Reconstitution of Metabolic Pathway in Nicotiana benthamiana

Reconstitution of metabolite biosynthesis pathway plays a pivotal role in functional characterization of biosynthesis enzymes and metabolite bioengineering. Traditionally, metabolic pathways are reconstituted in bacteria or yeast due to their ease for genetic manipulation and transformation. Many plant metabolite pathways involve multiple enzyme complexes channeled on plant endomembrane system, which is absent in bacteria and yeast. Nicotiana benthamiana is particularly suitable for reconstitution plant metabolite pathway involving enzymes associated with plant endomembrane systems. Compared with other plants, N. benthamiana can be easily transiently transformed by multiple genes simultaneously by a procedure called leaf agroinfiltration. The results of transient transformation can be analyzed in several days, compared with several months with other stable transformation procedures. In this chapter, we present a protocol for multiple-gene transformation by agroinfiltration, followed by UPLC MS analysis.