Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology

Chlamydomonas reinhardtii PsbS protein is functional and accumulates rapidly and transiently under high light.

International audienc

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR

International audienc

Alternative photosynthesis pathways drive the algal CO2-concentrating mechanism

International audienceGlobal photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption1. The high efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, which enhances CO2 fixation2. Although many cellular components involved in the transport and sequestration of inorganic carbon have been identified3,4, how microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains unknown4–6. Here we show that in the green alga Chlamydomonas reinhardtii, the combined action of cyclic electron flow and O2 photoreduction—which depend on PGRL1 and flavodiiron proteins, respectively—generate a low luminal pH that is essential for CCM function. We suggest that luminal protons are used downstream of thylakoid bestrophin-like transporters, probably for the conversion of bicarbonate to CO2. We further establish that an electron flow from chloroplast to mitochondria contributes to energizing non-thylakoid inorganic carbon transporters, probably by supplying ATP. We propose an integrated view of the network supplying energy to the CCM, and describe how algal cells distribute energy from photosynthesis to power different CCM processes. These results suggest a route for the transfer of a functional algal CCM to plants to improve crop productivity

Cyclic and pseudo-cyclic electron pathways play antagonistic roles during nitrogen deficiency in Chlamydomonas reinhardtii

Nitrogen (N) deficiency is a frequently encountered situation that constrains global biomass productivity. In response to N deficiency, cell division stops and photosynthetic electron transfer are downregulated, while carbon storage is enhanced. However, the molecular mechanism downregulating photosynthesis during N deficiency and its relationship with carbon storage are not fully understood. The Proton Gradient Regulator-like 1 (PGRL1)-involved in cyclic electron flow (CEF) and Flavodiiron proteins involved in pseudo-(CEF) are major players in the acclimation of photosynthesis. To determine the role of PGRL1 or FLV in photosynthesis under N deficiency, we measured photosynthetic electron transfer, oxygen gas exchange and carbon storage in the knockout of Chlamydomonas pgrl1 and flvB mutants. Under N deficiency, pgrl1 maintains higher net photosynthesis and O 2 photoreduction rates, while flvB shows similar responses compared to control strains. The amount of cytochrome b 6 f was maintained at a higher level in pgrl1 . The photosynthetic activity of pgrl1 flvB double mutants decreases in response to N deficiency similar to the control strains. Furthermore, the triacylglycerol content of pgrl1 was twice higher than the controls under N deficiency. Taken together, our results suggest that in the absence of PGRL1, FLV-mediated O 2 photoreduction through PCEF maintains net photosynthesis at a high level, resulting in increased triacylglycerol biosynthesis. This study reveals that PGRL1 and FLV play antagonistic roles during N deficiency. It further illustrates how nutrient status can affect the regulation of photosynthetic energy production in relation to carbon storage and provides new strategies for improving lipid productivity in algae. Significance statement Nitrogen (N) deficiency, an often-encountered phenomenon in nature, triggers growth arrest and massive lipid accumulation in microalgae. The downregulation of photosynthesis is necessary to ensure cell viability. We demonstrate that a well-conserved protein in chlorophytes, the Proton Gradient Regulator-like 1 (PGRL1) is a key (down) regulator of photosynthesis. In its absence, cells exhibited sustained photosynthesis and over-accumulated lipids thanks to the Flavodiiron (FLV) protein. We propose that both PGRL1 and FLV, by having antagonistic roles in N deficiency, manage the redox landscape, carbon storage and biomass production. Our work revolves around the current paradigm of photosynthesis regulation during N deficiency and provides a new framework for improving lipid accumulation in microalgae for biotechnological purposes

Alternative electron pathways of photosynthesis drive the algal CO 2 concentrating mechanism

Abstract Global photosynthesis consumes ten times more CO 2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption 1 . The great efficiency of algal photosynthesis relies on a mechanism concentrating CO 2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, thus enhancing CO 2 fixation 2 . While many cellular components involved in the transport and sequestration of inorganic carbon (C i ) have been uncovered 3,4 , the way microalgae supply energy to concentrate CO 2 against a thermodynamic gradient remains elusive 4-6 . Here, by monitoring dissolved CO 2 consumption, unidirectional O 2 exchange and the chlorophyll fluorescence parameter NPQ in the green alga Chlamydomonas , we show that the complementary effects of cyclic electron flow and O 2 photoreduction, respectively mediated by PGRL1 and flavodiiron proteins, generate the proton motive force ( pmf ) required by C i transport across thylakoid membranes. We demonstrate that the trans-thylakoid pmf is used by bestrophin-like C i transporters and further establish that a chloroplast-to-mitochondria electron flow contributes to energize non-thylakoid C i transporters, most likely by supplying ATP. We propose an integrated view of the CCM energy supply network, describing how algal cells distribute photosynthesis energy to power different C i transporters, thus paving the way to the transfer of a functional algal CCM in plants towards improving crop productivity. One sentence summary Photosynthetic alternative electron flows and mitochondrial respiration drive the algal CO 2 concentrating mechanis

Flavodiiron Proteins Promote Fast and Transient O 2 Photoreduction in Chlamydomonas

International audienceDuring oxygenic photosynthesis, the reducing power generated by light energy conversion is mainly used to reduce carbon dioxide. In bacteria and archae, flavodiiron (Flv) proteins catalyze O 2 or NO reduction, thus protecting cells against oxidative or nitrosative stress. These proteins are found in cyanobacteria, mosses, and microalgae, but have been lost in angiosperms. Here, we used chlorophyll fluorescence and oxygen exchange measurement using [ 18 O]-labeled O 2 and a membrane inlet mass spectrometer to characterize Chlamydomonas reinhardtii flvB insertion mutants devoid of both FlvB and FlvA proteins. We show that Flv proteins are involved in a photo-dependent electron flow to oxygen, which drives most of the photosynthetic electron flow during the induction of photosynthesis. As a consequence, the chlorophyll fluorescence patterns are strongly affected in flvB mutants during a light transient, showing a lower PSII operating yield and a slower nonphotochemical quenching induction. Photoautotrophic growth of flvB mutants was indistinguishable from the wild type under constant light, but severely impaired under fluctuating light due to PSI photo damage. Remarkably, net photosynthesis of flv mutants was higher than in the wild type during the initial hour of a fluctuating light regime, but this advantage vanished under long-term exposure, and turned into PSI photo damage, thus explaining the marked growth retardation observed in these conditions. We conclude that the C. reinhardtii Flv participates in a Mehler-like reduction of O 2 , which drives a large part of the photosynthetic electron flow during a light transient and is thus critical for growth under fluctuating light regimes