JPH104993A - Production of phosphoric acid derivative of glycerols - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject derivative useful as a synthetic intermediate for phospholipids and pharmaceuticals in high productivity by subjecting a specific yeast to osmotic pressure treatment and reacting with an inorganic phosphoric acid and a glycerol compound. SOLUTION: A yeast containing glycerol kinase and belonging to the genus Candida or Pichia is subjected to osmotic pressure treatment to impart the cell membrane with permeability to organic phosphoric acid compounds. The yeast is made to react with an inorganic phosphoric acid and a glycerol compound. The phosphoric acid derivative of glycerol compound synthesized in the yeast cell is secreted from the cell and accumulated in the reaction liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a phosphoric acid derivative of glycerol useful as a synthetic intermediate for phospholipids and pharmaceuticals.

[0002]

2. Description of the Related Art Glycerols, that is, derivatives such as glycerol, dihydroxyacetone, propanediol and α-chlorohydrin are one of important compounds widely used as intermediates of various medicinal and agricultural chemicals. Derivatives of glycerols are important as pharmaceutical and agricultural chemical intermediates. Glycerol has three hydroxyl groups in its molecule, and the hydroxyl groups at both ends are equivalent in organic synthesis reactivity, so it is complicated to synthesize optically active glycerol derivatives in organic synthesis. A proper synthesis route is required. On the other hand, it is known that a synthesis method using an enzyme can relatively easily synthesize an optically active glycerol derivative. For example, in the production of derivatives of optically active glycerols, JP
For example, JP-A-132196 is known. However, sn-glycerol-3 which is the basic skeleton of phospholipids
-As a method for producing an optically active phosphoric acid derivative of glycerol such as phosphoric acid (hereinafter abbreviated as G3P), a method that is industrially satisfactory has not yet been known. That is, as a method for producing a phosphate derivative of glycerol, a method of synthesizing a phosphate derivative of glycerol using alkaline phosphatase (European Patent No. 289416) is known. In this method, glycerol-1-phosphate is used. And G3
It is difficult to obtain a phosphoric acid derivative of glycerol, in which P is produced at the same time and has sufficient optical purity. Also, Whites
ide et al. reported that G3 from Glycerol and ATP (adenosine-5'-triphosphate) in a reaction using glycerokines.
The synthesis of P has been reported (J. Org. Chem. 48, 3130 (19
83)). In this case, although the optical purity is sufficient, the ATP required for the reaction is expensive. Therefore, the use of phosphoenolpyruvate and pyruvate kinase together with the reaction to regenerate ADP produced as a by-product into ATP is possible. Have gone. However, phosphoenolpyruvate, which is necessary for ATP regeneration, is also expensive, and this method is industrially advantageous because the reaction requires two types of purified enzymes to the extent that no contaminating reaction occurs. is not. Furthermore, Nakagiri (Lpn. J. Genet. 62, 415-424
(1982)) et al. Report that a mutant lacking glycerol-3-phosphate dehydrogenase accumulates G3P in cells, but the amount of G3P accumulated in cells is generally small, It is difficult to remove G3P from the cells, and it is still difficult to produce inexpensive G3P industrially by this method.

[0003] Usually, microorganisms such as yeast and bacteria do not have the membrane permeability of organophosphate compounds such as nucleotides. Therefore, production of nucleotides using microorganisms usually involves the cell membrane of microorganisms. Need to be modified so that the organic phosphoric acid compound can permeate. For this purpose, the prior art uses a drying treatment (J. Ferment. Technol., 45, 511 (19).
67), Amino Acid Nucleic Acid, 29, 59 (1974), ibid.
30, 62 (1974)), self-digestion treatment (U.S. Pat.
No. 038), surfactant treatment (Agr. Biol. Chem.,
36, 867 (1972)). Yeast cells treated in this manner are thought to have permeability to various nucleotides including ATP.
Nothing is known about the modification of the permeability of glycerols to phosphate compounds.

