毕赤酵母糖基化初步研究
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摘要
毕赤酵母表达系统有很多优点,已广泛应用于外源蛋白的表达。但是,毕赤酵母不适于糖蛋白的表达,其表达产物为高甘露糖型,容易在人体中产生免疫反应,限制了毕赤酵母作为宿主菌的使用。a-1,6-甘露糖转移酶(该酶由och1基因编码)的存在是导致高甘露糖形成的重要原因。对该基因的敲除,可有效阻断酵母对糖蛋白的翻译后高甘露糖化修饰,得到的糖蛋白为低甘露糖结构,可规避毕赤酵母高甘露糖修饰弊端,同时为毕赤酵母后续糖基化改造作基础。虽然目前对于毕赤酵母中och1基因敲除有了很多的研究,但是存在问题。och1基因基因敲除效率非常低;对och1基因敲除后的生理特性研究较少。本论文研究发现两步基因重组技术可以高效敲除ura3及och1基因,och1基因的敲除对酵母的细胞壁结构产生很大的影响,并进一步影响其对温度的敏感性。
两步基因重组技术可以在高效、定向敲除毕赤酵母目的基因的同时,还可以去除掉筛选标记,为后续改造保留筛选标记,同时没有为待改造菌株引入外源性的抗性类等基因。
利用两步基因重组技术成功敲除了毕赤酵母GS115中的ura3基因,该基因编码乳清酸核苷-5-磷酸脱羧酶,此酶在毕赤酵母尿嘧啶合成中起催化作用。GS115被敲除此基因后,突变菌株在尿嘧啶缺陷培养基中不能生长,补充尿嘧啶后生长旺盛,并且可稳定传代,为GS115菌株糖基化改造提供筛选标记,解决该菌株糖基化改造过程中筛选标记不足难题。同时还发现利用该技术可以高效敲除了毕赤酵母JC308中的负责高甘露糖合成的a-1,6-甘露糖转移酶基因(och1),其效率是文献报道的利用双交换同源重组的敲除同样菌株och1基因的20倍。
在och1基因的敲除对酵母生理特性影响方面,我们对毕赤酵母X-33 och1敲除株进行了研究,通过刚果红染色、扫描电镜等手段发现och1基因敲除株的细胞壁结构产生很大的影响,其形体较野生型的大,子细胞不易从母细胞完全分离。进一步的细胞活力实验、渗透压实验、生长特征研究等考察,发现其与野生型X-33在生理上有很大差异。och1基因敲除株对温度敏感,生长缓慢,菌体密度低,菌株容易聚集生长,培养过程中死亡率很高等。
P. pastorisis is a methylotrophic yeast that had already been used for production of a wide variety of heterologous proteins. Besides the advantages of protein processing, folding and post-translational modifications in eukaryotic cells, it offers the benefits of cost-effective and easy scale-up in E. coli. Additionally, it does not secrete too much intrinsic protein. This makes P. pastorisis as an excellent candidate for the production of protein-based therapeutic agents. Though series of profits come from pichia pastoris expression, but it is not suitable to express glycan proteins due to presence of yeast-specific outer-chain mannosylation. P. pastorisis N-glycans of the high mannose (Man) type out chain biosyntheses in yeast are initiated by a 1,6-mannosyltransferase encoded by och1 gene in P. pastorisis. Much work had already been done in och1 gene knokout in P. pastorisis, but there are still some problems during the genetic manipulation. Normally double homologus recombination was used to knockout och1, but it is highly inefficient. In this research, two steps of genetic recombination strategy was used to knockout ura3 and och1.
Two steps of genetic recombination knock out target gene efficiently. The method is that knocking out open reading frame first, then linking this fragment to the vector with screening marker. The first recombination is to input the plasmid into the yeast, checking whether the integration site is right, and the second is to knock out the target gene. At the same time, screening marker is removed.
The ura3 gene of GS115 was knocked out through this technology. The mutant of this strain cannot grow in the medium lack of uracil, but after adding uracil, grows well and goes down to the later generation stably. GS115 with ura3 mutant leads to additional selecting marker in glycosylation engineering and can be used as the initial strain for further research.
The same recombination method was used to remove och1 gene of JC308. In addtion, we also characterized the physical type of X-33 och1 mutant strain. The och1 gene is responsible for the high mannose of glycoprotein. Comparisoning growth and phenotype of both Pichia pastoris X-33 wild strain and X-33 och1 mutant strain, to lay foundation for further research. Compared with wild strain, the characteristic of och1 mutant lies in as follows: slow growth, decreased biomass, sensitive to temperature, which can be counterbalanced by the addtion of osmotic pressure, unabled to grow in culture containing congo red, the cell wall deficient in division, insufficient cell vitality, high death rate in cultivation process.
