Outdoor cultivation and metabolomics exploration of Chlamydomonas engineered for bisabolene production.

Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineeredC. reinhardtii,establishing it as a one-cell two-wells biorefinery.

Mechanistic transcriptome comprehension of Chlamydomonas reinhardtii subjected to black phosphorus.

Two-dimensional materials have recently gained significant awareness. A representative of such materials, black phosphorous (BP), earned attention based on its comprehensive application potential. The presented study focuses on the mode of cellular response underlying the BP interaction with Chlamydomonas reinhardtii as an algal model organism. We observed noticeable ROS formation and changes in outer cellular topology after 72 h of incubation at 5 mg/L BP. Transcriptome profiling was employed to examine C. reinhardtii response after exposure to 25 mg/L BP for a deeper understanding of the associated processes. The RNA sequencing has revealed a comprehensive response with abundant transcript downregulation. The mode of action was attributed to cell wall disruption, ROS elevation, and chloroplast disturbance. Besides many other dysregulated genes, the cell response involved the downregulation of GH9 and gametolysin within a cell wall, pointing to a shift to discrete manipulation with resources. The response also included altered expression of the PRDA1 gene associated with redox governance in chloroplasts implying ROS disharmony. Altered expression of the Cre-miR906-3p, Cre-miR910, and Cre-miR914 pointed to those as potential markers in stress response studies.

Elemental and macromolecular plasticity of Chlamydomonas reinhardtii (Chlorophyta) in response to resource limitation and growth rate.

With the ongoing differential disruption of the biogeochemical cycles of major elements that are essential for all life (carbon, nitrogen, and phosphorus), organisms are increasingly faced with a heterogenous supply of these elements in nature. Given that photosynthetic primary producers form the base of aquatic food webs, impacts of changed elemental supply on these organisms are particularly important. One way that phytoplankton cope with the differential availability of nutrients is through physiological changes, resulting in plasticity in macromolecular and elemental biomass composition. Here, we assessed how the green alga Chlamydomonas reinhardtii adjusts its macromolecular (e.g., carbohydrates, lipids, and proteins) and elemental (C, N, and P) biomass pools in response to changes in growth rate and the modification of resources (nutrients and light). We observed that Chlamydomonas exhibits considerable plasticity in elemental composition (e.g., molar ratios ranging from 124 to 971 for C:P, 4.5 to 25.9 for C:N, and 15.1 to 61.2 for N:P) under all tested conditions, pointing to the adaptive potential of Chlamydomonas in a changing environment. Exposure to low light modified the elemental and macromolecular composition of cells differently than limitation by nutrients. These observed differences, with potential consequences for higher trophic levels, included smaller cells, shifts in C:N and C:P ratios (due to proportionally greater N and P contents), and differential allocation of C among macromolecular pools (proportionally more lipids than carbohydrates) with different energetic value. However, substantial pools of N and P remained unaccounted for, especially at fast growth, indicating accumulation of N and P in forms we did not measure.

Promoting Photosynthetic Production of Dammarenediol-II in Chlamydomonas reinhardtii via Gene Loading and Culture Optimization.

Ginsenosides are major bioactive compounds found in Panax ginseng that exhibit various pharmaceutical properties. Dammarenediol-II, the nucleus of dammarane-type ginsenosides, is a promising candidate for pharmacologically active triterpenes. Dammarenediol-II synthase (DDS) cyclizes 2,3-oxidosqualene to produce dammarenediol-II. Based on the native terpenoids synthetic pathway, a dammarane-type ginsenosides synthetic pathway was established in Chlamydomonas reinhardtii by introducing P. ginseng PgDDS, CYP450 enzyme (PgCYP716A47), or/and Arabidopsis thaliana NADPH-cytochrome P450 reductase gene (AtCPR), which is responsible for producing dammarane-type ginsenosides. To enhance productivity, strategies such as "gene loading" and "culture optimizing" were employed. Multiple copies of transgene expression cassettes were introduced into the genome to increase the expression of the key rate-limiting enzyme gene, PgDDS, significantly improving the titer of dammarenediol-II to approximately 0.2 mg/L. Following the culture optimization in an opt2 medium supplemented with 1.5 mM methyl jasmonate under a light:dark regimen, the titer of dammarenediol-II increased more than 13-fold to approximately 2.6 mg/L. The C. reinhardtii strains engineered in this study constitute a good platform for the further production of ginsenosides in microalgae.

