1.   Introduction
Cellulosic biofuels are renewable liquid-fuel alternatives due to abundant feedstock availability and substantial CO2 emission reduction (Lynd, 2017). Saccharomyces cerevisiae plays an essential role in production of cellulosic biofuels by fermenting cellulosic sugars, mainly glucose and xylose, which requires engineering of the yeast via a heterologous xylose pathway (Kim et al., 2013c; Richa et al., 2019). Current efforts on the metabolic engineering of the yeast are still focused on improving the xylose fermentation yield and productivity under multiple stress conditions of lignocellulosic biomass hydrolysates (Park et al., 2020; Qin et al., 2020).
Previously, an efficient xylose-fermenting strain of S. cerevisiae (SR8) was developed through the introduction of a heterologous xylose pathway, optimization of its expression levels, and adaptive evolution, which resulted in a loss-of-function mutation on PHO13 (Kim et al., 2013d). Continued efforts have discovered that the deletion of PHO13 (pho13) resulted in transcriptional and metabolic changes favorable to xylose and other C5 sugar fermentation (Kim et al., 2015; Xu et al., 2016; Ye et al., 2019).  However, as PHO13 was first discovered as a knockout target to improve xylose fermentation (Ni et al., 2007; Van Vleet et al., 2008), detailed molecular mechanisms underlying the pho13-phositive phenotype remained unelucidated. The most advanced finding thus far is that pho13 results in the transcriptional activation of non-oxidative pentose phosphate pathway (PPP) genes, which therefore facilitates xylose metabolism (Xu et al., 2016).
However, through this study pho13-positive effect on xylose fermentation was seen as strain background-dependent, and one factor is associated with a loss of function mutation in GCR2 coding for a transcriptional activator of the genes in glycolysis. Gcr2 enhances the CT box-dependent transcriptional activation of a Rap1-Gcr1 complex required for the expression of glycolytic genes (Huie et al., 1992). As a transcriptional activation of complex, Rap1 and Gcr1 provide the specific DNA-binding and the activation of glycolytic and ribosomal genes, respectively (Sasaki et al., 2005; Uemura and Fraenkel, 1990). Function of Gcr2 is to provide an activation domain to the Gcr1p-Gcr2p complex mediating high level of glycolytic gene expression (Uemura and Jigami, 1992). However, it is unknown how the regulatory systems would function if new foreign pathways, such as the heterologous xylose-assimilating genes, are introduced.
Therefore, the aim of the current study was to understand the strain specific pho13-positive effect by investigating transcriptomic changes of gcr2 mutant during glucose and xylose fermentation. The result suggests that the native regulator system, primarily the transcriptional regulations, is highly associated with the suboptimal xylose fermentation by xylose-fermenting S. cerevisiae.
2.1. Strain construction
All S. cerevisiae strains used in the present study are listed in Table 1. To construct xylose-fermenting strains, the linear expression cassette of Scheffersomyces stipitis XYL1 , XYL2 , and XYL3 genes was used as described previously (Kim et al., 2013d). To constructpho13 mutants of the xylose-fermenting strains, thepho13 ::KanMX4 cassette was used as described previously (Kim et al., 2013d). To isolate spores from the KSM diploid strain, tetrad dissection was performed as described previously (Kim et al., 2017). To construct the gcr2 mutant, the gcr2 ::KanMX4 cassette was amplified from the genomic DNA of the BY4742 gcr2 strain (clone ID: 12013) of the Yeast Knockout Collection (Thermo Fisher Scientific, USA) by polymerase chain reaction (PCR) using SOO303/298 primers. The PCR product was purified and genome-integrated to the SR7 strain by the LiAc transformation method (Gietz and Schiestl, 2007). The resulting deletion mutant was selected on an agar medium containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose (YPD), 15 g/L agar, and 300 mg/mL G418 sulfate (GoldBio, St. Louis, MO, USA).