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. Terefore 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 (BBa_I719005) and Ptac ( BBa_K731500 ) promotors for this task. The T7 is at least suitable for a simple overexpression, but could cause the building of inclusion bodies. The resulting protein could be analysed by SDS-PAGE and MALDI-TOF-MS. This methods are suitable to show the synthesis of GlpX. Additionally there is an enzyme assay available to validate the function of this protein (Stolzenberger et al., 2013). The expression for this assay should be done under the controll of the Ptac promotor.

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). The evolutionary distance between these two species is huge and the codon usage is different. Therefore we ordered the synthesis of a codong optimized sequence for E. coli(BBa_K1465201). The function of this enzyme could be analysed by the toxicity of its product. Ribulose-1,5-bisphosphate is not metabolized in the wildtype of E. coli(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. To validate the enzyme function of the RubisCO we would like to coexpress it with the phosphoribulokinase. The toxicity of the PrkA should be avoided by the RubisCO activity. The produced ribulose-1,5-bisphosphate is no longer a metabolic dead end. The RubisCO should use this metabolite as substrate and thereby close this pathway. The RubisCO 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. We would like to compare the enzymatic activity inside the carboxysome and in the cytoplasm. The separation of enzymes from both compartments could be achieved by ultracentrifugation.

Carboxysome

The carboxysome ensures an efficient carbon fixation reaction. It separates the RubisCO from the aerobic cytoplasm. The concentration of carbon dioxide is selectively increased inside the carboxysome (Reinhold et al., 1991; Dou et al., 2008; Mangan and Brenner, 2014). We selected a carboxysome encoding plasmid from H. neapolitanus as template for our synthetic construct.
The gene cluster of the carboxysome contains many illegal restriction sites. Therefore we decided to order some synthtic sequences to replace special parts of pHnCBS1D. This sequences are free of illegal restriction sites. This synthesis also allowed an optimization of 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 carbon dioxide as sole carbon source. Nevertheless there is still a need for an energy source. We would like to address this issue by inventing an electroautotrophic E. coli strain. As a backup we think of feeding a pentose (e.g. xylose) to support the Calvin cycle in case the efficiency of the newly created electroautotrophic metabolism is not high enough.
To engineer an autotrophic E. coli strain we need to fullfill the following tasks:

• heterologous expression of glpX^C from B. methanolicus in E. coli
• heterologous expression of prkA from S.elongatus in E. coli
• construction of a plasmid encoding a carboxysome
• production of a functional carboxysome in E.coli
• heterologous expression of an RubisCO in E.coli
• knock down of pfkA to avoid an futile cycle
• invention of an energy feeding mechanisms (e.g. electric)

References

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