Phase partitioning of mercury, arsenic, selenium, and cadmium in Chlamydomonas reinhardtii and Arthrospira maxima microcosms.

As the global human population increases, demand for protein will surpass our current production ability without an increase in land use or intensification. Microalgae cultivation offers a high yield of protein, and utilization of wastewater from municipal or agricultural sources in place of freshwater for microalgae aquaculture may increase the sustainability of this practice. However, wastewater from municipal and agricultural sources may contain contaminants, such as mercury (Hg), cadmium (Cd), selenium (Se), and arsenic (As). Association of these elements with algal biomass may present an exposure risk to product consumers, while volatilization may present an exposure hazard to industry workers. Thus, the partitioning of these elements should be evaluated before wastewater can be confidently used in an aquaculture setting. This study explored the potential for exposure associated with Arthrospira maxima and Chlamydomonas reinhardtii aquaculture in medium contaminated with 0.33 µg Hg L-1, 60 µg As L-1, 554 µg Se L-1, and 30 µg Cd L-1. Gaseous effluent from microalgae aquaculture was analyzed for Hg, As, Se, and Cd to quantify volatilization. A mass balance approach was used to describe the partitioning of elements between the biomass, medium, and gas phases at the end of exponential growth. Contaminants were recovered predominantly in medium and biomass, regardless of microalgae strain. In the case of Hg, 48 ± 2% was associated with A. maxima biomass and 55 ± 8% with C. reinhardtii when Hg was present as the only contaminant, but this increased to 85 ± 11% in C. reinhardtii biomass when As, Se, and Cd were also present. A small and highly variable abiotic volatilization of Hg was observed in the gas phase of both A. maxima and C. reinhardtii cultures. Evidence presented herein suggests that utilizing wastewater containing Hg, Cd, Se, and As for microalgae cultivation may present health hazards to consumers.

Chlamydomonas reinhardtii: A Factory of Nutraceutical and Food Supplements for Human Health.

Chlamydomonas reinhardtii (C. reinhardtii) is one of the most well-studied microalgae organisms that revealed important information for the photosynthetic and metabolic processes of plants and eukaryotes. Numerous extensive studies have also underpinned its great potential as a biochemical factory, capable of producing various highly desired molecules with a direct impact on human health and longevity. Polysaccharides, lipids, functional proteins, pigments, hormones, vaccines, and antibodies are among the valuable biomolecules that are produced spontaneously or under well-defined conditions by C. reinhardtii and can be directly linked to human nutrition and diet. The aim of this review is to highlight the recent advances in the field focusing on the most relevant applications related to the production of important biomolecules for human health that are also linked with human nutrition and diet. The limitations and challenges are critically discussed along with the potential future applications of C. reinhardtii biomass and processed products in the field of nutraceuticals and food supplements. The increasing need for high-value and low-cost biomolecules produced in an environmentally and economy sustainable manner also underline the important role of C. reinhardtii.

Tuning photosynthetic oxygen for hydrogen evolution in synergistically integrated, sulfur deprived consortia of Coccomyxa chodatii and Rhodobium gokarnense at dim and high light.

