通用型毕赤酵母高密度培养策略的网络共享技术  

Network sharing technology of universal control strategy for Pichia pastoris high-density culture

作　　者：吕奎 贾禄强 戴京京 丁健[1] LYU Kui;JIA Luqiang;DAI Jingjing;DING Jian(School of Biotechnology,Jiangnan University,Wuxi 214122,China;School of Food Science and Engineering,Yangzhou University,Yangzhou 225009,China)

机构地区：[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

出　　处：《食品与发酵工业》2021年第5期92-98,共7页Food and Fermentation Industries

基　　金：国家自然科学基金资助项目(21606106)。

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-GCSF m)和甲醇利用慢型(methanol utilization slow phenotype,Mut S)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-GCSF m的质量浓度达到532 mg/L;Mut S型毕赤酵母诱导69 h后总酶活达到84032.0 U/mg,对应的比酶活力为52884.0 U/mg;HSA-GCSF m产量和hLYZ活力均达到较优水平。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-GCSF m and Mut S Pichia pastoris expressing hLYZ.The concentration of HSA-GCSF m reached 532 mg/L after 60 h of Mut+Pichia pastoris induction.The average total enzyme activity reached 84032.0 U/mg after 74 hours of Pichia Mut S induction,and the corresponding specific activity was 52884.0 U/mg.The hLYZ activity and HSA-GCSF m production reached a high level.

关 键 词：毕赤酵母 高密度培养 控制策略 Django框架 网络共享 

分 类 号：TP393.09[自动化与计算机技术—计算机应用技术] TQ920.1[自动化与计算机技术—计算机科学与技术]