Process control and physiological analysis of heterologous protein production by recombinant Pichia pastoris
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摘要: 毕赤酵母是近年来应用广泛、极受青睐的一种外源蛋白表达系统。除了构建高效、稳定的毕赤酵母生产菌株外,研究不同发酵控制条件下,细胞的生理和代谢状态变化,为毕赤酵母细胞提供最优的生长和环境条件并综合考虑发酵成本是成功实现外源蛋白规模化、商业化生产的保障。本文从培养基组分、外界环境条件、细胞比生长速率、生长期甘油流加策略和诱导期甲醇流加策略等方面阐述了发酵过程对外源蛋白表达的影响,并进一步从细胞生理和代谢层面深入讨论了不同发酵过程控制条件的影响机制。Abstract: Pichia pastoris was currently one of the most effective and versatile systems for the expression of heterologous proteins. Besides the construction of efficient and stable recombinant Pichia pastoris, fermentation process engineering technique, mainly covering the methods of control proper fermentation conditions and envirements, that is considered as another important route for successful production of recombinant protein.The components of culture medium, environment conditions, cell specific growth rate, glycerol feeding strategies in growth period and methanol feeding strategies in induction period were summarized in this review. In additon, cell physiological and metabolic funcations in different fermentation conditions were analyzed.
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Keywords:
- Pichia pastoris /
- process control /
- physiological analysis /
- protein secretion /
- stress response
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[1] Cos O, Ramón R, Montesinos J L, et al.Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters:A review[J].Microb Cell Fact, 2006, 5:17.
[2] Çelik E, Çalιk P.Production of recombinant proteins by yeast cells[J].Biotechnol Adv, 2012, 30 (5) :1108-1118.
[3] Idiris A, Tohda H, Kumagai H, et al.Engineering of protein secretion in yeast:strategies and impact on protein production[J].Appl Microbiol Biotechnol, 2010, 86 (2) :403-417.
[4] Gao M J, Shi Z P.Process control and optimization for heterologous protein production by methylotrophic Pichia pastoris[J].Chinese J Chem Eng, 2013, 21 (2) :216-226.
[5] Guan B, Chen F X, Lei J Y, et al.Constitutive expression of a rh IL-2-HSA fusion protein in Pichia pastoris using glucose as carbon source[J].Appl Biochem Biotechnol, 2013, 171 (7) :1792-1804.
[6] Yu X W, Lu X, Zhao L S, et al.Impact of NH4+nitrogen source on the production of rhizopus oryzae lipase in Pichia pastoris[J].Process Biochem, 2013, 48 (10) :1462-1468.
[7] Rumiantsev A M, Padkina M V, Sambuk E V.Effect of nitrogen source on gene expression of first steps of methanol utilization pathway in Pichia pastoris[J].Genetika, 2013, 49 (4) :454-460.
[8] Wang Y, Wang Z H, Xu Q L, et al.Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris[J].Process Biochem, 2009, 44 (9) :949-954.
[9] Jia D X, Liu L, Wang H L, et al.Over production of a truncated poly (vinyl alcohol) dehydrogenase in recombinant Pichia pastoris by low-temperature induction strategy and related mechanism analysis[J].Bioprocess Biosyst Eng, 2013, 36 (8) :1095-1103.
[10] Wu J M, Wang S Y, Fu W C.Lower temperature cultures enlarge the effects of vitreoscilla hemoglobin expression on recombinant Pichia pastoris[J].Int J Mol Sci, 2012, 13 (10) :13212-13226.
[11] Jin H, Liu G Q, Ye X F, et al.Enhanced porcine interferonalpha production by recombinant Pichia pastoris with a combinational control strategy of low induction temperature and high dissolved oxygen concentration[J].Biochem Eng J, 2010, 52 (1) :91-98.
[12] Zhao H L, Xue C, Wang Y, et al.Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in pmr1 gene by culture condition optimization[J].Appl Microbiol Biotechnol, 2008, 81 (2) :235-241.
[13] Jahic M, Wallberg F, Bollok M, et al.Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures[J].Microb Cell Fact, 2003, 2 (1) :6.