In this work, tuning oxygen tension was targeted to improve hydrogen evolution. To achieve such target, various consortia of the chlorophyte Coccomyxa chodatii with a newly isolated photosynthetic purple non-sulfur bacterium (PNSB) strain Rhodobium gokarnense were set up, sulfur replete/deprived, malate/acetate fed, bicarbonate/sulfur added at dim/high light. C. chodatii and R. gokarnense are newly introduced to biohydrogen studies for the first time. Dim light was applied to avoid the inhibitory drawbacks of photosynthetic oxygen evolution, values of hydrogen are comparable with high light or even more and thus economically feasible to eliminate the costs of artificial illumination. Particularly, the consortium of 2n- (n = 1.9 × 105 cell/ml, sulfur deprived) demonstrated its perfection for the target, i.e., the highest possible cumulative hydrogen. This consortium exhibited negative photosynthesis, i.e., oxygen uptake in the light. Most hydrogen in consortia is from bacterial origin, although algae evolved much more hydrogen than bacteria on per cell basis, but for only one day (the second 24 h), as kinetics revealed. The higher hydrogen in unibacterial culture or consortia results from higher bacterial cell density (20 times). Consortia evolved more hydrogen than their respective separate cultures, further enhanced when bicarbonate and sulfur were supplemented at higher light. The share of algae relatively increased as bicarbonate or sulfur were added at higher light intensity, i.e., PSII activity partially recovered, resulting in a transient autotrophic hydrogen evolution. The addition of acetic acid in mixture with malic acid significantly enhanced the cumulative hydrogen levels, mostly decreased cellular ascorbic acid indicating less oxidative stress and relief of PSII, relative to malic acid alone. Starch, however, decreased, indicating the specificity of acetic acid. Exudates (reducing sugars, amino acids, and soluble proteins) were detected, indicating mutual utilization. Yet, hydrogen evolution is limited; tuning PSII activity remains a target for sustainable hydrogen production.

Responses of Chlamydomonas reinhardtii during the transition from P-deficient to P-sufficient growth (the P-overplus response): The roles of the vacuolar transport chaperones and polyphosphate synthesis.

Phosphorus (P) assimilation and polyphosphate (polyP) synthesis were investigated in Chlamydomonas reinhardtii by supplying phosphate (PO43- ; 10 mg P·L-1 ) to P-depleted cultures of wildtypes, mutants with defects in genes involved in the vacuolar transporter chaperone (VTC) complex, and VTC-complemented strains. Wildtype C. reinhardtii assimilated PO43- and stored polyP within minutes of adding PO43- to cultures that were P-deprived, demonstrating that these cells were metabolically primed to assimilate and store PO43- . In contrast, vtc1 and vtc4 mutant lines assayed under the same conditions never accumulated polyP, and PO43- assimilation was considerably decreased in comparison with the wildtypes. In addition, to confirm the bioinformatics inferences and previous experimental work that the VTC complex of C. reinhardtii has a polyP polymerase function, these results evidence the influence of polyP synthesis on PO43- assimilation in C. reinhardtii. RNA-sequencing was carried out on C. reinhardtii cells that were either P-depleted (control) or supplied with PO43- following P depletion (treatment) in order to identify changes in the levels of mRNAs correlated with the P status of the cells. This analysis showed that the levels of VTC1 and VTC4 transcripts were strongly reduced at 5 and 24 h after the addition of PO43- to the cells, although polyP granules were continuously synthesized during this 24 h period. These results suggest that the VTC complex remains active for at least 24 h after supplying the cells with PO43- . Further bioassays and sequence analyses suggest that inositol phosphates may control polyP synthesis via binding to the VTC SPX domain.

Loss of two families of SPX domain-containing proteins required for vacuolar polyphosphate accumulation coincides with the transition to phosphate storage in green plants.

Phosphorus is an essential nutrient for plants. It is stored as inorganic phosphate (Pi) in the vacuoles of land plants but as inorganic polyphosphate (polyP) in chlorophyte algae. Although it is recognized that the SPX-Major Facilitator Superfamily (MFS) and VPE proteins are responsible for Pi influx and efflux, respectively, across the tonoplast in land plants, the mechanisms that underlie polyP homeostasis and the transition of phosphorus storage forms during the evolution of green plants remain unclear. In this study, we showed that CrPTC1, encoding a protein with both SPX and SLC (permease solute carrier 13) domains for Pi transport, and CrVTC4, encoding a protein with both SPX and vacuolar transporter chaperone (VTC) domains for polyP synthesis, are required for vacuolar polyP accumulation in the chlorophyte Chlamydomonas reinhardtii. Phylogenetic analysis showed that the SPX-SLC, SPX-VTC, and SPX-MFS proteins were present in the common ancestor of green plants (Viridiplantae). The SPX-SLC and SPX-VTC proteins are conserved among species that store phosphorus as vacuolar polyP and absent from genomes of plants that store phosphorus as vacuolar Pi. By contrast, SPX-MFS genes are present in the genomes of streptophytes that store phosphorus as Pi in the vacuoles. These results suggest that loss of SPX-SLC and SPX-VTC genes and functional conservation of SPX-MFS proteins during the evolution of streptophytes accompanied the change from ancestral polyP storage to Pi storage.

