Energy Conservation Model Based on Genomic and Experimental Analyses of a Carbon Monoxide-Utilizing, Butyrate-Forming Acetogen, Eubacterium limosum KIST612by Jiyeong Jeong, Johannes Bertsch, Verena Hess, Sunju Choi, In-Geol Choi, In Seop Chang, Volker Müller

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A model for energy conservation based on genomic and 1 experimental analyses in a carbon monoxide-utilizing, butyrate-2 forming acetogen, Eubacterium limosum KIST612 3 4

Running title: 5

Energy metabolism of E. limosum KIST612 6 7

Jiyeong Jeonga1, Johannes Bertschb1, Verena Hessb, Sunju Choia, In-Geol Choic, 8

In Seop Changa# and Volker Müllerb# 9 10 a School of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 11 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea 12 b Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang 13

Goethe University Frankfurt/Main, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany 14 c Computational and Synthetic Biology Laboratory, College of Life Sciences and Biotechnology, 15

Korea University, Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea 16 17 1Both authors contributed equally to the study 18 #To whom correspondence may be addressed. Email: or vmueller@bio.uni-19 20 21

AEM Accepted Manuscript Posted Online 8 May 2015

Appl. Environ. Microbiol. doi:10.1128/AEM.00675-15

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Jeong et al. Energy metabolism of E. limosum KIST612 2


Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from 23 carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate 24 formation, the enzymes involved and the potential coupling to ATP synthesis. Oxidation of 25

CO is catalyzed by the acetyl-CoA synthase/CO dehydrogenase and coupled to the reduction 26 of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na+-27 dependent. Consistent with the finding of a Na+-dependent Rnf complex is the presence of a 28 conserved Na+-binding motif in the c subunit of the ATP synthase. Butyrate formation is from 29 acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA and butyryl-CoA and is 30 consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The 31 activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to 32 butyryl-CoA with NADH as reductant was coupled to reduction of ferredoxin. We postulate 33 that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce 34 ferredoxin which is consistent with the finding of etfA and etfB genes next to it. The overall 35

ATP yield was calculated and is significantly higher than the one obtained with H2 + CO2. The 36 energetic benefit may be one reason that butyrate is only formed from CO but not from 37

H2 + CO2. 38

Jeong et al. Energy metabolism of E. limosum KIST612 3


Acetogenic bacteria are a phylogenetically diverse group of strictly anaerobic bacteria 40 able to reduce two molecules of CO2 to acetate by the Wood-Ljungdahl pathway (WLP) (1-41 4). Electrons may derive from molecular hydrogen (autotrophic growth), carbon monoxide or 42 from organic donors (heterotrophic growth) such as hexoses, pentoses, formate, lactate, 43 alcohols or methyl-group donors (1). The WLP not only provides the cell with organic 44 material for biomass formation, but the pathway is also coupled to energy conservation for 45

ATP supply by a chemiosmotic mechanism (2, 5). Every acetogen examined to date uses 46 reduced ferredoxin (Fd) as electron donor for an ion-translocating membrane protein complex 47 and acetogens can have either a Fd:NAD+ oxidoreductase (Rnf) or a Fd:H+ oxidoreductase 48 (Ech) complex for generation of an ion motive force (5). In both cases, the ion gradient can 49 either be a H+ or Na+ gradient. The electrochemical ion gradient thus established is then used 50 by a membrane bound, H+ or Na+ translocating F1FO ATP synthase (2). 51

Acetate production from CO2 proceeds via formate that is converted to formyl-52 tetrahydrofolate (THF) in an ATP-consuming reaction (6). Water is split off from formyl-53

THF to yield methenyl-THF which is reduced via methylene-THF to methyl-THF. The latter 54 is condensed with CO (derived from another molecule of CO2) and CoA to acetyl-CoA which 55 is the starting molecule for biosynthetic reactions (4, 7, 8). Acetyl-CoA is also the precursor 56 of the end product acetate that is produced by the enzymes acetyltransferase and acetate 57 kinase. ATP production in the acetate kinase reaction is of special importance for the WLP 58 since it regains the ATP spent in the activation of formate. 59

Since the energy available for biosynthetic reactions ultimately stems from the oxidation 60 of reduced ferredoxin, the number of redox reactions able to reduce ferredoxin as well as the 61 electron accepting reactions that do not use ferredoxin as electron donors determines the ATP 62 yield. In Acetobacterium woodii which only has an electron bifurcating hydrogenase to reduce 63

Jeong et al. Energy metabolism of E. limosum KIST612 4 ferredoxin (9) and that uses one mol H2, two mol NADH and one mol of reduced ferredoxin 64 for the four redox reactions of the WLP (10), only 0.5 mol reduced ferredoxin is available for 65 the Rnf complex and only 0.3 mol of ATP/acetate are formed by this chemiosmotic 66 mechanism (5). 67

A loss of the acetate kinase reaction would make the WLP highly energy consuming 68 (by -0.7 ATP) if no other reactions improve the ATP yield either by substrate level 69 phosphorylation or by reducing ferredoxin that then “fuels” the chemiosmotic mechanism. 70

Therefore, it is the more surprising that acetogens can also make a living from producing 71 ethanol that is produced by way of acetyl-CoA (1). Apparently, ethanol production must be 72 coupled to energy conservation but the mechanisms are obscure. Some acetogens may also 73 produce other reduced end products from acetyl-CoA and face the same energetic problem 74 (11). 75

The conversion of gases to acetate is of special interest since it is a promising way of 76 producing biofuels and biocommodities independent of carbohydrates. The conversion of 77 syngas, a mixture of H2, CO, and CO2, to ethanol by microbial fermentation is already 78 demonstrated in large industrial-scale pilot plants by companies like Coskata, INEOS Bio or 79