Introduction: The study purpose was to identify possible key metabolites that determine the adaptation of the Endomyces magnusii yeast to long-term cultivation (four weeks) using glycerol as an “oxidative” and glucose as a “fermentative” substrate. Methods: The metabolic profile was assayed using gas chromatography combined with mass spectrometry, followed by bioinformatic analysis (PARADISe, Golm metabolome database (GMD), MassBank, UniChrom). Results: PCA and PLS-DA analyses showed that the type of carbon source contributed significantly to the overall variability of the data, and the greatest variance was observed for the groups grown on different substrates for the first cultivation week. Growth on glycerol increased the chronological lifespan of E. magnusii due to the early launch of adaptive oxidative stress, the active use of lipids as an energy source, the accumulation of membrane sterols, osmo-protective polyols, organic acids (malic, methyl glycerinic, palmitic, linoleic), and some sugars (lyxose, galactose), which increased the overall resistance and maintained high cell survival. On the contrary, cultivation using glucose provoked a sharp substrate depletion, inducing passive storage of sugars (trehalose), diauxic shock, and less effective antioxidant protection, which provided lower cell survival upon prolonged growth. Conclusions: (1) Metabolic signs associated with prolonged culturing were identified in all the compounds classes tested (polyols, fatty acids, lactones); (2) some metabolites (in particular, dulcitol), being hypothetical biomarkers of aging, are at the same time protective agents involved in the adaptation of yeast cells to the deep stationary growth stages. Our data can serve as a basis for comparative studies of aging-related metabolism in other eukaryotic models.

Dihydroquercetin (taxifolin) is a natural bioflavonoid with diverse biological activities, including antioxidant, anti-inflammatory, antibacterial, antiviral, and geroprotective effects. The review summarizes current advances in the research of dihydroquercetin with a focus on its structural features, physicochemical properties, and biological functions. Particular attention is drawn to its low solubility and limited bioavailability, which have stimulated the development of nanotechnology-based delivery systems. In the paper, the systematization of the systems, namely nanoparticles, liposomes, hydrogels, nanofibers, nanoenzymes, and crystalline complexes, is presented. Moreover, some specific features of the antibacterial and antiviral action of the compound are described. Recent findings on the molecular mechanisms of action, including regulation of oxidative stress and cellular signaling pathways, highlight the therapeutic promise of dihydroquercetin. Taken together, the data support its potential as a valuable compound for pharmaceutical and biomedical applications, though further comprehensive investigations are required.

The changes in the proteomic profiles of pectoral and thigh muscles of Japanese quails (Coturnix japonica) were studied when a biological supplement based on the phytase from Obesumbacterium proteus, encapsulated in Yarrowia lipolytica yeast, was introduced into the diet compared to the commercial fungal phytase (Ladozym proxy) from Aspergillus ficuum. Both phytase preparations provided some changes in the histologic pattern of muscles and increased the protein amount. However, the encapsulated phytase from O. proteus acted more efficiently at normal levels of available phosphorus than the fungal phytase from A. ficuum. It provided a concomitant increase in troponin family proteins and a significant change in the contractility of skeletal muscles. Also, for all the groups using the phytases in the diet, we first demonstrated the acetylation of the N-terminal part of the molecule for four quails' proteins, namely, parvalbumin alpha isoform X2 (PVALB), l-lactate dehydrogenase A chain (LDHA), a fragment of PDZ, and LIM domain protein 5 isoform X9 (LOC107313615) that could be related to the changes in forming the contractile apparatus of the muscles and the redistribution of the metabolites between aerobic and anaerobic metabolic pathways. It could impact the meat quality via acetylation/deacetylation of some enzymes related to glycogenolysis/glycolysis.

Phytases of the PhyD class according to their pH optimum (7.0–7.8) and high thermal stability can claim to be used in the production of feed supplements. However, today they have no practical application in feed production because there are no suitable producers sufficient for its biotechnological production compared to the PhyA and PhyC class ones. Moreover, in most cases, the technologies with the enzymes produced in secretory form are preferable for the production of phytases, though upon microencapsulation in yeast-producing cells, the phytase thermal stability increases significantly compared to the extracellular form, which improves its compatibility with spray drying technology. In this study, we assayed the intracellular heterologous expression of PhyD phytase from Bacillus species in the Yarrowia lipolytica yeast cells. While the technology has been successfully used to synthesize PhyC phytase from Obesumbacterium proteus, PhyD phytase tends to aggregate upon intracellular accumulation. Furthermore, we evaluated the prospects for the production of encapsulated phytase of the PhyD class of high enzymatic activity when it accumulates in the cell cytoplasm of the Y. lipolytica extremophile yeast, a highly effective platform for the production of recombinant proteins.

