title: Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids.
abstract: Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
authors.forename: Miriam
authors.initials: M
authors.lastname: Amiram
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
authors.forename: Adrian D
authors.initials: AD
authors.lastname: Haimovich
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
authors.forename: Chenguang
authors.initials: C
authors.lastname: Fan
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
authors.forename: Yane-Shih
authors.identifier: http://orcid.org/0000-0003-2356-9577
authors.initials: YS
authors.lastname: Wang
authors.affiliation: Systems Biology Institute, Yale University, West Haven, Connecticut, USA.|Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, USA.
authors.forename: Hans-Rudolf
authors.identifier: http://orcid.org/0000-0001-9477-2949
authors.initials: HR
authors.lastname: Aerni
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois, USA.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Ntai
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Moonan
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Ma
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
authors.forename: Alexis J
authors.initials: AJ
authors.lastname: Rovner
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA.
authors.forename: Seok Hoon
authors.identifier: http://orcid.org/0000-0003-2401-6368
authors.initials: SH
authors.lastname: Hong
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois, USA.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.affiliation: Department of Microbial Pathogenesis and Microbial Sciences Institute, Yale University School of Medicine, New Haven, Connecticut, USA.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Goodman
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.|Department of Chemistry, Yale University, New Haven, Connecticut, USA.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.affiliation: Systems Biology Institute, Yale University, West Haven, Connecticut, USA.|Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, USA.
authors.forename: Jesse
authors.identifier: http://orcid.org/0000-0003-4839-4005
authors.initials: J
authors.lastname: Rinehart
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
authors.forename: Farren J
authors.initials: FJ
authors.lastname: Isaacs