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Biotechnol Biofuels. 2015 Feb 12;8:20. doi: 10.1186/s13068-015-0204-4. eCollection 2015.

Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum.

Author information

1
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; Current address: Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016 India.
2
BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; Thayer School of Engineering at Dartmouth College, Hanover, NH 03755 USA.
3
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; One Bethel Valley Road, Oak Ridge, TN 37831-6038 USA.

Abstract

BACKGROUND:

The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol.

RESULTS:

H2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in ∆hydG∆ech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ∆hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, ∆hydG and ∆hydG∆ech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum.

CONCLUSIONS:

The dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.

KEYWORDS:

Cellulosic ethanol; Clostridium thermocellum; Hydrogenase maturation; Metabolic engineering

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