Steven D. Brown1, Tatiana Karpinets1, Jonathan R. Mielenz1, Shihui Yang1, Dawn M. Klingeman1, Miriam L. Land1, Loren J. Hauser1, Babu Raman1, Miguel Rodriguez Jr.1, Tingfen Yan1, Tatiana A. Vishnivetskaya1, Herbert Strobel2, Ying Xu3, Phuongan Dam3, Lee R. Lynd4, and Martin Keller1. (1) Biosciences Division and BioEnergy Science Center, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, (2) Department of Animal and Food Sciences, University of Kentucky, Lexington, KY 40546, (3) Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, (4) Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755
Clostridium thermocellum is a gram-positive, anaerobic, thermophilic bacterium that can ferment cellulose at one of the highest growth rates directly to ethanol via a large extracellular enzyme complex termed the cellulosome. C. thermocellum is a candidate industrial biocatalyst for future lignocellulosic fuel production. The metabolic byproducts of fermentation can inhibit fermentation performance and lignocellulosic biomass pretreatment processes also produce a variety of inhibitory chemicals that can adversely affect the fermentation. Limited information is available on the mechanisms and responses of C. thermocellum to different inhibitors. The genetic differences between wild-type C. thermocellum and an ethanol tolerant mutant have been identified through microarray based comparative genome sequencing and 454-pyrosequencing. We detected more than 400 differences in the ethanol tolerant mutant compared to the C. thermocellum wild-type strain. The resequencing data were in agreement with published membrane proteomic data and identified new mutations in key genes such as alcohol dehydrogenase. Bioinformatics analyses identified 16 mutational hot-spots in the ethanol tolerant strain, with 7 out of 16 related to cellulose degradation and likely accounted for the strain’s decreased growth on cellulose. Further work to identify and verify important loci and physiological changes conferring tolerance to inhibitors will assist in the development of industrial strains for consolidated bioprocessing (CBP) of lignocellulosic biomass and therefore reduce biofuel production costs.
