Reconstitution of the unusual PE-III phycobiliprotein of Prochlorococcus marinus SS120 in Escherichia coli

The marine cyanobacterium Prochlorococcus sp. is known to be the smallest photosynthetic organism on this planet, nonetheless, its sheer abundance makes it ecological one of the most significant cyanobacteria in the ocean. Unlike other cyanobacteria, Prochlorococcus has abandoned the effective light harvesting complexes, called phycobilisomes (PBS). Instead, it relies on divinyl-chlorophyll-antenna, harvesting blue light very efficiently. Interestingly, Prochlorococcus kept small amounts of a remnant of PBSs in form of a single phycobiliprotein (PBP), phycoerythrin III (PE-III). This PE-III of low-light adapted P. marinus SS120 is composed of an α- and β-subunit (SU) and likely carries covalently attached phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores in a 1:3 ratio. Genes required for the assembly of PE-III are encoded in a ~10 kb gene cluster, encoding SUs and five putative PBP lyases for the proper stereochemical attachment of chromophores to apo-PE-III. In addition to the cluster, genes are found encoding biosynthetic enzymes for PEB and phycocyanobilin (PCB). The function of lyases and their role in the assembly of apo-PE-III were investigated by a heterologous E. coli expression system. To date, only the function of lyase CpeS was confirmed by this approach, ligating (3Z)-PEB to Cys82 on the β-SU CpeB. The other lyases remained uncharacterized mainly due to protein folding issues. A new approach with genes that replicate the codon usage pattern (harmonization) of the original organism in the heterologous expression host was able to bypass folding problems. With the help of this method, we identified MpeX as the first isomerase-lyase in Prochlorococcus, attaching PUB at Cys51/60 on CpeB. Interestingly, MpeX shows only activity in the presence of chaperon-like "helper-lyase" CpeZ and a prior attachment of PEB to Cys82 by CpeS. The remaining lyase CpeT will be used to presumably attach PEB to cysteine residue Cys163 on the β-SU, while CpeY might attach PEB to Cys73 on the α-SU CpeA. Isolated recombinant PBPs are examined by UV-Vis- and fluorescence spectroscopy, Zinc-blot, HPLC and MS to evaluate their composition. The ultimate goal is to reconstitute the whole PE-III in E. coli and on the long run, to transplant PE-III into the model cyanobacterium Synechocystis sp. PCC 6803 to investigate its light harvesting capabilities in a non-native host.