The strain of GLN1 overexpression associated with FPS1 deletion and GPD2 deletion produced 17.2 and 8.0% higher ethanol than control strain during xylose and glucose–xylose mixture medium. However, genetic manipulation did not increase the ethanol yield from xylose, and the xylitol yield of F106X was increased. This suggested that the ethanol production improvement was due to the xylose consumption rate increasing. To improve the ethanol production and decrease the xylitol formation, the recombinant strain should be further modified to solve the redox problem by protein engineering.