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  • 1
    Online Resource
    Online Resource
    Frontiers Media SA ; 2022
    In:  Frontiers in Plant Science Vol. 12 ( 2022-1-4)
    In: Frontiers in Plant Science, Frontiers Media SA, Vol. 12 ( 2022-1-4)
    Abstract: CRISPR/Cas9-mediated genome editing has been demonstrated in the model diatom P. tricornutum , yet the currently available genetic tools do not combine the various advantageous features into a single, easy-to-assemble, modular construct that would allow the multiplexed targeting and creation of marker-free genome-edited lines. In this report, we describe the construction of the first modular two-component transcriptional unit system expressing Sp Cas9 from a diatom episome, assembled using the Universal Loop plasmid kit for Golden Gate assembly. We compared the editing efficiency of two constructs with orthogonal promoter-terminator combinations targeting the StLDP gene, encoding the major lipid droplet protein of P. tricornutum . Multiplexed targeting of the StLDP gene was confirmed via PCR screening, and lines with homozygous deletions were isolated from primary exconjugants. An editing efficiency ranging from 6.7 to 13.8% was observed in the better performing construct. Selected gene-edited lines displayed growth impairment, altered morphology, and the formation of lipid droplets during nutrient-replete growth. Under nitrogen deprivation, oversized lipid droplets were observed; the recovery of cell proliferation and degradation of lipid droplets were impaired after nitrogen replenishment. The results are consistent with the key role played by StLDP in the regulation of lipid droplet size and lipid homeostasis.
    Type of Medium: Online Resource
    ISSN: 1664-462X
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2687947-5
    detail.hit.zdb_id: 2613694-6
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  • 2
    Online Resource
    Online Resource
    MDPI AG ; 2021
    In:  Journal of Fungi Vol. 7, No. 2 ( 2021-02-01), p. 100-
    In: Journal of Fungi, MDPI AG, Vol. 7, No. 2 ( 2021-02-01), p. 100-
    Abstract: The blastocladialean fungus Paraphysoderma sedebokerense parasitizes three microalgae species of economic interest: Haematococcus pluvialis, Chromochloris zofingiensis and Scenedesmus dimorphus. For the first time, we characterized the developmental stages of isolated fungal propagules in H. pluvialis co-culture, finding a generation time of 16 h. We established a patho-system to compare the infection in the three different host species for 48 h, with two different setups to quantify parameters of the infection and parameters of the parasite fitness. The prevalence of the parasite in H. pluvialis and C. zofingiensis cultures was 100%, but only 20% in S. dimorphus culture. The infection of S. dimorphus not only reached lower prevalence but was also qualitatively different; the infection developed preferentially on senescent cells and more resting cysts were produced, being consistent with a reservoir host. In addition, we carried out cross infection experiments and the inoculation of a mixed algal culture containing the three microalgae, to determine the susceptibility of the host species and to investigate the preference of P. sedebokerense for these microalgae. The three tested microalgae showed different susceptibility to P. sedebokerense, which correlates with blastoclad’s preference to the host in the following order: H. pluvialis 〉 C. zofingiensis 〉 S. dimorphus.
    Type of Medium: Online Resource
    ISSN: 2309-608X
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2784229-0
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  • 3
    Online Resource
    Online Resource
    MDPI AG ; 2023
    In:  Plants Vol. 12, No. 18 ( 2023-09-11), p. 3230-
    In: Plants, MDPI AG, Vol. 12, No. 18 ( 2023-09-11), p. 3230-
    Abstract: The blastocladialean fungus P. sedebokerense is a facultative parasite of economically important microalgae and for this reason it has gained a lot of interest. P. sedebokerense has a complex life cycle which includes vegetative and resting stages. The resting cysts were assumed to play an essential role in survival by resisting drought, but this ability was never tested and the factors that trigger their formation were not evaluated. This study was aimed to induce resting cyst formation and germination in P. sedebokerense. At first, we tested the survival of P. sedebokerense liquid cultures and found that infectivity is retained for less than two months when the cultures were stored on the bench at room temperature. We noticed that dry cultures retained the infectivity for a longer time. We, thus, developed a method, which is based on dehydration and rehydration of the biomass, to produce, maintain, and germinate resting cysts of P. sedebokerense in both saprophytic and parasitic modes of growth. When the dry cultures were rehydrated and incubated at 30 °C, resting cysts asynchronously germinated after 5 h and the “endosporangium” was protruding outside of the cyst. Our method can be used to preserve P. sedebokerense for research purposes with the advantage of no need for expensive equipment.