In this study, we used the Manchurian golden breed of quails. We assessed the efficacy of the food additives of the phytase from Obesumbacterium proteus encapsulated in the recombinant Yarrowia lipolytica yeast, which was supplied at a concentration of 500 phytase activity units per kg of the feed. One hundred fifty one-day-old quails were distributed into six treatment groups. The results showed that adding the O. proteus encapsulated phytase to the quails’ diets improved live weight, body weight gain, and feed conversion compared to those in the control groups and the groups using a commercial phytase from Aspergillus ficuum. The results obtained during the experiments indicate a high degree of assimilation of phytate-containing feeds if the encapsulated phytase was fed by the quails compared to that in the other groups. We can conclude that the class D encapsulated phytase is an expedient additive to the diets possessing better kinetic features compared to the PhyA and PhyC classes phytases when it acts inside the quail’s chyme.

In the example of a rat model with chronic hepatoencephalopathy (HE), changes in the organ morphology of rats affect the balance of metabolites of the tricarboxylic acid (TCA) cycle and metabolites of the glutamine–glutamate (Gln-Glu) cycle, namely α-ketoglutarate (αKG) and α-ketoglutaramate (αKGM), as well as the enzymes associated with them, ω-amidase (ωA) and glutamine transaminase (GTK). This model of rats was obtained as a result of 2–22 weeks of consumption by animals of hepatotoxin thioacetamide (TAA) added to drinking water at a concentration of 0.4 g/L. The control (n = 26) and TAA-induced (n = 55) groups of rats consisted of 11 cohorts each. The control cohorts consisted of 2–4 rats, and the TAA-induced cohorts consisted of 4–7 individuals. Every two weeks, samples of blood plasma, liver, kidney, and brain tissues were taken from the next cohort of rats (a total of 320 samples). By the end of the experiment, irreversible morphological changes were observed in the organs of rats: the weight of the animals was reduced up to ~45%, the weight of the kidneys up to 5%, the brain up to ~20%, and the weight of the liver increased up to ~20%. The analysis revealed: (i) a decrease in the activity of ωA and GTK in the tissues of the brain, kidneys, and liver of rats with chronic HE (by ~3, 40, and 65% and ~10, 60, and 70%, respectively); and (ii) the appearance of a significant imbalance in the content of metabolites of the Gln-Glu cycle, αKG, and αKGM. It is indicative that a ~1.5–12-fold increase in the level of αKG in the blood plasma and tissues of the organs of rats with chronic HE was accompanied by a synchronous, ~1.2–2.5-fold decrease in the level of αKGM. The data obtained indicate an essential involvement of the Gln-Glu cycle in the regulation of energy metabolism in rats under conditions of chronic HE. Attention is focused on the significance of the αKG/αKGM ratio, which can act as a potential marker for diagnosing the degree of HE development.

In this study, we first thoroughly assayed the response of the key enzymes of energy metabolism and the antioxidant system in Yarrowia lipolytica yeast at extreme pH. The activity of the tricarboxylic acid cycle enzymes, namely NAD-dependent isocitrate dehydrogenase, aconitate hydratase, NAD-dependent malate dehydrogenase, and fumarate hydratase, NADPH-producing enzymes of glucose-6-P dehydrogenase and NADP-dependent isocitrate dehydrogenase, and the enzymes of the glutathione system was assessed. All the enzymes that were tested showed a significant induction contrary to some decrease in the aconitate hydratase activity with acidic and alkaline stress. It is probable that a change in the enzyme activity in the mitochondria matrix is involved in the regulation of the cellular metabolism of Y. lipolytica, which allows the species to prosper at an extreme ambient pH. It distinguishes it from any other type of ascomycete. A close relationship between the induction of the Krebs cycle enzymes and the key enzymes of the glutathione system accompanied by an increased level of reduced glutathione was shown. The assumption that the increased activity of the Krebs cycle dehydrogenases and promotion of the pentose phosphate pathway at pH stress launches a set of events determining the adaptive response of Y. lipolytica yeast.

