Lipid Membrane Remodeling and Metabolic Response During Ethanol and Isobutanol Stress in Zymomonas mobilis
Julio Rivera Vazquez1,2* (email@example.com), Edna Trujillo1,2, Jonathan Williams1,2, Fukang She2, Melanie Callaghan1,2, Joshua J. Coon1,2, Daniel Amador-Noguez1,2, and Tim Donohue1,2
1Great Lakes Bioenergy Research Center; and 2University of Wisconsin–Madison
Zymomonas mobilis, an ethanologenic gram-negative bacterium, is currently being bioengineered to produce isobutanol. However, it has been observed that exposure to isobutanol elicits detrimental physiological changes, including a reduction in growth rate and glucose consumption. This project aims to systematically investigate the physiological response of Z. mobilis to isobutanol with a particular emphasis on changes in lipid membrane composition and proteome allocation.
Despite being a proficient ethanol producer, Z. mobilis experiences growth inhibition at high ethanol titers and is highly sensitive to isobutanol. It is known that bacteria can modulate lipid membrane composition to increase their tolerance to environmental stressors. In this study, researchers used liquid chromatography–mass spectrometry (MS)/MS-based lipidomics to measure changes in lipid membrane composition that occur when Z. mobilis is exposed to increasing concentrations of ethanol and isobutanol. Exposure to ethanol and isobutanol resulted in significant but distinct changes to the lipid and fatty acid composition. Affected lipid classes included cardiolipins, phosphatidylcholines, and phosphatidylethanolamines. The fatty acid composition was also significantly affected. Most notably, a substantial increase in C19 cyclopropane fatty acid content was observed when cells were grown at high ethanol concentrations, suggesting that the changes comprise a defense mechanism in response to solvent stress. Previous evidence showed that cyclopropane-ringed fatty acids modify membrane fluidity and act as a barrier to prevent detrimental molecules from entering the cell. To test the hypothesis that C19 cyclopropane fatty acids and derived lipids contribute to solvent resistance in Z. mobilis, researchers engineered a strain that overexpressed the Cyclopropane Fatty Acyl Synthase (CFA synthase) protein (ZMO1033) responsible for transforming unsaturated fatty acids into cyclopropane fatty acids. Analysis of the lipid membrane composition of the CFA synthase overexpressing strain showed a significant increase in C19 cyclopropane fatty acid content for all lipid classes. This increase correlated with significantly improved growth rates in the presence of high ethanol and isobutanol concentrations. These data demonstrate the importance of cyclopropane fatty acids to solvent stress resistance and the effects of isobutanol on protein activity in Z. mobilis. This data will allow engineering of strains that are more resistant to high ethanol and isobutanol concentrations.
This material is based upon work supported by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Biological and Environmental Research (BER) Program under Award Number DE-SC0018409.