Introduction

The Ribulose 1,5-bisphosphate Carboxylase Oxygenase (RuBisCO) is the most important enzyme in the Calvin cycle. It binds gaseous carbon dioxide to ribulose-1,5-bisphosphate (Ru-BP) generating two molecules of 3-phosphoglycerate (3-PGA). Therefore, it is responsible for the fixation of carbon dioxide. 3-PGA is further converted in the Calvin cycle to glycerinaldehyde-3-phosphate. This is an essential intermediate in the central metabolism as it plays a central role in glycolysis and gluconeogenesis. RuBisCO enzymes are chracterized as enzymes with slow reaction rates with a k_cat of approximately 20 (Sage, 2002). Furthermore they catalyze a side reaction with oxygen instead of carbon dioxide, deteriorating the catalytic efficiency and producing the toxic compound 2-phopshoglycolate. The inclusion of the RuBisCO in a carboxysome would significantly improve the efficiency of carbon fixation.
It was our aim to enable carbon fixation in E. coli for generating an semi-autotrophic organism. Implementation of the Calvin cycle in this heterotrophic model organism should be associated by the expression of the carboxysome. We would like to use a carboxysome to increase efficiency of RuBisCo by sequestering the enzyme from oxygen. As a carbon source for our experiments with E. coli, we choose the pentose xylose. This ensures that energy generation does not occur via glycolysis. Xylose is metabolized by the cells to ribulose-5-phosphate. This is the substrate for Phosphoribulokinase A from S. elongatus, which is recombinantly expressed by E. coli. The PrkA attaches a phosphate group to ribulose-5-phosphate generating ribulose-1,5-bisphophate. This again is used by RuBisCO to produce 3-phosphoglycerate. 3-phosphoglycerate can enter the glycolysis and pyruvate is build up as a product. The reaction mechanism is illustrated in figure 1.

Figure 1: Pathway of the D-xylose consumption in E. coli for the fixation of carbon dioxide by the RuBisCO from Halothiobacillus neapolitanus. For this approach the substrate ribulose 1,5-bisphosphate needs to be accumulated in the cell. This is realized by the PrkA from Snyechoccous elongatus.

Our first aim was to prove carbon dioxide fixation by RuBisCO. The functionality of RuBisCo is essential for our project as this is the basis of carbon dioxide fixation. Expression of RuBisCO together with PrkA which is generating the substrate for RuBisCo could provide carbon dioxide fixation in E. coli. Therefore, we investigated the functionality of the RuBisCO in vitro perfoming a RuBisCO activity assay.

RuBisCO activity assay

For the verification of RuBisCo expression, we analyzed protein expression of E. coli KRX containing the construct P_T7cbbLS_{H. neap.} and as a second verification, protein expression using the construct BBa_K1465213. Cultivations were carried out as described in Cultivation for Expression of recombinant proteins. Samples were generated using the protocol for Fast Cell Lysis for SDS-PAGE. The results are shown in figure 2 and 3.

Figure 2: Protein expression of RuBisCO_{H. neap.} over time by E. coli KRX carrying P_T7cbbLS_{H. neap.} after induction with 0.1 % rhamnose.

Figure 3: Protein expression over time of RuBisCO_{H. neap.} by E. coli carrying BBa_K1465213 after induction with 0.5 mM IPTG.

In both SDS-PAGEs there is a clearly increasing band over the duration of the cultivation. Analysis with MALDI-TOF proved that the band corresponding to a size of a little less than 55 kDa is the large subunit of RuBisCO from Halothiobacillus neapolitanus. The analysis was done via tryptic digestion and an in silico comparison of the measured peptide masses to the predicted peptide masses. For RuBisCO expressed under control of the T7 promoter seven matching peptide masses were found, the sequence coverage was 15.2 % (MS) and 15.2 % (MS/MS). For RuBisCO expressed via the P_tac promoter six matching peptide masses were found and the sequence coverage was 13.5 % (MS) and 13.5 % (MS/MS). The small unit of the RuBisCO could only be identified via MALDI-TOF in the samples expressed under the control of the T7 promoter. The analysis was performed as described above, showing three matching peptide masses and a sequence coverage of 35.5 % (MS) and 12.7 (MS/MS). The reason for the problem in identifying the small subunit stems from its small size of 12.8 kDa, making it hard to find in the SDS-PAGE. Still, these results correspond to the verification of protein expression from the plasmid pHnCBS1D which we used as the basis for purifying of carboxysomes. Expression of this plasmid gave only in one of three experiments the proof of expression the small subunit.

