RESUMO
This work demonstrates the ability of the yeast Yarrowia lipolytica cultivated on biodiesel waste to synthesize α-ketoglutaric acid with a minimal content of pyruvic acid as the main byproduct. The key factor promoting the microbial production of α-ketoglutaric acid from the waste is a strong deficiency of thiamine in the cultivation medium. The production of α-ketoglutaric acid by the yeast can be regulated by changing the concentration of nitrogen, iron, zinc, copper, and manganese in the medium, as well as by pH medium and the aeration rate. The optimization of these parameters in flask experiments allowed us to increase the concentration of α-ketoglutaric acid in the medium by 2.6 times and to shift the α-ketoglutaric acid/pyruvic acid ratio from 5:1 to 30:1. During cultivation in a fermentor under optimized conditions, Y. lipolytica produced 80.4 g/L α-ketoglutaric acid with a process selectivity of 96.7% and the product yield (YKGA) equal to 1.01 g/g. KEY POINTS: ⢠α-Ketoglutaric acid is commercially important biotechnological product. ⢠Biosynthesis of α-ketoglutaric acid from biodiesel waste. ⢠Optimization of cultivation medium and nutrition medium.
Assuntos
Yarrowia , Biocombustíveis , Meios de Cultura , Fermentação , Ácidos CetoglutáricosRESUMO
(2R,3S)-isocitric acid has long been used only as a specific biochemical reagent. However, there is ever increasing evidence that it can also be used as an original promising substance for prevention and treatment of some diseases. The review considers the results of longtime research in our laboratory and the data of other researchers related to microbial synthesis of (2R,3S)-isocitric acid, derivation and selection of active microbial producers, development of their cultivation conditions, as well as the results of study of the mechanism of acid overproduction, and regulation of enzymes involed in this process. The efficient processes of (2R,3S)-isocitric acid production with the aid of natural, mutant, and recombinant strains of Yarrowia lipolytica and methods of product isolation and purification to pharmacopeial standarts are also reviewed.
Assuntos
Isocitratos/isolamento & purificação , Isocitratos/metabolismo , Yarrowia/metabolismo , Meios de Cultura/química , Ácidos Graxos/química , Microbiologia Industrial , Óleos de Plantas/químicaRESUMO
Biosynthesis of Ds(+)-threo-isocitric acid from ethanol in the Yarrowia lipolytica batch and repeated-batch cultures was studied. Repeated-batch cultivation was found to provide for a good biosynthetic efficiency of the producer for as long as 748 h, probably due to maintenance of high activities of enzymes involved in the biosynthesis of isocitric acid. Under optimal repeated-batch cultivation conditions, the producer accumulated 109.6 g/L Ds(+)-threo-isocitric acid with a production rate of 1.346 g/L h. The monopotassium salt of isocitric acid isolated from the culture liquid and purified to 99.9% was found to remove neurointoxication, to restore memory, and to improve the learning of laboratory rats intoxicated with lead and molybdenum salts. Taking into account the fact that the neurotoxic effect of heavy metals is mainly determined by oxidative stress, the aforementioned favorable action of isocitric acid on the intoxicated rats can be explained by its antioxidant activity among other pharmacological effects.
Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Técnicas de Cultura Celular por Lotes/métodos , Isocitratos/metabolismo , Isocitratos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/isolamento & purificação , Reatores Biológicos/microbiologia , Etanol/metabolismo , Isocitratos/isolamento & purificação , Masculino , Metais Pesados/toxicidade , Ratos , Yarrowia/metabolismoRESUMO
The effect of biologically active form (threo-Ds-) of isocitric acid (ICA) on oxidative stress was studied using the infusorian Paramecium caudatum stressed by hydrogen peroxide and salts of some heavy metals (Cu, Pb, Zn, and Cd). ICA at concentrations between 0.5 and 10 mM favorably influenced the infusorian cells with oxidative stress induced by the toxicants studied. The maximal antioxidant effect of ICA was observed at its concentration 10 mM irrespective of the toxicant used (either H2O2 or heavy metal ions). ICA was found to be a more active antioxidant than ascorbic acid. Biologically active pharmaceutically pure threo-Ds-ICA was produced through cultivation of the yeast Yarrowia lipolytica and isolated from the culture liquid in the form of crystalline monopotassium salt with a purity of 99.9%.
