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

羧基体壳蛋白选择性渗透性的表征及其对 CO2 固定效率的影响

• 批准号: 289026613
• 负责人: Dr. Manuel Sommer
• 金额: --
• 依托单位: Institut für Biochemie der Pflanzen
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/289026613?language=en
• 关键词: Characterization; Selective; Permeability; Carboxysome; Shell

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.

光合作用通过利用光能从气态二氧化碳中产生糖来为地球上的生命提供动力。名为蓝细菌的光合自养原核生物对这种糖的生产方式做出了主要贡献，因为它们占海洋光合作用的60%。光合CO2同化的关键酶是RuBisCO。这种酶的羧化活性被CO2增强，在O2存在下被抑制，因此光合自养生物进化出碳浓缩机制（CCM）来改善富氧环境中的光合作用。蓝藻CCM抑制大多数细胞RuBisCO在蛋白质微区室称为carboxysomes，其中CO2富集和O2被排除在外。羧基体壳形成二十面体，其被六边形蛋白质寡聚体层限制。但是，尽管对羧基体结构有相当的了解，代谢物穿过羧基体壳的扩散途径还没有得到证实。结构分析表明，羧基体壳蛋白CcmK 3，CcmK 4和CcmP形成含有中心孔的低聚物。CcmK低聚物的孔径为4 μ m，CcmP低聚物的孔径为13 μ m，这对于RuBisCO反应的底物和产物是足够的。本项目的目的是分析CcmK 3，CcmK 4和CcmP的突变体对羧基体渗透性和碳固定效率的影响，以表征它们在蓝细菌细长聚球藻PCC 7942中的功能。为了达到这一目的，我将培养野生型，ccmK 3，ccmK 3/4和ccmP突变体的S。elongatus PCC 7942在中等光照下培养，并将培养物转换为a）高光和B）低CO2。我将比较野生型和突变体在转换过程中代谢组的变化，因为ccm突变很可能会影响碳代谢中间产物库的大小。为了显示CcmK 3、CcmK 4或CcmP突变对碳固定和其他代谢过程的影响，我将使用转录组学和蛋白质组学分析基因表达水平变化的反应。初步结果表明，生长表型的ccmK 3，ccmK 4和ccmP突变体，这取决于光强度。这表明这些基因的功能参与同化过程。我推测，CcmK 3，CcmK 4和CcmP参与代谢物运输的羧基体壳和功能丧失突变体显示壳的通透性的变化对RuBisCO底物和/或产品。突变体的代谢组学转移到高光或低CO2将揭示假定的羧基体运输的瓶颈。转录组学和蛋白质组学将有助于确定羧基体适应强光环境的动力学。此外，他们将展示基因表达对环境变化的反应如何受到突变体中羧基体新特性的影响。