    Type of Medium: Online Resource
    ISSN: 2223-7747
    Language: English
    Publisher: MDPI AG
    Publication Date: 2023
    detail.hit.zdb_id: 2704341-1
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  • 4
    In: Molecular Nutrition & Food Research, Wiley, Vol. 67, No. 6 ( 2023-03)
    Abstract: Microalgae are an emerging nutritional resource of biomolecules with potential to alleviate gut inflammation. The study explores the anti‐inflammatory and immunomodulatory potential of the microalga Lobosphaera incisa P127, which accumulates a rare omega‐6 LC‐PUFA dihomo‐ɣ‐linolenic acid (DGLA) under nitrogen starvation. The therapeutic potential of dietary supplementation with P127 is investigated in the zebrafish model of IBD (TNBS‐induced colitis). Methods and results Guts are sampled from zebrafish fed experimental diets for 4 weeks, before and 24 h after TNBS challenge. Diets containing 15% non‐starved (Ns) and 7.5% and 15% N‐starved (St) algal biomass significantly attenuate the severity of gut injury and goblet cell depletion. In contrast, diets containing 7.5% Ns and DGLA ethyl ester have no effect on gut condition. Fish fed 15% St, high‐DGLA biomass, have the fewest individuals with pathological alterations in the gut. Dietary inclusion of Ns and St distinctly modulates gut‐associated expression of the immune and inflammatory genes. Fish fed 15% Ns biomass display a coordinated boost in immune gene expression and show major changes in the gut microbiome prior challenge. Conclusion Dietary inclusion of L. incisa biomass at two physiological states, ameliorates TNBS‐induced gut inflammation, suggesting the synergistic beneficial effects of biomass components not limited to DGLA.
    Type of Medium: Online Resource
    ISSN: 1613-4125 , 1613-4133
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2023
    detail.hit.zdb_id: 2160372-8
    SSG: 12
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  • 5
    In: Separation and Purification Technology, Elsevier BV, Vol. 289 ( 2022-05), p. 120744-
    Type of Medium: Online Resource
    ISSN: 1383-5866
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2022
    detail.hit.zdb_id: 2022535-0
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  • 6
    Online Resource
    Online Resource
    Frontiers Media SA ; 2020
    In:  Frontiers in Plant Science Vol. 11 ( 2020-11-27)
    In: Frontiers in Plant Science, Frontiers Media SA, Vol. 11 ( 2020-11-27)
    Abstract: The green microalga Lobosphaera incisa accumulates triacylglycerols (TAGs) with exceptionally high levels of long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid (ARA) under nitrogen (N) deprivation. Phosphorous (P) deprivation induces milder changes in fatty acid composition, cell ultrastructure, and growth performance. We hypothesized that the resource-demanding biosynthesis and sequestration of ARA-rich TAG in lipid droplets (LDs) are associated with the enhancement of catabolic processes, including membrane lipid turnover and autophagic activity. Although this work focuses mainly on N deprivation, a comparative analysis of N and P deprivation responses is included. The results of lipidomic profiling showed a differential impact of N and P deprivation on the reorganization of glycerolipids. The formation of TAG under N deprivation was associated with the enhanced breakdown of chloroplast glycerolipids and the formation of lyso-lipids. N-deprived cells displayed a profound reorganization of cell ultrastructure, including internalization of cellular material into autophagic vacuoles, concomitant with the formation of LDs, while P-deprived