Assuntos
Isocitratos/metabolismo , Paramecium caudatum/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Meios de Cultura/metabolismo , Peróxido de Hidrogênio/metabolismo , Isocitratos/farmacologia , Metais Pesados/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Paramecium caudatum/efeitos dos fármacos , Yarrowia/metabolismoRESUMO
The basic tendency in the field of plant protection concerns with reducing the use of pesticides and their replacement by environmentally acceptable biological preparations. The most promising approach to plant protection is application of microbial metabolites. In the last years, bactericidal, fungicidal, and nematodocidal activities were revealed for citric, succinic, α-ketoglutaric, palmitoleic, and other organic acids. It was shown that application of carboxylic acids resulted in acceleration of plant development and the yield increase. Of special interest is the use of arachidonic acid in very low concentrations as an inductor (elicitor) of protective functions in plants. The bottleneck in practical applications of these simple, nontoxic, and moderately priced preparations is the absence of industrial production of the mentioned organic acids of required quality since even small contaminations of synthetic preparations decrease their quality and make them dangerous for ecology and toxic for plants, animals, and human. This review gives a general conception on the use of organic acids for plant protection against the most dangerous pathogens and pests, as well as focuses on microbiological processes for production of these microbial metabolites of high quality from available, inexpensive, and renewable substrates.
Assuntos
Anti-Infecciosos/farmacologia , Ácidos Carboxílicos/farmacologia , Ácidos Graxos/farmacologia , Praguicidas/química , Doenças das Plantas/prevenção & controle , Plantas , Anti-Infecciosos/metabolismo , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Ácido Cítrico/metabolismo , Ácido Cítrico/farmacologia , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Humanos , Agricultura Orgânica , Controle de Pragas/métodos , Praguicidas/toxicidade , Ácido Succínico/metabolismo , Ácido Succínico/farmacologiaRESUMO
The ability of taxonomically different yeasts to synthesize pyruvic acid (PA) from glucose was studied. The study showed that many yeasts are able to produce PA from glucose under the condition of growth limitation by thiamine. This ability was found in the yeast Blastobotrys adeninivorans for the first time. The production (oversynthesis) of PA in this yeast can be explained by disturbance in the function of thiamine-dependent pyruvate dehydrogenase. Namely, the partial inhibition of this enzyme brings about the excretion of PA from the yeast cells. Due to incomplete inhibition of pyruvate dehydrogenase, the formation of acetyl-CoA continues, although at a lower level, maintaining the synthesis of α-ketoglutaric acid (KGA) in the tricarboxylic acid (TCA) cycle. KGA is no longer oxidized in the TCA cycle, because thiamine limitation inhibits α-ketoglutarate dehydrogenase. As a result, KGA is excreted from the yeast cells as a byproduct of PA oversynthesis. Furthermore, the increased level of KGA in the yeast cells inhibits NAD-dependent isocitrate dehydrogenase in the TCA cycle and enhances the production and excretion of citric acid, another byproduct of PA oversynthesis. During cultivation in a fermentor, the strain Blastobotrys adeninivorans VKM Y-2677 produced 43.2 g l(-1) PA from glucose with a product yield (YPA) of 0.77 g PA/g glucose. The proportion of PA to byproducts was 18:1 for KGA and 8:1 for citric acid.
