Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (II) evaluation of TAG yield and productivity in controlled photobioreactors

Background Many microalgae accumulate carbohydrates simultaneously with triacylglycerol (TAG) upon nitrogen starvation, and these products compete for photosynthetic products and metabolites from the central carbon metabolism. As shown for starchless mutants of the non-oleaginous model alga Chlamydomonas reinhardtii, reduced carbohydrate synthesis can enhance TAG production. However, these mutants still have a lower TAG productivity than wild-type oleaginous microalgae. Recently, several starchless mutants of the oleaginous microalga Scenedesmus obliquus were obtained which showed improved TAG content and productivity. Results The most promising mutant, slm1, is compared in detail to wild-type S. obliquus in controlled photobioreactors. In the slm1 mutant, the maximum TAG content increased to 57¿±¿0.2% of dry weight versus 45¿±¿1% in the wild type. In the wild type, TAG and starch were accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The starchless mutant did not produce starch and the liberated photosynthetic capacity was directed towards TAG synthesis. This increased the maximum yield of TAG on light by 51%, from 0.144¿±¿0.004 in the wild type to 0.217¿±¿0.011 g TAG/mol photon in the slm1 mutant. No differences in photosynthetic efficiency between the slm1 mutant and the wild type were observed, indicating that the mutation specifically altered carbon partitioning while leaving the photosynthetic capacity unaffected. Conclusions The yield of TAG on light can be improved by 51% by using the slm1 starchless mutant of S. obliquus, and a similar improvement seems realistic for the areal productivity in outdoor cultivation. The photosynthetic performance is not negatively affected in the slm1 and the main difference with the wild type is an improved carbon partitioning towards TAG

Les théologiens des IIe-IIIe siècles: les Pères de l'Eglise mettent de l'ordre.

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Improved DNA/protein delivery in microalgae – A simple and reliable method for the prediction of optimal electroporation settings

Genetic transformation of microalgae remains a challenge due to poor intracellular delivery of exogenous molecules. This limitation is caused by the structure and composition of the cell wall and cell membrane of each species. Moreover, successful delivery of proteins or nucleic acids cannot be assessed by determining transformability since their functionality is not always known in the studied microorganism. We propose a quick and effective screening tool for the prediction and optimization of electroporation settings by monitoring cell permeability and viability using Sytox Green and propidium iodide respectively. We determined voltage settings for the microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, Neochloris oleoabundans and Acutodesmus obliquus. To evaluate the predicted settings, we delivered labelled DNA and proteins into the cells. We demonstrated that high transformation efficiencies can be accomplished when predicted values were applied with functional plasmids. Additionally, we increased transformation efficiencies by testing cell concentrations, light intensities and fragment sizes. This method can be used to determine suitable transformation conditions for non-transformed microalgae species and to increase the insight on established transformation protocols.</p

<i>Neochloris oleoabundans</i> is worth its salt: Transcriptomic analysis under salt and nitrogen stress

<div><p><i>Neochloris oleoabundans</i> is an oleaginous microalgal species that can be cultivated in fresh water as well as salt water. Using salt water gives the opportunity to reduce production costs and the fresh water footprint for large scale cultivation. Production of triacylglycerols (TAG) usually includes a biomass growth phase in nitrogen-replete conditions followed by a TAG accumulation phase under nitrogen-deplete conditions. This is the first report that provides insight in the saline resistance mechanism of a fresh water oleaginous microalgae. To better understand the osmoregulatory mechanism of <i>N</i>. <i>oleoabundans</i> during growth and TAG accumulating conditions, the transcriptome was sequenced under four different conditions: fresh water nitrogen-replete and -deplete conditions, and salt water (525 mM dissolved salts, 448mM extra NaCl) nitrogen-replete and -deplete conditions. In this study, several pathways are identified to be responsible for salt water adaptation of <i>N</i>. <i>oleoabundans</i> under both nitrogen-replete and -deplete conditions. Proline and the ascorbate-glutathione cycle seem to be of importance for successful osmoregulation in <i>N</i>. <i>oleoabundans</i>. Genes involved in Proline biosynthesis were found to be upregulated in salt water. This was supported by Nuclear magnetic resonance (NMR) spectroscopy, which indicated an increase in proline content in the salt water nitrogen-replete condition. Additionally, the lipid accumulation pathway was studied to gain insight in the gene regulation in the first 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen-deplete conditions in a comparable way in both fresh and salt water. The mechanism behind the biosynthesis of compatible osmolytes can be used to improve <i>N</i>. <i>oleoabundans</i> and other industrially relevant microalgal strains to create a more robust and sustainable production platform for microalgae derived products in the future.</p></div

Proline and GABA biosynthesis pathway.

