Characterization of the Selective Permeability of Carboxysome Shell Proteins and Implications for CO2 Fixation Efficiency

光合作用利用光能从气态二氧化碳中产生糖，从而为地球上的生命提供动力。被称为蓝藻的光自养原核生物为这种制糖方式贡献了很大一部分，因为它们占海洋光合作用的60%。光合作用中二氧化碳同化的关键酶是Rubisco。这种酶的羧化活性被CO2增强，而在O2存在时被抑制，因此光自养生物进化出碳浓缩机制(CCM)来改善充氧环境中的光合作用。蓝藻CCM将大多数细胞Rubisco限制在称为羧基体的蛋白质微室中，在那里CO2被浓缩，O2被排除在外。羧基体壳形成一个二十面体，它被一层层的六角形蛋白质低聚体所限制。但是，尽管对羧体结构有相当多的了解，代谢物穿过羧体外壳的扩散途径还没有被证明。结构分析表明，羧体壳蛋白CcmK3、CcmK4和CCMP形成了含有中心孔的低聚物。CcmK齐聚物的孔径为4？，CCMP齐聚物的孔径为13？，足以满足Rubisco反应的底物和产物的需要。本项目的目的是分析CcmK3、CcmK4和CCMP突变体对羧体通透性和固碳效率的影响，以表征它们在长聚球藻PCC7942中的功能。为了实现这一目标，我将在中光下培养长链霉菌Ccc7942野生型、ccmK3、ccmK3/4和ccmp突变体，并将培养条件转移到a)高光和b)低CO2。我将比较野生型和突变型代谢组在转变过程中的变化，因为CCM突变很可能会影响碳代谢中间体的池大小。为了揭示CcmK3、CcmK4或CCMP突变对碳固定和其他代谢过程的影响，我将利用转录组学和蛋白质组学来分析基因表达水平变化的反应。初步结果表明，ccmK3、ccmK4和CCMP突变体都有生长表型，这取决于光照强度。这表明这些基因的功能参与了同化过程。我推测CcmK3、CcmK4和CCMP参与了通过羧基体壳的代谢物运输，并且它们的功能丧失突变体显示了壳层对Rubisco底物和/或产物的渗透性的变化。突变株转移到高光或低二氧化碳的代谢组学将揭示推测的羧体运输瓶颈。转录组学和蛋白质组学将有助于确定羧体对高光环境的动态适应。此外，他们还将展示基因表达对环境变化的反应如何受到突变体中羧体的新特性的影响。

Photosynthesis powers life on earth by using light energy to produce sugars from gaseous CO2. Photoautotrophic prokaryotes named cyanobacteria contribute a major portion to this way of sugar production, as they account for 60% of marine photosynthesis. A key enzyme for photosynthetic CO2 assimilation is RuBisCO. The carboxylating activity of this enzyme is enhanced by CO2 and inhibited in the presence of O2 and thus photoautotrophs evolved carbon concentrating mechanisms (CCMs) to improve photosynthesis in oxygenic environments. The cyanobacterial CCM restrains the majority of the cells RuBisCO in proteinaceous microcompartments called carboxysomes, where CO2 is enriched and O2 is excluded. The carboxysome shell forms an icosahedron that is confined by layers of hexagonal protein oligomers. But despite fair knowledge about carboxysome structure, diffusion routes that metabolites take across the carboxysome shell have not been demonstrated. Structural analysis revealed that the carboxysome shell proteins CcmK3, CcmK4 and CcmP form oligomers that contain a central pore. The pore diameter is 4 Å in CcmK oligomers and 13 Å in CcmP oligomers, which is sufficient for the substrates and products of the RuBisCO reaction. The aim of this project is to analyze mutants of CcmK3, CcmK4 and CcmP regarding their effect on carboxysome permeability and carbon fixation efficiency in order to characterize their function in the cyanobacterium Synechococcus elongatus PCC 7942. To reach this aim, I will grow wildtype, ccmK3, ccmK3/4 and ccmP mutants of S. elongatus PCC 7942 in moderate light and shift the cultures to a) high light and b) low CO2. I will compare alterations of the metabolome of wildtype and mutants during the shift, since ccm mutation will most likely affect the pool size of carbon metabolism intermediates. In order to show implications on carbon fixation and other metabolic processes of CcmK3, CcmK4 or CcmP mutation, I will analyze the response to the shift on the gene expression level using transcriptomics and proteomics. Preliminary results show a growth phenotype of both ccmK3 ccmK4 and the ccmP mutant, which depends on the light intensity. This indicates that the function of these genes is involved in assimilatory processes. I hypothesize that CcmK3, CcmK4 and CcmP are involved in metabolite transport across the carboxysome shell and that their loss-of-function mutants display a change of the shells permeability towards RuBisCO substrates and/or products. Metabolomics of mutants shifted to high light or low CO2 will reveal putative bottlenecks in carboxysome transport. Transcriptomics and proteomics will help to determine the dynamics of carboxysome adaptation to high light environment. Furthermore, they will show how the response of gene expression to environmental changes is affected by the new properties of the carboxysome in the mutants.