酵母糖蛋白工程的研究进展

doi:10.14088/j.cnki.issn0439-8114.2022.13.032

摘要/Abstract

摘要： 以酵母作为宿主生产的蛋白在生活和医疗中有着广泛应用,但其特有的对蛋白进行高甘露糖基化修饰会造成蛋白免疫原性改变、半衰期缩短等负面影响。通过工程改造,能够减弱酵母中蛋白的甘露糖基化修饰程度,且在毕赤酵母（Pichia pastoris）中已经能够产生唾液酸化糖蛋白。介绍酵母系统在生产药物蛋白方面的应用,简述酵母N-糖基化修饰过程以及目前酵母人源化研究进展。

关键词: 酵母, N-糖基化, 人源化, 糖蛋白

Abstract: Proteins produced by yeast as hosts are widely used in life and medical treatment, but their unique modification of protein with high mannose glycosylation will cause negative effects such as changes in protein immunogenicity and shortening of half-life. Through engineering modification, the degree of mannose modification of proteins in yeast can be reduced, and sialic acid glycoproteins have been produced in Pichia pastoris. The application of the yeast system in the production of pharmaceutical proteins was introduced. The modification process of yeast N-glycosylation and the research progress of yeast humanization were reviewed.

Key words: yeast, N-glycosylation, humanization, glycoprotein

中图分类号:

Q936

李婉情, 张蕾. 酵母糖蛋白工程的研究进展[J]. 湖北农业科学, 2022, 61(13): 173-178.

LI Wan-qing, ZHANG Lei. Research progress of yeast glycoprotein engineering[J]. HUBEI AGRICULTURAL SCIENCES, 2022, 61(13): 173-178.

