Multi-Gene Transformation for Pathway Engineering of Secondary Metabolites

Genetic manipulation using multi-gene transformation is an essential technique for pathway engineering of secondary metabolites. We performed triterpene pathway engineering for the production of glycyrrhizin in Glycine max (soybean), which does not normally accumulate glycyrrhizin, using the multi-gene transformation technique. We constructed a vector plasmid harboringthreekeyP450genes, combining down-regulation of endogenous soybean β-amyrin 24-hydroxylase (CYP93E1) and overexpression of licorice β-amyrin 11-hydroxylase (CYP88D6) and β-amyrin 30-hydroxylase (CYP72A154) as heterologous genes. The three key gene cassettes, in which each gene was flanked by the Cauliflower Mosaic Virus (CaMV) 35S Promoter and dual terminators, the nopaline synthase (NOS) and Arabidopsis heat shock protein 18.2 (HSP) terminators, were connected in tandem, and the resulting plasmid constructs used for particle bombardment of soybean. This is a useful technique for creating simple plasmid constructs without requiring time-consuming steps. We describe herein the advanced protocol for plasmid construction used for multi-gene transformation and subsequent metabolite profiling of the transformed plants.