Genetical approach

Our goal is to bind carbon dioxide. There are different pathways in bacteria described. One option could be the 3-hydroxypropionate bicycle, but E.coli lacks many of the involved enzymes. That is why we decided to construct a bacterial version of the calvin cycle which is well understood in plants. In E.coli are only three enzymes missing to close this cycle.
The first missing enzyme in the Calvin-Cycle is the Sedoheptulose 1,7-bisphosphatase (SBPase). It was successfully cloned and characterized by Stolzenberger et al. in 2013. The origin of this enzyme is the thermophilic Bacillus methanolicus. We would like to use this enzyme in E. coli. 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