RESUMEN
Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.
Asunto(s)
Modelos Biológicos , Estramenopilos , Estramenopilos/metabolismo , Estramenopilos/genética , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Redes y Vías Metabólicas/genética , Aminoácidos/metabolismo , Biomasa , Metabolismo de los Lípidos , Nutrientes/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
The carotenogenic thraustochytrid Thraustochytrium sp. RT2316-16 was grown in batch and repeated-batch cultures using different feeds containing glucose, or glycerol, and yeast extract, for the production of lipids, phospholipids and carotenoids. RT2316-16 produced canthaxanthin, astaxanthin and ß-carotene. The effects of biotin, ascorbic acid, light and temperature were evaluated in some of the experiments. In 2-day-old batch cultures, the combined mass percentage of eicosapentaenoic acid and docosahexaenoic acid in total lipids was between 16.5% (glycerol-based medium in the dark; biomass concentration = 4.2 ± 1.1 g L-1) and 42.6% (glucose-based medium under light; biomass concentration = 3.3 ± 0.1 g L-1), decreasing to 3.8% and 6.1%, respectively, after day 4. In repeated-batch cultures, the total lipids in the biomass increased after glucose or glycerol was fed alone, whereas the total carotenoids (168 ± 7 µg g-1 dry weight (DW)) and phospholipids in the biomass increased after feeding with yeast extract. The biomass with the highest content of phospholipids (28.7 ± 4.3 mg g-1 DW) was obtained using a feed medium formulated with glycerol, yeast extract and ascorbic acid. Glycerol was the best carbon source for the production of a biomass enriched with total lipids (467 ± 45 mg g-1 DW). The composition of carotenoids depended strongly on the composition of the feed. Repeated-batch cultures fed with yeast extract contained canthaxanthin as the main carotenoid, whereas in the cultures fed only with glucose, the biomass contained mainly ß-carotene.
Asunto(s)
Técnicas de Cultivo Celular por Lotes , Estramenopilos , Ácido Ascórbico , Biomasa , Cantaxantina , Carotenoides , Glucosa , Glicerol , Fosfolípidos , beta CarotenoRESUMEN
The psychrophilic marine microorganism Thraustochytrium sp. RT2316-16 can produce carotenoids as well as lipids containing the omega-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid and docosahexaenoic acid. This work reports on the effects of the composition of the culture medium, including certain amino acids, on growth and lipid synthesis by RT2316-16. Compared with the culture on glutamate, the use of lysine, alanine, or serine, increased the content of the omega-3 PUFA in total lipids. In the media that contained yeast extract, glutamate, and glucose, lipid accumulation occurred when organic ammonium was exhausted earlier than glucose. In contrast, lipid mobilization was promoted if glucose was exhausted while organic ammonium (supplied by yeast extract and glutamate) remained in the medium. The total content of carotenoids in the lipid-free biomass decreased during the first 12 to 24 h of culture, simultaneously with a decrease in the total lipid content of the biomass. The experimental data suggested a possible interrelationship between the metabolism of carotenoids and lipids. A high content of omega-3 PUFA in the total lipids could be obtained by growing the thraustochytrid in a medium with a low glucose concentration (6 g L-1) and a high concentration of organic nitrogen (yeast extract 12 g L-1; glutamate 1.06 g L-1), after glucose was exhausted. These observations may guide the development of a strategy to enhance omega-3 PUFA in the biomass.
Asunto(s)
Ácidos Grasos Omega-3 , Estramenopilos , Nitrógeno/metabolismo , Ácidos Grasos Omega-3/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Estramenopilos/metabolismo , Carotenoides/metabolismo , Glucosa/metabolismo , Glutamatos/metabolismo , Ácidos Grasos/metabolismoRESUMEN
Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and carotenoids are needed as human dietary supplements and are essential components in commercial feeds for the production of aquacultured seafood. Microorganisms such as thraustochytrids are potential natural sources of these compounds. This research reports on the lipid and carotenoid production capacity of thraustochytrids that were isolated from coastal waters of Antarctica. Of the 22 isolates, 21 produced lipids containing EPA+DHA, and the amount of these fatty acids exceeded 20% of the total fatty acids in 12 isolates. Ten isolates were shown to produce carotenoids (27.4-63.9 µg/g dry biomass). The isolate RT2316-16, identified as Thraustochytrium sp., was the best producer of biomass (7.2 g/L in five days) rich in carotenoids (63.9 µg/g) and, therefore, became the focus of this investigation. The main carotenoids in RT2316-16 were ß-carotene and canthaxanthin. The content of EPA+DHA in the total lipids (34 ± 3% w/w in dry biomass) depended on the stage of growth of RT2316-16. Lipid and carotenoid content of the biomass and its concentration could be enhanced by modifying the composition of the culture medium. The estimated genome size of RT2316-16 was 44 Mb. Of the 5656 genes predicted from the genome, 4559 were annotated. These included genes of most of the enzymes in the elongation and desaturation pathway of synthesis of ω-3 polyunsaturated fatty acids. Carotenoid precursors in RT2316-16 were synthesized through the mevalonate pathway. A ß-carotene synthase gene, with a different domain organization compared to the gene in other thraustochytrids, explained the carotenoid profile of RT2316-16.
