## 3 Discussion
### Robust Pullulan Synthesis by Strain ZH27 after Optimization  
Pullulan titers generally depend on high concentrations of carbon sources [29]. In the quest for high pullulan production, we optimized several key factors. Notably, strain ZH27 exhibited a preference for 15% sucrose as the optimal carbon source, which is a concentration rarely reached by wild-type strains (Figure 2a,b). Interestingly, except for mutant M233-20, which evolved from whole-cell mutagenesis to be able to convert 20% glucose into pullulan [14], 15% sucrose was the highest concentration of single sugar (Figure 2b) used to produce pullulan by wild-type strains so far [1; 7]. It was found that the wild-type strain ZH27 showed tolerance to hyper-osmotic stress. Its pullulan accumulation was significantly and negatively influenced by (NH4)2 SO4 concentrations, which is sensitive to (NH4)2 SO4 concentrations due to occurrence of NCR at 0.12% (NH4)2 SO4 (Figure 2c), while _A. pullulans_ CCTCC M201225 and _A. melanogaster_ P16 exhibit NCR at 0.24-0.30% (NH4)2 SO4 [30; 31]. Thus, a low concentration of (NH4)2 SO4 (0.01%) was found to be preferable for pullulan production, redirecting carbon flux toward metabolite synthesis rather than biomass formation by activating some enzymes and enhancing the ATP supply [31]. K2 HPO4, which is a crucial factor known to influence pullulan production [19; 32], did not significantly impact strain ZH27 to produce pullulan, highlighting differences in sensitivities between different strains and their regulation to the genes involved in pullulan synthesis (Figure 2d) [2; 13]. The pullulan production of strain ZH27 was also sensitive to changes in NaCl concentrations, with the highest pullulan titer (67.8 \(\pm\) 2.5 g/L)observed at 0.4% NaCl and an increase of 11% compared with the control at 0.1% NaCl (Figure 2e). This change is similar to a previously reported increase of 26.7% at 0.3% NaCl [33]. Interestingly, the highest biomass of strain ZH27 occurred at 0.5% NaCl, likely due to the formation of small molecules (e.g., glycerol) for self-protection against high salinity and osmotic stress. Consequently, enhancing carbon flow toward biomass replaced pullulan synthesis [34]. Moreover, high expressions of some enzymes (e.g., amylases) induced by high NaCl concentrations probably contributed to the reduction in pullulan titers [33]. These findings highlight the complicated interplay of factors influencing pullulan production in strain ZH27. Many _Aureobasidum_ strains optimally produce pullulan within the temperature range of 24-30 \({}^{\circ}\)C [6]. Nevertheless, a temperature suitable for cell growth may not always favor metabolite synthesis. For instance, _A. pullulans_ CGMCC1234 grew well at 32 \({}^{\circ}\)C but favored producing pullulan at 26 \({}^{\circ}\)C, implying that a two-step temperature regulation might enhance pullulan production [35]. Interestingly, [PERSON] et al. (2019) reported that the optimal temperature for both cell growth and pullulan synthesis of _A. pullulans_ LB83 was the same (25 \({}^{\circ}\)C) [36]. Hence, temperature is considered an important regulator for pullulan synthesis. Indeed, the pullulan yield was remarkably restricted at lower initial pH values (<5.0) or higher ones (>7.0), indicating that initial pH values sensitively influence the pullulan-producing process of strain ZH27 (Figure 2g). At different pH values, pullulan-producing strains may exhibit specific cell types that are likely required for pullulan synthesis. These findings emphasize the importance of precise environmental conditions for efficient pullulan synthesis. Recently, a few wild-type strains have achieved pullulan levels over 100 g/L (Table 2). Several _A. melanogaster_ strains can produce high pullulan titers (110-122.3 g/L), but some of them have very low yields (<0.5 g/g) [10; 13; 18]. Therefore, it is still urgent to mine novel strains to produce pullulan with high titer, high yield, and high productivity.