n-Butanol has many advantages over ethanol, including a higher energy density due to two extra carbons, and can be used in gasoline engines without modification. n-Butanol is less hygroscopic and evaporative than ethanol and has been recently regarded as a more viable transportation biofuel than ethanol . Additionally, n-butanol is also a permitted artificial flavoring and is used in a wide range of industries, including the food and plastic industries . n-Butanol often occurs as a metabolic product of the microbial fermentation using sugars and other carbohydrates as carbon sources. However, during the production of n-butanol, its accumulation is known to be highly toxic to both natural producers and engineered hosts [3, 4]. This toxicity makes it difficult to produce large titers of n-butanol at levels needed for economic efficiency.
The cellular membrane is a vital factor that allows for cells to acclimate to external stresses and is also one of the components highly affected by organic solvents [5, 6]. Most toxicity researchers have proposed that the plasma membrane is the most affected target of organic solvents and plays a significant role in adapting to stress. Additionally, the length of the carbon backbone of organic solvents could alter the toxicity mechanism; increasing the hydrophobicity of the solvent could also raise the level of toxicity . The long- and short-chain alcohols are known to cause stress during biofuel production by changing membrane fluidity. Ethanol and n-butanol are known to respectively decrease and increase the membrane fluidity [6, 8, 9]. Understanding the membrane stress response to solvents and alcohols could facilitate engineer-ing microorganisms for improved toxin tolerance. As such, stress responses of organisms such as E. coli, to ethanol exposure has been widely studied , and information from these studies have been successfully adapted to engineering improved ethanologenic hosts . To understand the effect of n-butanol toxicity on the host, cell-wide studies have been conducted to obtain a global view of the n-butanol stress-response in transcript, protein, and metabolite levels. In Clostridium acetobutylicum, transcript analysis indicated that the primary response was an accumulation of transcripts encoding chaperones, proteases, and other heat shock-related proteins . In E. coli, several transcriptional analyses have been performed to understand the stress caused by alcohols including ethanol, n-butanol, and isobutanol [13–16]. Additionally, observations from fluorescent dye-staining indicated a large increase in reactive oxygen species during n-butanol stress . This increasing oxidative stress is a response of the cell to extracellular xenobiotics, which may mediate macromolecular damage. These free radicals could directly attack the membrane by lipid peroxidation .
ROS include molecules that are either oxidants (such as hydrogen peroxide, H2O2) or reductants (such as the superoxide anion, O2
˙−). All are typical side products of cellular aerobic metabolism. To decrease ROS-generated oxidative damage, microorganisms synthesize many antioxidant enzymes, including catalases, superoxide dismutases and glutathione peroxidase [18, 19]. Recently, metallothioneins (MTs), a beneficial antioxidant enzyme that widely occurs in mammals, plants and fungi, has been identified . MTs are heat-stable, low-molecular-weight and cysteine-rich intracellular proteins that are responsible for maintaining the homeostasis of essential metals, such as Cu2+, Zn2+ and for the detoxification of toxic metal ions, such as Cd2+ and Hg2+[20–22]. In addition, MTs also play a role as a defense system against oxidative stress through their ROS-targeted scavenging abilities . For example, the tilapia fish (Oreochromis mossambicus), which serves as a biomarker for the contamination level of aqueous environments, has the ability to survive in a highly polluted environment because of its MTs function [24, 25]. Furthermore, purified tilapia MT (TMT) has been shown to have a higher ability than glutathione (GSH) to scavenge both 2-diphenyil-1-picrylhydrazyl (DPPH●) and 2,2-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt (ABTS●+) . These observations have prompted us to postulate that TMT may serve as a good candidate for the purposes of metal absorption and free radicals scavenging in microorganisms during bio-fuel production.
It is known that the levels of intracellular reactive oxygen species increase in E. coli after exposure to n-butanol . In this study, we demonstrate that engineered E. coli strains expressing OmpC fused MTs could elevate n-butanol tolerance by scavenging intra- and extra-cellular free radicals and the fusion protein could still contribute in osmosis via either GB or glucose uptaking.