Assuntos
Complexo Piruvato Desidrogenase/metabolismo , Ácido Pirúvico/metabolismo , Saccharomycetales/metabolismo , Tiamina/metabolismo , Ácido Cítrico/metabolismo , Glucose/metabolismo , Ácidos Cetoglutáricos/metabolismoRESUMO
In this study, physiologo-biochemical characteristics of citrate-producing yeast Yarrowia lipolytica grown on glycerol-containing waste of biodiesel industry were studied by an investigation of growth dynamics, the consumption of glycerol, and the fatty acid fractions from waste as well as by measuring the activities of enzymes involved in the metabolism of waste. It was shown that Y. lipolytica realizes concurrent uptake of glycerol and the fatty acid fractions during conversion of glycerol-containing waste, although glycerol was utilized at a higher rate than fatty acids. Under optimal feeding of glycerol-containing waste by portions of 20 g l(-1), the citric acid production and the ratio between citric acid and isocitric acid depended on the strain used. It was revealed that wild strain Y. lipolytica VKM Y-2373 produced citrate and isocitrate with a ratio of 1.7:1, while the mutant strain Y. lipolytica NG40/UV7 synthesized presumably citric acid (122.2 g l(-1)) with a citrate-to-isocitrate ratio of 53:1 and the yield of 0.95 g g(-1).
Assuntos
Biocombustíveis/microbiologia , Citratos/metabolismo , Glicerol/metabolismo , Resíduos Industriais , Yarrowia/crescimento & desenvolvimento , Yarrowia/metabolismo , Ácidos Graxos/metabolismo , Yarrowia/enzimologiaRESUMO
The process of succinic acid (SA) production represents the combination of microbial synthesis of α-ketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by hydrogen peroxide to SA that leads to the production of 69.0 g l(-1) of SA and 1.36 g l(-1) of acetic acid. SA was isolated from the culture broth filtrate in a crystalline form. The SA recovery from the culture filtrate has certain difficulties due to the presence of residual triglycerides of rapeseed oil. The effect of different methods of the culture filtrate treatment and various sorption materials on the coagulation of triglycerides was studied, and as a result, the precipitation of residual triglycerides by acetone was chosen. The subsequent isolation procedures involved the decomposition of H2O2 in the filtrate followed by filtrate bleaching and acidification with a mineral acid, evaporation of filtrate, and SA extraction with ethanol from the residue. The purity of crystalline SA isolated from the culture broth filtrate achieved 97.6-100 %. The product yield varied from 62.6 to 71.6 % depending on the acidity of the supernatant.
Assuntos
Óleos de Plantas/metabolismo , Ácido Succínico/isolamento & purificação , Ácido Succínico/metabolismo , Yarrowia/metabolismo , Ácido Acético , Ácidos Graxos Monoinsaturados , Peróxido de Hidrogênio/metabolismo , Ácidos Cetoglutáricos/metabolismo , Óleo de Brassica napusRESUMO
The production of α-ketoglutaric acid by yeast Yarrowia lipolytica VKMY-2412 from ethanol and its subsequent chemical conversion to succinic acid (SA) were investigated. A highly effective and environmentally friendly process of α-ketoglutaric acid production was developed using a special pH-controlling strategy, in which the titration of the culture broth with KOH in the acid-formation phase was minimal, that allowed accumulation of only low amounts of inorganic wastes in the course of SA recovery. The culture broth filtrate containing α-ketoglutaric acid (88.7 g l(-1)) was directly employed for SA production; the amount of SA produced comprised 71.7 g l(-1) with the yield of 70% from ethanol consumed. SA was isolated from the culture broth filtrate in a crystalline form with the purity of 100%. The yield of isolated SA was as high as 72% of its amount in the culture broth filtrate. The antimicrobial and nematocidic effects of SA of microbial origin on pathogenic organisms that cause human and plant diseases were revealed for the first time.
Assuntos
Ácido Succínico/metabolismo , Yarrowia/metabolismo , Etanol/metabolismo , Ácidos Cetoglutáricos/metabolismoRESUMO
The possibility of using rapeseed oil as a carbon source for microbiological production of α-ketoglutaric acid (KGA) has been studied. Acid formation on the selective media has been tested in 26 strains of Yarrowia lipolytica yeast, and the strain Y. lipolytica VKM Y-2412 was selected as a prospective producer of KGA from rapeseed oil. KGA production by the selected strain was studied in dependence on thiamine concentration, medium pH, temperature, aeration, and concentration of oil. Under optimal conditions (thiamine concentration of 0.063 µg g cells(-1), pH 3.5, 30 °C, high dissolved oxygen concentration (pO2) of 50 % (of air saturation), and oil concentration in a range from 20 to 60 g l(-1)), Y. lipolytica VKM Y-2412 produced up to 102.5 g l(-1) of KGA with the mass yield coefficient of 0.95 g g(-1) and the volumetric KGA productivity (QKGA) of 0.8 g l(-1) h(-1).
