Energy Conservation Model Based on Genomic and Experimental Analyses of a Carbon Monoxide-Utilizing, Butyrate-Forming Acetogen, Eubacterium limosum KIST612

Abstract

c Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na ؉ dependent. Consistent with the finding of a Na ؉ -dependent Rnf complex is the presence of a conserved Na ؉ -binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H 2 ؉ CO 2 . The energetic benefit may be one reason that butyrate is formed only from CO but not from H 2 ؉ CO 2 .A cetogenic bacteria are a phylogenetically diverse group of strictly anaerobic bacteria able to reduce two molecules of CO 2 to acetate by the Wood-Ljungdahl pathway (WLP) (1-4). Electrons may derive from molecular hydrogen (autotrophic growth), from carbon monoxide, or from organic donors (heterotrophic growth) such as hexoses, pentoses, formate, lactate, alcohols, or methyl group donors (1). Not only does the WLP provide the cell with organic material for biomass formation, but it is also coupled to energy conservation for ATP supply by a chemiosmotic mechanism (2, 5). Every acetogen examined to date uses reduced ferredoxin (Fd) as the electron donor for an ion-translocating membrane protein complex, and acetogens can have either an Fd:NAD ϩ oxidoreductase (Rnf) or an Fd:H ϩ oxidoreductase (Ech) complex for generation of an ion motive force (5). In both cases, the ion gradient can be either an H ϩ or an Na ϩ gradient. The electrochemical ion gradient thus established is then used by a membrane bound, H ϩ -or Na ϩ -translocating F 1 F o ATP synthase (2).Acetate production from CO 2 proceeds via formate that is converted to formyl-tetrahydrofolate (THF) in an ATP-consuming reaction (6). Water is split off from formyl-THF to yield methenyl-THF, which is reduced via methylene-THF to methyl-THF. The latter is condensed with CO (derived from another molecule of CO 2 ) and coenzyme A (CoA) to acetyl-CoA, which is the starting molecule for biosynthetic reactions (4,7,8). Acetyl-CoA is also the precursor of the end product, acetate, that is produced by the enzymes acetyltransferase and acetate kinase. ATP production in the acetate kinase reaction is of special...

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

“…Despite the similarity at the genomic level between the E. limosum strains (ATCC 8486 and KIST612), butyrate production was not observed for wild type E. limosum ATCC 8486 under the CO condition, thus contradicting the previous report on E. limosum KIST612 (Chang et al, 1998). E. limosum KIST612 produced acetate and butyrate under CO condition (Jeong et al, 2015). For butyrate production, three additional reduction powers are required that recycle the excessive reducing equivalents, with potential ATP production (Kerby et al, 1997).…”

“…, at a pressure of 200 kPa and 50 mL of headspace filled with 0%, 20%, 40%, 60%, 80%, and 100% CO that is balanced using 100%, 80%, 60%, 40%, 20%, and 0% N 2 , respectively, for autotrophic growth conditions. To enhance the autotrophic growth rate during adaptation, 40 mM NaCl was supplemented to the media, which couples with sodium dependent ATP synthase (Jeong et al, 2015;Song et al, 2017).…”

A Na+ A1AO ATP synthase with a V‐type c subunit in a mesophilic bacterium

“…Inspection of genome sequences from mesophilic bacteria and archaea revealed one gene cluster in Eubacterium limosum (T opt = 37°C) potentially encoding an A 1 A O ATP synthase with an unusual V-type c subunit [17]. We have purified the enzyme from membranes of E. limosum and will demonstrate that it is a Na + -dependent A 1 A O ATP synthase containing a Vtype like c subunit capable of ATP synthesis.…”

Exploration of isoxanthohumol bioconversion from spent hops into 8-prenylnaringenin using resting cells of Eubacterium limosum

“…This pathway was thus suggested as the most likely metabolism for IX conversion (Fig. 1b) (Drake 1994;Studenik et al 2012;Jeong et al 2015). For methylotrophic reactions in Eubacterium limosum, inducible enzymes and complex patterns of diauxic growth and (co-) metabolic regulations have been described (Pacaud et al 1985(Pacaud et al , 1986Lindley et al 1987;Cocaign et al 1991;Loubiere and Lindley 1991;Loubiere et al 1992;Lebloas et al 1994Lebloas et al , 1996.…”

“…The major enzymes involved in O-demethylation of IX are methyltransferase I (MT I), corrinoid protein (CFeSP) with cobalt in the respective oxidation states Co (x), methyltransferase II (MT II), activating enzyme (AE), Coenzyme A (CoA), carbon monoxide dehydrogenase (CODH), acetyl-CoA synthase (ACS). Small adaptations were made from the schemes provided by Drake (1994), Sakamoto (2003), Studenik (2012), Jeong (2015). The green, red and blue parts in the figure may be important factors in the study of solubility of IX, toxicity of spent hops residuals and induction of conversion enzymes of Enzyme, Fermentation and Brewing Technology (Gent Belgium).…”