RESUMEN
The present study was designed to explore the possibility of improving lipid production in oleaginous filamentous fungus Mortierella alpina based on an autophagy regulation strategy. According to multiomics information, vacuolate-centered macroautophagy was identified as the main type of autophagy in M. alpina under nitrogen-limited conditions. Mutation of autophagy-related gene MAatg8 led to impaired fatty acid synthesis, while overexpression of both MAatg8 and phosphatidylserine decarboxylases (MApsd2) showed promoting effects on fatty acid synthesis. MAatg8 overexpression strain with external supply of ethanolamine significantly increased arachidonic acid (ARA)-rich triacylglycerol (TAG) and biomass synthesis in M. alpina, and the final fatty acid content increased by approximately 110% compared with that in the wild-type strain. Metabolomics and lipidomics analyses revealed that cell autophagy enhanced the recycling of preformed carbon, nitrogen, and lipid in mycelium, and the released carbon skeleton and energy were contributed to the accumulation of TAG in M. alpina. This study suggests that regulation of autophagy-related MAatg8-phosphatidylethanolamine (MAatg8-PE) conjugation system could be a promising strategy for attaining higher lipid production and biomass growth. The mechanism of autophagy in regulating nitrogen limitation-induced lipid accumulation elucidated in this study provides a reference for development of autophagy-based strategies for improving nutrient use efficiency and high value-added lipid production by oleaginous microorganism. IMPORTANCE Studies have indicated that functional oil accumulation occurs in oleaginous microorganisms under nitrogen limitation. However, until now, large-scale application of nitrogen-deficiency strategies was limited by low biomass. Therefore, the identification of the critical nodes of nitrogen deficiency-induced lipid accumulation is urgently needed to further guide functional oil production. The significance of our research is in uncovering the function of cell autophagy in the ARA-rich TAG accumulation of oleaginous fungus M. alpina and demonstrating the feasibility of improving lipid production based on an autophagy regulation strategy at the molecular and omics levels. Our study proves that regulation of cell autophagy through the MAatg8-PE conjugation system-related gene overexpression or exogenous supply of ethanolamine would be an efficient strategy to increase and maintain biomass productivity when high TAG content is obtained under nitrogen deficiency, which could be useful for the development of new strategies that will achieve more biomass and maximal lipid productivity.
Asunto(s)
Ácido Araquidónico/metabolismo , Autofagia , Mortierella/citología , Mortierella/metabolismo , Triglicéridos/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Metabolismo de los Lípidos , Mortierella/genéticaRESUMEN
An effective method for research of macro-morphological characterization and its kinetics was developed by studying the macro-morphological characteristics of Mortierella alpina, an oleaginous zygomycete widely used to produce lipids rich in PUFA, in function of culture medium composition and to link morphological features of fungus with the level of lipid production. A number of distinct morphological forms including hollow pellets, fluffy pellets and freely dispersed mycelia were obtained by changing the fermentation factors. By fitting a Logistic curve, the maximum specific growth rate (µmax)was obtained, which determined the final mycelia morphology. µmax of 0.6584 in three kind of morphological forms is the more appropriate. According to the Luedeking-Piret equation fitting, α≠0 and ß≠0, lipid production was partially associated with the hyphal growth, fluffy pellets which turn glucose into lipidwas more effective than the other two kinds of morphological forms.
Asunto(s)
Técnicas de Cultivo Celular por Lotes , Mortierella/citología , Mortierella/crecimiento & desarrollo , Medios de Cultivo , Fermentación/fisiología , Cinética , Metabolismo de los Lípidos , Mortierella/metabolismo , Micelio/citología , Micelio/crecimiento & desarrollo , Micelio/metabolismoRESUMEN
Oleaginous fungus Mortierella isabellina showed excellent lipid conversion on non-detoxified lignocellulosic hydrolysate. This study investigated the effects of inhibitory compounds (furfural, hydroxymethylfurfural, and ferulic and coumaric acids) in lignocellulosic hydrolysate on M. isabellina growth and lipid production. M. isabellina can tolerate furfural (â¼1 g/L), hydroxymethylfurfural (â¼2.5 g/L), ferulic (â¼0.5 g/L) and coumaric acid (â¼0.5 g/L) with normal growth rates. Synergistic effect of these inhibitors (2 g/L furfural, 0.4 g/L hydroxymethylfurfural, 0.02 g/L ferulic acid and 0.02 g/L coumaric acid) moderately reduces total fungal growth (by 28%), while the presence of these inhibitors has minor impact on cell lipid contents and lipid profiles. In the presence of inhibitory compounds, (13)C-tracing has revealed that M. isabellina can simultaneously utilize glucose and acetate, and acetate is mainly assimilated for synthesis of lipid and TCA cycle amino acids. The results also demonstrate that glucose has strong catabolite repression for xylose utilization for biomass and lipid production in the presence of inhibitors.
