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

Biotechnology for Biofuels
Ranjita BiswasAdam M Guss

Abstract

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. 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 tol...Continue Reading

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Citations

Mar 10, 2015·Current Opinion in Biotechnology·Daniel G OlsonLee R Lynd
Dec 23, 2015·Biotechnology for Biofuels·Kyle SanderSteven D Brown
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Methods Mentioned

BETA
genetic modification
genetic modifications
PCR
Assay

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