Calvin-Benson-Bassham Cycle Research Articles

Isotopically non-stationary 13 C metabolic flux analysis of two closely related fast-growing cyanobacteria Synechococcus elongatus PCC 11801 and 11802.

Synechococcus elongatus PCC 11801 and 11802 are closely related cyanobacterial strains that are fast-growing and tolerant to high light and temperature. These strains hold significant promise as chassis for photosynthetic production of chemicals from carbon dioxide. A detailed quantitative understanding of the central carbon pathways would be a reference for future metabolic engineering studies with these strains. We have used isotopic non-stationary 13 C-metabolic flux analysis (INST-MFA) to quantitively assess the metabolic potential of these two strains. This study highlights key similarities and differences in the central carbon flux distribution between these and other model/non-model strains. The two strains demonstrated a higher Calvin-Benson-Bassham (CBB) cycle flux coupled with negligible flux through the oxidative pentose phosphate (OPP), photorespiratory pathway, and lower anaplerosis fluxes under photoautotrophic conditions. Interestingly, PCC 11802 shows the highest CBB cycle and pyruvate kinase (PK) flux values among those reported in cyanobacteria. The unique tricarboxylic acid (TCA) cycle diversion in PCC 11801 makes it ideal for the large-scale production of TCA cycle-derived chemicals. Additionally, dynamic labeling transients were measured for intermediates of amino acid, nucleotide, and nucleotide-sugar metabolism. Overall, this study provides the first detailed metabolic flux maps of S. elongatus PCC 11801 and 11802 that may aid metabolic engineering efforts in these strains.

Ribulose-1,5-bisphosphate regeneration in the Calvin-Benson-Bassham cycle: Focus on the last three enzymatic steps that allow the formation of Rubisco substrate.

The Calvin-Benson-Bassham (CBB) cycle comprises the metabolic phase of photosynthesis and is responsible for carbon fixation and the production of sugar phosphates. The first step of the cycle involves the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which catalyzes the incorporation of inorganic carbon into 3-phosphoglyceric acid (3PGA). The following steps include ten enzymes that catalyze the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco. While it is well established that Rubisco activity acts as a limiting step of the cycle, recent modeling studies and experimental evidence have shown that the efficiency of the pathway is also impacted by the regeneration of the Rubisco substrate itself. In this work, we review the current understanding of the structural and catalytic features of the photosynthetic enzymes that catalyze the last three steps of the regeneration phase, namely ribose-5-phosphate isomerase (RPI), ribulose-5-phosphate epimerase (RPE), and phosphoribulokinase (PRK). In addition, the redox- and metabolic-based regulatory mechanisms targeting the three enzymes are also discussed. Overall, this review highlights the importance of understudied steps in the CBB cycle and provides direction for future research aimed at improving plant productivity.

Photorespiration is the solution, not the problem

The entry of carbon dioxide from the atmosphere into the biosphere is mediated by the enzyme Rubisco, which catalyzes the carboxylation of ribulose 1,5-bisphosphate (RuBP) as the entry reaction of the Calvin Benson Bassham cycle (CBBC), leading to the formation of 2 molecules of 3-phosphoglyceric acid (3PGA) per CO2 fixed. 3PGA is reduced to triose phosphates at the expense of NADPH + H+ and ATP that are provided by the photosynthetic light reactions. Triose phosphates are the principal products of the CBBC and the precursors for almost any compound in the biosphere.

Dark accumulation of downstream glycolytic intermediates initiates robust photosynthesis in cyanobacteria.

