Structure of cryptophyte photosystem II-light-harvesting antennae supercomplex.

Cryptophytes are ancestral photosynthetic organisms evolved from red algae through secondary endosymbiosis. They have developed alloxanthin-chlorophyll a/c2-binding proteins (ACPs) as light-harvesting complexes (LHCs). The distinctive properties of cryptophytes contribute to efficient oxygenic photosynthesis and underscore the evolutionary relationships of red-lineage plastids. Here we present the cryo-electron microscopy structure of the Photosystem II (PSII)-ACPII supercomplex from the cryptophyte Chroomonas placoidea. The structure includes a PSII dimer and twelve ACPII monomers forming four linear trimers. These trimers structurally resemble red algae LHCs and cryptophyte ACPI trimers that associate with Photosystem I (PSI), suggesting their close evolutionary links. We also determine a Chl a-binding subunit, Psb-γ, essential for stabilizing PSII-ACPII association. Furthermore, computational calculation provides insights into the excitation energy transfer pathways. Our study lays a solid structural foundation for understanding the light-energy capture and transfer in cryptophyte PSII-ACPII, evolutionary variations in PSII-LHCII, and the origin of red-lineage LHCIIs.

Optimization design of semi-open impeller based on thrombogenicity in a blood pump.

BACKGROUND: Blood clots are composed of aggregated fibrin and platelets, and thrombosis is the body's natural response to repairing injured blood vessels or stopping bleeding. However, when this process is activated abnormally, such as in a mechanical blood pump, it can lead to excessive thrombus formation. Therefore, how to avoid or reduce the probability of thrombus formation is an important indicator of the stable operation of a blood pump. METHODS: In this paper, Lagrangian particle tracking trajectories are simulated to study platelet transport in a blood pump. The design of the thrombus blood pump was optimized using an orthogonal design method based on three factors: inlet angle, outlet angle, and blade number. The effect of blood pump pressure, rotational speed, impeller outlet angle, inlet angle, and number of blades on thrombus formation was analysed using Fluent software. The thrombogenic potential was derived by analyzing the trajectory and flow parameters of platelet particles in the blood pump, as well as the statistical parameters of residence time and stress accumulation thrombus in the platelet pump. RESULTS: When the impeller inlet angle is 30°, the outlet angle is 20°, and the number of blades is 6, the probability of thrombus formation is minimized in the orthogonal design method, aligning with the requirements for blood pump performance. CONCLUSIONS: These design parameters serve as a numerical guideline for optimizing the geometry of the semi-open impeller in blood pumps and provide a theoretical foundation for subsequent in vitro experiments.

Conjugated Polymer/Recombinant Escherichia coli Biohybrid Systems for Photobiocatalytic Hydrogen Production.

Biohybrid photocatalysts are composite materials that combine the efficient light-absorbing properties of synthetic materials with the highly evolved metabolic pathways and self-repair mechanisms of biological systems. Here, we show the potential of conjugated polymers as photosensitizers in biohybrid systems by combining a series of polymer nanoparticles with engineered Escherichia coli cells. Under simulated solar light irradiation, the biohybrid system consisting of fluorene/dibenzo [b,d]thiophene sulfone copolymer (LP41) and recombinant E. coli (i.e., a LP41/HydA BL21 biohybrid) shows a sacrificial hydrogen evolution rate of 3.442 mmol g-1 h-1 (normalized to polymer amount). It is over 30 times higher than the polymer photocatalyst alone (0.105 mmol g-1 h-1), while no detectable hydrogen was generated from the E. coli cells alone, demonstrating the strong synergy between the polymer nanoparticles and bacterial cells. The differences in the physical interactions between synthetic materials and microorganisms, as well as redox energy level alignment, elucidate the trends in photochemical activity. Our results suggest that organic semiconductors may offer advantages, such as solution processability, low toxicity, and more tunable surface interactions with the biological components over inorganic materials.

A multilevel investigation to reveal the regulatory mechanism of lignin accumulation in juice sac granulation of pomelo.

