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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 (6): 39-50    DOI: 10.13523/j.cb.20160606
研究报告     
人工酵母后鲨烯路径基因对7-脱氢胆固醇合成的影响
张文倩1,2, 周晓1,2, 肖文海1,2, 王颖1,2
1. 天津大学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
Effect of Post-squalene Genes on the Synthesis of 7-Dehydrocholesterol in the Artificial Saccharomyces cerevisiae
ZHANG Wen-qian1,2, XIAO Wen-hai1,2, ZHOU Xiao1,2, WANG Ying1,2
1. Key Laboratory of Systems Bioengineering Ministry of Education, Tianjin University, Tianjin 300072, China;
2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China
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摘要:

酵母内源后鲨烯路径中的固醇类物质,是异源甾体类药物合成的重要前体。为了通过微调后鲨烯路径,与异源模块进行适配,以期达到提高异源甾体类化合物表达的目的,以维生素D3的直接前体—7-脱氢胆固醇(7-DHC)的合成为例,首先在固醇C-24甲基转移酶(ERG6)缺失的酿酒酵母BY4742中,通过导入人源固醇C-24 还原酶DHCR24,并过表达截短的羟甲基戊二酰辅酶A还原酶tHMGR,获得可以合成7-DHC的人工酵母。在此基础上,将后鲨烯路径分割并构建成ERG1、ERG7、ERG11、ERG24-25-26-27和ERG2-3这5个模块,分别在所构建的7-DHC合成菌株中过表达。通过GC-TOF/MS分析7-DHC以及后鲨烯路径中相关代谢中间体的含量,并结合主成分分析发现,过表达不同后鲨烯模块会引起后鲨烯路径上固醇组分的变化而最终影响7-DHC的产量:与出发菌株相比,过表达ERG11模块会显著强化其他固醇物质到酵母固醇的转化;而过表达ERG2-3模块则会减少鲨烯的积累,同时显著增加羊毛固醇及其之后的固醇组分的含量,并获得迄今为止7-DHC在微生物中摇瓶水平的最高产量。因此,对ERG11和ERG2-3的表达优化对7-DHC的合成以及后鲨烯路径代谢流的强化起到了显著的作用,是后续优化人工7-DHC合成酵母的潜在靶点。为研究后鲨烯路径与其他异源甾体合成模块间的适配,提供了可供参考的案例。

关键词: 后鲨烯路径合成生物学7-脱氢胆固醇酿酒酵母    
Abstract:

Steroids from endogenous post-squalene pathway are important precursors for heterologous sterol drugs biosynthesis in yeast. In order to enhance heterologous sterols synthesis, post-squalene pathway should be engineered to increase the compatibility with the heterologous modules. The construction of 7-dehydrocholesterol (7-DHC, the direct precursor of vitamin D3) biosynthesis pathway in Saccharomyces cerevisiae was took as the example to reveal the effect of post-squalene pathway on heterologous sterols production. To produce 7-DHC in Saccharomyces cerevisiae BY4742, ERG6 (encoding sterol C-24 methyltransferase) was knocked out and the Homo sapiens C-24 reductase (DHCR24) together with truncated hydroxymethylglutaryl-CoA reductase (tHMGR) were overexpressed, obtaining strain SyBE0019-Sc-002. Meanwhile, the post-squalene pathway was divided into five modules as ERG1, ERG7, ERG11, ERG24-25-26-27and ERG2-3.Each module was overexpressed in strain SyBE0019-Sc-002 individually. Through GC-TOF/MS analysis and principal components analysis (PCA), it was revealed that overexpression of post-squalene modules brought changes on the contents of the related steroids and effected 7-DHC production. To be specific, overexpression of module ERG11 significantly enhanced the conversion of other steroids to zymosterol; whereas overexpression of module ERG2-3 reduced the accumulation of squalene and significantly increase the synthesis of steroids from lanosterol to 7-DHC. Consequently the highest 7-DHC titer known in shake flask was achieved in the strain with module ERG2-3 overexpressed. Therefore, manipulation of the expression level of module ERG11 and ERG2-3 significantly affected the synthesis of 7-DHC and enhanced the metabolic flux of the whole post-squalene pathway, suggesting ERG11 and ERG2-3 were the potential targets for further optimization 7-DHC producing yeast. This provided a good reference for increasing the compatibility by means of engineering endogenous post-squalene pathway with heterologous sterols synthesis modules.

Key words: Synthetic biology    7-dehydrocholesterol    Saccharomyces cerevisiae    Post-squalene genes
收稿日期: 2015-11-25 出版日期: 2015-12-14
ZTFLH:  Q819  
通讯作者: 王颖     E-mail: ying.wang@tju.edu.cn
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引用本文:

张文倩, 周晓, 肖文海, 王颖. 人工酵母后鲨烯路径基因对7-脱氢胆固醇合成的影响[J]. 中国生物工程杂志, 2016, 36(6): 39-50.

ZHANG Wen-qian, XIAO Wen-hai, ZHOU Xiao, WANG Ying. Effect of Post-squalene Genes on the Synthesis of 7-Dehydrocholesterol in the Artificial Saccharomyces cerevisiae. China Biotechnology, 2016, 36(6): 39-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160606        https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I6/39

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