Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast

Abdullah, Mudabir; Greco, Brittany M.; Laurent, Jon M; Garge, Riddhiman K.; Boutz, Daniel R.; Vandeloo, Michelle; Marcotte, Edward M.; Kachroo, Aashiq H.

doi:10.1016/j.crmeth.2023.100464

Cited by 4 publications

(2 citation statements)

References 57 publications

(80 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The biochemical analysis reveals that several replacement-competent human β2c variants are proteolytically active. Further characterization of the incompatibilities associated with nonreplaceable human β subunits should identify a path to full humanization of the yeast proteasome core while distinguishing divergent ortholog functions ( Abdullah et al . 2023 ).…”

Section: Discussionmentioning

confidence: 99%

Species-specific protein–protein interactions govern the humanization of the 20S proteasome in yeast

Sultana

Abdullah

et al. 2023

Genetics

Self Cite

View full text Add to dashboard Cite

Yeast and humans share thousands of genes despite a billion years of evolutionary divergence. While many human genes can functionally replace their yeast counterparts, nearly half of the tested shared genes cannot. For example, most yeast proteasome subunits are “humanizable”, except subunits comprising the β-ring core, including β2c (HsPSMB7, a constitutive proteasome subunit). We developed a high-throughput pipeline to humanize yeast proteasomes by generating a large library of Hsβ2c mutants and screening them for complementation of a yeast β2 (ScPup1) knockout. Variants capable of replacing ScPup1 included (1) those impacting local protein-protein interactions (PPIs), with most affecting interactions between the β2c C-terminal tail and the adjacent β3 subunit, and (2) those affecting β2c proteolytic activity. Exchanging the full-length tail of human β2c with that of ScPup1 enabled complementation. Moreover, wild-type human β2c could replace yeast β2 if human β3 was also provided. Unexpectedly, yeast proteasomes bearing a catalytically inactive HsPSMB7-T44A variant that blocked precursor autoprocessing were viable, suggesting an intact propeptide stabilizes late assembly intermediates. In contrast, similar modifications in human β2i (HsPSMB10), an immuno-proteasome subunit and the co-ortholog of yeast β2, do not enable complementation in yeast, suggesting distinct interactions are involved in human immunoproteasome core assembly. Broadly, our data reveal roles for specific PPIs governing functional replaceability across vast evolutionary distances.

show abstract

Section: Discussionmentioning

confidence: 99%

Species-specific protein–protein interactions govern the humanization of the 20S proteasome in yeast

Sultana

Abdullah

et al. 2023

Genetics

Self Cite

View full text Add to dashboard Cite

show abstract

“…Yeast proteins for mass spectrometry were isolated using a previously described protocol ( Abdullah et al . 2023 ).…”

Section: Methodsmentioning

confidence: 99%

Systematic profiling of ale yeast protein dynamics across fermentation and repitching

Garge,

Geck,

Armstrong

et al. 2023

G3: Genes, Genomes, Genetics

View full text Add to dashboard Cite

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is amongst the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout two fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.

show abstract

Comprehensive plasmid toolkit for multipart assembly in the Dunaliella salina nuclear system

Chen,

Xie,

Xie

et al. 2024

Algal Research

View full text Add to dashboard Cite

Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast

Cited by 4 publications

References 57 publications

Species-specific protein–protein interactions govern the humanization of the 20S proteasome in yeast

Species-specific protein–protein interactions govern the humanization of the 20S proteasome in yeast

Systematic profiling of ale yeast protein dynamics across fermentation and repitching

Comprehensive plasmid toolkit for multipart assembly in the Dunaliella salina nuclear system

Contact Info

Product

Resources

About