In vivo and in silico production of mutants affecting photosynthetic pathways: experimental development and modeling of metabolic pathways that influence photosynthetic yields downstream of photosynthetic electron transfer in green algae. – ChloroPaths

Using a mutant strain devoid of RuBisCO, deltarbcL, for genetic transformation, we propose to screen for new mutants (double mutants) using a video imaging system of chlorophyll fluorescence. The deltarbcL mutant uses alternative metabolic pathways as exhaust valves for the excess electrons for accumulated ATP and NADPH not used by the Calvin cycle. This is a way to specifically target bypass metabolic reactions (generation of xxx deltarbcL double mutants). By crossing these double mutants with a wildtype strain we can place the mutant with disruption of this electron valve into a CO2 fixing context, where the obstruction of alternative pathways can be studied in relation to photosynthetic productivity. To analyse our mutants we will depend on many techniques that we have optimised in our laboratory (PCR-based techniqques for molecular analysis, protein targetting, protein accumulation, CO2 capture by PBR, growth tests on inorganic/organic C source, photosynthetic parameters by spectroscopic analysis), and others that will be outsourced (metabolite quantification). Metabolic flux analysis and constraint-based modeling techniques can be used to understand such a complex regulation of photosynthesis, respiration and carbon metabolism. Such
a systemic approach has been demonstrated to be well adapted for assessing those interactions for a
wildtype strain under light-limited growth conditions. To follow them in mutant backgrounds under
controlled conditions will then require expanding existing model representations and define new specific
constraints for model analysis to assess potential metabolic responses and identify where pools of
metabolites will accumulate and be shuttled. Databases are constructed and open source software programs chosen to feed the reactions and parameters. Specific PBRs are designed and tested in the modelling context for mutant characterisation where absorbed light can be measured and modelled more accurately.

The mutant library screening has been successfully completed : sixteen mutants have been identified from screening ten thousand insertional mutants with strong phenotypes deviating from the background and of these, we have been able to identify, using PCR based molecular techniques, the majority of the insertion sites.

The insertion sites of ten of these mutants have been identified in genes that broadly fall into two categories: 1. Transporters, enzymes and regulators of primary chloroplast/cytosolic metabolism (TPT5, MATE type efflux pump, pantothenate kinase, DET1, Soluble Starch Synthase 4, SRR16, 2OGFeDO-motif protein) ; 2. Conserved protein effectors that directly regulate the components of the photosynthetic chain (APE1, CGL11, HSCB-like FeS chaperone).

TPT5, APE1and CGL11 have been complemented by nuclear complementation experiments proving that the insertion site and interruption of these genes is the cause of the mutant phenotype. Linkage analysis is being completed for the other mutants with an insertion site identified, with complementation experiments planned until the end of the year.

The Photobioreactor set up using on line monitoring of CO2 uptake rates has proven a robust means to monitor photosynthesis and biomass production. We used published mutants to understand if absence of regulatory pathways affects photosynthetic productivity. We could show that while pgrl1 and npq4 are compensatory mechanisms for the adaptation to high light, pgrl1npq4 was unable to adapt and showed strong PSI photoinhibition.

A metabolic network of the wild-type strain of Chlamydomonas has been reconstructed from available database and current literature. It includes subcellular compartmentalization in its main components, namely chloroplast, mitochondrion and cytosol. The implementation of a metabolic model was initiated to calculate metabolic flux distributions of C. reinhardtii for various environmental and/or genetic conditions.

We have preliminary but convincing data that we have identified proteins that act as metabolic shuttles between the chloroplast and the mitochondria. Phenotypic, molecular and bioinformatic data based on homology searches, strongly suggest that we have identified a triose-phosphate transporter and either an organic acid or acetate transporter. Knock out of both of these proteins results in a strong flouresecence phenotype that proves its relation to photosynthetic electron transfer. We believe that we identified both the elusive triose-phosphate translocator and the malate valve in this screen.

Furthermore, work by the modelling team is revealing that these metabolic interactions between the chloroplast and the mitochondria could favorise biomass production under steady state photo-autorophic conditions and they are currently working to understand the physiological bottlenecks that limit these interactions to feed our studies towards future strain improvements.

From a different angle the mutants that have been isolated in this screen reveal to us that the deltarbcL mutant context that we have used as the genetic background responds to absence of Rubisco as would any cell that is placed under strong PSI acceptor side limitation which is the trigger for acclimation responses, for instance when we observe chlorophyll flourescence profiles during a dark light transition . A number of mutants we have identified in the screen appear to be previously unknown components of this response, including the lumenal green cut protein CGL11 and the PSII associated protein APE1 that we can now place as regulators alongside PGR5. This acclimation response termed 'photosynthetic control' is able to link electron transfer, proton gradient, ATP production and NPQ thus is central to acclimation. This opens up the perspectives in the field of
photosynthetic control and their manipulation in both green algae and plants to increase biomass yields.

