## 3 Results
### Identification of Upresulated Genes in DaMDHAR-Expressing Yeast Cells to FT2 Stress  
Transcriptome analysis using 3'-quant RNA-Seq was used to investigate whether heterologous _DaMDHAR_ expression contributes to stress tolerance in FT2 stress by upregulating single gene and gene cluster in transgenic yeast cells. A genetic network analysis was performed using a bioinformatic tool of ClueGO because it performs single cluster analysis, and the comparison of several clusters uses a Cytoscape plug-in that visualizes the non-redundant biological terms for large clusters of genes in a functionally grouped network [50; 53]. Among 197 upregulated genes, DM cells induced the expression of genes involved in metal ion homeostasis (_FIT1_, _GEX1_, _ISUI_, and _MAM3_), an integral component of mitochondrial membrane (_GEM1_, _MPC3_, and _YDL183C_), an integral component of PM (_GAP1_, _HXT2_, _MEP1_, _MMP1_, _MUP1_, _PHO84_, _PNS1_, and _UGA4_), carboxylic acid transport (_AT03_, _GAP1_, _MMP1_, _MPC3_, _MUP1_, _PUT4_, and _UGA4_), sulfate assimilation and reduction, methionine biosynthesis, sulfide biosynthetic process, cysteine biosynthesis, and amino acid metabolic process including aspartate (_MET1_, _MET10_, _MET14_, _MET16_, _MET3_, _MET12_, _STR3_, _XBP1_, and _SOK2_), NADP metabolic process (_ALD3_, _ALD4_, _ALD6_, _GPP2_, _NDE2_, and _GPD2_), PPP (_GND2_, _NQM1_, and _TKL2_), and \(\beta\)-alanine metabolism (_ALD4_, _ALD6_, and _ALD3_) (Figures 3 and 4; Table 1 and Tables S2-S4). In particular, _ALD4_, _ALD6_ and _ALD3_ genes of aldehyde dehydrogenase (ALD) were related to amino acids biosynthesis, including histidine, tryptophan, and pyruvate, and glycerolipid metabolism for glycerol synthesis (_GPP2_) as well as glycolysis/glucoseogenesis (Figure 4; Table S4). Interestingly, _CTT1_ encoding cytosolic CAT networked to the cluster of \(\beta\)-alanine metabolism via tryptophan metabolic process. Figure 2: Heat map (**A**) and gene cluster via genetic network analysis based on gene ontology (GO) (**B**) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (**C**) of twofold changed genes in _DaMDIAR_-expressing yeast cells under repeated FT (FT2) stress. Figure 4: ClueGO-based genetic network analysis of upregulated genes involved in sulfate assimilation, NADP metabolic process, and \(\beta\)-alanine metabolism in _DaMDHAR_-expressing yeast cells under repeated FT (FT2) stress. Figure 3: ClueGO-based genetic network analysis of upregulated genes associated with the pentose phosphate pathway, sulfur metabolism, and integral component of mitochondrial component and plasma membrane in _DaMDHAR_-expressing yeast cells under repeated FT (FT2) stress. Besides genetic network, the activated genes are as follows: _FRE8_ encoding putative ferric-chelate reductase, _GTO3_ encoding \(\omega\)-class GSH transferase, _PUT4_ encoding proline permease, _CTT1_ encoding cytosolic CAT, _YOL024W_ encoding thiol-sulfide oxidoreductaseate active site, _GEX1_ encoding proton-GSH exchanger, _SPG8_ encoding protein required for survival against heat shock, _GAP1_ encoding general amino acid permease, _ATO3_ and _MEP1_ encoding ammonium permease, _PDR15_ encoding ATP-binding cassette multidrug transporter, and _SIP18_ encoding phospholipid-binding hydrophilic (Table 1 and Table S2). By contrast, _DaMDHAR_ overexpression in DM cells upregulated transcription factors (_CIN5_ and _MSNA4_), putative maltose-responsive transcriptional factor (_YPR196W_), and transcription repressor (_XBP1_ and _ROX1_) under FT2 stress (Table 1). With respect to organelles, DM cells also upregulated multiple genes involved in the functionality and stabilization of mitochondria (_ALD4_, _CRC1_, _FMP45_, _GEM1_, _NDE2_, _MR54_, _SCM4_, _DP18_, _YDL183C_, _MBRI_, _ISUI1_, _MIN3_, _MAM3_, _SHH4_, and _MPC3_), PM (_YDR034W-B_, _BDH2_, _HSP12_, _ST2_), and PM (_YDR183C_, _MDRI_, _ISUI1_, _MIN3_, _MAM3_, _SHH4_, and _MPC3_).