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1.
Commun Biol ; 6(1): 19, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611062

RESUMO

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play important roles in the stability, dynamics, and regulation of condensates.


Assuntos
Plastídeos , Ribulose-Bifosfato Carboxilase , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismo
3.
Mar Drugs ; 21(1)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36662222

RESUMO

In this study, we studied the bioactive peptides produced by thermolysin hydrolysis of a water-soluble protein (WSP) from the red alga Gracilariopsis chorda, whose major components are phycobiliproteins and Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo). The results showed that WSP hydrolysate exhibited significantly higher ACE inhibitory activity (92% inhibition) compared to DPP-IV inhibitory activity and DPPH scavenging activity. The phycobiliproteins and RuBisCo of G. chorda contain a high proportion of hydrophobic (31.0-46.5%) and aromatic (5.1-46.5%) amino acid residues, which was considered suitable for the formation of peptides with strong ACE inhibitory activity. Therefore, we searched for peptides with strong ACE inhibitory activity and identified two novel peptides (IDHY and LVVER). Then, their interaction with human ACE was evaluated by molecular docking, and IDHY was found to be a promising inhibitor. In silico analysis was then performed on the structural factors affecting ACE inhibitory peptide release, using the predicted 3D structures of phycobiliproteins and RuBisCo. The results showed that most of the ACE inhibitory peptides are located in the highly solvent accessible α-helix. Therefore, it was suggested that G. chorda is a good source of bioactive peptides, especially ACE-inhibitory peptides.


Assuntos
Rodófitas , Ribulose-Bifosfato Carboxilase , Humanos , Simulação de Acoplamento Molecular , Peptídeos/química , Rodófitas/metabolismo , Ficobiliproteínas , Peptidil Dipeptidase A/química
4.
J Plant Physiol ; 280: 153899, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36566670

RESUMO

The photorespiratory repair pathway (photorespiration in short) was set up from ancient metabolic modules about three billion years ago in cyanobacteria, the later ancestors of chloroplasts. These prokaryotes developed the capacity for oxygenic photosynthesis, i.e. the use of water as a source of electrons and protons (with O2 as a by-product) for the sunlight-driven synthesis of ATP and NADPH for CO2 fixation in the Calvin cycle. However, the CO2-binding enzyme, ribulose 1,5-bisphosphate carboxylase (known under the acronym Rubisco), is not absolutely selective for CO2 and can also use O2 in a side reaction. It then produces 2-phosphoglycolate (2PG), the accumulation of which would inhibit and potentially stop the Calvin cycle and subsequently photosynthetic electron transport. Photorespiration removes the 2-PG and in this way prevents oxygenic photosynthesis from poisoning itself. In plants, the core of photorespiration consists of ten enzymes distributed over three different types of organelles, requiring interorganellar transport and interaction with several auxiliary enzymes. It goes together with the release and to some extent loss of freshly fixed CO2. This disadvantageous feature can be suppressed by CO2-concentrating mechanisms, such as those that evolved in C4 plants thirty million years ago, which enhance CO2 fixation and reduce 2PG synthesis. Photorespiration itself provided a pioneer variant of such mechanisms in the predecessors of C4 plants, C3-C4 intermediate plants. This article is a review and update particularly on the enzyme components of plant photorespiration and their catalytic mechanisms, on the interaction of photorespiration with other metabolism and on its impact on the evolution of photosynthesis. This focus was chosen because a better knowledge of the enzymes involved and how they are embedded in overall plant metabolism can facilitate the targeted use of the now highly advanced methods of metabolic network modelling and flux analysis. Understanding photorespiration more than before as a process that enables, rather than reduces, plant photosynthesis, will help develop rational strategies for crop improvement.


Assuntos
Dióxido de Carbono , Ribulose-Bifosfato Carboxilase , Ribulose-Bifosfato Carboxilase/metabolismo , Dióxido de Carbono/metabolismo , Fotossíntese , Plantas/metabolismo , Cloroplastos/metabolismo , Oxigênio/metabolismo
5.
Plant J ; 113(2): 416-429, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36479950

