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Researchers identify gene controlling ethanol intolerance in C. thermocellum; a basis for rational engineering of optimized microbial strains for cellulosic ethanol production

A team of researchers at the Department of Energy’s (DOE) BioEnergy Science Center (BESC) has identified the gene that controls ethanol intolerance in Clostridium thermocellum, a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing.

Among fermentation-based conversion processes of biomass, the use of cellulose-fermenting microorganisms without added enzymes—i.e., “consolidated bioprocessing”—has strong potential, and a variety of microorganisms are under development for such applications, the researchers note in an open access paper published in the Proceedings of the National Academy of Sciences.

C. thermocellum is a thermophilic bacterium that can rapidly solubilize biomass and use cellulose as a carbon and energy source. Wild-type (WT) strains produce ethanol as well as organic acids, but growth is inhibited by relatively low ethanol concentrations (<10 g/L). Although some cultures of C. thermocellum have been adapted to tolerate ethanol concentrations as high as 80 g/L, the highest concentration of ethanol production reported for this organism is <30 g/L.

Although scientists have studied C. thermocellum for decades, the genetic basis for its ability to tolerate higher concentrations of ethanol had not been determined. Rather than using just one technique or one approach, the research team that made the discovery was able to draw upon multiple experts spanning several scientific disciplines to contribute a broader set of analyses because of the BESC partnership.

Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays.

...It is clear from this study that approaches to genetically modify C. thermocellum and possibly other microorganisms for biofuel production from cellulosic feedstocks must be reconsidered. Indeed, recent deletion of the pta gene, required for acetate production, resulted in the elimination of acetate as a fermentation end product but did not increase ethanol yield. Hence, not only ethanol tolerance but also ethanol production might be limited by electron flow as the ethanol concentration begin to rise. The use of a C. thermocellum strain with altered ADH cofactor specificity might help overcome issues related to carbon and electron flow. Aside from ethanol, the breadth of compounds tolerated by C. thermocellum strains EA and adhE*(EA) is unclear.

Future determination of compounds resisted by these strains may reveal the selective pressures that led to evolution of altered cofactor specificity of AdhE and suggest further paths for metabolic engineering of this organism for industrial biofuel production. Finally, the ability to identify and characterize sets of biological components linked to desired phenotypes, such as the mutated AdhE gene in this study, or overexpression of endogenous genes offers the prospect for improved rational design of systems in the future that will be best suited to particular feedstocks and desired processes.

—Brown et al.

BESC is led by Oak Ridge National Laboratory and is one of three DOE Bioenergy Research Centers established by the DOE's Office of Science in 2007. The centers support multidisciplinary, multi-institutional research teams pursuing the fundamental scientific breakthroughs needed to make production of cellulosic biofuels, or biofuels from nonfood plant fiber, cost-effective on a national scale.


  • Steven D. Brown, Adam M. Guss, Tatiana V. Karpinets, Jerry M. Parks, Nikolai Smolin, Shihui Yang, Miriam L. Land, Dawn M. Klingeman, Ashwini Bhandiwad, Miguel Rodriguez, Jr., Babu Raman, Xiongjun Shao, Jonathan R. Mielenz, Jeremy C. Smith, Martin Keller, and Lee R. Lynd (2011) Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum. PNAS doi: 10.1073/pnas.1102444108



With all the activity in new alternatives, we have paid little attention to the state of biofuels. But they remain a critical transition fuel in electrification of transport. Particularly in fueling PHEVs and FFVs. Ethanol and other alcohols made from waste and cellulose are a good domestic alternative to fossil fuel.

We hope to see more of these type discoveries implemented in real world alcohol production. And to further the adoption of E85 as a national liquid fuel standard. We don't expect this to solve the energy crisis or replace petroleum fuels. But combined with higher CAFE, smaller engine sizing, and electrification - alcohol provides an immediate domestic alternative to foreign oil. Good for economy, good for security, good for ecology.

Tim Duncan

@ Reel$$, thanks for the clear eyed comments. We need all options to away from un gateful, unreliable and some flat out hateful trading partners.


Biofuels are, at best, a minor part of the solution to imported oil. Even the updated billion-ton study finds that biofuels can replace only about 1/3 of current motor fuel usage. Electrification is the proven route; even today Chevy Volt drivers are getting about 1000 miles per fillup (~100 MPG), which is the other 2/3.

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