两步基因重组技术可以在高效、定向敲除毕赤酵母目的基因的同时,还可以去除掉筛选标记,为后续改造保留筛选标记,同时没有为待改造菌株引入外源性的抗性类等基因。
利用两步基因重组技术成功敲除了毕赤酵母GS115中的ura3基因,该基因编码乳清酸核苷-5-磷酸脱羧酶,此酶在毕赤酵母尿嘧啶合成中起催化作用。GS115被敲除此基因后,突变菌株在尿嘧啶缺陷培养基中不能生长,补充尿嘧啶后生长旺盛,并且可稳定传代,为GS115菌株糖基化改造提供筛选标记,解决该菌株糖基化改造过程中筛选标记不足难题。同时还发现利用该技术可以高效敲除了毕赤酵母JC308中的负责高甘露糖合成的a-1,6-甘露糖转移酶基因(och1),其效率是文献报道的利用双交换同源重组的敲除同样菌株och1基因的20倍。
在och1基因的敲除对酵母生理特性影响方面,我们对毕赤酵母X-33 och1敲除株进行了研究,通过刚果红染色、扫描电镜等手段发现och1基因敲除株的细胞壁结构产生很大的影响,其形体较野生型的大,子细胞不易从母细胞完全分离。进一步的细胞活力实验、渗透压实验、生长特征研究等考察,发现其与野生型X-33在生理上有很大差异。och1基因敲除株对温度敏感,生长缓慢,菌体密度低,菌株容易聚集生长,培养过程中死亡率很高等。
P. pastorisis is a methylotrophic yeast that had already been used for production of a wide variety of heterologous proteins. Besides the advantages of protein processing, folding and post-translational modifications in eukaryotic cells, it offers the benefits of cost-effective and easy scale-up in E. coli. Additionally, it does not secrete too much intrinsic protein. This makes P. pastorisis as an excellent candidate for the production of protein-based therapeutic agents. Though series of profits come from pichia pastoris expression, but it is not suitable to express glycan proteins due to presence of yeast-specific outer-chain mannosylation. P. pastorisis N-glycans of the high mannose (Man) type out chain biosyntheses in yeast are initiated by a 1,6-mannosyltransferase encoded by och1 gene in P. pastorisis. Much work had already been done in och1 gene knokout in P. pastorisis, but there are still some problems during the genetic manipulation. Normally double homologus recombination was used to knockout och1, but it is highly inefficient. In this research, two steps of genetic recombination strategy was used to knockout ura3 and och1.
Two steps of genetic recombination knock out target gene efficiently. The method is that knocking out open reading frame first, then linking this fragment to the vector with screening marker. The first recombination is to input the plasmid into the yeast, checking whether the integration site is right, and the second is to knock out the target gene. At the same time, screening marker is removed.
The ura3 gene of GS115 was knocked out through this technology. The mutant of this strain cannot grow in the medium lack of uracil, but after adding uracil, grows well and goes down to the later generation stably. GS115 with ura3 mutant leads to additional selecting marker in glycosylation engineering and can be used as the initial strain for further research.
The same recombination method was used to remove och1 gene of JC308. In addtion, we also characterized the physical type of X-33 och1 mutant strain. The och1 gene is responsible for the high mannose of glycoprotein. Comparisoning growth and phenotype of both Pichia pastoris X-33 wild strain and X-33 och1 mutant strain, to lay foundation for further research. Compared with wild strain, the characteristic of och1 mutant lies in as follows: slow growth, decreased biomass, sensitive to temperature, which can be counterbalanced by the addtion of osmotic pressure, unabled to grow in culture containing congo red, the cell wall deficient in division, insufficient cell vitality, high death rate in cultivation process.
