Chlamydomonas Reinhardtii Strain Research Articles

Settling selection of Chlamydomonas reinhardtii for samarium uptake.

Samarium (Sm) is a rare-earth element recently included in the list of critical elements due to its vital role in emerging new technologies. With an increasing demand for Sm, microbial bioremediation may provide a cost-effective and a more ecologically responsible alternative to remove and recover Sm. We capitalized on a previously selected Chlamydomonas reinhardtii strain tolerant to Sm (1.33 × 10-4 M) and acidic pH and carried out settling selection to increase the Sm uptake performance. We observed a rapid response to selection in terms of cellular phenotype. Cellular size decreased and circularity increased in a stepwise manner with every cycle of selection. After four cycles of selection, the derived CSm4 strain was significantly smaller and was capable of sequestrating 41% more Sm per cell (1.7 × 10-05 ± 1.7 × 10-06 ng) and twice as much Sm in terms of wet biomass (4.0 ± 0.4 mg Sm · g-1) compared to the ancestral candidate strain. The majority (~70%) of the Sm was bioaccumulated intracellularly, near acidocalcisomes or autophagic vacuoles as per TEM-EDX microanalyses. However, Sm analyses suggest a stronger response toward bioabsorption resulting from settling selection. Despite working with Sm and pH-tolerant strains, we observed an effect on fitness and photosynthesis inhibition when the strains were grown with Sm. Our results clearly show that phenotypic selection, such as settling selection, can significantly enhance Sm uptake. Laboratory selection of microalgae for rare-earth metal bioaccumulation and sorption can be a promising biotechnological approach.

Unravelling the Mechanisms of Heavy Metal Tolerance: Enhancement in Hydrophilic Antioxidants and Major Antioxidant Enzymes Is Not Crucial for Long-Term Adaptation to Copper in Chlamydomonas reinhardtii.

Understanding of the mechanisms of heavy metal tolerance in algae is important for obtaining strains that can be applied in wastewater treatment. Cu is a redox-active metal directly inducing oxidative stress in exposed cells. The Cu-tolerant Chlamydomonas reinhardtii strain Cu2, obtained via long-term adaptation, displayed increased guaiacol peroxidase activity and contained more lipophilic antioxidants, i.e., α-tocopherol and plastoquinol, than did non-tolerant strain N1. In the present article, we measured oxidative stress markers; the content of ascorbate, soluble thiols, and proline; and the activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in N1 and Cu2 strains grown in the absence or presence of excessive Cu. The Cu2 strain displayed less pronounced lipid peroxidation and increased APX activity compared to N1. The amount of antioxidants was similar in both strains, while SOD and CAT activity was lower in the Cu2 strain. Exposure to excessive Cu led to a similar increase in proline content in both strains and a decrease in ascorbate and thiols, which was more pronounced in the N1 strain. The Cu2 strain was less tolerant to another redox-active heavy metal, namely chromium. Apparently other mechanisms, probably connected to Cu transport, partitioning, and chelation, are more important for Cu tolerance in Cu2 strain.

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.