Polyphenols are powerful natural antioxidants with numerous biological activities. They change cell membrane permeability, interact with receptors, intracellular enzymes, and cell membrane transporters, and quench reactive oxygen species (ROS). Yarrowia lipolytica yeast, being similar to mammalian cells, can be used as a model to study their survival ability upon long-lasting cultivation, assaying the effect of dihydroquercetin polyphenol (DHQ). The complex assessment of the physiological features of the population assaying cell respiration, survival, ROS detection, and flow cytometry was used. Y. lipolytica showed signs of chronological aging by eight weeks of growth, namely a decrease in the cell number, and size, increased ROS generation, a decrease in colony-forming unit (CFU) and metabolic activity, and decreased respiratory rate and membrane potential. An amount of 150 µM DHQ decreased ROS generation at the 6-week growth stage upon adding an oxidant of 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). Moreover, it decreased CFU at 1–4 weeks of cultivation, inhibited cell metabolic activity of the 24-h-old culture and stimulated that on 14–56 days of growth, induced the cell respiration rate in the 24-h-old culture, and blocked alternative mitochondrial oxidase at growth late stages. DHQ serves as a mild pro-oxidant on the first day of age-stimulating anti-stress protection. In the deep stationary stage, it can act as a powerful antioxidant, stabilizing cell redox status and reducing free radical oxidation in mitochondria. It provides a stable state of population. The hormetic effects of DHQ using lower eukaryotes of Y. lipolytica have been previously discussed, which can be used as a model organism for screening geroprotective compounds of natural origin.

An analysis is given of the dynamics of the activity of the phytase strain Yarrowia lipolytica that was transformed with the phytase genome from Obesumbacterium proteus based on the pUVLT2 vector with the VDAC (Voltage Dependent Anion Channel) mitochondrial porin promoter and induced under stress conditions when growing on low-value plant substrates. In the case of cultivation both in flasks and in a fermenter, an increase in the activity of intracellular phytase was observed when sunflower cake was used as a source of phytates. During the growth of the Y. lipolytica Po1f transformant under alkaline conditions, the VDAC promoter was activated as part of the pUV3-Op genetic construct, which was accompanied by an increase in the activity of intracellular phytase. The enzyme retained a high level of activity in Y. lipolytica pUV3-Op transformant cells after high-temperature treatment of the biomass in a spray dryer, which makes its use in the composition of feed additives for farm animals more effective.

The review describes the advances in the phytase immobilization for the past decade and their biotechnological applications. Different approaches for phytase immobilization are described including the process using organic and inorganic matrices and microbial cells, as well as nanostructures of various nature. Moreover, the immobilization of phytase-producing microbial cells and the use of cross-linked phytase aggregates have been under consideration. A detailed classification of various carriers for immobilization of phytases and the possibility of their applications are presented. A particular attention is drawn to a breakthrough approach of biotechnological significance to the design of microencapsulation of bacterial phytase from Obesumbacterium proteus in the recombinant extremophile of Yarrowia lipolytica.

This paper presents an analysis of the regulation activity of the partially purified preparations of cellular aconitate hydratase (AH) on the yeast Yarrowia lipolytica cultivated at extreme pH. As a result of purification, enzyme preparations were obtained from cells grown on media at pH 4.0, 5.5, and 9.0, purified by 48-, 46-, and 51-fold and having a specific activity of 0.43, 0.55 and 0.36 E/mg protein, respectively. The kinetic parameters of preparations from cells cultured at extreme pH demonstrated: (1) an increase in the affinity for citrate and isocitrate; and (2) a shift in the pH optima to the acidic and alkaline side in accordance with the modulation of the medium pH. The regulatory properties of the enzyme from cells subjected to alkaline stress showed increased sensitivity to Fe2+ ions and high peroxide resistance. Reduced glutathione (GSH) stimulated AH, while oxidized glutathione (GSSG) inhibited AH. A more pronounced effect of both GSH and GSSG was noted for the enzyme obtained from cells grown at pH 5.5. The data obtained provide new approaches to the use of Y. lipolytica as a model of eukaryotic cells demonstrating the development of a stress-induced pathology and to conducting a detailed analysis of enzymatic activity for its correction.