[0004]

An enzymatic reaction is advantageous for producing an optically active phosphoric acid derivative of glycerols, but for an industrially inexpensive production, the desired reaction is carried out. It is important that microbial cells with enzymes can be used directly in their synthesis reactions. However, it has been very difficult to produce a large amount of glycerol phosphate derivatives using microbial cells because the phosphate derivatives of glycerols cannot penetrate the cell membrane of the microbial cells and remain in the microbial cells. . As a method for imparting the permeability of the compound to the cell membrane of the microorganism, the drying treatment and the freezing treatment are troublesome and time-consuming, and are not necessarily industrially advantageous. In addition, in the surfactant treatment, the residue of the surfactant in the product may be a problem, and the purification of the product may be costly, and the surfactant or the like may inhibit the target enzyme reaction. There are many problems, such as that the surfactant is not inexpensive.

[0005]

As a result of various studies, the present inventors have found that yeast cells treated with an inexpensive and safe compound by a simple method secrete a phosphate derivative of glycerols outside the cell. We found the thing and arrived at the present invention. That is, the present invention provides a glycerol characterized in that yeast having glycerol kinase is subjected to osmotic pressure treatment and reacted with a reaction solution containing inorganic phosphoric acid, and a phosphate derivative of glycerol is recovered from the reaction solution. The present invention relates to a method for producing a phosphoric acid derivative.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. As the yeast preferably used in the present invention,
Candida guilliermondi
i) TR4580, GAD5, GAD6, GAD58 and Pichia pastoris IFO 1013. The yeast used in the present invention can be obtained by culturing using a well-known medium suitable for each yeast and using a well-known culture method. As the carbon source used for the medium, basically, any one can be used as long as yeast can grow well, but sugar alcohols such as sorbitol, polyols such as glycerol,
Good results are obtained with hydrocarbons such as paraffin or organic acids such as citric acid. In addition, the glycerol-sensitive mutant strain of the present invention is a cell in which the mutation has been induced by ordinary mutation treatment using ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfonic acid, nitrite, or the like. Among them, a microorganism strain whose growth is suppressed in a medium containing glycerol can be selected. Here, the glycerol-sensitive mutant may basically be a mutant having a growth ability lower than that of the parent strain in a medium containing glycerol, but the growth rate of the parent strain in a completely synthetic medium in which 1 g / l of glycerol is present. Mutants showing a growth degree of 50% or less can be suitably used as glycerol-sensitive strains. The yeast cells can be collected from the culture solution by a known method, for example, a method such as centrifugation.

The osmotic pressure is from 9.0 Pa / cm ² to 400.0 Pa / cm ²
Within this range, 24 Pa / cm ² to 80 Pa / cm ² can be more preferably used. The osmotic agent can be used as long as it is basically nontoxic to eukaryotic cells and does not destroy the basic structure of eukaryotic cells. For example, glucose, sorbitol, fructose, saccharides such as sucrose, glycerol, polyols such as polyethylene glycol, and at least one selected from inorganic salts such as NaCl and KCl dissolved in a solvent such as water are preferably used. . When a liquid polyol is used at the treatment temperature, it can be used as it is without dissolving it in a solvent.

[0008] The osmotic pressure of a liquid can be calculated by the following equation.

When the solute is a non-electrolyte, the osmotic pressure π (Pa /
cm ² ) = R × T × C When the solute is an electrolyte, the osmotic pressure π (Pa / cm ² ) = i ×
R = T × C where R = gas constant T = absolute temperature (K) C = molarity i = Fanthoff coefficient = φ × z φ = permeation coefficient z = number of ions ionized by solute For example, at 25 ° C. Since the osmotic pressure of a 1M aqueous sodium chloride solution is osmotic pressure of sodium chloride = 0.9355, osmotic pressure π (Pa / cm ² ) = i × R × T × C
= 0.9355 × 2 × 0.082 × (273 + 25) ×
1.0 × = 45.72 (Pa / cm ² ).

The osmotic treatment can be performed at a temperature higher than the temperature at which cells are damaged by freezing, and in this sense, usually 0 ° C.
As described above, when there is a freezing point drop, the treatment can be performed at 0 ° C. or lower. However, as the temperature decreases,
Long processing times tend to be required, and excessively low temperature processing is not necessarily industrially advantageous. Also, prolonged treatment at high temperatures is disadvantageous because it reduces the activity of the cells. Therefore, it is preferable to perform the osmotic treatment at 0 ° C. to 50 ° C. for 5 minutes or more, but by performing the osmotic treatment at 10 ° C. to 30 ° C. for 15 minutes to 3 hours, it is possible to obtain good osmotic pressure treated cells.

