Dataset Information

Identification of Sporopollenin as the Outer Layer of Cell Wall in Microalga Chlorella protothecoides.

ABSTRACT: 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.

SUBMITTER: He X

PROVIDER: S-EPMC4928020 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identification of Sporopollenin as the Outer Layer of Cell Wall in Microalga Chlorella protothecoides.

He Xi X Dai Junbiao J Wu Qingyu Q

Frontiers in microbiology 20160630

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 al ...[more]

PMID: 27446068

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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.

Project description:Over the last decades, microalgae have gained popularity due to demand for novel environmental green solutions and development of innovative mass-production sources for multiple processes, including animal feed and human diet, turning microalgae into an exquisite candidate for several ecofriendly technologies. Notwithstanding, there is a catch. Most species of microalgae, as the case of common Chlorella vulgaris (C. vulgaris) display a recalcitrant cell wall, characterized by a complex matrix of polysaccharides and glycoproteins, which constitutes a major barrier for monogastric species digestibility and extraction of inner valuable nutritional compounds. To overcome this limitation, the development of feed enzymes, in particular Carbohydrate-Active enZymes (CAZymes) with capacity to disrupt C. vulgaris cell wall may contribute to improve the bioavailability of these microalgae compounds in monogastric diets, namely at high levels of incorporation. In order to disclosure novel combination of feed enzymes to disrupt C. vulgaris cell wall, a lab protocol was implemented by our research team containing the following key steps: after microalgae cultivation and having available a repertoire of two hundred pre-selected CAZymes produced by high-throughput technology, the step 1 is the individual screening of the most functional enzymes on disrupting C. vulgaris cell wall (versus a control, defined as the microalgae suspension incubated with PBS) and the determination of reducing sugars released by the 3,5-dinitrosalicylic acid (DNSA) method; step 2 concerns on finding the best CAZymes cocktail, testing the synergistic effect of enzymes, to disrupt C. vulgaris cell wall (in parallel with running the control) along with characterization of each enzyme thermostability and resistance to proteolytic attack, to which feed enzymes are subjected in the animal gastrointestinal tract; step 3 is the assessment of C. vulgaris cell wall degradation degree by measuring the amount of reducing sugars released by the DNSA method, fatty acid analysis by gas chromatography (GC) with flame ionization detector (FID), oligosaccharides quantification by high performance liquid chromatography (HPLC) equipped with an electrochemical detector (ECD), protein content by the Kjeldahl method, and various pigments (chlorophylls a and b, and total carotenoids) in the supernatant. In the correspondent residue, we also assessed cellular counting using a Neubauer chamber by direct observation on a bright-field microscope and fluorescence intensity, after staining with Calcofluor White for both control and CAZymes cocktail treatments, on a fluorescence microscope. Beyond animal feed industry with impact on human nutrition, our lab protocol may increase the yield in obtaining valued constituents from C. vulgaris microalga for other biotechnological industries.

Project description:BackgroundMicroalgae 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.ResultsModel 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.ConclusionThe 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.

Dataset Information

Identification of Sporopollenin as the Outer Layer of Cell Wall in Microalga Chlorella protothecoides.

Publications

Identification of Sporopollenin as the Outer Layer of Cell Wall in Microalga Chlorella protothecoides.

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