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies have shown that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid dynamic changes (on a time scale of seconds) in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enabled estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicated that downstream glycolytic intermediates, including phosphoglycerate and phosphoenolpyruvate, accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant decreases in the initial oxygen evolution rate were observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathways. Accordingly, the degree of decrease in the initial oxygen evolution rate was proportional to the accumulated pool size of glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

The trade-off function of photorespiration in a changing environment

Abstract The photorespiratory pathway in plants comprises metabolic reactions distributed across several cellular compartments. It emerges from the dual catalytic function of Rubisco, i.e. ribulose-1,5-bisphosphate carboxylase/oxygenase. Rubisco either carboxylates or oxygenates ribulose-1,5-bisphosphate. Carboxylation reactions produce 3-phosphoglycerate molecules which are substrates for the central carbohydrate metabolism. However, oxygenation reactions additionally form 2-phosphoglycolate molecules which are (i) substrate for a multicompartmental recovery process, and (ii) inhibit several enzymes of the Calvin–Benson–Bassham cycle. Here, an approach of structural kinetic modelling is presented to investigate the extent of stabilization of the Calvin–Benson–Bassham cycle and carbohydrate metabolism by photorespiration. This method is based on a parametric representation of the Jacobian matrix of a metabolic system which offers a robust strategy for handling uncertainties associated with in vitro kinetic constants. Our findings indicate that oxygenation of ribulose-1,5-bisphosphate by Rubisco significantly stabilizes the Calvin–Benson–Bassham cycle. Hence, a trade-off function of photorespiration is suggested which reduces carbon assimilation rates but simultaneously stabilizes metabolism by increasing plasticity of metabolic regulation within the chloroplast. Furthermore, our analysis indicates that increasing carbon flux towards sucrose biosynthesis has a stabilizing effect. Finally, our findings shed light on the role of a multicompartmental metabolic pathway in stabilizing plant metabolism against perturbation induced by a dynamic environment.

Back to the future: Transplanting the chloroplast TrxF-FBPase-SBPase redox system to cyanobacteria.

Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) are two essential activities in the Calvin-Benson-Bassham cycle that catalyze two irreversible reactions and are key for proper regulation and functioning of the cycle. These two activities are codified by a single gene in all cyanobacteria, although some cyanobacteria contain an additional gene coding for a FBPase. Mutants lacking the gene coding for SBP/FBPase protein are not able to grow photoautotrophically and require glucose to survive. As this protein presents both activities, we have tried to elucidate which of the two are required for photoautrophic growth in Synechocystis sp PCC 6803. For this, the genes coding for plant FBPase and SBPase were introduced in a SBP/FBPase mutant strain, and the strains were tested for growth in the absence of glucose. Ectopic expression of only a plant SBPase gene did not allow growth in the absence of glucose although allowed mutation of both Synechocystis' FBPase genes. When both plant FBPase and SBPase genes were expressed, photoautrophic growth of the SBP/FBPase mutants was restored. This complementation was partial as the strain only grew in low light, but growth was impaired at higher light intensities. Redox regulation of the Calvin-Benson-Bassham cycle is essential to properly coordinate light reactions to carbon fixation in the chloroplast. Two of the best characterized proteins that are redox-regulated in the cycle are FBPase and SBPase. These two proteins are targets of the FTR-Trx redox system with Trx f being the main reductant in vivo. Introduction of the TrxF gene improves growth of the complemented strain, suggesting that the redox state of the proteins may be the cause of this phenotype. The redox state of the plant proteins was also checked in these strains, and it shows that the cyanobacterial redox system is able to reduce all of them (SBPase, FBPase, and TrxF) in a light-dependent manner. Thus, the TrxF-FBPase-SBPase plant chloroplast system is active in cyanobacteria despite that these organisms do not contain proteins related to them. Furthermore, our system opens the possibility to study specificity of the Trx system in vivo without the complication of the different isoforms present in plants.

Carbon fixation pathways across the bacterial and archaeal tree of life.

