Ribosome engineering enhances genetic code expansion in <i>Saccharomyces cerevisiae</i>.
Ribosome engineering in yeast can significantly enhance ncAA incorporation, but the impact on growth and translation may limit broader application.
Where it sits
this study against the rest of the glutathione (gsh) corpusSummary and findings
The study explores ribosome engineering in Saccharomyces cerevisiae to enhance the incorporation of noncanonical amino acids into proteins. A strain named ribo-hyper increased ncAA-dependent GFP production by 2.9-fold compared to the wild-type strain. The engineered ribosome affected translation activity and cellular growth, and altered proteomic profiles related to amino acid biosynthesis and stress-response pathways.
Abstract
Genetic code expansion enables the site-specific installation of noncanonical amino acids (ncAAs) into proteins, but its limited efficiency in eukaryotes remains a major barrier to broader application. Here we establish a visual, plug-and-play screening platform to evolve 18S ribosomal DNA in <i>Saccharomyces cerevisiae</i> and identify ribosomal variants that improve ncAA incorporation. The best-performing strain, designated ribo-hyper, increased ncAA-dependent GFP production by 2.9-fold relative to the wild-type rDNA strain and enhanced incorporation across distinct orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Characterization of ribo-hyper showed that global translation activity and cellular growth were moderately reduced. Proteomic analysis further revealed changes in amino acid biosynthesis, translation-related proteins and stress-response pathways, indicating that the engineered ribosome reshapes cellular translation homeostasis. Perturbation of translation quality-control pathways, including the ribosome-rescue factors Dom34 and Hbs1 and the core mRNA exosome component Ski6, reduced ncAA-containing protein output, whereas disruption of ribosome quality-control factor Rqc2 had little effect. These findings support a role for ribosome rescue and associated mRNA turnover in efficient ncAA incorporation in the ribo-hyper strain. Together, our results establish eukaryotic ribosome engineering as a viable strategy for improving genetic code expansion in yeast.
Background
The study addresses the challenge of efficiently incorporating noncanonical amino acids (ncAAs) into proteins in eukaryotic systems, which is a barrier to broader application of genetic code expansion. Previous research has shown limited efficiency in eukaryotes compared to prokaryotes. This research is significant as it explores ribosome engineering to potentially overcome these limitations in Saccharomyces cerevisiae.
Methods
The study utilized a visual, plug-and-play screening platform to evolve 18S ribosomal DNA in Saccharomyces cerevisiae. The researchers identified ribosomal variants that improved ncAA incorporation. The primary outcome was the increase in ncAA-dependent GFP production, while secondary outcomes included changes in global translation activity and cellular growth. Proteomic analysis was conducted to assess changes in amino acid biosynthesis and translation-related proteins.
Results
The ribo-hyper strain showed a 2.9-fold increase in ncAA-dependent GFP production compared to the wild-type strain. Global translation activity and cellular growth were moderately reduced in the ribo-hyper strain. Proteomic analysis revealed changes in amino acid biosynthesis, translation-related proteins, and stress-response pathways. Perturbation of translation quality-control pathways reduced ncAA-containing protein output, implicating ribosome rescue and mRNA turnover in efficient ncAA incorporation.
Interpretation
The findings suggest that ribosome engineering can enhance ncAA incorporation in yeast, potentially offering a strategy for genetic code expansion in eukaryotes. However, the moderate reduction in translation activity and cellular growth may limit the practical utility of this approach. The study's focus on yeast models raises questions about the generalizability of the results to other eukaryotic systems. Further research is needed to explore the clinical relevance and applicability of these findings.
Key findings
- 2.9-fold increase in ncAA-dependent GFP production in ribo-hyper strain.
- Moderate reduction in global translation activity and cellular growth in ribo-hyper.
- Changes in amino acid biosynthesis and translation-related proteins observed.
- Perturbation of translation quality-control pathways reduced ncAA-containing protein output.
- Ribosome-rescue factors Dom34 and Hbs1 involved in efficient ncAA incorporation.
Limitations
- yeast model only, not generalizable to other eukaryotes
- moderate reduction in cellular growth
- impact on global translation activity
- focus on ncAA-dependent GFP production