cells showed better cellular ultrastructural integrity. The expression of the hallmark autophagy protein ATG8 and the major lipid droplet protein (MLDP) genes were coordinately upregulated, but to different extents under either N or P deprivation. The expression of the Δ5-desaturase gene, involved in the final step of ARA biosynthesis, was coordinated with ATG8 and MLDP , exclusively under N deprivation. Concanamycin A, the inhibitor of vacuolar proteolysis and autophagic flux, suppressed growth and enhanced levels of ATG8 and TAG in N-replete cells. The proportions of ARA in TAG decreased with a concomitant increase in oleic acid under both N-replete and N-deprived conditions. The photosynthetic apparatus’s recovery from N deprivation was impaired in the presence of the inhibitor, along with the delayed LD degradation. The GFP-ATG8 processing assay showed the release of free GFP in N-replete and N-deprived cells, supporting the existence of autophagic flux. This study provides the first insight into the homeostatic role of autophagy in L. incisa and points to a possible metabolic link between autophagy and ARA-rich TAG biosynthesis.
    Type of Medium: Online Resource
    ISSN: 1664-462X
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2020
    detail.hit.zdb_id: 2687947-5
    detail.hit.zdb_id: 2613694-6
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  • 7
    In: Nutrients, MDPI AG, Vol. 12, No. 9 ( 2020-09-22), p. 2892-
    Abstract: Microalgae have been considered as a renewable source of nutritional, cosmetic and pharmaceutical compounds. The ability to produce health-beneficial long-chain polyunsaturated fatty acids (LC-PUFA) is of high interest. LC-PUFA and their metabolic lipid mediators, modulate key inflammatory pathways in numerous models. In particular, the metabolism of arachidonic acid under inflammatory challenge influences the immune reactivity of macrophages. However, less is known about another omega-6 LC-PUFA, dihomo-γ-linolenic acid (DGLA), which exhibits potent anti-inflammatory activities, which contrast with its delta-5 desaturase product, arachidonic acid (ARA). In this work, we examined whether administrating DGLA would modulate the inflammatory response in the RAW264.7 murine macrophage cell line. DGLA was applied for 24 h in the forms of carboxylic (free) acid, ethyl ester, and ethyl esters obtained from the DGLA-accumulating delta-5 desaturase mutant strain P127 of the green microalga Lobosphaera incisa. DGLA induced a dose-dependent increase in the RAW264.7 cells’ basal secretion of the prostaglandin PGE1. Upon bacterial lipopolysaccharide (LPS) stimuli, the enhanced production of pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin 1β (IL-1β), was affected little by DGLA, while interleukin 6 (IL-6), nitric oxide, and total reactive oxygen species (ROS) decreased significantly. DGLA administered at 100 µM in all forms attenuated the LPS-induced expression of the key inflammatory genes in a concerted manner, in particular iNOS, IL-6, and LxR, in the form of free acid. PGE1 was the major prostaglandin detected in DGLA-supplemented culture supernatants, whose production prevailed over ARA-derived PGE2 and PGD2, which were less affected by LPS-stimulation compared with the vehicle control. An overall pattern of change indicated DGLA’s induced alleviation of the inflammatory state. Finally, our results indicate that microalgae-derived, DGLA-enriched ethyl esters (30%) exhibited similar activities to DGLA ethyl esters, strengthening the potential of this microalga as a potent source of this rare anti-inflammatory fatty acid.
