Alternative Carbon Fixation Pathways in Cyanobacteria

Lead Research Organisation: Imperial College London
Department Name: Life Sciences


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.

[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


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011178/1 30/09/2015 29/09/2023
1814189 Studentship BB/M011178/1 30/09/2016 21/11/2019 Bethany Maria West
Description Extended a previously developed computational model for the cyanobacterium Synechocystis PCC. 6803, by adding the cyanobacterial carbon concentrating mechanism to the metabolic model. Used this model to explore the effects of knocking out the native Calvin cycle in silico, and replace it with previously unexplored natural and synthetic alternative carbon fixation pathways. This work was used to inform laboratory work. Also used this model to explore the efficiency of native and non-native photorespiratory bypasses in Synechocystis PCC. 6803. This model was further used to explore the predicted effects of growth rate in Synechocystis PCC. 6803, when using different sources of nitrogen, and also implementing the nitrogen fixation ability into the in silico model. A smaller, central carbon metabolism model was also developed de novo to confirm results from the full metabolic model, and also to more easily follow the flux differences in central carbon metabolism, when different carbon-fixation pathways were implemented in the model.

Experimentally a golden gate assembly kit is being developed for Synechocystis PCC. 6803, to facilitate a modular-based cloning strategy for future synthetic biology in this organism. This kit is also being developed to enable modular cloning of the 3-Hydroxypropionate bi-cycle into the cyanobacterium Synechocystis PCC. 6803 - to ascertain whether this alternative carbon fixation pathway is viable for use as a Calvin cycle replacement.
Exploitation Route Extension of the golden gate assembly kit to include more promoter / ribosome binding site combinations and both C and N terminal tags. Also extension of the integration site library for further transformations in Synechocystis PCC. 6803.

The modelling findings could be taken forward to plant-based work - both computational (to see if computational results hold true for plant metabolic models), and experimental.
Sectors Agriculture, Food and Drink,Environment

Description Educating the public on photosynthesis and Calvin Cycle research through the Imperial College Fringe event on 5th December 2017. Educating the public on ways to improve the efficiency of Carbon fixation to increase crop production through the Imperial College Fringe event on 5th December 2017. Educating the public on protein crystallography methods and the impact of solving protein structures at the Imperial Festival (28th - 29th April 2018). Encouraging young people to pursue STEM careers through talks and interactive demonstrations, such as the Imperial Festival (28th - 29th April 2018).
First Year Of Impact 2017
Sector Education
Impact Types Societal