1. Chaux F, Peltier G, Johnson X. A security network in PSI photoprotection: regulation of photosynthetic control, NPQ and O2 photoreduction by cyclic electron flow. Front Plant Sci. 2015 Oct 15;6:875. dx.doi.org/10.3389/fpls.2015.00875
2. Steinbeck J, Nikolova D, Weingarten R, Johnson X, Richaud P, Peltier G, Hermann M, Magneschi L, Hippler M. Deletion of Proton Gradient Regulation 5 (PGR5) and PGR5-Like 1 (PGRL1) proteins promote sustainable light-driven hydrogen production in Chlamydomonas reinhardtii due to increased PSII activity under sulfur deprivation. Front Plant Sci. 2015 Oct 27;6:892.
dx.doi.org/10.3389/fpls.2015.00892
3. Chaux F, Johnson X, Cuiné S, Auroy P, Beyly-Adriano A, Te I, Lucas PL, Peltier G. PGRL1 and LHCSR3 Can Play Compensatory Roles in the Control of Photosynthetic Electron Flow During High Light Acclimation to Sustain Carbon Dioxide Uptake and Avoid Photosystem I Photoinhibition. Under revision for Mol. Plant.
4. Dumas L, Chazaux M, Peltier G, Johnson X, Alric J. Cytochrome b6f function and localization, phosphorylation state of thylakoid membrane proteins and consequences on cyclic electron flow. Photosynth Res. 2016 Aug 17.
PMID: 27534565

1. Pawel Brzezowski, invited speaker at 17th International conference on the cell and molecular biology of Chlamydomonas, Kyoto Japan, 20 June 2016.
2. Pawel Brzezowski, poster presentation at 17th International conference on the cell and molecular biology of Chlamydomonas, Kyoto Japan, 20 June 2016.
3. Xenie Johnson, invited speaker at PS electron transfer Satellite conference, Arnhem, August, 2016
4. Marie Chazaux, poster presentation at 17th International conference on Photosynthesis, Maastricht, August, 2016.

1. Xenie Johnson invited speaker at Journees de la Photosynthese: Societe Francaise de la Photosynthese, Paris May 2015
2/3. Pawel Brzezowski and Marie Chazaux,, poster presentation at Journees de la Photosynthese: Societe Francaise de la Photosynthese, Paris May 2016

Microalgae are a promising renewable resource for the production of molecules with high added value. Until present they have been little used because crop plants have been the simpler choice due to their cultivation by traditional farming methods. Technological advances in process engineering now allow for the cultivation of microalgae from small to large-scale photo-bioreactors. While the output from heterotrophic cultures (microalgae fed with sugar) can achieve substantial yields, photoautotrophic production (with sunlight as the only energy intake), a strictly renewable culture condition, gives satisfactory but limited returns. We believe it is possible to increase the production yields of algal biomass by a better understanding of the limitations and metabolic energy constraints of these organisms. A future challenge is to domesticate microalgae for monoculture as we already have done for cereal crops in the past.
This research project offers a combination of genetic, metabolic and physiological studies to understand the inherent limitations in photosynthetic production of algal biomass, in an attempt to pinpoint and overcome these limitations. The Microalgae used for this study, Chlamydomonas reinhardtii is a model representative of the green lineage. It is well suited to both basic and applied research: a range of molecular and genetic characterization techniques are amenable to this species and photo- bioreactors have been designed solely for their large-scale production.
The principle of our scientific approach is very simple: it is based on the observation that the molecular mechanisms of photosynthesis consist of several interconnected pathways. The major pathway leads to the fixation of CO2 and thus growth or “increase in biomass’. Other, so-called alternative pathways, are involved in regulatory mechanisms, some of which are of crucial importance in natural conditions, but are required for energy dissipation and therefore represent a loss to potential biomass. In controlled culture conditions (average illumination and unlimited CO2), it is likely that the regulation of alternative paths becomes superfluous and it is possible to block certain to redirect the flow of energy to CO2 fixation without affecting the viability of the biological system. In doing this we will be contributing to our objective of domestication.
This project details an original method we have created to achieve these objectives. It has never been attempted before and is largely based on accumulated observations from working with the multitude of single and double photosynthetic mutants available in Chlamydmonas reinhardtii. The work detailed in ChloroPaths concentrates primarily on a mutant strain where the major pathway of CO2 fixation is blocked (mutant without Rubisco). In this strain, alternative routes thus become, by necessity, the major channels for dissipating photosynthetic electrons. Thus the mutant lacking Rubisco represents a good recipient strain to hunt for and knock out alternative pathways by the generation of double mutants. After a series of steps to characterize the double mutants in depth, described in detail in the project, we will then generate single mutants by placing the alternative pathway mutations into a wildtype genetic background. These single mutant strains will be tools to understand metabolite shuttling in photosynthetic species and will be tested for increased productivity.
All this work will be placed into the context of a functional model for growth and productivity that will allow us to understand the interaction between the metabolic pathways of carbon compounds. It will allow us to better describe the phenotype of mutants isolated by our genetic approach and in the future, to plan the most optimal synthetic biology projects for the rational improvement of microalgae.