JP3026190B2 - 5-Aminolevulinic acid-producing microorganism and method for producing 5-aminolevulinic acid using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a microorganism capable of producing and accumulating 5-aminolevulinic acid at a high concentration, and a method for producing 5-aminolevulinic acid using the microorganism.

[0002]

BACKGROUND OF THE INVENTION 5-Aminolevulinic acid is a compound that exists widely in the biosphere as a precursor of a tetrapyrrole compound and plays an important role in living organisms. 5-aminolevulinic acid is a herbicide,
Insects, plant growth regulators, show excellent action as a plant photosynthesis enhancer, etc., and also show no toxicity to humans,
It is a natural compound having excellent properties such as high decomposability and no persistence in the environment (JP-A-61-50281).
No. 4, JP-A-2-138201, etc.).

[0003] However, 5-aminolevulinic acid has a problem that its production cost is high and it is not practical for use in the above applications (CHEMICAL WEEK / OCTOBER, 29, 198).
Four).

For this reason, many chemical synthesis methods have been studied (for example, see Japanese Patent Application Laid-Open No. 2-76841).
No satisfactory method has yet been developed.

On the other hand, Rhodobacterium
m) genus, Propionibacterium
5 using microorganisms of the genus Methanobacterium, Methanosarcina, etc.
Methods for producing aminolevulinic acid are also being studied. However, methods using Propionibacterium, Methanobacterium, Methanosarcina and the like (see Japanese Patent Application Laid-Open No. 5-184376) have a very low production amount and are not satisfactory.

Microorganisms of red non-sulfur bacteria such as the genus Rhodobacterium and the genus Rhodopseudomonas are known to have a high ability to synthesize tetrapyrrole compounds using 5-aminolevulinic acid as a metabolic intermediate. However, using these microorganisms, the 5
-Aminolevulinic acid is metabolized to a tetrapyrrole compound, and there is a problem that a desired amount of 5-aminolevulinic acid is not accumulated.

[0007] Since the biosynthesis of 5-aminolevulinic acid is complicatedly controlled in vivo as described above, it is not easy to produce a strain that accumulates 5-aminolevulinic acid at a high concentration.

On the other hand, when glucose is used as a carbon source, 5
A method using a Rhodobacterium mutant capable of accumulating up to 14.3 mM of aminolevulinic acid has been proposed (JP-A-8-168391). However, this method needs to keep the dissolved oxygen concentration during culture at a low concentration, needs to mix nitrogen gas with air and ventilate the culture tank, and cannot be said to be an industrially advantageous method. Was something. In this method, oxygen is required when 5-aminolevulinic acid is accumulated aerobically using glucose as a carbon source. Therefore, it is conceivable that even when oxygen is sufficiently present, the microorganisms consume the dissolved oxygen, thereby lowering the dissolved oxygen concentration and efficiently producing 5-aminolevulinic acid. However, it has been impossible to efficiently produce 5-aminolevulinic acid using this microorganism under relatively high dissolved oxygen conditions.

Accordingly, an object of the present invention is to provide a microorganism capable of efficiently producing 5-aminolevulinic acid without introducing nitrogen gas or the like even under conditions where the dissolved oxygen concentration is relatively high, and a method for producing 5-aminolevulinic acid using the microorganism. It is to provide a manufacturing method.

[0010]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies in order to obtain a microorganism that efficiently produces 5-aminolevulinic acid. A method for selecting an extremely efficient 5-aminolevulinic acid-producing microorganism which can be varied to evaluate a large number of mutant strains has been found. By this method, 5-aminolevulinic acid can be produced at a high concentration even when the dissolved oxygen concentration is relatively high. The present inventors have found a microorganism that accumulates in the present invention and completed the present invention. Furthermore, the present inventors improve the production amount of 5-aminolevulinic acid by appropriately adding saccharides, glycine, levulinic acid, iron, and the like to the medium, and controlling the dissolved oxygen and the oxidation-reduction potential in the culture solution. It has been found that the present invention has been completed and the present invention has been completed.

[0011] That is, the present invention relates to a 5-aminolevulinic acid-producing microorganism belonging to Rhodobacter sphaeroides or a mutant strain thereof, wherein the microorganism has a dissolved oxygen concentration of 1.46 to 5.86 ppm under aerobic culture conditions. A 5-aminolevulinic acid-producing microorganism characterized by exhibiting a 5-aminolevulinic acid synthase activity of 3.5 to 5.6 (nmol / min / mg protein).

The present invention also provides a method for producing 5-aminolevulinic acid, which comprises culturing the 5-aminolevulinic acid-producing microorganism and collecting 5-aminolevulinic acid from the resulting culture. is there.

