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Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1
Author(s) -
Hong Seok Hoon,
Kwon YongChan,
Martin Rey W.,
Des Soye Benjamin J.,
de Paz Alexandra M.,
Swonger Kirsten N.,
Ntai Ioanna,
Kelleher Neil L.,
Jewett Michael C.
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402708
Subject(s) - cell free protein synthesis , escherichia coli , genome engineering , synthetic biology , lysis , genome , nuclease , gene , protein biosynthesis , effector , genome editing , biochemistry , biology , computational biology , strain (injury) , protein engineering , cell , chemistry , anatomy , enzyme
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.