RU2747583C1 - Method for synthesis of (2r, 3s)-isocitric acid from sunflower seed oil using yarrowia lipolytica yeast - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to the area of microbiological production of organic acids, particularly (2R,3S)-isocitric acid. Proposed are a yeast strain Yarrowia lipolytica VKM Y-3044D-producer of (2R,3S)-isocitric acid and a method of synthesis thereof involving cultivation of the yeast strain Yarrowia lipolytica VKM Y-3044D on a nutrient medium containing sunflower seed oil as a carbon source, a nitrogen source, trace elements, thiamine and Fe²⁺ ions in a defined amount, in conditions of aeration and stirring until the maximum accumulation of the target product in the medium. During the period from 20 to 24 hours of cultivation itaconic acid is introduced into the medium in form of sodium salt in a defined amount.

EFFECT: group of inventions allows raising the yield of a target product.

3 cl, 1 tbl, 3 ex

Description

Technology area

The invention relates to the field of microbiological production of organic acids, in particular, (2R, 3S) -isolymonic acid (traditional name: Tpeo-D _S -isolymonic acid), which has a hepatoprotective, actoprotective and antihypoxic effect, which can be used for sports nutrition and as a biologically active food supplement.

State of the art

Known methods for producing (2R, 3S) -isolytic acid using various strains of the yeast Yarrowia lipolytica. The acid is obtained by cultivating the above yeast in liquid mineral media containing sources of nitrogen, phosphorus and mineral salts.

Described is a method of obtaining (2R, 3S) -isolymonic acid in a medium with n-alkanes with Candida lipolytica IBPM Y-160 with a product yield of 68.3 g / l (109.8% of the used substrate) (Inventor's Certificate No. 849780 , 1983). However, the carcinogenicity of paraffins (Otto et al., 2011) and, therefore, the unacceptability of the product's use in the food industry and medicine limits the application of this method.

Known methods for the production of (2R, 3S) -isolytic acid from ethyl alcohol using the natural strain Yarrowia lipolytica VKM Y-2373 with a product yield of 90.5 g / l (77% of the substrate) under batch conditions (Kamzolova et al., 2018 ) and with a yield of 109.6 g / L (80% of the substrate) under the conditions of weaning-topping cultivation (Morgunov et al., 2019). The disadvantage of the above methods for producing (2R, 3S) -isolytic acid from ethyl alcohol is the low yield of the product and the complexity of fermentation due to the toxicity of high concentrations of ethanol for the cell.

A known method of producing (2R, 3S) -isolytic acid from glycerin-containing waste from biodiesel production. The genetically modified transformant Yarrowia lipolytica AJD pADUTGut1 / 2 with overexpression of the Gut1 and Gut2 genes was characterized by rapid growth on glycerol-containing waste and produced, along with citric acid, a significant amount of (2R, 3S) -isolymonic acid (42.5 g / L) (Rzechone et al., 2019). The disadvantage of this method is that it does not provide a sufficiently high yield of (2R, 3S) -isolytic acid.

Known methods for obtaining (2R, 3S) -isolymonic acid from rapeseed oil: using the natural strain Yarrowia lipolytica VKM Y-2373, a product yield of 70.6 g / l (82% of the substrate) was achieved (Kamzolova et al., 2013), and with the use of mutant strains Yarrowia lipolytica 704-UV4-A-NG50 (Kamzolova et al., 2013) and Yarrowia lipolytica UV / NG (Kamzolova et al., 2015), yields of 86 g / L (95% of the substrate) and 88 , 7 g / l (90% of the substrate), respectively. However, the presence of erucic acid (toxic to humans) in rapeseed oil limits the use of this substrate in the microbial synthesis of products for the food industry and medicine.

The closest to the expected one in terms of the achieved effect and a set of essential features is the method for obtaining (2R, 3S) -isolymonic acid from sunflower oil using the natural strain Yarrowia lipolytica VKM Y-2373 (Kamzolova et al., 2008). According to this method, yeast is grown on a medium containing sunflower oil as a source of carbon and energy, as well as sources of nitrogen, phosphorus, mineral salts and thiamine. Sunflower oil is added discretely - 20 g / l at the beginning and then as the oil concentration decreases below 5 g / l. The cultivation is carried out at 28 ° C, the concentration of dissolved oxygen is 55-60% of saturation, pH 6 (maintained by adding 10-20% NaOH). During the fermentation, 70 g / l of (2R, 3S) -isolymonic acid is accumulated, which is 75% of the used substrate. Also, good results on the accumulation of (2R, 3S) -isolytic acid in a medium with sunflower oil are obtained using the Yarrowia lipolytica EH 59 strain (Aurich et al., 2012, p. 406): 93 g / L (2R, 3S) -isolytic acid, which is 65% of the used substrate. The disadvantage of these methods is that they do not provide a sufficiently high yield of (2R, 3S) -isolytic acid.

