Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

Abstract

BackgroundCompounds 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.ResultsA 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.ConclusionsA 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0287-y) contains supplementary material, which is available to authorized users.

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

“…However, the titers were almost 10-fold lower than those observed using the H. thermocellum ADH [121]. Another T. pseudethanolicus ADH, annotated as a butanol dehydrogenase, increased the tolerance of C. bescii to furfurals, a byproduct of thermal and acid-pretreatment of plant biomass, by reducing furfurals to their cognate alcohols which are less toxic [134].…”

Progress in Second Generation Ethanol Production with Thermophilic Bacteria

“…A natural target for the strategy of converting a cellulolytic organism into a good ethanol producer would be members of the genus of Caldicellulosiruptor which has several cellulolytic members although none are good ethanol producers. Recent work with Caldicellulosiruptor bescii, a naturally cellulolytic organism, has produced ethanol producing strains [89][90][91][92][93].…”

“…It should be noted that this strain only used a small portion (4.4 mM of 29.2 mM cellobiose) provided and not produce ethanol above 65°C. Work by Cha [89] and Chung [93] introduced the alcohol dehydrogenase genes (adhB and adhE) from Thermoanaerobacter pseudoethanolicus into the ldh deficient strain. The two resultant strains yielded ethanol at temperatures greater than 65°C although titers were lower than the aforementioned strain JWCB032 (ldh − adhE + ).…”

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

“…Additional studies included engineeringimproved biomass utilization via heterologous expression of cellulose-degrading enzymes from other members of the Caldicellulosiruptor genus or other thermophilic cellulolytic organisms (8)(9)(10). Detoxification of furan aldehydes found in pretreated plant materials has also been addressed (11). Utilizing this genetic system also led to the discovery of the unexpected ability of C. bescii to utilize tungsten, a metal seldom used in biology (12).…”