Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenic Escherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible.
A butanol dehydrogenase encoding gene from Thermoanaerobacter pseudethanolicus 39E (Teth39_1597), previously shown to have furfural and 5-HMF reducing capabilities, was cloned into a suicide plasmid, pDCW171 and transformed into a lactate dehydrogenase mutant of Caldicellulosiruptor bescii. Integration of the gene into the C. bescii chromosome was verified via PCR amplification and stable expression was observed up to 75°C. Heterologous expression of the NADPH-dependent BdhA enzyme conferred increased resistance of the engineered strain to both furfural and 5-HMF relative to the wild-type and parental strains. Further, when challenged with 15 mM concentrations of either furan aldehyde, the ability to eliminate furfural or 5-HMF from the culture medium was significantly improved in the engineered strain.
A genetically engineered strain of C. bescii (JWCB044) has been constructed that shows both an improved tolerance to furan aldehydes and an improved ability to eliminate furfural and 5-HMF from the culture medium. The work presented here represents the first example of engineering furan aldehyde resistance into a CBP-relevant thermophile and further validates C. bescii as being a genetically tractable microbe of importance for lignocellulosic biofuel production.||