title: Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.
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.
authors: Department of Chemical Engineering, The University of Texas at Austin , 200 East Dean Keeton Street Stop C0400, Austin, Texas 78712, United States.
authors: Nathan C
authors: NC
authors: Crook
authors: Alexander C
authors: AC
authors: Schmitz
authors: Hal S
authors: HS
authors: Alper
mesh_terms: D004926:Escherichia coli; D015870:Gene Expression / Q000235:genetics*; D055785:Gene Knockdown Techniques; D060847:Metabolic Engineering / Q000379:methods*; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013386:Succinates / Q000032:analysis / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: D034741:RNA, Small Interfering; D013386:Succinates; C005229:itaconic acid