In accordance with the present invention, yeast cells that have been subjected to osmotic pressure treatment are imparted to their cell membranes with permeability to organic phosphate compounds such as phosphate derivatives of glycerols. That is, the organophosphate compounds synthesized in the yeast cells are secreted outside the cells and accumulate in the reaction solution using the yeast. Further, nucleotides such as AMP (adenosine-5'-monophosphate), ADP (adenosine-5'-diphosphate) and ATP added to the reaction solution using the osmotic-treated yeast are obtained from yeast cells. And is effectively used for the synthesis of phosphoric acid derivatives of glycerols,
It is possible to promote the synthesis of glycerol phosphate derivatives. As the phosphoric acid donor used in the production of the phosphoric acid derivative of glycerols of the present invention, inorganic phosphoric acid, polyphosphoric acid such as pyrophosphoric acid and triphosphoric acid and salts thereof can be used. In addition, sugar phosphates such as glucose-1-phosphate and glucose-6-phosphate can also be used.
Polyphosphoric acid is advantageous. In the production of the phosphoric acid derivative of glycerols of the present invention, aeration, that is, aggressive supply of oxygen is not always necessary. Active substances can be produced. This is because ATP, which is regenerated by osmotic eukaryotic cells,
Not only is it regenerated by phosphorylation at the substrate level as seen in the prior art, but it is also regenerated by the oxidative phosphorylation of mitochondria in eukaryotic cells (using the respiratory chain enzyme system). ing. As can be seen from the above, in the present invention, for example, citric acid, succinic acid, malic acid, fatty acids, etc. which are taken in and metabolized in the TCA cycle,
D (nicotinamide adenine dinucleotide (oxidized form))
To NADH (nicotinamide adenine dinucleotide)
(Reduced form)), such as, for example, ethanol, glycerol, and the like, and compounds that can be metabolized by a series of glycolytic enzymes, such as various sugars and metabolic intermediates, are added. It is possible to promote the synthesis of phosphoric acid derivatives of glycerols.

The phosphoric acid derivatives of glycerols in the present invention basically include all phosphoric acid derivatives formed by the reaction of glycerol kinase.

Glycerolkinase,
EC 2.7.1.30, hereinafter abbreviated as GK) is conventionally known as an enzyme that catalyzes the following reaction.

(Reaction) ATP + glycerol → ADP + G3P Glycerol kinase, however, can synthesize corresponding phosphoric acid derivatives using glycerol, dihydroxyacetone and other 25 or more glycerol analogs as reaction substrates. (J. Am. Chem. Soc., (1985) 107, 7008-70).
29). Therefore, according to the present invention, phosphoric acid derivatives of glycerols that can be produced as a result of the reaction of glycerol kinase as a reaction substrate can be produced industrially at low cost.

The phosphoric acid derivative of glycerol accumulated in the reaction solution can be collected by an ordinary method. For example, by adding BaCl ₂ to the reaction solution and then adding an alcohol such as ethanol, the reaction solution can be recovered as a white precipitate.

The details of the present invention are shown in the following Examples, in which the yeast used in the present invention, Candida gilia manday, is used.
(Candida guilliermondii) TR4580, GAD 5, GAD6, GAD
58 is FERM P-14601, FERM P-15590, FERM P, respectively
-15591, FERM P-15592, deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. Pichia pastoris IFO 1013 has been deposited with the Fermentation Research Institute (IFO).

[0017]

Hereinafter, the present invention will be described with reference to Examples.
The present invention is not limited to this.

