## 4 Discussion
On the other hand, the transcription levels of _FBA1/LOT1_, _TDH1/GLD3_, _TDH2/GLD2_, and _TDH3/GLD1_ at the borderline of preparatory and pay-off in glycolysis were depressed in WG+HLMU medium, strongly indicating that BY4741 could activate other metabolic pathways instead of the Embden-Meyerhof glycolysis pathway. [PERSON] et al. explained that eucaryotic cells could inactive TDH functions as a metabolic switch for rerouting the carbohydrate flux to counteract environmental changes [41], suggesting that _S. cerevisiae_ cells tried to shift metabolic flow from glucose to the pentose phosphate pathway via \(\beta\)-d-fructose-6P by replacing YNB with wheat bran. Therefore, a detailed evaluation of the transcription levels in the pentose phosphate pathway was profoundly desired. When wheat bran was used instead of YNB, the gene transcription levels of _PCKI/JPM2/ PPC1_ were enhanced, suggesting that carbon flow could be introduced from the TCA cycle to phosphoenolpyruvate-mediated glycolysis (Figure 4b). [PERSON] et al. indicated that PCK1/JPM2/ PPC1 could perform as a decarboxylase controlling the reactions related to gluconeogenesis, and the transcription of these genes could also be induced by glucoeneogenic substrates [42]. The strain might also induce gluconeogenesis when bran was used. Under the medium-condition using wheat bran instead of YNB, _S. cerevisiae_ could be about to begin carbon source starvation at the evaluation timing. Thus, the strain can improve growth and production by increasing the concentration of glucose to avoid carbon source starvation. In addition, the increase in the transcription levels of the isoenzymes _AC51/FUN44_ and _ACS2_ indicated the influx into the TCA cycle via acetate. This may indicate that the cells were trying to take in the previously produced ethanol as a carbon source. In fact, in 24 h fermentation, the concentration of ethanol in WG+HLMU medium was 69.5 wt% compared to that in YG+HLMU medium. To date, there have been many reports that BY4741 utilizes the ethanol produced by itself as a carbon source [43], and this acetic acid-mediated metabolic flow from ethanol could be likewise enhanced in the case of using wheat bran. In addition, since BY4741 is also able to regenerate NADH from NAD\({}^{+}\) in the metabolic pathway from acetaldehyde to acetic acid [44], the strain could use this alternative pathway instead of the pathway regulated by the repressed gene transcriptions of _TDH1/GLD3_, _TDH2/GLD2_, and _TDH3/GLD1_, which similarly control NADH production. Regarding ethanol production, the _ADH1_ transcription level decreased significantly. Although no significant difference in ethanol production efficiency was confirmed in this study, the significant difference between the _ADH_ transcription levels in WG+HLMU medium and YG+HLMU medium could affect metabolism because of ADH1's activity of regenerating NADH from NAD\({}^{+}\)[45; 46]. In the case of using wheat bran instead of YNB as the medium component, the gene transcription levels of _PYC1_ and _PYC2_ were high, with significant differences (Figure 5). Similarly, the transcription levels of _CIT1_ and _CIT3_ remained high, suggesting that carbon flow via pyruvate into the TCA cycle was induced. In particular, significantly high transcription levels of _PYC1_ and _PYC2_ possibly indicate that the cells wanted to introduce a carbon source not only into the TCA cycle but also into the amino acid synthesis pathway via aspartate. In terms of the amino acid synthesis pathway, by using wheat bran instead of YNB, the transcription levels around 2-oxoglutarate tended to be activated as a whole, indicating the possibility that amino acid synthesis such as glutamic acid was activated. To clarify the above, evaluation of the transcription levels of these pathways would be highly desirable. In the case of using wheat bran as an alternative nutrient to YNB, the gene transcription levels of isozyme genes _TKL_ were increased (Figure 6a).