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Figure S1. Vector map of the helper plasmid for CDT2 engineering, CDT-Eng-H.

Figure S2. A general scheme to engineer sugar transporters, using the colony size based screening method.

Figure S3. Characterization of the role of Q207H/F209I/N311H (HIH)mutations in cellobiose fermentation performance by creating all possible combinations, including Q207H, F209I, N311H, Q207H/F209I (HI), Q207H/N311H (HH), and F209I/N311H (IH).

Figure S4. Anaerobic cellobiose fermentation profiles of CDT2 mutants obtained in the first(HIH), second (HH), and third (HHT) round of directed evolution using the library screening conditions in rich media.

Figure S5. Mutant analysis by site-directed mutagenesis.

Figure S6. Cellobiose fermentation profiles of CDT2 on single copy plasmid (CEN/ARS Ori) and multiple copy plasmid (2µ Ori), respectively.

Figure S7. Aerobic cellobiose fermentation profiles of CDT1, CDT2, and CDT2 mutant (HHT).

Table SI. Oligonucleotides used in this study.

Table SII. Comparison of cellobiose fermentation performance of CDT1, CDT2, and CDT2 mutants obtained in the first(HIH), second(HH), and third(HHT) round of directed evolution using high cell density fermentation.

Table SIII. Saturation mutagenesis of CDT2 at the sites of Q207, N311, and I505.

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