Phase Separation-Mediated Multienzyme Assembly In Vivo

Area of Science:

• Synthetic biology
• Biocatalysis
• Protein engineering

Background:

• Multienzyme assembly is crucial for efficient biocatalysis.
• Phase-separated proteins offer novel platforms for enzyme complex formation.
• The ReverseTag/ReverseCatcher system enables specific protein-protein interactions.

Purpose of the Study:

• To investigate the integration of RGGRGG phase-separated protein condensates with the ReverseTag/ReverseCatcher system for multienzyme complex formation.
• To evaluate the efficacy of this strategy for enhancing metabolic pathway performance in vivo.

Main Methods:

• Coarse-grained simulations were used to study RGGRGG domain interactions and condensate formation.
• The ReverseTag/ReverseCatcher system was employed to anchor ReverseTagged proteins within ReverseCatcher_RGGRGG condensates.
• Enzymes for lycopene biosynthesis were engineered with ReverseTag and immobilized onto ReverseCatcher_RGGRGG condensates in cellular systems.

Main Results:

• RGGRGG domains were shown to form condensates capable of interfacing with the ReverseTag/ReverseCatcher system.
• ReverseTagged proteins successfully self-assembled within the ReverseCatcher_RGGRGG condensates.
• A 5.4-fold increase in lycopene production was achieved in engineered cellular systems compared to controls.

Conclusions:

• The combined RGGRGG and ReverseTag/ReverseCatcher system provides an effective strategy for constructing functional multienzyme complexes.
• This approach significantly enhances the production of target metabolites, demonstrating its potential for synthetic biology applications.
• The study opens new avenues for in vivo and in vitro multienzyme catalysis and metabolic engineering.