Non-Canonical Amino Acid Incorporation via Sense Codon Reassignment in E. coli

Suppression of nonsense codons leading to the incorporation non-canonical amino acids (ncAAs) using orthogonal tRNA and aminoacyl tRNA synthetase (aaRS) pairs has been the most commonly employed technology for the expansion of the genetic code. Nonsense suppression is limited due to competition with the termination machinery in the number of ncAAs that can be incorporated into a given protein and in the yield of protein containing ncAAs that can be obtained. Additionally, the number of nonsense codons is limited, presently enabling expansion to 21 amino acid genetic codes. We describe investigations of sense codon reassignment in E. coli: the incorporation of ncAAs into proteins in response to codons normally directing the incorporation of natural amino acids, as a means to expand the number of substitutions and yield of modified protein produced.

Variants of the orthogonal tyrosine tRNA/aaRS pair from Methanocaldococcus jannaschii have been evolved to aminoacylate nearly 100 different ncAAs onto forms of the M. jannaschii tyrosine tRNA directed to nonsense codons. In order to expand the utility of the suite of ncAA incorporating M. jannaschii aaRS variants presently available, we investigated the ability of already-evolved ncAA incorporating pairs to reassign sense codons in vivo. A goal of this work is to produce 21 and 22 amino acid genetics codes without eliminating any of the 20 canonical amino acids. By modifying the anticodon on the tRNA to decode a sense codon typically read through a wobble pairing interaction and directed evolution of improved variants, we have been able to reassign histidine, lysine and arginine sense codons to incorporate tyrosine with efficiencies between 10 and 70%. Using our improved lysine system the ncAA O-methyl-L-tyrosine can be incorporated in response to AAG codons with lower efficiencies. Further, we have also performed directed evolution experiments to increase this efficiency as well as investigate the transferability of modifications made between ncAA and sense codon reassignment systems, e.g. Do improvements to the Tyr-incorporating aaRS also increase the efficiency of para-aminophenylalanine incorporation?

Interactions between the orthogonal aaRSs and tRNAs are one of several factors that affect ncAA incorporation efficiencies. In attempts to reassign one of the two histidine codons in E. coli, the unintended modification of the orthogonal tRNA by an endogenous E. coli enzyme, tadA was observed. TadAâ??s essential function is to modify the A34 in the anticodon of the E. coli arginine tRNA. We observed the modification of A34 of the histidine-targeted M. jannaschii tRNA to inosine which produced a tRNA variant relatively incapable of discriminating between the two histidine codons. We identified variants of both the tRNA and synthetase that simultaneously increased the sense codon reassigning efficiency of the pair and eliminated post transcriptional modification of the M. jannaschii Tyr-tRNA bearing an AUG anticodon via directed evolution. The engineered variant is capable of reassigning the His CAU codon with 2.5-fold increased efficiency and has near perfect discrimination between the two histidine codons.