Exposure to the toxin thioacetamide (TAA) causes acute hepatic encephalopathy (HE), changes in the functioning of systemic organs, and an imbalance in a number of energy metabolites. The deferred effects after acute HE development are poorly understood. The study considers the balance of the tricarboxylic acid (TCA) cycle metabolites in the blood plasma, liver, kidneys, and brain tissues of rats in the post-rehabilitation period. The samples of the control (n = 3) and TAA-induced groups of rats (n = 13) were collected six days after the administration of a single intraperitoneal TAA injection at doses of 200, 400, and 600 mg/kg. Despite the complete physiological recovery of rats by this date, a residual imbalance of metabolites in all the vital organs was noted. The results obtained showed a trend of stabilizing processes in the main organs of the animals and permit the use of these data both for prognostic purposes and the choice of potential therapeutic agents.

(1) The study using the quails of the Manchurian golden breed was aimed at the efficacy of the food supplement based on micro-incapsulated phytase of Obessumbacterium proteus in the recombinant yeast producer Yarrowia lipolytica at a dose of 500 FYT/kg of the feed. (2) The effect of the supplement in the poultry diets on the productivity of young and adult quails was assayed; (3) It was found that the keeping the quails on the diets using the microencapsulated phytase O. proteus provided the highest live weight, the highest intensity of egg production, and the slaughter yield of animals, the lowest feed conversion compared to those in control groups and the groups getting a commercial phytase supplement based on Aspergillus ficuum. Taken together the data obtained in our experiments indicate a high degree of assimilation of phytate-containing products by the quails when using the encapsulated phytase compared to both the control and commercial preparation of phytase of the PhyA class. It can be concluded that it is a highly expedient and promising supplement based on the microencapsulated phytase of class D for feeding, which shows the best kinetic features compared to the phytases of PhyA and PhyC classes when working in the chyme of the poultry.

The delayed consequences of the influence of hepatic encephalopathy (HE) on the metabolism of animals have not been studied enough. We have previously shown that the development of acute HE under the influence of the thioacetamide (TAA) toxin is accompanied by pathological changes in the liver, an imbalance in CoA and acetyl CoA, as well as a number of metabolites of the TCA cycle. This paper discusses the change in the balance of amino acids (AAs) and related metabolites, as well as the activity of glutamine transaminase (GTK) and ω-amidase enzymes in the vital organs of animals 6 days after a single exposure to TAA. The balance of the main AAs in blood plasma, liver, kidney, and brain samples of control (n = 3) and TAA-induced groups (n = 13) of rats that received the toxin at doses of 200, 400, and 600 mg/kg was considered. Despite the apparent physiological recovery of the rats at the time of sampling, a residual imbalance in AA and associated enzymes persisted. The data obtained give an idea of the metabolic trends in the body of rats after their physiological recovery from TAA exposure and may be useful for prognostic purposes when choosing the necessary therapeutic agents.

The study presents the results of optimizing the medium components for the cultivation of the recombinant yeast of Yarrowia lipolytica Po1f pUV3-Op to increase the biomass concentration and its phytase activity. Glycerol, yeast extract, and the broth from low-value raw waste of the poultry farms were used as the substrates in the nutrient medium. During the cultivation on the developed nutrient medium in a 10-litre laboratory fermenter under the pilot conditions the optimal fermentation parameters were tested. The yield biomass under the optimized conditions reached 292 g of dried preparation with a phytase activity of 100.1 ± 1.07 mmol P i × min –1 × g –1 of dry biomass.

The study analyzes the dynamics in the lipid profile of the Endomyces magnusii yeast during the long-lasting cultivation using the substrates of “enzymatic” or “oxidative” type. Moreover, we studied its changes upon calorie restriction (CR) (0.5% glucose) and glucose depletion (0.2% glucose). Di-(DAGs), triacylglycerides (TAGs) and free fatty acids (FFAs) dominate in the storage lipid fractions. The TAG level was high in all the cultures tested and reached 80% of the total lipid amount. While being cultured on 2% substrates, the level of storage lipids decreased at the four-week stage, whereas upon CR their initially low amount doubled. Phosphatidylethanolamines (PE), sterols (St) (up to 62% of total lipids), phosphatidylcholines (PC), and phosphatidic acids (PA) (more than 40% of total lipids) were dominating in the membrane lipids of E magnusii. Upon CR at the late stationary growth stages (3–4 weeks), the total level of membrane lipid was two-fold higher than those on glycerol and 2% glucose. The palmitic acid C16:0 (from 10 to 23%), the palmitoleic acid C16:1 (from 4.3 to 15.9%), the oleic acid C18:1 (from 23.4 to 59.2%), and the linoleic acid C18:2 (from 10.8 to 49.2%) were the dominant fatty acids (FAs) of phospholipids. Upon glucose depletion (0.2% glucose), the total amount of storage and membrane lipids in the cells was comparable to that in the cells both on 2% and 0.5% glucose. High levels of PC and sphingolipids (SL) at the late stationary growth stages and an increased PA level throughout the whole experiment were typical for the membrane lipids composition upon the substrate depletion. There was shown a crucial role of St, PA, and a high share of the unsaturated FAs in the membrane phospholipids upon the adaptation of the E. magnusii yeast to the long-lasting cultivation upon the substrate restriction is shown. The autophagic processes in some fractions of the cell population provide the support of high level of lipid components at the late stages of cultivation upon substrate depletion under the CR conditions. CR is supposed to play the key role in regulating the lipid synthesis and risen resistance to oxidative stress, as well as its possible biotechnological application.

