Promoter engineering to optimize expression of the heterologous cellobiose-utilizing pathway. (A) Identification of strong promoters from transcription profiling of cellobiose-grown (C8) and glucose-grown (G8) cultures. (B) Verification of promoter strengths by measuring green fluorescent protein (GFP) fluorescence using flow cytometry. Anaerobically grown cells on cellobiose were harvested at mid-exponential phase and analyzed. The cell surface density of enhanced green fluorescent protein (eGFP)-tagged CDT-1 is shown. (C) Construction of a promoter library of 4 promoters and 2 gesnes for expression of the cellobiose-utilization pathway. The plasmids pRS316 (CEN URA) and pRS315 (CEN LEU) were used to express cdt-1 and a codon-optimized version of N. crassa gh1-1 (gh1-1a), respectively. (D) Comparison of cellobiose-consumption rates (qsmax) using strains expressing the cellobiose-utilization pathway from the promoter library. The starting OD600 of 1 was used. The promoters for each gene are shown, and the rates are color-coded by relative rates. Fermentation parameters were calculated from the fermentation profiles in Fig. S4D.