Proteomic investigation of Synechocystis SP. PCC 6803: S/T/Y phosphorylation and response of proteins to bioengineering tools based on the copper-regulated PETJ promoter

Cyanobacteria are unique prokaryotic organisms performing oxygenic photosynthesis. Their ability to transform sunlight and CO2 to sugar, together with the flexibility of metabolism, have made cyanobacteria interesting organisms for engineering the carbon and electrons fluxes toward exogenous pathways for the production of high value compounds. Understanding the complex mechanisms regulating photosynthesis and their dynamic interactions with cellular metabolism are crucial in order to improve the productivity of engineered strains. In this Thesis, reversible O-type phosphorylation on S/T/Y amino acids, a ubiquitous posttranslational regulatory mechanism, was investigated in Synechocystis 6803 using proteomics approaches. A global discovery-driven investigation of the phosphoproteome revealed that S/T/Y phosphorylation is extensive in Synechocystis 6803, and phosphoproteins are involved in various metabolic pathways. A large share of the identified phosphoproteins participate in photosynthesis-related processes. The quantitative targeted mass spectrometry method was set-up for (phospho)peptides of photosynthesis-related proteins. It was applied for screening a collection of S/T protein kinase mutants in Synechocystis 6803. The results revealed an intricate phosphoprotein-protein kinase network, including an interplay among several kinases and auxiliary proteins. In particular, the SpkG kinase was shown to phosphorylate the Fd5 protein, while Slr051 was revealed as an auxiliary protein regulating the balance between the phosphorylated and nonphosphorylated forms of Fd5. Moreover, the deletion of the SpkG kinase caused the induction of phosphorylation in several other peptides indicating a cross talk among the participants of the protein phosphorylation network. Further, the data obtained in this Thesis showed that the induction of exogenous pathways in synthetic biology approaches might cause background proteome changes due to the acclimation of the cells to the concentration of metal ions. Particularly, the Cu2+ treatment, used to regulate the petJ promoter, irreversibly affected DNA replication, transcription and translation machineries, cell wall proteins, transporters, signaling proteins and enzymes involved in lipid biosynthesis. These changes might consequently affect the expression of a pathway or the recovery of a high value product. In summary, the results presented in this Thesis provide a better understanding of Synechocystis 6803 regulatory mechanisms, which might support the optimization of engineered strains toward maximal productivity of high value compounds