Evolving Orthogonal Suppressor tRNAs To Incorporate Modified Amino Acids.
There have been considerable advancements in the incorporation of noncanonical amino acids (ncAA) into proteins over the last two decades. The most widely used method for site-specific incorporation of noncanonical amino acids, amber stop codon suppression, typically employs an orthogonal translation system (OTS) consisting of a heterologous aminoacyl-tRNA synthetase:tRNA pair that can potentially expand an organism's genetic code. However, the orthogonal machinery sometimes imposes fitness costs on an organism, in part due to mischarging and a lack of specificity. Using compartmentalized partnered replication (CPR) and a newly developed pheS negative selection, we evolved several new orthogonal Methanocaldococcus jannaschii (Mj) tRNA variants tRNAs with increased amber suppression activity, but that also showed up to 3-fold reduction in promiscuous aminoacylation by endogenous aminoacyl-tRNA synthetases (aaRSs). The increased orthogonality of these variants greatly reduced organismal fitness costs associated in part due to tRNA mischarging. Using these methods, we were also able to evolve tRNAs that supported the specific incorporation of 3-halo-tyrosines (3-Cl-Y, 3-Br-Y, and 3-I-Y) in E. coli.
Center for Systems and Synthetic Biology, University of Texas at Austin , Austin, Texas 78712, United States.
Andre C
AC
Maranhao
Center for Systems and Synthetic Biology, University of Texas at Austin , Austin, Texas 78712, United States.
Andrew D
AD
Ellington
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D063747:Methanocaldococcus / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D019642:RNA, Archaeal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013489:Suppression, Genetic; D058615:Synthetic Biology
D000596:Amino Acids; D019642:RNA, Archaeal; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases
aminoacyl-tRNA synthetase; noncanonical amino acids; orthogonal translation systems; tRNA