Structure of a PSI-LHCI-cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions.

Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)-light harvesting complex I (LHCI)-cytochrome (cyt) b6f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI-LHCI-LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b6f and fluorescent chlorophyll containing PSI-LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI-LHCI-cyt b6f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Δlhca2 knockout mutant has constitutively enhanced CEF.

RAZA: A Rapid 3D z-crossings algorithm to segment electron tomograms and extract organelles and macromolecules.

Resolving the 3D architecture of cells to atomic resolution is one of the most ambitious challenges of cellular and structural biology. Central to this process is the ability to automate tomogram segmentation to identify sub-cellular components, facilitate molecular docking and annotate detected objects with associated metadata. Here we demonstrate that RAZA (Rapid 3D z-crossings algorithm) provides a robust, accurate, intuitive, fast, and generally applicable segmentation algorithm capable of detecting organelles, membranes, macromolecular assemblies and extrinsic membrane protein domains. RAZA defines each continuous contour within a tomogram as a discrete object and extracts a set of 3D structural fingerprints (major, middle and minor axes, surface area and volume), enabling selective, semi-automated segmentation and object extraction. RAZA takes advantage of the fact that the underlying algorithm is a true 3D edge detector, allowing the axes of a detected object to be defined, independent of its random orientation within a cellular tomogram. The selectivity of object segmentation and extraction can be controlled by specifying a user-defined detection tolerance threshold for each fingerprint parameter, within which segmented objects must fall and/or by altering the number of search parameters, to define morphologically similar structures. We demonstrate the capability of RAZA to selectively extract subgroups of organelles (mitochondria) and macromolecular assemblies (ribosomes) from cellular tomograms. Furthermore, the ability of RAZA to define objects and their contours, provides a basis for molecular docking and rapid tomogram annotation.

Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii.

Photosynthetic organisms developed multiple strategies for balancing light-harvesting versus intracellular energy utilization to survive ever-changing environmental conditions. The light-harvesting complex (LHC) protein family is of paramount importance for this function and can form light-harvesting pigment protein complexes. In this work, we describe detailed analyses of the photosystem II (PSII) LHC protein LHCBM9 of the microalga Chlamydomonas reinhardtii in terms of expression kinetics, localization, and function. In contrast to most LHC members described before, LHCBM9 expression was determined to be very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g., when nutrient availability was limited. LHCBM9 was localized as part of PSII supercomplexes but was not found in association with photosystem I complexes. Knockdown cell lines with 50 to 70% reduced amounts of LHCBM9 showed reduced photosynthetic activity upon illumination and severe perturbation of hydrogen production activity. Functional analysis, performed on isolated PSII supercomplexes and recombinant LHCBM9 proteins, demonstrated that presence of LHCBM9 resulted in faster chlorophyll fluorescence decay and reduced production of singlet oxygen, indicating upgraded photoprotection. We conclude that LHCBM9 has a special role within the family of LHCII proteins and serves an important protective function during stress conditions by promoting efficient light energy dissipation and stabilizing PSII supercomplexes.

Challenges and opportunities for hydrogen production from microalgae.

The global population is predicted to increase from ~7.3 billion to over 9 billion people by 2050. Together with rising economic growth, this is forecast to result in a 50% increase in fuel demand, which will have to be met while reducing carbon dioxide (CO2 ) emissions by 50-80% to maintain social, political, energy and climate security. This tension between rising fuel demand and the requirement for rapid global decarbonization highlights the need to fast-track the coordinated development and deployment of efficient cost-effective renewable technologies for the production of CO2 neutral energy. Currently, only 20% of global energy is provided as electricity, while 80% is provided as fuel. Hydrogen (H2 ) is the most advanced CO2 -free fuel and provides a 'common' energy currency as it can be produced via a range of renewable technologies, including photovoltaic (PV), wind, wave and biological systems such as microalgae, to power the next generation of H2 fuel cells. Microalgae production systems for carbon-based fuel (oil and ethanol) are now at the demonstration scale. This review focuses on evaluating the potential of microalgal technologies for the commercial production of solar-driven H2 from water. It summarizes key global technology drivers, the potential and theoretical limits of microalgal H2 production systems, emerging strategies to engineer next-generation systems and how these fit into an evolving H2 economy.

Solution structure of the RNA-binding cold-shock domain of the Chlamydomonas reinhardtii NAB1 protein and insights into RNA recognition.

