Introduction

The Ribulose 1,5-bisphosphate Carboxylase Oxygenase (RuBisCO) is the most important enzyme in the Calvin cycle. It binds gaseous carbon dioxide to ribulose-1,5-bisphosphate (Ru-BP) generating two molecules of 3-phosphoglycerate (3-PGA). Therefore it is responsible for the fixation of carbon dioxide. 3-PGA is further converted in the Calvin cycle to glycerinaldehyde-3-phosphate. This is an essential intermediate in the central metabolism, as it plays a central role in glycolysis and gluconeogenesis. RuBisCO enzymes are chracterised as enzymes with slow reaction rates with a k_cat of approximately 20. Furthermore they catalyse a side reaction with oxygen instead of of carbon dioxide, deteriorating the catalytic efficiency. The inclusion of the RuBisCO in a carboxysome, would significantly improve the efficiency of carbon fixation.
It was our aim to enable carbon fixation in E. coli for generating an autotrophic organism. Implementation of the Calvin cycle in this heterotrophic model organism should be associated by the expression of the carboxysome. We would like to use a carboxysome to generate a higher efficiency. As a carbon source for our experimtens with E. coli, we choose the pentose xylose. Thereby could be ensured, that the glycolysis for the generation of energy could be avoided. Xylose is metabolized by the cells to ribulose-5-phosphate. This is the substrate for the phosphoribulokinase A from S. elongatus, which is recombinant expressed from E. coli. The PrkA attaches a phosphate group to ribulose-5-phosphate generating ribulose-1,5-bisphophate. This again is used by the RuBisCO to produce 3-phosphoglycerate. 3-phosphoglycerate can enter the glycolyses and pyruvat as a product is build up. The reaction mechanism is illustrated in figure 1.

Figure 1: Pathway of the D-xylose consumption in E. coli for the fixation of carbon dioxide by the RuBisCO from Halothiobacillus neapolitnaus. For this approach the substrate ribulose 1,5-bisphosphate needs to be accumulated in the cell. This is realzied be the PrkA from Snyechoccous elongatus.

Our first aim was to proof the carbon dioxide fixation by the RuBisCO. The functionality of the RuBisCo is essential for our project, as this is basic of carbon dioxide fixation. Expression of the RuBisCO together with the prkA, which is generating the substrate for the RuBisCo, could give the proof of carbon dioxide fixation in E. coli. Therefore, we investigated the functionality of the RuBisCO in vitro perfoming a RuBisCO activity assay.
Furthermore, the carbon dioxide fixation should be measured by cultivation of E. coli KRX in a bioreactor under high carbon dioxide concentrations measuring the content of CO₂ in the exhaust air. The wildtype KRX was compared with the KRX conaining ptac_Hneap RuBisCO together with the prkA.

RuBisCO activity assay

For the verification of RuBisCo expression, we analyzed protein expression of E. coli KRX containing the construct T7_Hneap RuBisCo and in a second verification, protein expression of BBa_K1465213. Therefore, cultivation was carried out as described in Cultivation for Expression of recombinant proteins. Samples were generated using the protocol for Fast Cell Lysis for SDS-PAGE. The results are shown in figure 2 and 3.

Figure 2: Proteinexpression of T7_Hneap RuBisCO induced with 0.1 % rhamnose in the time curve of the cultivation .

Figure 3: Proteinexpression of BBa_K1465213 induced with 0.5 mM IPTG in the time curve of the cultivation .

For the verification of RuBisCO activity, we performed an in vitro assay measuring ribulose-1,5-bisphosphate and 3-phosphoglycerate, substrate and product of the RuBisCo via HPLC. The methode for the in vitro assay is described


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and the measuring via HPLC in the protocol HPLC.

Figure 4: RuBisCO activity assay. The cell extract from E. coli KRX T7_Hneap RuBisCO and E. coli KRX wildtype was examined for RuBisCO activity. The substrate for the RuBisCO, ribulose-1,5-bisphosphate was added and the time curve of ribulose-1,5-bisphosphate and the product of the enzymatic reaction, 3-phosphoglycerate, was measured via HPLC in 5 minutes intervalls.

Figure 5: HPLC measurement of cell extract from E. coli KRX wildtype. In the first measurement, ribulose-1,5-bisphosphate was added. The second measurement was performed with the cell extract without adding ribulose-1,5-bisphosphate.

Figure 6: Standards for the RuBisCO activity assay. The first measurement was ribulose-1,5-bisphosphate, the second was 3-phosphoglycerate

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Cultivation

Bild Carbonat-Gleichgewicht
Bild Reaktor Schema
Bild Reaktor
Kalbriergerade

Figure x: Calibration by linear fit of the output signal of the qubit analyzer to determine the carbon dioxide fixation.

x = y - 1555,34754 / 4,10739
Kultivierung