引文
1 Ogata K, Nishikawa H, Ohsugi M. A yeast capable of utilizing methanol [J]. Agric Biol Chem, 1969, 33: 1519-1520
2 Cregg J M, Higgins D R. Pichia protocols[J]. Humana Press, 1998, 103: 95-107
3 Mattanovich D, Callewaert N, Rouze P, et al. Open access to sequence: Browsing the Pichia pastoris genome[J]. Microbial Cell Factories, 2009, 8: 53
4 Cereghino G P, Cereghino J L, Ilgen C, et al. Production of recombinant proteins in fermenter cultures of the yeast astoris[J]. Current Opinion in Biotechnology, 2002, 13: 329-332
5 Wildt S, Gerngross T U. The humanization of N-glycosylation pathways in yeast[J]. Nature Reviews Microbiology, 2005, 2: 119-128
6 Helenius A, Aebi M. Intracellular functions of N-linked glycans[J]. Science, 2001, 291: 2364-2369
7 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
8 Hamilton S R, Davidson R C, Sethuraman N, et al. Humanization of yeast to produce complex terminally sialylated glycoproteins[J]. Science, 2006, 313: 1441-1443
9 Nagasu T, Shimma Y I, Nakanishi Y, et al. Isolation of new temperature sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation[J]. Yeast, 2004, 10: 535-547
10 Nakayama K, Nagasu T, Shimma Y, et al. OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine linked oligosaccharides[J]. Embo J, 1992, 11: 2511-2519
11 Herscovics A, Orlean P. Glycoprotein biosynthesis in yeast[J]. FASEB J, 1993, 7 (6): 540- 550
12 Walsh G. Biopharmaceutical benchmarks[J]. Nature Biotechnology, 2003, 21 (8): 865-870
13 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: 173-178
14 Shindo Y N, 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]. J Biol Chem, 1993, 268 (35): 26338-26345
15 Chiba Y, Suzuki M, Yoshida S, et al. Production of human compatible high mannose type (Man5GlcNAc2) sugar chains in saccharomyces cerevisiae[J]. The Journal of Biological Chemistry, 1998, 273: 26298-26304
16 Choi B K, Bobrowicz P, Davidson R C, et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris[J]. PNAS, 2003, 100 (9): 5022- 5027
17 Hamilton S R, Bobrowiez P, Bobrowiez B, et al. Production of complex human glycoproteins in yeast[J]. Science, 2003, 301 (4): 1244-1246
18 Bobrowicz P, Davidson R C, Li H J, et al. Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose[J]. Glycobiology, 2004, 14 (9): 757-766
19 Li H, Sethuraman N, Stadheim T A, et al. Optimization of humanized IgGs in glycoengineered Pichia pastoris[J]. Nature Biotechnology, 2006, 24: 210-215
20 Hamilton S R, Davidson R C, Sethuraman N, et al. Humanization of yeast to produce complex terminally sialylated glycoproteins[J]. Science, 2006, 313: 1441-1443
21 Zhou J G, Zhang H C, Liu X W, et al. Influence of N-glycosylation on saccharomyces cerevisiae morphology: a Golgi glycosylation mutant shows cell division defects[J]. Current Microbiology, 2007, 55: 198-203
22 Liu B, Gong X, et al. Disruption of the och1 and mnn1 genes decrease N-glycosylation on glycoprotein expressed in kluyveromyces lactis[J]. Journal of Biotechnology, 2009, 143: 95-102
23 Kelly R, Miller S M, Kurtz M B. One- step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans[J]. Mol Gen Genet, 1988, 214: 24-31
24 Nett J H, Gerngross T U. Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris[J]. Yeast, 2003, 20: 1279-1290
25 Fonzi W A, Irwin M Y. Isogenic strain construction and gene mapping in Candida albicans[J]. Genetics, 1993, 134: 717-728
26刘波.乳酸克鲁维酵母糖基工程研究[D]:[博士学位论文].北京:军事医学科学院生物工程研究所, 2008
27 Wang Y, Gong X, et al. A Pichia pastoris withα-1,6-mannosyltransferases deletion and its use in the expression of HSA/GM-CSF chimera[J]. Chinese Journal of Biotechnology, 2007, 23: 907-914
28 Schmidt M, Strenk, et al. Importance of cell wall mannoproteins for septum formation insaccharomyces cerevisiae[J]. Yeast, 2005 (22): 715-723
29 Ballou CE, Isolation, characterization and properties of saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects[J]. Methods Enzymol, 1990, 185: 440-470
30 Zhou J G, Zhang H C, Liu X W, et al. Influence of N-glycosylation on saccharomyces cerevisiae morphology: a Golgi glycosylation mutant shows cell division defects[J]. Current Microbiology, 2007, 55: 198-203
31 Madeo F, Frohlich E, Frohlich K U. A yeast mutant showing diagnostic markers of early and late apoptosis[J]. Cell Biol, 1997, 139: 729-734
32 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