Granulation of juice sacs is a physiological disorder, which affects pomelo fruit quality. Here, the transcriptome and ubiquitinome of the granulated juice sacs were analyzed in Guanxi pomelo. We found that lignin accumulation in the granulated juice sacs was regulated at transcription and protein modification levels. In transcriptome data, we found that the genes in lignin biosynthesis pathway and antioxidant enzyme system of the granulated juice sacs were significantly upregulated. However, in ubiquitinome data, we found that ubiquitinated antioxidant enzymes increased in abundance but the enzyme activities decreased after the modification, which gave rise to reactive oxygen species (ROS) contents in granulated juice sacs. This finding suggests that ubiquitination level of the antioxidant enzymes is negatively correlated with the enzyme activities. Increased H2O2 is considered to be a signaling molecule to activate the key gene expressions in lignin biosynthesis pathway, which leads to the lignification in granulated juice sacs of pomelo. This regulatory mechanism in juice sac granulation of pomelo was further confirmed through the verification experiment using tissue culture by adding H2O2 or dimethylthiourea (DMTU). Our findings suggest that scavenging H2O2 and other ROS are important for reducing lignin accumulation, alleviating juice sac granulation and improving pomelo fruit quality.

Architecture of symbiotic dinoflagellate photosystem I-light-harvesting supercomplex in Symbiodinium.

Symbiodinium are the photosynthetic endosymbionts for corals and play a vital role in supplying their coral hosts with photosynthetic products, forming the nutritional foundation for high-yield coral reef ecosystems. Here, we determine the cryo-electron microscopy structure of Symbiodinium photosystem I (PSI) supercomplex with a PSI core composed of 13 subunits including 2 previously unidentified subunits, PsaT and PsaU, as well as 13 peridinin-Chl a/c-binding light-harvesting antenna proteins (AcpPCIs). The PSI-AcpPCI supercomplex exhibits distinctive structural features compared to their red lineage counterparts, including extended termini of PsaD/E/I/J/L/M/R and AcpPCI-1/3/5/7/8/11 subunits, conformational changes in the surface loops of PsaA and PsaB subunits, facilitating the association between the PSI core and peripheral antennae. Structural analysis and computational calculation of excitation energy transfer rates unravel specific pigment networks in Symbiodinium PSI-AcpPCI for efficient excitation energy transfer. Overall, this study provides a structural basis for deciphering the mechanisms governing light harvesting and energy transfer in Symbiodinium PSI-AcpPCI supercomplexes adapted to their symbiotic ecosystem, as well as insights into the evolutionary diversity of PSI-LHCI among various photosynthetic organisms.

Nanoengineering Carboxysome Shells for Protein Cages with Programmable Cargo Targeting.

Protein nanocages have emerged as promising candidates for enzyme immobilization and cargo delivery in biotechnology and nanotechnology. Carboxysomes are natural proteinaceous organelles in cyanobacteria and proteobacteria and have exhibited great potential in creating versatile nanocages for a wide range of applications given their intrinsic characteristics of self-assembly, cargo encapsulation, permeability, and modularity. However, how to program intact carboxysome shells with specific docking sites for tunable and efficient cargo loading is a key question in the rational design and engineering of carboxysome-based nanostructures. Here, we generate a range of synthetically engineered nanocages with site-directed cargo loading based on an α-carboxysome shell in conjunction with SpyTag/SpyCatcher and Coiled-coil protein coupling systems. The systematic analysis demonstrates that the cargo-docking sites and capacities of the carboxysome shell-based protein nanocages could be precisely modulated by selecting specific anchoring systems and shell protein domains. Our study provides insights into the encapsulation principles of the α-carboxysome and establishes a solid foundation for the bioengineering and manipulation of nanostructures capable of capturing cargos and molecules with exceptional efficiency and programmability, thereby enabling applications in catalysis, delivery, and medicine.

Structural diversity and modularity of photosynthetic RC-LH1 complexes.

Bacterial photosynthesis is essential for sustaining life on Earth as it aids in carbon assimilation, atmospheric composition, and ecosystem maintenance. Many bacteria utilize anoxygenic photosynthesis to convert sunlight into chemical energy while producing organic matter. The core machinery of anoxygenic photosynthesis performed by purple photosynthetic bacteria and Chloroflexales is the reaction center-light-harvesting 1 (RC-LH1) pigment-protein supercomplex. In this review, we discuss recent structural studies of RC-LH1 core complexes based on the advancement in structural biology techniques. These studies have provided fundamental insights into the assembly mechanisms, structural variations, and modularity of RC-LH1 complexes across different bacterial species, highlighting their functional adaptability. Understanding the natural architectures of RC-LH1 complexes will facilitate the design and engineering of artificial photosynthetic systems, which can enhance photosynthetic efficiency and potentially find applications in sustainable energy production and carbon capture.

Dynamic Changes in the Thylakoid Proteome of Cyanobacteria during Light-Regulated Thylakoid Membrane Development.

