Redistribution of metabolic fluxes in Chlorella protothecoides by variation of media nitrogen concentration.
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ABSTRACT:
SUBMITTER: Gopalakrishnan S
PROVIDER: S-EPMC8193236 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Microalgae have been proposed as potential platform to produce lipid-derived products, such as biofuels. Knowledge on the intracellular carbon flow distribution may identify key metabolic processes during lipid synthesis thus refining culture/genetic strategies to maximize cell lipid productivity. A kinetic metabolic model simulating cell metabolic behavior and lipid production was first applied in the microalgae platform Chlorella protothecoides under heterotrophic condition. It combines both physiology and flux information in a kinetic approach. Cell nutrition, growth, lipid production and almost 30 metabolic intermediates covering central carbon metabolism were included and simulated. RESULTS:Model simulations were shown to adequately agree with experimental data, which is suggesting that the proposed model copes with Chlorella protothecoides cells' biology. The dynamic metabolic flux analysis using the model showed a reversible starch flux from accumulation to decomposing when glucose reached depletion, while net lipid flux shows a quasi-constant rate. The sensitive flux parameters on starch and lipid metabolism suggested that starch synthesis is the major competing pathway that affects lipid accumulation in C. protothecoides. Flux analysis also demonstrated that high lipid yield under heterotrophic condition is accompanied with high lipid flux and low TCA activity. Meanwhile, the dynamic flux distribution also suggests a relatively constant ratio of glucose distributed to biomass, lipid, starch, nucleotides as well as pentose phosphate pathway. CONCLUSION:The model described not only experimental data, but also unraveled intracellular carbon flow distribution and identify key metabolic processes during lipid synthesis. Most of the metabolic kinetics also showed statistical significance for metabolic mechanism. Therefore, this study unravels the mechanisms of the glucose impact on the dynamic carbon flux distribution, thus improving our understanding of the links between carbon fluxes and lipid metabolism in C. protothecoides.
| S-EPMC6598345 | biostudies-literature
Project description:In this study, biodiesel production from microalgal oil by microwave-assisted transesterification was carried out to investigate its efficiency. Transesterification reactions were performed by using Chlorella protothecoides oil as feedstock, methanol, and potassium hydroxide as the catalyst. Methanol:oil ratio, reaction time and catalyst:oil ratio were investigated as process parameters affected methyl ester yield. 9:1 methanol/oil molar ratio, 1.5% KOH catalyst/oil ratio and 10 min were optimum values for the highest fatty acid methyl ester yield.
| S-EPMC4849035 | biostudies-literature
Project description:Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg(2+) binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.
| S-EPMC4663545 | biostudies-literature
Project description:Chlorella protothecoides has been put forth as a promising candidate for commercial biodiesel production. However, the cost of biodiesel remains much higher than diesel from fossil fuel sources, partially due to the high costs of oil extraction from algae. Here, we identified the presence of a sporopollenin layer outside the polysaccharide cell wall; this was evaluated using transmission electron microscopy, 2-aminoethanol treatment, acetolysis, and Fourier Transform Infrared Spectroscopy. We also performed bioinformatics analysis of the genes of the C. protothecoides genome that are likely involved in sporopollenin synthesis, secretion, and translocation, and evaluated the expression of these genes via real-time PCR. We also found that that removal of this sporopollenin layer greatly improved the efficiency of oil extraction.
| S-EPMC4928020 | biostudies-literature
Project description:To elucidate the lutein biosynthesis pathway in the lutein-producing alga, Chlorella protothecoides CS-41, the ζ-carotene desaturase gene (zds) was isolated from Chlorella protothecoides using the approach of rapid amplification of cDNA ends. The full-length cDNA sequence was 2031 bp and contained 1755 bp putative open reading frame which encodes a 584 amino acid deduced polypeptide whose computed molecular weight was 63.7 kDa. Sequence homology research indicated that the nucleotide and putative protein had sequence identities of 72.5% and 69.5% with those of the green alga Chlamydomonas reinhardtii, respectively. Phylogenetic analysis demonstrated that the ZDS from C. protothecoides CS-41 had a closer relationship with those of chlorophyta and higher plants than with those of other species. In addition, we also found that the zds gene expression was upregulated in response to light.
| S-EPMC3196254 | biostudies-literature
Project description:BACKGROUND: Microalgae-derived biodiesel is a promising substitute for conventional fossil fuels. In particular, the green alga Chlorella protothecoides sp. 0710 is regarded as one of the best candidates for commercial manufacture of microalgae-derived biofuel. This is due not only to its ability to live autotrophically through photosynthesis, but also to its capacity to produce a large amount of biomass and lipid through fermentation of glucose. However, until the present study, neither its genome sequence nor the platform required for molecular manipulations were available. RESULTS: We generated a draft genome for C. protothecoides, and compared its genome size and gene content with that of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169. This comparison revealed that C. protothecoides has a reduced genome size of 22.9 Mbp, about half that of its close relatives. The C. protothecoides genome encodes a smaller number of genes, fewer multi-copy genes, fewer unique genes, and fewer genome rearrangements compared with its close relatives. In addition, three Chlorella-specific hexose-proton symporter (HUP)-like genes were identified that enable the consumption of glucose and, consequently, heterotrophic growth. Furthermore, through comparative transcriptomic and proteomic studies, we generated a global perspective regarding the changes in metabolic pathways under autotrophic and heterotrophic growth conditions. Under heterotrophic conditions, enzymes involved in photosynthesis and CO2 fixation were almost completely degraded, either as mRNAs or as proteins. Meanwhile, the cells were not only capable of quickly assimilating glucose but also showed accelerated glucose catabolism through the upregulation of glycolysis and the tricarboxylic acid (TCA) cycle. Moreover, the rapid synthesis of pyruvate, upregulation of most enzymes involved in fatty acid synthesis, and downregulation of enzymes involved in fatty acid degradation favor the synthesis of fatty acids within the cell. CONCLUSIONS: Despite similarities to other Chlorella, C. protothecoides has a smaller genome than its close relatives. Genes involved in glucose utilization were identified, and these genes explained its ability to grow heterotrophically. Transcriptomic and proteomic results provided insight into its extraordinary ability to accumulate large amounts of lipid. The C. protothecoides draft genome will promote the use of this species as a research model.
