# Proteomics Analysis of _Zygosaccharomyces mellis_ in Response to Sugar Stress
## 3 Results
### Effect of Sugar Stress on the Growth of BY4741\(\triangle\)scFDH1 and BYzm4717 FDH1  
The OD values of BY4741, BY4741\(\triangle\)scFDH1 and BYzm4717 FDH1, which were grown in 20% and 60% glucose YPD for 3 d, respectively, were determined every 6 h (Figure 2A). The results revealed that there was no significant difference among these three strains in 20% glucose YPD at 24 h. The OD value of BY4741\(\triangle\)scFDH1 was significantly reduced compared with BY4741 in 60% glucose YPD (\(p<0.01\)) at 24 h, while BYmzFDH1 resumed its regular growth pattern, showing no difference from BY4741.  
\begin{table}
\begin{tabular}{c c c} \hline
**Protein\_ID** & **Description** & **60\% Glucose Group vs. 2\%** \\  & & **Glucose Group** \\ \hline Antioxidant & & \\ GCE98999.1 & superoxide dismutase (Mn), & \\ GCE99208.1 & mitochondrial & 1.21 \\ Ergosterol synthesis & & \\ GCE98125.1 & phosphomevalonate kinase & 0.64 \\ GCE98064.1 & diphosphopholipid:diacylglycerolglycerol & 0.68 \\ GCE98950.1 & acyltransferase & 0.77 \\ GCF01680.1 & squalene epoxidase & 0.8 \\ GCE99731.1 & erg10, acetyl-CoA C-acetyltransferase & 1.61 \\ GCE99856.1 & C-24(28) sterol reductase & 1.49 \\ GCE99284.1 & RNA polymerase C-22 sterol & 1.52 \\  & desaturase & \\ GCE97050.1 & farnesyl pyrophosphate synthetase & 0.66 \\ Cell wall & & \\ GCE99669.1 & UDP-glucose-4-epimerase & 0.48 \\ GCE98182.1 & UDP-\(N\)-acetylglucosamine & 0.73 \\ GCE99673.1 & chitin synthase, class 3 & 0.75 \\ GCE99446.1 & glutamine-fructose-6-phosphate & 0.77 \\  & transaminase & \\ GCE99630.1 & chitin synthase, class 1 & 0.82 \\ GCE97781.1 & Peroxin & 0.75 \\ NADH generation & & \\ GCE97061.1 & formate dehydrogenase 1 & 1.71 \\ GCE97099.1 & formate dehydrogenase 1 & 1.84 \\ GCE98400.1 & formate dehydrogenase 1 & 10 \\ \hline \end{tabular}
\end{table}
Table 3: Cont.  
As shown in Figure 2B,C, the intracellular glycerol and trehalose contents in yeasts under high sugar stress were measured. Our results showed that glycerol and trehalose in _Z. mellis_ accumulated in yeasts significantly during fermentation with 60% glucose YPD (Supplementary Figure S1). In comparison to BY4741, the amount of intracellular glycerol, as well as the activity of SOD, CAT and POD, decreased in BY4741\(\triangle\)scFDH1, but the amount of intracellular glycerol and trehalose, as well as POD activity, increased in BYzm4717 FDH1.  
Figure 1: Strain construction. (**A**): Schematic of construction of BY4741\(\triangle\)scFDH1 and BY4717 mzFDH1 strains. (**B**): PCR amplification. (1) Product of the homology arms; M: maker; down: down repair homologous arm; up: up repair homologous arm. (2) Confirmation of BY4741\(\triangle\)scFDH1 strain by PCR using primers FDH1-Up-F and FDH1-Down-R; M: marker; 1: negative colony; 2: positive colony. (3) Confirmation of BY4741\(\triangle\)scFDH1 strain by PCR using primers FDH1-KpnI-F and H1-BamHI-R; (4) PCR result of BYmzFDH1 strain; M: marker; 1: negative colony; 2: positive colony. (**C**): Colonies of BY4741, BY4741\(\triangle\)scFDH1 and BYmzFDH1 strains; M: marker; 1: negative colony. (**D**): The sites of the primers.  
As shown in Figure 3, the relative expression of _FDH1_ and the contents of ATP and NADH in yeasts were measured. The _FDH1_ gene was almost not expressed in BY4741\(\triangle\)scFDR11 but was expressed normally in BYzm4717 FDH1, indicating that _FDH1_ was successfully knocked out in BY4741\(\triangle\)scFDR11 and expressed in BYzm4717 FDH1. Compared with BY4741, the ATP and NADH contents decreased in BY4741\(\triangle\)scFDR11, but there was no difference in BYzm4717 FDH1, indicating that FDH1 has a significant effect on ATP and NADH synthesis.