Promoter Set for Heterologous Gene Expression in Clostridium thermocellum
For successful fermentation of biofuels and bioproducts from biomass, using microorganisms for which fewer genetic tools have been developed might be the most effective approach. To date, most metabolic engineering work in Clostridium thermocellum has focused on gene deletion, but many metabolic engineering strategies require well controlled heterologous gene expression, which requires a collection of well characterized and understood promoters. Researchers from the Department of Energy’s BioEnergy Science Center sought to identify new promoters for predictable gene expression in C. thermocellum. For this work, 17 different C. thermocellum promoters were tested with two different reporter genes (LacZ and AdhB) to ensure the activity of the target promoter was not gene-specific. Putative promoters were chosen by analyses of published C. thermocellum gene expression datasets. Promoter activity in both C. thermocellum and Escherichia coli were testedbecauseideally a promoter would not be strongly expressed in E. coli to avoid toxicity problems during cloning. Several useful promoters were identified (eno, cbp, cbp_2, 815, 966, 2638, and 2926), which showed high expression and high enzymatic activity of both reporter genes in C. thermocellum. Other promoters were not useful, showing no heterologous gene activity or negatively impacting plasmid stability. These results provide several new good promoters for C. thermocellum. This improved understanding of promoter function will enhance efforts to express heterologous genes important for improved biofuel production in C. thermocellum.
Olson, D. G., M. Maloney, A. A. Lanahan, S. Hon, L. J. Hauser, and L. R. Lynd. 2015. “Identifying Promoters for Gene Expression in Clostridium thermocellum,” Metabolic Engineering Communications, DOI: 10.1016/j.meteno.2015.03.002.