RU2097431C1 - Method of biosynthesis of ribonucleotides - Google Patents

Abstract

FIELD: biotechnology, biochemistry. SUBSTANCE: inoculum of the strain Corynebacterium ammoniagenes is grown on sterile medium. Biomass is treated with aprotonic lipid extractant and permeabilized biomass is separated. Part of the latter is suspended and grown on glucose-salt medium at constant stirring up to accumulation of ribose-5'-phosphate in medium in disodium form. Then the obtained product is added to the nutrient medium containing nitrogen base for growing another part of permeabilized biomass and the end product is isolated. EFFECT: improved method of biosynthesis. 2 cl, 4 dwg, 1 tbl

Description

 The invention relates to biotechnology, in particular to methods for the biosynthesis of ribonucleotides from cyclic fragments using microorganisms.

Known methods for the biosynthesis of ribonucleotides from exogenous purines and pyrimidines, in which nitrogenous bases, individually or in combination, are introduced into a late 48- or 72-hour periodic culture of a microorganism growing on a fermentation medium and incubated for accumulation in the medium of ribonucleotides for 48 -72 h at a temperature of 30 ^o C [1]
When carrying out known methods of biosynthesis in a native microbial culture, the concentration of ribonucleodites accumulating in the medium reaches only 5.0-6.0 mM due to the fact that metabolic processes in a normally functioning microbial cell are competitive for common intermediate substrates and cofactors for those reactions that participate in the condensation of exogenous purines and pyrimidines. For this reason, it is not possible to fully reveal the purine and pyrimidine-condensing potential of the used producers.

A known method of microbiological synthesis of nicotinamide adenine nucleotide, NAD ⁺ , consists in the condensation of exogenous adenine and nicotinamide with the help of xylene permeabilized Corynebacterium ammoniagenes cells LIA 0950 on the structure of endogenously formed ribose-5'-phosphate, which involves carrying out cell-mediated and separately [2] [3]
Known strain Corynebacterium species VSTI-301 nucleotide producer (ATP, ADP, AMP, GTP) [4] the closest analogue.

 For the cultivation of biomass Corynebacterim species VSTI-301, a well-known fermentation medium containing, in wt.

 glucose 10, magnesium sulfate, dilute 1, calcium chloride 0.01, mono- and disubstituted potassium phosphate 1, peptone 0.5, biotin 30 μg / l.

 For the growth of the inoculum, a known seed medium is used containing, in wt.

 peptone 1.5, glucose 2, monosubstituted potassium phosphate 0.1, magnesium sulphate 0.03, sodium chloride 0.3, ferrous sulphate 0.01, biotin 30 μg / L.

The duration of the procedure for growing the inoculum at a temperature of 30 ^o C is 24 hours, growing biomass 72 hours

 Fermentation is carried out on a circular shaker for three days and introduced into the flask of 30 mg of adenine and 0.15 cl xylene. Then the fermentation is continued for another day.

The yield of ATP, ADP, AMP is respectively 1.5, 055 and 1.5 mg / ml, GTP
0.42 mg / ml.

 The technical result achieved by the proposed method is to increase the yield of ribonucleotides.

