PT  - JOURNAL ARTICLE
AU  - Grasso, Katherine T.
AU  - Roy, Soumya Jyoti Singha
AU  - Yeo, Megan Jin Rae
AU  - Soni, Chintan
AU  - Osgood, Arianna O.
AU  - Hillenbrand, Christen M.
AU  - Chatterjee, Abhishek
TI  - A facile platform to engineer <em>E. coli</em> tyrosyl-tRNA synthetase adds new chemistries to the eukaryotic genetic code, including a phosphotyrosine mimic
AID  - 10.1101/2021.11.28.470256
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.11.28.470256
4099  - http://biorxiv.org/content/early/2021/11/28/2021.11.28.470256.short
4100  - http://biorxiv.org/content/early/2021/11/28/2021.11.28.470256.full
AB  - The E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform to genetically encode new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed for its engineering, has impeded its success in the past. Recently, we showed that EcTyrRS can be engineered using a facile E. coli based selection system, in a strain where the endogenous tyrosyl pair has been substituted with an archaeal counterpart. However, a significant cross-reactivity between the UAG-suppressing tRNACUAEcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyr that overcomes this cross-reactivity. Optimized selection systems using this tRNA enabled efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide-dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporate useful ncAAs in mammalian cells, including photo-affinity probes, bioconjugation handles, and a non-hydrolyzable mimic of phosphotyrosine.Competing Interest StatementA patent application on the EcTyrRS mutants and ncAAs reported here has been submitted. AC is a cofounder and senior advisor of BrickBio, Inc., which focuses on applications of the ncAA mutagenesis technology