Phase Separation-Mediated Multienzyme Assembly In Vivo.

Multienzyme assembly catalysis is a cornerstone of synthetic biology. In this study, we integrated the phase-separated protein RGGRGG with the ReverseTag/ReverseCatcher tagging system to investigate its potential for multienzyme complex formation. Initially, we utilized coarse-grained simulations to explore the interactions among RGGRGG domains, revealing their ability to form condensates and demonstrating how these condensates can interface with the ReverseTag/ReverseCatcher system for the assembly of multienzyme complexes. The ReverseTagged-proteins effectively self-assembled within ReverseCatcher_RGGRGG condensates. In cellular systems, we overexpressed six enzymes involved in lycopene biosynthesis and immobilized ReverseTag_Dxr, ReverseTag_Dxs, and ReverseTag_Idi by forming ester bonds with ReverseCatcher_RGGRGG. This approach resulted in a remarkable 5.4-fold enhancement in lycopene production compared to the control groups. This multienzyme assembly strategy holds significant promise for advancing the field of multienzyme catalysis, both in vivo and in vitro, and is expected to stimulate further research in this area.