressing multiple biosynthetic genes followed by GC-MS analysis, using production of the plant triterpene β-amyrin as an example. Our chapter provides a guideline to harness the potential of this versatile expression system in the natural product community for studying and engineering metabolic pathways.