Design of an improved Calvin-Benson Cycle for efficient CO2 fixation and photoproduction of biofuels and chemicals by microalgae and cyanobacteria – CalvinDesign

The objective of the CalvinDesign project is to use a synthetic and structural biology approach (genetic and metabolic engineering) to improve our knowledge of the structure, function and regulation of the Calvin-Benson cycle and to improve its functioning. This project focuses on the best characterized models of photosynthetic microorganisms, the cyanobacterium Synechocystis PCC6803 and the eukaryotic green microalga Chlamydomonas reinhardtii. The originality of this project is to apply the principles and methods of synthetic biology to meet the challenge of improving carbon fixation in photosynthetic microorganisms. The strains produced will be characterized under varying conditions, including conditions of production of molecules of industrial interest, to generate new knowledge. The strains generated by the CalvinDesign project will constitute unique microbial chassis for increased production of carbonaceous chemicals (alcohols, alkanes, lipids, sugars, pigments, terpenes, etc.). The knowledge obtained will offer great potential for coupling environmental protection, energy transition and bioeconomic growth.

Work on Chlamydomonas (P1) and Synechocystis (P2) was initiated as planned in the project.
At the beginning of the project, P1 finalized the MoClo (Modular Cloning) kit. In order to better understand the Calvin-Benson cycle we determined the atomic structure of all the enzymes of the Calvin-Benson cycle (11 enzymes) and of many regulators in Chlamydomonas. All structures have been deposited in the PDB. Some have been published (PRK, RPI, FBA, TRXf2, TRXh1, TRXz) and the other manuscripts are in preparation.
For metabolic engineering, we analyzed PRK KO mutants in both organisms (P1+P2). The mutants are heterotrophic and phototrophy can be restored by functional complementation by restoring PRK expression by transforming the mutant with a wild-type copy of the gene. We placed the PRK gene under the control of different promoters to obtain variable expression levels and thus determine the limitation imposed by PRK on the Calvin-Benson cycle. In Chlamydomonas (P1), our results indicate that PRK is not limiting (limitation between 50% and 80% of WT content) and overexpression (up to 3x) has no impact on carbon fixation measured via cell growth rate. The results are different in Synechocystis (P2). Mutant strains of cyanobacteria overproducing (constitutively or thermoregulated) various versions of Rubisco and PRK were constructed. These strains were combined with genetic circuits allowing the expression of different terpene synthases (C10 or C15). The farnesene yield (C15) is increased by a factor of 3.6 by overexpression of a PRK from Cyanothece PCC 7425 while expression of the rbc operon from Synechococcus PCC7002 allows a more limited increase (+85%). We also analyzed the regulators of the Calvin-Benson cycle enzymes. In Chlamydomonas (P1), the structural study of TRXs allowed us to understand that the surface charges constitute a molecular code determining the specificity of TRXs for their target. Naturally, TRXf2 activates Calvin cycle enzymes but not TRX x, y or h. We were able to demonstrate the importance of this code by modifying the selectivity of a TRX by substituting the charges of the surface residues and thus creating a TRXh1 able to activate the enzymes of the cycle (manuscript in preparation). In Synechocystis we have analyzed the role of the CP12 protein which plays a role of cycle inhibition. The absence of CP12 increases the production of limonene (C10) by a factor of 3 and of bisabolene (C15) by a factor of 2. We have also shown that photosynthesis is essential for the growth of the CP12 KO strain under mixotrophic conditions. We have also published several review articles on the synthetic biology of microalgae.

We were able to provide many new insights into the structure of Calvin-Benson cycle enzymes and their regulators. A remarkable result is the determination of the structure of all the enzymes of the cycle in Chlamydomonas, in particular PRK which had resisted structural analysis for more than 20 years. The structural data will allow us to analyze the supramolecular organization of the ring and to develop optimization strategies based on supramolecular engineering and regulatory engineering.
We obtained interesting results concerning the metabolic engineering of the Calvin-Benson cycle and in particular identified strategies allowing to significantly increase the production of carbonaceous molecules in Synechocystis. These strategies constitute interesting leads for the optimization of the photosynthetic production of molecules of industrial interest, in particular terpenes. We focused on the engineering of 3 enzymes of the cycle while the ambition was to analyze a larger number. The results will have to be completed by analyses on the other enzymes.
The CalvinDesign project has generated a lot of data on the enzymes of the Calvin-Benson cycle and their regulation. These data will be essential to design new optimization strategies. The metabolic engineering work has allowed to identify strategies in Synechocystis allowing to significantly increase the production of different terpenes of industrial interest.