To do this, in the method of biosynthesis of ribonucleotides, including condensation with Corynebacterium ammoniagenes LIA 0950 of nitrogen bases on the structure of ribose-5'-phosphate formed by cells from hexoses, and catalysis of these processes using permeabilized cells, condensation of nitrogenous bases and other fragments of ribonucleos - and triphosphates and their derivatives are carried out on the structure of exogenous ribose-5'-phosphate, carrying out the process by incubation with a suspension of Coryneb cells permeabilized with xylene or other aprotic lipid extractants acterium ammoniagenes LIA 0950 with a density (by dry biomass) of 100-105 mg / ml in a reaction mixture containing, in ribose-5'-phosphate, in disodium form 1.9 2.0, glucose 3.0 3.2, nitrogenous bases, separately or in combination, at 0.80 0.84, monosubstituted potassium phosphate 0.1 0.2, disubstituted potassium phosphate 1.4 1.5, magnesium carbonate 0.6 0.7 (pH 7.8), the rest is distilled water, for 24-30 hours at a temperature of 34 ^o C and constant stirring, until the cells completely consume glucose. At the same time, ribose-5'-phosphate specified in the reaction mixture is obtained by incubating a suspension of Corynebacterium ammoniagenes LIA 0950 cell lipid permeabilized with xylene or other aprotic extractants with a density (dry biomass) of 130 136 mg / ml in glucose-salt medium containing glucose 20.0 21.0, monosubstituted potassium phosphate 0.4 0.5, disubstituted potassium phosphate 2.44 2.6, magnesium carbonate 0.8 0.9 (pH 7.8), the rest is distilled water, for 24 30 h at a temperature of 34 ^o C and constant stirring, until the accumulation in the medium of ribose-5'-phosphate in quantity 25.0 mg / ml, followed by its release in the form of a preparation containing the main substance in disodium form in the amount of 87.0 93.0%. At the same time, the permeabilized cell mass of Corynebacterium ammoniagenes LIA 0950 suspended in the reaction mixture and in glucose-salt medium obtained by growing the cell inoculum in an amount of 10.0 vol. on a sterile growth medium containing, in glucose, molasses or another glucose-containing nutrient ingredient, equivalent to free glucose 5.6 5.9, yeast extract 0.5 0.6, urea 0.4 0.5, mono- and disubstituted salts potassium phosphate, 0.3 0.4 each, magnesium carbonate 0.20 0.3, the rest is distilled water, for 24 30 h at a temperature of 30 ^o C and aeration, for example in a fermenter, until the density of the cell population is reached (by dry biomass ) 12.0 13.0 mg / ml, followed by permeabilization of the cell mass with xylene or other aprotic lipid extractants and and obtaining it in the form of a concentrate with a mass fraction of moisture of 87.0 to 90.0% by mechanical separation from the medium by precipitation or filtration.

According to the results of a specially conducted study of processes catalyzed by xylene-permeabilized Corynebacterium ammoniagenes LIA 0950 cells suspended in glucose-salt medium, it was unexpectedly found that the processes occurring in the cells (in the same cell biomass) are endogenous biogenesis of ribose-5'-phosphate and exogenous the biosynthesis of ribonucleotides has a different physiological orientation (glucose catabolism, in addition to the biogenesis of ribose-5'-phosphate, simultaneously serves as a process of decomposition of hexoses to final products) and of different sizes Nost reaction rate, and for this reason it is incompatible to hold at the same cell biomass. The rate of glucose catabolization to end products, CO ₂ and lactate, which reaches 0.28 mM • mg ^-1 • h ^-1 in cells, exceeds the condensation rate of fragments, for example NAD ⁺ 0.02 mm • mg ^-1 • h ^{-1 by} almost whole order. It has been established that the ecogenic extracellular environment, ribose-5'-phosphate in disodium form, can penetrate inside the cell permeabilized with xylene and other aprotic lipid extractants and thereby be involved in the condensation reaction of ribonucleotide fragments when the cell is fed with a small amount of glucose, fructose and other digest as a source of energy; and also the fact that such cells suspended in a glucose-salt medium of a concentrated composition, but not containing nitrogen bases, are able to convert glucose with significant efficiency into ribose-5'-phosphate and accumulate it in the extracellular medium.

 These facts served as the basis for the separation of the reactions catalyzed by cells in the conversion of hexoses to ribose-5'-phosphate and the condensation of ribonucleotide fragments on its structure into two separately performed technological procedures, which at the same time comprise one general technological scheme of ribonucleotide biosynthesis from exogenous fragments of FIG. one.