Asunto(s)
Carbono/metabolismo , Lignina/farmacología , Mortierella/crecimiento & desarrollo , Mortierella/metabolismo , Aminoácidos/metabolismo , Carbono/farmacología , Isótopos de Carbono , Ácidos Cumáricos/farmacología , Ácidos Grasos/análisis , Fermentación/efectos de los fármacos , Furaldehído/análogos & derivados , Furaldehído/farmacología , Hidrólisis , Marcaje Isotópico , Cinética , Metabolismo de los Lípidos/efectos de los fármacos , Mortierella/citología , Mortierella/efectos de los fármacosRESUMEN
Mortierella sp. has been known to produce polyunsaturated fatty acids (PUFAs) such as GLA and AA under normal growth medium conditions. Similarly, under the stress condition, this fungus produces EPA and DHA in their mycelial biomass. Among the 67 soil samples screened from the Western Ghats of India, 11 Mortierella isolates showed the presence of omega-6 and omega-3 fatty acid, mainly GLA, AA, EPA, and DHA in starch, yeast-extract medium. Nile red and TTC strains were used for screening their qualitative oleaginesity. Among the representative isolates, when Mortierella sp. is grown in a fat-producing basal medium, a maximum lipid content of 42.0 ± 1.32% in its mycelia, 6.72 ± 0.5% EPA, and 4.09 ± 0.1% DHA was obtained. To understand the Mortierella sp. CFR-GV15, to the species level, its morphology was seen under the light microscope and scanning electron microscope, respectively. These microscopic observations showed that isolate Mortierella sp. CFR-GV15 produced coenocytic hyphae. Later on, its 18S rRNA and the internal transcribed spacer (ITS) sequences were cloned, sequenced, and analyzed phylogenetically to 18S rRNA and ITS1 and ITS4 sequences of related fungi. This newly isolated Mortierella alpina CFR-GV15 was found to be promising culture for the development of an economical method for commercial production of omega-3 fatty acid for food and therapeutical application.
Asunto(s)
Ácidos Grasos Omega-3/biosíntesis , Ácidos Grasos Omega-3/uso terapéutico , Alimentos , Mortierella/química , Biomasa , Hifa/citología , Hifa/ultraestructura , Datos de Secuencia Molecular , Mortierella/citología , Mortierella/crecimiento & desarrollo , Mortierella/aislamiento & purificación , Oxazinas/metabolismo , Filogenia , ARN Ribosómico 18S/genética , Microbiología del Suelo , Esporas Fúngicas/ultraestructura , Coloración y Etiquetado , TemperaturaRESUMEN
Hydrolytic enzymes were evaluated on the lipid accumulation via an oleaginous fungal species, Mortierella isabellina, cultivated on sugars released from soybean hulls. The weight loss of soybean hull, fungal growth, and lipid production were tested under different loads of hydrolytic enzymes. M. isabellina could not directly utilize cellulose and adding cellulase and ß-glucosidase significantly increased the cell growth and oil accumulation of M. isabellina on soybean hulls. The highest weight loss of soybean hulls was 47.80 % and the lipid production reached 0.14 g from 1 g of soybean hull when 12 U cellulase, 27.2 U ß-glucosidase, 2,278.56 U pectinase, and 15 U hemicellulase were added. Fatty acids (76.82 %) accumulated in M. isabellina were C16 and C18, which are suitable for biodiesel production. These results provide a new application for soybean hulls to be applied as the raw material for the production of biodiesel fuel, besides its traditional role as animal feed supplements.
Asunto(s)
Biocombustibles/microbiología , Fermentación , Glycine max/química , Lípidos/biosíntesis , Mortierella/metabolismo , Carbono/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Celulasa/metabolismo , Hidrólisis , Inmersión , Metabolismo de los Lípidos , Mortierella/citologíaRESUMEN
A filamentous fungus, Mortierella alpina 1S-4, was obtained, through extensive screening, as an potential producer of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid. With this discovery as a starting point, we conducted employing methods from metabolic engineering and molecular biology for controlling cultures and breeding mutant strains. These parental and mutant strains are now used for large-scale production of a variety of PUFAs.