1. Crozet P, Navarro FJ,..., Schroda M#, Smith AG#, Lemaire SD# (2018) Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology 7, 2074-2086. #Corresponding authors doi.org/10.1021/acssynbio.8b00251.
2. Lemaire SD, Tedesco D, Crozet P, Michelet L, Fermani S, Zaffagnini M, Henri J (2018) Crystal Structure of Chloroplastic Thioredoxin f2 from Chlamydomonas reinhardtii Reveals Distinct Surface Properties. Antioxidants 7, E171. doi.org/10.3390/antiox7120171.
3. Marchand CH, Fermani S, Rossi J, Gurrieri L, Tedesco D, Henri J, Sparla F, Trost P, Lemaire SD, Zaffagnini M. (2019) Structural and biochemical insights into the reactivity of thioredoxin h1 from Chlamydomonas reinhardtii. Antioxidants 8, E10. doi.org/10.3390/antiox8010010.
4. Gurrieri L, ..., Crozet P, Marchand CH, Henri J, Trost P, Lemaire SD, Sparla F, Fermani S. (2019) Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin-Benson cycle. Proc. Natl. Acad. Sci. USA. 116(16): 8048-8053. doi.org/10.1073/pnas.1820639116.
5. Vavitsas K, Crozet P, Hamborg Vinde M, Davies F, Lemaire SD, Vickers CE (2019) The synthetic biology toolkit for photosynthetic microorganisms. Plant Physiol. 181: 14-27. doi.org/10.1104/pp.19.00345
6. de Carpentier F, Le Peillet J, Boisset ND, Crozet P, Lemaire SD, Danon (2020) Blasticidin S deaminase: a new efficient selectable marker for Chlamydomonas reinhardtii. Front. Plant Sci. 11: 242. doi.org/10.3389/fpls.2020.00242.
7. Le Moigne T, Crozet P, Lemaire SD, Henri J (2020) High-Resolution Crystal Structure of Chloroplastic Ribose-5-Phosphate Isomerase from Chlamydomonas reinhardtii-An Enzyme Involved in the Photosynthetic Calvin-Benson Cycle (2020) Int J Mol Sci. 21(20):7787. doi.org/10.3390/ijms21207787
8. Le Moigne T, Gurrieri L, Crozet P, Marchand CH, Zaffagnini M, Sparla F, Lemaire SD, Henri J. (2021) Crystal structure of chloroplastic thioredoxin z defines a type-specific target recognition. Plant J. 107: 434-447. doi.org/10.1111/tpj.15300
9. Le Moigne T, Sarti E, Nourisson A, Zaffagnini M, Carbone A, Lemaire SD, Henri J. (2022) Crystal structure of chloroplast fructose-1,6-bisphosphate aldolase from the green alga Chlamydomonas reinhardtii. J Struct Biol. 214(3):107873. doi.org/10.1016/jsb.2022.107873
10. Cassier-Chauvat c, Blanc-Garin V, Chauvat F (2021) Genetic, Genomics, and Responses to Stresses in Cyanobacteria: Biotechnological Implications. Genes (Basel) 12(4):500. doi.org/10.3390/genes12040500.
11. Veaudor T, Blanc-Garin V, Chenebault C, Diaz-Santos E, Sassi JF, Cassier-Chauvat C, Chauvat F. (2020) Recent Advances in the Photoautotrophic Metabolism of Cyanobacteria: Biotechnological Implications. Life (Basel).10(5):71. doi.org/10.3390/life10050071

Irreversible depletion of traditional sources of fossil fuels coupled with accumulation of greenhouse gases produced by their combustion are an incentive to develop alternative forms of eco-responsible sources of reduced carbon for the production of fuels and chemicals needed by our society. Phototrophic microbes are regarded as promising organisms for the development of novel and highly innovative concepts based on their inherent ability to fix CO2, thereby producing various organic molecules via a sunlight-driven and sustainable process. Until today, only limited biotechnological work has been carried out for using green microalgae and cyanobacteria as production shuttles and green cell factories in biotechnology approaches. To allow these microorganisms to become efficient cell factories for commercially viable and sustainable biotechnological production of commodity chemicals, a major challenge is to improve their carbon-assimilating photoautotrophic metabolism. So far, metabolic engineering strategies have mainly focused on redirecting the assimilated carbon toward the production of specific chemicals (e.g. alcohols and lipids), whereas the challenging task of improving the photosynthetic process by optimizing carbon fixation has not been as aggressively pursued yet. This is the objective of the present CalvinDesign project.

Major energy losses during photosynthesis, that reduce the overall light conversion efficiency, can be directly attributed to kinetic bottlenecks within the CO2 fixing Calvin-Benson cycle (CBC). Many possibilities exist to improve the efficiency of the CBC, at least by changing the abundance of key enzymes. The aim of the CalvinDesign project is to combine systems biology (proteomics, metabolomics) and synthetic biology (genetic and metabolic engineering, pathway reconstitution, Design/Build/Test methodology) to develop highly efficient CO2 fixing strains of microalgae and cyanobacteria. This project focuses on the best-characterized models of photosynthetic microorganisms, the cyanobacterium Synechocystis PCC6803 and the eukaryotic green microalga Chlamydomonas reinhardtii, which are intensively used for improving the photoproduction of alcohols and lipids, respectively, and for the production of carbon-derived chemicals.

The groundbreaking nature of this project is to apply the principles and methods of synthetic biology to tackle the challenge of improving carbon-fixation in photosynthetic microorganisms. Pathway design will be based on our knowledge of bottlenecks limiting the CBC either already suggested (SBPase, Rubisco, TRX-dependent enzymes), or uncovered during this project through systems biology analyses (Test phase) or in vitro pathway reconstitution. Indeed, we will assemble a synthetic acellular CBC allowing fast testing for more efficient combinations of enzymes through metabolic control analyses. The major bottlenecks will be unlocked by introducing the new design through strain metabolic engineering using powerful molecular tools and DNA parts. The strains will be characterized under varying conditions including industrially relevant PBR-like conditions to generate new knowledge for a new cycle of analysis along the classical SynBio Cycle “Design, Build, Test & Learn”. The aim of the CalvinDesign project is to initiate the Synbio cycle by performing at least three iterations, generating new knowledge on the CBC as well as new strains. These strains will be tested to validate their overall gain of photosynthetic efficiency and CO2 fixation capacity. The highly efficient CO2 fixing strains generated by the CalvinDesign project will constitute unique microbial chassis for enhanced production of any carbon-based chemicals (alcohols, alkanes, lipids, sugars, pigments, terpenes, etc). These highly efficient synthetic strains of major industrial and commercial interest will offer great potential to couple environmental protection, energy transition and bioeconomic growth.