(Isolation of Glycerol-Sensitive Mutants) Candida guilliermondii TR
4580) FERM P-14601 with glucose 10 g / l, NH ₄ Cl 4 g / l,
K ₂ HPO ₄ 1 g / l, KH ₂ PO ₄ 0.5 g / l, yeast extract 2 g / l, MgSO ₄
Using 5 ml of a culture solution containing 0.5 g / l of 7H ₂ O (pH 6.5), the cells were cultured with shaking at 28 ° C. for 24 hours, and the obtained cells were collected by centrifugation (2000 rpm, 10 min). The recovered cells were treated with N-methyl-N′-nitro-N-nitrosoguanidine (300 μg / ml, 20 minutes at 25 ° C.) in a conventional manner, and then treated at 28 ° C. with 5 ml of the same medium as described above.
The cells were cultured with shaking for hours. After washing obtained cells aseptically with _{saline, KH 2 PO 4 0.8g / l} , K 2 HPO 4 0.8g / l, MgSO 4 · 7H 2
The mixture was shaken at 28 ° C. for 16 hours using 5 ml of a phosphate buffer (pH 5.0) consisting of 0.5 g / l of O, 0.1 g / l of CaCl _{2 and} 0.1 g / l of NaCl. 0.5 ml of the cell suspension was added to 5 g / l of NH ₄ SO ₄ and KH ₂ P
_{O 4 1g / l, MgSO 4} · 7H 2 O 0.5g / l, CaCl 2 · 2H 2 O 0.1g / l, NaCl
0.1g / l, metal mixture 1ml / l, vitamin mixture 200μl / l
5% of glycerol containing 10 g / l of glycerol as a sole carbon source was inoculated into 5 ml of a complete synthetic medium prepared by adding glycerol to a complete synthetic basal medium for yeast consisting of (pH 5.0), and cultured with shaking for 4 hours. And further continued the culture for 1 hour. The obtained culture solution was applied to an ethanol agar medium prepared by adding agar to a complete synthetic basal medium for yeast to which 10 g / l of ethanol had been added so as to form about 200 colonies per plate. 28
After culturing at 3 ° C. for 3 days, glycerol 10 g / l
A replica was added to a glycerol agar medium prepared by adding agar to a yeast complete synthetic basal medium supplemented with, and the above ethanol agar medium, and cultured at 28 ° C for 3 days. After cultivation, a colony that cannot grow on a glycerol agar medium and grows well on an ethanol agar medium was selected from the ethanol agar medium, and glycerol-sensitive strains Candida guilliermondii GAD5, GAD6, and GAD583 were selected. Got the strain. These microorganisms are, respectively, FERM P-15590, FERM P-15591, and FERM P-155
Deposited as 15592.

The metal mixture used in the complete synthetic basal medium for yeast was 500 mg / l H ₃ BO ₃ , 40 mg / CuSO ₄ .5H ₂ O / 40 mg / l.
_{l, MnCl 2 · 4H 2 0} 400mg / l, FeSO 4 · 7H 2 0 400mg / l, ZnSO 4 · 7H 2 O
It is an aqueous solution containing 400mg / l and KI 100mg / l. The vitamin mixture is folic acid 1g / l, p-aminobenzoic acid
1 g / l, riboflavin 1 g / l, nicotinic acid 1 g / l, pyrodoxin / hydrochloride 1 g / l, thiamine / hydrochloride 1 g / l, d-pantothenic acid 1 g / l, d-biotin 0.1 g / l is there.

(Assay for Glycerol Sensitivity) The microorganisms shown in Table 1 were subjected to glucose 10 g / l, NH ₄ Cl 4 g / l, K ₂ HPO ₄ 1 g /
_{l, KH 2 PO 4 0.5g /} l, yeast extract _{2g / l, MgSO 4 · 7H} 2 O 0.5g
/ l (pH 6.5) with 5 ml of a culture solution with shaking at 28 ° C for 24 hours, and centrifuging the obtained cells (2000 rpm, 10 min).
And the cells were washed twice by centrifugation (2000 rpm, 10 min) using 5 ml of physiological saline. As shown in Table 1, 100 μl of the washed cells were used as a glycerol sensitivity evaluation medium supplemented with 1 g / l glycerol (a complete synthetic basal medium for yeast supplemented with 10 g / l sorbitol).
After inoculation, the cells were cultured with shaking at 28 ° C. for 17 hours, and the growth of the cells was determined by measuring the turbidity of the cells at 660 nm. Table 1 shows the relative cell turbidity assuming that the cell turbidity in the glycerol sensitivity evaluation medium to which glycerol was not added was 100%. From this Table 1, it can be seen that Candida guilliermondii GAD
5. It can be seen that the GAD6 and GAD58 strains clearly have glycerol sensitivity.

