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中国生物工程杂志

China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (6): 39-50    DOI: 10.13523/j.cb.20160606
    
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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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 wordsSynthetic biology      7-dehydrocholesterol      Saccharomyces cerevisiae      Post-squalene genes     
Received: 25 November 2015      Published: 14 December 2015
ZTFLH:  Q819  
Cite this article:

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.

URL:

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

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