Isoflavonoids are phenolic secondary metabolites found almost exclusively in legumes. These compounds play key roles in many biotic and abiotic stress responses. The ubiquitous phenylpropanoid pathway provides substrates for isoflavonoid production. A set of legumespecific enzymes, including isoflavone synthase and Type II chalcone isomerase, act as a metabolic branch-point by converting flavanones to isoflavones. The production of flavonoids and isoflavonoids are differentially regulated in response to different environmental stresses. Exactly how cells direct the flow of common substrates towards each pathway under specific conditions is not clear. Previous studies demonstrated that key flavonoid and isoflavonoid biosynthetic enzymes form a macromolecular complex or a metabolon that channels metabolites into branches of the pathway. Multiple lines of evidence support that specific enzyme interactions are sometimes the main bottlenecks for metabolic engineering of isoflavone biosynthesis in non-legume plants. Recently, part of the flavonoid and isoflavonoid pathway has been reconstituted successfully in yeast by expressing key metabolic enzymes and supplying various substrates in the culture media. Surprisingly, the flow of flavonoid substrates in yeast was influenced by specific enzyme associations. This in vivo yeast system thus provides us a unique and interesting platform for studying the flux of metabolites and interactions of key enzymes. Using integrated biochemical, computational and cell biology approaches, this project aims to examine the impact of the metabolon on isoflavonoid biosynthesis in yeast and to explore the differential protein-protein interactions during biotic and abiotic stress responses in plants. It is very important to understand the structure and function of these putative multi-enzyme complexes because many other primary and secondary metabolic processes that require concerted action of multiple enzymes might well employ similar regulation mechanisms.
The objective of this project is to quantify the flux of flavonoid and isoflavonoid compounds in each branch of the pathway in transgenic yeasts, and to measure the effects of specific enzyme interactions on metabolite productions. Taking advantage of this unique heterologous system, the project seek to confirm the observed protein-protein interactions with standard in vitro and in vivo assays, and to establish a mathematical model to encompass the effects of gene expression, enzyme kinetics, and protein interactions.
Broader Impacts: The highly collaborative nature of this research project, which crosses between biochemistry, computational modeling, and cell biology, offers an environment for undergraduate students, postdoctoral associates, and visiting scholars to broaden as scientists. The proposed collaboration between faculties of the Danforth Center and the Maryville University is unique and complementary: It opens the up-to-date research experience to the teachers and students of a small liberal art college; at the same time, it enhances the educational and outreach component of the Danforth Center.
