Genome-wide RNAi screen reveals the E3 SUMO-protein ligase gene SIZ1 as a novel determinant of furfural tolerance in Saccharomyces cerevisiae

Abstract

BackgroundFurfural is a major growth inhibitor in lignocellulosic hydrolysates and improving furfural tolerance of microorganisms is critical for rapid and efficient fermentation of lignocellulosic biomass. In this study, we used the RNAi-Assisted Genome Evolution (RAGE) method to select for furfural resistant mutants of Saccharomyces cerevisiae, and identified a new determinant of furfural tolerance.ResultsBy using a genome-wide RNAi (RNA-interference) screen in S. cerevisiae for genes involved in furfural tolerance, we identified SIZ1, a gene encoding an E3 SUMO-protein ligase. Disruption of SIZ1 gene function by knockdown or deletion conferred significantly higher furfural tolerance compared to other previously reported metabolic engineering strategies in S. cerevisiae. This improved furfural tolerance of siz1Δ cells is accompanied by rapid furfural reduction to furfuryl alcohol and leads to higher ethanol productivity in the presence of furfural. In addition, the siz1Δ mutant also exhibited tolerance towards oxidative stress, suggesting that oxidative stress tolerance related proteins may be under the SUMO regulation of SIZ1p and responsible for furfural tolerance.ConclusionsUsing a genome-wide approach, we identified a novel determinant for furfural tolerance, providing valuable insights into the design of recombinant microbes for efficient lignocellulose fermentation.

Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration

“…Oligonucleotides used for gene amplification and pathway assembly are listed in Supplementary Table S2. HIS3 , TRP1 , LEU2 , URA3 , KanMX , and HygB expression cassettes with variable length of promoters were amplified from pRS423, pRS424, pRS425, pRS426, pUG6, and pXZ5 (Xiao and Zhao, ), respectively, and then cloned into pH5 linearized by Tth 111I (Supplementary Fig. S1).…”

Rational design and evolutional fine tuning of Saccharomyces cerevisiae for biomass breakdown

“…Both the generation of beneficial mutations and the removal of deleterious mutations in the genome affect the stress tolerance of yeasts. A recent study using RNAi-assisted genome evolution (RAGE) revealed that SIZ1, encoding an E3 small ubiquitin-like modifierprotein ligase, is a determinant of furfural tolerance in S. cerevisiae [21 ]. RAGE can enhance a desired trait by allowing the sequential introduction of tractable reduction-of-function modifications into the yeast genome.…”

“…Because these compounds negatively affect microbial growth, metabolism, and product yield, more robust strains have been developed using various molecular technologies, such as evolutionary engineering [17,18], mutagenesis [19], genome shuffling [19], meiotic recombination [20], and metabolic engineering [17,21 ]. Recent advances in 'multi-omics' techniques have enabled researchers to greatly enhance the understanding of the effects of environmental perturbations on microbial metabolism, facilitating the design of rational strategies for conferring stress tolerance on host strains.…”

Whole‐Genome Regulation for Yeast Metabolic Engineering

“…The 100% inhibitory HAc concentration for growth increased from 0.8% to 1.0%. RAGE has also been used successfully to increase furfural tolerance of yeast strains . These results showed that with iterative RAGE cycles, the traits of strains can be continuously improved by accumulating beneficial modifications.…”