Construction of a novel Saccharomyces cerevisiae spore surface display system based on chitosan-binding module

Abstract:

[Objective] Based on the existing problems in the current cell surface display systems, we aimed to establish a novel Saccharomyces cerevisiae spore surface display system with strong universality, better stress resistance, and higher efficiency and stability. [Methods] Firstly, according to the characteristics of immobilized enzymes in S. cerevisiae spores, we searched the potential chitosan-binding modules with high affinity with the chitosan layer of spore wall by referring to literature. Then, the binding module was fused and expressed with green fluorescent protein (GFP) and the affinity ability of the binding module with the spore wall was verified in vitro and in vivo. Finally, we selected α-galactosidase (MEL1) derived from S. cerevisiae AH109 to evaluate the efficacy of the novel display system. [Results] Firstly, we selected a carbohydrate binding module 32 (CBM32) derived from Paenibacillus sp. IK-5 chitosanase as the chitosan-binding module. Next, the fusion protein CBM32-GFP, which was expressed in Escherichia coli and purified, was co-incubated with dit1Δ spores, and the result showed that CBM32 exhibited good affinity ability with the spore wall in vitro by the localization and intensity of GFP fluorescence. Furthermore, the fluorescence localization and binding ability of CBM32-GFP in dit1Δ spores proved that CBM32 was tightly bound to the spore wall in vivo. Finally, we replaced GFP with MEL1 in this display system. Compared with those of spores only expressing MEL1, the activity of spores displaying CBM32-MEL1 was increased by 68.65%, with the highest specific activity reaching 460.59 U/g DCW (dry cell weight), and the reusability was also significantly improved. Moreover, the stability and operability of MEL1 were enhanced. [Conclusion] We constructed a novel S. cerevisiae spore surface display system based on the chitosan-binding module for the first time, which provided a reliable cell surface display platform for the eukaryotic proteins with multi-glycosylation sites and multi-subunit structures. It also provided the theoretical basis for the industrial application of immobilized enzymes in spores.