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Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum - PubMed

  • ️Sat Jan 01 2011

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Steven D Brown et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Clostridium thermocellum is a thermophilic, obligately anaerobic, gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Mutant C. thermocellum ADHs confers enhanced ethanol tolerance. (A–E) Growth of C. thermocellum DSM 1313 strains with different plasmids that provided a vector-only control, an additional copy of the WT version of adhE, and the mutant adhE gene was monitored by measuring culture turbidity (log10 OD600nm). Strains were grown at 55 °C in increasing amounts of added ethanol. (F) The final culture turbidities after 96 h of growth are presented as a function of ethanol added to the culture from the beginning the experiment. The graphs show the mean and the SE (bars) for three independent dose–response curves, and this experiment was repeated twice.

Fig. 2.
Fig. 2.

Homology/MD model of C. thermocellum AdhE double mutant (P704L, H734R). Mutation sites (Leu-704 and Arg-734) and the NAD cofactor and Fe are labeled. The configuration was taken from the end of a 10-ns MD trajectory. Figure was rendered by using VMD (57).

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