Asunto(s)
Mortierella/citología , Mortierella/metabolismo , Aceites/metabolismo , Ácido Araquidónico/biosíntesis , Ingeniería Genética , Mortierella/enzimología , Mortierella/genética , Mutación/genéticaRESUMEN
Using a flow-through chamber coupled with image analysis, the morphological parameters of 11 Mortierella species were quantified, and the relationship between micro- and macromorphology was investigated. On potato-dextrose-agar plates, 5 species formed rose petal-like colonies, 3 formed large round colonies, and 3 formed donut-like colonies. By observing micromorphology in a flow-through chamber, fungi were divided into 3 groups, classified according to morphological parameters: (i) a group with a high branch formation rate (q(b): tip/microm/h) and a low tip extension rate (q(tip): microm/tip/h); (ii) a group with a low branch formation rate and a high tip extension rate; and (iii) a group intermediate between the former and the latter groups. In suspension culture, group (i) fungi formed a hyphal bundle with a pulpy pellet-like morphology and a pellet core. In contrast, group (ii) fungi showed an aggregation of hyphae without the pellet core. In a narrow-specific hyphal growth rate (mu(l)) range (0.35-0.45 h(-1)), a higher branch formation rate led to increased hyphal branching, resulting in the formation of a hyphal bundle with a pulpy pellet-like morphology and a pellet core. When the branch formation rate was lower than 2 x 10(-3) tips/microm/h, the mycelia formed less branched but longer hypha. Our study surmises that a micromorphology consisting of a high hyphal growth rate (0.4 h(-1)), low tip extension rate (20 tips/microm/h), and high branch formation rate (8 x 10(-3) tips/microm/h) forms the suitable macromorphology for arachidonic acid production.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Mortierella/citología , Mortierella/crecimiento & desarrollo , Hifa/citología , Hifa/crecimiento & desarrollo , Microscopía , Morfogénesis , FotomicrografíaRESUMEN
Mortierella alpina was grown in a fed-batch culture using a 12-l jar fermenter with an initial 8-l working volume containing 20 g glucose l-1 and 10 g corn-steep powder l-1. Glucose was intermittently fed to give 32 g l-1 at each time. The pH of culture was maintained using 14% (v/v) NH4OH, which also acted as a nitrogen source. A final cell density of 72.5 g l-1 was reached after 12.5 days with a content of arachidonic acid (ARA) at 18.8 g l-1. These values were 4 and 1.8 times higher than the respective values in batch culture. Our results suggest that the combined feeding of glucose and NH4+ to the growth of M. alpina could be applied for the industrial scale production of ARA.
Asunto(s)
Ácido Araquidónico/biosíntesis , Hidróxidos/metabolismo , Mortierella/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Hidróxido de Amonio , Reactores Biológicos/microbiología , División Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Concentración de Iones de Hidrógeno , Hidróxidos/farmacología , Lípidos/biosíntesis , Mortierella/citología , Mortierella/efectos de los fármacos , Micelio/citología , Micelio/efectos de los fármacos , Micelio/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Factores de TiempoRESUMEN
We studied the regulation of lipid body biogenesis in the oleaginous fungus Mortierella ramanniana var. angulispora by investigating culture conditions to modulate lipid body size, which we found was affected by the carbon-to-nitrogen ratio (C/N ratio) in the culture medium. Increasing the nitrogen source or decreasing the C/N ratio from 38 to 9 induced the appearance of lipid bodies with diameters less than 2-3 micro m, which are usually found at a C/N ratio of 38 in this fungus. To determine factors regulating lipid body size, we compared lipid body fractions from fungal cells cultured at different C/N ratios. We found some differences in polypeptide profiles between lipid body fractions from fungal cells cultured at different C/N ratios for 2 days when the lipid bodies were enlarged at a C/N ratio of 38. We then compared the phosphorylation of lipid body proteins, since protein phosphorylation plays a pivotal role in various aspects of signal transduction. In vitro phosphorylation in the lipid body fraction indicated that protein kinase activity toward endogenous and exogenous substrates such as histone IIIS, VIIS, and myelin basic protein increased in the lipid body fraction at a C/N ratio of 9. Further analysis by in-gel protein kinase assay indicated the presence of at least three activated protein kinases with molecular masses of 75, 72, and 42 kDa, which were also autophosphorylated. These results indicate the presence of nutrient-regulated protein kinases and increased phosphorylation in lipid bodies, which correlate with the appearance of smaller lipid bodies in this fungus. Further studies to characterize these protein kinases at the molecular level should provide new insights into the link between nutrient sensing and lipid storage.
