Engineering a spermidine biosynthetic pathway in Clostridium thermocellum results in increased resistance to furans and increased ethanol production

Abstract

Polyamines are low molecular weight aliphatic nitrogen compounds found ubiquitously in microorganisms, plants, and animals. Spermidine is a common polyamine that plays a role in stabilizing chromatin, DNA replication, transcription, translation, as well as the regulation of cell growth and apoptosis in eukaryotes. Amines are also associated with defense to a number of environmental stresses including elevated temperature and have been shown to be involved in tolerance to fermentation inhibitors such as furan derivatives and acetic acid in Saccharomyces cerevisiae. While the tolerance and detoxifying mechanisms have been intensively studied, metabolic engineering efforts to construct tolerant and resistant strains have been few. Here we show that exogenously added spermidine confers enhanced tolerance to furans and acetic acid in the Gram-positive bacterium, Clostridium thermocellum. Deletion of the endogenous spermidine synthase resulted in a severe growth defect and hypersensitivity to both furans and acetic acid. Exogenously added spermidine rescued all three phenotypes. Overexpression of the endogenous spermidine synthase resulted in increased tolerance to these compounds without added spermidine. Increased tolerance to these fermentation inhibitors will facilitate the use of C. thermocellum, one of the most cellulolytic of all known bacterial species, for the production of fuels from plant biomass substrates.

Keywords: Biofuels; Clodstridium thermocellum; Fermentation inhibitors; Spermidine.