(798f) Facile Generation of Knockdown Phenotypes in Yeast Through RNA Interference

Microbial synthesis offers a promising approach for the sustainable production of fuels and chemicals.  These metabolic engineering efforts often rely upon a finite set of common genetic approaches such as gene deletions or overexpressions.  However, knockouts are time-consuming to construct and difficult to rapidly transfer to other strains, which greatly lowers the throughput of strain engineering programs.  Therefore, in order to facilitate the identification of valuable knockout candidates, a method enabling the rapid construction of gene knockdowns is needed.  Here, we develop a new technique for the facile generation of knockdown phenotypes in yeast through RNA interference, which is able to confer gene knockdowns using a simple, portable, plasmid-based system.  By importing the RNA interference pathway from S. castelli and optimizing the design of the siRNA cassette, we have been able to achieve up to 93% downregulation of genes from a wide variety of initial expression levels.  Finally, we demonstrated the utility of this technique for metabolic production phenotypes in yeast.   This approach will significantly expedite yeast strain development and greatly expand our understanding of how native metabolism interfaces with heterologous pathways.