In-Depth Two-Stage Transcriptional Reprogramming and Evolutionary Engineering of Saccharomyces cerevisiae for Efficient Bioethanol Production from Xylose with Acetate

In order to achieve rapid xylose utilization in the presence of acetate, improved yeast strains were engineered for higher bioethanol production. First, a six-gene cluster, including XYL1/XYL2/XKSI/TAL1/PYK1/MGT05196, was generated by using an in-depth two-stage (glucose and xylose) transcription reprogramming strategy in an evolutionary adapted strain of CE7, resulting in two improved engineered strains WXY46 and WXY53. Through a combined screening of xylose and glucose stage-specific promoters between tricarboxylic acid (TCA)/HSP and constitutive types, respectively, WXY46 with the constitutive promoters showed a much higher ethanol yield than that of WXY53 with the TCA/HSP promoters. Second, an optimized strain WXY74 was obtained by using more copies of a six-gene cluster, which resulted in a higher ethanol yield of 0.500 g/g total sugars with acetate conditions. At last, simultaneous saccharification and co-fermentation were performed by using the evolved WXY74 strain, which produced 58.4 g/L of ethanol from wheat straw waste and outperformed previous haploid XR-XDH strains.