Asunto(s)
Cuerpo Adiposo/metabolismo , Metabolismo de los Lípidos , Mortierella/metabolismo , Nitrógeno/farmacología , Proteínas Quinasas/metabolismo , Animales , Carbono/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Medios de Cultivo , Electroforesis , Cuerpo Adiposo/química , Lípidos/química , Peso Molecular , Mortierella/citología , Mortierella/efectos de los fármacos , Mortierella/ultraestructura , Nitrógeno/metabolismo , Fosforilación , Transducción de Señal , Factores de TiempoRESUMEN
Morphological parameters, such as hyphal growth rate, tip formation rate, tip extension rate and branch formation rate, of Mortierella alpina have been measured using a flow-through chamber under 25 different combinations of carbon and nitrogen concentrations. Morphological parameters were influenced not by C/N ratio but by carbon concentration in the medium. Specific rates of hyphal growth and tip formation both remained constant at a low carbon concentration of 5 g/l. Tip extension rate from one tip was 60 microm tip(-1) h(-1) at a carbon concentration below 15 g/l, and the branching formation rate was independent of carbon concentration. Tip extension rate was a function of specific hyphal growth rate, which in turn was linearly proportional to the specific tip formation rate, demonstrating that tip extension rate was exponentially proportional to the specific tip formation rate. Branch formation rate per hyphal element remained unchanged even at tip extension rates lower than 60 microm tip(-1) h(-1) and at specific hyphal growth rates lower than 0.83 h(-1), but decreased drastically at higher rates of tip extension and hyphal growth.
Asunto(s)
Ácido Araquidónico/biosíntesis , Mortierella/crecimiento & desarrollo , Carbono/metabolismo , Procesamiento de Imagen Asistido por Computador , Mortierella/citología , Mortierella/metabolismo , Nitrógeno/metabolismoRESUMEN
The specific activity of lipoxygenase from several strains of the zygomycete Mortierella varied from 1.02 to 2.02 microMol diene per min per mg protein). The enzyme equally used linoleic or arachidonic acid as a substrate. An increase in lipoxygenase activity was observed after adding corn oil to the culture medium. Tests with inhibitors having different chemical structures revealed that the lipoxygenase activity from Mortierella cells was inhibited only by esculetin, ethanol and nordihydroguaiaretic acid (NDGA). NDGA inhibited the enzyme in vitro (IC50 = 142 microM), but its addition in the exponential phase of growth activated the enzyme.
Asunto(s)
Lipooxigenasa/metabolismo , Mortierella/enzimología , Inhibidores de la Lipooxigenasa/farmacología , Mortierella/clasificación , Mortierella/citología , Mortierella/crecimiento & desarrollo , Especificidad de la EspecieRESUMEN
The intrastructure of mycelial pellets of Mortierella alpina, which accumulate fatty acids in mycelia, was visualized following labeling with fluorescein isothiocyanate (FITC) and Nile red using fluorescence microscopy. The pellet was an ellipse shape, but its intrastructure was shaped as a doughnut with a cave inside. Using three-dimensional image analysis, it was shown that the lipid was produced on the edge of the pellet, which corresponded to the area where the mycelial density was high. The cavity ratio of the pellet section was determined on the basis of the FITC fluorescence intensity, and in the early culture stage remained at 0.2 in a 10-kl fermentor culture, but finally increased to 0.35. Mycelial pellet volume paralleled the cavity ratio. Application of the technique used here allows analysis of the intrastructure of fungal pellets and new types of fungal biological study.
Asunto(s)
Lípidos/análisis , Mortierella/citología , Micelio/citología , Lípidos/biosíntesis , Mortierella/química , Micelio/químicaRESUMEN
Arachidonic acid (AA) production by Mortierella alpina 1S-4 was investigated using a 50-L fermentor. In order to optimize the dissolved oxygen (DO) concentration and to investigate the effect of DO on morphology, cultivation was carried out under constant DO at various levels in the range of 3-50 ppm. To maintain a DO concentration above 7 ppm, two methods, i.e., the oxygen-enrichment (OE) method (experimental range, 25-90% oxygen gas supplied) and the pressurization (PR) method (experimental range, 180-380 kPa headspace pressure), were used. As a result, the optimum DO concentration range was found to be 10-15 ppm. In this optimum DO concentration range, the AA yield was enhanced about 1.6-fold compared to that obtained at 7 ppm DO, and there was no difference in the AA productivity between the OE and PR methods. When the DO concentration was maintained at 20-50 ppm using the OE method, the morphology changed from filaments to pellets, and the AA yield decreased drastically because of stress due to the limited mass transfer through the pellet wall. When the DO concentration was maintained at 15-20 ppm using the PR method, the morphology did not change, and the AA yield decreased gradually.