Research Accomplishment Reports 2007

Proteomic Analysis of Enthanol Sensitivity and Tolerance in Thermophilic and Anaerobic Bacteria

H.J. Strobel, B. Lynn
Department of Animal and Food Sciences

 

Project Description

Domestic bio-ethanol offers promise for diversifying the national fuel portfolio, decreasing oil consumption, and improving environmental quality. Nearly all bio-ethanol in the United States is currently derived from fermentation of maize starch by yeast, but lifecycle analyses suggest that this production scheme offers little, if any, environmental advantage compared with petroleum-based fuels.

Bioconversion of lignocellulosic biomass by thermophilic anaerobic bacteria circumvents some of the problems associated with yeast fermentation of corn. But one of the technological barriers to efficient biomass conversion by bacteria is the relatively low ethanol tolerance that nearly all prokaryotes have when compared to yeast. Other microbial processes of commercial importance are also negatively impacted by fermentation end products, and this sensitivity constrains the technological application of many microorganisms.

There is still relatively little detailed information on the mechanisms responsible for physiological alterations that occur in response to ethanol and other solvents. Until recently, it was tedious and time-consuming to examine protein expression changes in cells responding to an environmental stress such as solvent exposure. However, state-of-the-art proteomic approaches are now available to efficiently identify protein expression changes.

Clostridium thermocellum is a thermophilic anaerobic bacterium that directly converts lignocellulosic feedstocks into ethanol and has been proposed as a bio-catalyst in bio-conversion processes. The genome of this bacterium has been sequenced and this bio-informatic resource can be directly used in proteomic analysis. Our hypothesis was that exposure of C. thermocellum to ethanol causes specific alterations in protein expression patterns, and the overall project goal was to identify and characterize these changes using proteomic approaches. We previously adapted a strain of C. thermocellum to tolerate up to 8% (w/v) ethanol and used this novel organism in proteomic investigations. Since our preliminary work indicated that bacterial membrane protein profiles were altered by exposure to ethanol, particular attention was given to this sub-cellular fraction. A combination of gel-based and two-dimensional liquid chromatography protein separation schemes coupled to tandem mass spectrometry (Multi-dimensional Protein Identification Technology; MudPIT) were employed to define alterations in protein expression for bacterial strains that were either sensitive or tolerant of ethanol. The results of these experiments provide information needed to overcome process limitations and to optimize microbial conversion of lignocellulosic biomass to ethanol, solvents and other useful products.

Impact

The protein expression patterns of ethanol sensitive and tolerant C. thermocellum strains were compared using sophisticated proteomic approaches. We first designed a novel gel-based separation method that circumvented problems associated with traditional membrane protein analysis. Approximately 60% of membrane proteins identified by mass spectrometry were differentially expressed. Most (73%) of these proteins were down-regulated in ethanol-tolerant cells, and a significant proportion were involved with carbohydrate transport and metabolism.

Overall, the results suggest that many membrane-associated proteins in the tolerant strain were either being synthesized in lower quantities or failed to properly incorporate into the membrane. Although our method was useful, protein resolution and detection are limited in gel-based separations, particularly for those molecules that are high molecular weight, very basic, or hydrophobic. Therefore, we developed a two-dimensional liquid chromatography separation scheme coupled to tandem mass spectrometry (Multi-dimensional Protein Identification Technology; MudPIT). This non-gel approach was refined to provide quantification by using a metabolic labeling strategy. Ethanol-sensitive cells were grown in the presence of isotopically labeled ammonium sulfate (15-N) while the tolerant strain was grown in medium containing non-labeled ammonium sulfate (14-N). The 15-N labeled cells served as internal standards, and this minimized experimental errors and provided better quantitative comparison of differences in protein levels. MudPIT analysis identified 168 and 172 proteins from membrane and cytosolic fractions, respectively. Many proteins were up-regulated in tolerant cells and a striking fraction (more than 20%) were ribosomal proteins. Significant up-regulation in tolerant cells was also noted for enzymes found in key energy transduction pathways, oxidation-reduction reactions, and those responsible for protein folding. Tolerant cells were quite different from sensitive cells when viewed via light microscopy. Subsequent analysis with electron microscopy indicated major changes in the ultra-structure of the cell envelope in the tolerant strain including an inability to properly divide. Interestingly, several cell division proteins were apparently down-regulated in the tolerant cells.

Based on these results, an integrative model was developed to describe the global changes that that occur in response to ethanol exposure. In summary, we have developed a reliable analysis platform that can be used to profile the C. thermocellum proteome, and this is the first quantitative proteomic analysis of the organism using the MudPIT approach. This project provides information needed to overcome present process limitations and to optimize microbial conversion of lignocellulosic biomass.

It is estimated that there are nearly 11 million tons of agricultural, forestry and urban-waste fibrous biomass that could be used each year for bio-fuel production in Kentucky. Bio-conversion of these feedstocks could yield 600 million gallons of ethanol and this could replace a significant quantity of gasoline utilized in the state.

Publications

Fu, Y- J., Strobel, H. J., and Lynn, B. C. (2007). Proteomic Analysis of Clostridium thermocellum sub-cellular fractions using 15N-metabolic labeling strategy. American Society of Mass Spectrometry Meeting. Indianapolis, In.

Williams, T. I., Combs, J. C., Lynn, B. C., and Strobel, H. J. (2007). Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum. Applied Microbiology and Biotechnology 74:422-432.