RESUMO

Crop photosynthesis (A) and productivity are often limited by a combination of nutrient stresses, such that changes in the availability of one nutrient may affect the availability of another nutrient, in turn influencing A. In this study, we examined the synergistic effects of phosphorus (P) and potassium (K) on leaf A in a nutrient amendment experiment, in which P and K were added individually or in combination to Brassica napus grown under P and K co-limitation. The data revealed that the addition of P gradually removed the dominant limiting factor (i.e. the limited availability of P) and improved leaf A. Strikingly, the addition of K synergistically improved the overall uptake of P, mainly by boosting plant growth, and compensated for the physiological demand for P by prioritizing investment in metabolic pools of P (P-containing metabolites and inorganic phosphate, Pi). The enlarged pool of metabolically active P was partially associated with the upregulation of Pi regeneration through release from triose phosphates rather than replacement of P-containing lipids. This process mitigated P restrictions on A by maintaining the ATP/NADPH and NADPH/NADP+ ratios and increasing the content and activity of Rubisco. Our findings demonstrate that sufficient K increased Pi-limited A by enhancing metabolic P fractions and Rubisco activity. Thus, ionic synergism may be exploited to mitigate nutrient-limiting factors to improve crop productivity.


Assuntos
Brassica napus , Fósforo , Fósforo/metabolismo , Fosfatos/metabolismo , Potássio/metabolismo , Brassica napus/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , NADP/metabolismo , Fotossíntese/fisiologia , Folhas de Planta/metabolismo
6.
New Phytol ; 237(1): 60-77, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36251512

RESUMO

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.


Assuntos
Dióxido de Carbono , Produtos Agrícolas , Citocromo P-450 CYP2B1 , Trifosfato de Adenosina/metabolismo , Dióxido de Carbono/metabolismo , Produtos Agrícolas/fisiologia , Citocromo P-450 CYP2B1/metabolismo , Fotossíntese , Ribulose-Bifosfato Carboxilase/metabolismo
7.
J Plant Physiol ; 280: 153889, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36493669

RESUMO

Photosynthetic acclimation to prolonged elevated CO2 could be attributed to the two limited biochemical capacity, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation and ribulose-1,5-bisphosphate (RuBP) regeneration, however, which one is the primary driver is unclear. To quantify photosynthetic acclimation induced by biochemical limitation, we investigated photosynthetic characteristics and leaf nitrogen allocation to photosynthetic apparatus (Rubisco, bioenergetics, and light-harvesting complex) in a japonica rice grown in open-top chambers at ambient CO2 and ambient CO2+200 µmol mol-1 (e [CO2]). Results showed that photosynthesis was stimulated under e [CO2], but concomitantly, photosynthetic acclimation obviously occurred across the whole growth stages. The content of leaf nitrogen allocation to Rubisco and biogenetics was reduced by e [CO2], while not in light-harvesting complex. Unlike the content, there was little effects of CO2 enrichment on the percentage of nitrogen allocation to photosynthetic components. Additionally, leaf nitrogen did not reallocate within photosynthetic apparatus until the imbalance of sink-source under e [CO2]. The contribution of biochemical limitations, including Rubisco carboxylation and RuBP regeneration, to photosynthetic acclimation averaged 36.2% and 63.8% over the growing seasons, respectively. This study suggests that acclimation of photosynthesis is mainly driven by RuBP regeneration limitation and highlights the importance of RuBP regeneration relative to Rubisco carboxylation in the future CO2 enrichment.


Assuntos
Oryza , Oryza/metabolismo , Dióxido de Carbono/farmacologia , Ribulose-Bifosfato Carboxilase/metabolismo , Fotossíntese , Aclimatação , Nitrogênio/farmacologia , Folhas de Planta/metabolismo
8.
Environ Pollut ; 318: 120906, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36549447

RESUMO

Submerged macrophytes play an important role in the global carbon cycle through diversified pathways of inorganic carbon (Ci) utilization distinct from terrestrial plants. However, the effects of silver nanoparticles (AgNPs), an emerging contaminant, were unknown on the Ci utilization of submerged macrophytes. In Ottelia alismoides, the only known submerged macrophyte with three pathways of Ci utilization, before absorption, AgNPs inhibited the external carbonic anhydrase activity thus reducing the capacity of the plant to use HCO3-. After entering the plant, AgNPs mainly aggregated at the cell wall and in the chloroplast. The internalized AgNPs inhibited ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) activity blocking CO2 fixation and disturbed C4 and crassulacean acid metabolism (CAM) by inhibiting phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphate dikinase (PPDK), and NAD-dependent malic enzyme (NAD-ME) activities to alter intracellular malate biosynthesis and decarboxylation. Overall, our findings indicate that the Ci utilization of the submerged macrophyte is a target of AgNPs toxicity that might affect the carbon cycle in aquatic systems.


