Methanolotrophic Pichia pastoris is an exogenous protein expression system widely used recently.In the previous research, we have constructed a series of general control strategies that can meet the process control requirements of most Pichia high-density fermentation processes:an improved DO-Stat glycerol feeding strategy for the cell culture period;the “high methanol concentration-low dissolved oxygen” induction control strategy in the methanol induction period;the “low methanol concentration-high dissolved oxygen” induction control strategy used in the methanol induction period.However, the rollout of these control strategies is costly in terms of time and labor.To this end, this article developed a server program based on the Django framework to realize the network sharing of the universal Pichia high-density cultivation strategy, and rely on the client program developed in the early stage to realize the docking between the control software package and different fermentation equipment.The network sharing function of the universal control strategy was verified in the experiments of Mut+ Pichia pastoris expressing HSA-GCSFm and MutS Pichia pastoris expressing hLYZ.The concentration of HSA-GCSFm reached 532 mg/L after 60 h of Mut+ Pichia pastoris induction.The average total enzyme activity reached 84 032.0 U/mg after 74 hours of Pichia MutS induction, and the corresponding specific activity was 52 884.0 U/mg.The hLYZ activity and HSA-GCSFm production reached a high level.
LYU Kui
,
JIA Luqiang
,
DAI Jingjing
,
DING Jian
. Network sharing technology of universal control strategy for Pichia pastoris high-density culture[J]. Food and Fermentation Industries, 2021
, 47(5)
: 92
-98
.
DOI: 10.13995/j.cnki.11-1802/ts.025803
[1] MACAULEY-PATRICK S, FAZENDA M L, MCNEIL B, et al.Heterologous protein production using the Pichia pastoris expression system[J].Yeast, 2005, 22(4):249-270.
[2] ELIK E, ALIK P.Production of recombinant proteins by yeast cells[J].Biotechnology Advances, 2012, 30(5):1 108-1 118.
[3] 武婕, 张晓雪, 余河水, 等.毕赤酵母工程菌高密度发酵研究与进展[J].中国生物工程杂志, 2016, 36(1):108-114.
WU J, ZHANG X X, YU H S, et al.Research progress of high density fermentation process of Pichia pastoris[J].China Biotechnology 2016, 36(1):108-114.
[4] FARINHA I, FREITAS F, REIS M A M.Implementation of a repeated fed-batch process for the production of chitin-glucan complex by Komagataella pastoris[J].New Biotechnol, 2017, 37:123-128.
[5] HELLWIG S, EMDE F, RAVEN N P G, et al.Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations[J].Biotechnol Bioeng, 2001, 74(4):344-352.
[6] 贾禄强.协同优化控制关键状态变量强化重组毕赤酵母在诱导期高效表达外源蛋白[D].无锡:江南大学, 2019.
JIA L Q.Enhanced heterologous proteins production by recombinant Pichia pastoris in induction phase by associately[D].Wuxi:Jiangnan University, 2019.
[7] LEE C Y, NAKANO A, SHIOMI N, et al.Effects of substrate feed rates on heterologous protein expression by Pichia pastoris in DO-stat fed-batch fermentation[J].Enzyme & Microbial Technology, 2003, 33(4):358-365.
[8] DING J, GAO M J, HOU G L, et al.Stabilizing porcine interferon-α production by Pichia pastoris with an ethanol on-line measurement based DO-Stat glycerol feeding strategy[J].Journal of Chemical Technology and Biotechnology, 2014, 89:1 948-1 953.
[9] JIN H, ZHENG Z Y, GAO M J, et al.Effective induction of phytase in Pichia pastoris fed-batch culture using an ANN pattern recognition model-based on-line adaptive control strategy[J].Biochemical Engineering Journal, 2007, 37(1):26-33.
[10] JIA L Q, GAO M J, YAN J, et al.Evaluation of the sub-optimal induction strategies for heterologous proteins production by Pichia pastoris Mut+/MutS strains and related transcriptional and metabolic analysis[J].World Journal of Microbiology & Biotechnology, 2018, 34(12).DOI:10.1007/s11274-018-2562-0.
[11] 韦立梅, 苏兵.Django框架下Python网站开发过程综述[J].电脑与电信, 2019(10):54-56.
WEI L M, SU B.Overview of python website development process under Django framework[J].Computer and Telecom, 2019(10):54-56.
[12] 涂庭勇, 贾禄强, 史仲平, 等.“低甲醇浓度-高溶解氧浓度”策略诱导毕赤酵母高效表达HSA-GCSFm及其转录组学机理分析[J].食品与发酵工业, 2017, 43(3):1-8.
TU T Y, JIA L Q, SHI Z P, et al.Enhanced HSA-GCSFm production by Pichia pastoris under “low methanol concentration-high dissolved oxygen” induction strategy and its transcriptome analysis[J].Food and Fermentation Industries, 2017, 43(3):1-8.
[13] 金虎.毕赤酵母高效发酵生产猪α干扰素过程的优化与代谢调控[D].无锡:江南大学, 2011.
JIN H.Fermentation optimization and metabolic regulation of porcine interferon-α expression by recombinant [D].Wuxi:Jiangnan University, 2011.
[14] GAO M J, LI Z, YU R S, et al.Methanol/sorbitol co-feeding induction enhanced porcine interferon-aproduction by P.pastoris associated with energy metabolism shift[J].Bioprocess and Biosystems Engineering, 2012, 35(7):1 125-1 136.
[15] GUAN B, CHEN F X, LEI J Y, et al.Constitutive expression of a rhIL-2-HSA fusion protein in Pichia pastoris using glucose as carbon source[J].Applied Biochemistry & Biotechnology, 2013, 171(7):1 792-1 804.
[16] 白昌盛.基于Django的Python Web开发[J].信息与电脑(理论版), 2019, 31(24):37-40.
BAI C S.Python web development based on Django[J].Information and Computer (Theoretical Edition), 2019, 31(24):37-40.
[17] PRASETVADI G, HANTORO U T , MUTIARA A B .Singkat:A keyword-based URL shortener and click tracker package for Django web application[J].International Journal of Advanced Computer Science and Applications, 2018, 9(9):118-122.
[18] 戴京京, 吕奎, 史仲平.BioJN发酵技术服务系统PC端的设计、开发及应用[J/OL].食品与发酵工业, 2020.DOI:10.13995/j.cnki.11-1802/ts.024716.
DAI J J, LYU K, SHI Z P.Design, development and application of BioJN fermentation technology service system PC terminal[J/OL].Food and Fermentation Industries, 2020.DOI:10.13995/j.cnki.11-1802/ts.024716.
