Enhance CO2 Fixation Research Articles

Enhancing CO2 fixation by microalgae in a Photobioreactor: Molecular mechanisms with exogenous carbonic anhydrase

Microalgae biotechnology holds great potential for mitigating CO2 emissions, yet faces challenges in commercialization due to suboptimal photosynthetic efficiency. This study presents an innovative approach to improve CO2 mass transfer efficiency in microalgae using carbonic anhydrase (CA) in an internal LED flexible air-lift photobioreactor. Optimal conditions initial inoculation with 3.55 × 106 cells/mL and 20 % CO2 concentration, complemented by white LED lighting in Chlorella sp. CA regulated intracellular composition, enhancing chlorophyll, lipid, and protein contents. Metabolomics revealed elevated malic and succinic acids, associated with increased Ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) and Acetoacetyl coenzyme A (Acetyl-CoA) activities, facilitating efficient carbon fixation. CA also mitigated cellular oxidative stress by reducing reactive oxygen species (ROS). Furthermore, CA improved extracellular electron acceptor with currents surpassed CK. This CA-based microalgae biotechnology provides a foundation for future commercial applications, addressing CO2 emissions.

Recent advances in engineering fast-growing cyanobacterial species for enhanced CO2 fixation

Atmospheric CO2 removal (CDR) is a fundamentally endergonic process. Performing CDR or Bioenergy with Carbon Capture and Storage (BECCS) at the gigatonne scale will produce a significant additional burden on the planet’s limited renewable energy resources irrespective of the technology employed. Harnessing photosynthesis to drive industrial-scale CO2 fixation has been of significant interest because of its minimal energy requirements and potential low costs. In this review, we evaluated the thermodynamic considerations of performing atmospheric carbon removal using microalgae and cyanobacteria versus physicochemical processes and explore the implications of these energetic costs on the scalability of each respective solution. We review the biomass productivities of recently discovered fast-growing cyanobacterial strains and discuss the prospects of genetically engineering certain metabolic pathways for channeling the fixed carbon into metabolic ‘carbon sinks’ to further enhance their CO2 capture while concurrently extracting value. We share our perspectives on how new highly productive chassis strains combined with advanced flux balance models, essentially coupling synthetic biology with industrial biotechnology, may unlock more favorable methods for CDR, both from an economic and thermodynamic perspective.

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP.

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.

Facile functionalization iron in zeolitic imidazole framework-67 under solvent-free conditions for enhancing CO2 fixation reaction

The heterometal functionalization on zeolitic-imidazole frameworks (ZIFs) is a strategy to modify properties over the homometal ZIFs. A green strategy for simultaneously functionalized iron atoms during the crystal formation of ZIF-67 (Fe@ZIF-67) through the solid-solid thermal (SST) method is newly developed. The single-step process and absent solvent conditions are beneficial advantages of this SST method over the early reported synthetic methods. The results show that the iron is well-dispersed and homogeneously coordinated in the structure of Fe@ZIF-67. Moreover, the optimal iron-functionalized ZIF-67 (20Fe@ZIF-67) significantly enhances the catalytic performance of CO2 cycloaddition epoxide by 2.3 times compared to pristine ZIF-67 under mild reaction conditions. The DFT calculation reveals that the iron atom induces the unsaturated sites or defect structure on the Fe@ZIF-67 catalyst, providing more reactive sites for the catalytic activity of the reaction.

Advances in succinic acid production: the enhancement of CO2 fixation for the carbon sequestration benefits.

Succinic acid (SA), one of the 12 top platform chemicals produced from biomass, is a precursor of various high value-added derivatives. Specially, 1mol CO2 is assimilated in 1mol SA biosynthetic route under anaerobic conditions, which helps to achieve carbon reduction goals. In this review, methods for enhanced CO2 fixation in SA production and utilization of waste biomass for SA production are reviewed. Bioelectrochemical and bioreactor coupling systems constructed with off-gas reutilization to capture CO2 more efficiently were highlighted. In addition, the techno-economic analysis and carbon sequestration benefits for the synthesis of bio-based SA from CO2 and waste biomass are analyzed. Finally, a droplet microfluidics-based high-throughput screening technique applied to the future bioproduction of SA is proposed as a promising approach.

Enhancing microbial CO2 electrocatalysis for multicarbon reduction in a wet amine-based catholyte.

