Please wait a minute...
 
 
食品与发酵工业  2021, Vol. 47 Issue (5): 92-98    DOI: 10.13995/j.cnki.11-1802/ts.025803
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
通用型毕赤酵母高密度培养策略的网络共享技术
吕奎1, 贾禄强2, 戴京京1, 丁健1*
1(江南大学 生物工程学院, 江苏 无锡, 214122)
2(扬州大学 食品科学与工程学院, 江苏 扬州, 225009)
Network sharing technology of universal control strategy for Pichia pastoris high-density culture
LYU Kui1, JIA Luqiang2, DAI Jingjing1, DING Jian1*
1(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
2(School of Food Science and Engineering, Yangzhou University, Yangzhou 225009, China)
下载:  HTML   PDF (3703KB) 
输出:  BibTeX | EndNote (RIS)      
摘要 甲醇营养型毕赤酵母是近年来广泛应用的一种外源蛋白表达系统。本课题组在前期研究中构建了一系列能够满足绝大多数毕赤酵母高密度发酵过程工艺控制需求的通用型控制策略:用于细胞培养期的改良型DO-Stat甘油流加策略;用于甲醇诱导期的“甲醇浓度受限-溶解氧浓度充足”诱导控制策略;用于甲醇诱导期的“甲醇浓度充足-溶氧浓度受限”诱导控制策略。然而, 这些控制策略的推广需要极高的时间和人力成本。为此, 本文开发了基于Django框架搭建的服务器程序, 以实现通用型毕赤酵母高密度培养策略的网络共享, 并依靠前期开发完成的客户端程序实现控制软件包与不同发酵设备之间的对接。在甲醇利用快型(methanol utilization plusphenotype, Mut+)毕赤酵母表达人血清白蛋白-人粒细胞集落刺激因子突变体融合蛋白(HSA-GCSFm)和甲醇利用慢型(methanol utilization slow phenotype, MutS)毕赤酵母表达人源溶菌酶(hLYZ)实验中分别验证了以上通用型控制策略的网络共享功能。结果表明, Mut+型毕赤酵母诱导60 h后HSA-GCSFm的质量浓度达到532 mg/L;MutS型毕赤酵母诱导69 h后总酶活达到84 032.0 U/mg, 对应的比酶活力为52 884.0 U/mg;HSA-GCSFm产量和hLYZ活力均达到较优水平。
服务
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章
吕奎
贾禄强
戴京京
丁健
关键词:  毕赤酵母  高密度培养  控制策略  Django框架  网络共享    
Abstract: 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.
Key words:  Pichia pastoris    high density culture    control strategy    Django framework    network sharing
收稿日期:  2020-10-01      修回日期:  2020-10-19                发布日期:  2021-03-31      期的出版日期:  2021-03-15
基金资助: 国家自然科学基金资助项目(21606106)
作者简介:  硕士研究生(丁健副教授为通讯作者, E-mail:dingjian@jiangnan.edu.cn)
引用本文:    
吕奎,贾禄强,戴京京,等. 通用型毕赤酵母高密度培养策略的网络共享技术[J]. 食品与发酵工业, 2021, 47(5): 92-98.
LYU Kui,JIA Luqiang,DAI Jingjing,et al. Network sharing technology of universal control strategy for Pichia pastoris high-density culture[J]. Food and Fermentation Industries, 2021, 47(5): 92-98.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.025803  或          http://sf1970.cnif.cn/CN/Y2021/V47/I5/92
[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.
[1] 高宇豪, 吴勇杰, 朱亚鑫, 付静, 徐建国, 王松涛, 徐国强, 张晓梅, 史劲松, 许正宏. 产谷胱甘肽毕赤酵母工程菌的构建及能量调控[J]. 食品与发酵工业, 2021, 47(7): 21-26.
[2] 孙媛媛, 崔树茂, 唐鑫, 毛丙永, 赵建新, 陈卫. 发酵乳杆菌的生长限制性因素分析及高密度培养工艺优化[J]. 食品与发酵工业, 2021, 47(6): 1-10.
[3] 钱晓芬, 吴涛, 赵理想, 孙杰, 汪钊, 魏春. 基因拷贝数对重组毕赤酵母的牛乳铁蛋白功能片段表达及细胞存活率的影响[J]. 食品与发酵工业, 2021, 47(4): 1-6.
[4] 叶德晓, 黄佳俊, 卢宇靖, 林育成, 李慧灵, 谭景航, 周金林. α-L-鼠李糖苷酶AnRhaE在毕赤酵母中的表达及应用[J]. 食品与发酵工业, 2021, 47(3): 25-30.
[5] 刘丽萍, 王力, 詹晓北, 朱莉, 郑志永, 高敏杰. 高果糖浆废液杂糖组分分析及其作为毕赤酵母发酵碳源的资源化利用[J]. 食品与发酵工业, 2020, 46(3): 8-13.
[6] 周海岩, 周建宝, 易晓男, 李勉, 柳志强. 来源于Rhodohalobacter barkolensis的昆布多糖酶RbLam16的重组表达及生产条件优化[J]. 食品与发酵工业, 2020, 46(21): 9-15.
[7] 蒋秋琪, 吕雪芹, 崔世修, 刘延峰, 堵国成, 刘龙. 代谢工程改造毕赤酵母发酵生产谷胱甘肽[J]. 食品与发酵工业, 2020, 46(17): 9-14.
[8] 梁鑫, 张仁怀, 吕自力, 艾华伟, 刘冬, 梁波, 单旭东, 陈浩然. 重组人Ⅲ型胶原蛋白的分离纯化[J]. 食品与发酵工业, 2020, 46(16): 159-163.
[9] 李鸿雁, 陆健, 李晓敏. 阿拉伯呋喃糖苷酶的重组表达及其发酵工艺优化[J]. 食品与发酵工业, 2020, 46(15): 14-20.
[10] 孟珊珊, 谭明, 肖冬光, 宋诙. 甜蛋白Brazzein在毕赤酵母中的表达及应用[J]. 食品与发酵工业, 2020, 46(15): 21-26.
[11] 李想, 陈瑜琦, 王子凡, 郑志永, 陈海琴, 高敏杰, 詹晓北. 螺旋筛板气升式反应器在真菌发酵中的应用[J]. 食品与发酵工业, 2020, 46(15): 93-99.
[12] 王莲哲, 江宏浩, 唐宜飞, 洪军. 新型抗菌肽Temporin-SHf在毕赤酵母中的表达及诱导条件优化[J]. 食品与发酵工业, 2020, 46(14): 98-102.
[13] 刘洁, 王宏涛, 钱和, 徐建中, 张伟国. 基于代谢工程构建产β-胡萝卜素重组毕赤酵母[J]. 食品与发酵工业, 2020, 46(11): 32-37.
[14] 徐富增, 王柯, 李善元, 毛忠贵, 张建华. 拟指数—DO-stat两阶段补料策略在糖蜜酵母高密度培养中的应用[J]. 食品与发酵工业, 2019, 45(7): 15-21.
[15] 于卓然, 洪厚胜. 功能性红曲Monacolin K产量控制策略的研究进展[J]. 食品与发酵工业, 2019, 45(3): 288-292.
No Suggested Reading articles found!
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
版权所有 © 《食品与发酵工业》编辑部
地址:北京朝阳区酒仙桥中路24号院6号楼111室
本系统由北京玛格泰克科技发展有限公司设计开发  技术支持:support@magtech.com.cn