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Updated: May 21, 2025

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Phase Separation-Mediated Multienzyme Assembly In Vivo.

Yao Chen1,2, Yi Shi1, Mengyu Li1

  • 1State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China.

Journal of Agricultural and Food Chemistry
|March 19, 2025
PubMed
Summary
This summary is machine-generated.

Synthetic biology advances multienzyme catalysis using RGGRGG condensates and ReverseTag/ReverseCatcher systems. This approach significantly boosted lycopene production by 5.4-fold in cellular systems.

Keywords:
RGGRGGReverseTag/ReverseCacthermultienzyme assembly

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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.