Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression

Max Z. Levine, Byungcheol So, Alissa C. Mullin, Rob Fanter, Kayla Dillard, Katharine R. Watts, Michael R. La Frano, Javin P. Oza

Research output: Contribution to journalArticlepeer-review

Abstract

Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, and education. The Escherichia coli cell extract-based platform has been broadly adopted due to its affordability and versatility. The upstream processing of cells to generate crude cell lysate remains time-intensive and technically nuanced, representing one of the largest sources of cost associated with the biotechnology. To overcome these limitations, we have improved the processes by developing a long-lasting autoinduction media formulation for CFPS that obviates human intervention between inoculation and harvest. The cell-free autoinduction (CFAI) media supports the production of robust cell extracts from high cell density cultures nearing the stationary phase of growth. As a result, the total mass of cells and the resulting extract volume obtained increases by 400% while maintaining robust reaction yields of reporter protein, sfGFP (>1 mg/mL). Notably, the CFAI workflow allows users to go from cells on a streak plate to completing CFPS reactions within 24 h. The CFAI workflow uniquely enabled us to elucidate the metabolic limits in CFPS associated with cells grown to stationary phase in the traditional 2× YTPG media. Metabolomics analysis demonstrates that CFAI-based extracts overcome these limits due to improved energy metabolism and redox balance. The advances reported here shed new light on the metabolism associated with highly active CFPS reactions and inform future efforts to tune the metabolism in CFPS systems. Additionally, we anticipate that the improvements in the time and cost-efficiency of CFPS will increase the simplicity and reproducibility, reducing the barriers for new researchers interested in implementing CFPS.

Original languageEnglish (US)
Pages (from-to)2765-2774
Number of pages10
JournalACS synthetic biology
Volume9
Issue number10
DOIs
StatePublished - Oct 16 2020
Externally publishedYes

Keywords

  • autoinduction
  • cell-free protein synthesis
  • in vitro transcription/translation
  • metabolomics
  • synthetic biology

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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