Holistic engineering of cell-free systems through proteome-reprogramming synthetic circuits

Area of Science:

• Synthetic Biology
• Systems Biology
• Molecular Engineering

Background:

• Traditional synthetic biology focuses on orthogonal genetic modules, separate from host cell machinery.
• A synthetic module can be designed to reprogram the host proteome, creating a more favorable environment for its own function.
• This holistic approach has not been extensively applied to cell-free systems.

Purpose of the Study:

• To engineer cell-free systems by reprogramming the host proteome through metabolic crosstalk.
• To enhance protein synthesis efficiency in cell-free environments.
• To achieve higher expression levels of synthetic products compared to conventional cell-free systems.

Main Methods:

• Engineered local modules expressing translation machinery to reprogram the bacterial proteome.
• Analyzed changes in protein expression levels, identifying over 700 affected proteins.
• Quantified the expression levels of synthesized fluorescent reporters, protein nanocages, and Cas9 nuclease in the engineered cell-free system.

Main Results:

• Demonstrated that local synthetic modules can reprogram the bacterial proteome.
• Observed significant changes in the expression levels of over 700 proteins.
• Achieved up to a 5-fold increase in the expression of fluorescent reporters, protein nanocages, and Cas9 in the developed cell-free system compared to classical systems.

Conclusions:

• A holistic synthetic biology approach, integrating synthetic and systems biology, can enhance cell-free system performance.
• Exploiting metabolic crosstalk and proteome reprogramming leads to improved protein synthesis environments.
• This strategy enables superior outcomes for producing complex biomolecules in cell-free systems.