The problem to be solved by the claimed invention is to obtain a new highly active producer strain (2R, 3S) -isolymonic acid. Another task is to increase the yield of (2R, 3S) -isolymonic acid during the growth of Yarrowia lipolytica yeast on a medium with sunflower oil.

The technical result that can be obtained with the implementation of the invention is to provide a high level of activity of the new strain of Yarrowia lipolytica, adapted to a high level of isolic acid during its production under industrial conditions for pharmaceutical and food use.

The essence of the invention

One aspect of the invention relates to the Yarrowia lipolytica strain VKM Y-3044D-producer of (2R, 3S) - isocitric acid.

Another aspect of the invention relates to a method for producing (2R, 3S) -isolymonic acid by aerobic cultivation using the Yarrowia lipolytica yeast strain VKM Y-3044D on a nutrient medium containing sunflower oil as a carbon and energy source. In order to increase the yield of the target product by increasing the degree of substrate utilization, in the period from 20-24 hours of cultivation, itaconic acid is introduced into the fermentation medium in the form of sodium salt in an amount of 3.0 to 4.5 g / l, preferably 3.9 g / l.

Description of the invention

With the improvement of industrial technology for the production of isocitric acid, technical problems were solved in the field of increasing the efficiency of the method of continuous cultivation of a new producer strain.

The Yarrowia lipolytica LAMM 704-9 strain was obtained by multi-stage adaptation of the natural Yarrowia lipolytica VKM Y-2373 strain to a high concentration of (2R, 3S) -isolytic acid. To carry out the adaptation, a stepwise mode of increasing the concentration of isocitric acid in the range from 0 to 1.4 g of isocitric acid per 1 g of cells in growth media with sunflower oil with a concentration of 30 g / L was chosen. The Yarrowia lipolytica LAMM 704-9 strain was deposited in the All-Russian collection of microorganisms at the I. G.K. Scriabin RAS under registration number VKM Y-3044D.

Storage conditions: the strain can be stored in a lyophilized state for several years and / or in test tubes on a slant agar medium Reeder with paraffin or on wort agar at + 4 ° C with obligatory reseeding at least once within 3-6 months.

The Yarrowia lipolytica strain VKM Y-3044D has the following characteristics:

Cultural and morphological characteristics

3-day culture in liquid wort and on agar wort is represented by oval, elongated-oval and rounded cells measuring 4.0-6.5 × 4.5-13 microns. Small cells of 2.0-2.5 × 3.0-3.5 microns are found. Some of the large cells have an irregular shape. The budding is polar or lateral. The cells are single or connected in chains of 2-3 cells. The line on wort agar (age 10 days) is solid, flat, cream-colored, smooth, pasty, smooth edges. Colonies on wort agar are round, the diameter of most of the colonies is 6 mm, there are small colonies with a diameter of 3 mm (5-6% of the population). Colonies are whitish-creamy, pasty, smooth edges, raised center of colonies. In 90% of the colonies, the surface is smooth, in 10% of the colonies it is wrinkled. Colonies grown on wort agar for 4 weeks are cream-colored, fringed edges, the surface of the colony is rough, pasty, the center of the colony is raised. On glass, in potato agar for 5 days, the culture forms a pseudomycelium of the Candida and Mycocandida types. Does not form a dispute. Strict aerobic.

Physiological and biochemical characteristics

Assimilates: rapeseed oil, sunflower oil, glucose, D-galactose (slow), L-sorbose, D-ribose, ethanol, glycerin, glycerin-containing waste from biodiesel production, erythritol, adonite, D-mannitol, sorbitol, milk, amber , citric, gluconic acid. Does not assimilate: sucrose, maltose, lactose, cellobiose, trehalose, melibiose, raffinose, melicytosis, inulin, starch, D-xylose, L- and D-arabinose, ramlose, dulcite, inositol, D-glucosamine, 2-glucuronoglucuronium 5-ketogluconic acid. Does not assimilate nitrates. Does not grow in a vitamin-free environment, requires thiamine, does not require biotin. It grows in an environment with 10-14% isocitric acid. The optimum pH value for growth is 4.0-6.0, for the synthesis of isocitric acid, 5.5-6.0. The optimum temperature is 29 ° C. The maximum growth temperature is 35 ° C. The strain does not grow at 37 ° C.

