Specialized metabolites constitute a group of diverse chemical compounds that contribute to plant fitness. Trp-derived indolic glucosinolates and phytoalexins are among well-studied specialized metabolites with roles in immunity of the model plant Arabidopsis thaliana and other Brassicaceae species. A. thaliana mutants deficient in particular pathway enzymes helped to reveal specific functions of particular Trp-derived metabolites in pre-invasive and post-invasive phases of fungal infection. However, it is still unclear if specific functions of these compounds are defined by their spatiotemporal accumulation patterns or by their unique biological activities.
Several members of Poaceae family, including the model grass Brachypodium distachyon, have been reported to produce serotonin and its derivatives as Trp-derived metabolites in response to infection. However, their involvement in plant immunity remains ambiguous. To explore these, we have engineered serotonin biosynthetic pathway in A. thaliana mutant unable to produce endogenous Trp-derived metabolites. To address the of impact spatiotemporal accumulation patterns on metabolite function we opted to use A. thaliana native promoters controlling Trp-metabolism during pre- and post-invasive phase of immune responses. To facilitate metabolic engineering, we expressed multiple pathway genes from B. distachyon under a single promoter using self-cleaving 2A viral sequences, and used artificial introns to enhance transgene expression. Metabolomic analysis confirmed the presence of tryptamine and serotonin together with several novel metabolites in transgenic A. thaliana lines. Overall, we have demonstrated the successful application of advanced synthetic biology and metabolic engineering tools in stable engineering of complex multistep biosynthetic pathways under native conditions.