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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (7): 48-55    DOI: 10.13523/j.cb.20190707
技术与方法     
新型重组毕赤酵母产人胰岛素前体的表达工艺研究 *
彭强强,刘启,徐名强,张元兴,蔡孟浩()
华东理工大学生物反应器工程国家重点实验室 上海 200237
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
 全文: PDF(1264 KB)   HTML
摘要：

以毕赤酵母为异源表达宿主合成人胰岛素前体,在实验室研究和工业生产中已有广泛应用。目前研究主要使用天然甲醇诱导型AOX1启动子,以甲醇为单一基础碳源进行胰岛素前体的诱导发酵生产。但在毕赤酵母高密度发酵生产过程中,甲醇代谢过程耗氧大、产热高,补料控制工艺复杂,限制了发酵生产的放大。基于前期对启动子AOX1的转录调控设计研究,提出以人工设计的高效组成型转录调控器件CSAD_5驱动胰岛素前体基因表达,开发了以葡萄糖为碳源的发酵生产工艺,以解决甲醇体系中的产热、耗氧及工艺控制问题。在此基础上,通过增强筛选压力提高异源基因拷贝,获得了一株胰岛素前体高表达重组毕赤酵母,利用优化的培养工艺在5L反应器水平发酵生产,胰岛素前体产量在108h达到1.85g/L,为目前报道以葡萄糖为碳源,生产人胰岛素前体的最高水平,为胰岛素前体的工业生产及毕赤酵母的应用提供了新的思路和方法。
关键词： 毕赤酵母;  胰岛素前体;  发酵工艺;  高产菌株筛选    
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.
Key words: Pichia pastoris    Insulin precursor    Fermentation process    High-producing strains
收稿日期: 2018-11-30 出版日期: 2019-08-05
ZTFLH:  Q815  
基金资助: * 国家自然科学基金面上项目(31870073);上海科技创新行动计划基础研究领域项目(17JC1402400);中央高校基本科研业务费(22221818014);上海市青年科技启明星计划资助项目(19QA1402700)
通讯作者: 蔡孟浩     E-mail: cmh022199@ecust.edu.cn
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彭强强
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引用本文:

彭强强,刘启,徐名强,张元兴,蔡孟浩. 新型重组毕赤酵母产人胰岛素前体的表达工艺研究 *[J]. 中国生物工程杂志, 2019, 39(7): 48-55.

Qiang-qiang PENG,Qi LIU,Ming-qiang XU,Yuan-xing ZHANG,Meng-hao CAI. Heterologous Expression of Insulin Precursor in A Newly Engineered Pichia pastoris. China Biotechnology, 2019, 39(7): 48-55.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190707    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I7/48

图1  CSAD_5-IP菌株在摇瓶中的胰岛素前体表达量
图2  不同的甲醇补料速率对生长及产物生成的影响
图3  不同的葡萄糖补料速率对生长及产物生成的影响
图4  WT-IP甲醇发酵工艺与CSAD_5-IP葡萄糖发酵工艺过程比较
图5  WT-IP甲醇发酵工艺与CSAD_5-IP葡萄糖发酵工艺通气量比较
图6  不同产量菌株在摇瓶中胰岛素前体表达量
图7  CSAD_5-IP10葡萄糖发酵工艺发酵过程
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