Genetical approach

Our goal is to bind carbon dioxide for which we searched for several pathways. We decided to work with the calvin cycle because there are only three enzymes missing to enable the whole cycle. The 3-Hydroxypropionate bicycle, would be also possible for our project but there are too many enzymes missing.
The first missing enzyme is the Sedoheptulose 1,7-bisphosphatase. It was successfully transformed by Stolzenberger et al. in 2013. The origin of this enzyme is Bacillus methanolicus. We aim to introduce this enzyme too. For the RuBisCO we decided to use the carboxysome of Halothiobacillus neapolitanus which was successfully transformed by Bonacchi et al. in 2011. The phosphoribulokinase is taken from Synechococcus elongatus which was functionally tested before by Parikh et al. in 2006.
The gene cluster of the carboxysome carries many illegal restriction sites in some sequence parts. Because of this we decided to synthesize some parts of the sequence which we will assemble with the original sequence. By synthesizing the sequence we are able to optimize the codon usage for E. coli.
In addition we want to compare the RuBisCO of H. neapolitanus with the RuBisCO of Synechococcus elongatus. By this comparison we want to identify the optimal enzyme for carbon dioxide fixation in E. coli.
If it is possible to enable the whole cycle in E. coli it should be able to grow with electricity and carbon dioxide. We think of feeding a pentacarbohydrate to feed the Calvin cycle if the efficiency is not high enough.

References

• Bonacci et al., 2011. Modularity of carbon-fixing protein organelle. PNAS, vol. 109, pp. 478-483
• Parikh et al., 2006. Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli. Protein Engineering, Design & Selection, vol. 19, pp. 113-119
• Stolzenberger et al., 2013. Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphate from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus. Journal of Bacteriology, Vol. 195, pp. 5112-5122