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| 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 |
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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.
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Received: 22 February 2020
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Corresponding Authors:
** yanlinliu@nwsuaf.edu.cn
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| About author:: *The authors who contribute equally |
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[1] 谢文平. 2015. 代谢工程改造酿酒酵母生物合成类胡萝卜素的研究[D]. 博士学位论文, 浙江大学,导师:于洪巍, pp. 43-44.
(Xie W P.2015. Study on the metabolic engineering transformation of Saccharomyces cerevisiae carotenoids[D]. Thesis for Ph.D, Zhejiang University, Supervisor: Yu H W, pp. 43-44.)
[2] 闫青, 朱凤妹, 彭利沙, 等. 2017.黑曲霉β-葡萄糖苷酶的活性位点和耐热结构预测[J]. 食品科学, 38(6): 87-93.
(Yan Q, Zhu F M, Peng L S, et al.2007. Prediction of the active site and heat-resistant structure of Aspergillus niger β-glucosidase[J]. Food Science, 38(6): 87-93.)
[3] Ahmad M, Hirz M, Pichler H, et al.2014. Protein expression in Pichia pastoris: Recent achievements and perspectives for heterologous protein production[J]. Applied Microbiology and Biotechnology, 98(12): 5301-5317.
[4] Andreu C, del Olmo M.2013. Yeast arming by the Aga2p system: Effect of growth conditions in galactose on the efficiency of the display and influence of expressing leucine-containing peptides[J]. Applied Microbiology and Biotechnology, 97(20): 9055-9069.
[5] Andreu C, del Olmo M l.2017. Development of a new yeast surface display system based on Spi1 as an anchor protein[J]. Applied Microbiology and Biotechnology, 101(1): 287-299.
[6] Bae J H, Sung B H, Kim H J, et al.2015. An efficient genome-wide fusion partner screening system for secretion of recombinant proteins in yeast[J]. Scientific Reports, 5: 12229-12229.
[7] Breinig F, Diehl B, Rau S, et al.2006. Cell surface expression of bacterial esterase a by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response[J]. Applied and Environmental Microbiology, 72(11): 7140-7147.
[8] Davison S A, den Haan R, van Zyl W H.2016. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains[J]. Applied Microbiology and Biotechnology, 100(18): 8241-8254.
[9] de Groot P W J, Hellingwerf K J, Klis F M.2003. Genome-wide identification of fungal GPI proteins[J]. Yeast, 20(9): 781-796.
[10] Fukuda T, Tsuchiyama K, Makishima H, et al.2010. Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system[J]. Biotechnology Letters, 32(5): 655-659.
[11] Inokuma K, Bamba T, Ishii J, et al.2016. Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide[J]. Biotechnology and Bioengineering, 113(11): 2358-2366.
[12] Jiang Z B, Song H T, Gupta N, et al.2007. Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris[J]. Protein Expression and Purification, 56(1): 35-39.
[13] Jones M E.1992. Analysis of algebraic weighted least-squares estimators for enzyme parameters[J]. The Biochemical Journal, 288(Pt2): 533-538.
[14] Karim A S, Curran K A, Alper H S.2013. Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications[J]. Fems Yeast Research, 13(1): 107-116.
[15] Kawai R, Yoshida M, Tani T, et al.2003. Production and characterization of recombinant phanerochaete chrysosporium β-glucosidase in the methylotrophic yeast Pichia pastoris bioscience[J]. Biotechnology and Biochemistry, 67(1): 1-7.
[16] Klis F M, Mol P, Hellingwerf K, et al.2002. Dynamics of cell wall structure in Saccharomyces cerevisiae[J]. Fems Microbiology Reviews, 26(3): 239-256.
[17] Lee C R, Sung B H, Lim K M, et al.2017. Co-fermentation using Recombinant Saccharomyces cerevisiae yeast strains hyper-secreting different cellulases for the production of cellulosic bioethanol[J]. Scientific Reports, 7(1): 4428.
[18] Lian J, HamediRad M, Hu S, et al.2017. Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system[J]. Nature Communications, 8(1): 1688-1688.
[19] Liu W S, Pan X X, Jia B, et al.2010. Surface display of active lipases Lip7 and Lip8 from Yarrowia Lipolytica on Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology, 88(4): 885-891.
[20] Murray P, Aro N, Collins C, et al.2004. Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii[J]. Protein Expression and Purification, 38(2): 248-257.
[21] Opassiri R, Hua Y, Wara-Aswapati O, et al.2004. Beta-glucosidase, exo-beta-glucanase and pyridoxine transglucosylase activities of rice BGlu1[J]. The Biochemical journal, 379(Pt 1): 125-131.
[22] Sitia R, Braakman I.2003. Quality control in the endoplasmic reticulum protein factory[J]. Nature, 426(6968): 891-894.
[23] Spellman P T, Sherlock G, Zhang M Q, et al.1998. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization[J]. Molecular Biology of the Cell, 9(12): 3273-3297.
[24] Yang N, Yu Z, Jia D, et al.2014. The contribution of Pir protein family to yeast cell surface display[J]. Applied Microbiology and Biotechnology, 98(7): 2897-2905.
[25] Zhang Y, Min Z, Qin Y, et al.2019. Efficient display of Aspergillus niger β-Glucosidase on Saccharomyces cerevisiae cell wall for aroma enhancement in wine[J]. Journal of Agricultural and Food Chemistry, 67(18): 5169-5176.
[26] Zhang Z, Moo-Young M, Chisti Y.1996. Plasmid stability in recombinant Saccharomyces cerevisiae[J]. Biotechnology Advances, 14(4): 401-435. |
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