Location of the photosynthetic carbon metabolism in microcompartments and separated phases in microalgal cells.

Carbon acquisition, assimilation and storage in eukaryotic microalgae and cyanobacteria occur in multiple compartments that have been characterised by the location of the enzymes involved in these functions. These compartments can be delimited by bilayer membranes, such as the chloroplast, the lumen, the peroxisome, the mitochondria or monolayer membranes, such as lipid droplets or plastoglobules. They can also originate from liquid-liquid phase separation such as the pyrenoid. Multiple exchanges exist between the intracellular microcompartments, and these are reviewed for the CO2 concentration mechanism, the Calvin-Benson-Bassham cycle, the lipid metabolism and the cellular energetic balance. Progress in microscopy and spectroscopic methods opens new perspectives to characterise the molecular consequences of the location of the proteins involved, including intrinsically disordered proteins.

Assembling Multiple Fragments: The Gibson Assembly.

The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.

Concentration-Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering.

Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana.

BACKGROUND: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. METHODS: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. RESULTS: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. CONCLUSIONS: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. Video Abstract.

ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum.

Chloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta- and tetraphosphate ((p)ppGpp). In the model flowering plant Arabidopsis, ppGpp acts as a potent regulator of plastid gene expression and influences photosynthesis, plant growth and development. However, little is known about ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here, we studied the function of ppGpp in the diatom Phaeodactylum tricornutum using transgenic lines containing an inducible system for ppGpp accumulation. We used these lines to investigate the effects of ppGpp on growth, photosynthesis, lipid metabolism and protein expression. We demonstrate that ppGpp accumulation reduces photosynthetic capacity and promotes a quiescent-like state with reduced proliferation and ageing. Strikingly, using nontargeted proteomics, we discovered that ppGpp accumulation also leads to the coordinated upregulation of a protein protection response in multiple cellular compartments. Our findings highlight the importance of ppGpp as a fundamental regulator of chloroplast function across different domains of life, and lead to new questions about the molecular mechanisms and roles of (p)ppGpp signalling in photosynthetic eukaryotes.

Overproduction of the Flv3B flavodiiron, enhances the photobiological hydrogen production by the nitrogen-fixing cyanobacterium Nostoc PCC 7120.

BACKGROUND: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H2) is a promising alternative for renewable, clean-energy production. However, the most recent, related studies point out that much improvement is needed for sustainable cyanobacterial-based H2 production to become economically viable. In this study, we investigated the impact of induced O2-consumption on H2 photoproduction yields in the heterocyte-forming, N2-fixing cyanobacterium Nostoc PCC7120. RESULTS: The flv3B gene, encoding a flavodiiron protein naturally expressed in Nostoc heterocytes, was overexpressed. Under aerobic and phototrophic growth conditions, the recombinant strain displayed a significantly higher H2 production than the wild type. Nitrogenase activity assays indicated that flv3B overexpression did not enhance the nitrogen fixation rates. Interestingly, the transcription of the hox genes, encoding the NiFe Hox hydrogenase, was significantly elevated, as shown by the quantitative RT-PCR analyses. CONCLUSION: We conclude that the overproduced Flv3B protein might have enhanced O2-consumption, thus creating conditions inducing hox genes and facilitating H2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H2 production.

Targeting TOR signaling for enhanced lipid productivity in algae.

Microalgae can produce large quantities of triacylglycerols (TAGs) and other neutral lipids that are suitable for making biofuels and as feedstocks for green chemistry. However, TAGs accumulate under stress conditions that also stop growth, leading to a trade-off between biomass production and TAG yield. Recently, in the model marine diatom Phaeodactylum tricornutum it was shown that inhibition of the target of rapamycin (TOR) kinase boosts lipid productivity by promoting TAG production without stopping growth. We believe that basic knowledge in this emerging field is required to develop innovative strategies to improve neutral lipid accumulation in oleaginous microalgae. In this minireview, we discuss current research on the TOR signaling pathway with a focus on its control on lipid homeostasis. We first provide an overview of the well characterized roles of TOR in mammalian lipogenesis, adipogenesis and lipolysis. We then present evidence of a role for TOR in controlling TAG accumulation in microalgae, and draw parallels between the situation in animals, plants and microalgae to propose a model of TOR signaling for TAG accumulation in microalgae.

RSH enzyme diversity for (p)ppGpp metabolism in Phaeodactylum tricornutum and other diatoms.

The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.

Hydrogenases and H2 metabolism in sulfate-reducing bacteria of the Desulfovibrio genus.

Hydrogen metabolism plays a central role in sulfate-reducing bacteria of the Desulfovibrio genus and is based on hydrogenases that catalyze the reversible conversion of protons into dihydrogen. These metabolically versatile microorganisms possess a complex hydrogenase system composed of several enzymes of both [FeFe]- and [NiFe]-type that can vary considerably from one Desulfovibrio species to another. This review covers the molecular and physiological aspects of hydrogenases and H2 metabolism in Desulfovibrio but focuses particularly on our model bacterium Desulfovibrio fructosovorans. The search of hydrogenase genes in more than 30 sequenced genomes provides an overview of the distribution of these enzymes in Desulfovibrio. Our discussion will consider the significance of the involvement of electron-bifurcation in H2 metabolism.

