Bin Gu

• Phycobilisomes (PBS) are the major light-harvesting complexes for the majority of cyanobacteria and allow these organisms to absorb in the so-called green gap. They consist of smaller units called phycobiliproteins (PBPs), which are composed of an α- and a β-subunit with covalently bound linear tetrapyrroles (phycobilins). The latter are attached to the apo-PBPs by phycobiliprotein lyases. Interestingly, cyanobacteria of the genus Prochlorococcus lack complete PBS and instead use prochlorophyte chlorophyll-binding proteins (Pcbs), which effectively utilize the energy of the blue light region. The low-light-adapted (LL) strain Prochlorococcus marinus SS120 has a single PBP, phycoerythrin-III (PE-III). It has been postulated that PE-III is chromophorylated with the phycobilins phycourobilin (PUB) and phycoerythrobilin (PEB) in a 3:1 ratio. Thereby, the function of PE-III remains unclear so far, so that light-gathering function and also photoreceptor function are discussed. The main goal of this work was to characterize the assembly of PE-III and thus the function of the six putative phycobiliprotein lyases of P. marinus SS120. Previous work found that the individual lyases could not be produced in soluble form, so we switched to a dual pDuet™ plasmid system in E. coli, which was successfully established. Investigation of the binding of PEB to Apo-PE revealed that the CpeS lyase specifically chromophorylated Cys82 with 3Z-PEB. Unfortunately, additional chromophorylation could not be observed using the pDuet system. Therefore, in a second part of the work, the entire PE gene cluster from P. marinus SS120 was to be introduced into E. coli and expressed. Although the gene cluster was successfully transcribed within E. coli, no translation was observed, possibly due to incompatible translation initiation between Prochlorococcus and E. coli. The introduction of a mini PE cluster (CpeAB) into the cyanobacterium Synechococcus sp. PCC 7002 was also successfully performed, in which case production of CpeB but not CpeA from Prochlorococcus was detected. Recombinant CpeB was also detected together with intrinsic PBP in Synechococcussp. 7002, indicating structural similarity and incorporation into PBS in Synechococcus sp. 7002. Overall, the obtained results suggest that a cyanobacterial host is a good option for the studies on the assembly of PE-III from P. marinus and, based on this, future work could aim at generating an artificial operon using synthetic biology to achieve efficient translation of all genes.