1. Introduction
Isocitric acid is a structural isomer of citric acid (CA), differing from the latter in the position of the hydroxyl group. Isocitric acid molecule has two asymmetric carbon atoms and hence exists in four stereoisomers, namely, threo-D
S-isocitric acid, threo-Ls-isocitric acid, erythro-D
S-isocitric acid, and erythro-L
S-isocitric acid [
1], of which only threo-D
S-isocitric acid (or (2
R,3
S)-isocitric acid according to IUPAC Organic Nomenclature) (conventionally named as ICA) is an intermediate of the tricarboxylic acid (TCA) cycle, a common metabolite formed by microorganisms, plants, and animals [
1].
ICA and its derivatives are widely used in industries producing pharmaceutical products, and cosmetics as surfactants, detergents, ion chelators, and biologically active additives to food products [
2,
3,
4,
5,
6,
7,
8,
9,
10]. It was found that ICA surpasses the classical antioxidant ascorbic acid in the model of oxidative stress induced by the action of hydrogen peroxide and ecotoxicants (Cu, Pb, Zn, Cd) on infusorian cells [
11]. In addition, ICA relieves the neurointoxication against the high concentration of lead and molybdenum salts, restores the memory and accelerates the learning [
12]. It was also reported the positive influence of ICA on the spatial component of memory [
13].
It is known that ICA accumulates in significant quantities in the juice of flowering showy stonecrop Hylotelephium spectabile, formerly called Sedum spectabile, which is sold at a price of 595 EUR per gram.
At present, microbiological production of ICA is more preferable for its applications in the medicine and food industries, because the resulting product contains only the natural isomer [
2,
4,
5,
9,
10].
The effective processes for ICA production were developed on the cultivation of different wild, mutant and genetically engineered strains of
Yarrowia lipolytica yeast on n-paraffin [
14], ethanol [
12,
15], plant oils [
3,
4,
6,
16,
17,
18,
19], and glycerol-containing waste of biodiesel industry [
13,
20,
21,
22]. It should be noted that CA accumulate simultaneously with ICA. The balance between CA and ICA can be shifted toward the overproduction of isocitrate by the activation of aconitate hydratase—enzyme, which isomerizes CA to ICA via
cis-aconitate [
14,
16]. For the operation of aconitase, iron ions are required [
14,
15,
17]. The ratio between citric acids toward the ICA production can be shifted by the inhibition of isocitrate lyase with itaconic and oxalic acid in
Y. lipolytica, grown on rapeseed oil [
23], while the genetically modified strain
Y. lipolytica with the inactivated
ICL1 gene exhibited only a small increase in the synthesis of ICA in media with glucose and glycerol [
24].
In recent years, sunflower oil has attracted increased interest as a renewable resource which can be obtained in any country. Sunflower oil is an excellent substrate for biotechnological processes because it provides for a high purity of the target product with the aim of its use in medicine and food industry. High ICA production from sunflower oil was revealed in wild and genetically modified yeast
Y. lipolytica [
3,
4,
6]. However, the knowledge on the physiology of isocitrate-producing strains
Y. lipolytica and the fermentation conditions essential for ICA production from sunflower oil is still limited.
The aim of the present work was the selection of producer and the evaluation of critical factors affecting Y. lipolytica growth and ICA production for the optimal design of fermentation process.
2. Materials and Methods
All chemicals were of analytical grade (Sigma-Aldrich, St. Louis, MO, USA). Sunflower oil “Golden Seed Oil” was purchased from the “YugRossii” Processing Plant (Rostov, Russia). The fatty acid profile of the sunflower oil was (%, by mass): C14:0, 0.1; C16:0, 6.2; C18:0, 4.1; C18:1, 13.7; C18:2, 75.9.
For screening, 30 wild-type yeast strains were obtained from the Collection of the Laboratory of Aerobic Metabolism of Microorganisms, the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences and from the All-Russia Culture Collection of Microorganisms, Russian Academy of Sciences (VKM). The strains were maintained at 4 °C on agar slants with 1% paraffin as the carbon source. The yeast strains were maintained on agar medium with paraffin at +4 °C. Yeasts were cultivated on an orbital shaker (180 ± 10 rpm) at 29 °C during 96 h in 750-mL shaking flasks with 50 mL of the Reader medium containing (g/L): sunflower oil, 10; (NH4)2SO4, 0.3; KH2PO4, 1.0; K2HPO4, 0.1; MgSO4·7H2O, 0.7; Ca(NO3)2·4H2O, 0.4; NaCl, 0.5; trace element solution containing (mg/L): KJ, 0.1; Na2B4O7·7H2O, 0.08; MnSO4·5H2O 0.05; ZnSO4·7H2O 0.04; CuSO4, 0.04; Na2MoO4, 0.03; FeSO4(NH4)2SO4·6H2O, 0.35; yeast extract “Difco”, 0.5. During cultivation, the pH of the medium was determined each 12 h and, if necessary, corrected by adding a certain volume of 10% solution of either KOH or H2SO4.
