(12f) Fine-Tuning of Gene Expression in Saccharomyces Cerevisiae Through RNA Interference

Si, T., University of Illinois at Urbana Champaign
Zhao, H., University of Illinois at Urbana-Champaign

Beyond gene overexpression or knockout, fine-tuning of gene expression is often required to optimize a target phenotype.  This requirement results from the need to balance the trade-off between catalytic activity and metabolic burden, or to avoid accumulation of toxic metabolic intermediates.  RNA interference (RNAi) is an effective gene silencing mechanism through specific degradation of target messenger RNAs (mRNAs).  But there is no functional RNAi pathway in the eukaryotic model S. cerevisiae until recently, when a heterologous RNAi pathway from Saccharomyces castellii was reconstituted.  Here we report the design of different RNAi expression constructs to achieve a continuum of gene knockdown levels in S. cerevisiae.  By varying the length and position of the anti-sense RNAs or the double-stranded RNAs expressed under the control of strong constitutive type II promoters, 10% to 80% gene silencing levels were achieved for green fluorescence protein (GFP).  We further modulated the gene expression of ptc6 by RNAi constructs for improved acetic acid (HAc) tolerance, a key trait for microbial production of chemicals and fuels.  An optimal knockdown level was identified which resulted in the highest acetic acid tolerance among the knockout, knockdown and wild type strains.  This is the first example in S. cerevisiae where RNAi is employed as a gene regulatory tool for metabolic engineering.  Our results also provided important insights on the design principles of RNAi in yeast.