## 3 Discussion
### Robust Pullulan Synthesis by Strain ZH27 after Optimization
The distinct features of vacuoles in the swollen cells of strain ZH27 compared with strain TN3-1 may account for the resistance to high osmotic stress and high levels of pullulan production [18]. It was found that _A. pullulans_ NG grew well under the nutrient-rich condition but produced mycelia and chlamospores under the nutrient-poor condition; while the osmotic stress prevented the differentiation of yeast-like cells into two types of the above-mentioned cells [41]. Both one type of cells (e.g., yeast-like cells [13], swollen cells [40], or arthoconiaia [18]) and two types of cells (e.g., yeast-like cells and swollen cells) [9] were conducive to pullulan synthesis. However, the exact cell type responsible for pullulan synthesis in _Aurebsidsimu_ strains remains unclear [1]. Moreover, the pH is identified as another factor that influences cell morphology and pullulan yield [42]. Yeast-like cells as core cells mainly occurred at the beginning of cultivation with higher pH values (\(\geq\)6.0), while low pH values induced the differentiation of yeast-like cells into swollen cells [22]. The regulation of pH values seems to be a crucial factor in determining the cell types during the cultivation period. Under high osmotic stress, genes related to the syntheses of pullulan and by-products in strain ZH27 underwent some significant changes [1] (Figure 6). First, enhanced expressions of glucose transporters and SUC2 under the OC suggest an efficient transport of sucrose and newly generated glucose into cells, which agrees with the way sucrose enters the cells of _A. pullulans_ for polymalate synthesis [43]. Then, all of the genes involved in pullulan synthesis were upregulated except PGM under the OC, which contributed to the high level of pullulan production (Table 1). For instance, the high expressions of _Apo_ and _Gltp_ (1.54-3.78 folds) responsible for the formation of Lph-glucose seemed to promote pullulan synthesis (Table 1). These observations are consistent with the previous report stating that the over-expression of _Apo_ resulted in an increase of \(\sim\)10% in pullulan titer [44]. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Strains**} & \multicolumn{3}{c}{**Fermentation**} & \multirow{2}{*}{**Production g/L**} & \multirow{2}{*}{**Yield (g/g)**} & \multirow{2}{*}{**References**} \\ \cline{2-2} \cline{4-6}  & **Carbon** & & & **Mode and Conditions** & **(g/L/h)** & **Yield (g/g)** & **References** \\ \hline _A. Pullulans_ M233-20 \({}^{\text{a}}\) & Glucose & Batch, 28 \({}^{\circ}\)C, 500 rpm, 20 L/min, 144 h & 162.3 (1.13) & 0.82 & [14] \\ _A. pullulans_ LB83 & SBH & Batch, 25.3 \({}^{\circ}\)C, 232 rpm, 96 h & 25.2 (0.28) & 0.48 & [36] \\ _A. melanogaster_ V6 \({}^{\text{a}}\)/F44 \({}^{\text{a}}\) & Sucrose & Batch, 28 \({}^{\circ}\)C, 180 rpm, 144 h & 102.3 (0.7)/ & 0.89/ & [38] \\ _A. melanogaster_ & A. melanogaster & A. melanogaster & Glucose & Batch, 28 \({}^{\circ}\)C, 300 rpm, 6.5 L/min, 120 h & 103.5 (0.86) & 0.75 & [39] \\ _A. melanogaster_ DG41 \({}^{\text{a}}\) & Glucose & Batch, 28 \({}^{\circ}\)C, 205 rpm, 8.0 L/min, 132 h & 64.9 (0.49) & 0.55 & [25] \\ _A. Pullulans_ & Sucrose & Fed-batch, 28 \({}^{\circ}\)C, 500 rpm, 120 h & 140.2 (1.17) & \(<\)0.5 & [11] \\ BL06 \(\Delta\)PMAs \({}^{\text{a}}\) & & Batch, 28 \({}^{\circ}\)C, 200–220 rpm, 4.5–5.5 & 115.4 (0.87) & 0.77 & This study \\ _A. melanogaster_ ZH27 & Sucrose & L/min, 132 h & 115.4 (0.87) & 0.77 & This study \\ \hline \hline \end{tabular}  
* Notes: SBH, sugarcane bagasse hydrolysate; \({}^{\text{a}}\), mutants.  
\end{table} 
Table 2: _Cont._The upregulated expression of _Ags2_ (3.7-fold) would probably accelerate the formation and transportation of pullulan precursors and facilitate pullulan synthesis [15]. During pullulan synthesis, UDPG transformed by _UGP_ could also synthesize several products (e.g., trehalose and glycogen), but it was the sole precursor for pullulan synthesis.