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.

Resources

• 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