Alternative Carbon Fixation Pathways in Cyanobacteria

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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