Plant secondary metabolism is structured spatiotemporally. In many plants and biosynthetic pathways, functional related enzymes in the same biosynthetic pathway tend to be associated with each other in cellular compartmentalization to form dynamic enzyme complexes through non-covalent weak bonds, e.g., Protein-Protein Interaction (PPI). The formation of enzyme complex is believed to provide diverse metabolic advantages and play crucial roles in plant secondary metabolism. A growing body of research has uncovered over a dozen of enzyme complexes in plant secondary metabolism and from diverse plants, including the dhurrin complex in sorghum, the lignin complex in Arabidopsis thaliana, and the bitter acid complex in hop. Furthermore, enzyme complexes likely widespread exist in all plant families. However, it remains challenging to identify enzyme complexes or characterize their metabolic functions due to technical challenges. Research in planta has long turnaround time and is limited by plant engineering tools, while in vitro studies might not be able to provide the appropriate cellular context and are hampered by difficulties in protein purification. All these limitations result in a huge knowledge gap. Here, we developed a microbial platform using baker’s yeast (Saccharomyces cerevisiae) to study dynamic plant enzyme complexes. Yeast has fast growth speed and massive genetic manipulation tools that enable rapid enzyme complex reconstruction. The membrane-bound organelles such as endoplasmic reticulum (ER) and the relatively simpler metabolic background in yeast provide a suitable intracellular environment for plant enzyme complex identification and characterization. My Ph.D work has been focused on developing synthetic biology tools to discover novel plant enzyme complexes via PPI screening and to characterize the metabolic functions of membrane-bound plant enzyme complexes. Furthermore, I am continuing to develop an in-planta proteomics-based method for complex discovery, which will be combined with the yeast-based platform to profile and characterize plant enzyme complexes efficiently. First, we developed orthogonal yeast-based PPI screening methods, including bimolecular fluorescence complementation (BiFC) and fluorescence co-localization, to capture PPIs from plants. Combining this method with plant multi-omics analysis, we discovered four enzyme complexes and their organized pathways from kratom, an alkaloid-producing plant. Second, we developed a yeast-based synthetic biology approach to elucidate the metabolic roles of PPIs between soybean isoflavone synthase (GmIFS2) and other cytosolic enzymes in isoflavonoid metabolism. By reconstructing multiple complex variants with a deactivated GmIFS2 in yeast, we found that GmIFS2-mediated PPIs can augment and redirect metabolic flux between two competing pathway branches producing deoxyisoflavonoids and isoflavonoids. Third, we developed a plant-based proximity labeling method, TurboID, in the non-model plant Catharanthus roseus. TurboID enables detection of weaker and transient PPIs in native plants, complementing yeast-based PPI methods for discovering plant enzyme complexes. This dissertation provides new strategies for plant enzyme complex discovery and functional characterization. It highlights the opportunity to leverage post-translational regulation features to uncover novel biosynthetic pathways for plant natural products. Ultimately, understanding the metabolic advantages of these complexes can inspire new strategies for plant natural product biomanufacturing and engineering.