Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.
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.
Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Tech E-136, Evanston, IL 60208 (USA).
Seok Hoon
SH
Hong
Yong-Chan
YC
Kwon
Rey W
RW
Martin
Benjamin J
BJ
Des Soye
Alexandra M
AM
de Paz
Kirsten N
KN
Swonger
Ioanna
I
Ntai
Neil L
NL
Kelleher
Michael C
MC
Jewett
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001709:Biotechnology; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry; D010454:Peptide Termination Factors / Q000172:deficiency* / Q000235:genetics; D014176:Protein Biosynthesis
D000596:Amino Acids; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C489005:prfA protein, E coli; D049452:Green Fluorescent Proteins
cell-free protein synthesis; genome engineering; non-standard amino acids; release factor 1; synthetic biology