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

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, H-2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H-2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol. Results: H-2 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. H-2 production in ΔhydGΔech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ΔhydG 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, ΔhydG and ΔhydGΔech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. Conclusions: Finally, themore » dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.« less

Authors:

Biswas, Ranjita ^[1]; Zheng, Tianyong ^[2]; Olson, Daniel G. ^[2]; Lynd, Lee R. ^[2]; Guss, Adam M. ^[1]

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+ Show Author Affiliations

1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Div. and BioEnergy Science Center
2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center; Dartmouth College, Hanover, NH (United States). Thayer School of Engineering

Publication Date:

Thu Feb 12 00:00:00 EST 2015

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1185431

Alternate Identifier(s):

OSTI ID: 1327623

Grant/Contract Number:  

AC05-00OR22725

Resource Type:

Accepted Manuscript

Journal Name:

Biotechnology for Biofuels

Additional Journal Information:

Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 1754-6834

Publisher:

BioMed Central

Country of Publication:

United States

Language:

English

Subject:

09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; Cellulosic ethanol; Clostridium thermocellum; Hydrogenase maturation; Metabolic engineering