Assuntos
Nanopartículas Metálicas , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , NAD/metabolismo , Fotossíntese , Plantas/metabolismo , Carbono/metabolismo , Dióxido de Carbono/farmacologia , Dióxido de Carbono/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo
9.
Commun Biol ; 5(1): 1290, 2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36434094

RESUMO

Bacteria and Eucarya utilize the non-oxidative pentose phosphate pathway to direct the ribose moieties of nucleosides to central carbon metabolism. Many archaea do not possess this pathway, and instead, Thermococcales utilize a pentose bisphosphate pathway involving ribose-1,5-bisphosphate (R15P) isomerase and ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Intriguingly, multiple genomes from halophilic archaea seem only to harbor R15P isomerase, and do not harbor Rubisco. In this study, we identify a previously unrecognized nucleoside degradation pathway in halophilic archaea, composed of guanosine phosphorylase, ATP-dependent ribose-1-phosphate kinase, R15P isomerase, RuBP phosphatase, ribulose-1-phosphate aldolase, and glycolaldehyde reductase. The pathway converts the ribose moiety of guanosine to dihydroxyacetone phosphate and ethylene glycol. Although the metabolic route from guanosine to RuBP via R15P is similar to that of the pentose bisphosphate pathway in Thermococcales, the downstream route does not utilize Rubisco and is unique to halophilic archaea.


Assuntos
Ribose , Ribulose-Bifosfato Carboxilase , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Ribose/metabolismo , Pentoses/metabolismo , Archaea/genética , Archaea/metabolismo , Guanosina/metabolismo , Fosfatos
10.
Environ Sci Technol ; 56(23): 17215-17226, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36375171

RESUMO

Continuous CO2 emissions from human activities increase atmospheric CO2 concentrations and affect global climate change. The carbon storage capacity of the ocean is 20-fold higher than that of the land, and diatoms contribute to approximately 40% of carbon capture in the ocean. Manganese (Mn) is a major driver of marine phytoplankton growth and the marine carbon pump. Here, we discovered self-assembled manganese oxides (MnOx) for CO2 fixation in a diatom-based biohybrid system. MnOx shared key features (e.g., di-µ-oxo-bridged Mn-Mn) with the Mn4CaO5 cluster of the biological catalyst in photosystem II and promoted photosynthesis and carbon capture by diatoms/MnOx. The CO2 capture capacity of diatoms/MnOx was 1.5-fold higher than that of diatoms alone. Diatoms/MnOx easily allocated carbon into proteins and lipids instead of carbohydrates. Metabolomics showed that the contents of several metabolites (e.g., lysine and inositol) were positively associated with increased CO2 capture. Diatoms/MnOx upregulated six genes encoding photosynthesis core proteins and a key rate-limiting enzyme (Rubisco, ribulose 1,5-bisphosphate carboxylase-oxygenase) in the Calvin-Benson-Bassham carbon assimilation cycle, revealing the link between MnOx and photosynthesis. These findings provide a route for offsetting anthropogenic CO2 emissions and inspiration for self-assembled biohybrid systems for carbon capture by marine phytoplankton.


Assuntos
Diatomáceas , Humanos , Carbono/metabolismo , Manganês , Dióxido de Carbono/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Fitoplâncton/metabolismo , Fotossíntese
11.
Int J Mol Sci ; 23(19)2022 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-36232645

RESUMO

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) functions as the initial enzyme in the dark reactions of photosynthesis, catalyzing reactions that extract CO2 from the atmosphere and fix CO2 into organic compounds. RuBisCO is classified into four types (isoforms I-IV) according to sequence-based phylogenetic trees. Given its size, the computational cost of accurate quantum-chemical calculations for functional analysis of RuBisCO is high; however, recent advances in hardware performance and the use of the fragment molecular orbital (FMO) method have enabled the ab initio analyses of RuBisCO. Here, we performed FMO calculations on multiple structural datasets for various complexes with the 2'-carboxylarabinitol 1,5-bisphosphate (2CABP) ligand as a substrate analog and investigated whether phylogenetic relationships based on sequence information are physicochemically relevant as well as whether novel information unobtainable from sequence information can be revealed. We extracted features similar to the phylogenetic relationships found in sequence analysis, and in terms of singular value decomposition, we identified residues that strongly interacted with the ligand and the characteristics of the isoforms for each principal component. These results identified a strong correlation between phylogenetic relationships obtained by sequence analysis and residue interaction energies with the ligand. Notably, some important residues were located far from the ligand, making comparisons among species using only residues proximal to the ligand insufficient.


