As a microbial host for cellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway. However, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (pho13) has been reported to be a crucial genetic perturbation for improving xylose fermentation. A confirmed mechanism of the pho13-positive effect on xylose fermentation is that the deletion of PHO13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the present study, we reported that a pho13-positive effect was not observed from a couple of engineered strains, among the many others we have examined. To extend our knowledge of pho13-mediated metabolic regulation, we performed genome sequencing of pho13-negative strains. We identified a loss-of-function mutation in GCR2 responsible for the pho13-negative phenotype. Gcr2 is a transcriptional activator of the lower glycolytic pathway. Thus, the deletion of GCR2 (gcr2) led to deactivation of lower glycolysis as confirmed by RNA-seq. Also, gcr2 resulted in the up-regulation of PPP genes, which explains the improved xylose fermentation of gcr2 mutants. As pho13 and gcr2 cause similar transcriptional changes with PPP genes, there was no synergistic effect between pho13 and gcr2 for improving xylose fermentation. The present study identified GCR2 as a new knockout target to improve xylose fermentation and cellulosic biofuel production. Now published in Frontiers in Bioengineering and Biotechnology doi: 10.3389/fbioe.2021.654177