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Comparative Study Highlights the Potential of Spectral Deconvolution for Fucoxanthin Screening in Live Phaeodactylum tricornutum Cultures.

ABSTRACT: Microalgal biotechnology shows considerable promise as a sustainable contributor to a broad range of industrial avenues. The field is however limited by processing methods that have commonly hindered the progress of high throughput screening, and consequently development of improved microalgal strains. We tested various microplate reader and flow cytometer methods for monitoring the commercially relevant pigment fucoxanthin in the marine diatom Phaeodactylum tricornutum. Based on accuracy and flexibility, we chose one described previously to adapt to live culture samples using a microplate reader and achieved a high correlation to HPLC (R² = 0.849), effectively removing the need for solvent extraction. This was achieved by using new absorbance spectra inputs, reducing the detectable pigment library and changing pathlength values for the spectral deconvolution method in microplate reader format. Adaptation to 384-well microplates and removal of the need to equalize cultures by density further increased the screening rate. This work is of primary interest to projects requiring detection of biological pigments, and could theoretically be extended to other organisms and pigments of interest, improving the viability of microalgae biotechnology as a contributor to sustainable industry.

SUBMITTER: Macdonald Miller S

PROVIDER: S-EPMC8780081 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Rapid Sorting of Fucoxanthin-Producing Phaeodactylum tricornutum Mutants by Flow Cytometry.

Project description:Fucoxanthin, which is widely found in seaweeds and diatoms, has many benefits to human health, such as anti-diabetes, anti-obesity, and anti-inflammatory physiological activities. However, the low content of fucoxanthin in brown algae and diatoms limits the commercialization of this product. In this study, we introduced an excitation light at 488 nm to analyze the emitted fluorescence of Phaeodactylum tricornutum, a diatom model organism rich in fucoxanthin. We observed a unique spectrum peak at 710 nm and found a linear correlation between fucoxanthin content and the mean fluorescence intensity. We subsequently used flow cytometry to screen high-fucoxanthin-content mutants created by heavy ion irradiation. After 20 days of cultivation, the fucoxanthin content of sorted cells was 25.5% higher than in the wild type. This method provides an efficient, rapid, and high-throughput approach to screen fucoxanthin-overproducing mutants.

| S-EPMC8072577 | biostudies-literature

Anti-Obesity Effect of Standardized Extract of Microalga Phaeodactylum tricornutum Containing Fucoxanthin.

Project description:Fucoxanthin (FX), a marine carotenoid found in macroalgae and microalgae, exhibits several beneficial effects to health. The anti-obesity activity of FX is well documented, but FX has not been mass-produced or applied extensively or commercially because of limited availability of raw materials and complex extraction techniques. In this study, we investigated the anti-obesity effect of standardized FX powder (Phaeodactylum extract (PE)) developed from microalga Phaeodactylum tricornutum as a commercial functional food. The effects of PE on adipogenesis inhibition in 3T3-L1 adipocytes and anti-obesity in high-fat diet (HFD)-fed C57BL/6J mice were evaluated. PE and FX dose-dependently decreased intracellular lipid contents in adipocytes without cytotoxicity. In HFD-fed obese mice, PE supplementation for six weeks decreased body weight, organ weight, and adipocyte size. In the serum parameter analysis, the PE-treated groups showed attenuation of lipid metabolism dysfunction and liver damage induced by HFD. In the liver, uncoupling protein-1 (UCP1) upregulation and peroxisome proliferator activated receptor γ (PPARγ) downregulation were detected in the PE-treated groups. Additionally, micro computed tomography revealed lower fat accumulation in PE-treated groups compared to that in the HFD group. These results indicate that PE exerts anti-obesity effects by inhibiting adipocytic lipogenesis, inducing fat mass reduction and decreasing intracellular lipid content, adipocyte size, and adipose weight.

| S-EPMC6562887 | biostudies-literature

Characterization of gene clusters encoding the fucoxanthin chlorophyll proteins of the diatom Phaeodactylum tricornutum.

