Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes

Simple item page
dc.citation.articleNumber111541en_US
dc.citation.issueNumber1en_US
dc.citation.journalTitleiScienceen_US
dc.citation.volumeNumber28en_US
dc.contributor.authorKar, Shaunaken_US
dc.contributor.authorGardner, Elizabeth C.en_US
dc.contributor.authorJavanmardi, Kamyaben_US
dc.contributor.authorBoutz, Daniel R.en_US
dc.contributor.authorShroff, Raghaven_US
dc.contributor.authorHorton, Andrew P.en_US
dc.contributor.authorSegall-Shapiro, Thomas H.en_US
dc.contributor.authorEllington, Andrew D.en_US
dc.contributor.authorGollihar, Jimmyen_US
dc.date.accessioned2025-01-09T20:16:58Zen_US
dc.date.available2025-01-09T20:16:58Zen_US
dc.date.issued2025en_US
dc.description.abstractT7 RNA polymerase (RNAP) has enabled orthogonal control of gene expression and recombinant protein production across diverse prokaryotic host chassis organisms for decades. However, the absence of 5′ methyl guanosine caps on T7 RNAP-derived transcripts has severely limited its utility and widespread adoption in eukaryotic systems. To address this shortcoming, we evolved a fusion enzyme combining T7 RNAP with the single subunit capping enzyme from African swine fever virus using Saccharomyces cerevisiae. We isolated highly active variants of this fusion enzyme, which exhibited roughly two orders of magnitude higher protein expression compared to the wild-type enzyme. We demonstrate the programmable control of gene expression using T7 RNAP-based genetic circuits in yeast and validate enhanced performance of these engineered variants in mammalian cells. This study presents a robust, orthogonal gene regulatory system applicable across diverse eukaryotic hosts, enhancing the versatility and efficiency of synthetic biology applications.en_US
dc.identifier.citationKar, S., Gardner, E. C., Javanmardi, K., Boutz, D. R., Shroff, R., Horton, A. P., Segall-Shapiro, T. H., Ellington, A. D., & Gollihar, J. (2025). Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes. iScience, 28(1). https://doi.org/10.1016/j.isci.2024.111541en_US
dc.identifier.digitalPIIS2589004224027688en_US
dc.identifier.doihttps://doi.org/10.1016/j.isci.2024.111541en_US
dc.identifier.urihttps://hdl.handle.net/1911/118110en_US
dc.language.isoengen_US
dc.publisherElsevieren_US
dc.rightsExcept where otherwise noted, this work is licensed under a Creative Commons Attribution-NonCommercial (CC BY-NC) license. Permission to reuse, publish, or reproduce the work beyond the terms of the license or beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0/en_US
dc.titleDirected evolution of an orthogonal transcription engine for programmable gene expression in eukaryotesen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
PIIS2589004224027688.pdf
Size:
2.72 MB
Format:
Adobe Portable Document Format
Download
Collections
Faculty Publications
Bioengineering Publications