Clostridial whole cell and enzyme systems for hydrogen production: current state and perspectives.

Strictly anaerobic bacteria of the Clostridium genus have attracted great interest as potential cell factories for molecular hydrogen production purposes. In addition to being a useful approach to this process, dark fermentation has the advantage of using the degradation of cheap agricultural residues and industrial wastes for molecular hydrogen production. However, many improvements are still required before large-scale hydrogen production from clostridial metabolism is possible. Here we review the literature on the basic biological processes involved in clostridial hydrogen production, and present the main advances obtained so far in order to enhance the hydrogen productivity, as well as suggesting some possible future prospects.

Phototrophic hydrogen production from a clostridial [FeFe] hydrogenase expressed in the heterocysts of the cyanobacterium Nostoc PCC 7120.

The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H2. As a result, their H2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H2 production activities, but their sensitivity to O2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O2. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 µmol-H2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H2 production by an O2-sensitive hydrogenase.

Viruses of parasites as actors in the parasite-host relationship: A "ménage à trois".

The complex parasite-host relationship involves multiple mechanisms. Moreover, parasites infected by viruses modify this relationship adding more complexity to the system that now comprises three partners. Viruses infecting parasites were described several decades ago. However, until recently little was known about the viruses involved and their impact on the resulting disease caused to the hosts. To clarify this situation, we have concentrated on parasitic diseases caused to humans and on how virus-infected parasites could alter the symptoms inflicted on the human host. It is clear that the effect caused to the human host depends on the virus and on the parasite it has infected. Consequently, the review is divided as follows: Viruses with a possible effect on the virulence of the parasite. This section reviews pertinent articles showing that infection of parasites by viruses might increase the detrimental effect of the tandem virus-parasite on the human host (hypervirulence) or decrease virulence of the parasite (hypovirulence). Parasites as vectors affecting the transmission of viruses. In some cases, the virus-infected parasite might facilitate the transfer of the virus to the human host. Parasites harboring viruses with unidentified effects on their host. In spite of recently renewed interest in parasites in connection with their viruses, there still remains a number of cases in which the effect of the virus of a given parasite on the human host remains ambiguous. The triangular relationship between the virus, the parasite and the host, and the modulation of the pathogenicity and virulence of the parasites by viruses should be taken into account in the rationale of fighting against parasites.

Interaction of Trypanosoma evansi with the plasminogen-plasmin system.

Trypanosoma evansi is a widely-distributed haemoflagellated parasite of veterinary importance that infects a variety of mammals including horses, mules, camels, buffalos, cattle and deer. It is the causal agent of a trypanosomiasis known as Surra which produces epidemics of great economic importance in Africa, Asia and South America. The main pathology includes an enlarged spleen with hypertrophy of lymphoid follicles, congested lungs, neuronal degeneration and meningoencephalitis, where migration of the parasites from the blood to the tissues is essential. Most cells, including pathogenic cells, use diverse strategies for tissue invasion, such as the expression of surface receptors to bind plasminogen or plasmin. In this work, we show that T. evansi is able to bind plasminogen and plasmin on its surface. The analysis of this binding revealed a high affinity dissociation constant (Kd of 0.080±0.009µM) and 1×10(5) plasminogen binding sites per cell. Also a second population of receptors with a Kd of 0.255±0.070µM and 3.2×10(4) plasminogen binding sites per cell was determined. Several proteins with molecular masses between ∼18 and ∼70kDa are responsible for this binding. This parasite-plasminogen interaction may be important in the establishment of the infection in the vertebrate host, where the physiological concentration of available plasminogen is around 2µM.

Fairy "tails": flexibility and function of intrinsically disordered extensions in the photosynthetic world.

Intrinsically Disordered Proteins (IDPs), or protein fragments also called Intrinsically Disordered Regions (IDRs), display high flexibility as the result of their amino acid composition. They can adopt multiple roles. In globular proteins, IDRs are usually found as loops and linkers between secondary structure elements. However, not all disordered fragments are loops: some proteins bear an intrinsically disordered extension at their C- or N-terminus, and this flexibility can affect the protein as a whole. In this review, we focus on the disordered N- and C-terminal extensions of globular proteins from photosynthetic organisms. Using the examples of the A2B2-GAPDH and the α Rubisco activase isoform, we show that intrinsically disordered extensions can help regulate their "host" protein in response to changes in light, thereby participating in photosynthesis regulation. As IDPs are famous for their large number of protein partners, we used the examples of the NAC, bZIP, TCP, and GRAS transcription factor families to illustrate the fact that intrinsically disordered extremities can allow a protein to have an increased number of partners, which directly affects its regulation. Finally, for proteins from the cryptochrome light receptor family, we describe how a new role for the photolyase proteins may emerge by the addition of an intrinsically disordered extension, while still allowing the protein to absorb blue light. This review has highlighted the diverse repercussions of the disordered extension on the regulation and function of their host protein and outlined possible future research avenues.

