Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen

Abstract

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNA Pyl pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.

High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

“…Previous selections in yeast 19,[52][53][54] and the sorts described above did not attempt to control ncAA substrate specificity preferences other than the preference for the target; such conditions can lead to polyspecific aaRSs. 33,55,56 For these experiments, we used a group of six structurally similar aromatic ncAAs: OmeY, p-acetyl-L-phenylalanine (AcF, 2), pazido-L-phenylalanine (AzF, 3), OPG, 4-azidomethyl-L-phenylalanine (AzMF, 5), and 4-iodo-Lphenylalanine (IPhe, 6). Based on our previous characterizations of EcTyrRS variant TyrAcFRS 57 and EcLeuRS variant LeuOmeRS, 58 we expected that similarities between these ncAAs would make it difficult for aaRS variants to selectively charge only a single ncAA without also charging others.…”

“…[26][27][28][29] While these findings were applied to the residue-specific replacement of methionine, later findings evaluating OTSs for site-specific applications appear to be consistent with these earlier studies. [30][31][32][33] Eppinger, Rueping, and coworkers evolved Methanosarcina barkeri pyrrolysyl-tRNA synthetases (PylRSs) using a high-throughput FACS screen in E. coli, identifying a polyspecific variant capable of supporting stop codon readthrough with up to 31 aliphatic, cyclic, or fluorinated ncAAs. 33 Isaacs and coworkers used multiplex automated genome engineering (MAGE) to evolve chromosomally-encoded OTSs with diversity at 12 residues in the amino acid binding pocket and five residues in the tRNA anticodon recognition domain of the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS).…”

“…[30][31][32][33] Eppinger, Rueping, and coworkers evolved Methanosarcina barkeri pyrrolysyl-tRNA synthetases (PylRSs) using a high-throughput FACS screen in E. coli, identifying a polyspecific variant capable of supporting stop codon readthrough with up to 31 aliphatic, cyclic, or fluorinated ncAAs. 33 Isaacs and coworkers used multiplex automated genome engineering (MAGE) to evolve chromosomally-encoded OTSs with diversity at 12 residues in the amino acid binding pocket and five residues in the tRNA anticodon recognition domain of the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS). Using a GFP reporter and FACS, they were able to isolate MjTyrRSs with 25-fold improvement of activity for two ncAAs, one of which they further evolved for specificity of a target ncAA and exclusion of 237 non-target ncAAs.…”

Broadening the toolkit for quantitatively evaluating noncanonical amino acid incorporation in yeast

“…Previous work has demonstrated the feasibility of using SPSs as alternatives to DPSs in both E. coli and mammalian cells. [15][16][17][18] SPSs are slightly less flexible than DPSs and larger plasmid sizes may impact transformation efficiency, but they provide access to genetic code expansion that is otherwise unattainable using DPSs. Additionally, with SPSs the burden of maintaining two separate plasmids is eliminated along with the risk of losing one of the two plasmids during cell propagation.…”

Encoded, click-reactive DNA-binding domains for programmable capture of specific chromatin segments

“…To discover proteins that are recruited to SATIII DNA during nuclear stress body formation, we next performed proteomics studies with cells grown under heat shock and normal conditions. To take advantage of our two-step biotinylation approach compared to potential direct incorporation of a biotin ncAA, 33 we blocked endogenous biotin sites before click-biotinylation to reduce background enrichment. Specically, we followed the protocol as before, but before the addition of biotin-tetrazine, we incubated the cells with avidin, added an excess of free biotin to block remaining binding sites at the avidin, and washed the cells.…”