Calredoxin regulates the chloroplast NADPH-dependent thioredoxin reductase in Chlamydomonas reinhardtii.

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas (Chlamydomonas reinhardtii) with a largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild-type cells compared with a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnect redox control with active photosynthetic electron transport and metabolism, as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning, as well as for efficient photosynthesis.

A PSII photosynthetic control is activated in anoxic cultures of green algae following illumination.

Photosynthetic hydrogen production from microalgae is considered to have potential as a renewable energy source. Yet, the process has two main limitations holding it back from scaling up; (i) electron loss to competing processes, mainly carbon fixation and (ii) sensitivity to O2 which diminishes the expression and the activity of the hydrogenase enzyme catalyzing H2 production. Here we report a third, hitherto unknown challenge: We found that under anoxia, a slow-down switch is activated in photosystem II (PSII), diminishing the maximal photosynthetic productivity by three-fold. Using purified PSII and applying in vivo spectroscopic and mass spectrometric techniques on Chlamydomonas reinhardtii cultures, we show that this switch is activated under anoxia, within 10 s of illumination. Furthermore, we show that the recovery to the initial rate takes place following 15 min of dark anoxia, and propose a mechanism in which, modulation in electron transfer at the acceptor site of PSII diminishes its output. Such insights into the mechanism broaden our understanding of anoxic photosynthesis and its regulation in green algae and inspire new strategies to improve bio-energy yields.

A two-phase protocol for ambient hydrogen production using Chlamydomonas reinhardtii.

H2 production from green-microalgae, for energy purposes, is the ultimate goal of large-scale production. Here, we present a two-phase protocol for hydrogen production assay under ambient conditions using Chlamydomonas reinhardtii, which eliminates steps used previously, including centrifugation and resuspension with sulfur-deprived media. We detail steps for Chlamydomonas reinhardtii culture, acetate supply replenishment, anaerobic induction, and H2 quantification. This protocol enables large-scale experiments in an easy and cost-effective method while maintaining cells vital, crucial factors for transition to industrial scales. For complete details on the use and execution of this protocol, please refer to Elman et al. (2022).

Advances and challenges in photosynthetic hydrogen production.

The vision to replace coal with hydrogen goes back to Jules Verne in 1874. However, sustainable hydrogen production remains challenging. The most elegant approach is to utilize photosynthesis for water splitting and to subsequently save solar energy as hydrogen. Cyanobacteria and green algae are unicellular photosynthetic organisms that contain hydrogenases and thereby possess the enzymatic equipment for photosynthetic hydrogen production. These features of cyanobacteria and algae have inspired artificial and semi-artificial in vitro techniques, that connect photoexcited materials or enzymes with hydrogenases or mimics of these for hydrogen production. These in vitro methods have on their part been models for the fusion of cyanobacterial and algal hydrogenases to photosynthetic photosystem I (PSI) in vivo, which recently succeeded as proofs of principle.

Photosystem I light-harvesting proteins regulate photosynthetic electron transfer and hydrogen production.

Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H2) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H2 production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H2 photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H2 photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H2 photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.

Phylloquinone is the principal Mehler reaction site within photosystem I in high light.

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy, and a part of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2•-) via the Mehler reaction, which occurs principally within photosystem I (PSI). For decades, O2 reduction within PSI was assumed to take place solely in the distal iron-sulfur clusters rather than within the two asymmetrical cofactor branches. Here, we demonstrate that under high irradiance, O2 photoreduction by PSI primarily takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O2 photoreduction rate constant in fully mature wild-type PSI from Chlamydomonas reinhardtii, complexes lacking iron-sulfur clusters, and a mutant PSI, in which phyllosemiquinone at the A-branch has a significantly longer lifetime. We suggest that the Mehler reaction at the phylloquinone site serves as a release valve under conditions where both the iron-sulfur clusters of PSI and the mobile ferredoxin pool are highly reduced.

Protection of Oxygen-Sensitive Enzymes by Peptide Hydrogel.