Genotype differences towards lead chloride harmful action

The aim of the study was to throw more light on the PbCl2 mode of action (MoA) depending on the genotype by the application of three model organisms and microbiological, biochemical, and molecular approaches. Three model systems – Chlamydomonas reinhardtii strain 137C – wild type (WT), Saccharomyces cerevisiae strain D7ts1, and Pisum sativum L. cultivar Ran1 and two experimental schemes – short- and long-term treatments were used. C. reinhardtii and S. cerevisiae cell suspensions (1×106 cells/ml) at the end of the exponential and the beginning of a stationary phase of growth were treated with various PbCl2 concentrations (0.45–3.6 mM) for 2 hours. Lower PbCl2 concentrations (0.03–0.22 mM) were also tested on C. reinhardtii 137C. Short-term treatment for up to 2 days with PbCl2 concentrations in the range of 0.45–3.6 mM and long-term treatment for up to 10 days with concentrations in the range of 0.45–2.7 mM was performed on P. sativum L. seeds and plants, respectively. Long-term treatment with a PbCl2 concentration of 3.6 mM was not tested because of the very strong toxic effect (plant death). The following endpoints were used – for C. reinhardtii: cell survival, “visible” mutations, DNA double-strand breaks (DSBs), malondialdehyde (MDA), intracellular peroxides (H2O2), and photosynthetic pigments; for S. cerevisiae – cell survival, gene conversion, reverse mutation, mitotic crossing-over, DSBs, superoxide anions, MDA and glutathione (GSH); P. sativum L. – germination and root length (short-term treatment), pro-oxidative markers – MDA, H2O2 and photosynthetic pigments (long-term treatment). Genotype differences between C. reinhardtii (0.047 mM) and S. cerevisiae (1.66 mM) were observed by two endpoints: concentrations inducing 50% lethality (LD50) and DSB induction. By contrast, no mutagenic effect was found for both unicellular test models. A slight toxic capacity of PbCl2, measured as inhibition of Pisum sativum L. seed germination and around 20% root length reduction was revealed after the treatment with concentrations equal to or higher than 1.8 mM. The variety of stress responses between the two plant test models was demonstrated by comparing MDA and H2O2. A dose-dependent increase in H2O2 levels and a minor increase of MDA levels (around 9–15%) were measured when C. reinhardtii cells were treated with concentrations in the range of LD20–LD80 (0.03–0.11 mM). Analyzing the kinetics of MDA and H2O2 in pea leaves, the most pronounced effect of concentration was shown for 2.7 mM. A decrease in the photosynthetic pigments was detected in the two experimental designs – short-term on C. reinhardtii and long-term on P. sativum treatments. The pro-oxidative potential was also proven in S. cerevisiae based on increased levels of MDA and superoxide anions and decreased GSH. New information is gained that PbCl2 can affect the DNA molecule and photosynthetic pigments via induction of oxidative stress. Our study revealed that the magnitude of stress response towards PbCl2 is genotype-specific. Our finding that Chlamydomonas reinhardtii is a sensitive test system towards PbCl2 contributes to good strategies for revealing very low levels of contaminants present chronically in main environmental matrices. This is the first report, as far as we know, affirming that PbCl2 can induce DSBs in Chlamydomonas reinhardtii and Saccharomyces cerevisiae.

Single-cell genetic models to evaluate orphan gene function: The case of QQS regulating carbon and nitrogen allocation.

We demonstrate two synthetic single-cell systems that can be used to better understand how the acquisition of an orphan gene can affect complex phenotypes. The Arabidopsisorphan gene, Qua-Quine Starch (QQS) has been identified as a regulator of carbon (C) and nitrogen (N) partitioning across multiple plant species. QQS modulates this important biotechnological trait by replacing NF-YB (Nuclear Factor Y, subunit B) in its interaction with NF-YC. In this study, we expand on these prior findings by developing Chlamydomonas reinhardtii and Saccharomyces cerevisiae strains, to refactor the functional interactions between QQS and NF-Y subunits to affect modulations in C and N allocation. Expression of QQS in C. reinhardtii modulates C (i.e., starch) and N (i.e., protein) allocation by affecting interactions between NF-YC and NF-YB subunits. Studies in S. cerevisiae revealed similar functional interactions between QQS and the NF-YC homolog (HAP5), modulating C (i.e., glycogen) and N (i.e., protein) allocation. However, in S. cerevisiae both the NF-YA (HAP2) and NF-YB (HAP3) homologs appear to have redundant functions to enable QQS and HAP5 to affect C and N allocation. The genetically tractable systems that developed herein exhibit the plasticity to modulate highly complex phenotypes.