Restoration of photosynthetic activity and supercomplexes from severe iron starvation in Chlamydomonas reinhardtii.

The eukaryotic alga Chlamydomonas (C.) reinhardtii is used as a model organism to study photosynthetic efficiency. We studied the organization and protein profile of thylakoid membranes under severe iron (Fe2+) deficiency condition and iron supplement for their restoration. Chlorophyll (Chl) a fluorescence fast OJIP transients were decreased in the severe Fe2+ deficient cells resulting in the reduction of the photochemical efficiency. The circular dichroism (CD) results from Fe2+ deficient thylakoid membranes show a significant change in pigment-pigment and pigment-protein excitonic interactions. The organization of super-complexes was also affected significantly. Furthermore, super-complexes of photosystem (PS) II and PSI, along with its dimers, were severely reduced. The complexes separated using sucrose gradient centrifugation shows that loss of super-complexes and excitonic pigment-pigment interactions were restored in the severely Fe2+ deficient cells upon Fe supplementation for three generations. Additionally, the immunoblots demonstrated that both PSII, PSI core, and their light-harvesting complex antenna proteins were differentially decreased. However, reduced core proteins were aggregated, which in turn proteins were unfold and destabilized the supercomplexes and its function. Interestingly, the aggregated proteins were insoluble after n-Dodecyl ß-D-maltoside solubilization. Further, they were identified in the pellet form. When Fe2+ was added to the severely deficient cells, the photosynthetic activity, pigment-proteins complexes, and proteins were restored to the level of control after 3rd generation.

Phosphorus Availability Regulates TORC1 Signaling via LST8 in Chlamydomonas.

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas (Chlamydomonas reinhardtii) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas.

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga.

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO2 ) fixation by toxic metals such as cadmium - classified as a priority pollutant - could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO2 and labelled 13 C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO2 fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (µXRF)/micro X-ray absorption near-edge structure (µXANES) at Cd LIII- edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the 13 C/12 C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO2 decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.

Chlamydomonas cell cycle mutant crcdc5 over-accumulates starch and oil.

The use of algal biomass for biofuel production requires improvements in both biomass productivity and its energy density. Green microalgae store starch and oil as two major forms of carbon reserves. Current strategies to increase the amount of carbon reserves often compromise algal growth. To better understand the cellular mechanisms connecting cell division to carbon storage, we examined starch and oil accumulation in two Chlamydomonas mutants deficient in a gene encoding a homolog of the Arabidopsis Cell Division Cycle 5 (CDC5), a MYB DNA binding protein known to be involved in cell cycle in higher plants. The two crcdc5 mutants (crcdc5-1 and crcdc5-2) were found to accumulate significantly higher amount of starch and oil than their corresponding parental lines. Flow cytometry analysis on synchronized cultures cultivated in a diurnal light/dark cycle revealed an abnormal division of the two mutants, characterized by a prolonged S/M phase, therefore demonstrating its implication in cell cycle in Chlamydomonas. Taken together, these results suggest that the energy saved by a slowdown in cell division is used for the synthesis of reserve compounds. This work highlights the importance in understanding the interplay between cell cycle and starch/oil homeostasis, which should have a critical impact on improving lipid/starch productivity.

TOR inhibition interrupts the metabolic homeostasis by shifting the carbon-nitrogen balance in Chlamydomonas reinhardtii.

