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

Alaksh Choudhury, C. Eric Hodgman, Mark J. Anderson, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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±0.28μgmL-1 in a 4h 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.

Original languageEnglish (US)
Pages (from-to)140-148
Number of pages9
JournalBiochemical Engineering Journal
Volume91
DOIs
StatePublished - Oct 15 2014

Keywords

  • Cell-free protein synthesis
  • Defined media
  • Enzyme biocatalysis
  • Fermentation
  • Growth kinetics
  • Modelling yeast

ASJC Scopus subject areas

  • Biotechnology
  • Environmental Engineering
  • Bioengineering
  • Biomedical Engineering

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