%0 Journal Article %A Jessica T. Stieglitz %A Priyanka Lahiri %A Matthew I. Stout %A James A. Van Deventer %T Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast %D 2022 %R 10.1101/2022.01.07.475408 %J bioRxiv %P 2022.01.07.475408 %X Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in E. coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remains limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUA support the incorporation of ncAAs into proteins produced in S. cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were translationally active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the translational activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using the yeast strain RJY100, and pairing these aaRSs with the M. barkeri tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS to the orthogonal translation machinery toolkit in yeast potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.Competing Interest StatementThe authors have declared no competing interest.aaRSaminoacyl-tRNA synthetaseAcKNε-acetyl-L-lysineAcrKacryloyl-L-lysineBFPblue fluorescent proteinBocKBoc-L-lysineBXGblue fluorescent protein fused to green fluorescent protein by a flexible linker containing a TAG codonBYGblue fluorescent protein fused to green fluorescent protein by a flexible linkerCbzKNε-benzyloxycarbonyl-L-lysineGFPgreen fluorescent proteinLysAlk2-Amino-6-(prop-2-ynoxycarbonylamino)hexanoic acidLysN3(S)-2-amino-6-((2-azidoethoxy)carbonylamino)hexanoic acidLysZNε-benzoyl-L-lysineMALDImatrix-assisted laser desorption ionizationMaPylRSMethanomethylophilus alvus pyrrolysyl-tRNA synthetaseMbPylRSMethanosarcina barkeri pyrrolysyl-tRNA synthetaseMmPylRSMethanosarcina mazei pyrrolysyl-tRNA synthetaseMMFmaximum misincorporation frequencyncAAnoncanonical amino acidNmH23-methyl-L-histidineOTSorthogonal translation systemPhK(S)-2-Amino-6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonylamino)hexanoic acidPylRSpyrrolysyl-tRNA synthetaseRRErelative readthrough efficiencytRNAtransfer RNA %U https://www.biorxiv.org/content/biorxiv/early/2022/01/08/2022.01.07.475408.full.pdf