Assuntos
Dióxido de Carbono , Ribulose-Bifosfato Carboxilase , Dióxido de Carbono/metabolismo , Ligantes , Oxigenases/metabolismo , Fotossíntese , Filogenia , Extratos Vegetais , Ribulose-Bifosfato Carboxilase/metabolismo
12.
Int J Mol Sci ; 23(19)2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36233343

RESUMO

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible carbon dioxide hydration reaction. Among the eight different CA classes existing in nature, the α-class is the largest one being present in animals, bacteria, protozoa, fungi, and photosynthetic organisms. Although many studies have been reported on these enzymes, few functional, biochemical, and structural data are currently available on α-CAs isolated from photosynthetic organisms. Here, we give an overview of the most recent literature on the topic. In higher plants, these enzymes are engaged in both supplying CO2 at the Rubisco and determining proton concentration in PSII membranes, while in algae and cyanobacteria they are involved in carbon-concentrating mechanism (CCM), photosynthetic reactions and in detecting or signaling changes in the CO2 level in the environment. Crystal structures are only available for three algal α-CAs, thus not allowing to associate specific structural features to cellular localizations or physiological roles. Therefore, further studies on α-CAs from photosynthetic organisms are strongly needed to provide insights into their structure-function relationship.


Assuntos
Anidrases Carbônicas , Animais , Dióxido de Carbono , Anidrases Carbônicas/metabolismo , Fotossíntese/fisiologia , Plantas/metabolismo , Prótons , Ribulose-Bifosfato Carboxilase
13.
Mar Pollut Bull ; 184: 114193, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36209535

RESUMO

Present study investigated composition and distribution of chromophytic phytoplankton in the Bohai Sea (BS) and the Yellow Sea (YS) by using rbcL genes. Bacillariophyceae, Haptophyceae and Pelagophyceae were the most abundant phytoplankton groups. Distinct phytoplankton communities were observed in the BS and the YS: offshore stations were dominated by bloom forming genera Thalassiosira and Skeletonema, while brown tide-forming species including Chrysochromulina spp. and Aureococcus anophagefferens were commonly found in the nearshore areas. Redundancy analysis showed that phosphate, temperature and silicic acid play key roles in structuring chromophytic phytoplankton, such as phytoplankton at nearshore stations were affected by nutrient runoff from adjacent rivers (Yellow River). Anthropogenic activities in the Bohai Sea and seasonal circulation of ocean currents may also contribute to shaping chromophytic phytoplankton communities. This study provides data support and foundational observations of chromophytic phytoplankton in the BS and the YS, and their responses to environmental gradients and human activities.


Assuntos
Diatomáceas , Estramenópilas , China , Fosfatos , Fitoplâncton/fisiologia , Ribulose-Bifosfato Carboxilase/genética , Rios , Ácido Silícico
14.
BMC Plant Biol ; 22(1): 486, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224553

RESUMO

BACKGROUND: The timing of bud break is very important for the flowering and fruiting of longan. To obtain new insights into the underlying regulatory mechanism of bud break in longan, a comparative analysis was conducted in three flower induction stages of two longan varieties with opposite flowering phenotypes by using isobaric tags for relative and absolute quantification (iTRAQ). RESULTS: In total, 3180 unique proteins were identified in 18 samples, and 1101 differentially abundant proteins (DAPs) were identified. "SX" ("Shixia"), a common longan cultivated variety that needs an appropriate period of low temperatures to accumulate energy and nutrients for flower induction, had a strong primary inflorescence, had a strong axillary inflorescence, and contained high contents of sugars, and most DAPs during the bud break process were enriched in assimilates and energy metabolism. Combined with our previous transcriptome data, it was observed that sucrose synthase 6 (SS6) and granule-bound starch synthase 1 (GBSSI) might be the key DAPs for "SX" bud break. Compared to those of "SX", the primary inflorescence, axillary inflorescence, floral primordium, bract, and prophyll of "SJ" ("Sijimi") were weaker. In addition, light, rather than a high sugar content or chilling duration, might act as the key signal for triggering bud break. In addition, catalase isozyme 1, an important enzyme in the redox cycle, and RuBisCO, a key enzyme in the Calvin cycle of photosynthetic carbon assimilation, might be the key DAPs for SJ bud break. CONCLUSION: Our results present a dynamic picture of the bud break of longan, not only revealing the temporal specific expression of key candidate genes and proteins but also providing a scientific basis for the genetic improvement of this fruit tree species.


