Modes of carbon dioxide fixation

Reductive citric acid cycle

The citric acid cycle [TCA cycle] (oxidative) is one of the main cycles used by all aerobic organisms to generate energy by the oxidation of pyruvate. The rTCA reverses the reactions of the oxidative citric acid cycle. The oxidative TCA is used to generate energy through oxidation of acetate which is derived from different substances like fats, carbohydrates and proteins and procues carbon dioxide and ATP. The reductive citric acid cycle runs in reverse. That means it uses two molecules of carbon dioxide and ATP to generate carbohydrates, fats and proteins from acetyl-CoA. It is used for autotrophic growth (Schauder et al., 1987).
The green sulfur bacterium Chlorobium thiosulfatophilum is the first organism where this cycle could be observed by Evans, Buchanan and Arnon 1966 (Arnon-Buchanan Cycle) (Evans et al., 1966). It has also been found in anaerobic and microaerobic bacteria.
There are three reactions of the oxidative citric acid cycle which are known as irreversible (Buchanan et al., 1990). The establishment of the reverse cycle from an oxidative TCA requires the replacement of three enzymes: The succinate dehydrogenase has to be replaced by the fumarate reductase, the NAD⁺-dependent 2-oxoglutarate dehydrogenase has to be replaced by the ferredoxin-dependent 2-oxoglutarat synthase and the citrate synthase has to be replaced by the ATP citrate lyase. The educt, Acetyl-CoA, of a constructed reverse TCA cycle which is further carboxylated to pyruvate is used by the cell. The electrons needed for the rTCA reactions are donated by ferredoxin and NAD(P)H and requires two ATP to form one pyruvate molecule.
We decided not to work with this cycle because we would have we had to use anaerobic cultivation conditions which we tried to avoid because of a lack of materials and equipment for this purpose.

Reductive acetyl CoA pathway

The reductive acetyl CoA pathway also called Wood-Ljungdahl pathway (1965) uses carbon dioxide as an electron acceptor and hydrogen as an electron donor for biosynthesis. The product of this pathway is acetyl-CoA which is used for several biological reactions, for example the oxidative TCA. The key enzyme is called CO dehydrogenase / acetyl-CoA synthase and represents a substantial portion of the whole cell protein. Acetyl-CoA is assimilated to pyruvate by the pyruvate synthase. The pathway works in both directions depending on the environmental condition (Schauder et al., 1988).
The pathway has been found in Clostridium thermoacetium which is a strictly anaerobic bacteria (acetogens) (Ragsdale et al., 2008). It is preferred by bacteria living close to the thermodynamic limit (Acetogens, methanogens) for the generation of energy.
Because this pathway, like the above mentioned rTCA, also only occurs under strict anoxic conditions we decided to not use it for our project. Besides the cycle depends strongly on metals (Mo or W, Co, Ni and Fe) which would increase the cost of the media needlessly.

3-Hydroxypropionate bicycle

The 3-hydroxypropionate bicycle produces 3-hydroxypropionate by consuming carbon dioxide. The bi-cycle allows coassimilation of different compounds, e.g. propionate, acetate and succinate. These substances are metabolized via acetyl-CoA. With seven molecules of ATP for one pyruvate and three additional for triose phosphates the energy costs of the bi-cycle are high (Strauss et al., 1993). In contrast to the above mentioned cycles the enzymes of this cycle are not especially oxygen sensitive. Additionally the enzymes are multifunctional which means there are only 13 enzymes for 17 reactions.
The cycle was discovered first in Chloroflexus aurantiacus by Helge Holo (Holo et al., 1986) and occurs in bacteria living under neutrophilic and alcalophilic conditions.
An exmaple of a hypothetical bi-cycle in E. coli is shown in figure 1. We decided to not establish this bicycle in E. coli because of the amount of missing enzymes. Additionally we had contact with Matthew Mattozzi who already tried to enable this cycle in E. coli which did not function.

The 3-hydroxypropionate (Fuchs-Holo) bicycle. 1. acetyl-CoA carboxylase, 2. malonyl-CoA reductase, 3. propionyl-CoA synthase, 4. propionyl-CoA carboxylase, 5. methylmalonyl-CoA epimerase, 6. methylmalonyl-CoA mutase, 7. succinyl-CoA:(S)-malate-CoA transferase, 8. succinate dehydrogenase, 9. fumarate hydratase, 10. (a,b,c) trifunctional (S)-malonyl-CoA a/(β)-methylmalonyl-CoA (b)/(S)-citramalyl-CoA lyase, 11. mesaconyl-C1-CoA hydratase, 12. mesaconyl-CoA C1-C4 CoA transferase, 13. mesaconyl-C4-CoA hydratase (Berg et al., 2011)

Calvin cycle

The Calvin cycle, also known as the reductive pentose pathway, is the light independent reaction of the photosynthesis. Photosynthesis is done by all plants and some sulfurbacteria. The products of the photosynthesis are ATP and NADPH which are used by the Calvin cycle to produce higher sugars by simultaneous incooperation of CO₂. We decided to work with the Calvin cycle and did further research on this pathway because the enzymatical and aerobic conditions as well as the number of missing enzymes fit our project idea to introduce a CO₂ fixation pathway.

The reductive pentose phosphate (Calvin-Benson-Bassham) cycle. 1. ribulose 1,5-bisphosphate carboxylase/oxygenase, 2. 3-phosphoglycerate kinase, 3. glyceraldehyde 3-phosphate dehydrogenase, 4. triose-phosphate isomerase, 5. fructose bisphosphate aldolase, 6. fructose-bisphosphate phosphatase, 7. transketolase, 8. sedoheptulose bisphosphate aldolase, 9. sedoheptulose bisphosphate phosphatase, 10. ribose-phosphate isomerase, 11. ribulose-phosphate epimerase, 12. phosphoribulokinase (Berg et al., 2011)

References

• Berg (2011) Ecological Aspects of the Distribution of Different Autotrophic CO₂ Fixation Pathways Applied and Environmental Microbiology, vol. 77, no. 6, pp. 1925-1936
• Evans et al., 1966. A new ferredoxin dependent carbon reduction cycle in a photosynthetic bacterium. Proc. Natl. Acad. Sci. U. S. A., vol. 55, pp.928-934
• Schauder et al., 1987. Carbon assimilation pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus. Arch Microbiol., vol. 148, pp.218-225
• Buchanan et al., 1990. A reverse KREBS cycle in photosynthesis: consensus at last . Photosynthesis Research, vol. 24, pp.47-53
• Schauder et al., 1988. Oxidative and reductive acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum . Archives of Microbiology, vol. 151, pp.84-89
• Ragsdale et al., 2008. Acetogenesis and the Wood-Ljungdahl pathway of CO₂ fixation. . Biochim. Biophys. Acta, vol. 1784, pp.1873-1898
• Holo et al., 1986. Autotrophic growth and CO₂ fixation of Chloroflexus aurantiacus. . Arch Microbiol., vol. 100, pp.5-24
• Strauss et al., 1993. Enzymes of a novel autotrophic CO₂ fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus, the 3-hydroxypropionate cycle . Arch Microbiol., vol. 100, pp.5-24