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Bioalcohol Production by a New Synthetic Route in a Hyperthermophilic Archaeon

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Bioalcohol Production by a New Synthetic Route in a Hyperthermophilic Archaeon COMMENTARY COMMENTARY Bioalcohol production by a new synthetic route in a hyperthermophilic archaeon Volker Müller1 Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt am Main, 60438 Frankfurt, Germany The still growing demand for bioethanol is establishing an electrochemical ion gradient currently met by two microbiological pro- across the cytoplasmic membrane that then cesses. The by far most important process is drives ATP synthesis by a membrane-bound the use of first- (sugar) or second- (lignocel- ATP synthase (5). One widespread, ferre- lulosic biomass) generation raw materials and doxin-fueled ion-translocating enyzme is + yeast as catalysts. Yeasts convert the sugars the ferredoxin:NAD oxidoreductase found via glycolysis to pyruvate, which is decar- in many bacteria and few archaea (6). In P. furiosus boxylated, and the resulting acetaldehyde is , the reduced ferredoxin is oxidized Fig. 1. Pathways for ethanol formation from glucose in then reduced to ethanol by a monofunctional by a different enzyme, a membrane-bound, yeasts (A) and bacteria (B). Adh1, alcoholdehydrogenase ethanol dehydrogenase. The second and in- multisubunit hydrogenase (Mbh) with evolu- 1; AdhE, bifunctional CoA-dependent ethanol/aldehyde dustrially less important is the bacterial pro- tionarysimilaritytothecomplexIofbacterial dehydrogenase. cess, in which pyruvate is oxidized to acetyl- and mitochondrial electron transport chains CoA, which is then reduced by a bifunctional (7). The Mbh is a proton-reducing (hydrogen aldehyde dehydrogenase/ethanol dehydroge- evolving) and sodium ion-extruding mem- used to drive acetate reduction and not the nase (AdhE) to ethanol. This pathway is brane protein complex (8, 9). generation of a chemiosmotic ion gradient, found in many fermenting bacteria (Fig. 1). The high-energy intermediate, reduced the overall energy balance still allows for There is also a growing interest in longer- ferredoxin, may also be used as driving force growth: net, two ATPs are still synthesized chain alcohols that have advantages over for other energy-dependent processes. Re- during acetate formation and 4 mol of re- ethanol in certain applications; for example, duction of aliphatic organic acids to the duced ferredoxin (each carrying one elec- butanol is superior to ethanol as an additive corresponding aldehydes is a highly energy- tron) are still available for chemiosmotic to gasoline. Pathways for production of bu- consuming, endergonic reaction (10). For ex- energy conservation. tanol are found in specific groups, such as in ample, reduction of acetate to acetaldehyde The labeling experiment revealed that Clostridia ′ = , and the encoding genes have has a standard redox potential of E0 externally added acetate was apparently taken been implemented in industrial production −580 mV and, thus, oxidation of NADH up by the cells and converted to ethanol. This ′ + = − strains, such as yeast (1). Bacteria that pro- (E0 NADH/NAD 320 mV) cannot drive finding opened the avenue for Basen et al. (2) duce even higher alcohols are rare. this reduction. However, it has long been to explore whether other carboxylic acids are In PNAS, Basen et al. (2) report a com- known that certain microbes can catalyze ac- taken up and reduced as well. Indeed, the pletely different and novel metabolic route etate reduction to acetaldehyde and that they recombinant strain of P. furiosus reduced ′ ∼− generated by insertion of a single gene into use reduced ferredoxin (E0 500 mV) as an butyrate, propionate, isobutyrate, valerate, the archaeon Pyrococcus furiosus (Fig. 2). electron donor for this reduction. The en- isovalerate, caproate, or phenylacetate to their This hyperthermophile grows optimally near zyme catalyzing this reaction is the alde- corresponding alcohols. This series of experi- 100 °C and ferments sugars to acetate, carbon hyde:ferredoxin oxidoreductase (AOR). ments alone are already outstanding because dioxide, and hydrogen. The pathway used for Luckily enough for Basen et al. (2), P. fur- they proved that P. furiosus is able to reduce sugar breakdown is a modification of glycoly- iosus has such an enzyme indicating that, a number of carboxylic acids quantitatively to sis in which the oxidation of glycerinaldehyde- in principal, this archaeon should be able their corresponding alcohols. However, elec- 3-phosphate is not coupled to the reduction of to reduce acetate (generated from sugars) + trons for the reduction process ultimately NAD but to that of ferredoxin (3), a small to acetaldehyde, but this has never been come from the sugars oxidized by P. furiosus iron-sulfur containing