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Improvement of n-butanol tolerance in Escherichia coli by membrane-targeted tilapia metallothionein

Wei-Chih Chin1, Kuo-Hsing Lin2, Jui-Jen Chang34 and Chieh-Chen Huang15*

Author Affiliations

1 Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan

2 Vaccine Research and Development Center, National Institute of Infectious Disease and Vaccinology, NHRI, Miaoli, Taiwan

3 Department of Medical Research, China Medical University Hospital, Taichung 402, Taiwan

4 Biodiversity Research Center, Academia Sinica, Taipei 11529, Taiwan

5 Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan

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Biotechnology for Biofuels 2013, 6:130  doi:10.1186/1754-6834-6-130

Published: 11 September 2013



Though n-butanol has been proposed as a potential transportation biofuel, its toxicity often causes oxidative stress in the host microorganism and is considered one of the bottlenecks preventing its efficient mass production.


To relieve the oxidative stress in the host cell, metallothioneins (MTs), which are known as scavengers for reactive oxygen species (ROS), were engineered in E. coli hosts for both cytosolic and outer-membrane-targeted (osmoregulatory membrane protein OmpC fused) expression. Metallothioneins from human (HMT), mouse (MMT), and tilapia fish (TMT) were tested. The host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural, and nickel. The same strain also allowed for an increased growth rate of recombinant E. coli under n-butanol stress. Further experiments indicated that the TMT-fused OmpC protein could not only function in ROS scavenging but also regulate either glycine betaine (GB) or glucose uptake via osmosis, and the dual functional fusion protein could contribute in an enhancement of the host microorganism’s growth rate.


The abilities of scavenging intracellular or extracellular ROS by these engineering E. coli were examined, and TMT show the best ability among three MTs. Additionally, the membrane-targeted fusion protein, OmpC-TMT, improved host tolerance up to 1.5% n-butanol above that of TMT which is only 1%. These results presented indicate potential novel approaches for engineering stress tolerant microorganism strains.

Metallothionein; n-butanol; OmpC; Tilapia; E. coli; Oxidative stress