[14] Dragosits M, Stadlmann J, Albiol J, et al.The effect of temperature on the proteome of recombinant Pichia pastoris[J].J Proteome Res, 2009, 8 (3) :1380-1392.
[15] Jiang F, Kongsaeree P, Schilke K, et al.Effects of p H and temperature on recombinant manganese peroxidase production and stability[J].Appl Biochem Biotechnol, 2008, 146:15-27.
[16] Charoenrat T, Khumruaengsri N, Promdonkoy P, et al.Improvement of recombinant endoglucanase produced in Pichia pastoris KM71 through the use of synthetic medium for inoculum and ph control of proteolysis[J].J Biosci Bioeng, 2013, 116 (2) :193-198.
[17] Çalιk P, Bayraktar E, Inankur B, et al.Influence of p H on recombinant human growth hormone production by Pichia pastoris[J].J Chem Technol Biotechnol, 2010, 85 (12) :1628-1635.
[18] Soyaslan E S, Çalιk P.Enhanced recombinant human erythropoietin production by Pichia pastoris in methanol fedbatch/sorbitol batch fermentation through p H optimization[J].Biochem Eng J, 2011, 55 (1) :59-65.
[19] Carnicer M.Metabolic flux profiling of Pichia pastoris expressing an antibody fragment under different oxygen conditions[J].New Biotechnol, 2009, 25:325-325.
[20] Min C K, Lee J W, Chung K H, et al.Control of specific growth rate to enhance the production of a novel disintegrin, saxatilin, in recombinant Pichia pastoris[J].J Biosci Bioeng, 2010, 110 (3) :314-319.
[21] Maurer M, Kuehleitner M, Gasser B, et al.Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris[J].Microb Cell Fact, 2006, 5:37.
[22] Jacobs P P, Inan M, Festjens N, et al.Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate[J].Microb Cell Fact, 2010, 9:93.
[23] Schenk J, Balazs K, Jungo C, et al.Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut (+) strain[J].Biotechnol Bioeng, 2008, 99 (2) :368-377.
[24] Wu D, Chu J, Hao Y Y, et al.Influence of specific growth rate on specific productivity and intermolecular disulfide bond of recombinant protein produced by a Pichia pastoris Mut (+) strain[J].J Biotechnol, 2010, 150:S540.
[25] Rebnegger C, Graf A B, Valli M, et al.In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response[J].Biotech J, 2014, 9 (4) :511-525.
[26] Wei C, Zhou X S, Zhang Y X.Improving intracellular production of recombinant protein in Pichia pastoris using an optimized preinduction glycerol-feeding scheme[J].Appl Microbiol Biotechnol, 2008, 78 (2) :257-264.
[27] Ferreira A R, Ataide F, Von Stosch M, et al.Application of adaptive DO-stat feeding control to Pichia pastoris x33 cultures expressing a single chain antibody fragment (sc Fv) [J].Bioprocess Biosystems Eng, 2012, 35 (9) :1603-1614.
[28] Woo S H, Park S H, Lim H K, et al.Extended operation of a pressurized 75-L bioreactor for sh Lkn-1 production by Pichia pastoris using dissolved oxygen profile control[J].J Ind Microbiol Biotechnol, 2005, 32 (10) :474-480.
[29] Gao M J, Zheng Z Y, Wu J R, et al.Improvement of specific growth rate of Pichia pastoris for effective porcine interferonalpha production with an on-line model-based glycerol feeding strategy[J].Appl Microbiol Biotechnol, 2012, 93 (4) :1437-1445.
[30] Ding J, Gao M J, Hou G L, et al.Stabilizing porcine interferon-αproduction by Pichia pastoris with an ethanol online measurement based DO-stat glycerol feeding strategy[J].J Chem Technol Biot, 2014, 89:10-16.
[31] Wu D, Yu X W, Wang T C, et al.High yield Rhizopus chinenisis prolipase production in Pichia pastoris:impact of methanol concentration[J].Biotechnol Bioprocess Eng, 2011, 16 (2) :305-311.
[32] Ding J, Zhang C L, Gao M J, et al.Enhanced porcine circovirus Cap protein production by Pichia pastoris with a fuzzy logic do control based methanol/sorbitol co-feeding induction strategy[J].J Biotechnol, 2014, 177:35-44.