Microbial CO2 electroreduction (mCO2ER) offers a promising approach for producing high-value multicarbon reductants from CO2 by combining CO2 fixing microorganisms with conducting materials (i. e., cathodes). However, the solubility and availability of CO2 in an aqueous electrolyte pose significant limitations in this system. This study demonstrates the efficient production of long-chain multicarbon reductants, specifically carotenoids (~C40 ), within a wet amine-based catholyte medium during mCO2ER. Optimizing the concentration of the biocompatible CO2 absorbent, monoethanolamine (MEA), led to enhanced CO2 fixation in the electroautotroph bacteria. Molecular biological analyses revealed that MEA in the catholyte medium redirected the carbon flux towards carotenoid biosynthesis during mCO2ER. The faradaic efficiency of mCO2ER with MEA for carotenoid production was 4.5-fold higher than that of the control condition. These results suggest the mass transport bottleneck in bioelectrochemical systems could be effectively addressed by MEA-assissted mCO2ER, enabling highly efficient production of valuable products from CO2 .

When green carbon plants meet synthetic biology

AbstractRecycling carbon dioxide (CO2) into chemicals or fuels presents a promising avenue for mitigating carbon emissions and addressing the energy crisis. Plants serve as the ideal platform for the production of materials and chemicals, thanks to their innate capacity to directly use CO2 in the synthesis of various organic compounds. While conventional methods for enhancing plant CO2 fixation may reach their limits, novel technological solutions are imperative. Synthetic biology has illuminated the potential for biosynthesising multiple carbon sources through artificial CO2 fixation pathways in vitro. Recent breakthroughs in photorespiratory bypasses and artificial carboxylation modules offer significant promise for engineering plants to improve carbon fixation, guiding the design and development of plants with more efficient CO2 utilisation. In this context, we begin by summarising recent progress in designing or engineering in vitro CO2 fixation pathways, as well as those solely established in microbes. Subsequently, we delineate strategies employed to enhance CO2 fixation in plants. Finally, we explore potential methods for introducing artificial CO2 fixation pathways into plants. These advancements are critical in advancing synthetic biology's efforts to tackle future challenges related to food and energy scarcity.

How to Efficiently Produce the Selenium-Enriched Cucumber Fruit with High Yield and Qualities via Hydroponic Cultivation? The Balance between Selenium Supply and CO2 Fertilization

Hydroponic-producing selenium (Se)-biofortified vegetables in a greenhouse is a convenient and effective way to provide Se-enriched food and overcome hidden hunger. CO2 fertilization is commonly implemented to increase vegetable yield in greenhouse production. However, this application accompanies decreased mineral concentrations in the edible parts. Here we investigated the effects of [CO2] and Se supply on the growth, gas exchange, and cucumber fruit quality. A hydroponic experiment with two CO2 concentrations ([CO2]) (C1: 410, and C2: 1200 μmol mol−1) and four Se supply levels (Se0: 0, Se1: 0.125, Se2: 0.250, and Se3: 0.500 mg Se L−1) was carried out. A low level of Se supply (Se1: 0.125 mg Se L−1) protected the photosynthetic pigments and stimulated the stomatal opening, especially under [CO2] fertilization. It leads to a higher net photosynthesis rate (Pn) and transpiration rate (Tr) than other Se treatments. The most significant changes in dry weight, fruit yield, and soluble sugar concentration were also obtained in Se1 under CO2 fertilization due to the enhanced CO2 fixation. Meanwhile, the Se concentration in fruit was 0.63 mg kg−1 FW in C2Se1, with the highest Se accumulation and use efficiency. According to the recommended dietary allowance of 55 μg Se day−1 for adults, an intake of 87 g of cucumber grown in C2Se1 is sufficient. Because of the improved Tr and better root structure in Se1, the uptake of mineral nutrients through mass flow and interception was well maintained under CO2 fertilization. So, the concentrations of N, P, K, Ca, and Mn in cucumber fruits were not significantly decreased by elevated [CO2] in Se1. However, the concentrations of soluble proteins, S, Mg, Fe, and Zn in cucumber fruits in C2Se1 were lower than those in C1Se1, which was mainly attributed to the dilution effects under CO2 fertilization. Therefore, a selenite supply of 0.125 mg Se L−1 was found to be the optimal dosage for producing Se-enriched cucumber fruits with high yield and better qualities under CO2 fertilization (1200 μmol mol−1).