Cyanobacteria were among the oldest organisms to undertake oxygenic photosynthesis and have an essential impact on the atmosphere and carbon/nitrogen cycles on the planet. The thylakoid membrane of cyanobacteria represents an intricate compartment that houses a variety of multi-component (pigment-)protein complexes, assembly factors, and regulators, as well as transporters involved in photosynthetic light reactions, and respiratory electron transport. How these protein components are incorporated into membranes during thylakoid formation and how individual complexes are regulated to construct the functional machinery remains elusive. Here, we carried out an in-depth statistical analysis of the thylakoid proteome data obtained during light-induced thylakoid membrane biogenesis in the model cyanobacterium Synechococcus elongatus PCC 7942. A total of 1581 proteins were experimentally quantified, among which 457 proteins demonstrated statistically significant variations in abundance at distinct thylakoid biogenesis stages. Gene Ontology and KEGG enrichment analysis revealed that predominantly photosystems, light-harvesting antennae, ABC transporters, and pathway enzymes involved in oxidative stress responses and protein folding exhibited notable alternations in abundance between high light and growth light. Moreover, through cluster analysis the 1581 proteins were categorized into six distinct clusters that have significantly different trajectories of the change in their abundance during thylakoid development. Our study provides insights into the physiological regulation for the membrane integration of protein components and functionally linked complexes during the cyanobacterial TM biogenesis process. The findings and analytical methodologies developed in this study may be valuable for studying the global responses of TM biogenesis and photosynthetic acclimation in plants and algae.

Making the connections: physical and electric interactions in biohybrid photosynthetic systems.

Biohybrid photosynthesis systems, which combine biological and non-biological materials, have attracted recent interest in solar-to-chemical energy conversion. However, the solar efficiencies of such systems remain low, despite advances in both artificial photosynthesis and synthetic biology. Here we discuss the potential of conjugated organic materials as photosensitisers for biological hybrid systems compared to traditional inorganic semiconductors. Organic materials offer the ability to tune both photophysical properties and the specific physicochemical interactions between the photosensitiser and biological cells, thus improving stability and charge transfer. We highlight the state-of-the-art and opportunities for new approaches in designing new biohybrid systems. This perspective also summarises the current understanding of the underlying electron transport process and highlights the research areas that need to be pursued to underpin the development of hybrid photosynthesis systems.

Chemoautotrophic production of gaseous hydrocarbons, bioplastics and osmolytes by a novel Halomonas species.

BACKGROUND: Production of relatively low value, bulk commodity chemicals and fuels by microbial species requires a step-change in approach to decrease the capital and operational costs associated with scaled fermentation. The utilisation of the robust and halophilic industrial host organisms of the genus Halomonas could dramatically decrease biomanufacturing costs owing to their ability to grow in seawater, using waste biogenic feedstocks, under non-sterile conditions. RESULTS: We describe the isolation of Halomonas rowanensis, a novel facultative chemoautotrophic species of Halomonas from a natural brine spring. We investigated the ability of this species to produce ectoine, a compound of considerable industrial interest, under heterotrophic conditions. Fixation of radiolabelled NaH14CO3 by H. rowanensis was confirmed in mineral medium supplied with thiosulfate as an energy source. Genome sequencing suggested carbon fixation proceeds via a reductive tricarboxylic acid cycle, and not the Calvin-Bensen-Bassham cycle. The mechanism of energy generation to support chemoautotrophy is unknown owing to the absence of an annotated SOX-based thiosulfate-mediated energy conversion system. We investigated further the biotechnological potential of the isolated H. rowanensis by demonstrating production of the gaseous hydrocarbon (bio-propane), bioplastics (poly-3-hydroxybutyrate) and osmolytes (ectoine) under heterotrophic and autotrophic CO2 fixation growth conditions. CONCLUSIONS: This proof-of-concept study illustrates the value of recruiting environmental isolates as industrial hosts for chemicals biomanufacturing, where CO2 utilisation could replace, or augment, the use of biogenic feedstocks in non-sterile, industrialised bioreactors.

Intrinsically disordered CsoS2 acts as a general molecular thread for α-carboxysome shell assembly.

Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In α-carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineer α-carboxysome shells using minimal shell components and determine cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal "molecular thread" stitching through multiple shell protein interfaces. We further uncover in CsoS2 a highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-governed carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.

