[Biosynthesis of isocitric acid by the yeast yarrowia lipolytica and its regulation].

We studied the biosynthesis of isocitric acid from rapeseed (canola) oil by the yeast Yarrowia lipolytica and its regulation. We determined a fundamental possibility for directed biosynthesis of isocitric acid by Y lipolytica yeast, with only minimal amounts of citric acid byproduct, when grown on a medium containing canola oil. Wild type strains of Y lipolytica were mutagenized by UV irradiation and treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NG). Subsequent selection on media with acetate and isocitrate resulted in isolation of a UV/NG Y lipolytica UV/NG mutant that synthesized isocitrate and citrate at a ratio of 2.7:1. In the parent strain, this ratio is 1:1. Inhibition of isocitrate lyase, a key enzyme in the metabolism of isocitric acid, by the addition of itaconic acid resulted in increased synthesis of isocitrate with a ratio of isocitrate to citrate reaching 6:1. Culturing of the Y lipolytica UV/NG mutant in a pilot industrial fermenter in the presence of itaconic acid resulted in the production of 88.7 g/L of isocitric acid with a yield of 90%.

Burst of succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in concert with the expression of genes coding for respiratory chain proteins underlies short-term beneficial physiological stress in mitochondria.

Conditions for the realization in rats of moderate physiological stress (PHS) (30-120 min) were selected, which preferentially increase adaptive restorative processes without adverse responses typical of harmful stress (HST). The succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH) activity and the formation of reactive oxygen species (ROS) in mitochondria were measured in lymphocytes by the cytobiochemical method, which detects the regulation of mitochondria in the organism with high sensitivity. These mitochondrial markers undergo an initial 10-20-fold burst of activity followed by a decrease to a level exceeding the quiescent state 2-3-fold by 120 min of PHS. By 30-60 min, the rise in SDH activity was greater than in KDH activity, while the activity of KDH prevailed over that of SDH by 120 min. The attenuation of SDH hyperactivity during PHS occurs by a mechanism other than oxaloacetate inhibition developed under HST. The dynamics of SDH and KDH activity corresponds to the known physiological replacement of adrenergic regulation by cholinergic during PHS, which is confirmed here by mitochondrial markers because their activity reflects these two types of nerve regulation, respectively. The domination of cholinergic regulation provides the overrestoration of expenditures for activity. In essence, this phenomenon corresponds to the training of the organism. It was first revealed in mitochondria after a single short-time stress episode. The burst of ROS formation was congruous with changes in SDH and KDH activity, as well as in ucp2 and cox3 expression, while the activity of SDH was inversely dependent on the expression of the gene of its catalytic subunit in the spleen. As the SDH activity enhanced, the expression of the succinate receptor decreased with subsequent dramatic rise when the activity was becoming lower. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaption and therapy.

[Obtaining of the mutant Yarrowia lipolytica strains producing citric acid from glucose].

The possibility of obtaining mutant yeasts Yarrowia lipolytica VKM Y-2373 with increased ability to synthesize citric acid from glucose by using UV irradiation and N-methyl-NT-nitro-N-nitrosoguanidine was studied. Of 1500 colonies of the Y. lipolytica treated with either UV or N-methyl-NT-nitro-N-nitrosoguanidine, three mutants were selected that displayed higher (by 23%) biosynthetic ability as compared with the initial strain. Additionally, three mutants were selected from 1000 colonies of the Y. lipolytica exposed to a combined action of UV and N-methyl-NT-nitro-N-nitrosoguanidine; their biosynthetic activity exceeded that of the initial strain by 43.9%. The selective media with citrate and acetate were developed for a rapid selection of mutants as well as the express methods for the detection of active citric acid producers on the solid media with chalk and bromocresol containing a limiting concentration of amine nitrogen and an excess of glucose.

Increase in the contribution of transamination to the respiration of mitochondria during arousal.

The involvement of transamination in the respiration of liver mitochondria in ground squirrels during hibernation and arousal has been studied. It was shown by HPLC that, in the presence of glutamate and malate, the formation of alpha-ketoglutarate (KGL), a transamination marker, and fumarate, a product of succinate oxidation, takes place. During arousal, the formation of KGL increased fourfold, and the respiration sensitive to the inhibitor of aspartate transaminase aminooxyacetate (AOA) increased threefold. The function of transamination upon arousal is related to the elimination of oxaloacetate, an endogenous inhibitor of succinate dehydrogenase. In addition, being more resistant to oxidative stress than oxidation, transamination is probably involved in the antioxidant defense required during the rapid rise of body temperature upon arousal. Our experiments showed an increase in the concentration of malonic dialdehyde (MDA), an end product of lipid peroxidation, in liver mitochondria in this state, which can reduce the activity of the enzymes of the tricarboxylic acids cycle. Under these conditions, the transamination contributes to the maintenance of a high respiration rate necessary for arousal.

[Synthesis citric and isocitric acids by Yarrowia lipolytica during yeasts growth on rapeseed oils].