参考文献

[1] RADER R A.(Re)defining biopharmaceutical[J]. Nature biotechnology, 2008,26(7):743-751.
[2] GOFFEAU A, BARRELL B G, BUSSEY H, et al.Life with 6000 genes[J]. Science, 1996,274(5287):563-567.
[3] SCHUTTER K D, LIN Y C, TIELS P, et al.Genome sequence of the recombinant protein production host Pichia pastoris[J]. Nature biotechnology, 2009,27(6):561-566.
[4] WINZELER E A,SHOEMAKER D D,ASTROMOFF A,et al.Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis[J]. Science,1999,285(5429):901-906.
[5] SCHERENS B, GOFFEAU A.The uses of genome-wide yeast mutant collections[J]. Genome biology, 2004,5(7):229.
[6] DONG U K, JACQUELINEH, KIM D, et al.Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe[J]. Nature biotechnology, 2010,28(6):617-623.
[7] GHAEMMAGHAMI S,HUH W K, BOWER K, et al.Global analysis of protein expression in yeast[J]. Nature, 2003,425(6959):737-741.
[8] YAMANISHI M, MATSUYAMA T.A modified cre-lox genetic switch to dynamically control metabolic flow in Saccharomyces cerevisiae[J]. ACS synthetic biology, 2012,1(5):172-180.
[9] DICARLO J E,NORVILLE J E,PRASHANT M,et al.Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems[J]. Nucleic acids research, 2013,41(7):4336-4343.
[10] SHI S, LIANG Y, ZHANG M M, et al.A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae[J]. Metabolic engineering, 2016,33:19-27.
[11] WACHTER C D,LANDUYT L V, CALLEWAERT N.Engineering of yeast glycoprotein expression[J]. Advances in biochemical engineering/biotechnology, 2021,175:93-135.
[12] FLYNN G C.High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans[J]. Glycobiology, 2011,21(7):949-959.
[13] ALESSANDRI L, OUELLETTE D, ACQUAH A, et al.Increased serum clearance of oligomannose species present on a human IgG1 molecule[J]. MABs, 2012,4(4):509-520.
[14] FERRER-MIRALLES N,DOMINGO-ESPÍN J,CORCHERO J L, et al. Microbial factories for recombinant pharmaceuticals[J]. Microbial cell factories, 2009,8(1):17.
[15] NOLAN R P,LEE K.Dynamic model for CHO cell engineering[J]. Journal of biotechnology, 2012,158(1-2):24-33.
[16] COX M M J. Recombinant protein vaccines produced in insect cells[J]. Vaccine, 2012,30(10):1759-1766.
[17] KESIK-BRODACKA M.Progress in biopharmaceutical development[J]. Biotechnology and applied biochemistry, 2018,65(3):306-322.
[18] HORWITZ A A,WALTER J M, SCHUBERT M G, et al.Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via crispr-cas[J]. Cell systems,2015,1(1):88-96.
[19] LIN Q, JIA B,MITCHELL L A, et al.Radom, an efficient in vivo method for assembling designed dna fragments up to 10 kb long in Saccharomyces cerevisiae[J]. ACS synthetic biology, 2015,4(3):213-220.
[20] WALSH G.Biopharmaceutical benchmarks 2018[J]. Nature biotechnology, 2018,36(12):1136-1145.
[21] CHUNG C, MAJEWSKA N I, WANG Q, et al.Snapshot: N-glycosylation processing pathways across kingdoms[J]. Cell, 2017,171(1):258.
[22] NAKAYAMA K, NAGASU T, SHIMMA Y, et al.OCH1 encodes a novel membrane bound mannosyltransferase: Outer chain elongation of asparagine-linked oligosaccharides[J]. The EMBO journal, 1992,11(7):2511-2519.
[23] TANG H, WANG S, WANG J, et al.N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae[J]. Scientific reports, 2016,6:25654.
[24] CHOI B, BOBROWICZ P, DAVIDSON R C, et al.Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris[J]. Proceedings of the National academy of sciences of the United States of America,2003,100(9):5022-5027.
[25] HAMILTON S R, DAVIDSON R C, SETHURAMAN N, et al.Humanization of yeast to produce complex terminally sialylated glycoproteins[J]. Science, 2006,313(5792):1441-1443.
[26] HAN M, WANG X, DING H, et al.The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris[J]. Enzyme and microbial technology, 2014,54:32-37.
[27] HOSHIDA H, FUJITA T, CHA-AIM K, et al.N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae[J]. Applied microbiology and biotechnology, 2013,97(12):5473-5482.
[28] GRAHAM T R, EMR S D.Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant[J]. Journal of cell biology, 1991,114(2):207-218.
[29] BORODINA I, KILDEGAARD K R, JENSEN N B, et al.Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine[J]. Metabolic engineering, 2015,27:57-64.
[30] KOSTOVA Z, RANCOUR D M, MENON A K, et al.Photoaffinity labelling with P3-(4-azidoanilido)uridine 5'-triphosphate identifies gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyses formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis[J]. The biochemical journal, 2000,350(Pt 3):815-822.
[31] HELENIUS J, NG D T W, MAROLDA C L, et al. Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein[J]. Nature, 2002,415(6870):447-450.
[32] LEHLE L, STRAHL S, TANNER W.Protein glycosylation, conserved from yeast to man: A model organism helps elucidate congenital human diseases[J]. Angewandte chemie international edition, 2006,45(41):6802-6818.
[33] PETER O.Architecture and biosynthesis of the Saccharomyces cerevisiae cell wall[J]. Genetics, 2012,192(3):775-818.
[34] LAMBOU K, PERKHOFER S, FONTAINE T, et al.Comparative functional analysis of the OCH1 mannosyltransferase families in Aspergillus fumigatus and Saccharomyces cerevisiae[J]. Yeast, 2010,27(8):625-636.
[35] LUSSIER M, SDICU A, CAMIRAND A, et al.Functional characterization of the YUR1, KTR1, and KTR2 genes as members of the yeast KRE2/MNT1 mannosyltransferase gene family[J]. Journal of biological chemistry, 1996,271(18):11001-11008.
[36] NAKANISHI-SHINDO Y,NAKAYAMA K,TANAKA A,et al.Structure of the N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1,och1 mnn1,and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae.[J]. Journal of biological chemistry,1993,268(35):26338-26345.
[37] LIU Y, XIE W, YU H.Enhanced activity of rhizomucor miehei lipase by deglycosylation of its propeptide in pichia pastoris[J]. Current microbiology, 2014,68(2):186-191.
[38] CHIBA Y,SUZUKI M,YOSHIDA S,et al.Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae[J]. Journal of biological chemistry,1998, 273(41):26298-26304.
[39] PAQUES F, HABER J E.Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.[J]. Microbiol Mol Biol Rev, 1999,63(2):349-404.
[40] VERVECKEN W, KAIGORODOV V,CALLEWAERT N, et al.In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris[J]. Applied and environmental microbiology, 2004,70(5):2639-2646.
[41] KRAINER F W,GMEINER C,NEUTSCH L,et al.Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris[J]. Scientific reports, 2013,3:3279.
[42] DAVIDSON R C, NETT J H, RENFER E, et al.Functional analysis of the ALG3 gene encoding the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris[J]. Glycobiology, 2004,14(5):399-407.
[43] CIPOLLO J F,TRIMBLE R B.The accumulation of man6GlcNAc2- PP-dolichol in the Saccharomyces cerevisiae Δalg9 mutant reveals a regulatory role for the Alg3p α1,3-Man Middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing[J]. Journal of biological chemistry,2000,275(6):4267-4277.
[44] 贺铁凡, 徐沙, 张阁元, 等. 重构酿酒酵母N-糖基化途径生产人源化糖蛋白[J]. 微生物学报, 2014(5):509-516.
[45] ABE H, TAKAOKA Y, CHIBA Y, et al.Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins[J]. Glycobiology, 2009, 19(4):428-436.
[46] HELENIUS J, NG D T W, MAROLDA C L, et al. Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein[J]. Nature, 2002,415(6870):447-450.
[47] CALLEWAERT N,LAROY W, CADIRGI H, et al.Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-α-D-mannosidase for N-glycan engineering in Pichia pastoris[J]. FEBS letters, 2001,503(2-3):173-178.
[48] HAMILTON S R, GERNGROSS T U.Glycosylation engineering in yeast: The advent of fully humanized yeast[J]. Current opinion in biotechnology, 2007,18(5):387-392.