Carbon fixation is a critical process for our planet; however, its distribution across the bacterial and archaeal domains of life has not been comprehensively studied. Here, we performed an analysis of 52,515 metagenome-assembled genomes and discover carbon fixation pathways in 1,007 bacteria and archaea. We reveal the genomic potential for carbon fixation through the reverse tricarboxylic acid cycle in previously unrecognized archaeal and bacterial phyla (i.e. Thermoplasmatota and Elusimicrobiota) and show that the 3-hydroxypropionate bi-cycle is not, as previously thought, restricted to the phylum Chloroflexota. The data also substantially expand the phylogenetic breadth for autotrophy through the dicarboxylate/4-hydroxybutyrate cycle and the Calvin-Benson-Bassham cycle. Finally, the genomic potential for carbon fixation through the 3-hydroxypropionate/4-hydroxybutyrate cycle, previously exclusively found in Archaea, was also detected in the Bacteria. Carbon fixation thus appears to be much more widespread than previously known, and this study lays the foundation to better understand the role of archaea and bacteria in global primary production and how they contribute to microbial carbon sinks.

History of the Study of the Genus Thiothrix: From the First Enrichment Cultures to Pangenomic Analysis.

Representatives of the genus Thiothrix are filamentous, sulfur-oxidizing bacteria found in flowing waters with counter-oriented sulfide and oxygen gradients. They were first described at the end of the 19th century, but the first pure cultures of this species only became available 100 years later. An increase in the number of described Thiothrix species at the beginning of the 21st century shows that the classical phylogenetic marker, 16S rRNA gene, is not informative for species differentiation, which is possible based on genome analysis. Pangenome analysis of the genus Thiothrix showed that the core genome includes genes for dissimilatory sulfur metabolism and central metabolic pathways, namely the Krebs cycle, Embden–Meyerhof–Parnas pathway, glyoxylate cycle, Calvin–Benson–Bassham cycle, and genes for phosphorus metabolism and amination. The shell part of the pangenome includes genes for dissimilatory nitrogen metabolism and nitrogen fixation, for respiration with thiosulfate. The dispensable genome comprises genes predicted to encode mainly hypothetical proteins, transporters, transcription regulators, methyltransferases, transposases, and toxin–antitoxin systems.

Evolution of gene regulatory network of C4 photosynthesis in the genus Flaveria reveals the evolutionary status of C3-C4 intermediate species

C4 photosynthesis evolved from ancestral C3 photosynthesis by recruiting pre-existing genes to fulfill new functions. The enzymes and transporters required for the C4 metabolic pathway have been intensively studied and well documented; however, the transcription factors (TFs) that regulate these C4 metabolic genes are not yet well understood. In particular, how the TF regulatory network of C4 metabolic genes was rewired during the evolutionary process is unclear. Here, we constructed gene regulatory networks (GRNs) for four closely evolutionarily related species from the genus Flaveria, which represent four different evolutionary stages of C4 photosynthesis: C3 (F. robusta), type I C3-C4 (F. sonorensis), type II C3-C4 (F. ramosissima), and C4 (F. trinervia). Our results show that more than half of the co-regulatory relationships between TFs and core C4 metabolic genes are species specific. The counterparts of the C4 genes in C3 species were already co-regulated with photosynthesis-related genes, whereas the required TFs for C4 photosynthesis were recruited later. The TFs involved in C4 photosynthesis were widely recruited in the type I C3-C4 species; nevertheless, type II C3-C4 species showed a divergent GRN from C4 species. In line with these findings, a 13CO2 pulse-labeling experiment showed that the CO2 initially fixed into C4 acid was not directly released to the Calvin–Benson–Bassham cycle in the type II C3-C4 species. Therefore, our study uncovered dynamic changes in C4 genes and TF co-regulation during the evolutionary process; furthermore, we showed that the metabolic pathway of the type II C3-C4 species F. ramosissima represents an alternative evolutionary solution to the ammonia imbalance in C3-C4 intermediate species.

Improving plant productivity by re-tuning the regeneration of RuBP in the Calvin-Benson-Bassham cycle.