Glyceraldehyde-3-phosphate dehydrogenase is regulated by ferredoxin-NADP reductase in the diatom Asterionella formosa.

Diatoms are a widespread and ecologically important group of heterokont algae that contribute c. 20% to global productivity. Previous work has shown that regulation of their key Calvin cycle enzymes differs from that of the Plantae, and that in crude extracts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be inhibited by nicotinamide adenine dinucleotide phosphate reduced (NADPH) under oxidizing conditions. The freshwater diatom, Asterionella formosa, was studied using enzyme kinetics, chromatography, surface plasmon resonance, mass spectrometry and sequence analysis to determine the mechanism behind this GAPDH inhibition. GAPDH interacted with ferredoxin-nicotinamide adenine dinucleotide phosphate (NADP) reductase (FNR) from the primary phase of photosynthesis, and the small chloroplast protein, CP12. Sequences of copurified GAPDH and FNR were highly homologous with published sequences. However, the widespread ternary complex among GAPDH, phosphoribulokinase and CP12 was absent. Activity measurements under oxidizing conditions showed that NADPH can inhibit GAPDH-CP12 in the presence of FNR, explaining the earlier observed inhibition within crude extracts. Diatom plastids have a distinctive metabolism, including the lack of the oxidative pentose phosphate pathway, and so cannot produce NADPH in the dark. The observed down-regulation of GAPDH in the dark may allow NADPH to be rerouted towards other reductive processes contributing to their ecological success.

Extracellular functions of glycolytic enzymes of parasites: unpredicted use of ancient proteins.

In addition of their usual intracellular localization where they are involved in catalyzing reactions of carbohydrate and energy metabolism by glycolysis, multiple studies have shown that glycolytic enzymes of many organisms, but notably pathogens, can also be present extracellularly. In the case of parasitic protists and helminths, they can be found either secreted or attached to the surface of the parasites. At these extracellular localizations, these enzymes have been shown to perform additional, very different so-called "moonlighting" functions, such as acting as ligands for a variety of components of the host. Due to this recognition, different extracellular glycolytic enzymes participate in various important parasite-host interactions such as adherence and invasion of parasites, modulation of the host's immune and haemostatic systems, promotion of angiogenesis, and acquisition of specific nutrients by the parasites. Accordingly, extracellular glycolytic enzymes are important for the invasion of the parasites and their establishment in the host, and in determining their virulence.

Hysteresis and positive cooperativity as possible regulatory mechanisms of Trypanosoma cruzi hexokinase activity.

In Trypanosoma cruzi, the causal agent of Chagas disease, the first six or seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Hexokinase (HK), the first enzyme in the glycolytic pathway, has been an important research object, particularly as a potential drug target. Here we present the results of a specific kinetics study of the native HK from T. cruzi epimastigotes; a sigmoidal behavior was apparent when the velocity of the reaction was determined as a function of the concentration of its substrates, glucose and ATP. This behavior was only observed at low enzyme concentration, while at high concentration classical Michaelis-Menten kinetics was displayed. The progress curve of the enzyme's activity displays a lag phase of which the length is dependent on the protein concentration, suggesting that HK is a hysteretic enzyme. The hysteretic behavior may be attributed to slow changes in the conformation of T. cruzi HK as a response to variations of glucose and ATP concentrations in the glycosomal matrix. Variations in HK's substrate concentrations within the glycosomes may be due to variations in the trypanosome's environment. The hysteretic and cooperative behavior of the enzyme may be a form of regulation by which the parasite can more readily adapt to these environmental changes, occurring within each of its hosts, or during the early phase of transition to a new host.

Parasitism in optima forma: exploiting the host fibrinolytic system for invasion.

The interaction of pathogenic bacteria with the host fibrinolytic system through the plasminogen molecule has been well documented. It has been shown, using animal models, to be important in invasion into the host and establishment of the infection. From a number of recent observations with parasitic protists and helminths, emerges evidence that also in these organisms the interaction with plasminogen may be important for infection and virulence. A group of molecules that act as plasminogen receptors have been identified in parasites. This group comprises the glycolytic enzymes enolase, glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-biphosphate aldolase, in common with the plasminogen receptors known in prokaryotic pathogens. The interaction with the fibrinolytic system may arm the parasites with the host protease plasmin, thus helping them to migrate and cross barriers, infect cells and avoid clot formation. In this context, plasminogen receptors on the parasite surface or as secreted molecules, may be considered virulence factors. A possible evolutionary scenario for the recruitment of glycolytic enzymes as plasminogen receptors by widely different pathogens is discussed.