[33] Maghsoudi A, Hosseini S, Shojaosadati S A, et al.A new methanol-feeding strategy for the improved production of betagalactosidase in high cell-density fed-batch cultures of Pichia pastoris mut (+) strains[J].Biotechnol Bioprocess Eng, 2012, 17 (1) :76-83.
[34] Niu H, Jost L, Pirlot N, et al.A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris mut (+) /p AOX1-lac Z strain[J].Microb Cell Fact, 2013, 12:33.
[35] Jorda J, Suarez C, Carnicer M, et al.Glucose-methanol coutilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis[J].Bmc Syst Biol, 2013, 7:17.
[36] Çalιk P, Bozkurt B, Zerze G H, et al.Effect of co-substrate sorbitol different feeding strategies on human growth hormone production by recombinant Pichia pastoris[J].J Chem Technol Biotechnol, 2013, 88 (9) :1631-1640.
[37] Paulova L, Hyka P, Branska B, et al.Use of a mixture of glucose and methanol as substrates for the production of recombinant trypsinogen in continuous cultures with Pichia pastoris mut (+) [J].J Biotechnol, 2012, 157 (1) :180-188.
[38] Arnau C, Casas C, Valero F.The effect of glycerol mixed substrate on the heterologous production of a rhizopus oryzae lipase in Pichia pastoris system[J].Biochem Eng J, 2011, 57:30-37.
[39] Gao M J, Li Z, Yu R S, et al.Methanol/sorbitol co-feeding induction enhanced p IFN-αproduction by P.pastoris associated with energy metabolism shift[J].Bioprocess Biosyst Eng, 2012, 35 (7) :1125-1136.
[40] Zhu T C, Hang H F, Chu J, et al.Transcriptional investigation of the effect of mixed feeding to identify the main cellular stresses on recombinant Pichia pastoris[J].J Ind Microbiol Biotechnol, 2013, 40 (2) :183-189.
[41] Baumann K, Carnicer M, Dragosits M, et al.A multi-level study of recombinant Pichia pastoris in different oxygen conditions[J].Bmc Syst Biol, 2010, 4:141.
[42] Gasch A P, Werner Washburne M.The genomics of yeast responses to environmental stress and starvation[J].Funct Integr Genomics, 2002, 2:181-192.
[43] Vanz A L, Luensdorf H, Adnan A, et al.Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen:catabolic adaptation, stress responses, and autophagic processes[J].Microb Cell Fact, 2012, 11:103.
[44] Carnicer M, Ten Pierick A, Van Dam J, et al.Quantitative metabolomics analysis of amino acid metabolism in recombinant Pichia pastoris under different oxygen availability conditions[J].Microb Cell Fact, 2012, 11:83.
[45] Dragosits M, Stadlmann J, Graf A, et al.The response to unfolded protein is involved in osmotolerance of Pichia pastoris[J].BMC Genomics, 2010, 11:207.
[46] Jorda J, Jouhten P, Camara E, et al.Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:Methanol mixtures[J].Microb Cell Fact, 2012, 11:57.
[47] Sola A, Jouhten P, Maaheimo H, et al.Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates[J].Microbiology, 2007, 153:281-290.
[48] Jorda J, De Jesus S S, Peltier S, et al.Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived13C-labelling data from proteinogenic amino acids[J].New Biotechnol, 2014, 31 (1) :120-132.
[49] Unrean P.Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins[J].Biotechnol Prog, 2014, 30 (1) :28-37.
[50] 关波, 金坚, 李华钟.改良毕赤酵母分泌表达外源蛋白能力的研究进展[J].微生物学报, 2011, 51 (7) :851-857. [51] Zhu T, Guo M, Zhuang Y, et al.Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes[J].Appl Microbiol Biotechnol, 2011, 89 (4) :1127-1135.
[52] Lin X Q, Liang S L, Han S Y, et al.Quantitative ITRAQ LC-MS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastaris[J].J Proteomics, 2013, 91:58-72.
[53] Liberek K, Lewandowska A, Zietkiewicz S.Chaperones in control of protein disaggregation[J].EMBO J, 2008, 27 (2) :328-335.
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