For the verification of RuBisCO activity, we performed an in vitro assay measuring variances for ribulose-1,5-bisphosphate and 3-phosphoglycerate, substrate and product of the RuBisCo, in the cell extract of KRX wildtype and KRX carrying the construct P_T7 cbbLS_H.neap.. The method for the in vitro assay is described in RuBisCO activity assay and the measuring via HPLC in the protocol for HPLC.
The first measurement with HPLC was made to identify substrate and product of the RuBisCO, Ru-BP and 3-PGA. Therefore, standards containing just one of the substances were measured (figure 4). The substances are clearly separable with a retention time of 14.4 min for Ru-BP and 12,6 min for 3-PGA.

Figure 4: Standards for the RuBisCO activity assay. The first measurement was ribulose-1,5-bisphosphate, the second was 3-phosphoglycerate.

To show that Ru-BP does not occur in the cell extract of E. coli wildtype, we performed the assay with just the cell extract of the wildtype without addition of Ru-BP. As a control, we did a second assay containing the cell extract and Ru-BP was added. The samples were taken after 30 min, to demonstrate, that there is no unspecific degradation of Ru-BP. The results are shown in figure 5. Interestingly there is a small peak found in both assays that corresponds to 3-PGA. No significant increase of the 3-PGA peak was observed in the cell extract when Ru-BP was added. While a number of cellular reactions yields 3-PGA, thus explaining the small amount of 3-PGA in both cell extracts, conversion of Ru-BP to 3-PGA is a specific reaction catalyzed by RuBisCO. This nicely confirmed by the lack of 3-PGA accumulation in the assay containing Ru-BP.

Figure 5: HPLC measurement of cell extract from E. coli KRX wildtype. In the first measurement, ribulose-1,5-bisphosphate was added. The second measurement was performed with the cell extract without adding ribulose-1,5-bisphosphate. Samples were taken after 30 min.

In the third experiment we performed the assay with the cell extract of the wildtype and KRX carrying the construct P_T7 cbbLS_H.neap. taking samples every 5 min to demonstrate the time response of the reaction. Two biological replicates each were performed, which showed both the same results. In figure 6 the course of the reaction is shown.

Figure 6: RuBisCO activity assay. The cell extract from E. coli KRX carrying P_tac cbbLS_H.neap. and E. coli KRX wildtype was examined for RuBisCO activity. The substrate for the RuBisCO, ribulose-1,5-bisphosphate was added and the time curve of ribulose-1,5-bisphosphate and the product of the enzymatic reaction, 3-phosphoglycerate, was measured via HPLC in 5 minutes intervals.

In the assay containing the wildtype cell extract the peak for Ru-BP remains practically constant over time. In contrast, in the assay containing the cell extract of KRX P_T7 cbbLS_H.neap. shows a clearly decrease of the substrate Ru-BP and a increase of 3-phosphoglycerate over time (Figure 6). It appears that the reaction is not completed after 15 min because there is still some Ru-BP detectable. This may be due to the low reaction rate and high Km of RuBisCO (Sage, 2002).
It can be concluded, that the RuBisCO is active, showing the highly specific conversion of Ru-BP to 3-PGA over time course. Therefore, we successfully proved the activity of the RuBisCO.

Summary

Our aim was to transform two different RuBisCOs. We were able to transform the RuBisCO from Halothiobacillus neapolitanus. The activity should be characterized through a thin layer chromatography. The spots on the layer did not separate during the procedure. Because of that we decided to do an enzyme assay. We measured the activity through the decrease of ribulose 1,5-bisphosphate and increase of 3-phosphoglycerate in the cell extract over a time period.

References

Sage, Rowan F. „Variation in the K(cat) of Rubisco in C(3) and C(4) Plants and Some Implications for Photosynthetic Performance at High and Low Temperature“. Journal of Experimental Botany 53, no. 369 (2002): 609–20.
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