Unravelling the Roles of Integral Polypeptides in Excitation Energy Transfer of Photosynthetic RC-LH1 Supercomplexes.

Elucidating the photosynthetic processes that occur within the reaction center-light-harvesting 1 (RC-LH1) supercomplexes from purple bacteria is crucial for uncovering the assembly and functional mechanisms of natural photosynthetic systems and underpinning the development of artificial photosynthesis. Here, we examined excitation energy transfer of various RC-LH1 supercomplexes of Rhodobacter sphaeroides using transient absorption spectroscopy, coupled with lifetime density analysis, and studied the roles of the integral transmembrane polypeptides, PufX and PufY, in energy transfer within the RC-LH1 core complex. Our results show that the absence of PufX increases both the LH1 → RC excitation energy transfer lifetime and distribution due to the role of PufX in defining the interaction and orientation of the RC within the LH1 ring. While the absence of PufY leads to the conformational shift of several LH1 subunits toward the RC, it does not result in a marked change in the excitation energy transfer lifetime.

Engineering α-carboxysomes into plant chloroplasts to support autotrophic photosynthesis.

The growth in world population, climate change, and resource scarcity necessitate a sustainable increase in crop productivity. Photosynthesis in major crops is limited by the inefficiency of the key CO2-fixing enzyme Rubisco, owing to its low carboxylation rate and poor ability to discriminate between CO2 and O2. In cyanobacteria and proteobacteria, carboxysomes function as the central CO2-fixing organelles that elevate CO2 levels around encapsulated Rubisco to enhance carboxylation. There is growing interest in engineering carboxysomes into crop chloroplasts as a potential route for improving photosynthesis and crop yields. Here, we generate morphologically correct carboxysomes in tobacco chloroplasts by transforming nine carboxysome genetic components derived from a proteobacterium. The chloroplast-expressed carboxysomes display a structural and functional integrity comparable to native carboxysomes and support autotrophic growth and photosynthesis of the transplastomic plants at elevated CO2. Our study provides proof-of-concept for a route to engineering fully functional CO2-fixing modules and entire CO2-concentrating mechanisms into chloroplasts to improve crop photosynthesis and productivity.

Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome.

Carboxysomes are proteinaceous bacterial microcompartments that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, encapsulating several enzymes, including ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), responsible for the first step of the Calvin-Benson-Bassham cycle. Despite their significance in carbon fixation and great bioengineering potentials, the structural understanding of native carboxysomes is currently limited to low-resolution studies. Here, we report the characterization of a native α-carboxysome from a marine cyanobacterium by single-particle cryoelectron microscopy (cryo-EM). We have determined the structure of its RuBisCO enzyme, and obtained low-resolution maps of its icosahedral shell, and of its concentric interior organization. Using integrative modeling approaches, we have proposed a complete atomic model of an intact carboxysome, providing insight into its organization and assembly. This is critical for a better understanding of the carbon fixation mechanism and toward repurposing carboxysomes in synthetic biology for biotechnological applications.

Million-atom molecular dynamics simulations reveal the interfacial interactions and assembly of plant PSII-LHCII supercomplex.

Protein-protein interface interactions dictate efficient excitation energy transfer from light-harvesting antennas to the photosystem II (PSII) core. In this work, we construct a 1.2 million atom-scale model of plant C2S2-type PSII-LHCII supercomplex and perform microsecond-scale molecular dynamics (MD) simulations to explore the interactions and assembly mechanisms of the sizeable PSII-LHCII supercomplex. We optimize the nonbonding interactions of the PSII-LHCII cryo-EM structure using microsecond-scale MD simulations. Binding free energy calculations with component decompositions reveal that hydrophobic interactions predominantly drive antenna-core association and the antenna-antenna interactions are relatively weak. Despite the positive electrostatic interaction energies, hydrogen bonds and salt bridges mainly provide directional or anchoring forces for interface binding. Analysis of the roles of small intrinsic subunits of PSII suggests that LHCII and CP26 first interact with small intrinsic subunits and then bind to the core proteins, whereas CP29 adopts a one-step binding process to the PSII core without the assistance of other factors. Our study provides insights into the molecular underpinnings of the self-organization and regulation of plant PSII-LHCII. It lays the framework for deciphering the general assembly principles of photosynthetic supercomplexes and possibly other macromolecular structures. The finding also has implications for repurposing photosynthetic systems to enhance photosynthesis.

Synthetic engineering of a new biocatalyst encapsulating [NiFe]-hydrogenases for enhanced hydrogen production.

