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.

Thin Layer Chromatography

Figure 2: Proteinexpression of T7_Hneap RuBisCO induced with 0.1 % rhamnose .

Figure 3: Proteinexpression of BBa_K1465213 induced with 0.5 mM IPTG .

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.