Assuntos
Proteômica , Sintase do Amido , Acrilatos , Carbono , Catalase/genética , Flores/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoenzimas/genética , Ribulose-Bifosfato Carboxilase/genética , Sapindaceae , Sintase do Amido/genética , Açúcares
15.
J Microbiol Biotechnol ; 32(10): 1292-1298, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36224752

RESUMO

Soil autotrophic bacterial communities play a significant role in the soil carbon (C) cycle in paddy fields, but little is known about how rhizosphere soil microorganisms respond to different long-term (35 years) fertilization practices under double rice cropping ecosystems in southern China. Here, we investigated the variation characteristics of rhizosphere soil RubisCO gene cbbL in the double rice ecosystems of in southern China where such fertilization practices are used. For this experiment we set up the following fertilizer regime: without any fertilizer input as a control (CK), inorganic fertilizer (MF), straw returning (RF), and organic and inorganic fertilizer (OM). We found that abundances of cbbL, 16S rRNA genes and RubisCO activity in rhizosphere soil with OM, RF and MF treatments were significantly higher than that of CK treatment. The abundances of cbbL and 16S rRNA genes in rhizosphere soil with OM treatment were 5.46 and 3.64 times higher than that of CK treatment, respectively. Rhizosphere soil RubisCO activity with OM and RF treatments increased by 50.56 and 45.22%, compared to CK treatment. Shannon and Chao1 indices for rhizosphere soil cbbL libraries with RF and OM treatments increased by 44.28, 28.56, 29.60, and 23.13% compared to CK treatment. Rhizosphere soil cbbL sequences with MF, RF and OM treatments mainly belonged to Variovorax paradoxus, uncultured proteobacterium, Ralstonia pickettii, Thermononospora curvata, and Azoarcus sp.KH33C. Meanwhile, cbbL-carrying bacterial composition was obviously influenced by soil bulk density, rhizosphere soil dissolved organic C, soil organic C, and microbial biomass C contents. Fertilizer practices were the principal factor influencing rhizosphere soil cbbL-carrying bacterial communities. These results showed that rhizosphere soil autotrophic bacterial communities were significantly changed under conditions of different long-term fertilization practices Therefore, increasing rhizosphere soil autotrophic bacteria community with crop residue and organic manure practices was found to be beneficial for management of double rice ecosystems in southern China.


Assuntos
Fertilizantes , Oryza , Fertilizantes/análise , Solo/química , Oryza/microbiologia , Rizosfera , Ecossistema , Dióxido de Carbono , Ribulose-Bifosfato Carboxilase/genética , RNA Ribossômico 16S/genética , Microbiologia do Solo , Bactérias/genética , China , Agricultura/métodos
16.
ACS Synth Biol ; 11(10): 3405-3413, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36219726

RESUMO

Carbon-neutral production of valuable bioproducts is critical to sustainable development but remains limited by the slow engineering of photosynthetic organisms. Improving existing synthetic biology tools to engineer model organisms to fix carbon dioxide is one route to overcoming the limitations of photosynthetic organisms. In this work, we describe a pipeline that enabled the deletion of a conditionally essential gene from the Shewanella oneidensis MR-1 genome. S. oneidensis is a simple bacterial host that could be used for electricity-driven conversion of carbon dioxide in the future with further genetic engineering. We used Flux Balance Analysis (FBA) to model carbon and energy flows in central metabolism and assess the effects of single and double gene deletions. We modeled the growth of deletion strains under several alternative conditions to identify substrates that restore viability to an otherwise lethal gene knockout. These predictions were tested in vivo using a Mobile-CRISPRi gene knockdown system. The information learned from FBA and knockdown experiments informed our strategy for gene deletion, allowing us to successfully delete an "expected essential" gene, gpmA. FBA predicted, knockdown experiments supported, and deletion confirmed that the "essential" gene gpmA is not needed for survival, dependent on the medium used. Removal of gpmA is a first step toward driving electrode-powered CO2 fixation via RuBisCO. This work demonstrates the potential for broadening the scope of genetic engineering in S. oneidensis as a synthetic biology chassis. By combining computational analysis with a CRISPRi knockdown system in this way, one can systematically assess the impact of conditionally essential genes and use this knowledge to generate mutations previously thought unachievable.