Project description:We are studying the multigene family encoding the fucoxanthin-chlorophyll binding proteins (fcp genes) that constitute the major component of the photosystem II-associated light harvesting complex in diatoms and brown algae. The characteristics of clusters of fcp genes on the genome of the diatom Phaeodactylum tricornutum are described. Sequence analysis of two genomic clones, PT5 and PT4, has demonstrated the presence of four fcp genes (fcpA, fcpB, fcpC, fcpD) on the former and two fcp genes (fcpE, fcpF) on the latter. The proteins encoded by the six characterized fcp genes range in similarity from 86% to 99%. The genes within each cluster are separated by short intergenic sequences (between 0.5 to 1.1 kb). None of these genes contain introns and all appear to be transcribed with short 5' transcribed, untranslated leader sequences; the transcription initiation sites were mapped 26 to 48 bases upstream of the ATG translation start site. Small conserved motifs are found among all of the genes just upstream of both the translation and the transcription start sites. The codon bias is similar in all of the fcp genes, with a predominance of pyrimidines in the third positions of codons of the four codon families. The two fcp genes that are most similar are fcpC and fcpD, and might represent a recent gene duplication. Southern analyses using fcp cDNAs as hybridization probes suggest that there may be additional sequences on the P. tricornutum genome that resemble the characterized fcp sequences.

| S-EPMC311176 | biostudies-other

Exogenous Arachidonic Acid Affects Fucoxanthin Biosynthesis and Photoprotection in <i>Phaeodactylum tricornutum</i>.

Project description:Fucoxanthin is an oxygenated carotenoid component that has been reported to play important roles in anti-oxidation, anti-obesity and anti-cancer in the human body. Fucoxanthin-chlorophyll protein (FCP) complexes participate in light harvesting and photoprotection in diatom. In order to better understand the change of fucoxanthin content and its role in photoprotection, the growth, fucoxanthin biosynthesis and photosynthetic phenotypes were studied in the diatom Phaeodactylum tricornutum under the treatment of exogenous arachidonic acid (AA). Our results demonstrated that even low concentration of AA at 0.1 mg/L strongly induced fucoxanthin accumulation in algal cells to a maximum of 1.1 mg/g dry weight (DW), which was 36.6% higher than that in the untreated ones. By principal component analysis (PCA), we also identified a close correlation between fucoxanthin accumulation and the expression of genes involved in fucoxanthin biosynthesis, especially phytoene synthase (PSY), suggesting that AA change the metabolism of fucoxanthin by inducing carotenoid metabolic enzymes at the transcriptional level. Furthermore, we found that the exogenous application of AA affected non-photochemical quenching (NPQ) and photoinhibition, which resulted from the changed diadinoxanthin (DD) and diatoxanthin (DT) cycle, and thus played an important role in photoprotection.

| S-EPMC9605588 | biostudies-literature

Fucoxanthin, A Carotenoid Derived from Phaeodactylum tricornutum Exerts Antiproliferative and Antioxidant Activities In Vitro.

Project description:Microalgae contain a multitude of nutrients and can be grown sustainably. Fucoxanthin, a carotenoid from Phaeodactylum tricornutum, could have beneficial health effects. Therefore, we investigated the anti-inflammatory, antioxidative and antiproliferative effects of fucoxanthin derived from this diatom in vitro. The effects of purified fucoxanthin on metabolic activity were assessed in blood mononuclear cells and different cell lines. In cell lines, caspase 3/7 activity was also analyzed. Nitrogen monoxide release and mRNA-expression of proinflammatory cytokines were measured. For antioxidant assays, cell free assays were conducted. Additionally, the antioxidant effect in neutrophils was quantified and glutathione was determined in HeLa cells. The results show that neither did fucoxanthin have anti-inflammatory properties nor did it exert cytotoxic effects on mononuclear cells. However, the metabolic activity of cell lines was decreased up to 58% and fucoxanthin increased the caspase 3/7 activity up to 4.6-fold. Additionally, dose-dependent antioxidant effects were detected, resulting in a 63% decrease in chemiluminescence in blood neutrophils and a 3.3-fold increase in the ratio of reduced to oxidized glutathione. Our studies show that fucoxanthin possesses antiproliferative and antioxidant activities in vitro. Hence, this carotenoid or the whole microalgae P. tricornutum could be considered as a food or nutraceutical in human nutrition, showcasing beneficial health effects.

| S-EPMC6617271 | biostudies-literature

Overexpression of PtVDL1 in <i>Phaeodactylum tricornutum</i> Increases Fucoxanthin Content under Red Light.

