Metabolic engineering of natural product synthesis

Title: Transfer of the biosynthetic pathway for the cyanogenic glucoside dhurrin from Sorghum bicolor to Arabidopsis using gene technology

Danish title: Overførsel af syntesevejen for det cyanogene glucosid dhurrin fra sorghum til Arabidopsis ved hjælp af gensplejsning

 

Arabidopsis is the preferred model plant for molecular genetics. Accordingly, we decided to transfer the entire pathway for cyanogenic glucoside synthesis from sorghum to Arabidopsis using genetic engineering. This would enable a study the effect of introduction of a cyanogenic glucoside into a plant species that in nature would not be cyanogenic.
Focused and non-targeted approaches were used to assess the impact associated with the introduction of the new high flux pathway for cyanogenic glucoside synthesis in Arabidopsis thaliana. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleiotrophic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific UV protectants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. The accumulation of new glucosides in the plants expressing CYP79A1 and CYP71E1, was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B1. These results demonstrate that insertion of a high flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome. The presence of dhurrin in the transgenic Arabidopsis plants conferred resistance to the flea beetle Phyllotreta nemorum, which is a natural pest of other members of the crucifer group. Further characterization of the transgenic plants with respect to possible alterations of their primary metabolism and altered interactions and responses to microbes and insects are in progress.

 

Researchers involved: Marc Morant, Charlotte Jørgensen, Søren Bak, Birger Lindberg Møller

 

Foreign collaborators: Alistair Fernie, Max-Planck Institute, Golm, Germany

 

Financial support: Danish National Research Foundation, Director Ib Henriksens Foundation, Marie Curie Training Grant