(530c) Use Saccharomyces Cerevisiae to Functionally Characterize the Dynamic Enzyme Complex from Soybean

Dynamic enzyme complexes are important post-translational regulatory machinery in plant specialized metabolism, which assembles and regulates functionally related enzymes in a pathway by non-covalent weak bound, namely protein-protein interactions (PPIs). Enzyme complexes in plants are essential for regulating various cellular processes. They not only enhance enzyme activities but also alter their specificities and prevent the accumulation of toxic intermediates. Despite the importance of enzyme complexes, the biochemical function characterization of distinct enzyme complexes in diverse plants can be challenging and time-consuming task due to the difficulties in plant genetic engineering. The presence of multiple compounds and metabolites in plant tissues can interfere with the detection of targeted products. Here, we used an efficient microbial host, baker’s yeast (Saccharomyces cerevisiae), to develop orthogonal PPI identification methods and functional characterization methods. Yeast has fast growth speed, massive genetic manipulation tools and relatively simpler metabolic background, which benefit enzyme complexes reconstruction and biochemical function studies. A previously identified enzyme complex centered in the endoplasmic reticulum (ER) membrane-bound isoflavone synthase (IFS) from soybean roots was used to validate the methods we developed. We used multiple methods of PPI identification, including yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and fluorescence co-localization assays, to validate the formation of the complex in yeast. We have demonstrated that the enzyme complex reconstructed in yeast exhibits similar interactions among the enzymes as observed in soybean. We further found the formation of this enzyme complex resulted in a higher flux towards the biosynthesis of daidzein, leading to its increased accumulation. This accumulation of daidzein could potentially be converted to more downstream products upon induction. Taken together, this study highlights the potential of reconstructing and functionally characterizing plant enzyme complexes in the efficient microbial host yeast. Ultimately, the new insights gained from studying these enzyme complexes in yeast can benefit the development of novel bioengineering strategies.