The study on the influence of different glucose concentrations (2%, 0.5%, and 0.2%) and glycerol (1%) on the morphological and physiological features, as well as the composition of soluble carbohydrates, was performed using Endomyces magnusii yeast. Two-factor analysis of variance with repetitions to process the data of the cell size changes showed that the substrate type affected cell size the most. The cells with 2% glucose were 30–35% larger than those growing on glycerol. The decrease in the initial glucose concentration up to 0.5–0.2% slightly changed the cell length. However, even in the logarithmic growth phase pseudo-mycelium of two to four cells appeared in the cultures when using low glucose, unlike those using glycerol. Throughout the whole experiment, more than 90% of the populations remained viable on all of the substrates tested. The ability for colony formation decreased during aging. Nevertheless, at the three-week stage, upon substrate restriction (0.2% glucose), it was twice higher than those under the other conditions. The respiration rate also decreased and exceeded not more than 10% of that in the logarithmic phase. By the end of the experiment, the cyanide-sensitive respiration share decreased up to 40% for all types of substrates. The study of soluble cytosol carbohydrates showed that the cultures using 2% glucose and 1% glycerol contained mainly arabitol and mannitol, while at low glucose concentrations they were substituted for inositol. The formation of inositol is supposed to be related to pseudo-mycelium formation. The role of calorie restriction in the regulation of carbohydrate synthesis and the composition in the yeast and its biotechnological application is under consideration.

Phytases are the largest group of feed enzymes increasing the accessibility of organic phosphorus for the animals. Feed phytases are usually sold as dried powder of secreting producers, mainly micellar fungi. We proposed a new technology for producing phytase from Obesumbacterium proteus (OPP) in yeast Yarrowia lipolytica as cytosolic protein (encapsulated OPP), where the capsule (yeast cell) protects the enzyme from unfavourable factors (acid medium and active proteolysis in stomachs) and releases it along with the substrate in the duodenum only. Here we report results of testing the encapsulated OPP on the model of a broiler chicken in comparison to a conventional phytase from Aspergillus ficuum. The encapsulated OPP at a dosage of 30 FYT/kg provided the maximum body weight of the chicken in the end of experiment equal or somewhat higher than in the control group, where the available phosphorus deficit was complemented with a mineral phosphorus supply. In contrast, the conventional soluble phytase at a dosage of 100 or 1000 FYT/kg was not able to compensate for the phosphorus deficit in the diet, although chemical analysis demonstrated much phosphorus in the diet in a non-accessible form. The encapsulated OPP decreased the residual Pi in the chicken faeces by 2.1 times in comparison to the control when added to the diet, whereas the conventional phytases negligibly affected this parameter regardless of the dosage.

The study of the redox and mitochondria state, as well as some other hallmarks of cell aging of extremophilic yeast Yarrowia lipolytica W29, was performed during long-term cultivation for 120 hours. An increase in the ROS level upon the transition to the stationary growth phase was accompanied by a decrease in the cell respiratory rates, an increase in the alternative oxidase share. Moreover, the activities of superoxide dismutase and catalase increased. During stationary growth phase, the ratio of glutathione/glutathione disulfide increased two to three times. Cytometric studies of the populations in vivo stained with a potentiometric mitotracker showed a decrease in mitochondrial potential as the culture gets old. At the late stationary growth stage, some lysosomal processes in the Y. lipolytica cells were induced that apparently increased the resistance to oxidative stress and allowed the culture keep the survival rate at the level of 80%.