Molecular oxygen (O2) is a highly reactive oxidizing agent and is harmful to many biological and industrial systems. Although O2 often interacts via metals or reducing agents, a binding mechanism involving an organic supramolecular structure has not been described to date. In this work, the prominent dipeptide hydrogelator fluorenylmethyloxycarbonyl-diphenylalanine is shown to encage O2 and significantly limit its diffusion and penetration through the hydrogel. Molecular dynamics simulations suggested that the O2 binding mechanism is governed by pockets formed between the aromatic rings in the supramolecular structure of the gel, which bind O2 through hydrophobic interactions. This phenomenon is harnessed to maintain the activity of the O2-hypersensitive enzyme [FeFe]-hydrogenase, which holds promising potential for utilizing hydrogen gas for sustainable energy applications. Hydrogenase encapsulation within the gel allows hydrogen production following exposure to ambient O2. This phenomenon may lead to utilization of this low molecular weight gelator in a wide range of O2-sensitive applications.

Bi-directional electron transfer between H2 and NADPH mitigates light fluctuation responses in green algae.

The metabolism of green algae has been the focus of much research over the last century. These photosynthetic organisms can thrive under various conditions and adapt quickly to changing environments by concomitant usage of several metabolic apparatuses. The main electron coordinator in their chloroplasts, nicotinamide adenine dinucleotide phosphate (NADPH), participates in many enzymatic activities and is also responsible for inter-organellar communication. Under anaerobic conditions, green algae also accumulate molecular hydrogen (H2), a promising alternative for fossil fuels. However, to scale-up its accumulation, a firm understanding of its integration in the photosynthetic apparatus is still required. While it is generally accepted that NADPH metabolism correlates to H2 accumulation, the mechanism of this collaboration is still vague and relies on indirect measurements. Here, we investigated this connection in Chlamydomonas reinhardtii using simultaneous measurements of both dissolved gases concentration, NADPH fluorescence and electrochromic shifts at 520-546 nm. Our results indicate that energy transfer between H2 and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations. At light onset, NADPH consumption initially eventuates in H2 evolution, which initiates the photosynthetic electron flow. Later on, as illumination continues the majority of NADPH is diverted to the Calvin-Benson-Bassham cycle. Dark onset triggers re-assimilation of H2, which produces NADPH and so, enables initiation of dark fermentative metabolism.

Juggling Lightning: How Chlorella ohadii handles extreme energy inputs without damage.

The green alga Chlorella ohadii was isolated from a desert biological soil crust, one of the harshest environments on Earth. When grown under optimal laboratory settings it shows the fastest growth rate ever reported for a photosynthetic eukaryote and a complete resistance to photodamage even under unnaturally high light intensities. Here we examined the energy distribution along the photosynthetic pathway under four light and carbon regimes. This was performed using various methodologies such as membrane inlet mass spectrometer with stable O2 isotopes, variable fluorescence, electrochromic shift and fluorescence assessment of NADPH level, as well as the use of specific inhibitors. We show that the preceding illumination and CO2 level during growth strongly affect the energy dissipation strategies employed by the cell. For example, plastid terminal oxidase (PTOX) plays an important role in energy dissipation, particularly in high light- and low-CO2-grown cells. Of particular note is the reliance on PSII cyclic electron flow as an effective and flexible dissipation mechanism in all conditions tested. The energy management observed here may be unique to C. ohadii, as it is the only known organism to cope with such conditions. However, the strategies demonstrated may provide an insight into the processes necessary for photosynthesis under high-light conditions.

Re-routing photosynthetic energy for continuous hydrogen production in vivo.

BACKGROUND: Hydrogen is considered a promising energy vector that can be produced from sustainable resources such as sunlight and water. In green algae, such as Chlamydomonas reinhardtii, photoproduction of hydrogen is catalyzed by the enzyme [FeFe]-hydrogenase (HydA). Although highly efficient, this process is transitory and thought to serve as a release valve for excess reducing power. Up to date, prolonged production of hydrogen was achieved by the deprivation of either nutrients or light, thus, hindering the full potential of photosynthetic hydrogen production. Previously we showed that the enzyme superoxide dismutase (SOD) can enhance HydA activity in vitro, specifically when tied together to a fusion protein. RESULTS: In this work, we explored the in vivo hydrogen production phenotype of HydA-SOD fusion. We found a sustained hydrogen production, which is dependent on linear electron flow, although other pathways feed it as well. In addition, other characteristics such as slower growth and oxygen production were also observed in Hyd-SOD-expressing algae. CONCLUSIONS: The Hyd-SOD fusion manages to outcompete the Calvin-Benson cycle, allowing sustained hydrogen production for up to 14 days in non-limiting conditions.