The native strain Yarrowia lipolytica VKM Y-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapeseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were observed in the glycerol concentration, the proportions of the major free fatty acids formed via oil hydrolysis, or the fatty acid composition of oil. Under nitrogen limitation of cell growth, the accumulation of citric acids reached 77.1 g/l with predominance of isocitric acid at pH 6.0, whereas at pH 4.5, almost equal amounts of citric and isocitric acids were produced. Cultivation of the mutant strain Y. lipolytica N 1 at pH 4.5 resulted in the predominant accumulation of citric acid (66.6 g/l) with an insignificant amount of isocitric acid. In the period of intense acid synthesis, high production of lipase was observed.

[Organic acid production by the yeast Yarrowia lipolytica (a review)].

The review sums up the results of studies of (1) physiological growth characteristics of the yeast Yarrowia lipolytica, cultured in the presence of diverse carbon sources (n-alkanes, glucose, and glycerol), and (2) superhigh synthesis of organic acids, which were performed at the Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences. Microbiological processes of obtaining alpha-ketoglutaric, pyruvic, isocitric, and citric acids are discussed.

[The isolation, purification, and some properties of NAD-dependent isocitrate dehydrogenase from the organic acid-producing yeast Yarrowia lipolytica]. / Vydelenie, ochistka i nekotorye svoistva NAD-zavisimoi izotsitratdegidrogenazy drozhzhei Yarrowia lipolytica--produtsenta organicheskikh kislot.

The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The Km for NAD+ is 136 microM, and that for isocitrate is 581 microM. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD+) in the overproduction of citric and keto acids is discussed.

Regulation of NAD(+)-dependent isocitrate dehydrogenase in the citrate producing yeast Yarrowia lipolytica.

The mechanism of the increased accumulation (overproduction) of citric acids in the yeast Yarrowia lipolytica while growing in the presence of glucose under nitrogen deficiency was investigated. The limitation of the yeast growth by the source of nitrogen decreases the total content of nucleotides and increases the ratios of ATP/AMP and NADH/NAD+. NAD+-Dependent isocitrate dehydrogenase, an enzyme of the tricarboxylic acid cycle playing a key role in the regulation of biosynthesis of citric and isocitric acids, was isolated from Y. lipolytica. The molecular weights of the native enzyme and its subunits were found to be 412 and 52 kD, respectively. It is concluded that the enzyme is a homo-oligomer consisting of eight subunits. Investigation of the effect of some intermediates of the tricarboxylic acid cycle on the activity of this enzyme suggests that the enhanced excretion of citric acids can be caused by the inhibition of NAD+-dependent isocitrate dehydrogenase due to the decrease in the content of AMP and increase in the NADH/NAD+ ratio in the cells of Y. lipolytica under depletion of nitrogen.

Biosynthesis of citric and isocitric acids from ethanol by mutant Yarrowia lipolytica N 1 under continuous cultivation.

The effect of ethanol, zinc, and iron (Fe(2+) and Fe(3+)) concentration and of oxygen supply on cell growth and the production of citric acid (CA) and isocitric acid (ICA) from ethanol by mutant Yarrowia lipolytica N 1 was studied under continuous cultivation. The following peculiarities of Y. lipolytica metabolism were found: (1) intensive CA production occurred under yeast growth limitation by nitrogen; (2) inhibition of yeast growth by ethanol was accompanied by significant alterations in fatty acid composition of lipids; (3) the production of CA and ICA from ethanol required high concentrations of zinc and iron ions; (4) the intracellular iron concentration determined whether CA or ICA was predominantly formed; (5) the cell's requirement for oxygen depended on the intracellular iron concentration. The events taking place in the production of CA and ICA were evaluated through the activities of enzyme systems involved in the metabolism of ethanol and CA in this strain.

Purification and characterization of citrate synthase from Methylobacterium extorquens--a methylotrophic producer of polyhydroxybutyrate.

Citrate synthase (citrate oxaloacetate-lyase, CoA-acetylating; EC 4.1.3.7, CS) was isolated and purified to homogeneity from a methylotrophic producer of polyhydroxybutyrate (PHB), Methylobacterium extorquens 15. The purification procedure includes streptomycin sulfate treatment of cell-free extract, ammonium sulfate fractionation, two steps of hydrophobic chromatography, and ion-exchange chromatography. The specific activity of the final enzyme preparation was 24 U/mg protein. The enzyme has apparent molecular weight 260 kD and consists of four 66-kD subunits. The enzyme shows a sigmoid saturation curve with CoASA (h = 1.3). Kinetic parameters are: K(m) = 84 microM for CoASA; K(m) = 12 microM for oxaloacetate; Vmax = 29.7 mumoles/min per mg protein. KCl at concentrations up to 80 mM activates the CS. ATP exerts a significant inhibitory effect on the enzyme activity, whereas NAD(P)H, isocitrate, alpha-ketoglutarate, ADP, acetoacetyl-CoA, glyoxylate, and glutamate have no influence. A possible role of the CS in coordinated control of CoASA transformation through the tricarboxylic acid cycle and PHB biosynthesis in this methylotroph is discussed.