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.

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.

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.

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.

Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

With a rising world population, demand will increase for food, energy and high value products. Renewable production systems, including photosynthetic microalgal biotechnologies, can produce biomass for foods, fuels and chemical feedstocks and in parallel allow the production of high value protein products, including recombinant proteins. Such high value recombinant proteins offer important economic benefits during startup of industrial scale algal biomass and biofuel production systems, but the limited markets for individual recombinant proteins will require a high throughput pipeline for cloning and expression in microalgae, which is currently lacking, since genetic engineering of microalgae is currently complex and laborious. We have introduced the recombination based Gateway® system into the construction process of chloroplast transformation vectors for microalgae. This simplifies the vector construction and allows easy, fast and flexible vector design for the high efficiency protein production in microalgae, a key step in developing such expression pipelines.

RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii.

Single cell green algae (microalgae) are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA) is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels). Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3) in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%), LHCBM2 (81.2% ±0.037%) and LHCBM3 (41.4% ±0.05%) compared to 100% control levels, and improved light to H2 (180%) and biomass (165%) conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m(-2) s(-1)) and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface; 2) improved light distribution in the reactor; 3) reduced photoinhibition; 4) early onset of HYDA expression and 5) reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems.

Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins.

Plastid transformation has become an attractive tool in biotechnology. Because of the prokaryotic nature of the plastid's gene expression machinery, expression elements (promoters and untranslated regions) that trigger high-level foreign protein accumulation in plastids usually also confer high expression in bacterial cloning hosts. This can cause problems, for example, when production of antimicrobial compounds is attempted. Their bactericidal activity can make the cloning of the corresponding genes in plastid transformation vectors impossible. Here, we report a general solution to this problem. We have designed a strategy (referred to as toxin shuttle) that allows the expression in plastids of proteins that are toxic to Escherichia coli. The strategy is based on blocking transcription in E. coli by bacterial transcription terminators upstream of the gene of interest, which subsequently are excised in planta by site-specific recombination. We demonstrate the applicability of the strategy by the high-level expression in plastids (to up to 30% of the plant's total soluble protein) of 2 phage-derived protein antibiotics that are toxic to E. coli. We also show that the plastid-produced antibiotics efficiently kill pathogenic strains of Streptococcus pneumoniae, the causative agent of pneumonia, thus providing a promising strategy for the production of next-generation antibiotics in plants.

Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic.

Plastids (chloroplasts) possess an enormous capacity to synthesize and accumulate foreign proteins. Here we have maximized chloroplast protein production by over-expressing a proteinaceous antibiotic against pathogenic group A and group B streptococci from the plastid genome. The antibiotic, a phage lytic protein, accumulated to enormously high levels (>70% of the plant's total soluble protein), and proved to be extremely stable in chloroplasts. This massive over-expression exhausted the protein synthesis capacity of the chloroplast such that the production of endogenous plastid-encoded proteins was severely compromised. Our data suggest that this is due to translational rather than transcriptional limitation of gene expression. We also show that the chloroplast-produced protein antibiotic efficiently kills the target bacteria. These unrivaled expression levels, together with the chloroplast's insensitivity to enzymes that degrade bacterial cell walls and the elimination of the need to remove bacterial endotoxins by costly purification procedures, indicate that this is an effective plant-based production platform for next-generation antibiotics, which are urgently required to keep pace with rapidly emerging bacterial resistance.