Assuntos
Genes Essenciais , Shewanella , Dióxido de Carbono/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Shewanella/genética , Shewanella/metabolismo , Deleção de Genes
17.
Science ; 378(6616): 137-138, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36227972

RESUMO

Analysis of Rubisco evolution could inform how to engineer a better enzyme.


Assuntos
Dióxido de Carbono , Ribulose-Bifosfato Carboxilase , Fotossíntese , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo
18.
Science ; 378(6616): 155-160, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36227987

RESUMO

The evolution of ribulose-1,5-bisphosphate carboxylase/oxygenases (Rubiscos) that discriminate strongly between their substrate carbon dioxide and the undesired side substrate dioxygen was an important event for photosynthetic organisms adapting to an oxygenated environment. We use ancestral sequence reconstruction to recapitulate this event. We show that Rubisco increased its specificity and carboxylation efficiency through the gain of an accessory subunit before atmospheric oxygen was present. Using structural and biochemical approaches, we retrace how this subunit was gained and became essential. Our work illuminates the emergence of an adaptation to rising ambient oxygen levels, provides a template for investigating the function of interactions that have remained elusive because of their essentiality, and sheds light on the determinants of specificity in Rubisco.


Assuntos
Dióxido de Carbono , Domínio Catalítico , Evolução Molecular , Ribulose-Bifosfato Carboxilase , Dióxido de Carbono/química , Oxigênio/química , Fotossíntese , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Especificidade por Substrato , Domínio Catalítico/genética , Metagenoma , Firmicutes/enzimologia
19.
Microsc Res Tech ; 85(11): 3694-3706, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36250444

RESUMO

Calcium oxalate (CaOx) crystals in plants are formed in crystal idioblasts cells and have specific geometric shapes. Their proposed functions include calcium homeostasis and carbon source, among others. Amaranth is a plant that presents high tolerance to abiotic stresses and accumulates considerable amounts of CaOx crystals; however, few studies have focused on characterizing the crystals ultrastructure and none is related to identifying proteins bound to them. This information is of great interest to understand the mechanisms related to CaOx crystal formation and to support their proposed functions. Thus, this work aimed to characterize CaOx crystals in amaranth leaves. Crystals were purified and the proteins bound to them were isolated and identified by nLC-MS/MS. Leaf sections were analyzed by light and electron microscopy. The identified proteins were related to the chloroplast such as ATPb synthase, RuBisCO large subunit, and cell wall-related proteins, which were validated by immunohistochemistry and immunogold labeling. In addition, it was observed that CaOx crystal idioblasts were formed from parenchyma cells associated with mesophyll and veins, in which the thylakoid membranes of degraded chloroplasts turned into crystal chambers. These results significantly advance our understanding of the mechanisms of CaOx crystal formation and the potential function as an alternative carbon source in leaves.


Assuntos
Oxalato de Cálcio , Cálcio , Oxalato de Cálcio/química , Carbono , Cloroplastos/metabolismo , Cristalização , Ribulose-Bifosfato Carboxilase , Espectrometria de Massas em Tandem
20.
PLoS One ; 17(9): e0272822, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36125987

RESUMO

Polar microalgae face two major challenges: 1- growing at temperatures (-1.7 to 5°C) that limit enzyme kinetics; and 2- surviving and exploiting a wide range of irradiance. The objective of this study is to understand the adaptation of an Arctic diatom to its environment by studying its ability to acclimate to changes in light and temperature. We acclimated the polar diatom Chaetoceros neogracilis to various light levels at two different temperatures and studied its growth and photosynthetic properties using semi-continuous cultures. Rubisco content was high, to compensate for low catalytic rates, but did not change detectably with growth temperature. Contrary to what is observed in temperate species, in C. neogracilis, carbon fixation rate (20 min 14C incorporation) equaled net growth rate (µ) suggesting very low or very rapid (<20 min) re-oxidation of the newly fixed carbon. The comparison of saturation irradiances for electron transport, oxygen net production and carbon fixation revealed alternative electron pathways that could provide energy and reducing power to the cell without consuming organic carbon which is a very limiting product at low temperatures. High protein contents, low re-oxidation of newly fixed carbon and the use of electron pathways alternative to carbon fixation may be important characteristics allowing efficient growth under those extreme environmental conditions.


Assuntos
Diatomáceas , Carbono/metabolismo , Oxigênio , Ribulose-Bifosfato Carboxilase/metabolismo , Temperatura
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