Steffen Heck

• A variety of organisms, including cyanobacteria, red algae and cryptophytes, perform oxygenic photosynthesis using chlorophyll-containing protein complexes and phycobiliproteins for light-harvesting. The most prominent phycobiliproteins described in literature assemble to macromolecular antenna complexes, called the phycobilisomes, to enhance oxygenic photosynthesis. Phycobiliproteins are categorized according their chromophore composition, with phycocyanins and allophycocyanins binding phycocyanobilin, while phycoerythrins can bind various bilins, such as 15,16-dihydrobiliverdin and phycoerythrobilin. Investigations of phycoerythrins often involves heterologous expression systems based on E. coli but only allow for biochemical characterization. In contrast, the utilization of expression systems in cyanobacteria, such as Synechocystis sp. PCC 6803, offer the opportunity to study phycoerythrins on a physiological level. Consequently, this work aimed at providing a platform for heterologous phycoerythrin assembly using the cyanobacterium Synechocystis sp. PCC 6803, which naturally synthesizes allophycocyanin and phycocyanin with covalently attached phycocyanobilin. As a necessary prerequisite, phycoerythrobilin biosynthesis was established to provide the chromophore for phycoerythrin assembly. The impact of heterologous phycoerythrobilin production on the cellular physiology was assessed by growth curve experiments, oxygen evolution measurements as well as nutrient- and phycobiliprotein content determination. Despite a reduction in the growth rate, physiological effects were negligible. In addition, the influence of phycoerythrobilin formation on the biochemical properties of endogenous phycobiliproteins and its consequences for phycobilisome assembly were evaluated and show covalent attachment of phycoerythrobilin to CpcA of phycocyanin. The modified phycocyanin is unable to assemble into the phycobilisome complex leading to free cytosolic phycobiliproteins. Eventually, the remaining genes necessary for the assembly of the P. marinus MED4 phycoerythrin were introduced in Synechocystis sp. PCC 6803. Although in a rather low abundance, evidence for recombinant protein production is provided. However, proper assembly of the phycoerythrin was not confirmed in this study which motivates for further optimization strategies to increase the yield of target proteins. In summary, this work demonstrates the amenability of Synechocystis sp. PCC 6803 to modifications in its biosynthetic phycobilin pathways by heterologously producing the chromophore phycoerythrobilin. Consequently, a promising foundation for the assembly of phycoerythrins is provided. Although proper phycoerythrin assembly was not confirmed in this study, the remaining challenges are most likely of technical nature. These findings still strengthen the potential of utilizing cyanobacteria as a platform for the biochemical and physiological evaluation of phycoerythrins from otherwise inaccessible oxygenic phototrophs. Consequently, using such a platform may strongly contribute to the understanding of the structure and the mechanism of accessory light-harvesting machineries contributing to oxygenic photosynthesis.