redox protein with a observed. Because P. furiosus does not en- and any alcohol formation process requires ′ = − rather low redox potential (E0 480 mV). code an alcohol dehydrogenase, Basen stoichiometric use of sugars as reductant. Oxidation of pyruvate to acetyl-CoA and car- et al. (2) rationalized that addition of a gene This limitation was again elegantly over- bon dioxide is also coupled to the reduction encoding an alcohol dehydrogenase may come by Basen et al. (2). Carbon monoxide P. furiosus of ferredoxin. switch the metabolism of from dehydrogenase (CODH) is an enzyme known Reduction of acetyl-CoA to acetate is cata- an acetate to an ethanol producer. to oxidize CO with concomitant reduction of lyzed by the unusual, ATP-forming acetyl- Insertion of the alcohol dehydrogenase ferredoxin. Unfortunately, P. furiosus does not adhA Thermoanaerobacter CoA synthetase. Reduced ferredoxin is a high- gene from the contain a CODH but Basen et al. rationalized energy intermediate used in the energy me- strain X514 into P. furiosus indeed led to tabolism of many anaerobes (4). In recent years a complete shift from acetate to ethanol for- it turned out that many anaerobes generate mation. Elegant labeling experiments re- Author contributions: V.M. wrote the paper. ATP by a chemiosmotic mechanism; that is, vealed that ethanol was indeed generated The author declares no conflict of interest. an exergonic redox reaction is used to drive out from acetate and not directly from pyruvate. See companion article 10.1073/pnas.1413789111. + + ions (H or Na ) from the cytoplasm, thus Although part of the reduced ferredoxin was 1Email: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1420385111 PNAS Early Edition | 1of2 Downloaded by guest on October 1, 2021 Now, a hyperthermophile can be used that reduces the risk of contamination and low- ers costs for cooling and distillation. The AOR/AdhA pathway could not only use electrons derived from sugar oxidation but also after insertion of a second gene (clus- ter) from CO oxidation. Oxidation of CO led to reduction of ferredoxin and subse- quently to chemiosmotic ATP synthesis with concomitant hydrogen production via the Mbh. Reduced ferredoxin is used as a re- ductant for the AOR, whereas H2 [via NAD (P)H] is used as a reductant for AdhA. This result opens the possibility to use synthesis gas (syngas), a mixture of hydrogen, car- bon dioxide, and carbon monoxide, which is often seen as third-generation feedstock for bioalcohol production, for acid reduc- tion (11). The use of the process on an industrial scale remains to be explored, but the new and unprecedented metabolic route for bioalcohol formation established by Basen et al. (2) by very elegant experi- ments opens a new door to bioalcohol for- mation. The biochemistry and bioenergetics of the process are well understood and the road to application is paved. Fig. 2. Bioenergetics and biochemistry of a synthetic pathway for ethanol production in P. furiosus. Glucose is oxidized by Basen et al.’s report (2) is also interesting a variation of the Embden–Meyerhof–Parnas pathway: The glycerinaldehyde-3-phosphate dehydrogenase is not coupled to from an evolutionary perspective. How could ATP production and NAD reduction, but the energy is used to reduce the low potential electron acceptor ferredoxin. energy conservation processes in living cells Pyruvate is oxidized by the pyruvate:ferredoxin oxidoreductase and acetyl CoA is reduced to acetate by the unusual acetyl- have evolved? It turns out that reduced fer- CoA synthetase, leading to the formation of ATP. Ferredoxin is indicated to transfer one electron. Four electrons are fed into ’ the Mbh to produce molecular hydrogen and to generate a transmembrane electrochemical sodium ion potential that then redoxin is a key player. Ferredoxin soxida- drives ATP synthesis by a sodium ion translocating A1AO ATP synthase. Stoichiometries for the membrane-bound reactions tion by the Rnf complex or the Mbh are not known. The other 4 mol of reduced ferredoxin are used to reduce the 2 mol of acetate to acetalydehyde, and four complex or similar enzymes leads to chemi- more electrons derived from molecular hydrogen via NAD(P)H are used to reduce the 2 mol of acetalydehyde to ethanol. osmotic energy conservation (ATP synthe- The electrons may also originate from oxidation of carbon monoxide by carbon monoxide dehydrogenase (CODH) coupled sis). H2 (in the case of Mbh) or NADH are to reduction of ferredoxin and its reoxidation by the ion-translocating Mbh. Cells apparently also take up different carboxylic acids and convert them by the AOR/AdhA pathway to the corresponding alcohol. the end products of this respiration, like wa- ter is the end product of the oxygenic elec- tron transport chains. The waste products H2 that insertion of the CODH-encoding genes
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