 Technological processes combined in block 2 of the reaction of transformation of hexoses into ribose-5'-phosphate, and in block 3 of condensation of exogenous fragments of ribonucleotides (Fig. 1) are separately catalyzed using the same (obtained by the same method) permeabilized cell biomass (PCB) ; a flexible technology for the production of ribonucleotides (block 3) is based on the use of exogenous ribose-5'-phosphate (P5F) of the same type of sugar phosphate fragment, on the structure of which, depending on the needs, nitrogen and other fragments (AF) are condensed; To obtain cell biomass at any scale, a uniform growth medium is used.

 Example 1. Obtaining a catalytic bioagent (biomass of permeabilized cells of Corynebacterium ammoniagenes LIA 0950).

Inoculum freshly grown on growth medium in an amount of 300 ml, containing 12.0 mg cells / ml (by dry biomass), is introduced aseptically into a fermenter with a working volume of 5 l, filled with 3 l of sterile growth medium prepared in distilled water and containing molasses , based on free glucose 5.6; yeast extract 0.5, urea (30% sterile solution is added fractionally) 0.4, salts of mono- and disubstituted potassium phosphate, 0.3 each, magnesium carbonate 0.2 (pH 7.4), establish aeration using mixers with a speed of 900 rpm, a medium temperature of 30 ^o C and grown 24 30 h, controlling the density of the cell population nephelometric at λ = 630 nm.
Upon reaching a population density of 12.0 mg / ml by dry biomass (according to the calibration curve), 30 ml of xylene (a mixture of the o-, m- and p-isomers) is introduced into the culture and incubated to extract lipids from the cell membrane (cell permeabilization) for 0.5 hours with stirring using a mixer with a speed of 200 rpm The permeabilized in this way, the cellular biomass is separated from the medium by centrifugation at a rotor speed of 5000 rpm. Get biomass containing 31 g of dry cells.

 Example 2. Obtaining ribose-5'-phosphate.

Permeabilized with xylene, the cell biomass in the amount of 26.0 g (dry weight) is suspended in 200 ml of glucose-salt medium in a 500 ml flask containing, in distilled water, 20.0 glucose; monosubstituted potassium phosphate 0.4; disubstituted potassium phosphate 2.4; magnesium carbonate 0.8 (pH 7.8). The resulting suspension is incubated on a circular shaker at a temperature of 34 ^o C for 24-30 hours, controlling the accumulation of ribose-containing substances in the medium (colorimetrically using an orcine reagent) in an amount of 25.0 mg / ml.

The isolation of ribose-5'-phosphate from the medium is carried out using ion exchange chromatography on resin AB-17. Received in an amount of 2.0 g of the preparation of ribose-5'-phosphate contains the main substance in disodium form in the amount of 87.0%
Example 3. The biosynthesis of adenosine mono-, di - and triphosphates (purine ribonucleotides).

Permeabilized with xylene, the cell biomass in an amount of 1.0 g (by dry weight) is suspended in 10 ml of the reaction mixture in a 100 ml flask containing, in distilled water, in ribose-5'-phosphate (87.0%, in disodium form, in the base material) 1.9; glucose 3.0; adenine 0.8; monosubstituted potassium phosphate 0.1; disubstituted potassium phosphate - 1.4; magnesium carbonate 0.6 (pH 7.8). The resulting suspension is incubated on a circular shaker at a temperature of 34 ^o C for 24-30 hours, monitoring on the device "Exan-G" the moment of complete consumption of glucose by cells.

 The content of adenine nucleotides accumulated in the medium is: ATP 14.5 mg / ml, ADP 5.6 mg / ml, AMP 2.1 mg / ml.

 Example 4. The biosynthesis of uridine monophosphate (pyrimidine ribonucleotides).

 The biosynthesis process and the method of its implementation are carried out in the same manner as in example 3, only the reaction mixture instead of adenine contained an equimolar amount of uracil.

 The content of UMF accumulated in the medium is 20.2 mg / ml.

