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

Daehwan Chung, Tobin J. Verbeke, Karissa L. Cross, Janet Westpheling, James G. Elkins

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

Background: 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. Results: 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. Conclusions: 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.

Original languageEnglish
Article number102
JournalBiotechnology for Biofuels
Volume8
Issue number1
DOIs
StatePublished - Jul 22 2015

Funding

Karissa L. Cross was supported by the U.S. Department of Energy, Office of Science, Office of Workforce Development for Teachers and Scientists (WDTS) under the Science Undergraduate Laboratory Internship (SULI) program. This work was supported by the BioEnergy Science Center (BESC), which is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science, Oak Ridge National Laboratory. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/ doe-public-access-plan).

FundersFunder number
BioEnergy Science Center
Office of Workforce Development for Teachers
U.S. Department of Energy
U.S. Department of Energy Bioenergy Research Center
U.S. Department of Energy
Office of Science
Biological and Environmental Research
Oak Ridge National LaboratoryDE-AC05-00OR22725

    Keywords

    • 5-hydroxymethylfurfural
    • Biofuel
    • Butanol dehydrogenase
    • Caldicellulosiruptor bescii
    • Furfural
    • Genetic engineering
    • Inhibitor
    • Lignocellulose
    • Pretreatment
    • Thermophile

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