<p>For the figure legend refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194834#pone.0194834.g002" target="_blank">Fig 2</a>. Abbreviations: GSA: glutamate-semialdehyde. P5C: L-1-Pyrroline-5-carboxylate. GABA: 4-aminobutanoate. P5CR: P5C reductase. P5CS1: glutamate-5-kinase. P5CS2: GSA dehydrogenase. GDH: glutamate dehydrogenase (EC:1.4.1.3/4). GST: glutamate synthase (EC:1.4.1.13/14). GSTT: glutamine synthetase. GDC: glutamate decarboxylase. OAT: ornithine aminotransferase. PRODH: Proline dehydrogenase. *This reaction occurs non-enzymatically.</p

Biomass composition of <i>N</i>. <i>oleoabundans</i> in the four tested conditions.

<p>Upper panel: Confocal laser scanning microscope images of <i>N</i>. <i>oleoabundans</i> under four different cultivation conditions. (A) Nitrogen-replete fresh water (FN+). (B) Nitrogen-deplete fresh water conditions (FN-). (C) Nitrogen-replete salt water conditions (SN+). (D) Nitrogen-deplete salt water conditions (SN-). Chlorophyll autofluorescence is shown in red and the BODIPY stain is shown in yellow. The bar represents 20 μm. Lower panel: (E) Lipid and starch content. TFA (dark grey), TAG (light grey), and starch (white) 24 hours after medium replacement. The height of the bars represents the average of the two independent measurements. Error bars represent distance of the sample values to the average value. TAG, TFA and starch are given as a percentage of total dry weight. (F) Fatty acid profile under the different conditions expressed as percentage of TFA.</p

Carbon metabolism in <i>N</i>. <i>oleoabundans</i>.

<p>For the figure legend refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194834#pone.0194834.g002" target="_blank">Fig 2</a>. Codes in the white boxes represent the corresponding EC numbers. Abbreviations: PEP phosphoenol pyruvate; LysoPA Lysophosphatidic acid; PA Phosphatidic acid; DAG 1,2-Diacylglycerol; TAG Triacylglycerol. EC:2.3.1.12 could not be annotated from the transcriptome of <i>N</i>. <i>oleoabundans</i>.</p

Glutathione biosynthesis pathway and glutathione-ascorbate cycle.

<p>For the figure legend refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194834#pone.0194834.g002" target="_blank">Fig 2</a>. Abbreviations: SOD Superoxide dismutase; CAT Catalase; APX Ascorbate peroxidase (not annotated); MDHA monodehydroascorbate; MDHAR monodehydroascorbate reductase; DHA dehydroascorbate; DHAR dehydroascorbate reductase; GSH (reduced) Glutathione; GSSG oxidised glutathione; GSHR Glutathione reductase; Glu Glutamate; Cys Cysteine; Gly Glyceine; γ-GCSTT γ-glutamylcystein Synthetase; γ-Glu-Cys γ-glutamylcystein; GSHSTT Glutathione Synthetase; GSH-S-T glutathione S-transferase; PCST Phytochelatin synthase. * This reaction can occur enzymatically or non-enzymatically.</p

Biosynthesis pathway of sucrose and starch.

<p>The values that are shown in the following figures refer to the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for each condition. The white left-most box represents the FPKM value of the respective gene in the fresh water nitrogen-replete reference condition. This is followed by three colored boxes that represent the log2 fold change (LFC) of the other conditions compared to the reference condition. The order of the remaining three boxes are from left to right, FN-, SN+, SN- respectively. Abbreviations: G1PUT: glucose-1-phosphate uridylyltransferase; UTP: Uridine triphosphate. SucPS: sucrose-phosphate synthase. SucST: sucrose synthase. G1PAT: glucose-1-phosphate adenylyltransferase. αTrPS: alpha,alpha-trehalose-phosphate synthase. αTrS: alpha,alpha-trehalose synthase. TrP: trehalose-phosphatase. StS:starch synthase. GlBE: glycogen branching enzyme. SucPP: sucrose-phosphate phosphatase. SucGH: sucrose glucohydrolase. β-am: β-amylase. D-enzyme: 4-alpha-glucanotransferase. StarchP: Starch phosphorylase.</p