Advances in RNAi-Assisted Strain Engineering in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is a widely used eukaryotic model and microbial cell factory. RNA interference (RNAi) is a conserved regulatory mechanism among eukaryotes but absent from S. cerevisiae . Recent reconstitution of RNAi machinery in S. cerevisiae enables the use of this powerful tool for strain engineering. Here we first discuss the introduction of heterologous RNAi pathways in S. cerevisiae , and the design of various expression cassettes of RNAi precursor reagents for tunable, dynamic, and genome-wide regulation. We then summarize notable examples of RNAi-assisted functional genomics and metabolic engineering studies in S. cerevisiae . We conclude with the future challenges and opportunities of RNAi-based approaches, as well as the potential of other regulatory RNAs in advancing yeast engineering.

Genome-Scale Screening and Combinatorial Optimization of Gene Overexpression Targets to Improve Cadmium Tolerance in Saccharomyces cerevisiae

“…In the presence of 200 µM cadmium, synergistic interaction was noted between CRS5 and CUP1 when both expressed episomally (Figure 4B). Synergistic improvement confirmed the necessity for multiplex, combinatorial optimization of individual engineering targets (Wang et al, 2009;Si et al, 2015aSi et al, , 2017Cao et al, 2020;Chen et al, 2020).…”

“…Chemical tolerance is a complex phenotype with hundreds of genetic determinants (Almeida et al, 2007). Due to the lack of mechanistic understanding, it often requires genome-wide screening to identify mutations that confer resistance (Warner et al, 2010;Si et al, 2015b;Chen et al, 2020;Liu et al, 2020). For such endeavors, gene-deletion strain libraries are often utilized in S. cerevisiae, but most identified mutations lead to cadmium sensitivity (Jin et al, 2008;Ruotolo et al, 2008;Serero et al, 2008;Thorsen et al, 2009).…”

Metabolic Engineering: Methodologies and Applications

“…Gene repression in S. cerevisiae can be achieved using RNA interference (RNAi), which is a post-transcriptional, gene-silencing mechanism present in eukaryotic organisms. 171 RNAi employs an RNA-induced silencing complex to degrade mRNA transcripts. In particular, Dicer (Dcr) cleaves a doublestranded RNA to form smaller guide RNAs that target the gene of interest.…”

“…Gene downregulation can be beneficial when the target gene is essential, and thus complete deletion is lethal. Gene repression in S. cerevisiae can be achieved using RNA interference (RNAi), which is a post-transcriptional, gene-silencing mechanism present in eukaryotic organisms . RNAi employs an RNA-induced silencing complex to degrade mRNA transcripts.…”