新型重组毕赤酵母产人胰岛素前体的表达工艺研究
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摘要
以毕赤酵母为异源表达宿主合成人胰岛素前体,在实验室研究和工业生产中已有广泛应用。目前研究主要使用天然甲醇诱导型AOX1启动子,以甲醇为单一基础碳源进行胰岛素前体的诱导发酵生产。但在毕赤酵母高密度发酵生产过程中,甲醇代谢过程耗氧大、产热高,补料控制工艺复杂,限制了发酵生产的放大。基于前期对启动子AOX1的转录调控设计研究,提出以人工设计的高效组成型转录调控器件CSAD_5驱动胰岛素前体基因表达,开发了以葡萄糖为碳源的发酵生产工艺,以解决甲醇体系中的产热、耗氧及工艺控制问题。在此基础上,通过增强筛选压力提高异源基因拷贝,获得了一株胰岛素前体高表达重组毕赤酵母,利用优化的培养工艺在5L反应器水平发酵生产,胰岛素前体产量在108h达到1. 85g/L,为目前报道以葡萄糖为碳源,生产人胰岛素前体的最高水平,为胰岛素前体的工业生产及毕赤酵母的应用提供了新的思路和方法。
Pichia pastoris is one of the most widely used heterologous expression host in the synthesis of human insulin precursors both in laboratory research and industrial production. Currently, the induction fermentation production of insulin precursors is carried out mainly by using the naturally methanol-inducible AOX1 promoter and utilizing methanol as the sole carbon source. High oxygen consumption, heat release and complicated feeding process control caused by methanol catabolism usually bring difficulties to industrial scale-up and limited application for protein expression by high cell densities fermentation in Pichia pastoris. A synthetic efficient constitutive transcriptional signal amplification device of CSAD _ 5 was constructed to drive insulin precursor gene expression based on the transcriptional regulation design of the AOX1 promoter in our previous work. In order to reduce the oxygen consumption,heat generation and bioprocess regulation during fermentation process,a glucose supported fermentation process is established. Through increasing module-specific antibiotic stress to optimize the biosynthetic gene dosages,a recombinant Pichia pastoris strain producing 1. 85 g/L insulin precursor was screened in 108 h in a 5-L bioreactor,which is the highest productivity to produce human insulin precursor by utilizing glucose as a carbon source according to reports. Our findings provide a new alternative choice to the high-yield production of insulin precursor in industrial application and reveal the potential of Pichia pastoris to produce a wide range of compounds.
Pichia pastoris is one of the most widely used heterologous expression host in the synthesis of human insulin precursors both in laboratory research and industrial production. Currently, the induction fermentation production of insulin precursors is carried out mainly by using the naturally methanol-inducible AOX1 promoter and utilizing methanol as the sole carbon source. High oxygen consumption, heat release and complicated feeding process control caused by methanol catabolism usually bring difficulties to industrial scale-up and limited application for protein expression by high cell densities fermentation in Pichia pastoris. A synthetic efficient constitutive transcriptional signal amplification device of CSAD _ 5 was constructed to drive insulin precursor gene expression based on the transcriptional regulation design of the AOX1 promoter in our previous work. In order to reduce the oxygen consumption,heat generation and bioprocess regulation during fermentation process,a glucose supported fermentation process is established. Through increasing module-specific antibiotic stress to optimize the biosynthetic gene dosages,a recombinant Pichia pastoris strain producing 1. 85 g/L insulin precursor was screened in 108 h in a 5-L bioreactor,which is the highest productivity to produce human insulin precursor by utilizing glucose as a carbon source according to reports. Our findings provide a new alternative choice to the high-yield production of insulin precursor in industrial application and reveal the potential of Pichia pastoris to produce a wide range of compounds.
引文
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[22]王锦佳.新型重组毕赤酵母中外源蛋白的诱导表达新工艺.上海:华东理工大学,2017.Wang J J.Novel strategies for induction expression of heterologous protein in a new type of recombinant Pichia pastoris.Shanghai:East China University of Science&Technology,2017.
[23]刘一奇.基于甲醇/乙醇底物的洛伐他汀及莫纳可林J异源生物合成.上海:华东理工大学,2018.Liu Y Q.Heterologous biosynthesis of lovastatin and monacolin Jon methanol and ethanol by engineered Pichia pastoris.Shanghai:East China University of Science&Technology,2018.
[24]Wang J J,Wang X L,Shi L,et al.Reduced methanol input induces increased protein output by AOX1 promoter in a trans-acting elements engineered Pichia pastoris.Journal of Industrial Microbiology&Biotechnology,2018,45(1):25-30.
[25]Garcia-Ortega X,Ferrer P,Montesinos J L,et al.Fed-batch operational strategies for recombinant Fab production with Pichia pastoris,using the constitutive GAP promoter.Biochemical Engineering Journal,2013,79(41):172-181.