Light-harvesting complex (LHC) proteins are among the most abundant proteins on Earth and play critical roles in photosynthesis, both in light capture and in photoprotective mechanisms. The Chlamydomonas reinhardtii nucleic acid-binding protein 1 (NAB1) is a negative regulator of LHC protein translation. Its N-terminal cold-shock domain (CSD) binds to a 13-nt element [CSD consensus sequence (CSDCS)] found in the mRNA of specific LHC proteins associated with Photosystem II (PSII), an interaction which regulates LHC expression and, consequently, PSII-associated antenna size, structure and function. In the present study, we elucidated the solution structure of the NAB1 CSD as determined by heteronuclear NMR. The CSD adopts a characteristic five-stranded anti parallel ß-barrel fold. Upon addition of CSDCS RNA, a large number of NMR chemical shift perturbations were observed, corresponding primarily to surface-exposed residues within the highly conserved ß2- and ß3-strands in the canonical RNA-binding region, but also to residues on ß-strand 5 extending the positive surface patch and the overall RNA-binding site. Additional chemical shift perturbations that accompanied RNA binding involved buried residues, suggesting that transcript recognition is accompanied by conformational change. Our results indicate that NAB1 associates with RNA transcripts through a mechanism involving its CSD that is conserved with mechanisms of sequence-specific nucleic acid recognition employed by ancestrally related bacterial cold-shock proteins (CSPs).

Sulphur responsiveness of the Chlamydomonas reinhardtii LHCBM9 promoter.

MAIN CONCLUSION: A 44-base-pair region in the Chlamydomonas reinhardtii LHCBM9 promoter is essential for sulphur responsiveness. The photosynthetic light-harvesting complex (LHC) proteins play essential roles both in light capture, the first step of photosynthesis, and in photoprotective mechanisms. In contrast to the other LHC proteins and the majority of photosynthesis proteins, the Chlamydomonas reinhardtii photosystem II-associated LHC protein, LHCBM9, was recently reported to be up-regulated under sulphur deprivation conditions, which also induce hydrogen production. Here, we examined the sulphur responsiveness of the LHCBM9 gene at the transcriptional level, through promoter deletion analysis. The LHCBM9 promoter was found to be responsive to sulphur deprivation, with a 44-base-pair region between nucleotide positions -136 and -180 relative to the translation start site identified as essential for this response. Anaerobiosis was found to enhance promoter activity under sulphur deprivation conditions, however, alone was unable to induce promoter activity. The study of LHCBM9 is of biological and biotechnological importance, as its expression is linked to photobiological hydrogen production, theoretically the most efficient process for biofuel production, while the simplicity of using an S-deprivation trigger enables the development of a novel C. reinhardtii-inducible promoter system based on LHCBM9.

Inducible release of particulates from liposomes using the mechanosensitive channel of large conductance and L-α-lysophosphatidylcholine.

The mechanosensitive channel of large conductance (MscL) from Escherichia coli is a prototype for the mechanosensitive class of ion channels and opens one of the largest known gated transmembrane pores. As such, MscL offers the structural framework for the development of liposomal nanovalves for biotechnological applications. Here we incorporated MscL into liposomes and investigated the effects of L-α-lysophosphatidylcholine (LPC) with varying acyl chain lengths or saturation on its pore gating. This was measured by the efflux of encapsulated 5,6-carboxyfluorescein (CF) from the MscL proteoliposomes. Efflux improved in the presence of shorter and double-bonded LPC acyl chains. It was also dependent on the detergent concentration employed during MscL purification. MscL purified in 2 mM dodecyl ß-D-maltopyranoside (DDM) had a marked increase in CF efflux compared to MscL purified in 1 mM DDM when treated with LPC. The purification conditions also resulted in increased efflux from proteoliposomes containing the G22C-MscL pore mutant channel, which requires higher membrane tension for its activation compared to WT-MscL.

Functional similarities between heterogeneously and homogenously expressed MscL constructs.

The mechanosensitive channel of large conductance MscL is a well-characterized mechanically gated non-selective ion channel, which often serves as a prototype mechanosensitive channel for mechanotransduction studies. However, there are some discrepancies between MscL constructs used in these studies, most notably unintended heterogeneous expression from some MscL expression constructs. In this study we investigate the possible cause of this expression pattern, and compare the original non-homogenously expressing constructs with our new homogeneously expressing one to confirm that there is little functional difference between them. In addition, a new MscL construct has been developed with an improved molar extinction coefficient at 280 nm, enabling more accurate protein quantification.

