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To investigate the role of the sugar transporter MAL31 on pullulan biosynthesis, the coding gene mal31 was respectively disrupted and overexpressed in the parental strain A. pullulans CCTCC M 2012259 to construct mutants of A. pullulans Δmal31 and A. pullulans Mal31. Batch pullulan production significantly decreased by 69.1 % in A. pullulans Δmal31 but increased by 15.9 % in A. pullulans Mal31, as compared to the parental strain. We performed kinetics analysis, assays of key enzymes, determination of intracellular UDPG, NADH, and ATP contents, and measurement of transcriptional levels of genes associated with pullulan biosynthesis and excretion. The results confirmed that the mal31 disruption decreased the glucose consumption rate, decreased the formation rate and titer of pullulan, but increased the intracellular UDPG supply for β-glucan accumulation. In contrast, the mal31 overexpression increased the transcriptional levels of genes associated with pullulan biosynthesis, and accelerated the rates of glucose consumption and pullulan formation, thereby increased pullulan production. Our findings revealed that MAL31 is involved in the transport of precursors for pullulan biosynthesis. This study provides an accurate operating site for genetic modification of A. pullulans for improving pullulan production and also presents a feasible technique route for the overproduction of other polysaccharides.

In this study, cost-effective substrates such as cassava starch, corn steep liquor (CSL) and soybean meal hydrolysate (SMH) were used for pullulan production by Aureobasidium pullulans CCTCC M 2012259. The medium was optimized using response surface methodology (RSM) and artificial neural network (ANN) approaches, and analysis of variance indicated that the ANN model achieved higher prediction accuracy. The optimal medium predicted by ANN was used to produce high molecular weight pullulan in high yield. SMH substrates increased both biomass and pullulan titer, while CSL substrates maintained higher pullulan molecular weight. Results of kinetic parameters, key enzyme activities and intracellular uridine diphosphate glucose contents revealed the physiological mechanism of changes in pullulan titer and molecular weight using different substrates. Economic analysis of batch pullulan production using different substrates was performed, and the cost of nutrimental materials for CSL and SMH substrates was decreased by 46.1% and 49.9%, respectively, compared to the control using glucose and yeast extract as substrates, which could improve the competitiveness of pullulan against other polysaccharides in industrial applications.

The effects of representative surfactants on the growth of Saccharomyces cerevisiae WSH-J701 and glutathione (GSH) production were studied. The anionic surfactant sodium dedecyl sulfate (SDS) and the cationic surfactant cetyltrimethyl ammonium bromide (CTAB) had critical inhibitory concentrations of 0.5 and 0.025 g/l, respectively, to cell growth during cultivation. Above these concentrations, cell growth was greatly inhibited or even ceased. Nonionic surfactants such as polyoxyethylene lauryl ether (Brij30) and polyoxyethylene sorbitan monooleate (Tween80) could also affect cell growth only when their concentrations reached much higher levels. Under the critical concentration, SDS and CTAB increased the extracellular GSH concentration by 10 and 15%, but above this level, they improved extracellular GSH concentration by 35 and 60%, respectively. Otherwise, total GSH concentration and intracellular GSH content decreased slightly. The effect of Brij30 was somewhat like ionic surfactants, extracellular GSH concentration could be increased by 50% but the intracellular GSH content was reduced by 33%. Tween80 seemed to have no effect on extracellular GSH accumulation, but total GSH concentration and intracellular GSH content decreased gradually by increasing its concentration.