 Example 5. The biosynthesis of 6-thioguanine ribonucleotide (purine nucleotide analogues).

 The biosynthesis process and the method of its implementation are carried out in the same manner as in example 3, only the reaction mixture instead of adenine contained an equimolar amount of 6-thioguanine.

 The content of 6-thio GMF accumulated in the medium is 12.3 mg / ml.

 Example 6. The biosynthesis of 5-fluorouracilribonucleotide (pyrimidine nucleotide analogues).

 The biosynthesis process and its method are carried out in the same manner as in example 3, only the reaction mixture instead of adenine contained an equimolar amount of 5-fluorouracil.

 The content of 5-fluoro UMF accumulated in the medium is 14.7 mg / ml.

 Example 7. The biosynthesis of nicotinamide adenine dinucleotide (adenine nucleotide coenzymes).

 The biosynthesis process and the method of its implementation is carried out in the same manner as in example 3, only the reaction mixture in combination with adenine contained an equimolar amount of nicotinamide.

The content of NAD ⁺ coenzyme accumulated in the medium is 19.6 mg / ml.

 Example 8. Permeabilization of cell biomass.

The biosynthesis of NAD ⁺ and its method are carried out in the same manner as in example 7, only the cell suspension, instead of biomass lipids permeabilized with xylene and other aprotic extractants, contained lipid permeabilized with proton extractants, for example, dimethyl sulfoxide, biomass.

The content of NAD ⁺ accumulated in the medium is trace amounts.

The values of the economic growth coefficient of cell biomass Y g / g (the amount of biomass, in grams, relative to the amount of glucose consumed, in grams) and the NAD ⁺ -synthetic activity of xylene-permeabilized biomass (determined as in Example 7), established populations of Corynebacterium ammoniagenes LIA 0950 cells grown on fermentation and claimed growth media for 24, 48, 72 and 96 hours are summarized in the attached table.

 As can be seen from the above results, in the examples of a specific embodiment, the nutrient medium optimal for growing cell biomass and the reactions catalyzed by it was used and the optimal age of the grown cell populations was used.

 In FIG. 2 is shown in the growth medium of the ingredients (the concentration of ingredients in the growth medium used in example 1 is taken as 100%).

 It can be seen from it that the population density (in mg dry biomass per ml of medium) grown over 24 hours on media with a low concentration of ingredients is lower than in the control; on media with a higher concentration of ingredients, its dependence curve goes to a plateau. In order to avoid excessive consumption of ingredients for growing biomass, a growth medium containing 100 105% concentration of ingredients was selected.

потенциала клеточной мембраны, необходимого для энергообеспечения акта экскреции (выбрасывания из клеток наружу) данных фосфороорганических веществ.For permeabilization of biomass, more than 15 proton (with zero dipole moment) lipid extractants were tested. The most effective for excretion from cells of ribose-5'-phosphate and ribonucleotides were aprotic lipid extractants, such as benzene, toluene, o-, m- and p-isomers of xylene, from the surface-active substances cetylpyridinium chloride, providing minimal dispersion of proton

the potential of the cell membrane necessary for the energy supply of the act of excretion (throwing out the cells outward) of these organophosphorus substances.

 In FIG. Figure 3 shows the dependence of the accumulation in the environment of ribose-5'-phosphate on the concentration of the components of the suspensions in the glucose-salt medium (the concentration of the components of the suspension used in Example 2 is taken as 100%).

 As can be seen from these results, for an equal period of time (24 h), incubation in suspensions having a lower concentration of components than in the control (biomass density, concentration of glucose and other ingredients), the content of ribose-5'-phosphate accumulating in the medium is lower than in control; in suspensions having higher component concentrations than in the control, the accumulation curve of ribose-5'-phosphate reaches a plateau.

 In order to avoid overspending of the components of the suspension, such concentration values are selected that are 100 105% of the control.

