title: Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.
abstract: The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208-3120.
authors.forename: Rui
authors.initials: R
authors.lastname: Gan
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208-3120.
authors.forename: Jessica G
authors.initials: JG
authors.lastname: Perez
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208-3120.
authors.forename: Erik D
authors.initials: ED
authors.lastname: Carlson
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208-3120.|Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Ntai
authors.affiliation: Systems Biology Institute, Yale University, West Haven, Connecticut.|Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut.
authors.forename: Farren J
authors.initials: FJ
authors.lastname: Isaacs
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208-3120.|Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208.|Department of Molecular Biosciences, Northwestern University, Evanston, Illinois.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208-3120.|Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208.|Interdisciplinary Biological Sciences Program, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208-0001.|Northwestern Institute on Complex Systems, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208.|Simpson Querry Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics / Q000502:physiology; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D011506:Proteins; D010444:Peptide Elongation Factor Tu; D000604:Amino Acyl-tRNA Synthetases
keywords: aminoacyl-tRNA synthetase; directed evolution; elongation factor Tu; genomically recoded organism; noncanonical amino acid; orthogonal translation system; synthetic biology