In experiments on optimization of ICA production by the selected strain
Y. lipolytica VKM Y-2373, five mineral media were used: (1) Reader medium; (2) nitrogen-deficient; (3) phosphorus-deficient; (4) sulfur-deficient; (5) magnesium-deficient. The first medium contained (g/L): (NH
4)
2SO
4, 12.0; MgSO
4·7H
2O, 1.4; Ca(NO
3)
2, 0.8; NaCl, 0.5; KH
2PO
4, 2.0; K
2HPO
4, 0.2; double volume of Burkholder microelement solution; yeast autolysate, 8 mL/L; thiamine, 100 µg/L. Limitation of the yeast growth was performed by decreasing concentration of the salt containing limiting component. The second medium contained nitrogen, phosphorus, sulfur and magnesium in concentrations of 630, 492, 362 and 280 mg/L, respectively. The third medium decreased (almost 30-fold) the amount of phosphorus; concentrations of nitrogen, phosphorus, sulfur and magnesium were 1960, 16.4, 362 and 280 mg/L. In the fourth medium the sulfur amount was decreased by almost 30 times; concentrations of nitrogen, phosphorus, sulfur and magnesium were 1960, 492, 12 and 280 mg/L, respectively. The fifth medium decreased (almost 50-fold) the amount of magnesium; concentrations of nitrogen, phosphorus, sulfur and magnesium were 1960, 492, 362 and 5.6 mg/L. The initial concentration of oil was 20 g/L; pulsed additions of oil (by 20 g/L) at 24, 48 and 72 h. The fermentation conditions were maintained automatically at the constant level: temperature (29 °C); dissolved oxygen concentration (pO
2) (55–60% from air saturation); pH 6.0 was adjusted with 20% KOH. These fermentation parameters were selected on the basis of data on optimization of the ICA synthesis from rapeseed oil by
Y. lipolytica [
17]. Cultivation was performed for 96 h.
The effect of inhibitors on the metabolization and production of ICA and CA was studied in experiments carried out in test tubes and in a fermenter. In the first case, Y. lipolytica VKM Y-2373 were cultivated on an orbital shaker (180 ± 10 rpm) at 29 °C during 48 h in large test tubes (20 cm long, 2 cm in diameter) with 5 mL Reader medium containing sunflower oil (5 g/L) as carbon substrate. In the second case, Y. lipolytica was cultivated in the 10-L fermenter with 5 L of medium containing (g/L): (NH4)2SO4, 3; MgSO4·7H2O, 1.4; Ca(NO3)2, 0.8; NaCl, 0.5; KH2PO4, 2.0; K2HPO4, 0.2; double volume of Burkholder microelement solution; yeast autolysate, 8 mL/L; thiamine, 100 µg/L and FeSO4·7H2O, 7.5 mg/L. The fermentation conditions were maintained automatically at the constant level: temperature (29 °C); dissolved oxygen concentration (pO2) (55–60% from air saturation); pH 6.0 was adjusted with 20% KOH. Cultivation was performed for 96 h.
The amount of biomass, oil was determined as described earlier [
17].
Concentration of organic acids (pyruvic acid, citric acid, α-ketoglutaric acid, fumaric acid, malic acid, oxaloacetic acid, itaconic acid, oxalic acid) was determined using high-performance liquid chromatography (HPLC) with an HPLC chromatograph (Pharmacia, LKB, Uppsala, Sweden) on an Inertsil ODS-3 reversed-phase column (250 × 4 mm, Elsiko, Moscow, Russia) at 210 nm; 20 mM phosphoric acid was used as a mobile phase with the flow rate of 1.0 mL/min; the column temperature was maintained at 35 °C. Acids were also identified using the standard solutions (Roche Diagnostics GmbH. Germany). Glucose 6-phosphate was determined by enzymatic assay with glucose-6-phosphate dehydrogenase. Fructose 1,6-bisphosphate was determined by fructose based on the Seliwanoff color reaction.
For the enzyme assay, yeast cells were collected by centrifugation at 3000× g for 10 min (4 °C) and washed with an ice-cold 0.9% NaCl solution. The cell pellet was suspended in a proportion of 1:10 in 100 mM phosphate buffer (pH 7.4) supplemented with 1 mM EDTA. Cells in the suspension were disrupted with Ballotini glass beads (d = 150–250 μ) on a planetary mixer for 3 min at 1000 rpm (0 °C). The cell homogenate was centrifuged at 5000 g for 30 min (4 °C), and the supernatant was used for the assay of citrate synthase (CS), aconitate hydratase (AH), NAD-isocitrate dehydrogenase (NAD-ICDH) and isocitrate lyase (ICL). CS was assayed in the reaction mixture containing 0.25 mM oxaloacetate, 0.25 mM acetyl CoA, 0.1 mM 5.5-dithiobis(2-nitrobenzoate), and 100 mM Tris-HCl buffer (pH 8.5). AH was assayed in the reaction mixture containing 5 mM ICA and 50 mM potassium phosphate buffer (pH 7.5). NAD-ICDH was assayed in the reaction mixture containing 0.25 mM ICA, 4 mM NAD, 0.5 mM AMP, 10 mM MgCl2, 2 mM antimycin, and 50 mM Tris-HCl buffer (pH 7.5). ICL was assayed in the reaction mixture containing 4 mM ICL, 8 mM phenylhydrazine-HCl, 4 mM cysteine-HCl, 10 mM MgCl2, and 75 mM potassium phosphate buffer (pH 6.85). The amount of enzyme catalyzing conversion of 1 μmol of substrate per min per mg of protein was taken as one unit of enzyme activity (U/mg of protein).
The mass yield coefficient of ICA production (Y
ICA), expressed in g of ICA per g of sunflower oil consumed, was calculated from:
The volume productivity (Q
ICA), expressed in g/L·h, was calculated from:
where P is the total amount of ICA in the culture liquid at the end of cultivation (g), S is the total amount of sunflower oil consumed (g), V is the initial volume of culture liquid (L), t is the fermentation duration (h).
All the data presented are the means values of three experiments and two measurements for each experiment; standard deviations were calculated (S.D. < 10%). The ICA/CA ratio, the mass yield coefficient of ICA production (YICA) and the volume productivity (QICA) are calculated using the mean value of ICA.