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.

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.

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.

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.

Algae biofertilisers promote sustainable food production and a circular nutrient economy - An integrated empirical-modelling study.

Agriculture has radically changed the global nitrogen (N) cycle and is heavily dependent on synthetic N-fertiliser. However, the N-use efficiency of synthetic fertilisers is often only 50% with N-losses from crop systems polluting the biosphere, hydrosphere and atmosphere. To address the large carbon and energy footprint of N-fertiliser synthesis and curb N-pollution, new technologies are required to deliver enhanced energy efficiency, decarbonisation and a circular nutrient economy. Algae fertilisers (AF) are an alternative to synthetic N-fertiliser (SF). Here microalgae were used as biofertiliser for spinach production. AF production was evaluated using life-cycle analyses. Over 4 weeks, AF released 63.5% of N as bioavailable ammonium and nitrate, and 25% of phosphorous (P) as phosphate to the growth substrate; SF released 100% N and 20% P. To maximise crop N-use and minimise N-leaching, we explored AF and SF dose-response-curves with spinach in glasshouse conditions. AF-grown spinach produced 36% less biomass than SF-grown plants due to AF's slower and linear N-release; SF exhibited 5-times higher N-leaching than AF. Optimised AF:SF blends yielded greater synchrony between N-release and crop-uptake, boosting crop yields and minimising N-loss. Additional benefits of AF included greener leaves, lower leaf nitrate concentration, and higher microbial diversity and water holding capacity of the growth substrate. An integrated techno-economic and life-cycle-analysis of scaled-up microalgae systems (+/- wastewater) normalised to the application dose showed that replacing the most effective SF-dose with AF lowered the annual carbon footprint of fertiliser production from 3.644 kg CO2 m-2 (C-producing) to -6.039 kg CO2 m-2 (C-assimilation). N-loss from growth substrate was lowered by 54%. Embodied energy for AF:SF blends could be reduced by 29% when cultivating microalgae on wastewater. Conclusions: (i) microalgae offer a sustainable alternative to synthetic N-fertiliser for spinach production and potentially other crop systems, (ii) microalgae biofertilisers support the circular-nutrient-economy and several UN-Sustainable-Development-Goals.

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.

Microalgal nanocellulose - opportunities for a circular bioeconomy.

Over 3 billion years, photosynthetic algae have evolved complex uses for cellulose, the most abundant polymer worldwide. A major cell-wall component of lignocellulosic plants, seaweeds, microalgae, and bacteria, cellulose can be processed to nanocellulose, a promising nanomaterial with novel properties. The structural diversity of macro- and microalgal nanocelluloses opens opportunities to couple low-impact biomass production with novel, green-chemistry processing to yield valuable, sustainable nanomaterials for a multitude of applications ranging from novel wound dressings to organic solar cells. We review the origins of algal cellulose and the applications and uses of nanocellulose, and highlight the potential for microalgae as a nanocellulose source. Given the limited state of current knowledge, we identify research challenges and strategies to help to realise this potential.

Microalgal Aquafeeds As Part of a Circular Bioeconomy.

Photosynthetic microalgae are unicellular plants, many of which are rich in protein, lipids, and bioactives and form an important part of the base of the natural aquatic food chain. Population growth, demand for high-quality protein, and depletion of wild fishstocks are forecast to increase aquacultural fish demand by 37% between 2016 and 2030. This review highlights the role of microalgae and recent advances that can support a sustainable 'circular' aquaculture industry. Microalgae-based feed supplements and recombinant therapeutic production offer significant opportunities to improve animal health, disease resistance, and yields. Critically, microalgae in biofloc, 'green water', nutrient remediation, and integrated multitrophic aquaculture technologies offer innovative solutions for economic and environmentally sustainable development in line with key UN Sustainability Goals.

Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin.

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 Å, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.

Green Bioplastics as Part of a Circular Bioeconomy.

