Chromophore composition and assembly of phycoerythrin III of Prochlorococcus marinus CCMP1375

海洋原绿球藻（Prochlorococcus marinus）是海洋中数量最多、具有重要生态意义的光合自养生物之一。与共存的聚球藻物种相反，除了二乙烯基叶绿素天线之外，P. marinus仅具有以称为藻红蛋白III（PE III）的单一藻胆蛋白形式的捕光藻胆体的残余。在低光适应的生态型P. marinus CCMP 1375中，该PE III由具有连接的开链四吡咯发色团（藻胆素）的α-和β-亚基组成。光谱学研究已经假定，这种PE III与藻胆素藻红胆素（PEB）和藻胆素（PUB）以1：3的比例发色。然而，到目前为止，这还没有得到生物化学证实，主要是由于生物化学研究的低生长产量。PE组装所需的大多数基因编码在海原藻CCMP 1375中的~ 10 kb基因簇中。该簇不仅包含分别用于α-和β-亚基的基因，而且还包含编码藻胆蛋白裂解酶的基因。这些蛋白质可能参与藻胆素与亚基的翻译后连接。此外，在基因簇之外，可以找到编码藻胆素PEB和藻蓝胆素的生物合成酶的基因，以及另外一种推定的藻胆蛋白裂解酶。在此资助计划中，我们希望探索PE III的发色团化状态和组装机制。首先，将通过PE III富集，然后结合紫外-可见光谱和荧光光谱以及质谱，验证天然PE III的发色裂解。使用最近描述的TREX系统，在使用随机转座将其整合到大肠杆菌BL 21（DE 3）基因组中之后，整个基因簇将从侧翼T7启动子双向表达。另外，藻胆素生物合成基因将以附加型共表达。成功整合和表达后，PE III将被纯化，并进行紫外-可见和荧光光谱，然后结合藻胆素的详细表征。在平行方法中，我们将利用采用pDuet™载体系统的模块化表达。在这里，所有参与α-或β-亚基组装的推定基因将被共表达。由于一些推定的藻胆蛋白裂解酶还没有表征的同系物，不同的组合进行了测试。此外，第二种方法能够将组装过程分解为各个子单元。该项目的总体目标是确定PE III的发色团化状态，并进一步鉴定和表征所涉及的藻胆蛋白裂解酶。因此，我们也打算确定新的藻胆蛋白裂解酶的活动，这将有助于理解光捕获结构组装和适应这些特定的光制度。

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.