[0013]

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The 5-aminolevulinic acid-producing microorganism of the present invention is obtained, for example, by treating a wild-type red non-sulfur bacterium or its mutant strain as a parent strain and subjecting it to mutation treatment, and then having a dissolved oxygen concentration of 1.46 to 1. Under the condition of 5.86 ppm, 5-aminolevulinic acid synthase activity is 3.5 to 5.6 (nmol / m2).
in / mg protein).

More specifically, it is obtained by the following method.

First, a liquid medium in which the parent strain can grow is prepared in a test tube, sterilized, inoculated with the parent strain, and cultured with shaking. After the grown cells are washed with a buffer, a mutation operation is performed.

For this mutation operation, ordinary mutation means can be used. For example, a parental strain on an agar medium is irradiated with a physical mutagen such as ultraviolet light or ionizing radiation, or ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (NTG), or ethyl nitrosourea. A method of culturing the parent strain in a buffer to which a chemical mutagen such as an alkylating agent such as (ENU) or a base analog such as bromodeoxyuridine (BrdUrd) is added can be used.

The cells treated by the mutation means as described above are further washed with a buffer, spread on an agar medium or the like, and cultured. In order to select a strain showing the above properties from the mutants grown by this culture, the following steps are performed.

The obtained mutant is cultured in a test tube or the like, and after the cells are grown, levulinic acid and glycine are added. After adding them, the amount of accumulated 5-aminolevulinic acid in the culture solution may be measured to select a strain having a large amount of 5-aminolevulinic acid. In order to culture and evaluate more microorganisms, it is efficient and preferable to use a microtiter plate. Further, if the measurement of 5-aminolevulinic acid is performed by the Erich reaction, the amount of accumulated 5-aminolevulinic acid can be visually recognized, so that the selection can be performed efficiently.

First, about 1-100 strains having high productivity of 5-aminolevulinic acid are selected from about 15,000 strains of mutant strains obtained by one mutation treatment, and then,
These strains are subcultured on a test tube or an agar plate, and the strain is selected by further selecting strains having stable growth and productivity.
Also at this time, it is preferable to use an Erich reaction or a microtiter plate.

The above-described mutation operation and selection are repeated using the obtained strain as a parent strain. The optimal addition concentration of levulinic acid may change as the mutation is repeated and the productivity of 5-aminolevulinic acid increases, so it is preferable to appropriately change the addition concentration of levulinic acid. In this manner, a strain having repeated mutation breeding and capable of accumulating a significant amount of 5-aminolevulinic acid under aerobic conditions has enhanced 5-aminolevulinic acid synthetase among metabolism-related enzymes of 5-aminolevulinic acid. It has the characteristic. Further, even under the condition that the dissolved oxygen concentration in the culture solution exceeds 2 ppm, it is difficult to obtain 5-
Inhibition of aminolevulinic acid synthase activity is less likely to occur.

The parent strain used first in the above method is a wild-type strain of Rhodocobacter spheroides or a mutant thereof.
Specifically, Rhodobacter spheroides CR-0
02 strain (FERM P-15312) is a preferred parent strain.

The strain obtained by the above mutation and selection is Rhodobacter sphaeroides CR-007.
The one named 2009 and deposited as FERM BP-6320 is preferred. This strain is obtained by repeatedly mutating and selecting the above-mentioned Rhodobacter sphaeroides CR-002 strain, and is a strain capable of accumulating a significant amount of 5-aminolevulinic acid.

Rhodobacter sphaeroides CR-0
The 072009 strain was obtained by mutagenesis from the strain Rhodobacter sphaeroides CR-002 as described above.
Same as for R-002 strain. Hereinafter, the bacteriological properties of the Rhodobacter sphaeroides CR-0072009 strain will be described.

A Morphological characteristics Cell size 0.5 × 1 to 2.5 μm, bacilli, several cells are connected in the longitudinal direction. Spore presence / absence None b. Growth on agar medium Circular colonies in the medium, shiny, reddish brown c Physiological properties-Gram stainability--Reduction of nitrate +-Oxidase +-Glucose fermentability--Growth range pH 4.0 to 9.0, 10C to 40C d Chemical taxonomic properties-G / C content of DNA (mol%) 68-Quinone type Q-10-Carotenoid + (Main components: chloroxanthine, methylchloroxanthine)-Bacteriochlorophyll + e Other growth conditions, etc.-Photosynthetic growth Very weak in the medium of Table 1. The origin bacteria are photosynthetic bacteria, but their photosynthetic viability is reduced. -Glucose assimilation (aerobic) +-Sodium acetate assimilation (aerobic) +-5-Aminolevulinic acid dehydratase activity In aerobic culture, 1 compared to the original bacterium (CR-002 strain) / 3 or less

