Chromophore composition and assembly of phycoerythrin III of Prochlorococcus marinus CCMP1375

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The cyanobacterium Prochlorococcus marinus is one of the most abundant and ecologically important photosynthetic autotrophs in the ocean. In contrast to the co-occuring Synechococcus species, P. marinus only possesses remnants of a light-harvesting phycobilisome in form of a single phycobiliprotein termed phycoerythrin III (PE III) in addition to a divinyl-chlorophyll antenna. In the low –light adapted ecotype P. marinus CCMP1375, this PE III consist of an alpha- and beta-subunit with linked open-chain tetrapyrrole chromophores (phycobilins). Spectroscopic studies have postulated that this PE III is chromophorylated with the phycobilins phycoerythrobilin (PEB) and phycourobilin (PUB) at a 1:3 ratio. However, thus far this has not yet been confirmed biochemically mainly due to low growth yields for biochemical studies. Most of the genes required for PE assembly are encoded in a ~ 10 kb gene cluster in P. marinus CCMP1375. This cluster not only contains the genes for the alpha- and beta- subunit respectively, but also genes encoding phycobiliprotein lyases. These are proteins likely to be involved in the posttranslational attachment of phycobilins to the subunits. Furthermore, outside the gene cluster, genes encoding the biosynthetic enzymes for the phycobilins PEB and phycocyanobilin and an additional putative phycobiliprotein lyase can be found. Within this grant proposal, we wish to explore the chromophorylation state and mechanism of assembly of PE III. First, chromophorlyation of native PE III will be validated by PE III enrichment followed by a combination of UV-Vis and fluorescence spectroscopy and mass spectrometry. Using the recently described TREX system, the whole gene cluster will be bidirectionally expressed from flanking T7 promoters following its integration into the Escherichia coli BL21 (DE3) genome using randomized transposition. Additionally, the phycobilin biosynthesis genes will be co-expressed episomally. After successful integration and expression, PE III will be purified and subjected to UV-Vis and fluorescence spectroscopy, followed by detailed characterization of the bound phycobilins. In a parallel approach we will make use of modular expression employing the pDuet™ vector system. Here, all putative genes involved in either alpha- or beta-subunit assembly will be co-expressed. Since some of the putative phycobiliprotein lyases have no characterized homologs yet, different combinations have to be tested. Furthermore, this second approach enables the dissection of the assembly process into the individual subunits. The overall goal within this project is to define the chromophorylation state of PE III and furthermore identify and characterize the involved phycobiliprotein lyases. Thereby we also intent to identify novel phycobiliprotein lyase activities, which will contribute to the understanding of light-harvesting structure assembly and adaptation of those to specific light regimes.