Alternative Carbon Fixation Pathways in Cyanobacteria

含氧光合作用的羧化步骤是由Rubisco催化的，根据卡尔文（CBB）循环将RuBP、CO2和H2O转化为2分子3-PGA。Rubisco还与O2催化副反应（光呼吸），产生3- pga和2PG各一个分子[2,3]。这种反应是不可取的，因为2PG回收的能量成本很高[3,4]，并且抑制中心代谢反应[2,5,6,7]，降低了含氧光合作用的生产率。O2是CO2的竞争性抑制剂[8,9]，因此大气中CO2的增加应该有利于Rubisco羧化反应。然而，随着光呼吸速率随着温度的升高而增加，由于全球变暖，碳同化的效率正在降低。这对农业产生了影响，加剧了污染、荒漠化和人口增长对粮食安全的影响。在目前的大气二氧化碳水平下，光呼吸速率约为25%。因此，即使找到了解决全球变暖的办法，提高光合作用生产力的能力仍然存在。已知有六种自然发生的碳固定途径。其中，只有3-羟丙酸双环（3HP）是耐氧的[13,14,15]，因此适合取代CBB循环。目前正在开发耐氧合成固碳途径。一种蓝藻模式生物（聚胞菌）。由于易于基因操作和生长速度快，本项目选择了6803)。由于聚囊藻和植物以相似的方式进行光合作用，这项工作是一个概念的证明，可以适应于植物。然而，缺乏羧基体[17]的植物，不能产生对3HP双循环功能至关重要的维生素B12，可能会限制对[18]翻译的研究。利用通量平衡分析（Flux Balance Analysis， FBA）将3HP双循环建模为联胞菌，并预测与CBB循环相比，其生长速度将提高30%[19,20]。需要进一步的工作来验证结果。为了更好地研究光呼吸-温度关系，将建立Rubisco的热力学模型。比较基因组学方面的工作将用于预测新的碳固定途径。本项目将使用Golden-Gate模块化克隆系统将3HP双循环植入synnechocystis。该方法的灵活设计特性应有助于解决在先前研究中发现的问题[19]，并使解决方案能够解决潜在的下游问题。由于在以前的工作b[21]中，金门系统适应聚胞虫的局限性，吉布森组装方法将并行使用。参考书目[b]巴萨姆等。1954。JACS.76, 1160 - 1170。[2] Anderson.1971。工商管理硕士-酶学。235,237-244。[10]鲍威等。植物学报，15(5):330-336。[qh]Maurino & Peterhansel.2010。植物学报，26(3):449 - 456。[5] Busch.2013。植物学报，36(5):648-655。[qh]Peterhansel & Maurino.2011。植物生理学报，28(2):449 - 455。b[7]凯利和拉茨科，1976。《财经快报》，68,55-58。[b]鲍斯和奥格伦，1972。JBC.247, 2171年。[10]彼得汉塞尔等。2010。ASPB。8, e0130。库和爱德华兹，1977。植物生理学报，36(5):986-990。[11] Busch.2013。植物学报，36(5):648-655。b[12]Bar-Even et al. 2010。PNAS.107, 8889 - 8894。[10]Herter等。2002。[j] .中国生物医学工程学报，2004,19(4):593 - 596。[10]Herter等。2002。JBC.277, 20277 - 20283。[10]Zarzycki等。PNAS.106, 21317 - 21322。[10]Schwander等。2017。Science.354, 900 - 904。[b]Kaplan & Reinhold.1999。植物物理学报，26(5):539- 557。[b] Helliwell等。2011。MBE.28(10) 2921 - 2933。[19] Cotton.2016。在聚胞藻sp. pcc6803中引入另一种固定碳循环。哲学博士（未出版）。伦敦帝国理工学院。[10]蔡国强等。2017。Synechocystis sp. PCC 6803不同固碳途径和光呼吸的通量平衡分析。公共科学图书馆(提交)。[21] West.2017。蓝藻中不同的碳固定途径。研究硕士(未出版)。伦敦帝国理工学院

The carboxylation step of oxygenic photosynthesis is catalysed by Rubisco - converting RuBP, CO2, and H2O into 2 molecules of 3-PGA as per the Calvin (CBB) cycle [1]. Rubisco also catalyses a side-reaction with O2 (photorespiration), producing one molecule each of 3-PGA and 2PG [2,3]. This reaction is undesirable as 2PG is energetically costly to recycle [3,4] and inhibits central metabolic reactions [2,5,6,7], reducing the productivity of oxygenic photosynthesis.O2 is a competitive inhibitor of CO2 [8,9], hence increases to atmospheric CO2 should favour the Rubisco carboxylation reaction. However, as the rate of photorespiration increases with temperature [10], carbon assimilation is becoming less efficient due to global warming. This has impacts for agriculture - compounding the effects of pollution, desertification, and population growth on food security. At current atmospheric CO2 levels, photorespiration rates are approximately 25% [11]. Therefore, even if solutions to global warming are found, the capacity to improve on photosynthetic productivity remains. There