Abstract
The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Intolerance to stresses routinely encountered during industrial fermentations may hinder the commercial development of this organism. In this study, C. thermocellum was grown to mid-exponential phase and treated with ethanol, furfural or heat to a final concentration of 3.9 g.L-1, 4 g.L-1 or 68°C respectively to investigate physiological and regulatory responses. Samples were taken at 10, 30, 60 and 120 min post-shock, and from untreated control fermentations for transcriptomic analyses and selected extracellular metabolite determinations. In total 1,539 genes were differentially expressed significantly in at least a single time point for a given stress and 197 were differentially expressed for all treatments. Genes affected by all treatments included DNA repair enzymes, Hsp20, a sulfate transporter and enzymes in the sulfate assimilatory pathway. Changes in metabolite profiles correlated well with gene expression data, particularly the gene expression patterns of enzymes in the acetate and ethanol pathway correlating with altered yields. A search for cis regulatory motifs in the promoter regions of putative operons differentially expressed in at least one of the stress analyses identified six potential regulatory motifs with possible candidate regulators identified by database comparisons. This study has identified C. thermocellum gene regulatory motifs and aspects of physiology and gene regulation for further study with systems biology approaches and newly developed genetic tools.