Project description:Phaeodactylum tricornutum is a model diatom with significant biotechnological applications, including enhancing biomass, biofuel, and carotenoid production. Specifically, owing to the capacity of this organism to serve as a valuable source of essential raw materials for pharmaceuticals and nutraceuticals, ongoing research is actively focused on enhancing its productivity. One of the genes involved in various stages of fucoxanthin (Fx) biosynthesis, violaxanthin de-epoxidase like 1 (VDL1), has recently been identified. To validate the intracellular function of this gene and boost Fx production through overexpression, we established and examined three transgenic P. tricornutum lines characterized by elevated P. tricortunum VDL1 ( PtVDL1) expression and evaluate their cell growth and Fx productivity. These transgenic lines exhibited substantially increased PtVDL1 mRNA and protein levels compared to the wild type (WT). Notably, the enzyme substrate violaxanthin was entirely depleted and could not be detected in the transformants, whereas it remained at constant levels in the WT. Interestingly, under standard white light conditions, Fx productivity in the transformants remained unchanged; however, but after 48 h of exposure to red light, it increased by up to 15%. These results indicate that PtVDL1-overexpressing P. tricornutum has industrial potential, particularly for enhancing Fx production under red light conditions.

| S-EPMC10840463 | biostudies-literature

A key gene, violaxanthin de-epoxidase-like 1, enhances fucoxanthin accumulation in Phaeodactylum tricornutum.

Project description:BackgroundFucoxanthin has been widely investigated owing to its beneficial biological properties, and the model diatom Phaeodactylum tricornutum, possessing fucoxanthin (Fux) chlorophyll proteins as light-harvesting systems, is considered to have the potential to become a commercial cell factory for the pigment production.ResultsHere, we compared the pigment contents in 10 different P. tricornutum strains from the globe, and found that strain CCMP631 (Pt6) exhibited the highest Fux content but with a low biomass. Comparison of mRNA levels revealed that higher Fux content in Pt6 was related with the higher expression of gene violaxanthin de-epoxidase-like (VDL) protein 1 (VDL1), which encodes the enzyme catalyzing the tautomerization of violaxanthin to neoxanthin in Fux biosynthesis pathway. Single nucleotide variants of VDL1 gene and allele-specific expression in strains Pt1 (the whole genome sequenced strain CCMP632) and Pt6 were analyzed, and overexpressing of each of the 4 VDL1 alleles, two from Pt1 and two from Pt6, in strain Pt1 leads to an increase in downstream product diadinoxanthin and channels the pigments towards Fux biosynthesis. All the 8 VDL1 overexpression (OE) lines showed significant increases by 8.2 to 41.7% in Fux content without compromising growth, and VDL1 Allele 2 OE lines even exhibited the higher cell density on day 8, with an increase by 24.2-28.7% in two Pt1VDL1-allele 2 OE lines and 7.1-11.1% in two Pt6VDL1-allele 2 OE lines, respectively.ConclusionsThe results reveal VDL1, localized in the plastid stroma, plays a key role in Fux over-accumulation in P. tricornutum. Overexpressing VDL1, especially allele 2, improved both the Fux content and growth rate, which provides a new strategy for the manipulation of Fux production in the future.

| S-EPMC10986045 | biostudies-literature

A Lipophilic Fucoxanthin-Rich Phaeodactylum tricornutum Extract Ameliorates Effects of Diet-Induced Obesity in C57BL/6J Mice.