3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity.

Toxin complex (Tc) proteins are a class of bacterial protein toxins that form large, multisubunit complexes. Comprising TcA, B, and C components, they are of great interest because many exhibit potent insecticidal activity. Here we report the structure of a novel Tc, Yen-Tc, isolated from the bacterium Yersinia entomophaga MH96, which differs from the majority of bacterially derived Tcs in that it exhibits oral activity toward a broad range of insect pests, including the diamondback moth (Plutella xylostella). We have determined the structure of the Yen-Tc using single particle electron microscopy and studied its mechanism of toxicity by comparative analyses of two variants of the complex exhibiting different toxicity profiles. We show that the A subunits form the basis of a fivefold symmetric assembly that differs substantially in structure and subunit arrangement from its most well characterized homologue, the Xenorhabdus nematophila toxin XptA1. Histopathological and quantitative dose response analyses identify the B and C subunits, which map to a single, surface-accessible region of the structure, as the sole determinants of toxicity. Finally, we show that the assembled Yen-Tc has endochitinase activity and attribute this to putative chitinase subunits that decorate the surface of the TcA scaffold, an observation that may explain the oral toxicity associated with the complex.

Proteomic and electron microscopy survey of large assemblies in macrophage cytoplasm.

Many cellular processes are carried out by large macromolecular assemblies. We systematically analyzed large macromolecular assemblies in the cytoplasm of mouse macrophages (RAW264.7 cell line), cells with crucial roles in immunity and inflammation. Fractionation of the cytoplasmic fraction was performed using sucrose density gradient centrifugation, and individual fractions were subjected in parallel to (i) identification of constituent proteins by mass spectrometry and (ii) structural visualization by electron microscopy. Macromolecular assemblies present in the fractions were analyzed by integrating available data using bioinformatic approaches. We identified 368 unique proteins in our sample. Among these are components of some well-characterized assemblies involved in diverse cellular processes and structures including translation, proteolysis, protein folding, metabolism, and the cytoskeleton, as well as less characterized proteins that may correspond to additional components of known assemblies or other homo- or hetero-oligomeric structures. Single-particle analysis of electron micrographs of negatively stained samples allowed the identification of clearly distinguishable two-dimensional projections of discrete protein assemblies. Among these, we can identify small ribosomal subunits and preribosomal particles, the 26S proteasome complex and small ringlike structures resembling the molecular chaperone complexes. In addition, a broad range of discrete and different complexes were seen at size ranges between 11 to 38 nm in diameter. Our procedure selects the assemblies on the basis of abundance and ease of isolation, and therefore provides an immediately useful starting point for further study of structure and function of large assemblies. Our results will also contribute toward building a molecular cell atlas.

Environmental and nuclear influences on microalgal chloroplast gene expression.

Microalgal chloroplasts, such as those of the model organism Chlamydomonas reinhardtii, are emerging as a new platform to produce recombinant proteins, including industrial enzymes, diagnostics, as well as animal and human therapeutics. Improving transgene expression and final recombinant protein yields, at laboratory and industrial scales, require optimization of both environmental and cellular factors. Most studies on C. reinhardtii have focused on optimization of cellular factors. Here, we review the regulatory influences of environmental factors, including light (cycle time, intensity, and quality), carbon source (CO2 and organic), and temperature. In particular, we summarize their influence via the redox state, cis-elements, and trans-factors on biomass and recombinant protein production to support the advancement of emerging large-scale light-driven biotechnology applications.

Promoting cell growth and characterizing partial symbiotic relationships in the co-cultivation of green alga Chlamydomonas reinhardtii and Escherichia coli.