Asunto(s)
Carotenoides/química , Ácidos Grasos Omega-3/química , Estramenopilos , Animales , Regiones Antárticas , Organismos AcuáticosRESUMEN
Seaweed processing generates liquid fraction residual that could be used as a low-cost nutrient source for microbial production of metabolites. The Rhodotorula strain is able to produce antimicrobial compounds known as sophorolipids. Our aim was to evaluate sophorolipid production, with antibacterial activity, by marine Rhodotorula rubra using liquid fraction residual (LFR) from the brown seaweed Macrocystis pyrifera as the nutrient source. LFR having a composition of 32% w/w carbohydrate, 1% w/w lipids, 15% w/w protein and 52% w/w ash. The best culture condition for sophorolipid production was LFR 40% v/v, without yeast extract, artificial seawater 80% v/v at 15 °C by 3 growth days, with the antibacterial activity of 24.4 ± 3.1 % on Escherichia coli and 21.1 ± 3.8 % on Staphylococcus aureus. It was possible to identify mono-acetylated acidic and methyl ester acidic sophorolipid. These compounds possess potential as pathogen controllers for application in the food industry.
Asunto(s)
Macrocystis/química , Ácidos Oléicos/química , Extractos Vegetales/farmacología , Rhodotorula/efectos de los fármacos , Organismos Acuáticos/química , Ácidos Oléicos/farmacología , Extractos Vegetales/química , Rhodotorula/patogenicidadRESUMEN
Oblongichytrium RT2316-13 synthesizes lipids rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The content of these fatty acids in the total lipids depended on growth temperature. Sequencing technology was used in this work to examine the thraustochytrid's response to a decrease in growth temperature from 15 °C to 5 °C. Around 4% (2944) of the genes were differentially expressed (DE) and only a few of the DE genes (533 upregulated; 206 downregulated) had significant matches to those in the SwissProt database. Most of the annotated DE genes were related to cell membrane composition (fatty acids, sterols, phosphatidylinositol), the membrane enzymes linked to cell energetics, and membrane structure (cytoskeletal proteins and enzymes). In RT2316-13, the synthesis of long-chain polyunsaturated fatty acids occurred through ω3- and ω6-pathways. Enzymes of the alternative pathways (Δ8-desaturase and Δ9-elongase) were also expressed. The upregulation of the genes coding for a Δ5-desaturase and a Δ5-elongase involved in the synthesis of EPA and DHA, explained the enrichment of total lipid with these two long-chain fatty acids at the low temperature. This molecular response has the potential to be used for producing microbial lipids with a fatty acids profile similar to that of fish oils.
Asunto(s)
Organismos Acuáticos/genética , Eucariontes/genética , Regulación de la Expresión Génica , Metabolismo de los Lípidos/genética , Temperatura , Transcriptoma , Regiones Antárticas , Organismos Acuáticos/crecimiento & desarrollo , Organismos Acuáticos/metabolismo , delta-5 Desaturasa de Ácido Graso , Eucariontes/crecimiento & desarrollo , Eucariontes/metabolismo , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados/biosíntesisRESUMEN
Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.
Asunto(s)
Organismos Acuáticos/metabolismo , Biotecnología/métodos , Medios de Cultivo/química , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Fermentación , Estramenopilos/metabolismo , Regiones Antárticas , Organismos Acuáticos/crecimiento & desarrollo , Organismos Acuáticos/aislamiento & purificación , Biomasa , Frío , Ácidos Docosahexaenoicos/análisis , Ácido Eicosapentaenoico/análisis , Estramenopilos/crecimiento & desarrollo , Estramenopilos/aislamiento & purificaciónRESUMEN
Thraustochytrids have been isolated from different aquatic systems; however, few studies have reported their occurrence in Antarctica. In this study, 13 strains close to strains belonging to the genera Oblongichytrium, Thraustochytrium, and Aurantiochytrium were isolated from seawater samples collected near the Antarctic Base Professor Julio Escudero (S 62°12'57' E 58°57'35â³). Docosahexaenoic acid (DHA) was found in the total lipids of all the isolates; DHA content of the biomass (dry weight) varied between 3.3 and 33 mg/g under the growth conditions for isolation. Five of the Antarctic thraustochytrids were able to accumulate lipids at levels higher than 20% w/w. Two strains, RT2316-7 and RT2316-13, were selected to test the effect of the incubation temperature (at 5°C for 14 days and at 15°C for 5 days). Incubation temperature had little effect on the lipid content and biomass yield; however, its effect on the fatty acid composition was significant (p < .05). The low incubation temperature favored the accumulation of eicosapentaenoic acid (EPA), palmitic acid and stearic acid in the total lipids of RT2316-7. Percentage of EPA, DHA and the omega-6 fatty acid dihomo-γ-linolenic acid of total fatty acids of RT2316-13 was higher at the low incubation temperature. RT2316-13 accumulated the highest lipid content (30.0 ± 0.5%) with a carbon to nitrogen mass ratio equal to 16.9. On the contrary, lipid accumulation in RT2316-7 occurred at high concentration of the nitrogen sources (monosodium glutamate or yeast extract). The capability to accumulate lipids with a fatty acid profile that can be tuned through cultivation temperature make the Antarctic thraustochytrid RT2316-13 a candidate for the production of lipids with different uses.
Asunto(s)
Reactores Biológicos/microbiología , Ácidos Grasos Omega-3/biosíntesis , Estramenopilos/metabolismo , Ácido 8,11,14-Eicosatrienoico/análisis , Regiones Antárticas , Membrana Celular/fisiología , Ácidos Docosahexaenoicos/análisis , Ácido Eicosapentaenoico/análisis , Ácido Palmítico/análisis , Agua de Mar , Ácidos Esteáricos/análisis , Estramenopilos/clasificación , Estramenopilos/crecimiento & desarrollo , Estramenopilos/aislamiento & purificación , TemperaturaRESUMEN
Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.