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Heterologous Expression of Insulin Precursor in A Newly Engineered Pichia pastoris |
Qiang-qiang PENG,Qi LIU,Ming-qiang XU,Yuan-xing ZHANG,Meng-hao CAI() |
State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China |
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Abstract 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 1.85g/L insulin precursor was screened in 108h 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.
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Received: 30 November 2018
Published: 05 August 2019
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Corresponding Authors:
Meng-hao CAI
E-mail: cmh022199@ecust.edu.cn
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[1] |
Tsukita S, Yamada T, Takahashi K , et al. MicroRNAs 106b and 222 improve hyperglycemia in a mouse model of insulin-deficient diabetes via pancreatic β-cell proliferation. EBioMedicine, 2017,15(C):163-172.
doi: 10.1016/j.ebiom.2016.12.002
|
|
|
[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.
doi: 10.1186/s12934-014-0141-0
|
|
|
[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.
doi: 10.1016/j.biotechadv.2017.07.009
|
|
|
[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.
doi: 10.1186/s12934-016-0432-8
|
|
|
[7] |
Nordén K, Agemark M, Danielson J Å , 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.
|
|
|
[8] |
Huang P, Yan L P, Zhang N , et al. Constitutive expression of human goose-type lysozyme 2 in Pichia pastoris using the GAP promoter. 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.
doi: 10.1016/S0378-1119(96)00672-5
|
|
|
[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.
doi: 10.1186/1475-2859-13-23
|
|
|
[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.
doi: 10.1074/jbc.M115.692053
|
|
|
[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.
doi: 10.1186/s12934-016-0578-4
|
|
|
[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.
|
|
|
[17] |
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.
doi: 10.1128/AEM.02913-08
|
|
|
[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.
|
|
|
[21] |
Li H L, Chen Y, Chen H R , et al. Increase of expression level of human proinsulin in Pichia pastoris by double promoter co-expression 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.
|
|
|
[22] |
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.
|
|
|
[23] |
Liu Y Q . Heterologous biosynthesis of lovastatin and monacolin J on 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.
doi: 10.1016/j.bej.2013.07.013
|
|
|
[26] |
Jungo C, Marison I, Stockar U V . Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures:A quantitative study based on concentration gradients in transient continuous cultures. Journal of Biotechnology, 2007,128(4):824-837.
doi: 10.1016/j.jbiotec.2006.12.024
|
|
|
[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.
doi: 10.1038/nbt0294-181
|
|
|
[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.
|
|
|
[28] |
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.
doi: 10.13523/j.cb.20150510
|
|
|
[29] |
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.
doi: 10.13523/j.cb.20150510
|
|
|
[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.
doi: 10.1002/yea.v26:9
|
|
|
[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.
doi: 10.1016/j.pep.2018.08.002
|
|
|
[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.
|
|
|
[33] |
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.
doi: 10.1186/s12934-018-0970-3
|
|
|
[35] |
Sreenivas S, Krishnaiah S M, Shyam Mohan A H , et al. Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain insulin glargine in Pichia pastoris. Protein Expression and Purification, 2016,118:1-9.
doi: 10.1016/j.pep.2015.10.002
|
|
|
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