Assuntos
Ácidos Cetoglutáricos/metabolismo , Óleos de Plantas/metabolismo , Yarrowia/metabolismo , Aerobiose , Meios de Cultura/química , Ácidos Graxos Monoinsaturados , Concentração de Íons de Hidrogênio , Óleo de Brassica napus , Temperatura , Tiamina/metabolismoRESUMO
The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen, phosphorus, or sulfur), 28 °C, pH 5.0, dissolved oxygen concentration (pO2) of 50 % (of air saturation), and pulsed addition of glycerol from 20 to 80 g L⻹ depending on the rate of medium titration. Under optimal conditions of fed-batch cultivation, in the medium with pure glycerol, strain Y. lipolytica NG40/UV7 produced 115 g L⻹ of CA with the mass yield coefficient of 0.64 g g⻹ and isocitric acid (ICA) amounted to 4.6 g L⻹; in the medium with raw glycerol, CA production was 112 g L⻹ with the mass yield coefficient of 0.90 g g⻹ and ICA amounted to 5.3 g L⻹. Based on the activities of enzymes involved in the initial stages of raw glycerol assimilation, the tricarboxylic acid cycle and the glyoxylate cycle, the mechanism of increased CA yield from glycerol-containing substrates in Y. lipolytica yeast was explained.
Assuntos
Ácido Cítrico/metabolismo , Glicerol/metabolismo , Yarrowia/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Redes e Vias Metabólicas , Oxigênio/metabolismo , TemperaturaRESUMO
The replacement of chemical synthesis by environmentally friendly energy-efficient technologies for production of valuable metabolites is a principal strategy of developing biotechnological industry all over the world. In the present study, we develop a method for α-ketoglutaric acid (KGA) production from rapeseed oil with the use of Yarrowia lipolytica yeast. Sixty strains of Y. lipolytica yeasts were tested for their ability to produce KGA, and the strain Y. lipolytica 212 (Y. lipolytica VKM Y-2412) was selected as a promising KGA producer. Using a three-stage pH controlling, in which pH was 4.5 in the growth phase, then since 72 to 144 h, pH was maintained at 3.5 and in the later phase of acid production, the titration by KOH was switch off, selected strain produced 106.5 g l(-1) of KGA with mass yield of 0.95 g g(-1). KGA in the form of monopotassium salt was isolated from the culture broth and purified. The isolation procedure involved separation of biomass, extraction of residual triglycerides, filtrate bleaching, and acidification with mineral acid (to pH 2.8-3.4), concentration, precipitation of mineral salts, and crystallization of the product. The purity of KGA isolated from the culture filtrate reached 99.1 %.
Assuntos
Meios de Cultura/química , Ácidos Cetoglutáricos/metabolismo , Yarrowia/crescimento & desenvolvimento , Yarrowia/metabolismo , Biotecnologia/métodos , Ácidos Graxos Monoinsaturados , Concentração de Íons de Hidrogênio , Óleos de Plantas/metabolismo , Óleo de Brassica napusRESUMO
Production of D S-threo-isocitric acid (ICA) by yeast meets serious difficulties since it is accompanied by a simultaneous production of citric acid (CA) in significant amounts that reduces the yield of desired product. In order to develop an effective process of ICA production, 60 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis, and Yarrowia) were tested for their ability to produce ICA from rapeseed oil; as a result, wild-type strain Yarrowia lipolytica VKM Y-2373 and its mutant Y. lipolytica 704-UV4-A/NG50 were selected as promising ICA producers. The effects of temperature, pH, aeration, and concentrations of rapeseed oil, iron, and itaconic acid on ICA production by selected strains were studied. Under optimal conditions (pH 6.0; aeration 50-55 %; rapeseed oil concentration of 20-60 gl(-1), iron ion concentration of 1.2 mg l(-1), and itaconic acid amount of 30 mM), selected strains of Y. lipolytica produced predominantly ICA with a low amount of a by-product, CA.