[0021]

[Table 1]

[0022]

Example 1 Candida Gilia Monday
G3P production using guilliermondii) and the effect of osmotic pressure treatment Candida guilliermondii
TR 4580 (FERM P-14601) and Candida guilliermondii GAD58 (FERM P-1559)
2), sorbitol 10 g / l, NH ₄ Cl 4 g / l, K ₂ HPO ₄ 1 g /
l, KH ₂ PO ₄ 0.5 g / l, yeast extract 2 g / l, MgSO ₄・ 7H ₂ O 0.5
The cells were shake-cultured at 28 ° C. for 16 hours in a culture solution containing g / l (pH 6.5). After collecting cells by centrifugation (2000rpm, 10min),
The suspension was suspended in 1 M NaCl and subjected to osmotic treatment for 20 minutes. After the cells were collected by centrifugation again, they were suspended in deionized water to obtain a cell-treated solution having a dry cell weight of 40 mg / ml. When the osmotic treatment was not performed, the same treatment was performed using physiological saline instead of 1M NaCl to obtain a 40 mg / ml bacterial cell treatment solution. The cell concentration was calculated from the previously prepared dry cell concentration and a calibration curve of the absorbance at 550 nm by measuring the absorbance at 550 nm.

ATP 2.5 mM, MgCl ₂ 10 mM, pH 7.4 potassium-phosphate buffer (KPB) 300 mM, glycerol 300 mM
M, NAD 1.2mM, citric acid 100mM, and the resulting yeast osmotic treated product per reaction solution for G3P biosynthesis reaction solution
After adding 8 mg dry cells / ml, the mixture was aerobically reacted at a reaction temperature of 25 ° C. under shaking conditions. 28 hours after the start of the reaction, the G3P concentration in the reaction solution supernatant was measured. The concentration of G3P in the obtained reaction solution was glycerol-3-phosphate dehydrogenase (GP) capable of specifically quantifying only G3P.
DH). Ie 100
0.1 ml of the diluted reaction solution was added to 1 ml of glycine hydrazine buffer (0.5 M glycine-0.4 M hydrazine (pH 9.5)), and NAD + was 0.1 mM in final concentration, GPDH (EC 1.1.1.8).
Rabbit muscle-derived Sigma product) at a final concentration of 1 unit / ml
And reacted at 30 ° C. for 1 hour. After the reaction, the increase in NADH was determined by measuring the absorbance at 340 nm, and quantified from a calibration curve prepared using a standard G3P (a product of Beohringermennheim). The results are shown in Table 2.

[0024]

[Table 2]

As is clear from Table 2, the glycerol-sensitive strain Candida guilli Monday
illiermondii) GAD58 (FERM P-15592) is the parent strain of Candida guilliermondii TR458.
Compared with 0 (FERM P-14601), G3P production ability is clearly superior. In addition, when osmotic pressure treatment is not performed, G3P is hardly generated when either microorganism is used, indicating that the osmotic pressure treatment is effective in generating G3P.

[0026]

Example 2 Pichia pastoris (Pichia pastori)
s) Production of G3P using IFO 1013 Pichia pastoris IFO 1013 was
The cells were shake-cultured at 28 ° C. for 24 hours in a complete synthetic basal medium for yeast supplemented with 10 g / l of glucose, citric acid or paraffin. The cells were collected by centrifugation (2000 rpm, 10 min) and subjected to osmotic pressure treatment and G3P biosynthesis reaction exactly as in Example 3 except that the reaction time was changed to 42 hours. Table 3 shows the results of quantification of G3P in the same manner as in Example 3.

[0027]

[Table 3]

From these results, it was found that glucose was used as a carbon source,
G3P generation was observed when either citric acid or paraffin was used as a carbon source, but the productivity of G3P was higher when citric acid or paraffin was used as a carbon source than when glucose was used as a carbon source. It was excellent.

[0029]

[Example 3] G3P production by yeast cultured in a medium supplemented with polyol Candida guilliermondi
i) GAD58 (FERM P-15592) was added to sorbitol 10 g / l, NH ₄
Cl 4g / l, K ₂ HPO ₄ 1g / l, KH ₂ PO ₄ 0.5g / l, yeast extract 2g
Using a medium consisting of 0.5 g / l of MgSO ₄ .7H ₂ O (pH 6.5) and shaking culture at 28 ° C. for 24 hours, a pre-culture liquid was obtained. The precultured solution thus obtained was inoculated with 5% (v / v) of a complete synthetic basal medium for yeast supplemented with 10 g / l of sorbitol and cultured with shaking at 28 ° C. for 16 hours. It was a cell.