33吴军,刘波. N-糖基工程研究进展[J].生物产业技术, 2009, 4: 47-52
2 Cregg J M, Higgins D R. Pichia protocols[J]. Humana Press, 1998, 103: 95-107
3 Mattanovich D, Callewaert N, Rouze P, et al. Open access to sequence: Browsing the Pichia pastoris genome[J]. Microbial Cell Factories, 2009, 8: 53
4 Cereghino G P, Cereghino J L, Ilgen C, et al. Production of recombinant proteins in fermenter cultures of the yeast astoris[J]. Current Opinion in Biotechnology, 2002, 13: 329-332
5 Wildt S, Gerngross T U. The humanization of N-glycosylation pathways in yeast[J]. Nature Reviews Microbiology, 2005, 2: 119-128
6 Helenius A, Aebi M. Intracellular functions of N-linked glycans[J]. Science, 2001, 291: 2364-2369
7 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
8 Hamilton S R, Davidson R C, Sethuraman N, et al. Humanization of yeast to produce complex terminally sialylated glycoproteins[J]. Science, 2006, 313: 1441-1443
9 Nagasu T, Shimma Y I, Nakanishi Y, et al. Isolation of new temperature sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation[J]. Yeast, 2004, 10: 535-547
10 Nakayama K, Nagasu T, Shimma Y, et al. OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine linked oligosaccharides[J]. Embo J, 1992, 11: 2511-2519
11 Herscovics A, Orlean P. Glycoprotein biosynthesis in yeast[J]. FASEB J, 1993, 7 (6): 540- 550
12 Walsh G. Biopharmaceutical benchmarks[J]. Nature Biotechnology, 2003, 21 (8): 865-870
13 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: 173-178
14 Shindo Y N, 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]. J Biol Chem, 1993, 268 (35): 26338-26345
15 Chiba Y, Suzuki M, Yoshida S, et al. Production of human compatible high mannose type (Man5GlcNAc2) sugar chains in saccharomyces cerevisiae[J]. The Journal of Biological Chemistry, 1998, 273: 26298-26304
16 Choi B K, Bobrowicz P, Davidson R C, et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris[J]. PNAS, 2003, 100 (9): 5022- 5027
17 Hamilton S R, Bobrowiez P, Bobrowiez B, et al. Production of complex human glycoproteins in yeast[J]. Science, 2003, 301 (4): 1244-1246
18 Bobrowicz P, Davidson R C, Li H J, et al. Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose[J]. Glycobiology, 2004, 14 (9): 757-766
19 Li H, Sethuraman N, Stadheim T A, et al. Optimization of humanized IgGs in glycoengineered Pichia pastoris[J]. Nature Biotechnology, 2006, 24: 210-215
20 Hamilton S R, Davidson R C, Sethuraman N, et al. Humanization of yeast to produce complex terminally sialylated glycoproteins[J]. Science, 2006, 313: 1441-1443
21 Zhou J G, Zhang H C, Liu X W, et al. Influence of N-glycosylation on saccharomyces cerevisiae morphology: a Golgi glycosylation mutant shows cell division defects[J]. Current Microbiology, 2007, 55: 198-203
22 Liu B, Gong X, et al. Disruption of the och1 and mnn1 genes decrease N-glycosylation on glycoprotein expressed in kluyveromyces lactis[J]. Journal of Biotechnology, 2009, 143: 95-102
23 Kelly R, Miller S M, Kurtz M B. One- step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans[J]. Mol Gen Genet, 1988, 214: 24-31
24 Nett J H, Gerngross T U. Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris[J]. Yeast, 2003, 20: 1279-1290
25 Fonzi W A, Irwin M Y. Isogenic strain construction and gene mapping in Candida albicans[J]. Genetics, 1993, 134: 717-728
26刘波.乳酸克鲁维酵母糖基工程研究[D]:[博士学位论文].北京:军事医学科学院生物工程研究所, 2008
27 Wang Y, Gong X, et al. A Pichia pastoris withα-1,6-mannosyltransferases deletion and its use in the expression of HSA/GM-CSF chimera[J]. Chinese Journal of Biotechnology, 2007, 23: 907-914
28 Schmidt M, Strenk, et al. Importance of cell wall mannoproteins for septum formation insaccharomyces cerevisiae[J]. Yeast, 2005 (22): 715-723
29 Ballou CE, Isolation, characterization and properties of saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects[J]. Methods Enzymol, 1990, 185: 440-470
30 Zhou J G, Zhang H C, Liu X W, et al. Influence of N-glycosylation on saccharomyces cerevisiae morphology: a Golgi glycosylation mutant shows cell division defects[J]. Current Microbiology, 2007, 55: 198-203
31 Madeo F, Frohlich E, Frohlich K U. A yeast mutant showing diagnostic markers of early and late apoptosis[J]. Cell Biol, 1997, 139: 729-734
32 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
33吴军,刘波. N-糖基工程研究进展[J].生物产业技术, 2009, 4: 47-52