Assuntos
Isocitratos/metabolismo , Óleos de Plantas/metabolismo , Yarrowia/metabolismo , Meios de Cultura/metabolismo , Ácidos Graxos Monoinsaturados , Óleo de Brassica napus , Leveduras/metabolismoRESUMO
Isocitric acid (ICA) refers to a group of promising regulators of energy metabolism which has antistress, antihypoxic, and antioxidant activities. In this paper, we reported a process of ICA production from rapeseed oil using yeast Yarrowia lipolytica VKM Y-2373 in a 500-L fermentor. The producer synthesized 64.1 g/L ICA with a product yield of 0.72 g/g and a productivity 0.54 g/L·h. We also developed an effective purification method, including a cell separation, clarification, concentration, acidification, and crystallization process, which resulted in the formation of the crystals of monopotassium salt of ICA with a purity of 99.0-99.9%. To the best of our knowledge, this is the first report on an ICA production process at an upscaled bioreactor level.
RESUMO
The yeast Yarrowia lipolytica VKM Y-2412 was selected as a prospective producer of α-ketoglutaric acid (KGA) from ethanol. The following peculiarities were found: (1) the intensive KGA production occurred only under the limitation of cell growth by thiamine and the excess of ethanol and nitrogen, (2) the production of KGA from ethanol required increased amount of zinc and iron ions, and (3) KGA production increased significantly with a high aeration at pH medium equal to 3.5. Under optimal conditions, the Y. lipolytica VKM Y-2412 produced up to 172 g l(-1) of KGA with the mass yield coefficient of 0.70 g g(-1).
Assuntos
Regulação Fúngica da Expressão Gênica , Ácidos Cetoglutáricos/metabolismo , Redes e Vias Metabólicas/genética , Yarrowia/genética , Yarrowia/metabolismo , Etanol/metabolismo , Ferro/metabolismo , Nitrogênio/metabolismo , Tiamina/metabolismo , Zinco/metabolismoRESUMO
Yarrowia lipolytica yeast is well known to be able to synthesize citric acid (CA) in large amounts. This study deals with CA biosynthesis, the production of biomass, as well as the accumulation and composition of proteins and lipids in Y. lipolytica VKM Y-2373 grown in media with glucose at different concentrations of ammonium sulfate (from 2 to 10 g/L). It was found that these concentrations of nitrogen source are limiting for the growth of Y. lipolytica and that nitrogen deficiency is the main cause of CA excretion. At the high concentration of (NH4)2SO4 (10 g/L), the accumulation of cell biomass, biomass yield (YX/S), and protein concentration was higher than in the medium with 2 g/L ammonium sulfate by 4.3 times, 143%, and 5.1 times, respectively. CA was accumulated in meaningful quantities only in media containing 3-10 g/L (NH4)2SO4 with the maximum concentration of CA (99.9 g/L) at 4 g/L ammonium sulfate. Also of interest is the technological mode with 6 g/L (NH4)2SO4, which is characterized by high productivity (1.11 g/L × h). It should be noted that biomass contains large amounts of essential amino acids and unsaturated fatty acids and can be used in food biotechnologies and agriculture.
Assuntos
Yarrowia , Ácido Cítrico , Sulfato de Amônio/metabolismo , Nitrogênio/metabolismo , Glucose/metabolismo , Lipídeos , Aminoácidos Essenciais/metabolismoRESUMO
Yarrowia lipolytica A-101-1.22 produces high citric acid (112 g l(-1)) with a yield of 0.6 g g(-1) and a productivity of 0.71 g l(-1) h(-1) during batch cultivation in the medium with glycerol-containing waste of biodiesel industry. However, it was observed that the specific citric acid production rate, which was maximal at the beginning of the biosynthesis, gradually decreases in the late production phase and it makes continuation of the process over 100 h pointless. The cell recycle and the repeated batch regimes were performed as ways for prolongation of citric acid synthesis by yeast. Using cell recycle, the active citric acid biosynthesis (96-107 g l(-1)) with a yield of 0.64 g g(-1) and a productivity of 1.42 g l(-1) h(-1) was prolongated up to 300 h. Repeated batch culture remained stable for over 1000 h; the RB variant of 30% feed every 3 days showed the best results: 124.2 g l(-1) citric acid with a yield of 0.77 g g(-1) and a productivity of 0.85 g l(-1) h(-1).
