Genetical approach

Introduction and motivation

Our goal is to bind carbon dioxide in organic molecules. There are many different pathways known for carbon dioxide fixation in bacteria. Most suitable for our application are the 3-hydroxypropionat bicycle and the reductive pentose phosphate cycle also called Calvin cycle. We were looking for aerobic possibilities of carbon dioxide fixation, because of the desired energy source. Electric power shuld be the only source of energy for our bacteria. Therefor an aerobic cultivation is required. The electrons should pass through the bacterial respiratory chain. Oxygen is needed as the final acceptor of electrons at the end of the respiratory chain. Finally we decided to construct a bacterial version of the calvin cycle which is a well understood carbon dioxide fixation mechanism in plants Winkel, 2004; Raines, 2003. One reason for our choice was the existence of almost all essentiell enzymes in E. coli. Only enzymes for three steps are missing. The implementation of the 3-hydroxypropionate bicycle would require the heterologous expression of genes for nearly all involved enzymes. All in all would this sum up to at least 12 genes. Nevertheless we developed plans for the construction of both pathways in E. coli.

Sedoheptulose-1,7-bisphosphatase

The first missing enzyme of the Calvin cycle in E. coli is the sedoheptulose-1,7-bisphosphatase (SBPase). While this enzyme is common in plants only a few bacterial versions of it are known. One bacterial SBPase was recently identified and characterized (Stolzenberger et al. in 2013). The origin of this sequence is the thermophilic Bacillus methanolicus. B. methanolicus lives at 50°C and therefore the temperature optimum of this enzyme is also at 50°C (Stolzenberger et al., 2013). We would like to use this enzyme at 37°C in E. coli. It had not escaped our notice that this temperature difference could cause a significant reduction in activity (Peterson et al., 2007). To our best knowledge there are no better fitting bacterial enzymes available.

Phosphoribulokinase

The next critical step of the Calvin cycle is the phosphorylation of riublose-5-phosphate. This reaction is catalyzed by the phosphoribulokinase. E. coli lacks such an enzyme. Therefore a phosphoribulokinase encoding sequence is taken from Synechococcus elongatus. It was functionally tested before (Parikh et al., 2006).

RubisCO

The most important step is the actual carbon dioxide fixing reaction. It is catalyzed by ribulose-1,5-bisphosphate-carboxylase/oxygenase (RubisCO) in plants. We looked for a bacterial ezymes and selected two different RuBisCOs. One is an enzyme from the carboxysome of Halothiobacillus neapolitanus (Bonacchi et al., 2011). The other one is from Synechococcus elongatus (Long et al., 2007). We decided to express both in E. coli and to make a comparison between them. The enzyme from H. neapolitanus is usually located in a special microcompartiment called "carboxysome"(Bonacchi et al. in 2011). Therefore we decided to construct a carboxysome in E. coli.

Carboxysome

The carboxysome ensures an efficient carbon fixation reaction under aerobic conditions. The concentration of carbon dioxide is slectively increased inside the carboxysome (Reinhold et al., 1991; Dou et al., 2008; Mangan and Brenner, 2014). The gene cluster of the carboxysome contains many illegal restriction sites. Because of this we decided to order synthesis of some parts of the sequence to delete this recognition sites. By synthesizing we are able to optimize the codon usage for expression in E. coli.

Summary

If it is possible to enable the whole cycle in E. coli the cells should be able to grow with electricity as energy source and carbon dioxide as sole carbon source. As a backup we think of feeding a pentose to support the Calvin cycle in case the efficiency of the electroautotrophic metabolism is not high enough.

References

• Winkel, B. S. J. „Metabolic Channeling in Plants“. Annual Review of Plant Biology 55 (2004): 85–107. doi:10.1146/annurev.arplant.55.031903.141714.
• Raines, C. A. „The Calvin Cycle Revisited“. Photosynthesis Research 75, Nr. 1 (2003): 1–10. doi:10.1023/A:1022421515027.
• Stolzenberger, Jessica, Steffen N. Lindner, Marcus Persicke, Trygve Brautaset, und Volker F. Wendisch. „Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus Methanolicus“. Journal of Bacteriology 195, Nr. 22 (November 2013): 5112–22. doi:10.1128/JB.00672-13.
• Peterson, Michelle E., Roy M. Daniel, Michael J. Danson, und Robert Eisenthal. „The dependence of enzyme activity on temperature: determination and validation of parameters“. Biochemical Journal 402, Nr. Pt 2 (1. März 2007): 331–37. doi:10.1042/BJ20061143.
• Bonacci, Walter, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver, und David F. Savage. „Modularity of a Carbon-Fixing Protein Organelle“. Proceedings of the National Academy of Sciences 109, Nr. 2 (1. Oktober 2012): 478–83. doi:10.1073/pnas.1108557109.
• Long, Benedict M., Murray R. Badger, Spencer M. Whitney, und G. Dean Price. „Analysis of Carboxysomes from Synechococcus PCC7942 Reveals Multiple Rubisco Complexes with Carboxysomal Proteins CcmM and CcaA“. Journal of Biological Chemistry 282, Nr. 40 (10. Mai 2007): 29323–35. doi:10.1074/jbc.M703896200.
• Reinhold et al., 1990. A model for inorganic carbon fluxes and photosynthesis in cyanobacterial carboxysomes. Canadian Journal of Botany, vol. 69, pp. 984-988
• Dou, Zhicheng, Sabine Heinhorst, Eric B. Williams, C. Daniel Murin, Jessup M. Shively, und Gordon C. Cannon. „CO2 Fixation Kinetics of Halothiobacillus Neapolitanus Mutant Carboxysomes Lacking Carbonic Anhydrase Suggest the Shell Acts as a Diffusional Barrier for CO2“. The Journal of Biological Chemistry 283, Nr. 16 (18. April 2008): 10377–84. doi:10.1074/jbc.M709285200.
• Mangan, Niall M, und Michael P Brenner. „Systems analysis of the CO2 concentrating mechanism in cyanobacteria“. eLife 3 (29. April 2014). doi:10.7554/eLife.02043.