Creation of master transcription factors to regulate plant metabolic pathways

Key steps of biosynthetic pathways of plant secondary metabolites are regulated by multiple transcription factors (TFs). The ability to create single "master" TFs capable of regulating the expression of all genes in a particular pathway would be a powerful tool for metabolic pathway engineering. To test the feasibility, directed evolution technologies were applied to modify the properties of a family of plant bHLH-like TFs involved in flavonoid biosynthesis. Directed evolution utilizes genomic information and incorporates Darwinian principles of mutation and selection into experimental strategies for improving gene and protein functions. We have demonstrated that the transcriptional activity and promoter specificity of the bHLH TFs can be improved through recursive evolution. TF variants with increased transactivational activity and broadened promoter specificity have been generated. Ectopic expression of an evolved bHLH TF in transgenic tobacco resulted in increased anthocyanin accumulation compared to plants expressing the wild-type gene. Analysis of the genetic diversity of the evolved variants reveals the potential cooperation between functional domains of the bHLH TFs. Our study substantiates the potential of utilizing master TFs for regulation of important traits of plants.