    Type of Medium: Online Resource
    ISSN: 2072-6643
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2518386-2
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  • 8
    In: Journal of Phycology, Wiley, Vol. 56, No. 6 ( 2020-12), p. 1642-1663
    Abstract: Lipid droplets (LDs) are an organelle conserved amongst all eukaryotes, consisting of a neutral lipid core surrounded by a polar lipid monolayer. Many species of microalgae accumulate LDs in response to stress conditions, such as nitrogen starvation. Here, we report the isolation and proteomic profiling of LD proteins from the model oleaginous pennate diatom Phaeodactylum tricornutum , strain Pt4 (UTEX 646). We also provide a quantitative description of LD morphological ontogeny, and fatty acid content. Novel cell disruption and LD isolation methods, combined with suspension‐trapping and nanoflow liquid chromatography coupled to high resolution mass spectrometry, yielded an unprecedented number of LD proteins. Predictive annotation of the LD proteome suggests a broad assemblage of proteins with diverse functions, including lipid metabolism and vesicle trafficking, as well as ribosomal and proteasomal machinery. These proteins provide mechanistic insights into LD processes, and evidence for interactions between LDs and other organelles. We identify for the first time several key steps in diatom LD‐associated triacylglycerol biosynthesis. Bioinformatic analyses of the LD proteome suggests multiple protein targeting mechanisms, including amphipathic helices, post‐translational modifications, and translocation machinery. This work corroborates recent findings from other strains of P. tricornutum , other diatoms, and other eukaryotic organisms, suggesting that the fundamental proteins orchestrating LDs are conserved, and represent an ancient component of the eukaryotic endomembrane system. We postulate a comprehensive model of nitrogen starvation‐induced diatom LDs on a molecular scale, and provide a wealth of candidates for metabolic engineering, with the potential to eventually customize LD contents.
    Type of Medium: Online Resource
    ISSN: 0022-3646 , 1529-8817
    URL: Issue
    RVK:
    Language: English
    Publisher: Wiley
    Publication Date: 2020
    detail.hit.zdb_id: 281226-5
    detail.hit.zdb_id: 1478748-9
    SSG: 12
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  • 9
    Online Resource
    Online Resource
    Elsevier BV ; 2020
    In:  Fungal Biology Vol. 124, No. 6 ( 2020-06), p. 612-
    In: Fungal Biology, Elsevier BV, Vol. 124, No. 6 ( 2020-06), p. 612-
    Type of Medium: Online Resource
    ISSN: 1878-6146
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2020
    detail.hit.zdb_id: 2532164-X
    SSG: 12
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  • 10
    In: Biology, MDPI AG, Vol. 10, No. 2 ( 2021-02-16), p. 157-
    Abstract: Several green algae can divide by multiple fission and spontaneously synchronize their cell cycle with the available light regime. The yields that can be obtained from a microalgal culture are directly affected by cell cycle events. Chromochloris zofingiensis is considered as one of the most promising microalgae for biotechnological applications due to its fast growth and the flexible trophic capabilities. It is intensively investigated in the context of bio-commodities production (carotenoids, storage lipids); however, the pattern of cell-cycle events under common cultivation strategies was not yet characterized for C. zofingiensis. In this study, we have employed fluorescence microscopy to characterize the basic cell-cycle dynamics under batch and continuous modes of phototrophic C. zofingiensis cultivation. Staining with SYBR green—applied in DMSO solution—enabled, for the first time, the clear and simple visualization of polynuclear stages in this microalga. Accordingly, we concluded that C. zofingiensis divides by a consecutive pattern of multiple fission, whereby it spontaneously synchronizes growth and cell division according to the available illumination regime. In high-light continuous culture or low-light batch culture, C. zofingiensis cell-cycle was completed within several light-dark (L/D) cycles (14 h/10 h); however, cell divisions were synchronized with the dark periods only in the high-light continuous culture. In both modes of cultivation, daughter cell release was mainly facilitated by division of 8 and 16-polynuclear cells. The results of this study are of both fundamental and applied science significance and are also important for the development of an efficient nuclear transformation system for C. zofingiensis.
    Type of Medium: Online Resource
    ISSN: 2079-7737
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2661517-4
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