The rapid accumulation of plastic waste is driving international demand for renewable plastics with superior qualities (e.g., full biodegradability to CO2 without harmful byproducts), as part of an expanding circular bioeconomy. Higher plants, microalgae, and cyanobacteria can drive solar-driven processes for the production of feedstocks that can be used to produce a wide variety of biodegradable plastics, as well as bioplastic-based infrastructure that can act as a long-term carbon sink. The plastic types produced, their chemical synthesis, scaled-up biorefinery concepts (e.g., plant-based methane-to-bioplastic production and co-product streams), bioplastic properties, and uses are summarized, together with the current regulatory framework and the key barriers and opportunities.

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.

High-throughput optimisation of light-driven microalgae biotechnologies.

Microalgae biotechnologies are rapidly developing into new commercial settings. Several high value products already exist on the market, and systems development is focused on cost reduction to open up future economic opportunities for food, fuel and freshwater production. Light is a key environmental driver for photosynthesis and optimising light capture is therefore critical for low cost, high efficiency systems. Here a novel high-throughput screen that simulates fluctuating light regimes in mass cultures is presented. The data was used to model photosynthetic efficiency (PEµ, mol photon-1 m2) and chlorophyll fluorescence of two green algae, Chlamydomonas reinhardtii and Chlorella sp. Response surface methodology defined the effect of three key variables: density factor (Df, 'culture density'), cycle time (tc, 'mixing rate'), and maximum incident irradiance (Imax). Both species exhibited a large rise in PEµ with decreasing Imax and a minimal effect of tc (between 3-20 s). However, the optimal Df of 0.4 for Chlamydomonas and 0.8 for Chlorella suggested strong preferences for dilute and dense cultures respectively. Chlorella had a two-fold higher optimised PEµ than Chlamydomonas, despite its higher light sensitivity. These results demonstrate species-specific light preferences within the green algae clade. Our high-throughput screen enables rapid strain selection and process optimisation.

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.

Trading Off Global Fuel Supply, CO2 Emissions and Sustainable Development.

The United Nations Conference on Climate Change (Paris 2015) reached an international agreement to keep the rise in global average temperature 'well below 2°C' and to 'aim to limit the increase to 1.5°C'. These reductions will have to be made in the face of rising global energy demand. Here a thoroughly validated dynamic econometric model (Eq 1) is used to forecast global energy demand growth (International Energy Agency and BP), which is driven by an increase of the global population (UN), energy use per person and real GDP (World Bank and Maddison). Even relatively conservative assumptions put a severe upward pressure on forecast global energy demand and highlight three areas of concern. First, is the potential for an exponential increase of fossil fuel consumption, if renewable energy systems are not rapidly scaled up. Second, implementation of internationally mandated CO2 emission controls are forecast to place serious constraints on fossil fuel use from ~2030 onward, raising energy security implications. Third is the challenge of maintaining the international 'pro-growth' strategy being used to meet poverty alleviation targets, while reducing CO2 emissions. Our findings place global economists and environmentalists on the same side as they indicate that the scale up of CO2 neutral renewable energy systems is not only important to protect against climate change, but to enhance global energy security by reducing our dependence of fossil fuels and to provide a sustainable basis for economic development and poverty alleviation. Very hard choices will have to be made to achieve 'sustainable development' goals.

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.

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.

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.

Automated nutrient screening system enables high-throughput optimisation of microalgae production conditions.

BACKGROUND: Microalgae provide an excellent platform for the production of high-value-products and are increasingly being recognised as a promising production system for biomass, animal feeds and renewable fuels. RESULTS: Here, we describe an automated screen, to enable high-throughput optimisation of 12 nutrients for microalgae production. Its miniaturised 1,728 multiwell format allows multiple microalgae strains to be simultaneously screened using a two-step process. Step 1 optimises the primary elements nitrogen and phosphorous. Step 2 uses Box-Behnken analysis to define the highest growth rates within the large multidimensional space tested (Ca, Mg, Fe, Mn, Zn, Cu, B, Se, V, Si) at three levels (-1, 0, 1). The highest specific growth rates and maximum OD750 values provide a measure for continuous and batch culture. CONCLUSION: The screen identified the main nutrient effects on growth, pairwise nutrient interactions (for example, Ca-Mg) and the best production conditions of the sampled statistical space providing the basis for a targeted full factorial screen to assist with optimisation of algae production.

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).