[26]Jungo C,Marison I,Stockar U V.Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures:Aquantitative study based on concentration gradients in transient continuous cultures.Journal of Biotechnology,2007,128(4):824-837.
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[29]钱凯,张晶晶,吴素平,等.利用GAP启动子在毕赤酵母中表达与纯化GLP-1类似物.中国生物工程杂志,2015,35(5):66-73.Qian K,Zhang J J,Wu S P,et al.Constituted expression and purification of glucagon-like peptide-1 analogue in Pichia pastoris using GAP promoter.China Biotechnology,2015,35(5):66-73.
[30]Celik E,Calik P,Oliver S G.Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol.Yeast,2009,26(9):473-484.
[31]Wang J J,Wang X L,Shi L,et al.Methanol-independent protein expression by AOX1 promoter with trans-acting elements engineering and glucose-glycerol-shift induction in Pichia pastoris.Scientific Reports,2017,7:41850.
[32]Sawanan T,Tanapati P,Nanthika K,et al.Expression purification and biological activity of monomeric insulin precursors from methylotrophic yeasts.Protein Expression and Purification,2019,153(1):35-43.
[33]梁晨晨,王立,罗秋玲,等.一种增加毕赤酵母生产胰岛素前体的方法.生物工程学报,2017,33(7):1178-1189.Liang C C,Wang L,Luo Q L,et al.A method to increase the production of insulin precursor in Pichia pastoris.Chinese Journal of Biotechnology,2017,33(7):1178-1189.
[34]Gustavo R,Vanz A L,Lünsdorf H,et al.Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris.Microbial Cell Factories,2018,17(1):123.
[35]Sreenivas S,Krishnaiah S M,Shyam Mohan A H,et al.Disruption of KEX1 gene reduces the proteolytic degradation of secreted twochain insulin glargine in Pichia pastoris.Protein Expression and Purification,2016,118:1-9.
[2]Langley J,Adams G.Insulin-based regimens decrease mortality rates in critically ill patients:a systematic review.Diabetes/Metabolism Research and Reviews,2010,23(3):184-192.
[3]Baeshen N A,Baeshen M N,Sheikh A,et al.Cell factories for insulin production.Microbial Cell Factories,2014,13(1):141.
[4]Macauley-Patrick S,Fazenda M L,Mcneil B,et al.Heterologous protein production using the Pichia pastoris expression system.Yeast,2010,22(4):249-270.
[5]Schwarzhans J P,Luttermann T,Geier M,et al.Towards systems metabolic engineering in Pichia pastoris.Biotechnology Advances,2017,35(6):681.
[6]Aw R,Polizzi K M.Liquid PTVA:a faster and cheaper alternative for generating multi-copy clones in Pichia pastoris.Microbial Cell Factories,2016,15(1):29.
[7]Nordén K,Agemark M,Danielson J,et al.Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris.BMC Biotechnology,2011,11(1):47.
[8]黄鹏,阎丽萍,张宁,等.利用GAP启动子在毕赤酵母中组成型表达人鹅型溶菌酶2.中国生物工程杂志,2018,38(10):55-63.Huang P,Yan L P,Zhang N,et al.Constitutive expression of human goose-type lysozyme 2 in Pichia pastoris using the GAPpromoter.China Biotechnology,2018,38(10):55-63.
[9]Sreekrishna K,Brankamp R G,Kropp K E,et al.Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.Gene,1997,190(1):55-62.
[10]Ciofalo V,Barton N,Kreps J,et al.Safety evaluation of a lipase enzyme preparation,expressed in Pichia pastoris,intended for use in the degumming of edible vegetable oil.Regulatory Toxicology&Pharmacology Rtp,2006,45(1):1-8.
[11]Wang Y,Liang Z H,Zhang Y S,et al.Secretory expression of human insulin in methylotrophic yeast Pichia pastoris.Acta Biochimica Et Biophysica Sinica,1999,31(5):587-589.
[12]Kjeldsen T,Pettersson A F,Hach M.Secretory expression and characterization of insulin in Pichia pastoris.Biotechnology&Applied Biochemistry,2011,29(1):79-86.
[13]Ana L V,Nimtz M,Rinas U.Decrease of UPR-and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures.Microbial Cell Factories,2014,13(1):23.
[14]Wang X L,Wang Q,Wang J J,et al.Mit1 transcription factor mediates methanol signaling and regulates the alcohol oxidase 1(AOX1)promoter in Pichia pastoris.Journal of Biological Chemistry,2016,291(12):6245-6261.
[15]Shen W,Xue Y,Liu Y Q,et al.A novel methanol-free Pichia pastoris system for recombinant protein expression.Microbial Cell Factories,2016,15(1):178.
