title: Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli.
abstract: N-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.
  
  
  
  The 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.
  
  
  
  These 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.
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Wei-Chih
authors.initials: WC
authors.lastname: Chin
authors.affiliation: Center of Cold Chain Logistics Certification, College of Management, National Kaohsiung First University of Science and Technology, Kaohsiung, Taiwan.
authors.forename: Kuo-Hsing
authors.initials: KH
authors.lastname: Lin
authors.affiliation: Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, 402, Taiwan.
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Liu
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Tsuge
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan. cchuang@dragon.nchu.edu.tw.
authors.forename: Chieh-Chen
authors.initials: CC
authors.lastname: Huang
mesh_terms: D020001:1-Butanol / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D015398:Signal Transduction / Q000235:genetics; D017210:Tilapia / Q000235:genetics / Q000378:metabolism*
chemical_list: C113724:OmpC protein; D018272:Porins; D020001:1-Butanol; D008668:Metallothionein
keywords: E. coli; OmpC; Oxidative stress; Tilapia metallothionein; Transcriptomic analysis; n-butanol