3.2. Loss-of-function mutation in GCR2 is responsible for the pho13-negative phenotype
To identify the molecular mechanism of the pho13 -negative phenotype of the YSX3 strain, genome sequencing of the YSX3 strain and its parental strain (L2612) was performed, and non-synonymous SNPs inYEN1 and GCR2 were identified (Fig. 1b). From the diploid of YSX3 and a D452-2 derivative, four haploid spores were dissected out, and two spores showed improved xylose fermentation by pho13 , whereas the other two spores did not. Sanger sequencing of the spores revealed that both pho13 -positive spores had wild-typeGCR2 , whereas pho13 -negative spores had the mutantGCR2 gene. Because the mutation in GCR2 resulted in the truncation of the protein (Glu204*), we assumed its loss-of-function mutation. When the deletion of GCR2 (gcr2 ) was tested in the SR7 strain, gcr2 mutant showed faster xylose consumption and higher ethanol yield compared to the control strain, but the double deletion of gcr2 and pho13 was not synergistic (Fig. 1c–e). These results suggested that the loss-of-function mutation inGCR2 is responsible for the pho13 -negative phenotype of the YSX3 strain. Also, it can be concluded that GCR2 is a novel deletion target to improve xylose fermentation.