Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1 |
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Authors: | Dr. Seok Hoon Hong Dr. Yong‐Chan Kwon Rey W. Martin Benjamin J. Des Soye Alexandra M. de Paz Kirsten N. Swonger Dr. Ioanna Ntai Prof. Neil L. Kelleher Prof. Michael C. Jewett |
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Affiliation: | 1. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Tech E‐136, Evanston, IL 60208 (USA);2. Interdisciplinary Biological Sciences, Northwestern University, 2205 Tech Drive, Hogan 2‐100, Evanston, IL 60208 (USA);3. Department of Chemistry, Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL 60208 (USA) |
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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. |
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Keywords: | cell‐free protein synthesis genome engineering non‐standard amino acids release factor 1 synthetic biology |
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