Solving the Riddle of the Evolution of Shine-Dalgarno Based Translation in Chloroplasts.

Chloroplasts originated from an ancient cyanobacterium and still harbor a bacterial-like genome. However, the centrality of Shine-Dalgarno ribosome binding, which predominantly regulates proteobacterial translation initiation, is significantly decreased in chloroplasts. As plastid ribosomal RNA anti-Shine-Dalgarno elements are similar to their bacterial counterparts, these sites alone cannot explain this decline. By computational simulation we show that upstream point mutations modulate the local structure of ribosomal RNA in chloroplasts, creating significantly tighter structures around the anti-Shine-Dalgarno locus, which in-turn reduce the probability of ribosome binding. To validate our model, we expressed two reporter genes (mCherry, hydrogenase) harboring a Shine-Dalgarno motif in the Chlamydomonas reinhardtii chloroplast. Coexpressing them with a 16S ribosomal RNA, modified according to our model, significantly enhances mCherry and hydrogenase expression compared with coexpression with an endogenous 16S gene.

Binding of ferredoxin NADP+ oxidoreductase (FNR) to plant photosystem I.

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 µM and ∆H of -20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ∆CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Å2. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.

Paradigm Shift in Algal H2 Production: Bypassing Competitive Processes.

Hydrogen is a promising energy carrier, but producing it sustainably remains a challenge. Green algae can produce hydrogen photosynthetically using their efficient but oxygen-sensitive hydrogenases. Recent strategies aiming to bypass competing processes provide a promising route for scaling up algal hydrogen production.

Prediction and large-scale analysis of primary operons in plastids reveals unique genetic features in the evolution of chloroplasts.

While bacterial operons have been thoroughly studied, few analyses of chloroplast operons exist, limiting the ability to study fundamental elements of these structures and utilize them for synthetic biology. Here, we describe the creation of a plastome-specific operon database (link provided below) achieved by combining experimental tools and predictive modeling. Using a Reverse-Transcription-PCR based method and published data, we determined the transcription-state of 213 gene pairs from four plastomes of evolutionary distinct organisms. By analyzing sequence-based features computed for our dataset, we were able to highlight fundamental characteristics differentiating between operon pairs and non-operon pairs. These include an interesting tendency toward maintaining similar messenger RNA-folding profiles in operon gene pairs, a feature that failed to yield any informative separation in cyanobacteria, suggesting that it catches unique traits of operon gene expression, which have evolved post-endosymbiosis. Subsequently, we used this feature set to train a random-forest classifier for operon prediction. As our results demonstrate the ability of our predictor to obtain accurate (84%) and robust predictions on unlabeled datasets, we proceeded to building operon maps for 2018 sequenced plastids. Our database may now present new opportunities for promoting metabolic engineering and synthetic biology in chloroplasts.

ChimeraUGEM: unsupervised gene expression modeling in any given organism.

MOTIVATION: Regulation of the amount of protein that is synthesized from genes has proved to be a serious challenge in terms of analysis and prediction, and in terms of engineering and optimization, due to the large diversity in expression machinery across species. RESULTS: To address this challenge, we developed a methodology and a software tool (ChimeraUGEM) for predicting gene expression as well as adapting the coding sequence of a target gene to any host organism. We demonstrate these methods by predicting protein levels in seven organisms, in seven human tissues, and by increasing in vivo the expression of a synthetic gene up to 26-fold in the single-cell green alga Chlamydomonas reinhardtii. The underlying model is designed to capture sequence patterns and regulatory signals with minimal prior knowledge on the host organism and can be applied to a multitude of species and applications. AVAILABILITY AND IMPLEMENTATION: Source code (MATLAB, C) and binaries are freely available for download for non-commercial use at http://www.cs.tau.ac.il/~tamirtul/ChimeraUGEM/, and supported on macOS, Linux and Windows. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Image-Processing Software for High-Throughput Quantification of Colony Luminescence.

