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Figure S1. DNA sequences of the two promoters used in the cellobiose utilizing pathway in the wild type and evolved strains. Mutations are highlighted in cyan.

Figure S2. Cellobiose fermentation profiles of six recombinant yeast strains with defined promoter strengths for both cellobiose transporter and β-glucosidase. Four ENO2 promoters (100%, 8%, 14%, and 149% of the wild type promoter strength) and three PDC1 promoters (100%, 6%, and 3% of the wild type promoter strength) were selected from a library of promoter mutants and sorted using green fluorescent protein (GFP) as a reporter in glucose cultivation. Each of the six different cellobiose utilizing pathways was assembled into the SalI–NotI digested single copy plasmid pRS-kanMX. Culture conditions: recombinant yeast strains were cultured in YPAD medium to the exponential phase to prepare seed cells. The resulting cells were washed with cold distilled water2 times, directly transferred into 10 mL YPAC medium (8% cellobiose) in a 50 mL un-baffled shake flask, and shaken with 100 rpm at 30°C. No YPAC pre-culture was performed before the main cultivation to avoid any host adaptation. Significant lag phases were observed from this cultivation.

Figure S3. Evaluation of cellobiose toxicity. A: Cultivation of the Classic Turbo Yeast strain containing the wild type cellobiose utilizing pathway in YPAD (4% glucose) and YPADC medium (YPA medium supplemented with 4% glucose and 4% cellobiose). G418 was added in both mediums to ensure the selection pressure. B: Cultivation of the Classic Turbo Yeast strain containing both the cellobiose utilizing pathway (WT) and xylose utilizing pathway in YPAC (YPA medium supplemented with 4% cellobiose and G418), YPAX (YPA medium supplemented with 4% xylose and G418) and YPAXC (YPA medium supplemented with 4% xylose and 4% cellobiose and G418) media.

Figure S4. Quantitative measurements of intracellular and extracellular cellobiose and glucose in evolved strains cultured in YPAC medium.

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