Fig. 3: iMAGIC for the construction of a furfural tolerant yeast strain.

a Furfural tolerance of the engineered strains identified in each round of iMAGIC screening, R1, R2, and R3. The cell densities of the engineered strains were normalized to the wild-type (WT) strain under the specified conditions (red bars for 7.5 mM furfural, blue for 12.5 mM, and purple for 17.5 mM). b Verification of gain-of-function and reduction-of-function mutations by qPCR. The expression level of each target (SIZ1, NAT1, and PDR1) was compared before (NC, red) and after (INT, blue) CRISPRa or CRISPRi cassette integration. Fermentation profiles including cell density c, glucose consumption d, ethanol production e, as well as furfural and furfuryl alcohol (FfOH) concentration f of WT (black square and blue triangle) and R3 (red circle and purple diamond) in synthetic medium with (blue triangle and purple diamond) or without (black square and red circle) the supplementation of 17.5 mM furfural (Ff). A single colony of WT or R3 was inoculated into 3 mL SED/G418 medium and cultured until saturation, which was then transferred into 50 mL fresh SED/G418 medium with or without the supplementation of 17.5 mM furfural in a 250 mL un-baffled shaker flask. Fermentation was performed under oxygen-limited conditions (30 °C and 100 rpm), and samples were taken every 24 h. The decrease of furfural concentration in WT might result from evaporation, as no growth and furfuryl alcohol production were observed. Notably, the cell density (biomass accumulation) in f was determined by measuring the absorbance at 600 nm using a UV–vis spectrometer. Error bars represent the mean ± s.d. of biological triplicates. The source data are provided as a Source Data file.