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In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli

Authors

  • Fayin Zhu,

    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, PR China
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  • Xiaofang Zhong,

    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, PR China
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  • Mengzhu Hu,

    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, PR China
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  • Lei Lu,

    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, PR China
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  • Zixin Deng,

    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, PR China
    2. Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan, PR China
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  • Tiangang Liu

    Corresponding author
    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, PR China
    2. Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan, PR China
    • Correspondence to: T. Liu

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ABSTRACT

Approaches using metabolic engineering and synthetic biology to overproduce terpenoids, such as the precursors of taxol and artemisinin, in microbial systems have achieved initial success. However, due to the lack of steady-state kinetic information and incomplete understanding of the terpenoid biosynthetic pathway, it has been difficult to build a highly efficient, universal system. Here, we reconstituted the mevalonate pathway to produce farnesene (a precursor of new jet fuel) in vitro using purified protein components. The information from this in vitro reconstituted system guided us to rationally optimize farnesene production in E. coli by quantitatively overexpressing each component. Targeted proteomic assays and intermediate assays were used to determine the metabolic status of each mutant. Through targeted engineering, farnesene production could be increased predictably step by step, up to 1.1 g/L (∼2,000 fold) 96 h after induction at the shake-flask scale. The strategy developed to release the potential of the mevalonate pathway for terpenoid overproduction should also work in other multistep synthetic pathways. Biotechnol. Bioeng. 2014;111: 1396–1405. © 2014 Wiley Periodicals, Inc.

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