产青蒿二烯的人工酵母细胞的构建及发酵优化

doi:10.11949/j.issn.0438-1157.20140956

化工学报 ›› 2015, Vol. 66 ›› Issue (1): 378-385.DOI: 10.11949/j.issn.0438-1157.20140956

产青蒿二烯的人工酵母细胞的构建及发酵优化

郭睿, 丁明珠, 元英进

系统生物工程教育部重点实验室, 天津大学化工学院制药工程系, 天津化学化工协同创新中心, 天津 300072

收稿日期:2014-06-24 修回日期:2014-09-26 出版日期:2015-01-05 发布日期:2015-01-05
通讯作者: 丁明珠
基金资助:
国家高技术研究发展计划项目(2012AA02A701)。

Construction of artificial yeast cell for producing amorphadiene, and optimization of fermentation

GUO Rui, DING Mingzhu, YUAN Yingjin

Key Laboratory of Systems Bioengineering, Ministry of Education; Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, 300072, China

Received:2014-06-24 Revised:2014-09-26 Online:2015-01-05 Published:2015-01-05
Supported by:
supported by the National High Technology Research and Development Program of China(2012AA02A701).

摘要/Abstract

摘要：

为了异源合成抗疟疾药物青蒿素重要前体青蒿二烯, 以酿酒酵母作为底盘细胞, 利用基因工程手段构建功能人工酵母细胞。为提高基因拷贝数, 并增加重组菌株基因型的稳定性, 选择酵母基因组中多拷贝位点Delta为整合点, 实现酿酒酵母内源基因tHMGR和ERG20的过表达以及外源基因ADS的整合。过表达tHMGR和ERG20基因增加了酵母体内半萜类物质共同前体法尼基焦磷酸FPP的积累量;而导入外源基因ADS, 实现了酵母生产青蒿二烯。经过摇瓶发酵优化实验, 人工酵母菌株青蒿二烯产量为225.3 mg·L^-1;为了进一步提高青蒿二烯产量, 经过发酵过程优化和补料策略, 人工酵母菌株在5 L发酵罐中青蒿二烯产量达到1.05 g·L^-1。

关键词: 合成生物学, 青蒿二烯, 酿酒酵母, 基因工程, 发酵

Abstract:

Amorphadiene, the role precursor of anti-malaria drug artemisinin, was produced by artificial Saccharomyces cerevisiae cells that were constructed under guidance of gene engineering methods. Homologous genes tHMGR and ERG20 as well as heterogenous gene ADS were integrated into yeast genome at the multiple copies Delta sites. Two advantages of this strategy were to increase copy number of genes and to enhance the stability of recombinant strains genotype. Overexpression of tHMGR and ERG20 genes increased accumulation of FPP, the common precursor for sesquiterpenoids. The artificial yeast strains could produce amorphadiene by introducing heterogenous gene ADS. The production of amorphadiene was increased to 225.3 mg·L^-¹in 50 ml flask fermentation through optimizing fermentation condition. In 5 L fermentor, production of amorphadiene was increased to 1.05 g·L^-1 after optimization of fermentation process and the fed-batch feeding strategy.

Key words: synthetic biology, amorphadiene, Saccharomyces cerevisiae, gene engineering, fermentation

中图分类号:

Q819

郭睿, 丁明珠, 元英进. 产青蒿二烯的人工酵母细胞的构建及发酵优化[J]. 化工学报, 2015, 66(1): 378-385.

GUO Rui, DING Mingzhu, YUAN Yingjin. Construction of artificial yeast cell for producing amorphadiene, and optimization of fermentation[J]. CIESC Journal, 2015, 66(1): 378-385.

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产青蒿二烯的人工酵母细胞的构建及发酵优化

Construction of artificial yeast cell for producing amorphadiene, and optimization of fermentation

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