Dec 18, 2013

Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering

ACS Synthetic Biology
Nathan C CrookHal S Alper

Abstract

Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.

  • References16
  • Citations24

References

  • References16
  • Citations24

Citations

Mentioned in this Paper

Metabolic Process, Cellular
Biochemical Pathway
RNA Conformation
Alkalescens-Dispar Group
Gene Knockdown Techniques
Saccharomyces cerevisiae allergenic extract
Cosuppression
Transcription, Genetic
Gene Expression
Organism

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