The allocation of nutrient resources to growth and metabolism is an essential function for controlling biomass accumulation in photoautotrophic organisms. One essential protein complex involved in this process is the target of rapamycin (TOR) kinase. It has been shown that the inhibition of TOR leads to a considerable upsurge in the amino acid levels. This molecular phenotype relies mainly on the availability of light, carbon (C) and nitrogen (N). To validate the time-resolved response of C and N metabolites, we used a targeted gas chromatography mass spectrometery (GC-MS)-based metabolomic approach, where we examined the response of Chlamydomonas reinhardtii upon TOR inhibition under C-limited condition, namely extended darkness. Contrary to C-supplemented conditions, the rapid increase in the amino acid levels is suppressed almost completely 4 h after TOR inhibition, confirming that C supply is essential to raise the amino acid levels mediated by their de novo synthesis. An exception to this observation was the levels of aspartate, which is presumably synthesized via the anaplerotic pathway. In agreement with previous reports, TOR repression, under these C-limited conditions, leads to a significant reduction in the C/N ratio, corroborating with the crucial role of the pathway in maintaining the metabolic balance of the cells and consequently propelling growth.

Lipid remodeling regulator 1 (LRL1) is differently involved in the phosphorus-depletion response from PSR1 in Chlamydomonas reinhardtii.

The elucidation of lipid metabolism in microalgae has attracted broad interest, as their storage lipid, triacylglycerol (TAG), can be readily converted into biofuel via transesterification. TAG accumulates in the form of oil droplets, especially when cells undergo nutrient deprivation, such as for nitrogen (N), phosphorus (P), or sulfur (S). TAG biosynthesis under N-deprivation has been comprehensively studied in the model microalga Chlamydomonas reinhardtii, during which TAG accumulates dramatically. However, the resulting rapid breakdown of chlorophyll restricts overall oil yield productivity and causes cessation of cell growth. In contrast, P-deprivation results in oil accumulation without disrupting chloroplast integrity. We used a reverse genetics approach based on co-expression analysis to identify a transcription factor (TF) that is upregulated under P-depleted conditions. Transcriptomic analysis revealed that the mutants showed repression of genes typically associated with lipid remodeling under P-depleted conditions, such as sulfoquinovosyl diacylglycerol 2 (SQD2), diacylglycerol acyltransferase (DGTT1), and major lipid droplet protein (MLDP). As accumulation of sulfoquinovosyl diacylglycerol and TAG were suppressed in P-depleted mutants, we designated the protein as lipid remodeling regulator 1 (LRL1). LRL1 mutants showed slower growth under P-depletion. Moreover, cell size in the mutant was significantly reduced, and TAG and starch accumulation per cell were decreased. Transcriptomic analysis also suggested the repression of several genes typically upregulated in adaptation to P-depletion that are associated with the cell cycle and P and lipid metabolism. Thus, our analysis of LRL1 provides insights into P-allocation and lipid remodeling under P-depleted conditions in C. reinhardtii. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The sequencing data were made publicly available under the BioProject Accession number PRJDB6733 and an accession number LC488724 at the DNA Data Bank of Japan (DDBJ). The data is available at https://trace.ddbj.nig.ac.jp/BPSearch/bioproject?acc=PRJDB6733; http://getentry.ddbj.nig.ac.jp/getentry/na/LC488724. The metabolome data were made publicly available and can be accessed at http://metabolonote.kazusa.or.jp/SE195:/; http://webs2.kazusa.or.jp/data/nur/.

Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.

Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii.

Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are considered to be one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organisms is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism. In this review, we summarize recent advances in the development of genetic manipulation tools for Chlamydomonas, from gene delivery methods to state-of-the-art genome-editing technologies and fluorescent dye-based high-throughput mutant screening approaches. Furthermore, we discuss practical strategies and toolkits that enhance transgene expression, such as choice of expression vector and background strain. We then provide examples of how advanced genetic tools have been used to increase oil content in Chlamydomonas. Collectively, the current literature indicates that microalgal oil content can be increased by overexpressing key enzymes that catalyze lipid biosynthesis, blocking lipid degradation, silencing metabolic pathways that compete with lipid biosynthesis and modulating redox state. The tools and knowledge generated through metabolic engineering studies should pave the way for developing a synthetic biological approach to enhance lipid productivity in microalgae.

