Structural Robustness Affects the Engineerability of Aminoacyl-tRNA Synthetases for Genetic Code Expansion

Abstract

The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the sitespecific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii-derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The Escherichia coli-derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs have been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the structural robustness of EcTyrRS being lower than that of MjTyrRS. We show that the thermostability of engineered TyrRS mutants is generally significantly lower than those of their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homologue from a thermophilic bacterium, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.

Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast

“…Moreover, while general performance trends are expected to hold upon transferring ncAA incorporation systems between organisms, quantitative differences between organisms can occur, necessitating rigorous characterizations if further SPS implementation is performed in E. coli, mammalian cells, or cells from other organisms. , Despite these important considerations, our results provide several initial demonstrations that, in yeast, SPSs are a comparable format to DPSs.…”

Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains

“…First, the target enzyme should have low levels of the desired new activity, which in case of PylRS means enzymatic activity toward ncAAs that are highly divergent from the native substrate [ 21 , 22 ]. Second, sufficient stability is required to buffer destabilizing mutations necessary for active site remodeling [ 23 , 24 , 25 ]. Unfortunately, PylRS is marginally stable under standard cultivation conditions in Escherichia coli ( E. coli ) [ 26 ], which is also reflected by the low in vitro solubility of the enzyme [ 20 , 27 ].…”

A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic

“…We recently reported that mutants of EcTyrRS often exhibit low thermostabilities. 34 In particular, pBPARS-1 was found to be largely insoluble under ambient conditions, which likely explains its poor activity. Interestingly, a large fraction of pBPARS-3.1 was found in the soluble fraction of the E. coli cell-free extract, while nearly all of pBPARS-1 was found in the insoluble fraction ( Figure S4C ).…”