Genetical approach

Introduction and motivation

Our aim is to bind carbon dioxide in organic molecules using E. coli. There are many different pathways known for carbon dioxide fixation in bacteria. Most suitable for our application are the 3-hydroxypropionate 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 should be the only source of energy for our bacteria. Therefore 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. Tierefore we decided to clone the coding sequence of glpX^C into pSB1C3. This BioBrick should then be combined with a strong promotor to enable a heterologous overexpression in E. coli. We selected the T7 and Ptac promotors for this task. The T7 is at least suitable for a simple overexpression. The resulting protein could be analysed by SDS-PAGE and MALDI-TOF-MS. This methods are suitable to proof the synthesis of GlpX. There is also an enzyme assay to validate the function of protein.