Many microbiological assays include colonies that produce a luminescent or fluorescent (here generalized as "luminescent") signal, often in the form of luminescent halos around the colonies. These signals are used as reporters for a trait of interest; therefore, exact measurements of the luminescence are often desired. However, there is currently a lack of high-throughput methods for analyzing these assays, as common automatic image analysis tools are unsuitable for identifying these halos in the presence of the inherent biological noise. In this work, we have developed CFQuant-automatic, high-throughput software for the analysis of images from colony luminescence assays. CFQuant overcomes the problems of automatic identification by relying on the luminescence halo's expected shape and provides measurements of several features of the colonies and halos. We examined the performance of CFQuant using one such colony luminescence assay, where we achieved a high correlation (R = 0.85) between the measurements of CFQuant and known protein expression levels. This demonstrates CFQuant's potential as a fast and reliable tool for analysis of colony luminescence assays.IMPORTANCE Luminescent markers are widely used as reporters for various biologically interesting traits. In colony luminescence assays, the levels of luminescence around each colony can be used to compare the levels of traits of interest for different strains, treatments, etc., using quantitative measurements of the luminescence. However, automatic methods of obtaining this data are underdeveloped, making this a laborious manual process, especially in analyzing large numbers of colonies. The significance of this work is in developing an automatic, high-throughput tool for quantitative analysis of colony luminescence assays, which will allow fast collection of qualitative data from these assays and thus increase their overall usability.

The Integration of Multiple Nuclear-Encoded Transgenes in the Green Alga Chlamydomonas reinhardtii Results in Higher Transcription Levels.

The integration of genes into the nuclear genome of Chlamydomonas reinhardtii is mediated by Non-Homologous-End-Joining, thus resulting in unpredicted insertion locations. This phenomenon defines 'the position-effect', which is used to explain the variation of expression levels between different clones transformed with the same DNA fragment. Likewise, nuclear transgenes often undergo epigenetic silencing that reduces their expression; hence, nuclear transformations require high-throughput screening methods to isolate clones that express the foreign gene at a desirable level. Here, we show that the number of integration sites of heterologous genes results in higher mRNA levels. By transforming both a synthetic ferredoxin-hydrogenase fusion enzyme and a Gaussia-Luciferase reporter protein, we were able to obtain 33 positive clones that exhibit a wide range of synthetic expression. We then performed a droplet-digital polymerase-chain-reaction for these lines to measure their transgene DNA copy-number and mRNA levels. Surprisingly, most clones contain two integration sites of the synthetic gene (45.5%), whilst 33.3% contain one, 18.1% include three and 3.1% encompass four. Remarkably, we observed a positive correlation between the raw DNA copy-number values to the mRNA levels, suggesting a general effect of which transcription of transgenes is partially modulated by their number of copies in the genome. However, our data indicate that only clones harboring at least three copies of the target amplicon show a significant increment in mRNA levels of the reporter transgene. Lastly, we measured protein activity for each of the reporter genes to elucidate the effect of copy-number variation on heterologous expression.

Green Algal Hydrogenase Activity Is Outcompeted by Carbon Fixation before Inactivation by Oxygen Takes Place.

Photoproduction of hydrogen by green algae is considered a transitory release valve of excess reducing power and a potential carbon-free source of sustainable energy. It is generally accepted that the transitory production of hydrogen is governed by fast inactivation of hydrogenase by oxygen. However, our data suggest that photosynthetic electron loss to competing processes, mainly carbon fixation, stops hydrogen production, supports hydrogen uptake, and precedes the inevitable inactivation by oxygen. Here, we show that when transitioning from dark anaerobiosis to light, hydrogen production ceases within 2 min, regardless of the presence of oxygen. Simultaneous monitoring of the active hydrogenase pool size shows that it remains entirely intact up to 4 min after illumination and is inactivated only later. Thus, our data reveal a window of 4 min in which the hydrogenase pool is not being degraded by oxygen. Furthermore, we show that electron loss, prominently to carbon fixation, outcompetes hydrogen production and leads to hydrogen uptake. Indeed, supplying additional reducing power to hydrogenase at the cessation point regenerates the accumulation of hydrogen. Our results imply the fast cessation of hydrogen production is governed by electron loss rather than oxygen inactivation, which takes place minutes later.