Influence of Cell Wall on Biomolecules Biosynthesis in Chlamydomonas reinhardtii Strains Exposed to Magnetic Fields

The application of magnetic fields (MF) has attracted the attention of researchers due to their efficiency to change cell metabolism. Chlamydomonas reinhardtii is a biotechnologically useful microalga with versatile metabolism that may be a valuable organism to study the effects of the MF in biology. Therefore, two C. reinhardtii strains, one with cell wall (2137) and other which lacks the cell wall (Wt-S1-cc4694), were evaluated that a new sensitivity factor in the analysis could be included. Comparative studies were undertaken with the two C. reinhardtii strains under the MF intensities of 0.005 mT (terrestrial MF - control), 11 and 20mT. Results indicated that the physical cell wall barrier protected cells against the MF applied during the assays. Only with the highest MF applied (20mT) a slight increase in lipid concentration in the cell wall strain was detected. The lowest growth of the strain that lacks cell wall (Wt-S1) indicated that these cells are under a negative effect. To cope with the two MF stresses conditions, Wt-S1 cells produced more pigments (chlorophylls and carotenoids) and lipids and enhanced the antioxidant defense system. The raise of these compounds under MF could potentially have a positive biotechnological impact on algal biomass.

The cytotoxicity of nano- and micro-sized graphene oxides on microalgae depends on the characteristics of cell wall and flagella

Cytotoxic effects of emerging contaminants in aquatic environments have been widely studied using diverse microalgal species. However, the role of microalgal characteristics such as presence/absence of cell wall or flagella on cytotoxicity of contaminants was not elucidated yet. In this study, four different Chlamydomonas reinhardtii strains that have different characteristics were used to confirm how these characteristics affect toxicity of contaminants, nano-/micro-sized graphene oxide (GO). The nano-sized GO inhibited the growth of cell wall-deficient strains and reduced the photosynthetic activity. The micro-sized GO inhibited the growth of all strains, but the inhibition efficiency was higher in flagella-deficient strains, indicating that cell wall and flagella have different roles in response to contaminant exposure. The electron microscopy analysis demonstrated that nano-sized GO caused the cell rupture in cell wall-deficient strains. In flagella-deficient strains, the nano- and micro-sized GOs were parallelly attached on the surface of cells, covering the cells. The wrapping of flagella-deficient cells by GO led to the increase of reactive oxygen species (ROS) contents. These results indicate main cytotoxic mechanism of nano-sized GO was the membrane damage of cells, and the presence of cell wall can protect the cells from the attack of nano-sized GO. On the one hand, the presence of flagella might help to avoid the attachment of GO while the cell proliferation and photosynthesis were inhibited in flagella-deficient cells due to the GO wrapping. Overall, given that different microalgal species have different characteristics and these characteristics might affect the cytotoxic effect of the contaminants, it is of great importance to consider the characteristics of test microalgal species when evaluating the cytotoxic mechanism of the nano-/micro-sized pollutants.

Gamete dimorphism of the isogamous green alga (Chlamydomonas reinhardtii), is regulated by the mating type-determining gene, MID

The gametes of chlorophytes differ morphologically even in isogamy and are divided into two types (α and β) based on the mating type- or sex-specific asymmetric positioning of the mating structure (cell fusion apparatus) with respect to the flagellar beat plane and eyespot, irrespective of the difference in gamete size. However, the relationship between this morphological trait and the mating type or sex determination system is unclear. Using mating type-reversed strains of the isogamous alga Chlamydomonas reinhardtii, produced by deletion or introduction of the mating type-determining gene MID, we revealed that the positioning of the mating structure is associated with conversion of mating types (mt– and mt+), implying that this trait is regulated by MID. Moreover, the dominant mating type is associated with the type β phenotype, as in the chlorophyte species Ulva prolifera. Our findings may provide a genetic basis for mating type- or sex-specific asymmetric positioning of the chlorophyte mating structure.

Safety Evaluation of a Novel Algal Feed Additive for Poultry Production.

