Identification and characterization of a highly thermostable crotonase from Meiothermus ruber

Butanol is deemed a second generation biofuel due to its enhanced energy content and improved hydrophobicity compared to ethanol. The state of the art production is the Clostridia based anaerobic acetone, butanol and ethanol (ABE) fermentation process. However, the classical ABE fermentation is capped at 2% (v/v) butanol yield, due to end-product toxicity effects. By contrast, cell-free bio-butanol production systems based on designed enzyme cascades hold the promise of higher butanol yields and simplified end-product recovery. Crotonases catalyze the reversible hydration of crotonyl-CoA and are key enzymes in butanol biosynthesis. In this study, we present the isolation, heterologous expression and characterization of a new highly thermostable crotonase (Crt) derived from the bacterium Meiothermus ruber (Mr). Mr-Crt displays a broad activity range of 50-70 °C, with optimal reactivity at pH 7.0 and 55 °C. Moreover, Mr-Crt displays an extended half-life of over 1 month (IT₅₀ (50 °C) = 743 ± 0.7 h) at elevated temperatures. Robust enzyme activities, such as Mr-Crt, with high temperature and solvent tolerance will ultimately contribute to an improved cell-free butanol process.