New Insight into the Role of the Calvin Cycle: Reutilization of CO2 Emitted through Sugar Degradation.
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ABSTRACT:
SUBMITTER: Shimizu R
PROVIDER: S-EPMC4486974 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Photosynthetic microalgae are emerging as potential biomass feedstock for sustainable production of biofuels and value-added bioproducts. CO2 biomitigation through these organisms is considered as an eco-friendly and promising alternative to the existing carbon sequestration methods. Nonetheless, the inherent relatively low photosynthetic capacity of microalgae has hampered the practical use of this strategy for CO2 biomitigation applications. RESULTS:Here, we demonstrate the feasibility of improving photosynthetic capacity by the genetic manipulation of the Calvin cycle in the typical green microalga Chlorella vulgaris. Firstly, we fused a plastid transit peptide to upstream of the enhanced green fluorescent protein (EGFP) and confirmed its expression in the chloroplast of C. vulgaris. Then we introduced the cyanobacterial fructose 1,6-bisphosphate aldolase, guided by the plastid transit peptide, into C. vulgaris chloroplast, leading to enhanced photosynthetic capacity (~ 1.2-fold) and cell growth. Molecular and physiochemical analyses suggested a possible role for aldolase overexpression in promoting the regeneration of ribulose 1,5-bisphosphate in the Calvin cycle and energy transfer in photosystems. CONCLUSIONS:Our work represents a proof-of-concept effort to enhance photosynthetic capacity by the engineering of the Calvin cycle in green microalgae. Our work also provides insights into targeted genetic engineering toward algal trait improvement for CO2 biomitigation uses.
| S-EPMC5629779 | biostudies-literature
Project description:Feeding 14CO2 was crucial to uncovering the path of carbon in photosynthesis. Feeding 13CO2 to photosynthesizing leaves emitting isoprene has been used to develop hypotheses about the sources of carbon for the methylerythritol 4-phosphate pathway, which makes the precursors for terpene synthesis in chloroplasts and bacteria. Both photosynthesis and isoprene studies found that products label very quickly (<10 min) up to 80-90% but the last 10-20% of labeling requires hours indicating a source of 12C during photosynthesis and isoprene emission. Furthermore, studies with isoprene showed that the proportion of slow label could vary significantly. This was interpreted as a variable contribution of carbon from sources other than the Calvin-Benson cycle (CBC) feeding the methylerythritol 4-phosphate pathway. Here, we measured the degree of label in isoprene and photosynthetic metabolites 20 min after beginning to feed 13CO2. Isoprene labeling was the same as labeling of photosynthesis intermediates. High temperature reduced the label in isoprene and photosynthesis intermediates by the same amount indicating no role for alternative carbon sources for isoprene. A model assuming glucose, fructose, and/or sucrose reenters the CBC as ribulose 5-phosphate through a cytosolic shunt involving glucose 6-phosphate dehydrogenase was consistent with the observations.
| S-EPMC7666771 | biostudies-literature
Project description:In this study, an enzymatic pathway has been developed to replicate the Calvin Cycle by creating the individual steps of the carbon cycle in a bioreactor. The technology known as "artificial photosynthesis" converts CO2 emissions into a variety of intermediates that serve as precursors to high-value products. CO2, light, water, and electricity were used as feedstock. An electrochemical reactor was also studied for the regeneration of active NADH operating at constant electrode potential. Initially, a batch electrochemical reactor containing 80 mL of 0.2 mM NAD+ in Tris-buffer (pH 7.40) was used to evaluate the electrode material operating at normal temperature and pressure. The results showed that the cathode is highly electrocatalytically efficient and selective to regenerate 97.45±0.8% of NADH from NAD+ at electrode potential of -2.3 V vs. mercury standard electrode (MSE). The NADH regeneration system was then integrated with ATP regeneration system and bioreactor containing Ribulose bisphosphate carboxylase/oxygenase (RuBisCO). NADH was regenerated successfully during the process electrochemically and then was used by the enzymatic reaction to produce triose phosphate and 3-Phosphoglycerate (3GPA).
| S-EPMC7497995 | biostudies-literature
Project description:BackgroundModeling the Calvin-Benson cycle has a history in the field of theoretical biology. Anyone who intends to model this system will look at existing models to adapt, refine and improve them. With the goal to study the regulation of carbon metabolism, we investigated a broad range of relevant models for their suitability to provide the basis for further modeling efforts. Beyond a critical analysis of existing models, we furthermore investigated the question how adjacent metabolic pathways, for instance photorespiration, can be integrated in such models.ResultsOur analysis reveals serious problems with a range of models that are publicly available and widely used. The problems include the irreproducibility of the published results or significant differences between the equations in the published description of the model and model itself in the supplementary material. In addition to and based on the discussion of existing models, we furthermore analyzed approaches in PGA sink implementation and confirmed a weak relationship between the level of its regulation and efficiency of PGA export, in contrast to significant changes in the content of metabolic pool within the Calvin-Benson cycle.ConclusionsIn our study we show that the existing models that have been investigated are not suitable for reuse without substantial modifications. We furthermore show that the minor adjacent pathways of the carbon metabolism, neglected in all kinetic models of Calvin-Benson cycle, cannot be substituted without consequences in the mass production dynamics. We further show that photorespiration or at least its first step (O2 fixation) has to be implemented in the model if this model is aimed for analyses out of the steady state.
