Effects of Different Promoters and Anchoring Proteins on β-glucosidase Activity Displayed on the Surface of Saccharomyces cerevisiae
ZHOU Hong1,*, ZHANG Yang1,2,*, SONG Yu-Yang1, LI Ying1, DU Qing1, ZHANG Hui-Ning3, LIU Yan-Lin1,**
1 College of Enology, Northwest A & F University, Yangling 712100, China; 2 Moutai Institute, Renhuai 564500, China; 3 Yangzhou Hospitality Institute, Yangzhou 225002, China
Abstract：β-glucosidase plays a vital role as a rate-limiting enzyme in the field of bioenergy and food industries. In this study, different promoters and anchor proteins were screened in the surface display system of Saccharomyces cerevisiae to explore the effects of different promoters and anchor proteins on the BGL activity from Aspergillus niger. pYES2/CT/α-factor was used as the starting plasmid, green fluorescent protein (GFP) as the reporter protein and mating agglutinin protein (Sag1p), suppression of exponential defect protein (Sed1p) and cell wall protein 2 (Cwp2p) as anchors protein, to construct 3 different surface display plasmids of anchor protein GAL1-eGFP-Sag1/Sed1/Cwp2 and yeast surface display system. The original promoter GAL1 of plasmid pYES2/CT/α-factor was replaced with constitutive promoters promoter of glycerol 3-phos‐phate dehydrogenase (GPD) and promoter of suppression of exponential defect protein (SED1) by In-Fusion technology. Plasmids pYES2-GPD and pYES2-SED were constructed and digested , then they were separately ligated with the digested GAL1-eGFP-Sag1/Sed1/Cwp2 constructed above to construct 6 recombinant plasmids GAP/SED1-eGFP-Sag1/Sed1/Cwp2. Finally, the eGFP on the plasmids GPD/SED1-eGFP-Sag1/Sed1/Cwp2 were replaced with the target protein gene bgl1 derived from A. niger and 6 recombinant yeasts, PBy-GBSa, PBy-GBSe, PBy-GBCw, PBy-SBSa, PBy-SBSe, PBy-SBCw, were successfully constructed to explore the effects of different promoters (SED1 and GPD) and anchoring proteins (Sag1p, Sed1p and Cwp2p) on BGL activity. Green fluorescent protein was observed by laser confocal microscope and was found to be located on the cell surface of S. cerevisiae, revealing that the surface display platform of S. cerevisiae was successfully established. The activities of BGL displayed on the surface of 6 recombinant yeasts were compared with each other, which showed that the GPD promoter performed better than the SED1 promoter; no matter what kind of promoter, Sag1p showed the highest enzyme activity among the 3 anchor proteins (Sag1p, Sed1p, Cwp2p); When GPD was used as the promoter, the differences in enzyme activity between different anchor proteins were more obvious. The BGL recombinant strain PBy-GBSa was able to display BGL more efficiently, and the enzyme activity reached a maximum of (18.29±1.05) U/g when cultured for 24 h. The types of promoters and anchoring proteins had important effects on the surface display of BGL in S. cerevisiae, which would provide a theoretical basis for the more efficient and stable surface display of BGL in S. cerevisiae and the industrial application of BGL whole-cell catalysts.
周红, 张阳, 宋育阳, 李莹, 杜青, 张会宁, 刘延琳. 不同启动子及锚定蛋白对酿酒酵母表面展示β-葡萄糖苷酶活性的影响[J]. 农业生物技术学报, 2020, 28(10): 1849-1861.
ZHOU Hong, ZHANG Yang, SONG Yu-Yang, LI Ying, DU Qing, ZHANG Hui-Ning, LIU Yan-Lin. Effects of Different Promoters and Anchoring Proteins on β-glucosidase Activity Displayed on the Surface of Saccharomyces cerevisiae. 农业生物技术学报, 2020, 28(10): 1849-1861.
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