Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1

Seok Hoon Hong, Yong Chan Kwon, Rey W. Martin, Benjamin J. Des Soye, Alexandra M. De Paz, Kirsten N. Swonger, Ioanna Ntai, Neil L. Kelleher, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA- endA-), we synthesized 550±40 μg mL-1 of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ∼1300 μg mL-1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.

Original languageEnglish (US)
Pages (from-to)844-853
Number of pages10
JournalChemBioChem
Volume16
Issue number5
DOIs
StatePublished - Mar 23 2015

Keywords

  • cell-free protein synthesis
  • genome engineering
  • non-standard amino acids
  • release factor 1
  • synthetic biology

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

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