BACKGROUND: By co-culturing selected microalgae and heterotrophic microorganisms, the growth rate of microalgae can be improved even under atmospheric conditions with a low CO2 concentration. However, the detailed mechanism of improvement of proliferative capacity by co-culture has not been elucidated. In this study, we investigated changes in the proliferative capacity of the green alga Chlamydomonas reinhardtii by co-culturing with Escherichia coli. MAIN METHODS AND MAJOR RESULTS: In the co-culture, the number of C. reinhardtii cells reached 2.22 × 1010  cell/L on day 14 of culture. This was about 1.9 times the number of cells (1.16 × 1010  cell/L) on day 14 compared to C. reinhardtii cells in monoculture. The starch content per cell in the co-culture of C. reinhardtii and E. coli on the 14th day (2.09 × 10-11  g/cell) was 1.3 times higher than that in the C. reinhardtii monoculture (1.59 × 10-11  g/cell), and the starch content per culture medium improved 2.5 times with co-cultivation. By analyzing the gene transcription profiles and key media components, we clarified that E. coli produced CO2 from the organic carbon in the medium and the organic carbon produced by photosynthesis of C. reinhardtii, and this CO2 likely enhanced the growth of C. reinhardtii. CONCLUSIONS: Consequently, E. coli plays a key role in promoting the growth of C. reinhardtii as well as the accumulation of starch which is a valuable intermediate for the production of a range of useful chemicals from CO2 .

Sustainable Production of Pigments from Cyanobacteria.

Pigments are intensely coloured compounds used in many industries to colour other materials. The demand for naturally synthesised pigments is increasing and their production can be incorporated into circular bioeconomy approaches. Natural pigments are produced by bacteria, cyanobacteria, microalgae, macroalgae, plants and animals. There is a huge unexplored biodiversity of prokaryotic cyanobacteria which are microscopic phototrophic microorganisms that have the ability to capture solar energy and CO2 and use it to synthesise a diverse range of sugars, lipids, amino acids and biochemicals including pigments. This makes them attractive for the sustainable production of a wide range of high-value products including industrial chemicals, pharmaceuticals, nutraceuticals and animal-feed supplements. The advantages of cyanobacteria production platforms include comparatively high growth rates, their ability to use freshwater, seawater or brackish water and the ability to cultivate them on non-arable land. The pigments derived from cyanobacteria and microalgae include chlorophylls, carotenoids and phycobiliproteins that have useful properties for advanced technical and commercial products. Development and optimisation of strain-specific pigment-based cultivation strategies support the development of economically feasible pigment biorefinery scenarios with enhanced pigment yields, quality and price. Thus, this chapter discusses the origin, properties, strain selection, production techniques and market opportunities of cyanobacterial pigments.

Delivering impactful solutions for the bioeconomy.

We are increasingly challenged to operate within our planetary boundaries, while delivering on United Nations (UN) Sustainable Development Goal (SDG) 2030 targets, and net-zero emissions by 2050. Failure to solve these challenges risks economic, social, political, climate, food, water, and fuel security. Therefore, new, scalable, and adoptable circular economy solutions are urgently required. The ability of plants to use light, capture CO2, and drive complex biochemistry is pivotal to delivering these solutions. However, harnessing this capability efficiently also requires robust accompanying economic, financial, market, and strategic analytics. A framework for this is presented here in the Commercialization Tourbillon. It supports the delivery of emerging plant biotechnologies and bio-inspired light-driven industry solutions within the critical 2030-2050 timeframe, to achieve validated economic, social, and environmental benefits.

Structure of the dengue virus glycoprotein non-structural protein 1 by electron microscopy and single-particle analysis.

The flavivirus non-structural protein 1 (NS1) is a glycoprotein that is secreted as a soluble hexameric complex during the course of natural infection. Growing evidence indicates that this secreted form of NS1 (sNS1) plays a significant role in immune evasion and modulation during infection. Attempts to determine the crystal structure of NS1 have been unsuccessful to date and relatively little is known about the macromolecular organization of the sNS1 hexamer. Here, we have applied single-particle analysis to images of baculovirus-derived recombinant dengue 2 virus NS1 obtained by electron microscopy to determine its 3D structure to a resolution of 23 Å. This structure reveals a barrel-like organization of the three dimeric units that comprise the hexamer and provides further insights into the overall organization of oligomeric sNS1.

Circular biomanufacturing through harvesting solar energy and CO2.

Using synthetic biology, it is now time to expand the biosynthetic repertoire of plants and microalgae by utilizing the chloroplast to augment the production of desired high-value compounds and of oil-, carbohydrate-, or protein-enriched biomass based on direct harvesting of solar energy and the consumption of CO2. Multistream product lines based on separate commercialization of the isolated high-value compounds and of the improved bulk products increase the economic potential of the light-driven production system and accelerate commercial scale up. Here we outline the scientific basis for the establishment of such green circular biomanufacturing systems and highlight recent results that make this a realistic option based on cross-disciplinary basic and applied research to advance long-term solutions.