The Calvin-Benson-Bassham (CBB) cycle is arguably the most important pathway on earth, capturing CO2 from the atmosphere and converting it into organic molecules, providing the basis for life on our planet. This cycle has been intensively studied over the 50 yr since it was elucidated, and it is highly conserved across nature, from cyanobacteria to the largest of our land plants. Eight out of the 11 enzymes in this cycle catalyse the regeneration of ribulose-1-5 bisphosphate (RuBP), the CO2 acceptor molecule. The potential to manipulate RuBP regeneration to improve photosynthesis has been demonstrated in a number of plant species, and the development of new technologies, such as omics and synthetic biology provides exciting future opportunities to improve photosynthesis and increase crop yields.

Sulfur and methane oxidation by a single microorganism

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic-anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, 'Methylovirgula thiovorans' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox-rDsr pathway and the S4I system. Strain HY1 employed the Calvin-Benson-Bassham cycle for CO2 fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic-anoxic interface environments.

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12.

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.

Chloroplast pH Homeostasis for the Regulation of Photosynthesis

The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants.

Phylogeny and Metabolic Potential of the Methanotrophic Lineage MO3 in Beijerinckiaceae from the Paddy Soil through Metagenome-Assembled Genome Reconstruction.

Although the study of aerobic methane-oxidizing bacteria (MOB, methanotrophs) has been carried out for more than a hundred years, there are many uncultivated methanotrophic lineages whose metabolism is largely unknown. Here, we reconstructed a nearly complete genome of a Beijerinckiaceae methanotroph from the enrichment of paddy soil by using nitrogen-free M2 medium. The methanotroph labeled as MO3_YZ.1 had a size of 3.83 Mb, GC content of 65.6%, and 3442 gene-coding regions. Based on phylogeny of pmoA gene and genome and the genomic average nucleotide identity, we confirmed its affiliation to the MO3 lineage and a close relationship to Methylocapsa. MO3_YZ.1 contained mxaF- and xoxF-type methanol dehydrogenase. MO3_YZ.1 used the serine cycle to assimilate carbon and regenerated glyoxylate through the glyoxylate shunt as it contained isocitrate lyase and complete tricarboxylic acid cycle-coding genes. The ethylmalonyl-CoA pathway and Calvin–Benson–Bassham cycle were incomplete in MO3_YZ.1. Three acetate utilization enzyme-coding genes were identified, suggesting its potential ability to utilize acetate. The presence of genes for N2 fixation, sulfur transformation, and poly-β-hydroxybutyrate synthesis enable its survival in heterogeneous habitats with fluctuating supplies of carbon, nitrogen, and sulfur.

Interactions Between Carbon Metabolism and Photosynthetic Electron Transport in a Chlamydomonas reinhardtii Mutant Without CO2 Fixation by RuBisCO.

A Chlamydomonas reinhardtii RuBisCO-less mutant, ΔrbcL, was used to study carbohydrate metabolism without fixation of atmospheric carbon. The regulatory mechanism(s) that control linear electron flow, known as “photosynthetic control,” are amplified in ΔrbcL at the onset of illumination. With the aim to understand the metabolites that control this regulatory response, we have correlated the kinetics of primary carbon metabolites to chlorophyll fluorescence induction curves. We identify that ΔrbcL in the absence of acetate generates adenosine triphosphate (ATP) via photosynthetic electron transfer reactions. Also, metabolites of the Calvin Benson Bassham (CBB) cycle are responsive to the light. Indeed, ribulose 1,5-bisphosphate (RuBP), the last intermediate before carboxylation by Ribulose-1,5-bisphosphate carboxylase-oxygenase, accumulates significantly with time, and CBB cycle intermediates for RuBP regeneration, dihydroxyacetone phosphate (DHAP), pentose phosphates and ribose-5-phosphate (R5P) are rapidly accumulated in the first seconds of illumination, then consumed, showing that although the CBB is blocked, these enzymes are still transiently active. In opposition, in the presence of acetate, consumption of CBB cycle intermediates is strongly diminished, suggesting that the link between light and primary carbon metabolism is almost lost. Phosphorylated hexoses and starch accumulate significantly. We show that acetate uptake results in heterotrophic metabolism dominating phototrophic metabolism, with glyoxylate and tricarboxylic acid (TCA) cycle intermediates being the most highly represented metabolites, specifically succinate and malate. These findings allow us to hypothesize which metabolites and metabolic pathways are relevant to the upregulation of processes like cyclic electron flow that are implicated in photosynthetic control mechanisms.