In FIG. Figure 4 shows the dependence of the accumulation in the NAD ⁺ medium on the concentration of the components of the suspensions in the reaction mixture (the components of the suspensions used in Examples 3–8 are taken as 100%).

As seen in FIG. 4 in suspensions having a lower concentration of components than in the control, the content of NAD ⁺ accumulated in the medium over 24 hours is lower than in the control; in suspensions having higher component concentrations than in the control, the accumulation curve of NAD ⁺ in the medium also goes to a plateau.

 For reasons of the effective use of the components of the suspensions, such concentration ranges are selected that are 100 105% of the control.

 Thus, the claimed invention together allows to fully reveal the hexose-transforming and, based on it, nucleotide synthesizing potentials of Corynebacterium ammoniaqenes LIA 0950 (VSTI 404), to obtain ribose-5'-phosphate in a preparative amount and to conduct on its basis preparative synthesis of almost the entire purine-nomenclature and pyrimidinribonucleoside mono-, di and triphosphates and their derivatives of natural and synthetic origin. The invention allows to accumulate in the reaction mixture ribonucleotides in an amount of 28 30 mm.

Information sources:
1. Tsyrenov V.Zh. Microbiological synthesis of nucleoside phosphates. M. Science, 1990, p. 200.

 2. Gonchikov G. G. Lenkhoboev V. D. Abrosimova E.V. Barkhutova D.D. Characterization of NAD synthesizing activity of producer Corynebacterium ammoniagenes VSTI 404. Abstracts of conferences "Problems of the microbial synthesis of vitamins and their derivatives" Tashkent, 1990, p. 12.

 3. US patent N 3705080, class. C 12 D 13/06, 1968.

 4. SU, copyright certificate, N 726161, cl. C 12 P 19/30, 1980.

Claims (2)

 1. The method of biosynthesis of ribonucleotides, involving the cultivation of seed material of corynebacteria, introducing the obtained inoculum into a fermentation medium containing sources of carbon, nitrogen, phosphorus, mineral salts and cultivation under aeration conditions, characterized in that Corynebacterium ammoniagenes strain LIA 0950, whose inoculum is used contribute to the fermentation medium containing the following components, wt. Glucose or molasses in an amount equivalent to free glucose 5.6 5.9
Yeast extract 0.5 0.6
Urea 0.4 0.5
Salts of mono- and disubstituted potassium phosphate 0.3 0.4 each
Magnesium Carbonate 0.2 0.3
Distilled Water Else
and cultivation is carried out until the density of the cell population in the dry biomass is 12.0 13.0 mg / ml, then the biomass is treated with an aprotic lipid extractant and the permeabilized biomass with a mass fraction of moisture of 87.0 90.0% is separated, then part of the permeabilized biomass with density in the dry biomass 130 136 mg / ml are suspended and grown in glucose-salt medium containing the following components, wt. Glucose 20.0 21.0
Monosubstituted potassium phosphate 0.4 0.5
Disubstituted potassium phosphate 2.4 2.6
Magnesium Carbonate 0.8 0.9 (pH 7.8)
Distilled Water Else
at a temperature of 34 ^o With constant stirring until the accumulation in the medium of ribose-5'-phosphate in disodium form in an amount of 25.0 mg / ml of medium, then the resulting product is introduced into a nutrient medium for growing another part of the permeabilized biomass carried out under the same conditions on a nutrient medium of the following composition, wt. Disodium ribose-5'-phosphate 1.9 2.0
Glucose 3.0 3.2
Nitrogen base, alone or in combination .80 0.80
Monosubstituted potassium phosphate 0.1 0.2
Disubstituted potassium phosphate 1.4 1.5
Magnesium Carbonate 0.6 0.7
until complete consumption of glucose, then the target product is isolated.  2. The method according to claim 1, characterized in that benzene, xylene, toluene, cetylpyridinium chloride are used as the aprotic lipid extractant.

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