The citric acid cycle [TCA cycle] (oxidative) is one of the main cycles used by all aerobic organisms. The cycle reverses the reactions of the oxidative citric acid cycle. It is used to generate energy through oxidation of acetate which is derived from different substances like fats, carbohydrates and proteins over carbon dioxide and ATP. The reductive citric acid cycle runs in reverse. That means it uses two molecules carbon dioxide and ATP to generate carbohydrates, fats and proteins from acetyl-CoA.
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 the oxidative citric acid cycle which are known irreversible. The replacements to reverse the cycle are: succinate dehydrogenase by fumarate reductase, NAD⁺-dependent 2-oxoglutarate dehydrogenase by ferredoxin-dependent 2-oxoglutarat synthase and citrate synthase by ATP citrate lyase. The product, Acetyl-CoA, is further carboxylated to pyruvate which is used by the cell. As electron donors ferredoxin and NAD(P)H are used. The cycle uses two ATP to form one pyruvate molecule.
We decided not to work with this cycle because by using it we had to use anaerobic cultivation conditions which we tried to avoid.

The reductive citric acid cycle (Arnon-Buchanan). 1. ATP-citrate lyase, 2. malate dehydrogenase, 3. fumarate hydratase, 4. fumarate reductase, 5. succinyl-CoA synthetase, 6. ferredoxin dependent 2-oxoglutarate synthase, 7. isocitrate dehydrogenase, 8. aconitate hydratase, 9. ferredoxin dependent pyruvate synthase, 10. phosphoenolpyruvate synthase, 11. phosphoenolpyruvate carboxylase (Berg et al., 2011)

The reductice acetyl CoA pathway also called Wood-Ljungdahl pathway (1965) uses carbon dioxide as electron acceptor and hydrogen as electron donor for biosynthesis. The product of this pathway is acetyl-CoA which is used for several biological reactions. The key enzyme is called CO dehydrogenase / acetyl-CoA synthase and represents a substantial part of the cell protein. Acetyl-CoA is assimilated to pyruvate by pyruvate synthase.
The pathway has been found in Clostridium thermoacetium which is a strictly anaerobic bacteria (acetogens). It is preferred by bacteria living cloes to the thermodynamic limit (Acetogens, methanogens). There the cycle is also used for energy generation.
Because this pathway also occurs only 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).

The reductive acetyl coA (Wood-Ljungdahl) pathway. 1. formate dehydrogenase, 2. formyl-tetrahydroforlate synthetase, 3. formyl-methanofuran dehydrogenase, 4. formyl-methanofuran:tetrahydromethanopterin formyltransferase, 5. methenyl-tetrahydroforlate cyclohydrolase, 6. methenyl-tetrahydromethanopterin cyclohydrolase, 7. methylene-tetrahydroforlate dehydrogenase, 8. methylene-tetrahydromethanopterin dehydrogenase, 9. methylen-tetrahydroforlate reductase, 10. methylen-tetrahydromethanopterin reductase, 11. CO, dehydrogenase / acetyl-CoA synthase (Berg et al., 2011)

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. The energy costs of the cycle are high with seven molecules ATP for one pyruvate and three aditional for triose phosphates.The enzymes of this cycle are not especially oxygen sensitive. Also 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. It occurs in bacteria living under neutrophilic and alakliphilic conditions.
We decided to did some research about this cycle but our main focus lies on the Calvin cycle.

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)

The Calvin cycle is the light independent reaction of the photosynthesis. Photosynthesis is done by all plants and some sulfurbacteria. The products of the photosynthesis ATP and NADPH are used for the Calvin cycle. By using ATP and NADPH carbohydrates were produced. In the cycle carbon dioxide is taken up and higher sugars were produced.

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)

Reaction of sedoheptulose 1,7-bisphosphatase

The SBPase is one of enzymes needed for the Calvin cycle. It catalyzes the reaction from sedoheptulose 1,7-bisphosphate to sedoheptulose 7-phosphate. The enzyme is characteristic for the part of regeneration in the Calvin-cycle. It was shown before that oveerexpression of the SBPase in tobacco results in enhanced carbon assimilation and crop yield (Rosenthal et al., 2011).
It does not occur in E.coli which makes it a target to transform for enabling the whole cycle.