The Yarrowia lipolytica strain VKM Y-3044D is cultivated under aerobic conditions on a nutrient medium containing sunflower oil, a source of nitrogen, phosphorus, mineral salts and thiamine as a source of carbon and energy. The cultivation is carried out in fermenters. Sunflower oil is added discretely during cultivation as it is consumed from the environment. To increase efficiency, iron ions are additionally introduced into the culture medium - 1.2 mg / l (activator of aconitate hydratase) and itaconic acid (inhibitor of isocitrate lyase). When growing yeast without itaconic acid at a temperature of 29 ° C and pH 6.0, the yield of (2R, 3S) -isolymonic acid is 95.7 g / l, i.e. 85.8% of the substrate, and with itaconic acid - 109.2 g / l, i.e. 99.8% of the substrate (see table 1).

The possibility of using the invention is illustrated by examples, which do not limit the scope and essence of the claims associated with them.

Example 1. Storing the strain

The strain is stored on an agar mineral Reeder medium of the following composition (g / l): paraffin - 20; (NH ₄ ) ₂ SO ₄ - 3.0; MgSO ₄ ⋅7H ₂ O 0.7; Ca (NO ₃ ) ₂ - 0.4; NaCl - 0.5; KH ₂ PO ₄ 1.0; K ₂ HPO ₄ - 0.1; agar-agar -25.0; yeast autolysate - 2 ml; Burkholder mixture - the composition of the mixture of trace elements according to the Burkholder recipe, mg / ml: KJ - 0.1; B ³⁺ - 0.01; Mn ²⁺ - 0.01; Zn ²⁺ - 0.01; Cu ²⁺ - 0.01; Mo ²⁺ - 0.01; Fe ²⁺ - 0.05; distilled water - up to 1 liter.

Example 2. Preparation and incubation of inoculum Yeast is grown on shoals of agar medium for three days at 28-30 ° C.

Cells from two schools are suspended in 5 ml of sterile distilled medium and transferred into a 750 ml flask containing 100 ml of medium of the following composition (g / l): sunflower oil - 20.0; (NH ₄ ) ₂ SO ₄ - 3.0; MgSO ₄ ⋅7H ₂ O - 0.7; Ca (NO ₃ ) ₂ - 0.4; NaCl - 0.5; KH ₂ PO ₄ 1.0; K ₂ HPO ₄ - 0.1; trace elements (double Burkholder mixture); thiamine - 500 mcg, distilled water.

Incubation of inoculum is carried out on a shaker (220 rpm) for 20-24 hours at 28-30 ° C. The grown cells (at the rate of 100 mg / L by dry weight) are transferred to another batch of flasks with the medium of the same composition as for the inoculum and incubated for 48 h at 28-30 ° C. At 24 hours and 48 from the beginning of cultivation, the pH of the culture medium is measured and the medium is titrated with a sterile solution of 10% KOH to a pH of 4.5-6.0. At the end of cultivation, the grown culture (5-6 flasks) is poured into a flask with a shoot and used for inoculation; dry weight of seed - 3-4 g / l (for a fermenter with a working volume of 6 l).

Example 3. Cultivation of the Yarrowia lipolytica strain VKM Y-3044D with sunflower oil.

The fermentation medium has the following composition (g / l): (NH ₄ ) ₂ SO ₄ - 3.0; MgSO ₄ ⋅7H ₂ O 1.4; Ca (NO ₃ ) ₂ - 0.8; NaCl - 0.5; KH ₂ PO ₄ 2.0; K ₂ HPO ₄ 0.2; yeast autolysate - 6 ml; doubled Burkholder mix; Fe ²⁺ - 1.2 mg / l; distilled water.

Before sowing, thiamine is introduced into the fermenter - 0.5 mg / l. Sunflower oil is added discretely - 20 g / l at the beginning and then at the moment when the concentration of dissolved oxygen increases by 10% in comparison with a stable level of oxygen saturation. This increase in dissolved oxygen concentration indicates complete consumption of the previously present substrate by the yeast.

The cultivation is carried out at 29 ° C, the concentration of dissolved oxygen is 55-60% of saturation, pH 6 (maintained by adding a 20% KOH solution). During the fermentation, 95.7 g / L of (2R, 3S) -isolymonic acid is accumulated for 144 h, which is 85.8% of the used substrate.

Example 4. Cultivation of the Yarrowia lipolytica strain VKM Y-3044D with the addition of itaconic acid.