| S-EPMC3257313 | biostudies-literature
Project description:Sugar-derived reactive carbonyls (RCs), including methylglyoxal (MG), are aggressive by-products of oxidative stress known to impair the functions of multiple proteins. These advanced glycation end-products accumulate in patients with diabetes mellitus and cause major complications, including arteriosclerosis and cardiac insufficiency. In the glycolytic pathway, the equilibration reactions between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (GAP) have recently been shown to generate MG as a by-product. Because plants produce vast amounts of sugars and support the same reaction in the Calvin cycle, we hypothesized that MG also accumulates in chloroplasts. Incubating isolated chloroplasts with excess 3-phosphoglycerate (3-PGA) as the GAP precursor drove the equilibration reaction toward MG production. The rate of oxygen (O2) evolution was used as an index of 3-PGA-mediated photosynthesis. The 3-PGA- and time-dependent accumulation of MG in chloroplasts was confirmed by HPLC. In addition, MG production increased with an increase in light intensity. We also observed a positive linear relationship between the rates of MG production and O2 evolution (R = 0.88; P < 0.0001). These data provide evidence that MG is produced by the Calvin cycle and that sugar-derived RC production is inevitable during photosynthesis. Furthermore, we found that MG production is enhanced under high-CO2 conditions in illuminated wheat leaves.
| S-EPMC3913449 | biostudies-literature
Project description:SignificancePhotosynthesis metabolites are quickly labeled when 13CO2 is fed to leaves, but the time course of labeling reveals additional contributing processes involved in the metabolic dynamics of photosynthesis. The existence of three such processes is demonstrated, and a metabolic flux model is developed to explore and characterize them. The model is consistent with a slow return of carbon from cytosolic and vacuolar sugars into the Calvin-Benson cycle through the oxidative pentose phosphate pathway. Our results provide insight into how carbon assimilation is integrated into the metabolic network of photosynthetic cells with implications for global carbon fluxes.
| S-EPMC8931376 | biostudies-literature
Project description:CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).
| S-EPMC23388 | biostudies-literature
Project description:The nucleophilic attack of hydrogen sulfide (HS-) on six different chloroacetanilide herbicides was evaluated theoretically using the dispersion-corrected hybrid functional wB97XD and the 6-311++G(2d,2p) Pople basis sets. The six evaluated substrates were propachlor (A), alachlor (B), metolachlor (C), tioacetanilide (D), β-anilide (E), and methylene (F). Three possible mechanisms were considered: (a) bimolecular nucleophilic substitution (SN²) reaction mechanism, (b) oxygen assistance, and (c) nitrogen assistance. Mechanisms based on O- and N-assistance were discarded due to a very high activation barrier in comparison with the corresponding SN² mechanism, with the exception of compound F. The N-assistance mechanism for compound F had a free activation energy of 23.52 kcal/mol, which was close to the value for the corresponding SN² mechanism (23.94 kcal/mol), as these two mechanisms could occur in parallel reactions with almost 50% of each one. In compounds A to D, an important electron-withdrawing effect of the C=O and C=S groups was seen, and consequently, the activation free energies in these SN² reactions were smaller, with a value of approximately 18 kcal/mol. Instead, compounds E and F, which have a CH₂ group in the β-position, presented a higher activation free energy (≈22 kcal/mol). Good agreement was found between experimental and theoretical values for all cases, and a reaction force analysis was performed on the intrinsic reaction coordinate profile in order to gain more details about the reaction mechanism. Finally, from the natural bond orbital (NBO) analysis, it was possible to evaluate the electronic reorganization through the reaction pathway where all the transition states were early in nature in the reaction coordinate (δBav < 50%); the transition states corresponding to compounds A to D turned out to be more synchronous than those for compounds E and F.
| S-EPMC6213056 | biostudies-literature
Project description:Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term "phosphoglycolate salvage." Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2 We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2 We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.
| S-EPMC7486775 | biostudies-literature
Project description:ChuW, ChuX, and ChuY are contiguous genes downstream from a single promoter that are expressed in the enteric pathogen Escherichia coli O157:H7 when iron is limiting. These genes, and the corresponding proteins, are part of a larger heme uptake and utilization operon that is common to several other enteric pathogens, such as Vibrio cholerae. The aerobic degradation of heme has been well characterized in humans and several pathogenic bacteria, including E. coli O157:H7, but only recently was it shown that ChuW catalyzes the anaerobic degradation of heme to release iron and produce a reactive tetrapyrrole termed "anaerobilin". ChuY has been shown to function as an anaerobilin reductase, in a role that parallels biliverdin reductase. In this work we have employed biochemical and biophysical approaches to further interrogate the mechanism of the anaerobic degradation of heme. We demonstrate that the iron atom of the heme does not participate in the catalytic mechanism of ChuW and that S-adenosyl-l-methionine binding induces conformational changes that favor catalysis. In addition, we show that ChuX and ChuY have synergistic and additive effects on the turnover rate of ChuW. Finally, we have found that ChuS is an effective source of heme or protoporphyrin IX for ChuW under anaerobic conditions. These data indicate that ChuS may have dual functionality in vivo. Specifically, ChuS serves as a heme oxygenase during aerobic metabolism of heme but functions as a cytoplasmic heme storage protein under anaerobic conditions, akin to what has been shown for PhuS (45% sequence identity) from Pseudomonas aeruginosa.
| S-EPMC7189753 | biostudies-literature