Hydrogenases are microbial metalloenzymes capable of catalyzing the reversible interconversion between molecular hydrogen and protons with high efficiency, and have great potential in the development of new electrocatalysts for renewable fuel production. Here, we engineered the intact proteinaceous shell of the carboxysome, a self-assembling protein organelle for CO2 fixation in cyanobacteria and proteobacteria, and sequestered heterologously produced [NiFe]-hydrogenases into the carboxysome shell. The protein-based hybrid catalyst produced in E. coli shows substantially improved hydrogen production under both aerobic and anaerobic conditions and enhanced material and functional robustness, compared to unencapsulated [NiFe]-hydrogenases. The catalytically functional nanoreactor as well as the self-assembling and encapsulation strategies provide a framework for engineering new bioinspired electrocatalysts to improve the sustainable production of fuels and chemicals in biotechnological and chemical applications.

Structural basis and evolution of the photosystem I-light-harvesting supercomplex of cryptophyte algae.

Cryptophyte plastids originated from a red algal ancestor through secondary endosymbiosis. Cryptophyte photosystem I (PSI) associates with transmembrane alloxanthin-chlorophyll a/c proteins (ACPIs) as light-harvesting complexes (LHCs). Here, we report the structure of the photosynthetic PSI-ACPI supercomplex from the cryptophyte Chroomonas placoidea at 2.7-Å resolution obtained by crygenic electron microscopy. Cryptophyte PSI-ACPI represents a unique PSI-LHCI intermediate in the evolution from red algal to diatom PSI-LHCI. The PSI-ACPI supercomplex is composed of a monomeric PSI core containing 14 subunits, 12 of which originated in red algae, 1 diatom PsaR homolog, and an additional peptide. The PSI core is surrounded by 14 ACPI subunits that form 2 antenna layers: an inner layer with 11 ACPIs surrounding the PSI core and an outer layer containing 3 ACPIs. A pigment-binding subunit that is not present in any other previously characterized PSI-LHCI complexes, ACPI-S, mediates the association and energy transfer between the outer and inner ACPIs. The extensive pigment network of PSI-ACPI ensures efficient light harvesting, energy transfer, and dissipation. Overall, the PSI-LHCI structure identified in this study provides a framework for delineating the mechanisms of energy transfer in cryptophyte PSI-LHCI and for understanding the evolution of photosynthesis in the red lineage, which occurred via secondary endosymbiosis.

Cryo-EM structure of a monomeric RC-LH1-PufX supercomplex with high-carotenoid content from Rhodobacter capsulatus.

In purple photosynthetic bacteria, the photochemical reaction center (RC) and light-harvesting complex 1 (LH1) assemble to form monomeric or dimeric RC-LH1 membrane complexes, essential for bacterial photosynthesis. Here, we report a 2.59-Å resolution cryoelectron microscopy (cryo-EM) structure of the RC-LH1 supercomplex from Rhodobacter capsulatus. We show that Rba. capsulatus RC-LH1 complexes are exclusively monomers in which the RC is surrounded by a 15-subunit LH1 ring. Incorporation of a transmembrane polypeptide PufX leads to a large opening within the LH1 ring. Each LH1 subunit associates two carotenoids and two bacteriochlorophylls, which is similar to Rba. sphaeroides RC-LH1 but more than one carotenoid per LH1 in Rba. veldkampii RC-LH1 monomer. Collectively, the unique Rba. capsulatus RC-LH1-PufX represents an intermediate structure between Rba. sphaeroides and Rba. veldkampii RC-LH1-PufX. Comparison of PufX from the three Rhodobacter species indicates the important residues involved in dimerization of RC-LH1.

In situ visualization of Braun's lipoprotein on E. coli sacculi.

Braun's lipoprotein (Lpp) plays a major role in stabilizing the integrity of the cell envelope in Escherichia coli, as it provides a covalent cross-link between the outer membrane and the peptidoglycan layer. An important challenge in elucidating the physiological role of Lpp lies in attaining a detailed understanding of its distribution on the peptidoglycan layer. Here, using atomic force microscopy, we visualized Lpp directly on peptidoglycan sacculi. Lpp is homogeneously distributed over the outer surface of the sacculus at a high density. However, it is absent at the constriction site during cell division, revealing its role in the cell division process with Pal, another cell envelope-associated protein. Collectively, we have established a framework to elucidate the distribution of Lpp and other peptidoglycan-bound proteins via a direct imaging modality.

Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.