| S-EPMC4111847 | biostudies-literature
Project description:Clostridium thermocellum and Thermoanaerobacterium saccharolyticum were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. C. thermocellum produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of T. saccharolyticum, ethanol was the main fermentation product. Enzyme assays confirmed that in C. thermocellum, glycolysis proceeds via pyrophosphate (PPi)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK), as well as a malate shunt for the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase activity was not detectable. In T. saccharolyticum, ATP but not PPi served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present, whereas the activities of a malate shunt enzymes were low in T. saccharolyticum In C. thermocellum, glycolysis via PPi-PFK and PPDK obeys the equation glucose?+?5 NDP?+?3?PPi???2 pyruvate?+?5 NTP?+?Pi (where NDP is nucleoside diphosphate and NTP is nucleoside triphosphate). Metabolic flux analysis of chemostat data with the wild type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PPi-generating mechanism must be present that operates according to ATP?+?Pi???ADP?+?PPi Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type C. thermocellum revealed a bottleneck in pyruvate oxidation, as large amounts of pyruvate and amino acids, mainly valine, were excreted; up to 50% of the nitrogen consumed was excreted again as amino acids.IMPORTANCE This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum, could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP?+?Pi???ADP?+?PPi.
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Project description:The studies of rice nitrogen concentration on the expression of miRNA so far are mostly limited to the exogenous nitrogen, leaving the effect of endogenous nitrogen largely unexplored. OsNAR2.1 is a high-affinity nitrate transporter partner protein which plays a central role in nitrate absorption and translocation in rice. The expression of OsNAR2.1 could influence the concentration of the endogenous nitrogen in rice. We showed that the expression and production of miRNA in rice can be influenced by manipulating the endogenous nitrogen concentration via OsNAR2.1 transgenic lines. The small RNA expressed differently in two transgenic rice lines (nitrogen efficient line with overexpression of OsNAR2.1 (Ov199), nitrogen-inefficient line with knockdown OsNAR2.1 by RNAi (RNAi)) compared to the wild-type (NP). Comparative hierarchical clustering expression pattern analysis revealed that the expression profiles of mature miRNA in both transgenic lines were different from NP.
2023-06-23 | GSE224933 | GEO
Project description:Microalgae can accumulate large proportions of their dry cell weight as storage lipids when grown under appropriate nutrient limiting conditions. While a high ratio of carbon to nitrogen is often cited as the primary mode of triggering lipid accumulation in microalgae, fast optimization strategies to increase lipid production for mixotrophic cultivation have been difficult to developed due to the low cell densities of algal cultures, and consequently the limited amount of biomass available for compositional analysis. Response surface methodologies provide a power tool for assessing complex relationships such as the interaction between the carbon source and nitrogen source. A 15 run Box-Behnken design performed in shaker flasks was effective in studying the effect of carbon, nitrogen, and magnesium on the growth rate, maximum cell density, lipid accumulation rate, and glucose consumption rate. Using end-point dry cell weight and total lipid content as assessed by direct transesterification to FAME, numerical optimization resulted in a significant increase in lipid content from 18.5?±?0.76% to 37.6?±?0.12% and a cell density of 5.3?±?0.1?g/L to 6.1?±?0.1?g/L between the centre point of the design and the optimized culture conditions. The presented optimization process required less than 2 weeks to complete, was simple, and resulted in an overall lipid productivity of 383?mg/L·d.
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Project description:The fibrin clot is gelatinous matter formed upon injury to stop blood loss and is later destroyed by fibrinolysis, an enzymatic cascade with feedback. Pharmacological fibrinolysis stimulation is also used to destroy pathological, life-threatening clots and thrombi (thrombolysis). The regulation of the nonlinear spatially nonuniform fibrinolytic process in thrombolysis is not currently well understood. We developed a reaction-diffusion-advection model of thrombolysis by tissue plasminogen activator (TPA) in an occluded vessel with a pressure gradient. Sensitivity-analysis-based model reduction was used to reveal the critical processes controlling different steps of thrombolysis. The propagation of thrombolysis in the system without flow was predominantly controlled by TPA diffusion, whereas transport of other active components was rendered nonessential either by their high fibrin-binding parameters and short lifetimes or their initial uniform distribution. The concentration of the main TPA inhibitor plasminogen activator inhibitor 1 (PAI-1) controlled both the extent of lysis propagation and the shape of fibrin spatial distribution during lysis. Interestingly, PAI-1 remained important even when its concentration was an order of magnitude below that of TPA because of its role at the edge of the diffusing TPA front. The system was robust to reaction rate constant perturbations. Using these data, a reduced model of thrombolysis was proposed. In the presence of flow, convection of TPA was the critical controlling process; although the role of PAI-1 concentration was much less in the presence of flow, its influence became greater in the presence of collateral bypassing vessels, which sufficiently reduced TPA flux through the thrombus. Flow bypass through the collateral vessel caused a decrease in TPA flux in the clotted vessel, which increased the PAI-1/TPA ratio, thus making PAI-1-induced inhibition relevant for the regulation of spatial lysis up to its arrest.
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