Feed additives are critical components for poultry health and the economic viability of antibiotic-free poultry production. The aim of the present study is to evaluate the safety of a novel algal-derived feed additive, a dried biomass powder produced from Chlamydomonas reinhardtii strain crAL082, modified to express an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and a lysozyme-type enzyme (EC 3.2.1.17). A 42-day oral toxicity study showed that the crAL082 dried biomass powder was fully tolerated by broiler chicken based on the lack of detrimental effects found in performance, mortality, hematology, blood clinical chemistry, and histopathologic results compared with those of a nontreated control group, resulting in a "No Observed Adverse Effect Level" of 5000 ppm, the highest dose tested. The study demonstrates the first-ever safety result of a C. reinhardtii microalgae dried biomass powder used as a feed additive in broiler chickens. Furthermore, safety is shown for the two additional enzymes expressed within the C. reinhardtii crAL082 strain and ingested by the birds.

Measurement of dielectric properties of cells at single-cell resolution using electrorotation.

Dielectric properties of a cell are biophysical properties of high interest for various applications. However, measuring these properties accurately is not easy, which can be exemplified by the large variations in reported dielectric properties of the same cell types. This paper presents a method for measuring the dielectric properties of cells at high frequency, especially lipid-producing microalgae, at single-cell resolution, by integrating an electrorotation-based dielectric property measurement method with a negative dielectrophoretic (nDEP) force-based single-cell trapping method into a single device. In this method, a four-electrode nDEP structure was used to trap a single cell in an elevated position in the center of another four-electrode structure that can apply electrorotational force. By measuring the speed of cell rotation under different applied electrorotation frequencies and fitting the results into a theoretical core-shell cell model, the dielectric properties of cells, including membrane capacitance and cytoplasm conductivity, could be obtained. This system was applied to measure the dielectric properties of lipid-accumulating microalga Chlamydomonas reinhardtii strain Sta6 by applying an electrorotation signal of up to 100MHz. By utilizing a broad frequency range and expanding the measurement spectra to a high frequency region, increased accuracy in fitting the dielectric parameters to a theoretical model was possible, especially the cytoplasm conductivity. The developed method can be used in various applications, such as screening microalgae based on their lipid production capabilities, separating cells of different dielectric properties, identifying different cell types, as well as conducting basic biophysical analyses of cellular properties.

On the mode of action of Origanum vulgare spp. hirtum methanolic extract and essential oil on Chlamydomonas reinhardtii

Aim: To reveal whether methanolic extract and essential oil from Origanum vulgare subsp. hirtum in doses causing even low levels of mortality in aphids, would have harmful effects on plants-genotoxic, mutagenic and/or DNA damaging. Materials and methods: Aerial parts of Origanum vulgare ssp. hirtum from the ex-situ collection of IBER, BAS during flowering were collected. Extraction and isolation procedures, as well as GC/MS analysis of essential oil and methanolic extract were performed by standard protocols. The components were identified by comparing their relative retention times to the retention times of authentic standards, and with mass spectra with the NIST. Test system: Chlamydomonas reinhardtii strain 137 C+ (WT). Endpoints: “clonal” assay, the test of “visible mutations”, constant field gel electrophoresis. Statistics: GraphPad Prism version 6.04 (San Diego, USA) and One-way Analysis of Variance ANOVA with multiple comparisons using the Tukey method. Results: A good correlation was observed between chemical composition of essential oil and methanolic extract, and their mode of action. Our genotoxic and double strand breaks results demonstrated mild genotoxic and statistically non-significant DNA damaging potential of methanolic extract and concentration-dependent well - expressed genotoxic and DNA damaging potential of essential oil. A good relationship between increased double strand breaks levels and decreased survival might be related to one of the main constituents of essential oil, suspected to be carvacrol. No mutagenic effect for ME and EO was found. Conclusion: Well-expressed toxic/genotoxic capacity of essential oil, as well as its capacity to damage DNA inducing double strand breaks, but the absence of mutagenic potential, could be considered as a good reason to recommend Origanum vulgare subsp. hirtum essential oil as a promising candidate for purposes of “green” technologies.

