Graduate Students Mini Symposium I 2021

Program:

16:00 Pascal Pfister, AG Erb

Establishing a synthetic photorespiratory bypass in Synechococcus elongatus PCC7942

The Calvin-Benson-Bassham cycle is a wide spread CO₂ assimilation pathway, present in cyanobacteria, algae and plants. Its key enzyme RuBisCO (Ribulose-1-5-bisphosphate carboxylase/oxygenase) is known to perform a deleterious side reaction with O₂, leading to the toxic intermediate 2-phosphoglycolate (2PG). The metabolic process recycling 2PG to central carbon metabolites is called photorespiration. The photorespiratory reactions lead to the release CO₂ and ammonia. It has been estimated that in C3-plants up to 30% of the carbon is lost due to this process. Photorespiration as well as the refixation of the lost carbon requires additional input of ATP and reducing equivalents. Previously engineered photorespiratory bypasses were only able to circumvent the loss of nitrogen, but still resulted in (even higher) CO₂ release. In contrast, our approach aims at direct CO₂ fixation via an additional carboxylase reaction. We proposed a cyclic bypass that is based on the 3-Hydroxypropionate Bi-Cycle, an alternative CO₂ fixation pathway found in the thermophilic phototroph Chloroflexus aurantiacus. All enzymes required for the bypass could be produced in cyanobacteria and showed activity in cell extracts. The major bottleneck was found to be acetyl-CoA carboxylation. We investigated on the posttranslational modification of the acetyl-CoA carboxylase (ACC) by the biotin ligase. Furthermore we want to deliberate the ACC from other regulators like the nitrogen stress regulator PII to reach maximal ACC activities.

16:30 Nicola Bellotto, AG Sourjik

Diffusional properties of Escherichia coli cytoplasm

The cytoplasm of bacterial cells is highly crowded with macromolecules that give rise to the phenomenon of macromolecular crowding. Macromolecular crowding in turns affects protein diffusion, which is a crucial factor in determining biochemical reaction rates and protein-protein interactions. To perform a systematic analysis of the dependence of diffusion on protein properties, in particular their molecular mass, we created a library of proteins endogenous of the cytoplasm of Escherichia coli tagged with sfGFP. Furthermore, we are exploring how cytoplasmic viscosity, the energetic state of the cytoplasm and the treatment with antibiotic influence diffusion by using selected fusions from our library.