High-throughput screening of Aureobasidium pullulans for high β-glucan production using UV mutagenesis combined with artificial fluorescent labeling

Abstract

Aureobasidium pullulans (A. pullulans) is a yeast-like fungus that produces β-glucan, an exopolysaccharide, during its fermentation. However, there is currently a lack of high-throughput screening and breeding methods for high β-glucan-producing A. pullulans. The present study aimed to construct a highly specific fluorescent screening system focused on β-glucan. Notably, the carbohydrate-binding module-4 (CBM4) domain exhibited remarkable substrate specificity towards β-1,3-glucan. When used in bimolecular fluorescence complementation, CBM4 effectively bound to its substrate and generated fluorescence signals. This innovative fluorescent probe system facilitated high-throughput screening of β-glucan-producing strains. After UV mutagenesis, the resulting strains were labeled with fluorescent proteins, and flow cytometry was used for high-throughput screening of strains exhibiting enhanced fluorescence. The A. pullulans strains acclimated in a high-glucose medium to identify and select high-yielding strains. The findings revealed that, following multiple rounds of mutagenesis and domestication, the yield of A. pullulans D7 increased by 65.18 % from 2.93 g/L to 4.84 g/L compared to the original A. pullulans M3. After 6 days of fermentation, the molecular weight of β-glucan decreased from approximately 7500 to 5500 kDa. The method constructed in this study effectively improved the activity of A. pullulans, and a mutant strain with a significantly increased yield of β-glucan was identified.

Keywords: Aureobasidium pullulans; Fusion protein; High-throughput screening; UV mutagenesis; β-Glucan.