Reaction of phosphoribulokinase

The phosphoribulokinase A is the enzyme which catalyzes the reaction from ribulose 5-phosphate to ribulose 1,5-bisphosphate. This step needs ATP.

Reaction of phosphoribulokinase

The Ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) is the most abundant enzyme in the world. Because of its key role in carbon fixation metabolism, it is found in nearly all autotrophic organisms like plants, but also in cyanobacteria and fotosynthetic bacteria in high concentrations (Andersson, 2008). RuBisCo catalyses the fixation of atmospheric carbon dioxide by generating two tricarbohydrates out of one pentacarbohydrate. So you could say it is responsible for conversion of carbon dioxide in biomass or with other words for incorporation of inorganic carbon dioxide to form organic molecules. To give some facts, more than 10¹¹ tons of atmospheric carbon dioxide is fixed per year baesd on RuBisCo activity (Field et al., 1998).
RuBisCo catalyses the rate limiting step in the Calvin cycle. The Calvin cycle is the light independent reaction of photosynthesis. In this cycle, carbon dioxide is fixed to build up energy-rich substrates. RuBisCo catalyses the fixation of one molecule carbon dioxide to ribulose-1,5-bisphosphate (RuBP). The product is instabile and decays directly in two molecules 3-D-phosphoglycerate (3-PGA)(Andersson, 2008, Parikh et al. 2006). 3-PGA is further converted in the Calvin cycle to glycerinaldehyde-3-phosphate and metabolized by the cells. Furthermore 3-D-phosphoglycerate is an essential intermediate in the central metabolism, as it plays a central role in glycolysis and gluconeogenesis. The carboxylation of RuBP is a multiple step reaction. In detail, the firt step is activation of the RuBisCo by carbamylation of the amino group from a Lysin in the active centre (Lysin 201). The activated RuBisCo is then stabilized by magnesium ions, a cofactor for activity. In the carboxylation of RuBP the first step is enolisation of the substrate by H₂O. The enediolate reacts then with either carbon dioxide oder oxygen. If carbon dioxide is bound by the enediolate, water is split up from the molecule and the instabile intermediate
Beside the carbon fixation reaction of the RuBisCo, the enzyme catalyses numerous side reactions. An alternative substrate to carbon dioxide is atmospheric oxygen. When the oxygenation of RuBP ist catalyzed instead of carboxylation, the product is 2-phosphoglycolate. This product can not be used by the metabolism of the cells and the fixed carbon has to be regenerated by the metabolic pathway photorespiration, a high-energy consuming pathway. This metabolic stress for the cells reduces the effizienz of carbon dioxide fixation about 20 - 50 % (Mann, 1999, Andersson, 2008). The higher affinity of RuBisCo to carbon dioxide, nearly by a factor 100 higher than to oxygen, still does not have a positive effect for the efficiency, because of the concentration from both gases in the atmosphere. Oxygen has a percentage of 20 % (v/v) whereas carbon dioxide accounts for only 0,04 % (v/v). The oxygenation side reaction of the RuBisCo is one reason for the inefficiency of this enzyme.

The Ribulose 1,5-bisphosphate carboxylase oxygenase is the most abundant enzyme of the world because it occurs in every plant in a high concentration. The reaction of this enzyme is essential for the functionality of the Calvin cycle because it uses the atmospheric carbon dioxide to generate two tricarbohydrates out of one pentacarbohydrate. The problem of the RuBisCO is that is also accepts oxygen with a higher percentage as cofactor. The following reaction results in one dicarbohydrate and one tricarbohydrate.
The RuBisCO consists of two subunits, a small and a large subunit. In higher plants the RuBisCO is formed out of four large and four small subunits. In smaller organisms the RuBisCO is only formed out of two proteins each.
We aim to use the RuBisCO from two different organisms which are mentioned below.