Native-state imaging of calcifying and noncalcifying microalgae reveals similarities in their calcium storage organelles.

Calcium storage organelles are common to all eukaryotic organisms and play a pivotal role in calcium signaling and cellular calcium homeostasis. In most organelles, the intraorganellar calcium concentrations rarely exceed micromolar levels. Acidic organelles called acidocalcisomes, which concentrate calcium into dense phases together with polyphosphates, are an exception. These organelles have been identified in diverse organisms, but, to date, only in cells that do not form calcium biominerals. Recently, a compartment storing molar levels of calcium together with phosphorous was discovered in an intracellularly calcifying alga, the coccolithophore Emiliania huxleyi, raising a possible connection between calcium storage organelles and calcite biomineralization. Here we used cryoimaging and cryospectroscopy techniques to investigate the anatomy and chemical composition of calcium storage organelles in their native state and at nanometer-scale resolution. We show that the dense calcium phase inside the calcium storage compartment of the calcifying coccolithophore Pleurochrysis carterae and the calcium phase stored in acidocalcisomes of the noncalcifying alga Chlamydomonas reinhardtii have common features. Our observations suggest that this strategy for concentrating calcium is a widespread trait and has been adapted for coccolith formation. The link we describe between acidocalcisomal calcium storage and calcium storage in coccolithophores implies that our physiological and molecular genetic understanding of acidocalcisomes could have relevance to the calcium pathway underlying coccolithophore calcification, offering a fresh entry point for mechanistic investigations on the adaptability of this process to changing oceanic conditions.

Organic selenium, selenate, and selenite accumulation by lake plankton and the alga Chlamydomonas reinhardtii at different pH and sulfate concentrations.

Selenium (Se) concentrations measured in lake planktonic food chains (microplankton <64 µm, copepods, and Chaoborus larvae) were strongly correlated with the concentrations of dissolved organic Se. These correlations were strengthened slightly by adding the concentrations of dissolved selenate to those of organic Se. To better understand the role of Se species and the influence of water chemistry on Se uptake, we exposed the green alga Chlamydomonas reinhardtii to selenite, selenate, or selenomethionine at various H+ ion and sulfate concentrations under controlled laboratory conditions. At low sulfate concentrations, inorganic Se species (selenate >> selenite) were more readily accumulated by this alga than was selenomethionine. However, at higher sulfate concentrations the uptake of selenite was higher than that of selenate, whereas the uptake of selenomethionine remained unchanged. Although the pH of the exposure water did not influence the uptake of selenate by this alga, the accumulation of selenomethionine and selenite increased with pH because of their relative pH-related speciation. The Se concentrations that we measured in C. reinhardtii exposed to selenomethionine were 30 times lower than those that we measured in field-collected microplankton exposed in the same laboratory conditions. This difference is explained by the taxa present in the microplankton samples. Using the present laboratory measurements of Se uptake in microplankton and of natural Se concentrations in lake water allowed us to model Se concentrations in a lake pelagic food chain. Environ Toxicol Chem 2018;37:2112-2122. © 2018 SETAC.

Green biologics: The algal chloroplast as a platform for making biopharmaceuticals.

Most commercial production of recombinant pharmaceutical proteins involves the use of mammalian cell lines, E. coli or yeast as the expression host. However, recent work has demonstrated the potential of eukaryotic microalgae as platforms for light-driven synthesis of such proteins. Expression in the algal chloroplast is particularly attractive since this organelle contains a minimal genome suitable for rapid engineering using synthetic biology approaches; with transgenes precisely targeted to specific genomic loci and amenable to high-level, regulated and stable expression. Furthermore, proteins can be tightly contained and bio-encapsulated in the chloroplast allowing accumulation of proteins otherwise toxic to the host, and opening up possibilities for low-cost, oral delivery of biologics. In this commentary we illustrate the technology with recent examples of hormones, protein antibiotics and immunotoxins successfully produced in the algal chloroplast, and highlight possible future applications.