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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2016, Vol. 36 Issue (7): 83-91    DOI: 10.13523/j.cb.20160712
技术与方法     
高产β-胡萝卜素酿酒酵母菌株的设计与构建
王瑞钊, 潘才惠, 王颖, 肖文海, 元英进
1 天津大学化工学院制药工程系 系统生物工程教育部重点实验室 天津 300072;
2 天津化学化工协同创新中心合成生物学平台 天津 300072
Design and Construction of highβ-carotene Producing Saccharomyces cerevisiae
WANG Rui-zhao, PAN Cai-hui, WANG Ying, XIAO Wen-hai, YUAN Ying-jin
1 Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University;Key Laboratory of Systems Bioengineering(Ministry of Education, Tianjin 300072, China;
2 SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and technology, Tianjin University, Tianjin 300072, China
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摘要:

酿酒酵母是一种重要的食品安全的工业微生物宿主。如何精确地控制代谢路径中相关基因的表达强度,是在酿酒酵母体内合成β-胡萝卜素的关键因素。通过在Delta位点整合红发夫酵母来源的β-胡萝卜素合成基因(crtE、crtI、crtYB),构建β-胡萝卜素生产菌库。从中挑选28株颜色较黄的菌株,通过96孔细胞培养板及摇瓶发酵,发现这些菌株β-胡萝卜素产量存在显著差异(产量从5.70mg/L到61.88mg/L动态分布)。然后以其中β-胡萝卜素产量最高的菌为出发菌(SyBE_Sc118012),在敲除该菌株内源基因ypl062w的同时,在此位点过表达tHMG1和BTS1-ERG20融合蛋白以增加前体供应,最终使β-胡萝卜素的产量提高1.65倍,达到162.1mg/L,是目前已知的摇瓶水平最高发酵产量。因此,通过Delta位点整合和增加前体供应结合的策略来强化酿酒酵母中的异源表达具有重要的指导意义。

关键词: 合成生物学酿酒酵母Delta位点β-胡萝卜素前体供应基因拷贝数目    
Abstract:

Saccharomyces cerevisia is generally regarded as safe and widely used in industrial fields. Fine-tuning the expression level of key genes involved inβ-carotene biosynthetic pathway is the key issue for β-carotene production in S. cerevisia. β-carotene producing strain library was constructed by integrating heterologous genes (crtE, crtI, crtYB) from Xanthophyllomyces dendrorhous through Delta site integration.28 strains in darker yellow were picked out and the titer was testified in 96 well plate coupled with shake flask. There were significant difference inβ-carotene titers, ranging from 5.70mg/L to 61.88mg/L. Then the strain with highest titer(SyBE_Sc118012)was selected for further engineering. By means of integrate one copy of tHMG1 and fusion gene BTS1-ERG20 at endogenous ypl062w gene site, the production ofβ-carotene was improved by 1.65 fold, up to 162.1mg/L, which is the highest reported titer at shake flask level. The research highlighted the importance of Delta site integration together with precursor enhancement strategy in heterologous biosynthesis in S. cerevisia.

Key words: β-carotene    site    Precursor supply    Saccharomyces cerevisiae    Synthetic biology    &delta    Gene copy number
收稿日期: 2016-01-04 出版日期: 2016-03-16
ZTFLH:  Q753  
基金资助:

国家“863”计划资助项目(2012AA02A701)

通讯作者: 肖文海     E-mail: wenhai.xiao@tju.edu.cn
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引用本文:

王瑞钊, 潘才惠, 王颖, 肖文海, 元英进. 高产β-胡萝卜素酿酒酵母菌株的设计与构建[J]. 中国生物工程杂志, 2016, 36(7): 83-91.

WANG Rui-zhao, PAN Cai-hui, WANG Ying, XIAO Wen-hai, YUAN Ying-jin. Design and Construction of highβ-carotene Producing Saccharomyces cerevisiae. China Biotechnology, 2016, 36(7): 83-91.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160712        https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I7/83

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