Global regulator engineering significantly improved Escherichia coli tolerances toward inhibitors of lignocellulosic hydrolysates

Authors

  • Jianqing Wang,

    1. National Engineering Laboratory for Industrial Enzymes, Department of Chemical Engineering, Tsinghua University, 1 Tsinghua Garden Road, Beijing 100084, China; telephone: +86-10-6279-4403; fax: +86 (10) 6277-0304
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  • Yan Zhang,

    1. National Engineering Laboratory for Industrial Enzymes, Department of Chemical Engineering, Tsinghua University, 1 Tsinghua Garden Road, Beijing 100084, China; telephone: +86-10-6279-4403; fax: +86 (10) 6277-0304
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  • Yilu Chen,

    1. College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, China
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  • Min Lin,

    Corresponding author
    1. Key Laboratory of Crop Biotechnology of Ministry of Agriculture, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 South Zhongguancun Street, Beijing 100081, China; telephone: +86-10-8210-6145; fax: +86 (10) 8210-6142
    • Key Laboratory of Crop Biotechnology of Ministry of Agriculture, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 South Zhongguancun Street, Beijing 100081, China; telephone: +86-10-8210-6145; fax: +86 (10) 8210-6142
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  • Zhanglin Lin

    Corresponding author
    1. National Engineering Laboratory for Industrial Enzymes, Department of Chemical Engineering, Tsinghua University, 1 Tsinghua Garden Road, Beijing 100084, China; telephone: +86-10-6279-4403; fax: +86 (10) 6277-0304
    • National Engineering Laboratory for Industrial Enzymes, Department of Chemical Engineering, Tsinghua University, 1 Tsinghua Garden Road, Beijing 100084, China; telephone: +86-10-6279-4403; fax: +86 (10) 6277-0304
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Abstract

Lignocellulosic biomass is regarded as the most viable source of feedstock for industrial biorefinery, but the harmful inhibitors generated from the indispensable pretreatments prior to fermentation remain a daunting technical hurdle. Using an exogenous regulator, irrE, from the radiation-resistant Deinococcus radiodurans, we previously showed that a novel global regulator engineering (GRE) approach significantly enhanced tolerances of Escherichia coli to alcohol and acetate stresses. In this work, an irrE library was subjected to selection under various stresses of furfural, a typical hydrolysate inhibitor. Three furfural tolerant irrE mutants including F1-37 and F2-1 were successfully obtained. The cells containing these mutants reached OD600 levels of 4- to 16-fold of that for the pMD18T cells in growth assay under 0.2% (v/v) furfural stress. The cells containing irrE F1-37 and F2-1 also showed considerably reduced intracellular oxygen species (ROS) levels under furfural stress. Moreover, these two irrE mutants were subsequently found to confer significant cross tolerances to two other most common inhibitors, 5-hydroxymethyl-2-furaldehyde (HMF), vanillin, as well as real lignocellulosic hydrolysates. When evaluated in Luria–Bertani (LB) medium supplemented with corn stover cellulosic hydrolysate (prepared with a solid loading of 30%), the cells containing the mutants exhibited lag phases markedly shortened by 24–44 h in comparison with the control cells. This work thus presents a promising step forward to resolve the inhibitor problem for E. coli. From the view of synthetic biology, irrE can be considered as an evolvable “part” for various stresses. Furthermore, this GRE approach can be extended to exploit other exogenous global regulators from extremophiles, and the native counterparts in E. coli, for eliciting industrially useful phenotypes. Biotechnol. Bioeng. 2012; 109: 3133–3142. © 2012 Wiley Periodicals, Inc.

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