[16]Mateles R I.Calculation of the oxygen required for cell production.Biotechnology&Bioengineering,1971,13(4):581-582.
[17]战春君,李翔,刘国强,等.巴斯德毕赤酵母甘油转运体的发现及功能研究.中国生物工程杂志,2017,37(7):48-55.Zhan C J,Li X,Liu G Q,et al.Identification of glycerol transporter in Pichia pastoris and function research.China Biotechnology,2017,37(7):48-55.
[18]Xiao A F,Zhou X S,Zhou L,et al.Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation.Applied Microbiology&Biotechnology,2006,72(4):837-844.
[19]Ahn J,Hong J,Park M,et al.Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter.Applied and Environmental Microbiology,2009,75(11):3528-3534.
[20]Menendez J,Valdes I,Cabrera N.The ICL1 gene of Pichia pastoris,transcriptional regulation and use of its promoter.Yeast,2010,20(13):1097-1108.
[21]李红亮,陈勇,陈海容,等.应用双启动子共表达体系提高人胰岛素原在毕赤酵母中的表达量.中国生物制品学杂志,2012,25(4):422-425.Li H L,Chen Y,Chen H R,et al.Increase of expression level of human proinsulin in Pichia pastoris by double promoter coexpression system.Chinese Journal of Biologicals,2012,25(4):422-425.
[22]王锦佳.新型重组毕赤酵母中外源蛋白的诱导表达新工艺.上海:华东理工大学,2017.Wang J J.Novel strategies for induction expression of heterologous protein in a new type of recombinant Pichia pastoris.Shanghai:East China University of Science&Technology,2017.
[23]刘一奇.基于甲醇/乙醇底物的洛伐他汀及莫纳可林J异源生物合成.上海:华东理工大学,2018.Liu Y Q.Heterologous biosynthesis of lovastatin and monacolin Jon methanol and ethanol by engineered Pichia pastoris.Shanghai:East China University of Science&Technology,2018.
[24]Wang J J,Wang X L,Shi L,et al.Reduced methanol input induces increased protein output by AOX1 promoter in a trans-acting elements engineered Pichia pastoris.Journal of Industrial Microbiology&Biotechnology,2018,45(1):25-30.
[25]Garcia-Ortega X,Ferrer P,Montesinos J L,et al.Fed-batch operational strategies for recombinant Fab production with Pichia pastoris,using the constitutive GAP promoter.Biochemical Engineering Journal,2013,79(41):172-181.
[26]Jungo C,Marison I,Stockar U V.Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures:Aquantitative study based on concentration gradients in transient continuous cultures.Journal of Biotechnology,2007,128(4):824-837.
[27]Scorer C A,Clare J J,Mccombie W R,et al.Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.Nature Biotechnology,1994,12(2):181-184.
[28]宁光.中国糖尿病防治的现状及展望.中国科学:生命科学,2018,48(8):810-811.Ning G.Status quo and prospect of prevention and control of diabetes in China.Scientia Sinica Vitae,2018,48(8):810-811.
[29]钱凯,张晶晶,吴素平,等.利用GAP启动子在毕赤酵母中表达与纯化GLP-1类似物.中国生物工程杂志,2015,35(5):66-73.Qian K,Zhang J J,Wu S P,et al.Constituted expression and purification of glucagon-like peptide-1 analogue in Pichia pastoris using GAP promoter.China Biotechnology,2015,35(5):66-73.
[30]Celik E,Calik P,Oliver S G.Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol.Yeast,2009,26(9):473-484.
[31]Wang J J,Wang X L,Shi L,et al.Methanol-independent protein expression by AOX1 promoter with trans-acting elements engineering and glucose-glycerol-shift induction in Pichia pastoris.Scientific Reports,2017,7:41850.
[32]Sawanan T,Tanapati P,Nanthika K,et al.Expression purification and biological activity of monomeric insulin precursors from methylotrophic yeasts.Protein Expression and Purification,2019,153(1):35-43.
[33]梁晨晨,王立,罗秋玲,等.一种增加毕赤酵母生产胰岛素前体的方法.生物工程学报,2017,33(7):1178-1189.Liang C C,Wang L,Luo Q L,et al.A method to increase the production of insulin precursor in Pichia pastoris.Chinese Journal of Biotechnology,2017,33(7):1178-1189.
[34]Gustavo R,Vanz A L,Lünsdorf H,et al.Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris.Microbial Cell Factories,2018,17(1):123.
[35]Sreenivas S,Krishnaiah S M,Shyam Mohan A H,et al.Disruption of KEX1 gene reduces the proteolytic degradation of secreted twochain insulin glargine in Pichia pastoris.Protein Expression and Purification,2016,118:1-9.