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Module II - Carbon Dioxide (CO₂) Fixation

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 approach to enable carbon fixation in E. coli for generating an autotrophic organism. Implemention of the calvin cycle in the heterotrophic model organism E. coli should be associated by expression of the carboxysome, to generate a higher efficiency. As a carbon source for our experimtens E. coli, we choose a pentose, Xylose. Thereby could be ensured, that the glycolysis for energy generating could be avoided. Xylose is metabolized by the cells to ribulose-5-phosphate. This is the substrate for the phosphoribulokinase A from Snyechoccous elongatus, which generates ribulose-1,5-bisphophate. This again is used by the RuBisCO, producing 3-phosphoglycerate.

Figure1: 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.