The bHLH family NITROGEN‐REPLETION INSENSITIVE1 represses nitrogen starvation‐induced responses in Chlamydomonas reinhardtii

The use of nitrogen (N) fertilizers has contributed to a quadrupling of agricultural production. However, > 50% of N fertilizers remain unabsorbed by crops and leak into the environment, demanding strategies to improve the N use efficiency. To identify the regulatory mechanisms of N assimilation, we performed a genetic screen using a Chlamydomonas reinhardtii strain whose motility depends upon the transcription of nitrate reductase-encoding NIT1, which is upregulated in response to N starvation. A persistently swimming mutant, nitrogen repletion insensitive 1 (nri1), was isolated and found to express N starvation-induced genes in N-replete culture. NRI1, previously reported as NSG17 (nitrogen starvation-induced gene 17), encodes a basic helix-loop-helix (bHLH) family transcription factor homologous to BES-interacting myc-like (BIM) in plants. Transcriptome analysis of N-replete nri1 culture revealed a concerted upregulation of the genes involved in the uptake and assimilation of external N sources. Many transcription factor-encoding genes in the bHLH and SBP families were also upregulated in nri1 culture. The DNA-binding sites of bHLH and SBP proteins were enriched in the promoters of the upregulated genes in nri1, suggesting that NRI1 suppresses transcriptional activators to regulate N starvation-induced genes. Consistent with the ammonium-insensitive gene expression pattern, N-starved nri1 gametes could not dedifferentiate back to vegetative cells when incubated in ammonium-containing media. Taken together, we conclude that NRI1 represses N starvation-specific responses when sufficient N is supplied in C. reinhardtii. Further investigation of how cellular N conditions regulate NRI1 activity will unravel the elusive mechanisms by which plant cells sense and respond to N starvation.

Characterisation of a simple ‘hanging bag’ photobioreactor for low‐cost cultivation of microalgae

AbstractBACKGROUNDMicroalgae are a diverse group of photosynthetic microorganisms of significant interest to the biotechnology industry, either as a sustainable source of natural compounds, or as light‐driven cell factories to produce recombinant metabolites and proteins. Their ability to utilise light, CO2, and basic nutrients leads to a simple and low‐cost phototrophic cultivation process. This is particularly relevant to low‐ and middle‐income countries, all of which require a cultivation system that is cheap, technically simple to operate, readily scalable, and can meet basic Good Manufacturing Practice requirements. A disposable ‘hanging bag’‐type photobioreactor operated as a bubble column fits these criteria.RESULTSIn this study, the characterisation and design modifications to improve the performance of a 15 L hanging bag is reported. The bubble behaviour using different sparger designs was investigated together with gas hold‐up, mixing time, and mass transfer coefficient of CO2. A gas flow rate of 5 L min−1 using a new sparger design and a modified height‐to‐diameter ratio of the bag led to a two‐fold improvement in algal biomass productivity when culturing the green microalga Chlorella sorokiniana. The cultivation of a luciferase‐expressing Chlamydomonas reinhardtii strain in the modified hanging bag also demonstrated an 11% increase in luciferase content.CONCLUSIONThis is the first attempt to characterise this simple hanging bag system that brings the industry‐favoured single‐use bag concept into the research field of photobioreactor technology. The hanging bag with modified sparger and dimensions improves microalgal biomass productivity and demonstrates the potential of simple and low‐cost systems for microalgal cultivation. © 2021 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system

The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes.