Phylogeny and Metabolic Potential of the Candidate Phylum SAR324.

Simple SummarySAR324, newly proposed as its own candidate phylum, is a diverse and globally abundant bacterial group living in a wide range of environments, from deep-sea hydrothermal vents and brine pools to the epipelagic regions of the global oceans and terrestrial aquifers. The different SAR324 clades harbor a diverse array of genes and pathways well adapted to their respective environments. This metabolic flexibility explains the ubiquitous presence and the importance of SAR324 in global biogeochemical cycles.The bacterial SAR324 cluster is ubiquitous and abundant in the ocean, especially around hydrothermal vents and in the deep sea, where it can account for up to 30% of the whole bacterial community. According to a new taxonomy generated using multiple universal protein-coding genes (instead of the previously used 16S rRNA single gene marker), the former Deltaproteobacteria cluster SAR324 has been classified since 2018 as its own phylum. Yet, very little is known about its phylogeny and metabolic potential. We downloaded all publicly available SAR324 genomes (65) from all natural environments and reconstructed 18 new genomes using publicly available oceanic metagenomic data and unpublished data from the waters underneath the Ross Ice Shelf. We calculated a global SAR324 phylogenetic tree and identified six clusters (namely 1A, 1B, 2A, 2B, 2C and 2D) within this clade. Genome annotation and metatranscriptome read mapping showed that SAR324 clades possess a flexible array of genes suited for survival in various environments. Clades 2A and 2C are mostly present in the surface mesopelagic layers of global oceans, while clade 2D dominates in deeper regions. Our results show that SAR324 has a very versatile and broad metabolic potential, including many heterotrophic, but also autotrophic pathways. While one surface water associated clade (2A) seems to use proteorhodopsin to gain energy from solar radiation, some deep-sea genomes from clade 2D contain the complete Calvin–Benson–Bassham cycle gene repertoire to fix carbon. This, in addition to a variety of other genes and pathways for both oxic (e.g., dimethylsulfoniopropionate degradation) and anoxic (e.g., dissimilatory sulfate reduction, anaerobic benzoate degradation) conditions, can help explain the ubiquitous presence of SAR324 in aquatic habitats.

The reliance of glycerol utilization by Cupriavidus necator on CO2 fixation and improved glycerol catabolism

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.

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.

Biosensor-informed engineering of Cupriavidus necator H16 for autotrophic D-mannitol production

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO2)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO2 utilising precursors derived from the CBB cycle.

Photosynthetic assimilation of CO2 regulates TOR activity

The target of rapamycin (TOR) kinase is a master regulator that integrates nutrient signals to promote cell growth in all eukaryotes. It is well established that amino acids and glucose are major regulators of TOR signaling in yeast and metazoan, but whether and how TOR responds to carbon availability in photosynthetic organisms is less understood. In this study, we showed that photosynthetic assimilation of CO2 by the Calvin-Benson-Bassham (CBB) cycle regulates TOR activity in the model single-celled microalga Chlamydomonas reinhardtii Stimulation of CO2 fixation boosted TOR activity, whereas inhibition of the CBB cycle and photosynthesis down-regulated TOR. We uncovered a tight link between TOR activity and the endogenous level of a set of amino acids including Ala, Glu, Gln, Leu, and Val through the modulation of CO2 fixation and the use of amino acid synthesis inhibitors. Moreover, the finding that the Chlamydomonas starch-deficient mutant sta6 displayed disproportionate TOR activity and high levels of most amino acids, particularly Gln, further connected carbon assimilation and amino acids to TOR signaling. Thus, our results showed that CO2 fixation regulates TOR signaling, likely through the synthesis of key amino acids.