The interplay of proton, electron, and metabolite supply for photosynthetic H2 production in Chlamydomonas reinhardtii.

To obtain a detailed picture of sulfur deprivation-induced H(2) production in microalgae, metabolome analyses were performed during key time points of the anaerobic H(2) production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H(2) production process. This work reports on the differential analysis of metabolic profiles of the high H(2)-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H(2) production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H(2) production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast.

The structure of bacterial RNA polymerase in complex with the essential transcription elongation factor NusA.

There are three stages of transcribing DNA into RNA. These stages are initiation, elongation and termination, and they are well-understood biochemically. However, despite the plethora of structural information made available on RNA polymerase in the last decade, little is available for RNA polymerase in complex with transcription elongation factors. To understand the mechanisms of transcriptional regulation, we describe the first structure, to our knowledge, for a bacterial RNA polymerase in complex with an essential transcription elongation factor. The resulting structure formed between the RNA polymerase and NusA from Bacillus subtilis provides important insights into the transition from an initiation complex to an elongation complex, and how NusA is able to modulate transcription elongation and termination.

Three-dimensional structure of AAA ATPase Vps4: advancing structural insights into the mechanisms of endosomal sorting and enveloped virus budding.

Vps4 is a AAA ATPase that mediates endosomal membrane protein sorting. It is also a host factor hijacked by a diverse set of clinically important viruses, including HIV and Ebola, to facilitate viral budding. Here we present the three-dimensional structure of the hydrolysis-defective Vps4p(E233Q) mutant. Single-particle analysis, multiangle laser light scattering, and the docking of independently determined atomic models of Vps4 monomers reveal a complex with C6 point symmetry, distinguishing between a range of previously suggested oligomeric states (8-14 subunits). The 3D reconstruction also reveals a tail-to-tail subunit organization between the two rings of the complex and identifies the location of domains critical to complex assembly and interaction with partner proteins. Our refined Vps4 structure is better supported by independent lines of evidence than those previously proposed, and provides insights into the mechanism of endosomal membrane protein sorting and viral envelope budding.

Techno-economic evaluation of microalgae high-density liquid fuel production at 12 international locations.

BACKGROUND: Microalgae-based high-density fuels offer an efficient and environmental pathway towards decarbonization of the transport sector and could be produced as part of a globally distributed network without competing with food systems for arable land. Variations in climatic and economic conditions significantly impact the economic feasibility and productivity of such fuel systems, requiring harmonized technoeconomic assessments to identify important conditions required for commercial scale up. METHODS: Here, our previously validated Techno-economic and Lifecycle Analysis (TELCA) platform was extended to provide a direct performance comparison of microalgae diesel production at 12 international locations with variable climatic and economic settings. For each location, historical weather data, and jurisdiction-specific policy and economic inputs were used to simulate algal productivity, evaporation rates, harvest regime, CapEx and OpEx, interest and tax under location-specific operational parameters optimized for Minimum Diesel Selling Price (MDSP, US$ L-1). The economic feasibility, production capacity and CO2-eq emissions of a defined 500 ha algae-based diesel production facility is reported for each. RESULTS: Under a for-profit business model, 10 of the 12 locations achieved a minimum diesel selling price (MDSP) under US$ 1.85 L-1 / US$ 6.99 gal-1. At a fixed theoretical MDSP of US$ 2 L-1 (US$ 7.57 gal-1) these locations could achieve a profitable Internal Rate of Return (IRR) of 9.5-22.1%. Under a public utility model (0% profit, 0% tax) eight locations delivered cost-competitive renewable diesel at an MDSP of < US$ 1.24 L-1 (US$ 4.69 gal-1). The CO2-eq emissions of microalgae diesel were about one-third of fossil-based diesel. CONCLUSIONS: The public utility approach could reduce the fuel price toward cost-competitiveness, providing a key step on the path to a profitable fully commercial renewable fuel industry by attracting the investment needed to advance technology and commercial biorefinery co-production options. Governments' adoption of such an approach could accelerate decarbonization, improve fuel security, and help support a local COVID-19 economic recovery. This study highlights the benefits and limitations of different factors at each location (e.g., climate, labour costs, policy, C-credits) in terms of the development of the technology-providing insights on how governments, investors and industry can drive the technology forward.