A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology

In the future, algae biotechnology could play an important role in sustainable development, especially with regard to the production of valuable chemicals. Among the established laboratory strains with efficient transgene expression, there are none that have demonstrated the required robustness for industrial applications, which generally require growth at larger scale. Here, we created a robust and mating-competent cell line of the green microalga Chlamydomonas reinhardtii, which also possesses a high transgene expression capacity. This strain shows a comparably high resistance to shear stress by accumulating increased amounts of biomass under these conditions. As a proof-of-concept, a high phototrophic productivity of cadaverine from CO2 and nitrate was demonstrated by efficiently expressing a bacterial l-lysine decarboxylase. In contrast to other established strains, this novel chassis strain for phototrophic production schemes is equipped with the traits required for industrial applications, by combining mating-competence, cell wall-mediated robustness and high level transgene expression.

Strain-Specific Biostimulant Effects of Chlorella and Chlamydomonas Green Microalgae on Medicago truncatula.

Microalgae have been identified to produce a plethora of bioactive compounds exerting growth stimulating effects on plants. The objective of this study was to investigate the plant-growth-promoting effects of three selected strains of eukaryotic green microalgae. The biostimulatory effects of two Chlorella species (MACC-360 and MACC-38) and a Chlamydomonas reinhardtii strain (cc124) were investigated in a Medicago truncatula model plant grown under controlled greenhouse conditions. The physiological responses of the M. truncatula A17 ecotype to algal biomass addition were characterized thoroughly. The plants were cultivated in pots containing a mixture of vermiculite and soil (1:3) layered with clay at the bottom. The application of live algae cells using the soil drench method significantly increased the plants’ shoot length, leaf size, fresh weight, number of flowers and pigment content. For most of the parameters analyzed, the effects of treatment proved to be specific for the applied algae strains. Overall, Chlorella application led to more robust plants with increased fresh biomass, bigger leaves and more flowers/pods compared to the control and Chlamydomonas-treated samples receiving identical total nutrients.

Transcriptome analysis of <italic>Chlamydomonas reinhardtii</italic> under the co-regulation of three genes

In order to study the regulatory roles of multiple genes in microalgal lipid metabolism, a Chlamydomonas reinhardtii strain DLC is constructed with overexpression of DOF transcription regulator and silencing of acs2 and cis1. Transcriptome sequencing and analysis are conducted and 749 differentially expressed genes (DEGs) are identified. According to the result of KEGG (Kyoto encyclopedia of genes and genomes) classification, 51 and 15 DEGs are involved in carbohydrate metabolism and lipid metabolism respectively. These DEGs mainly play key roles in glyceride metabolism, fatty acid degradation, glycolysis/gluconeogenesis and starch and sucrose metabolism. In addition, triacylglycerol (TAG) and starch content are analyzed by BODIPY505/515 fluorescence analysis and absorbance detection, which shows that intracellular TAG increases by 193% as compared with wild-type, while starch content decreases by 45%. These results further indicate that the co-regulation of the three genes not only enhances the lipid anabolism, but also reorients the carbon flux in the saccharide metabolism. The research reveals the molecular mechanism of lipid overproduction under co-regulation of three genes and provides a theoretical reference for the genetic engineering of microalgae lipid production.

Comparative secretome analysis between salinity-tolerant and control Chlamydomonas reinhardtii strains.

