## 2 Results
### Probable Reasons for Hyper-Production of Pullulan
#### 2.5.2 Expression Changes of the Genes in Pullulan Synthesis and Carbon Metabolism  
Compared with the transcriptional gene expression of strain ZH27 under the IC, quantitative real-time PCR (qPCR) tests revealed significant transcriptional changes in its genes involved in carbon metabolism and pullulan synthesis under the OC. During the stage where sucrose entered the cells, three genes encoding glucose transporters and _SUC2_ encoding the sucrose-proton symporter were significantly upregulated under the OC (Table 1). Most genes associated with carbon metabolism, pullulan synthesis and regulation showed upregulated expressions, including _GluK_ encoding glucose kinase, _UGP_ encoding UDP-pyrophosphoryylase, _UGT1_ encoding UDP-glucoseyltransferase, _Ags2_ encoding \(\alpha\)-glucan synthase 2, and _PUL1_ encoding pullulan synthetase, which were upregulated by 18.4-, 2.15-, 46.4-, 3.7-, and 4.4-fold, respectively (Table 1). Additionally, the transcriptional levels of glycolipid transfer protein genes _Apo_ and _Gltp_ were also upregulated by 1.54- and 3.78-fold, respectively (Table 1). These findings indicate enhanced gene expression levels in most steps of the proposed pullulan synthesis pathway. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Genes**} & \multirow{2}{*}{**Contents/Functions**} & \multicolumn{2}{c}{**Relative Gene Expressions of Strain ZH27 (\%)**} & \multicolumn{2}{c}{**Changes in Gene**} \\ \cline{3-5}  & & **IC** & **OC** & **Expressions under the OC** \\ \hline _GluT1_ & Glucose transporter & 100 & \(70.9\pm 14.3\) * & Downregulation \\ _GluT2_ & Glucose transporter & 100 & \(1386.1\pm 485.8\) ** & Upregulation \\ _GluT3_ & Glucose transporter & 100 & \(69.5\pm 3.3\) ** & Downregulation \\ _GluT4_ & Glucose transporter & 100 & \(857.1\pm 126\) ** & Upregulation \\ _GluT5_ & Glucose transporter & 100 & \(2894.9\pm 750.2\) ** & Upregulation \\ _SUC2_ & Sucrose-proton symporter 2 & 100 & \(1432.9\pm 364.1\) ** & Upregulation \\ _GluK_ & Glucose kinase & 100 & \(1844.6\pm 499.4\) ** & Upregulation \\ _PGM_ & Phosphoglucues mutase & 100 & \(118.6\pm 20.3\) & No significance \\ _UGP_ & UDPG-pyrophosphoryylase & 100 & \(215.4\pm 29.9\) ** & Upregulation \\ _UGT1_ & UDP-glucoseyltransferase & 100 & \(4742.8\pm 508.5\) ** & Upregulation \\ _PUL1_ & Pullulan synthetase & 100 & \(438.9\pm 70.7\) ** & Upregulation \\ _Ags2_ & Alpha-glucan synthase 2 & 100 & \(367.5\pm 77.4\) ** & Upregulation \\ _Msn2_ & Stress-responsive transcriptional & 100 & \(228.6\pm 13.5\) ** & Upregulation \\ _Apo_ & Glycolipid transfer protein & 100 & \(153.5\pm 32.4\) * & Upregulation \\ _Gltp_ & Glycolipid transfer protein & 100 & \(377.7\pm 98.6\) ** & Upregulation \\ _CrrA_ & Regulation of gene transcription & 100 & \(222.9\pm 22\) ** & Upregulation \\ \(\alpha\)-amylase gene & Hydrolysis of \(\alpha\)-1,4-linkage & 100 & \(3938.8\pm 200\) ** & Upregulation \\ Glucamylase & Hydrolysis of \(\alpha\)-1,4-and & 100 & \(2040.8\pm 281.3\) ** & Upregulation \\ gene & \(\alpha\)-1,6-linkages & & & \\ Pullulanase gene & Hydrolysis of \(\alpha\)-1,6-linkage & 100 & \(1262.1\pm 174\) ** & Upregulation \\ _TPS1_ & \(\alpha\)-trehalose-6-phosphate & 100 & \(496.4\pm 50.1\) ** & Upregulation \\ _TPP_ & Trehalose-6-phosphate & 100 & \(278.7\pm 42.5\) ** & Upregulation \\ _GPP_ & Glycerol-3-phosphatase & 100 & \(1341.7\pm 278.3\) ** & Upregulation \\ _GPD_ & Glycerol-3-phosphate & 100 & \(376.3\pm 56.4\) ** & Upregulation \\ _Hgg1_ & Hyper-osmotic glycerol & 100 & \(256.7\pm 33.7\) ** & Upregulation \\ _Sho1_ & High osmolarity signaling & 100 & \(161.7\pm 4.0\) ** & Upregulation \\ _FF1_ & Fructosyltransferase & 100 & \(373.4\pm 174.8\) ** & Upregulation \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Comparison of transcriptional levels of the genes involved in the syntheses of pullulan, trehalose, glycerol, and metabolism in strain ZH27 between the OC and IC. Moreover, the expressions of genes related to the synthesis of by-products and metabolism also underwent some changes.