Salicylic acid and thiourea mitigate salinity and drought stress in wheat (Triticum aestivum)

Water and salinity stresses are the major environmental concerns limiting crop production, particularly in arid and semi-arid ecologies. Focused research is required to develop mitigating strategies to manage such stresses. Application of plant-bioregulators (PBRs) may be an effective option to manage water and salinity stresses. Therefore, a field experiment was conducted during 2016–17 and 2017–18 at semi-arid saline site of NW (north-western) India to assess the effectiveness of salicylic acid and thiourea in easing out the adverse effect of variable water deficit and irrigation water salinity regimes. Increasing water deficit and its salinity significantly (P<0.001) reduced all the studied growth parameters, viz. plant height, crop stand, no. of tillers, leaf area index and relative growth rate; and yield attributes like no. of effective tillers, spike length, grains/spike, grain weight/spike, 1000-grain weight, specific weight. Application of either salicylic acid or thiourea significantly improved growth parameters in comparison to control and thiourea proved more effective. Thiourea application improved grain yield by 3.96 and 17.36%, biological yield by 4.21 and 14.82%, effective no. of tillers by 3.77 and 11.91%, weight/spike by 2.83 and 15.17%, 1000-grain weight by 3.72 and 12.66% and specific weight by 2.04 and 8.84% compared to salicylic acid and control, respectively. This might be due to better nutrient uptake, water relations, enhanced CO2 fixation and effective partitioning of assimilates. Thus, thiourea and salicylic acid application in wheat found better as compared to control under saline water stress.

Synergistic effects of carbon cycle metabolism and photosynthesis in Chinese cabbage under salt stress

Chinese cabbage (Brassica rapa ssp. pekinensis) has a long cultivation history and is one of the vegetable crops with the largest cultivation area in China. However, salt stress severely damages photosynthesis and hormone metabolism, nutritional balances, and results in ion toxicity in plants. To better understand the mechanisms of salt-induced growth inhibition in Chinese cabbage, RNA-seq and physiological index determination were conducted to explore the impacts of salt stress on carbon cycle metabolism and photosynthesis in Chinese cabbage. Here, we found that the number of thylakoids and grana lamellae and the content of starch granules and chlorophyll in the leaves of Chinese cabbage under salt stress showed a time-dependent response, first increasing and then decreasing. Chinese cabbage increased the transcript levels of genes related to the photosynthetic apparatus and carbon metabolism under salt stress, probably in an attempt to alleviate damage to the photosynthetic system and enhance CO2 fixation and energy metabolism. The transcription of genes related to starch and sucrose synthesis and degradation were also enhanced; this might have been an attempt to maintain intracellular osmotic pressure by increasing soluble sugar concentrations. Soluble sugars could also be used as potential reactive oxygen species (ROS) scavengers, in concert with peroxidase (POD) enzymes, to eliminate ROS that accumulate during metabolic processes. Our study characterizes the synergistic response network of carbon metabolism and photosynthesis under salt stress.

Enhancement of Carbon Assimilates and Macronutrients in Legumes under Elevated CO2 Concentration

Impact of elevated carbon dioxide (Free Air Carbon dioxide Enrichment) was studied on the plant chlorophyll, plant growth, plant macronutrients, total starch, total carbohydrate and the activity of phosphoenol pyruvate carboxylase (Pep C) enzymatic assays in leaves, pods and seeds in leguminous plant Cyamopsis tetragonoloba. Plants of Cyamopsis tetragonoloba (C3) were exposed to different atmospheric CO2 concentrations 420 ± 20 ppm (ambient) and 550 ± 20ppm (elevated). An average increase in the plant total chlorophyll (+39.17%), total starch (+43.73%, +25.44% and +26.35% in leaves, pods, and seeds), sucrose (+69.77%, +22.27% and +33.77% in leaves, pods and seed) and total carbohydrate (+58.88%, +30.54% and +28.38 % in leaves, pods and seeds) content were found in plant grown under elevated condition when compared to ambient counterpart. More over overall plant growth (+40% height and +25% biomass) increased in [CO2] concentration. Plant total nitrogen (N) content decreased (−12.55% in leaves) under the elevated condition where as total phosphorus (P) decreased (−3.15% in leaves) along with total potassium (K) (−46.63% in leaves). In soil, total potassium (+60.23%) and phosphorous (+48.88%) were found to increase with (−16%) decrease in soil nitrogen content. In seed total nitrogen content increased (+18.15%) on an average with no significant change in total potassium and phosphorus content under e[CO2]. Phosphoenolpyruvate carboxylase enzyme (Pep C) (+139.5% in leaves) activity and total organic carbon (TOC) (+19.12%, +17.85% in leaves and seeds) increases in elevated concentration thus promoting and indicating higher photosynthesis via enhanced CO2 fixation. Thus our studies showed that [CO2] positively promotes sugars, carbohydrates synthesis, translocation and partitioning in plant tissues and enhanced macronutrients level in leaves and seeds tissue which is contradictory to other C3 plants. Thus [CO2] works as the boon for Cyamopsisvarieties and the seeds are nutritionally rich, healthy, balanced in proteins and carbohydrates (C/N) and so these varieties have future implication for industrial use in the agricultural country like India.