The Candida guilliermondii GAD58 (FERM P-155) obtained in the same manner.
92) The pre-culture solution was inoculated with 5% (v / v) to a complete synthetic basal medium for yeast supplemented with 10 g / l of sorbitol,
After culturing with shaking for 16 hours, glycerol as a polyol was added at a concentration of 2 g / l, followed by shaking culturing at 28 ° C. for 6 hours to obtain glycerol cultured cells. The sorbitol cultured cells and glycerol cultured cells were subjected to osmotic pressure treatment and G3P generation reaction in exactly the same manner as in Example 3 except that the reaction time was changed to 20 hours. After completion of the reaction, the G3P accumulation concentration was measured in the same manner as in Example 3. Table 4 shows the results. From these results, it can be seen that G3P productivity is superior when yeast cells cultured with glycerol added are used.

[Table 4]

[0031]

Example 4 Isolation of G3P formed Candida guilliermondi
i) Add GAD 58 (FERM P-15592) to 10 ml of sorbitol 1
_{0g / l, NH 4 Cl 4g} / l, K 2 HPO 4 1g / l, KH 2 PO 4 0.5g / l, yeast extract 2 g / l, the _{MgSO 4 · 7H 2 O 0.5g /} l (pH.6.5) A precultured solution was obtained by inoculating the medium into a medium containing the medium and shaking the culture at 28 ° C. for 24 hours. This was inoculated into 200 ml of a complete synthetic basal medium for yeast supplemented with 10 g / l of sorbitol, and cultured with shaking at 28 ° C. for 24 hours using a 1 liter Sakaguchi flask. Collect cells by centrifugation (2,000 rpm, 10 min),
The cells were suspended in 5 ml of distilled water to prepare osmotic treated cells in the same manner as in Example 3. The G3P biosynthesis reaction was performed using the reaction composition shown in Example 3 and a reaction scale of 100 ml. 2
After 4 hours, the reaction was terminated, and the reaction solution was centrifuged (15,000 rp).
m, 20 min) to remove bacterial cells to obtain 87.7 ml of a supernatant. The supernatant contained 22.2 mM G3P. From the supernatant, the method of Crans et al. (J. Am. Chem.
Soc. 107, 7019-7027 (1985)), G3P was collected. That is, 0.5 g of activated carbon was added to the supernatant, and the mixture was stirred at room temperature for 1 hour, and then the activated carbon was removed by filtration. The obtained filtrate is concentrated to about 10 ml, and 24 mmol of BaC
l2 was added, and the formed precipitate was removed by filtration to obtain 14.2 ml of a clear filtrate. To this filtrate, 2 mmol of BaCl2 was added and dissolved well, and then 2 volumes of ethanol was added.
After standing at room temperature for 1 hour, the formed precipitate was separated by filtration and sufficiently dried by a vacuum drier to obtain 0.61 g of a barium salt of G3P as a white powder. G3
Assay using P-dehydrogenase revealed 81.5%.
The purification yield was 82.5%.

[0032]

EFFECT OF THE INVENTION By using the yeast having the osmotically treated glycerokinase of the present invention, a phosphate derivative of glycerols useful as a pharmaceutical intermediate can be produced at low cost.

Claims (6)

[Claims] An osmotic treatment of a yeast belonging to the genus Candida or the genus Pichia having glycerol kinase, and reacting it with inorganic phosphoric acid and glycerols. For producing phosphoric acid derivatives of glycerols, comprising recovering phosphoric acid derivatives of glycerol ^2. The method for producing a phosphoric acid derivative of glycerols according to claim 1, wherein the osmotic treatment is performed by an osmotic pressure of 9.0 Pa / cm ² to 400.0 Pa / cm ^2. 3. The method for producing a phosphoric acid derivative of glycerol according to claim 2, wherein an inorganic salt is used for the osmotic treatment. 4. The method for producing a phosphoric acid derivative of glycerol according to claim 1, wherein the phosphoric acid derivative of glycerol is sn-glycerol-3-phosphate. 5. A yeast grown using at least one compound selected from polyols, hydrocarbons, fatty acids and organic acids as a carbon source.
The method for producing a phosphoric acid derivative of glycerols according to any one of the above 6. The method according to claim 1, wherein a yeast sensitive to glycerol is used.
PROCESS FOR PRODUCING PHOSPHATE DERIVATIVES OF GLYCEROLS