De novo genetic engineering of the camalexin biosynthetic pathway

•Elucidation of in planta biosynthesis of camalexin through synthetic biology.•Biochemical evidence for CYP71A12 conducting the same reaction as CYP71A13 in the camalexin pathway.•Engineering plant defense as a tool for screening yet unidentified candidate genes.•De novo engineering of the camalexin pathway in Nicotiana benthamiana.•Transient expression in N. benthamiana as a powerful in planta system for the assessment of gene function.

Camalexin is a tryptophan-derived phytoalexin that is induced in the model plant Arabidopsis thaliana upon pathogen attack. Only few genes in the biosynthetic pathway of camalexin remain unidentified, however, investigation of candidate genes for these steps has proven particularly difficult partly because of redundancy in the genome of Arabidopsis. Here we describe metabolic engineering of the camalexin biosynthetic pathway in the transient Nicotiana benthamiana expression system. Camalexin accumulated in levels corresponding to what is seen in induced Arabidopsis thaliana. We have used this system to evaluate candidate genes suggested to be involved in the camalexin pathway. This has provided biochemical evidence for CYP71A12 conducting same reaction as CYP71A13 in the pathway. We discuss the prospects of using metabolic engineering of camalexin, both with respect to engineering plant defense and as a tool for screening yet unidentified candidate genes in the camalexin pathway.