Multi-objective tailored optimization deciphering carbon partitioning and metabolomic tuning in response to elevated CO2 levels, organic carbon and sparging period.

Microalgae have garnered much contemplation as candidates to fix CO2 into valuable compounds. Although microalgae have been studied to produce various metabolites, they have not yet proved successful for commercialization. Since, handling such problems practically requires satisfying multiple parameters simultaneously, we put forth a multi-parameter optimization strategy to manipulate the carbon metabolism of Scenedesmus sp. to improve biomass production and enhance CO2 fixation to increase the production of fuel-related metabolites. The Box-Behnken design method was applied with CO2 concentration, CO2 sparging time and glucose concentration as independent variables; biomass and total fatty acid methyl ester (total FAME) content were analyzed as response variables. The strain is supplemented with both CO2 and glucose with an aim to enhance carbon flux and rechannel it towards carbon fixation. As per the results obtained in this study, Scenedesmus sp. could effectively exploit high CO2 concentration (15%) for longer duration under high concentration of glucose supplementation (9g/L) producing a biomass of 635.24±39.9μg/mL with a high total fatty acid methyl ester (FAME) content of 71.29±4.2μg/mg, significant acetyl-CoA carboxylase enzyme activity and a favorable fatty acid profile: 35.8% palmitic acid, 10.5% linoleic acid and 30.6% linolenic acid. The carbohydrate content was maximum at 10% CO2 sparged for the longest duration of 90min under glucose concentration of 9g/L. This study puts forth an optimal design that can provide evidence on comprehending the carbon assimilation mechanism to enhance production of biomass and biofuels and provide conditions to microalgal species to tolerate CO2 rich flue gas.

Dissolved Inorganic Carbon-Accumulating Complexes from Autotrophic Bacteria from Extreme Environments.

In nature, concentrations of dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO2 fixation. Such mechanisms have been well studied in Cyanobacteria, but much remains to be learned about their activity in other phyla. Novel multisubunit membrane-spanning complexes capable of elevating intracellular DIC were recently described in three species of bacteria. Homologs of these complexes are distributed among 17 phyla in Bacteria and Archaea and are predicted to consist of one, two, or three subunits. To determine whether DIC accumulation is a shared feature of these diverse complexes, seven of them, representative of organisms from four phyla, from a variety of habitats, and with three different subunit configurations, were chosen for study. A high-CO2-requiring, carbonic anhydrase-deficient (ΔyadF ΔcynT) strain of Escherichia coli Lemo21(DE3), which could be rescued via elevated intracellular DIC concentrations, was created for heterologous expression and characterization of the complexes. Expression of all seven complexes rescued the ability of E. coli Lemo21(DE3) ΔyadF ΔcynT to grow under low-CO2 conditions, and six of the seven generated measurably elevated intracellular DIC concentrations when their expression was induced. For complexes consisting of two or three subunits, all subunits were necessary for DIC accumulation. Isotopic disequilibrium experiments clarified that CO2 was the substrate for these complexes. In addition, the presence of an ionophore prevented the accumulation of intracellular DIC, suggesting that these complexes may couple proton potential to DIC accumulation. IMPORTANCE To facilitate the synthesis of biomass from CO2, autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multisubunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, raising the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found. This study shows that DIC accumulation is a trait shared by complexes with various subunit structures, from organisms with diverse physiologies and taxonomies, suggesting that this trait is universal among them. Successful expression in E. coli suggests the possibility of their expression in engineered organisms synthesizing compounds of industrial importance from CO2.

Mixed electron donors synergistically enhance CO2 fixation of non-photosynthetic microorganism communities through optimizing community structure to promote cbb gene transcription

Studies have shown that mixed electron donors (MEDs) can enhance the CO2-fixing efficiency of non-photosynthetic microbial communities (NPMCs), even up to the level of fixation observed when H2 is used as an electron donor. However, this promotion effect is not stable because its mechanism remains unclear. To elucidate the mechanisms involved, allowing further regulation and optimization of the MED system for improving the CO2-fixing efficiency of NPMCs consistently, cbb gene transcription level and efficiency, extracellular free organic carbon (EFOC) content as well as microbial structure of NPMCs under MED and other electron donor systems were investigated. MEDs synergistically promoted CO2 fixation efficiency of NPMCs, even producing levels seen when H2 was used as the electron donor. Subsequent experiments revealed that the cbb gene abundance and transcription level in the MED system were high compared with those in other single-electron donor systems; the concentration of EFOC per unit cell was relatively lower than that in any other electron donor system; and the system developed a large number of dominant heterotrophic bacteria such as Enterobacteriaceae and Vibrionaceae. Data analysis revealed a high negative correlation between EFOC concentration per unit cell and cbb gene abundance as well as gene transcription level. These results implied that MEDs can promote a complex microbial community structure enriched with high-efficiency heterotrophic bacteria, which can effectively reduce excessive EFOC generated by NPMCs in the CO2 fixation process, promoting overall cbb gene abundance and transcription level within the NPMC and thus enhancing CO2 fixation.

