Single gene insertion drives bioalcohol production by a thermophilic archaeon

Basen, Mirko; Schut, Gerrit J.; Nguyen, Duy; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

doi:10.1073/pnas.1413789111

Cited by 86 publications

(84 citation statements)

References 38 publications

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“…(19). "AdhA" was also used to describe the Pyrococcus furiosus short-chain dehydrogenase (adh_short) (20,21) (Fig. 3) and the PQQdependent alcohol dehydrogenases from Frateuria aurantia (22) (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…3) into the hyperthermophilic archaeon P. furiosus, which has a unique aldehyde ferredoxin oxidoreductase (AOR) enzyme that reduces acetate to acetaldehyde but no native alcohol dehydrogenase. Wild-type P. furiosus does not produce ethanol; however, as a result of the adhA insertion, the engineered strain was able to generate ethanol from acetaldehyde and produced Ͼ20 mM ethanol from 5 g/liter cellobiose (21), demonstrating that the iron-containing AdhA can be used for ethanol production. Here, we show that adhA is important for ethanol production in a native adhA host.…”

Section: Discussionmentioning

confidence: 99%

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Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

et al. 2017

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Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at about 90% of the theoretical maximum yield (2 ethanol molecules per glucose equivalent) and a titer of 70 g/liter. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here, we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ϳ50%, whereas multiple gene deletions of all annotated alcohol dehydrogenase genes except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked alcohol dehydrogenase (ADH) activity (acetaldehyde-reducing direction) by 93%.IMPORTANCE In this study, we set out to identify the alcohol dehydrogenases necessary for high ethanol production in T. saccharolyticum. Based on previous work, we had assumed that adhE was the primary alcohol dehydrogenase gene. Here, we show that both adhA and adhE are needed for high ethanol yield in the engineered strain LL1049. This is the first report showing adhA is important for ethanol production in a native adhA host, which has important implications for achieving higher ethanol yields in other microorganisms.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

et al. 2017

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“…The ability of C. bescii to express this AOR demonstrates that it has a very active tungsten utilization pathway, an important factor to consider for future engineering strategies in this organism. For example, the recently discovered alcohol production pathway from organic acids, involving AOR and an aldehyde dehydrogenase, AdhA (35), might be applicable in this organism and other Caldicellulosiruptor species.…”

Section: Discussionmentioning

confidence: 99%

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria

Scott

Rubinstein

Lipscomb

et al. 2015

Appl Environ Microbiol

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bCaldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C 5 and C 6 sugars primarily to hydrogen gas, lactate, acetate, and CO 2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.T hermophilic bacteria of the genus Caldicellulosiruptor are currently under intense investigation due to their ability to decompose lignocellulosic plant biomass anaerobically at high temperature, thereby potentially mitigating costly thermochemical pretreatment steps (1, 2). One of these species, Caldicellulosiruptor bescii, has an optimal growth temperature of 78°C and is the most thermophilic cellulose degrader known to date. It is able to ferment high concentrations of cellulosic feedstock primarily to hydrogen gas, lactate, acetate, and CO 2 (3, 4). Species from this genus can degrade cellulose (and also xylan), using novel multidomain glycosyl hydrolases, representing a new paradigm in cellulose conversion by anaerobic thermophiles (2). Moreover, the recent development of a genetic system for C. bescii creates potential for using this and related species for consolidated biomass processing in the production of liquid fuels (5).Developing metabolic engineering strategies for any microorganism obviously requires an in-depth understanding of their primary metabolism. Evidence that C. bescii may have a completely uncharacterized aspect to its primary redox metabolism came from an analysis of its genome sequence for molybdoenzymes (6). These are present in virtually all forms of life, serving diverse roles in primary metabolism of carbon, nitrogen and sulfur (7). As expected, we found that the C. bescii genome contains genes necessary for the synthesis of the pyranopterin cofactor that coordinates molybdenum (Mo) in such enzymes (7). Accordin...

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“…With a recombinant strain of Pyrococcus furiosus it was recently directly demonstrated that the acetaldehyde:ferredoxin oxidoreductase is involved in ethanol formation from acetic acid (82).…”

Section: H 2 Activationmentioning

confidence: 99%

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

et al. 2015

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Most acetogens can reduce CO 2 with H 2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO 2 and H 2 . How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H 2 /CO 2 -grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD ؉ oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H 2 /CO 2 -grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H 2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO 2 and H 2 . IMPORTANCEEthanol formation from syngas (H 2 , CO, and CO 2 ) and from H 2 and CO 2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H 2 and CO 2 , considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H 2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

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Single gene insertion drives bioalcohol production by a thermophilic archaeon

Cited by 86 publications

References 38 publications

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

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Single gene insertion drives bioalcohol production by a thermophilic archaeon

Cited by 86 publications

References 38 publications

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

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Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation