JPS6257314B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing coenzyme Q ₁₀ by microorganisms. More specifically, the present invention relates to carotenoid synthesis, which is obtained by further mutating a mutant strain lacking or deteriorating carotenoid synthesis ability obtained by mutating a bacterium belonging to the genus Pseudomonas that has the ability to produce carotenoids together with coenzyme _Q10 . This invention relates to a method for producing coenzyme Q ₁₀ by culturing using a revertant mutant.

It is already known that coenzyme Q ₁₀ has remarkable pharmacological effects on various diseases, and it is desired to develop a method for industrially advantageously producing this substance. The present inventors previously revealed for the first time that strains of bacteria belonging to the Pseudomonas family lacking or deficient in carotenoid synthesis ability have excellent coenzyme Q ₁₀ production ability ["Agricultural Biological
Chemistry, Vol. 43, pp. 797-801 (1979)]. As a result of further intensive studies, the present inventors discovered that some mutant strains with carotenoid synthesis defective or degraded carotenoid synthesis ability reversion were found to have significantly enhanced coenzyme Q ₁₀ production ability. .

In the first place, coenzyme Q ₁₀ is biosynthesized through mevalonic acid and isopentenyl pyrophosphate, after isoprenyl pyrophosphate is condensed with p-hydroxybenzoic acid.
It is synthesized through further biochemical changes. On the other hand, carotenoids and triterpenoids are similarly biosynthesized using isoprenyl pyrophosphate as a precursor. In order to effectively utilize such a common precursor for the synthesis of the target substance, coenzyme Q ₁₀ , it is desirable to suppress the production of by-products such as carotenoids. In carotenoid-producing microorganisms, the carotenoid synthesis ability of individual cells can be determined very easily by macroscopically observing colonies of individual cells grown on an agar medium. Based on the above principles, the present inventors mutated Pseudomonas N842 (Feikoken Bacteria No. 3154) and selected Pseudomonas M157 as a colony with a decreased degree of coloration that appeared on the agar medium, that is, a mutant strain with impaired carotenoid synthesis ability. did.

Many of the thus obtained mutant strains lacking or deteriorating the carotenoid synthesis ability were found to have double the coenzyme Q ₁₀ production ability, but among the mutant strains that were further mutated to restore the carotenoid synthesis ability. In addition to those that repair the carotenoid biosynthesis system that has already been damaged, we can find cases where the mevalonic acid and isoprenyl pyrophosphate biosynthesis systems are significantly activated.

In order to improve the coenzyme Q productivity of a coenzyme Q-producing microorganism that has carotenoid production ability by mutation, a mutant strain lacking or deteriorating the ability to synthesize carotenoids is induced, and a mutant strain with reversion to the ability to synthesize carotenoid is further induced. This allows highly efficient coenzyme Q-producing mutant strains to be collected. The above principle improves the coenzyme Q ₁₀ production capacity of various microorganisms that have the ability to biosynthesize carotenoids together with coenzyme Q, such as Pseudomonas, Rhodoseudomonas, Rhodospirillum, and Sporobolomyces. It can be applied as an extremely effective means for this purpose.

Based on this discovery, the present invention induces mutants with reversion to carotenoid synthesis ability from mutant strains of coenzyme Q ₁₀ -producing strains belonging to the genus Pseudomonas that are defective or have degraded carotenoid synthesis ability, and improves the growth of these mutant strains. This is a method for producing coenzyme Q ₁₀ using a microorganism, which is characterized by culturing in a medium containing necessary nutrients and collecting coenzyme Q ₁₀ from the culture.

Such revertant strains are exposed to ultraviolet rays, X-rays,
It is induced by applying radioactivity such as radiation or treatment with chemicals such as sodium nitrite, nitrogen mustard, nitrosoguanidine, and ethylmethylsulfonate. An example of the mutation method for Pseudomonas M157 used in the present invention will be described.

Pseudomonas M157 (Microtechnical Research Institute No. 3153)
is cultured in a medium (PH 6.5) containing 2% glucose, 1% peptone and 1% yeast extract, and then the cells are collected by centrifugation in the logarithmic growth phase. The bacterial cells thus obtained were dissolved in a buffer solution (KH ₂ PO ₄
75ppm, K ₂ HPO ₄ 61ppm, MgSO ₄・
_7H2O20ppm , _CaCl23ppm , _FeCl30.24ppm ,
MnCl ₂・4H ₂ O 0.3ppm, Na ₂ MoO ₄・2H ₂ O
After washing several times with 0.1 ppm and NH ₄ Cl (67 ppm, pH 6.8), 10 ⁸ to 10 ⁹ cells were suspended in 1 ml of the same buffer, and N-methyl-N′-nitro-N-nitrosoguanidine was added to the suspension. Add 2 ml of the same buffer containing 6 mg,
and hold at 30°C for 20-60 minutes. After treatment, wash several times with the same buffer, dilute appropriately, and add glucose 2
%, peptone 1%, yeast extract 1% and agar
1.5% (PH6.5) and cultured at 30° C. for about one week. Among the colonies generated after culturing, the original strain (Pseudomonas
A revertant strain (Pseudomonas M157-B3) that is deficient in carotenoid synthesis ability or a degraded mutant strain can be obtained by picking up and purifying colonies that are clearly more colored than Pseudomonas M157).

This Pseudomonas M157-B3
The mycological properties of No. 5706) are as follows.

Morphological findings: 0.8 to 1.0 x 1.5 to 2.0μ Gram-negative bacilli, motile, 1 polar flagellum Growth on broth, broth agar, and potato dextrose agar Weak glucose 2%, peptone 1%, yeast extract 1 Colonies are pink and round, the periphery is all round, and the ridge is smooth and convex in the center Physiological properties Growth pH 5.3-7.5 (optimum pH 6.5) Growth temperature 25-37℃ (optimum (Temperature 30℃) Oxygen requirement Aerobic gelatin liquefaction ability None Starch liquefaction ability None Catalase Positive cytochrome oxidase Positive nitrate reduction Positive litmus milk Alkalinization methyl red test Negative VP test Positive Indole No hydrogen sulfide formation No ammonia formation A pigment that does not produce acid or gas from glucose under both aerobic and anaerobic conditions Ammonium sulfate, ammonium chloride, and sodium nitrate are used as water-insoluble pigment-producing nitrogen sources Utilization of carbon sources Used: glucose, galactose ,
Xylose, glycerin, succinic acid, malic acid, fumaric acid, gluconic acid, citric acid, methanol, formic acid, alanine, asparagine, aspartic acid, glutamine, glutamic acid Unused: fructose, maltose, lactose, mannitol, sorbitol, mannose, raffinose , trehalose, rhamnose, inulin, inositol,
Dextrin, starch, tartaric acid, glycolate, acetic acid, propionic acid, serine, glycine, formaldehyde, benzene, p-hydroxybenzoic acid In the present invention, a mutant strain deficient in carotenoid synthesis ability or a strain with impaired carotenoid synthesis ability from Pseudomonas N842 is used. Pseudomonas M157
The Pseudomonas M157-B3 strain was obtained by further mutating Pseudomonas N842 (Feikoken Bacterial Serial No. 3153), but the mycological properties of Pseudomonas N842 are that the carotenoid production ability is 1.3 times higher than that of Pseudomonas M157-B3. The mycological properties of Pseudomonas M157 are also almost the same as the other two, except that its carotenoid production ability is lower than that of Pseudomonas N842.

Carbon sources for the mutants thus obtained include, for example, glucose, starch hydrolysates, glycerin, methanol, ethanol, etc., and nitrogen sources include, for example, ammonium salts,
Nitrates, cornstarch liquor, peptone,
Inorganic or organic nitrogen-containing substances such as meat extract, yeast extract, etc. are used. In addition, calcium salts,
Inorganic salts such as magnesium salts, phosphates, potassium salts, and sulfates, vitamins, and other growth-promoting factors may be added. The above-mentioned revertant mutant of the carotenoid synthesis defective or degraded mutant strain was inoculated into such a medium, and the culture was incubated under aerobic conditions at a pH of 5 to 8.
Culture at 40°C for 2-10 days. Bacterial cells are separated from the culture solution by centrifugation or filtration using conventional methods.
The cells obtained here contain abundant coenzyme Q ₁₀ , so after appropriately treating the cells, coenzyme Q ₁₀ can be used together with the cells in nutrients, medicines, feeds, etc. You can also do it. In addition, the starting material for the isolation of coenzyme Q ₁₀ is a live bacterial cell, a dried bacterial cell,
Any form of the bacterial cell treatment product can be used.

An example of a method for isolating coenzyme _Q10 is to add a mixture of methanol, caustic soda and pyrogallol to a solution containing bacterial cells, and to
Heat extraction under reflux for ~2 hours. The extract is then extracted with a solvent such as n-hexane, the solvent layer is washed with water, dehydrated, concentrated, and developed on a silica gel column with benzene. The obtained coenzyme Q ₁₀ fraction is purified with Diaion HP20AG and crystallized from ethanol to obtain pure crystals of coenzyme Q ₁₀ .

The present invention will be described below with reference to Examples.

Example 1 In a medium (PH6.5) containing 2% glucose, 1% peptone, and 1% yeast extract, Pseudomonas M157-B3, a mutant strain with reversion to carotenoid synthesis ability derived from Pseudomonas M157, a mutant strain with impaired carotenoid synthesis ability, was added. A culture solution prepared by pre-cultivating Koken Bacteria No. 5706) in 300 ml of the same medium for 48 hours is inoculated as a seed. The cells were cultured with aeration at 30° C. for 48 hours using a jar fermenter, and a wet bacterial cell paste was obtained by centrifugation. This product weighed 70g (as dry bacterial cells) and contained 6.8mg of coenzyme _Q10 per 1g of dry matter. Mix with 300g of sodium oxide,
The mixture was then heated to reflux at 85°C for 1 hour. After cooling, the mixture was extracted twice with n-hexane (2), and the n-hexane layer was taken, washed with water, dried over Glauber's salt, and concentrated to dryness. The acetone-soluble portion of the dried solid product is taken, the acetone is distilled off, and the residue is dissolved in n-hexane.

This solution is developed on a silica gel column with n-hexane/ether and a fraction containing coenzyme Q ₁₀ is collected. The solvent is distilled off from this eluate, and the residue is dissolved in a small amount of acetone. This is developed with acetone/methanol using Diaion HP20AG and a fraction containing coenzyme Q ₁₀ is collected. The solvent is distilled off from this eluate, and the residue is dissolved in a small amount of ethanol and left in a cool place to produce coenzyme Q ₁₀ .
crystals are obtained. Further recrystallization from ethanol was repeated to obtain 210 mg of coenzyme Q ₁₀ crystals.

N-propanol/water (4:
1) gives a single spot in reversed-phase paper chromatography using acetone/water (95:5) as a developing solvent and reversed-phase thin layer chromatography using acetone/water (95:5) as a developing solvent, and also provides IR, NMR, and mass spectra. were completely consistent with those of the Coenzyme Q ₁₀ standard.

Comparative Example Pseudomonas M157 was cultured in the same manner to obtain a bacterial cell paste (72 g as dry bacterial cells) containing 2.3 mg of coenzyme Q ₁₀ per 1 g of dry bacterial cells. This bacterial cell paste was treated by the method described in Example 1 to obtain 110 mg of coenzyme Q ₁₀ crystals.

Claims (1)

[Claims] 1. A carotenoid synthesis-deficient mutant strain or a carotenoid-synthesis-reverting mutant strain of a mutant strain with impaired carotenoid synthesis ability that belongs to the genus Pseudomonas and has the ability to produce coenzyme Q ₁₀ is cultured in a medium, and coenzyme Q ₁₀ is collected from the culture. A method for producing coenzyme Q ₁₀ , characterized by: