Metabolic Engineering via RNAi-Assisted Genome Evolution (RAGE)

Tong Si, Han Xiao, Xiong Xiong, and Huimin Zhao

Departments of Chemical and Biomolecular Engineering, Chemistry, and Biochemistry, Bioengineering, Institute for Genomic Biology, and Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801

Engineering complex traits such as inhibitor tolerance and temperature tolerance remains a main challenge in metabolic engineering. In this talk we report the development of RNA interference (RNAi)-assisted genome evolution (RAGE) as a generally applicable method for engineering complex traits in yeast. Through iterative cycles of creating a library of RNAi induced reduction- of-function mutants coupled with high throughput screening or selection, RAGE can continuously improve target trait(s) by accumulating multiplex beneficial genetic modifications in an evolving yeast genome. We demonstrated RNAi screening in Saccharomyces cerevisiae for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation yku70Î?. We then showed the application of RAGE for improved acetic acid tolerance, a key trait for microbial production of chemicals and fuels. Three rounds of RAGE led to the identification of three gene knockdown targets that acted synergistically to confer an engineered yeast strain with one of the highest reported levels of acetic acid tolerance. In addition, we used RAGE to improve the furfural tolerance, another key trait for microbial production of chemicals and fuels from cellulosic materials. We discovered that disruption of SIZ1 gene encoding an E3 SUMO-protein ligase by knockdown or deletion conferred significantly higher furfural tolerance compared to other previously reported metabolic engineering strategies in S. cerevisiae. As a third example, we used RAGE to engineer a thermotolerant S. cerevisiae strain that grows well under 42C, a temperature at which the wild type strain grows poorly. Finally, coupled with in vivo biosensors, we used RAGE to improve the titers of polyhydroxybutyrate (PHB) and fatty acids, respectively. Taken together, RAGE represents a novel high throughput genome-scale engineering tool for engineering complex traits and improving production of chemicals and fuels in yeast.