Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis

• Evaluated fermentation on chemically defined media for crude extract preparation.
• Realized a 4-fold increase in protein synthesis yields with optimized protocol.
• Analyzed the effects of growth environment changes on cell-free metabolism.
• Varied fermentation to modify metabolic activity present in CFPS.

Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL−1 in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.

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