Plant natural product biosynthesis through metabolon engineering

Area of Science:

• Plant biochemistry and molecular biology.
• Metabolic engineering and synthetic biology.

Background:

• Metabolons, or multienzyme complexes, are increasingly recognized for their role in regulating plant metabolic pathways.
• These complexes facilitate substrate channeling, protect reactive intermediates, and enable efficient metabolic flux control.

Purpose of the Study:

• To define criteria for identifying true metabolons and synthesize evidence for their role across various plant pathways.
• To present an integrated workflow for studying metabolon composition, dynamics, and function in vivo.
• To outline engineering strategies for creating programmable metabolons for biotechnological applications.

Main Methods:

• Affinity purification mass spectrometry (AP-MS)/Co-immunoprecipitation (Co-IP) and proximity labeling to determine complex composition.
• Bimolecular fluorescence complementation (BiFC)/Förster resonance energy transfer (FRET)/Split-luciferase assays to study protein-protein interactions and dynamics.
• Isotope-dilution metabolomics to analyze in vivo substrate channeling.
• AI-assisted generative models for designing enzyme binders and interfaces.

Main Results:

• Evidence for metabolon involvement in cyanogenic glucoside, phenylpropanoid/flavonoid, alkaloid, terpenoid, polyamine, sporopollenin, and auxin pathways was synthesized.
• An integrated workflow combining multiple techniques was presented for comprehensive metabolon analysis.
• Design rules for engineering metabolons, including membrane anchoring and compartment targeting, were distilled.
• AI was proposed for optimizing enzyme arrangement within metabolons.

Conclusions:

• Metabolons represent a powerful, deployable technology for programmable metabolic flux in plants.
• Engineering metabolons can lead to safer handling of reactive intermediates and increased production of valuable natural products.
• This work provides a framework for advancing metabolon research and application in plant biotechnology.