Characterizing and alleviating substrate limitations for improved in vitro ribosome construction

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biochemistry

Background:

• Cell-free synthesis of ribosomes is crucial for minimal cell projects and engineering novel ribosome functions.
• The integrated synthesis, assembly, and translation (iSAT) method enables in vitro construction and activity assessment of Escherichia coli ribosomes.

Purpose of the Study:

• To investigate the causes of iSAT reaction termination and improve its efficiency and product yields.
• To enhance the iSAT method for increased superfolder green fluorescent protein (sfGFP) production.

Main Methods:

• Analyzed the degradation of energy substrates (phosphoenolpyruvate - PEP) and nucleoside triphosphates (NTPs) during iSAT reactions.
• Assessed the impact of inorganic phosphate accumulation on iSAT performance.
• Implemented fed-batch and semi-continuous feeding strategies for substrate replenishment and byproduct removal.

Main Results:

• Identified rapid degradation of PEP and NTPs, leading to energy depletion and protein synthesis termination.
• Demonstrated that inorganic phosphate accumulation inhibits iSAT.
• Fed-batch addition of PEP and magnesium glutamate extended reaction time by 2-fold and increased sfGFP yield by ~75%.
• Semi-continuous iSAT prolonged reaction duration 5-fold and increased sfGFP yield 7-fold, achieving 7.5 ± 0.7 μmol L(-1).

Conclusions:

• Energy substrate availability and management are critical for sustained iSAT reactions.
• Optimizing metabolic processes, particularly substrate depletion and byproduct inhibition, is key to advancing cell-free synthetic biology.
• The developed semi-continuous iSAT method represents a significant advancement in achieving high-yield cell-free ribosome synthesis.