Growing yeast on an agar medium, preparing inoculum, preparing the medium for fermentation, the period of sunflower oil supply, as well as setting the parameters of temperature, dissolved oxygen concentration and pH of the medium, are carried out as in examples 2-3. During the period of 20-24 hours of cultivation of the Yarrowia lipolytica strain VKM Y-3044D, itaconic acid is introduced into the fermentation medium in the form of sodium salt in an amount of 3.0 to 4.5 g / l, preferably 3.9 g / l. Itaconic acid is an inhibitor of isocitrate lyase, a key enzyme in the metabolism of isocitric acid.

During the fermentation, 109.2 g / l of (2R, 3S) -isolymonic acid is accumulated for 144 h, which is 99.8% of the used substrate.

The results of testing the methods are presented in table 1.

Industrial applicability

The present invention describes a new method for producing (2R, 3S) -isolytic acid, in which in order to increase the yield of the target product from sunflower oil by increasing the degree of substrate utilization, a new Yarrowia lipolytica strain VKM Y-3044D is used as a producing yeast. At the same time, itaconic acid is introduced into the fermentation medium during cultivation from 20-24 hours. The use of a new strain Yarrowia lipolytica in an improved cultivation method allows achieving a synergistic effect from the use of a new strain and an improved method of cultivating highly active and non-toxic (2R, 3S) -isolymonic acid for the food industry and medicine. This makes it possible to use it as a component of hepatoprotective, actoprotective and antihypoxic agents used in sports nutrition and in biologically active food supplements. The proposed method allows you to obtain the yield of (2R, 3S) -isolymonic acid when cultivated on a medium with sunflower oil 1.22-1.42 times higher than the previously known prototype method on sunflower oil.

Information sources

1. Illarionova V.I. and other Method of obtaining isocitric acid. Copyright certificate No. SU 849780 (1983).

2. Otto Ch. et al. Overproduction and secretion of α-ketoglutaric acid by microorganisms // Appl Microbiol Biotechnol 92, pp. 689-695 (2011).

3. Kamzolova S.V. et al. Fermentation conditions and media optimization for isocitric acid production from ethanol by Yarrowia lipolytica // Biomed Research International, Feb 7; 2018: 2543210 (2018).

4. Morgunov I.G. et al. Biosynthesis of isocitric acid in repeated-batch culture and testing of its stress-protective activity // Appl Microbiol Biotechnol 103 (8), pp. 3549-3558 (2019).

5. Rzechonek D.A. et al. Aseptic production of citric and isocitric acid from crude glycerol by genetically modified Yarrowia lipolytica // Bioresour Technol 271: pp. 340-344 (2019).

6. Kamzolova S.V. et al. Isocitric acid production from rapeseed oil by Yarrowia lipolytica yeast // Appl Microbiol Biotechnol, V. 97, pp. 9133-9144 (2013).

7. Kamzolova S.V. et al. Biosynthesis of isolic acid by yeast Yarrowia lipolytica and its regulation // Applied biochemistry and microbiology, vol. 51, no. 2, p. 251-257 (2015).

8. Kamzolova S.V. et al. Microbiological production of citric and isocitric acid from sunflower oil // Food Technol. Biotechnol. V. 46, no. 1, pp. 51-59 (2008).

9. Aurich A. et al. Microbiologically produced carboxylic acids used as building blocks in organic synthesis. In: Wang X., Chen J., Quinn P. (eds) Reprogramming Microbial Metabolic Pathways // Subcellular Biochemistry, vol 64. Springer, Dordrecht, pp. 391-423 (2012).

Claims (3)

1. A method of obtaining (2R, 3S) -isolytic acid, including the cultivation of a yeast strain Yarrowia lipolytica in a nutrient medium containing sunflower oil, a source of nitrogen, salt, trace elements and a vitamin - thiamine, as a source of carbon and energy, under aeration and stirring to maximum accumulation of the target product in the medium, characterized in that the Yarrowia lipolytica VKM Y-3044D strain is used as a producer strain of (2R, 3S) -isolytic acid, where during the cultivation period from 20-24 hours itaconic acid is added to the fermentation medium the form of sodium salt in an amount of 3.0 to 4.5 g / l, preferably 3.9 g / l. 2. The method according to claim 1, characterized in that Fe ²⁺ ions are additionally introduced into the fermentation medium in an amount of 1.2 mg / l. 3. Strain Yarrowia lipolytica VKM Y-3044D (All-Russian collection of microorganisms of the Federal State Budgetary Institution of Science "Federal Research Center" Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences "(FRC PSCBI RAS)) - producer of (2R, 2S) -isolymonic acid.