Enhanced CO2 fixation in the biocatalytic carboxylation of resorcinol: Utilization of amines for amine scrubbing and in situ product precipitation

Abstract Reversible enzymatic decarboxylation reactions are known for their thermodynamic limitations regarding the carboxylation. Significant thermodynamic and kinetic improvements were achieved for the carboxylation of resorcinol by the 2,6-dihydroxybenzoic acid decarboxylase from Rhizobium sp. utilizing the amine scrubber triethanolamine for CO2 mediation. In a first approach, maximal yields of up to 50 % are achieved and are found to correlate with the CO2 loading capacity of triethanolamine. In a second approach, application of quaternary amines for in situ carboxylation product removal by precipitation improves the achievable yields depending on utilized amine concentration. This demonstrates the possibility for a reusable system. By variation of hydrated CO2 species ratio and concentration, CO2 and alternatively H2CO3 is identified acting as second substrate.

Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intent of engineering BMC-based metabolic modules for new functions in synthetic biology.

Enhanced CO2 fixation and lipid production of Chlorella vulgaris through the carbonic anhydrase complex

Photosynthesis of C. vulgaris shows slow growth and low lipid production due to the low solubility of CO2, and it is thus necessary to increase the dissolved inorganic carbon source to solve this problem. In this study, carbonic anhydrase (CA) was fused with dockerin to form a CA complex by cohesion-dockerin interaction. The CA complex was displayed on the surface of C. vulgaris by a cellulose binding module. The CA complex increased activity and stability compared to those of a single enzyme. Additionally, C. vulgaris showed an average of 1.6-fold rapid growth during log phase through the influence of the CA complex. The bicarbonate produced by the CA complex increased the lipid production about 1.7-fold (23.3%), compared to 13.6% for the control group. The present results suggest that the CA complex successfully enhances the CO2 fixation, which should be an essential study for 4th generation biofuels.

Enhancing Scenedesmus obliquus biofilm growth and CO2 fixation in a gas-permeable membrane photobioreactor integrated with additional rough surface

Microalgae biofilm photobioreactors (PBRs) have been gaining more and more attention worldwide attributed to their advantages of lower water requirement and simpler harvesting process. In this work, aiming at enhancing microalgae biofilm growth and gas-liquid mass transfer based on previous biofilm PBRs, a gas-permeable membrane PBR integrated with additional rough surface (GMPBR-RS) was designed. By attaching an additional piece of stainless steel mesh onto the upper surface of the newly prepared gas-permeable membrane, the roughness of the substratum surface for Scenedesmus obliquus biofilm adhesion was enhanced and therefore resulted in a 28.27% improvement in areal biomass density of S. obliquus relative to the control PBR. Moreover, responses of S. obliquus biofilm adsorption growth and CO2 fixation in the GMPBR-RS to various liquid flow rates and gas flow rates were investigated. Notably, the maximum areal biomass density of S. obliquus biofilm and CO2 removal efficiency of the GMPBR-RS reached 31.44 g m−2 and 65.05%, respectively.

Crystal structure of pentameric shell protein CsoS4B of Halothiobacillus neapolitanus α-carboxysome

Carboxysome, encapsulating an enzymatic core within an icosahedral-shaped semipermeable protein shell, could enhance CO2 fixation under low CO2 conditions in the environment. The shell of Halothiobacillus neapolitanus α-carboxysome possesses two 38% sequence-identical pentameric proteins, namely CsoS4A and CsoS4B. However, the functions of two paralogous pentameric proteins in α-carboxysome assembly remain unknown. Here we report the crystal structure of CsoS4B at 2.15 Å resolution. It displays as a stable pentamer, each subunit of which consists of a β-barrel core domain, in addition to an insertion of helix α1 that forms the central pore. Structural comparisons and multiple-sequence alignment strongly indicate that CsoS4A and CsoS4B differ from each other in interacting with various components of α-carboxysome, despite they share a similar overall structure. These findings provide the structural basis for further investigations on the self-assembly process of carboxysome.

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment.

The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO2 fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry, and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single β-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization, and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.