Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

BMC Biotechnology

Research Article

Kuo-Hsing Lin¹  Wei-Chih Chin²  Chieh-Chen Huang²  Kenji Tsuge³  Chun-Chi Liu⁴ 

[1] Center of Cold Chain Logistics Certification, College of Management, National Kaohsiung First University of Science and Technology, Kaohsiung, Taiwan;Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan;Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan;Institute of Genomics and Bioinformatics, National Chung Hsing University, 402, Taichung, Taiwan;

关键词: Tilapia metallothionein;    OmpC;    n-butanol;    E. coli;    Oxidative stress;    Transcriptomic analysis;   

DOI  :  10.1186/s12896-017-0356-3

 received in 2016-10-27, accepted in 2017-03-22,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundN-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell.ResultsThe host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis.ConclusionsThese results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD+ and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD+ redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.

【 授权许可】

CC BY   
© The Author(s). 2017