are six known naturally occurring carbon fixation pathways [12]. Of these, only the 3-Hydroxypropionate bi-cycle (3HP) is oxygen tolerant [13,14,15] - and thus suitable to replace the CBB cycle. Oxygen tolerant synthetic carbon-fixation pathways are being developed [16].A cyanobacterium model organism (Synechocystis PCC. 6803) has been selected for this project, for ease of genetic manipulation and it's fast growth rate. As Synechocystis and plants perform photosynthesis in a similar way, this work is a proof of concept that can be adapted into plants. However, plants lacking carboxysomes [17], and not producing vitamin B12, vital to 3HP bi-cycle function, are could cause limitations to research translation [18]. The 3HP bi-cycle was modelled into Synechocystis using Flux Balance Analysis (FBA) and predicted a 30% growth rate improvement compared to the CBB cycle [19,20]. Further work is needed to validate results. A thermodynamic model for Rubisco will be produced to better study the photorespiration-temperature relationship [10]. A comparative genomics aspect of the work will be used to predict novel carbon-fixation pathways. This project will use the Golden-Gate modular cloning system to implement the 3HP bi-cycle into Synechocystis. The flexible design properties of this method should help address problems found during previous studies [19], and enable solutions to potential downstream problems. Due to limitations in adapting Golden Gate systems to Synechocystis in previous work [21], the Gibson assembly method will be used in parallel. Bibliography[1]Bassham, et al.1954. JACS.76,1160-1170. [2]Anderson.1971. BBA - Enzymology.235,237-244. [3]Bauwe et al.2010. Trends Plant Sci.15,330-336. [4]Maurino & Peterhansel.2010. Curr Opin Plant Biol.13, 249-256. [5]Busch.2013. Plant Biol.15, 648-655. [6]Peterhansel & Maurino.2011. Plant Physiol.155, 49-55. [7]Kelly & Latzko.1976. FEBS Lett.68, 55-58. [8]Bowes & Ogren.1972. JBC.247, 2171. [9]Peterhansel, et al.2010. ASPB. 8,e0130. [10]Ku & Edwards.1977. Plant Physiol.59, 986-990. [11]Busch.2013. Plant Biol.15, 648-655. [12]Bar-Even et al. 2010. PNAS.107, 8889-8894. [13]Herter et al.2002. J. Bacteriol.184, 5999-6006. [14]Herter et al.2002. JBC.277, 20277-20283. [15]Zarzycki et al.2009. PNAS.106, 21317-21322. [16]Schwander et al.2017. Science.354, 900-904. [17]Kaplan & Reinhold.1999. Annu Rev Plant Physiol Plant Mol Biol.50, 539-570. [18] Helliwell et al.2011. MBE.28(10), 2921-2933. [19]Cotton.2016. Introduction of an Alternative Carbon Fixation Cycle into Synechocystis sp. PCC 6803. Doctor of Philosophy(unpublished). Imperial College London. [20]Chua et al.2017. Flux Balance Analysis of Alternative Carbon Fixation Pathways and Photorespiration in Synechocystis sp. PCC 6803. PLOS(submission). [21] West.2017. Alternative Carbon Fixation Pathways in Cyanobacteria. MRes(unpublished). Imperial College London