Project description:Phaeodactylum tricornutum (P. tricornutum) comprise several lipophilic constituents with proposed anti-obesity and anti-diabetic properties. We investigated the effect of an ethanolic P. tricornutum extract (PTE) on energy metabolism in obesity-prone mice fed a high fat diet (HFD). Six- to eight-week-old male C57BL/6J mice were switched to HFD and, at the same time, received orally placebo or PTE (100 mg or 300 mg/kg body weight/day). Body weight, body composition, and food intake were monitored. After 26 days, blood and tissue samples were collected for biochemical, morphological, and gene expression analyses. PTE-supplemented mice accumulated fucoxanthin metabolites in adipose tissues and attained lower body weight gain, body fat content, weight of white adipose tissue (WAT) depots, and inguinal WAT adipocyte size than controls, independent of decreased food intake. PTE supplementation was associated with lower expression of Mest (a marker of fat tissue expandability) in WAT depots, lower gene expression related to lipid uptake and turnover in visceral WAT, increased expression of genes key to fatty acid oxidation and thermogenesis (Cpt1, Ucp1) in subcutaneous WAT, and signs of thermogenic activation including enhanced UCP1 protein in interscapular brown adipose tissue. In conclusion, these data show the potential of PTE to ameliorate HFD-induced obesity in vivo.

| S-EPMC6521120 | biostudies-literature

Production of Fucoxanthin from Phaeodactylum tricornutum Using High Performance Countercurrent Chromatography Retaining Its FOXO3 Nuclear Translocation-Inducing Effect.

Project description:Phaeodactylum tricornutum is a rich source of fucoxanthin, a carotenoid with several health benefits. In the present study, high performance countercurrent chromatography (HPCCC) was used to isolate fucoxanthin from an extract of P. tricornutum. A multiple sequential injection HPCCC method was developed combining two elution modes (reverse phase and extrusion). The lower phase of a biphasic solvent system (n-heptane, ethyl acetate, ethanol and water, ratio 5/5/6/3, v/v/v/v) was used as the mobile phase, while the upper phase was the stationary phase. Ten consecutive sample injections (240 mg of extract each) were performed leading to the separation of 38 mg fucoxanthin with purity of 97% and a recovery of 98%. The process throughput was 0.189 g/h, while the efficiency per gram of fucoxanthin was 0.003 g/h. Environmental risk and general process evaluation factors were used for assessment of the developed separation method and compared with existing fucoxanthin liquid-liquid isolation methods. The isolated fucoxanthin retained its well-described ability to induce nuclear translocation of transcription factor FOXO3. Overall, the developed isolation method may represent a useful model to produce biologically active fucoxanthin from diatom biomass.

| S-EPMC8466784 | biostudies-literature

Fucoxanthin from microalgae Phaeodactylum tricornutum inhibits pro-inflammatory cytokines by regulating both NF-κB and NLRP3 inflammasome activation.

Project description:Pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α are mediated by the activation of various kinds of signaling pathways in the innate immune system. Particularly, NF-κB and NLRP3 inflammasome signaling are involved in the production and secretion of these cytokines. Each signaling is participated in the two steps necessary for IL-1β, a representative pro-inflammatory cytokine, to be processed into a form secreted by cells. In the priming step stimulated by LPS, pro-IL-1β is synthesized through NF-κB activation. Pro-IL-1β cleavages into mature IL-1β by formed NLRP3 inflammasome in the activation step induced by ATP. The mature form of IL-1β is subsequently secreted out of the cell, causing inflammation. Moreover, IL-6 and TNF-α are known to increase in NLRP3 inflammasome-mediated conditions. Here, we found that fucoxanthin, one of the major components of Phaeodactylum tricornutum, has an inhibitory effect on NF-κB and NLRP3 inflammasome activation induced by the combination of LPS and ATP in bone marrow-derived immune cells as well as astrocytes. Fucoxanthin, which is abundant in the EtOH fraction of Phaeodactylum tricornutum extracts, has shown to have less cell toxicity and found to decrease the production of major pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. Fucoxanthin has also shown to suppress the expression of cleaved caspase-1 and the oligomerization of ASC, which are the main components of the NLRP3 inflammasome. Furthermore, phosphorylated IκBα and pro-IL-1β expression decreased in the presence of fucoxanthin, suggesting that fucoxanthin can negatively regulate the priming step of inflammasome signaling. Thus, our results provide reliable evidence that fucoxanthin may serve as a key candidate in the development of potential therapeutic agents for inflammatory diseases as well as neurodegenerative diseases caused by NF-κB and NLRP3 inflammasome activation.

| S-EPMC7803995 | biostudies-literature

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