Alternative Carbon Fixation Pathways in Cyanobacteria

根据卡尔文（CBB）循环[1]，产氧光合作用的羧化步骤由Rubisco催化-将RuBP、CO2和H2O转化为2分子3-PGA。Rubisco还催化与O2的副反应（光呼吸），产生3-PGA和2 PG各一个分子[2，3]。这种反应是不希望的，因为2 PG的再循环是能量昂贵的[3，4]，并抑制中心代谢反应[2，5，6，7]，降低产氧光合作用的生产力。O2是CO2的竞争性抑制剂[8，9]，因此增加大气CO2应该有利于Rubisco羧化反应。然而，随着光呼吸速率随着温度的增加而增加[10]，由于全球变暖，碳同化的效率越来越低。这对农业产生了影响，加重了污染、荒漠化和人口增长对粮食安全的影响。在目前的大气CO2水平下，光呼吸率约为25% [11]。因此，即使找到了全球变暖的解决方案，提高光合生产力的能力仍然存在。已知有六种天然存在的碳固定途径[12]。其中，只有3-羟基丙酸双循环（3 HP）是耐氧的[13，14，15] -因此适合替代CBB循环。正在开发耐氧合成碳固定途径[16]。6803）已被选定为这个项目，为易于遗传操作和它的快速增长速度。由于集胞藻和植物以类似的方式进行光合作用，这项工作是可以适应植物的概念证明。然而，缺乏羧化酶体的植物[17]，并且不产生对3 HP双循环功能至关重要的维生素B12，可能会限制研究翻译[18]。使用通量平衡分析（FBA）将3 HP双循环建模为集胞藻，并预测与CBB循环相比生长率提高30%[19，20]。需要进一步开展工作来验证成果。将建立Rubisco的热力学模型，以更好地研究光呼吸-温度关系[10]。这项工作的比较基因组学方面将用于预测新的碳固定途径。该项目将使用Golden-Gate模块化克隆系统将3 HP双循环实施到集胞藻中。该方法的灵活设计特性应有助于解决以前研究中发现的问题[19]，并能够解决潜在的下游问题。由于在以前的工作中使Golden Gate系统适应集胞藻的局限性[21]，将并行使用吉布森组装方法。参考文献[1]Bassham，et al.1954. JACS.76，1160 -1170。[2]Anderson.1971. BBA -酶学235，237 -244. [3]Bauwe et al.2010.《植物科学趋势》，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.植物生理学，155，49-55。[7]KELLY & LATZKO.1976. FEBS Lett.68，55-58. [8] 1972. JBC.247，2171. [9]Peterhansel，et al.2010. ASPB。8，e0130. [10]Ku & Edwards.1977.植物生理学59，986-990。[11]Busch.2013. Plant Biol.15，648-655. [12]Bar-Even等人，2010年。PNAS.107，8889-8894。[13]Herter等人，2002年。J.Bacteriol.184，5999-6006。[14]Herter等人，2002年。JBC.277，20277-20283。[15]Zarzycki等人，2009年。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等人，2011年。28（10），2921-2933. [19]第十九话集胞藻6803的碳固定循环研究哲学博士（未出版）。帝国理工学院伦敦。[20]Chua et al.2017.集胞藻6803交替固碳途径和光呼吸的通量平衡分析。PLOS（呈件）。[21] West.2017.蓝细菌的碳固定途径。MRES（未发表）。帝国理工学院伦敦

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