Furan aldehyde detoxification by a heat-stable alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E
Tuesday, April 29, 2014
Exhibit/Poster Hall, lower level (Hilton Clearwater Beach)
James G. Elkins1, Nicolletta L. Cuthbert2, Sonya M. Clarkson1 and Adam M. Guss1, (1)BioEnergy Science Center, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, (2)Virginia Wesleyan College, Norfolk, VA
The reactivity of lignocellulosic biomass for digestion is generally improved when chemical and physical pretreatments are applied. However, these processes may also generate inhibitory compounds that negatively impact subsequent microbial fermentations. Typical inhibitors include furfural (FA) and 5-hydroxymethylfurfural (5-HMF), which are generated from the dehydration of xylose and glucose, respectively. Alcohol dehydrogenases with FA and 5-HMF reductase activity have been identified and overexpressed in ethanologens such as Saccharomyces cerevisiae to improve tolerance to pretreatment inhibitors. Similar strategies for improving inhibitor tolerance in thermophilic, cellulolytic microbes have not been investigated. We have identified an Fe-dependent alcohol dehydrogenase (ADH) from Thermoanaerobacter pseudethanolicus 39E, encoded by Teth39_1597, that readily reduces FA and 5-HMF to their respective alcohols. Teth39_1597 was cloned, overexpressed and purified from E. coli cell-free lysates for further characterization. In vitro assays showed that NADPH was the preferred cofactor and that the enzyme functions in the presence of oxygen. The enzyme was active at temperatures between 40o and 80oC with maximum activity at 75oC. Activity was also retained after 10 minutes of incubation at 70oC. The optimal buffer pH was 8.0 with a range between pH 6.0 and 9.0. The Teth39_1597 orf was also cloned into a shuttle vector, pAMG216, for heterologous expression in C. thermocellum DSM1313. Preliminary results indicate that expression of Teth39_1597 increased furfural reductase activity in cell-free extracts of C. thermocellum 2-fold. Optimized expression of Teth39_1597 with resulting phenotypic characterization will enable a rational engineering approach toward improving C. thermocellum fermentation performance in the presence of pretreatment inhibitors.