Overcoming Energetic Barriers in Acetogenic C1 Conversion

Abstract

Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO₂-based bioeconomy and a H₂-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H₂ cycle and thus prime candidates as driving forces in a H₂- and CO₂-bioeconomy. Naturally, they convert two molecules of CO₂ via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO₂, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.

Keywords: biofuels; biohydrogen; carbon capture; electron transport; hydrogen storage.

Figure 1

The Wood-Ljungdahl pathway of CO₂ reduction. Substrates fed directly into the pathway are shown to the left and right. Acetyl-CoA is the precursor of biomass and biocommodities (dotted arrows). [H], reducing equivalents; THF, tetrahydrofolic acid; [CO], enzyme-bound CO; large dashed arrows, oxidative direction of the pathway.

Figure 2

Respiratory enzyme complexes Rnf and Ech in acetogens. Acetogens are classified in Rnf- (A) or Ech-containing (B) organisms. Exergonic electron transfer leads to the translocation of H⁺/Na⁺ across the cytoplasmic membrane and the electrochemical H⁺/Na⁺ potential is then the driving force for ATP synthesis. Reducing equivalents are oxidized via different pathways, results in different product formations. Rnf, model of A. woodii; Ech, model of T. kivui; [H], reducing equivalents; FMN, flavin mononucleotide.

Figure 3

Modularity of acetogenesis. Depending on the redox potential of the substrate, redox carriers with different redox potentials are reduced. The WLP requires species-specific, fixed sets of distinct electron carriers with different redox potentials. To switch electron carriers between the substrate oxidation module and the WLP, a third module that adjusts the electron carriers is required; it contains enzymes like Rnf, Ech, electron-bifurcating hydrogenase, Nfn/Stn or the electron-bifurcating hydrogenase/formate dehydrogenase complex. THF, tetrahydrofolic acid; Hyd, electron-bifurcating hydrogenase; FDH/Hyl, electron-bifurcating formate dehydrogenase; Nfn, electron-bifurcating, ferredoxin-dependent transhydrogenase; Stn, Sporomusa type Nfn.

Figure 4

Bioenergetics of acetate formation from H₂ + CO₂ and CO in A. woodii. The reducing equivalents for the reductive steps in the WLP (A) are provided by an H₂-oxidizing, electron-bifurcating hydrogenase which reduces ferredoxin and NAD⁺. The reducing equivalents for the reductive steps during CO oxidation (B) are provided by the CO-oxidizing CODH/ACS which reduces ferredoxin. Excess Fd²⁻ is oxidized by the Rnf complex which reduces NAD⁺ and builds up a Na⁺ gradient. This gradient drives ATP synthesis via the Na⁺-dependent ATP synthase. In total, 0.3 ATP from H₂ + CO₂ and 1.5 ATP from CO can be synthesized per acetate produced. CODH/ACS, CO dehydrogenase/acetyl coenzyme A synthase; THF, tetrahydrofolic acid.

Figure 5

Bioenergetics of acetate formation from H₂ + CO₂ and CO in C. autoethanogenum. The reducing equivalents for the reductive steps in the WLP (A,C) are provided by an H₂-oxidizing, electron-bifurcating hydrogenase/formate dehydrogenase complex (HytA-E/FDH) which reduces Fd, NADP⁺ and CO₂. The reducing equivalents for the reductive steps during CO oxidation (B,D) are provided by the CO-oxidizing CODH/ACS which reduces Fd. The Nfn complex is transferring electrons between Fd, NADH and NADPH. The methylene-THF reductase is assumed to be electron bifurcating in (A,B). In (C,D) the methylene-THF reductase is not electron bifurcating. Excess Fd²⁻ is oxidized by the Rnf complex which reduces NAD⁺ and builds up a H⁺ gradient. This gradient drives ATP synthesis via the H⁺-dependent ATP synthase. In total, 0.4/1 ATP from H₂ + CO₂ and 1/1.5 ATP from CO (depending on the MTHFR reaction) can be synthesized per acetate produced. CODH/ACS, CO dehydrogenase/acetyl coenzyme A synthase; THF, tetrahydrofolic acid; Nfn, electron-bifurcating and ferredoxin-dependent transhydrogenase.

Figure 6

Metabolic pathways of acetyl-CoA reduction. Acetyl-CoA is synthesized via the Wood–Ljungdahl pathway and can be converted to lactate, 2,3 butanediol, ethanol, acetate, butanol, isoprene, acetone or isobutene. Cofactors involved in different metabolic pathways are indicated.

Figure 7

Ech gene-cluster of representative mesophilic anaerobes. Ech genes are highly distributed in the mesophilic Butyrivibrio clade. Typical anaerobes are for example Pseudobutyrivibrio ruminis, Butyrivibrio proteoclasticus, Pseudobutyrivibrio xylanivorans or Butyrivibrio fibrisolvens. Here shown are the Ech gene-cluster-comparison against representative archea M. mazei and M. barkeri. Ech gene-cluster of mesophilic anaerobes show similar genetic organization.