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食品与发酵工业  2020, Vol. 46 Issue (17): 9-14    DOI: 10.13995/j.cnki.11-1802/ts.023602
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
代谢工程改造毕赤酵母发酵生产谷胱甘肽
蒋秋琪1, 吕雪芹1,2, 崔世修1, 刘延峰1, 堵国成1, 刘龙1*
1(工业生物技术教育部重点实验室(江南大学),江苏 无锡,214122)
2(江南大学 生物工程学院,江苏 无锡,214122)
Metabolic engineered Pichia pastoris for synthesis of glutathione
JIANG Qiuqi1, LYU Xueqin1,2, CUI Shixiu1, LIU Yanfeng1, DU Guocheng1, LIU Long1*
1(Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)
2(College of Bioengineering, Jiangnan University, Wuxi 214122, China)
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摘要 谷胱甘肽(glutathione,GSH)作为一种具有多种生物学功能的含硫化合物,有广泛的市场应用价值。该研究旨在通过代谢工程改造巴斯德毕赤酵母,从而增加GSH的产量。首先,通过使用高浓度的G418抗生素筛选高拷贝的GSH合成酶Ⅰ(GshI)和GSH合成酶Ⅱ(GshⅡ),使GSH摇瓶产量增加了60%,达到1.6 g/L。其次,使用CRISPR/Cas9技术分别敲除GSH降解途径中的3个基因DUG1(YFR044c),DUG2(YBR281c)和DUG3(YNL191w)。其中,敲除DUG3基因的毕赤酵母GS115-No.10菌株的GSH产量从160 mg/g提高到了224 mg/g。最后,通过发酵培养基中葡萄糖和3种前体氨基酸(L-谷氨酸、L-半胱氨酸和L-甘氨酸)初始添加量的优化,将GSH摇瓶产量进一步提高至1.7 g/L,为GSH的工业生产奠定良好的基础。
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蒋秋琪
吕雪芹
崔世修
刘延峰
堵国成
刘龙
关键词:  谷胱甘肽  基因拷贝数  基因敲除  发酵优化  毕赤酵母    
Abstract: Glutathione (GSH) is a sulfur-containing compound with various biological functions. This work aimed to increase the production of GSH by metabolic engineered Pichia pastoris. First, by screening the GSH synthetase I (GshI) and GSH synthetase II (GshII) gene copy numbers with high concentration of G418 antibiotic, GSH production increased by 60% (1.6 g/L). Three genes DUG1(YFR044c), DUG2(YBR281c) or DUG3(YNL191w) in the GSH degradation pathway was knocked out using CRISPR/Cas9. The GSH titer of the strain P. pastoris GS115-No.10 in which DUG3 was knocked out, was improved from 160 to 224 mg/L. The GSH titer was further increased to 1.7 g/L by optimization of the additions of glucose and three precursor amino acids (L-glutamic acid, L-cysteine and L-glycine). This work establishes a good start point for the industrial production of GSH in the future.
Key words:  glutathione    gene copy numbers    gene knockout    fermentation optimization    Pichia pastoris
收稿日期:  2020-02-12      修回日期:  2020-05-12           出版日期:  2020-09-15      发布日期:  2020-10-14      期的出版日期:  2020-09-15
基金资助: 中国国家自然科学基金项目(31622001,31671845);中国江苏省关键技术研发计划项目(BE2016638);中央高校基本科研业务费专项资金项目(JUSRP51612A,JUSRP51713B)
作者简介:  硕士研究生(刘龙教授为通讯作者,E-mail:longliu@jiangnan.edu.cn)
引用本文:    
蒋秋琪,吕雪芹,崔世修,等. 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.
JIANG Qiuqi,LYU Xueqin,CUI Shixiu,et al. Metabolic engineered Pichia pastoris for synthesis of glutathione[J]. Food and Fermentation Industries, 2020, 46(17): 9-14.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.023602  或          http://sf1970.cnif.cn/CN/Y2020/V46/I17/9
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