Abstract: Plant metabolism is increasingly being demonstrated to be partially controlled by dynamically assembled metabolons—multienzyme complexes that enable substrate channeling, insulate reactive intermediates, and permit rapid, low-energy flux control. Rigorous criteria are defined to distinguish true metabolons from generic assemblies, and evidence is synthesized across cyanogenic glucoside, phenylpropanoid/flavonoid, alkaloid, terpenoid, polyamine, sporopollenin, and auxin pathways. A practical workflow is presented in which AP-MS (Affinity purification mass spectrometry)/Co-IP (Co-immunoprecipitation), proximity labeling, BiFC (Bimolecular fluorescence complementation)/FRET (Förster resonance energy transfer)/Split-luciferase, and isotope-dilution metabolomics are integrated to resolve composition, dynamics, and direct channeling in vivo. In enzyme-based substrate channeling engineering, design rules are distilled for membrane anchoring, modular scaffolds, compartment targeting, and inducible/optogenetic control, and limitations such as metabolic burden, stoichiometry, and leakiness are noted. An AI-assisted loop is outlined in which structure-aware generative models produce binders/interfaces that are coupled to spatial optimization of enzyme order, orientation, and distance. Together, these advances reposition metabolons as a deployable technology for programmable flux in plants, enabling safer handling of labile intermediates and higher titers of valuable natural products.

Key words: metabolon engineering, plant natural products, substrate channeling, synthetic biology