Secretome analysis of a salt-tolerant and control Chlamydomonas reinhardtii revealed 514 differentially expressed proteins. Membrane transport and trafficking, signal transduction and channel proteins were up-regulated in the ST secretome. Salinity is a major abiotic stress that limits crop production worldwide. Multiple adverse effects have been reported in many living organisms exposed to high-saline concentrations. Chlamydomonas reinhardtii is known for secreting proteins in response to many environmental stresses. A salinity-tolerant (ST) strain of Chlamydomonas has been developed, whose cells were able to grow at 300mM NaCl. The current study analyzed the secretomes of ST grown in TAP medium supplemented with 300mM NaCl and the laboratory strain CC-503 grown in TAP medium without NaCl supplement. In total, 514 secreted proteins were identified of which 203 were up-regulated and 110 were down-regulated. Bioinformatic analysis predicted 168 proteins to be secreted or in the conventional secretory pathway. Out of these, 70 were up-regulated, while 51 proteins were down-regulated. Proteins involved in membrane transport and trafficking, signal transduction and channel proteins were altered in their expression in the ST secretome, suggesting the response of saline stress acts toward not only the intracellular pool of proteins but also the extracellular proteins. This also suggested that the secreted proteins might have roles in the extracellular space. Signal peptide (SP) prediction revealed that almost 40% of the predicted secreted proteins contained a signal peptide; however, a high proportion of proteins lacked an SP, suggesting that these proteins might be secreted through an unconventional protein secretion pathway.

Methyl Jasmonate and Methyl-β-Cyclodextrin Individually Boost Triterpenoid Biosynthesis in Chlamydomonas Reinhardtii UVM4.

The commercialisation of valuable plant triterpenoids faces major challenges, including low abundance in natural hosts and costly downstream purification procedures. Endeavours to produce these compounds at industrial scale using microbial systems are gaining attention. Here, we report on a strategy to enrich the biomass of the biotechnologically-relevant Chlamydomonas reinhardtii strain UVM4 with valuable triterpenes, such as squalene and (S)-2,3-epoxysqualene. C. reinhardtii UVM4 was subjected to the elicitor compounds methyl jasmonate (MeJA) and methyl-β-cyclodextrine (MβCD) to increase triterpene yields. MeJA treatment triggered oxidative stress, arrested growth, and altered the photosynthetic activity of the cells, while increasing squalene, (S)-2,3-epoxysqualene, and cycloartenol contents. Applying MβCD to cultures of C. reinhardtii lead to the sequestration of the two main sterols (ergosterol and 7-dehydroporiferasterol) into the growth medium and the intracellular accumulation of the intermediate cycloartenol, without compromising cell growth. When MβCD was applied in combination with MeJA, it counteracted the negative effects of MeJA on cell growth and physiology, but no synergistic effect on triterpene yield was observed. Together, our findings provide strategies for the triterpene enrichment of microalgal biomass and medium.

Biosorption of cyanate by two strains of Chlamydomonas reinhardtii: evaluation of the removal efficiency and antioxidants activity

Two strains of the chlorophyte Chlamydomonas reinhardtii, a wild type (WT) and a transgenic strain (C.CYN) contained an exogenous cyanase gene (CYN), were used to investigate the growth and cyanate biosorption capability through the analysis of the adsorption equilibrium isotherm. The potential antioxidants activity of the algal strains was also investigated under cyanate concentration. The antioxidants activity of both C.CYN and WT were enhanced by the application of cyanate. Two adsorption isotherm models and the sorption kinetics were used to check the efficiency of the cyanate removal process. The results showed the biosorbent efficiency of Chlamydomonas in the removal of KCNO from aqueous solution. The C.CYN strain has great efficiency to remove cyanate as compared to the WT. The maximum percentage of cyanate removal was 83.75% for the C.CYN and 50% for the WT as treated with 0.8 mg.ml−1 KCNO. The data were adapted to the nonlinear Langmuir model on the basis of the coefficient of determination. The calculated qmax was 0.54 and 0.42 µg.mg−1 for C.CYN and WT which correlated to the experimental one (0.67 and 0.4 µg.mg−1, respectively). Our data highlight the application of the transgenic algal strain toward the removal of highly toxic materials as cyanate. Novelty statement The main objective of this work is to find out an efficient genetically-modified Chlamydomonas strain to remove the highly toxic cyanate compound from contaminated area. Moreover, to evaluate the biosorption ability of this transgenic strain with its wild one via two adsorption isotherm (the Langmuir and Freundlich) models. Also, to estimate the antioxidants activity of these strains under the cyanate toxicity through four different assays.