Summary and Outlook

The aim module II was to establish the whole Calvin cycle in E. coli. We were able to transform the RuBisCO from Halothiobacillus neapolitanus and tested the functionality in an in vitro assay via HPLC. The HPLC showed that the transformed RuBisCO is able to catalyze the reaction from ribulose 1,5-bisphosphate to 3-phosphoglycerate. The second transformed enzyme was the sedoheptulose 1,7-bisphosphatase (SBPase) which could be purified via His-tag purification. Together with two other purified enzymes the functionality could be verified in an in vitro assay. The original SBPase was taken from Bacillus methanolicus which has an temperature optimum of 50°C. We also verified that the enzyme is functional at 37°C albeit with a lower efficiency. The third enzyme is the phosphoribulokinase A which we were able to transform into E. coli. An enzyme assay on a crude cell extract did not show a functionality although we could identify the protein via MALDI-TOF. It was already shown before that a phosphoribulokinase could be functionally expressed in E. coli (Parikh et al., 2006).
To increase the functionality of the RuBisCO, we started to transform a carboxysome which is a microcompartment. The RuBisCO is located in the microcompartment together with the carbonic anhydrase. We were able to transform the empty carboxysome into E. coli and showed the assembly via GFP fusion. The shell associated protein CsoS2 was identified to be crucial for the correct folding of the RuBisCO which could be shown via microscopy. Besides we were able to show that the shell proteins form filaments if they were overexpressed with a T7 promoter as had been described before.
In conclusion we can state that we nearly established the whole Calvin cycle in E. coli. Within the next steps the target would be to show the functionality of the PrkA. Through the transformation of the carboxysome we provided the possibility to enhance the activity of carbon fixation. The carboxysome is a biotechnologically relevant microcompartment because of its constraints of a high carbon dioxide concentration within the shell. To our best knowledge, we are the first team which was able to send in a functional microcompartiment BioBrick which can be further developed in the next years.