Alternative Carbon Fixation Pathways in Cyanobacteria

氧化光合作用的羧化步骤是由Rubisco催化的，按照Calvin(CBB)循环将RuBP、CO2和H2O转化为2个3-PGA分子[1]。Rubisco还催化与O2(光呼吸)的副反应，分别产生一个3-PGA和2PG[2，3]分子。这个反应是不可取的，因为2PG回收[3，4]的能量成本很高，并且抑制了中心代谢反应[2，5，6，7]，降低了氧合光合作用的生产率。O2是CO2的竞争性抑制剂[8，9]，因此增加到大气中的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升高而增加[10]，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%[11]。因此，即使找到了全球变暖的解决方案，提高光合作用生产率的能力仍然存在。已知的自然固碳途径有六种[12]。其中，只有3-羟基丙酸双环(3HP)耐氧[13，14，15]-，因此适合取代CBB循环。耐氧合成固碳途径正在开发中[16]。蓝藻模式生物(聚球藻PCC)。6803)被选入该项目，是因为它的遗传操作简单，生长速度快。由于聚球藻和植物进行光合作用的方式相似，这项工作是对可以适应植物的概念的证明。然而，缺乏羧基体的植物[17]，以及不能产生对3HP双循环功能至关重要的维生素B12的植物，可能会对研究翻译造成限制[18]。利用通量平衡分析(FBA)将3HP双周期模拟为集胞藻，并预测与CBB周期相比，增长率将提高30%[19，20]。还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[10]。这项工作的比较基因组学方面将被用来预测新的固碳途径。本项目将使用金门模块克隆系统将3HP双周期实施到集胞体中。这种方法灵活的设计特性应该有助于解决以前研究中发现的问题[19]，并使潜在的下游问题能够得到解决。由于以前的工作[21]中在使金门系统适应聚球藻方面的局限性，吉布森组装方法将被并行使用。参考文献[1]Bassham等人，1954。JACS.76,1160-1170。[2]安德森1971年。BBA-酶学.235,237-244.[3]Bauwe等人，2010年。趋势植物科学15,330-336。[4]Maurino&Peterhansel.2010。《植物生物学》13,249-256。[5]Busch.2013。植物生物学。15,648-655。[6]Peterhansel&Maurino.2011。植物生理学。155，49-55。[7]Kelly&Latzko.1976。2月信报68，55-58。[8]Bowes&Ogren1972年。JBC.247,2171.[9]Peterhansel等人，2010年。ASPB。8，e0130。[10]Ku&Edwards.1977。植物生理学。59,986-990。[11]Busch.2013。植物生物学。15,648-655。[12]Bar-Even等人。2010年。PNAS.107,8889-8894。[13]Herter等人，2002年。细菌.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等人2017年。科学。354,900-904。[17]卡普兰和莱因霍尔德1999年。植物生理学年鉴.50,539-570.[18]Helliwell等人，2011年。MBE.28(10)，2921-2933[19]科顿。2016。在集胞藻中引入替代固碳循环。PCC 6803。哲学博士(未出版)。伦敦帝国理工学院。[20]Chua et al.2017年。聚球藻交替固碳途径与光呼吸的通量平衡分析PCC 6803。公共科学图书馆(提交)。[21]西部。2017。蓝藻的另一种碳固定途径。MRE(未发表)。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