Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast

View ORCID ProfileJessica T. Stieglitz, View ORCID ProfilePriyanka Lahiri, Matthew I. Stout, View ORCID ProfileJames A. Van Deventer
doi: https://doi.org/10.1101/2022.01.07.475408
Jessica T. Stieglitz
†Chemical and Biological Engineering Department, Tufts University, Medford, Massachusetts 02155, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Jessica T. Stieglitz
Priyanka Lahiri
†Chemical and Biological Engineering Department, Tufts University, Medford, Massachusetts 02155, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Priyanka Lahiri
Matthew I. Stout
†Chemical and Biological Engineering Department, Tufts University, Medford, Massachusetts 02155, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
James A. Van Deventer
†Chemical and Biological Engineering Department, Tufts University, Medford, Massachusetts 02155, USA
‡Biomedical Engineering Department, Tufts University, Medford, Massachusetts 02155, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for James A. Van Deventer
  • For correspondence: James.Van_Deventer@tufts.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

Abstract

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.

Figure
  • Download figure
  • Open in new tab

Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations

    aaRS
    aminoacyl-tRNA synthetase
    AcK
    Nε-acetyl-L-lysine
    AcrK
    acryloyl-L-lysine
    BFP
    blue fluorescent protein
    BocK
    Boc-L-lysine
    BXG
    blue fluorescent protein fused to green fluorescent protein by a flexible linker containing a TAG codon
    BYG
    blue fluorescent protein fused to green fluorescent protein by a flexible linker
    CbzK
    Nε-benzyloxycarbonyl-L-lysine
    GFP
    green fluorescent protein
    LysAlk
    2-Amino-6-(prop-2-ynoxycarbonylamino)hexanoic acid
    LysN3
    (S)-2-amino-6-((2-azidoethoxy)carbonylamino)hexanoic acid
    LysZ
    Nε-benzoyl-L-lysine
    MALDI
    matrix-assisted laser desorption ionization
    MaPylRS
    Methanomethylophilus alvus pyrrolysyl-tRNA synthetase
    MbPylRS
    Methanosarcina barkeri pyrrolysyl-tRNA synthetase
    MmPylRS
    Methanosarcina mazei pyrrolysyl-tRNA synthetase
    MMF
    maximum misincorporation frequency
    ncAA
    noncanonical amino acid
    NmH2
    3-methyl-L-histidine
    OTS
    orthogonal translation system
    PhK
    (S)-2-Amino-6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonylamino)hexanoic acid
    PylRS
    pyrrolysyl-tRNA synthetase
    RRE
    relative readthrough efficiency
    tRNA
    transfer RNA
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
    Back to top
    PreviousNext
    Posted January 08, 2022.
    Download PDF

    Supplementary Material

    Email

    Thank you for your interest in spreading the word about bioRxiv.

    NOTE: Your email address is requested solely to identify you as the sender of this article.

    Enter multiple addresses on separate lines or separate them with commas.
    Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast
    (Your Name) has forwarded a page to you from bioRxiv
    (Your Name) thought you would like to see this page from the bioRxiv website.
    CAPTCHA
    This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
    Share
    Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast
    Jessica T. Stieglitz, Priyanka Lahiri, Matthew I. Stout, James A. Van Deventer
    bioRxiv 2022.01.07.475408; doi: https://doi.org/10.1101/2022.01.07.475408
    Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
    Citation Tools
    Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast
    Jessica T. Stieglitz, Priyanka Lahiri, Matthew I. Stout, James A. Van Deventer
    bioRxiv 2022.01.07.475408; doi: https://doi.org/10.1101/2022.01.07.475408

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
    • Tweet Widget
    • Facebook Like
    • Google Plus One

    Subject Area

    • Synthetic Biology
    Subject Areas
    All Articles
    • Animal Behavior and Cognition (3514)
    • Biochemistry (7364)
    • Bioengineering (5341)
    • Bioinformatics (20316)
    • Biophysics (10038)
    • Cancer Biology (7769)
    • Cell Biology (11346)
    • Clinical Trials (138)
    • Developmental Biology (6445)
    • Ecology (9977)
    • Epidemiology (2065)
    • Evolutionary Biology (13351)
    • Genetics (9369)
    • Genomics (12603)
    • Immunology (7724)
    • Microbiology (19083)
    • Molecular Biology (7458)
    • Neuroscience (41125)
    • Paleontology (300)
    • Pathology (1235)
    • Pharmacology and Toxicology (2142)
    • Physiology (3174)
    • Plant Biology (6873)
    • Scientific Communication and Education (1276)
    • Synthetic Biology (1900)
    • Systems Biology (5324)
    • Zoology (1091)