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 subsequently to chemiosmotic ATP synthesis with concomitant hydrogen production via the Mbh. Reduced ferredoxin is used as a reductant 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, carbon dioxide, and carbon monoxide, which is often seen as third-generation feedstock for bioalcohol production, for acid reduction (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 experiments opens a new door to bioalcohol formation. 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 electron transport chains. The waste products H2 that insertion of the CODH-encoding genes an acetate into an ethanol producer and also or NADH must be disposed of, and this is may transform P. furiosus to a CO oxidizer. enabled the reduction of other acids to their why cells have evolved reductive pathways, This result was indeed observed after insertion corresponding alcohols. This process allows such as the AOR/AdhA pathway, which of the CODH genes from a close relative, the use of the recombinant as a catalyst for merely serve as “garbage cans”; but some- Thermococcus onnurineus.Therecombinant acid reduction at high temperatures. So far, all times the garbage can be recycled to pro- not only produced carbon dioxide and molec- other ethanol fermentation pathways operate duce valuable compounds. Bioenergetics ular hydrogen, but also reduced the acids to at moderate (30 °C) or high temperatures. drives biotechnology! the corresponding alcohols. The outstanding feature of the use of carbon monoxide as reductant is the uncoupling of oxidative from 1 Branduardi P, de Ferra F, Longo V, Porro D (2014) Microbial 7 Schut GJ, Boyd ES, Peters JW, Adams MW (2013) The modular reductive metabolism. Although they are n-butanol production from Clostridia to non-Clostridial hosts. Eng respiratory complexes involved in hydrogen and sulfur metabolism by Life Sci 14(1):16–26. heterotrophic hyperthermophilic archaea and their evolutionary strictly coupled in the sugar-based system, ox- 2 Basen M, et al. (2014) Single gene insertion drives bioalcohol implications. FEMS Microbiol Rev 37(2):182–203. idation of CO gives reduced ferredoxin and, production by a thermophilic archaeon. Proc Natl Acad Sci USA, 8 Sapra R, Bagramyan K, Adams MWW (2003) A simple energy- subsequently, molecular hydrogen (via the 10.1073/pnas1413789111. conserving system: Proton reduction coupled to proton translocation. 3 Siebers B, Schönheit P (2005) Unusual pathways and enzymes of Proc Natl Acad Sci USA 100(13):7545–7550. Mbh). Thus, by supplying the electrons via central carbohydrate metabolism in Archaea. Curr Opin Microbiol 9 Lim JK, Mayer F, Kang SG, Müller V (2014) Energy conservation by the gas phase, alcohol production is only 8(6):695–705. oxidation of formate to carbon dioxide and hydrogen via a sodium limited by factors such as acid or alcohol 4 Buckel W, Thauer RK (2013) Energy conservation via electron ion current in a hyperthermophilic archaeon. Proc Natl Acad Sci USA (+) tolerance, and not by electron availability. bifurcating ferredoxin reduction and proton/Na translocating 111(31):11497–11502. ferredoxin oxidation. Biochim Biophys Acta 1827(2):94–113. 10 Simon H, White H, Lebertz H, Thanos I (1987) Reduction of Proof-of-principle experiments revealed the 5 Mayer F, Müller V (2014) Adaptations of anaerobic archaea to life 2-enoates and alkanoates with carbon monoxide or formate, production of as much as 70 mM isobutanol under extreme energy limitation. FEMS Microbiol Rev 38(3):449–472. viologens, and Clostridium thermoaceticum to saturated acids and from isobutyrate with CO as reductant. 6 Biegel E, Schmidt S, González JM, Müller V (2011) Biochemistry, unsaturated and saturated alcohols. Angew Chem Int Ed Engl 26(8): adhA evolution and physiological function of the Rnf complex, a novel ion- 785–787. Insertion of a single gene trans- motive electron transport complex in prokaryotes. Cell Mol Life Sci 11 Bengelsdorf FR, Straub M, Dürre P (2013) Bacterial synthesis gas formed the hyperthermophile P. furiosus from 68(4):613–634. (syngas) fermentation. Environ Technol 34(13-16):1639–1651.

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