Assuntos
Biocombustíveis/análise , Reatores Biológicos/microbiologia , Ácido Cítrico/metabolismo , Glicerol/metabolismo , Microbiologia Industrial/métodos , Resíduos Industriais/análise , Yarrowia/metabolismo , FermentaçãoRESUMO
The decrease in metabolism is one of mechanisms for hibernating animals to resist hypoxia and oxidative stress. Assuming that the inhibition of mitochondria; respiration in torpor and its activation upon arousal are accompanied by changes in the content of mitochondrial substrates, we estimated the levels of endogenous metabolites of the tricarboxylic acid (TCA) cycle in the liver, brown adipose tissue, and the brain of the arctic ground squirrels as possible indicators of mitochondrial processes. The level of lactate in the same tissues and serum was determined as marker of hypoxia. It was found that the isocitrate (ISC) concentration in all tissues was one order of magnitude higher than that of alpha-ketoglutarate (KGL), while succinate was not detected in any of tissues, indicating the inhibition at the initial stages of the TCA cycle. During the torpor, the concentrations of ISC, KGL and lactate predominantly decreased in tissues. Serum lactate decreased five-fold in torpor and was restored in a temperature-dependent manner with a long period of persistence of stable concentration in the range of body temperature between 12 and 27°C upon arousal. The data obtained indicate the development of metabolic depression rather than hypoxia in these tissues.
Assuntos
Hibernação/fisiologia , Mitocôndrias/metabolismo , Sciuridae/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Nível de Alerta/fisiologia , Encéfalo/metabolismo , Ciclo do Ácido Cítrico , Ácido Láctico/metabolismo , Mitocôndrias Hepáticas/metabolismo , Estresse OxidativoRESUMO
Within this work, the microbial synthesis of (2R,3S)-isocitric acid (ICA), a metabolite of the nonconventional yeast Yarrowia lipolytica, from biodiesel waste, has been studied. The selected strain Y. lipolytica VKM Y-2373 synthesized ICA with citric acid (CA) as a byproduct. This process can be regulated by changing cultivation conditions. The maximal production of ICA with the minimal formation of the byproduct was provided by the use of a concentration of (NH4)2SO4 (6 g/L); the addition of biodiesel waste to cultivation medium in 20-60 g/L portions; maintaining the pH of the cultivation medium at 6, and degree of aeration between 25% and 60% of saturation. Itaconic acid at a concentration of 15 mM favorably influenced the production of ICA by the selected strain. The optimization of cultivation conditions allowed us to increase the concentration of ICA in the culture liquid from 58.32 to 90.2 g/L, the product yield (Y) by 40%, and the ICA/CA ratio from 1.1:1 to 3:1. Research on laboratory animals indicated that ICA counteracted the negative effect of ammonium molybdate (10-5 Ð) and lead diacetate (10-7 Ð) on the learning and spatial memory of rats, including those exposed to emotional stress.
RESUMO
A new two-step process of production of succinic acid (SA) has been developed, which includes the microbial synthesis of alpha-ketoglutaric acid by the yeast Yarrowia lipolytica (step 1) and subsequent oxidation of the acid by hydrogen peroxide to SA (step 2). The maximum concentration of SA and its yield were found to be 63.4 g l(-1) and 58% of the ethanol consumed, respectively. The purity of the SA isolated from the culture liquid filtrate reached 100%. The yield of SA was as high as 82% of its amount in the culture liquid filtrate. The quality of the SA produced by the invented method meets the biochemical grade definitions, as is evident from the respiratory and other relevant parameters of rat liver mitochondria upon the oxidation of this SA.