## 3 Discussion
### Robust Pullulan Synthesis by Strain ZH27 after Optimization
Consequently, the upregulation of the _UGP_ expression (2.2-fold) probably facilitated the efficient pullulan synthesis by strain ZH27 [2]. During pullulan synthesis under high sucrose concentrations, _Aureobasidium_ strains also produce by-products, mainly including FOSs, trehalose, and glycerol [20; 45]. FOSs synthesized by _Aureobasidium_ cells using sucrose were considered to lower its extracellular osmotic stress and pullulan titer [46]. However, FOSs would be subsequently hydrolyzed at late stages, contributing to pullulan accumulation [47]. The ability of strain ZH27 to hydrolyze FOSs could be attributed to the high expressions of hydrolases (e.g., \(\alpha\)-amylase, glucoamylase, and pullulanase) (Table 1). The hydrolyzed FOSs increased pullulan titers but reduced pullulan viscosity or molecular weight (Table S3), suggesting an efficient utilization of FOSs. These changes are consistent with the findings in previous report [47]. On the other hand, trehalose and glycerol are crucial osmolytes for intracellular osmotic stress alleviation [48]. They might be highly accumulated in the cells of strain ZH27 under the OC because of significantly upregulated expressions of its related genes (Table 1). This is similar to the upregulation of three genes (_GPP_, _TPS1_, and _TPP_) in strain TN3-1 (110 g/L pullulan) in comparison with strain P16 (65.3 g/L pullulan) [18]. These findings demonstrate that the strains of _Aureobasidium_ producing high levels of pullulan contained more glycerol and trehalose compared with those with a low pullulan-producing ability. Figure 6: Sensing and utilization of sucrose by strain ZH27 under the high osmotic stress for producing pullulan. HXT, hexose transporter; Gys, glycogen synthase; DHPA, dihydroxacetone phosphate. Other enzymes and regulatory factors are mentioned above in this study. [PERSON] and red arrows indicate activation and inhibition, respectively. However, their high expressions could divert the carbon flux away from pullulan synthesis. Consequently, a delicate balance may be maintained to ensure the optimal pullulan yield and simultaneously facilitate the production of necessary by-products. Furthermore, both pullulan and melanin secreted by _Aureobasidium_ cells are known to resist high osmotic stress [9]. More interestingly, melanin was not observed during the whole process of pullulan synthesis by strain ZH27 under the OC. The synthesis of metabolites (such as pullulan) and cell growth of _Aureobasidium_ strains are energy-intensive processes [16]. The upregulation of key enzymes (e.g., GluK, PFK, PYK, and PYD) involved in glycolysis in strain ZH27 under the OC (Table 1) indicates an enhanced glycolytic activity, potentially providing more acetyl-CoA for the tricarboxylic acid (TCA) cycle. This metabolic shift supports the energy-intensive processes of pullulan synthesis, cell growth, and by-product accumulation under the OC. Nevertheless, it contrasts with the effect of zinc sulfate addition, where the metabolic flux shifted away from glycolysis and the TCA cycle [49]. Pullulan synthesis, which is sensitive to high carbon source concentrations (Figure 2b), is suppressed by the glucose repressor CreA. Interestingly, the _CreA_ gene was upregulated under the OC (Table 1), whereas pullulan production was exactly improved (Figure 4). By contrast, when _CreA_ was downregulated in _A. melanogaster_ strains, the pullulan production was over 100 g/L [13; 18]. This difference may probably be due to evolutionarily genetic variations and different tolerances of strains to high osmotic stress. Therefore, we believe that strain ZH27 may produce a significantly higher pullulan titer under the OC after deleting _CreA_. AreA and AreB play direct roles in NCR and regulating pullulan synthesis [31]. The upregulation of _AreA_ and downregulation of _AreB_ in strain ZH27 under the OC indicate an efficient nitrogen utilization due to high C/N ratios under the OC compared with the IC (Figure 2b,c), which is consistent with those observed in strain P16 [31].