Hereinafter, the production conditions of 5-aminolevulinic acid using the 5-aminolevulinic acid-producing microorganism of the present invention will be described with respect to Rhodobacter sphaeroides CR-0072.
The description will be made mainly on the case where the 09 strain is used. For the production conditions of 5-aminolevulinic acid, ordinary microorganism culture conditions can be applied. For example, sugars such as glucose or acids such as acetic acid, malic acid and succinic acid can be used as the carbon source, and saccharides are particularly advantageous in terms of price. As the nitrogen source, an inorganic nitrogen source such as an ammonium nitrogen compound such as ammonium sulfate or ammonium salt or a nitrate nitrogen compound such as sodium nitrate; or an organic nitrogen source such as glycine, urea, polypeptone, yeast extract, or casamino acid is used. be able to. Among them, it is preferable to add glycine for improving the production of 5-aminolevulinic acid. The amount of glycine to be added is preferably in the range of usually 10 to 1000 mM, particularly preferably in the range of 10 to 400 mM. The amount of glycine to be added at one time is preferably 10 to 200 mM, and it is preferable to add glycine several times. If necessary, components such as vitamins and inorganic salts may be added to the medium.

It is known that microorganisms belonging to red non-sulfur bacteria such as the genus Rhodobacterium and Rhodopseudomonas generally have 5-aminolevulinic acid dehydratase and metabolize the generated 5-aminolevulinic acid.
However, since the 5-aminolevulinic acid dehydratase activity of the strain of the present invention is significantly reduced, 5-aminolevulinic acid accumulates outside the cells without adding a 5-aminolevulinic acid dehydratase inhibitor such as levulinic acid. However, the addition of a small amount of an inhibitor is effective in improving the ability to produce 5-aminolevulinic acid. In this case, the amount of addition is preferably in the range of 0.01 to 20 mM, particularly 0.1 to 10 mM. As the culturing conditions, all the conditions under which the Rhodobacter sphaeroides CR-0072009 strain can be grown can be used.
0 ° C., particularly preferably 20-35 ° C., and the pH of the medium
Is preferably 4 to 9, particularly preferably 5 to 8. If the pH changes during the culture, sodium hydroxide, ammonia, an alkaline solution such as sodium hydroxide, hydrochloric acid,
It is preferable to adjust to the above range with an acid such as sulfuric acid or phosphoric acid.

The production of 5-aminolevulinic acid can be carried out simultaneously with the growth of the microorganism, but can also be carried out independently. The microorganism used in this case may be either a growing cell or a quiescent cell and can be used directly for the production of 5-aminolevulinic acid. However, the microorganism is collected by a device such as a centrifugal separator, and a medium, a phosphate buffer, etc. Can be used with a higher bacterial concentration, such as resuspension in an appropriate solution of

Further, in order to further improve the productivity of 5-aminolevulinic acid, the amount of dissolved oxygen in the culture solution is reduced to 0.001.
It is preferable to control the oxidation-reduction potential in the range of -220 mV to 50 mV, and more preferably in the range of 0.001 to 1 ppm and -200 mV to 0 mV, respectively. As a control method at this time, a method of changing the number of revolutions and the amount of aeration, a method of adding a saccharide or a yeast extract to activate the respiration of microorganisms, or a combination thereof can be used.

In order to more stably control dissolved oxygen or oxidation-reduction potential, it is effective to increase the growth rate of microorganisms and to add an iron component to the medium in order to stabilize respiratory activity. The iron component used here is preferably a salt thereof, and the valence of iron in this case is not particularly limited. Examples of the iron component include ferric chloride, ferric sulfate, and iron citrate. When a natural component such as yeast extract or corn steep liquor used as a medium component contains a necessary amount of iron, it can be used as an iron component. The iron component is 5-500 as iron in the medium.
It is preferable to add so as to be μM. This is 5μ
If it is less than M, the growth promoting effect and the effect of stabilizing respiratory activity cannot be obtained, and if it exceeds 500 μM, the growth of microorganisms may be inhibited. A particularly preferred concentration of the iron component is 10 to 300 μM, and a more preferred concentration is 20 to 200 μM.

The produced 5-aminolevulinic acid can be separated and purified as required by a conventional method such as an ion exchange method, a chromatography method, and an extraction method.

[0032]

The microorganism of the present invention has excellent productivity of 5-aminolevulinic acid, and the method for producing 5-aminolevulinic acid using the microorganism does not require introduction of nitrogen gas even if the dissolved oxygen concentration is relatively high. It is industrially advantageous to produce 5-aminolevulinic acid.

[0033]

The present invention will now be described by way of examples, which are provided for illustrative purposes only, and are not intended to limit the present invention.

Example 1 Glutamate / glucose medium (medium 1) 1 shown in Table 1
0 ml was dispensed into a 21 mmφ test tube, sterilized at 121 ° C. for 15 minutes, and allowed to cool. One platinum loop of Rhodobacter sphaeroides CR-002 strain (FERM P-15312) was inoculated into this, followed by shaking culture at 30 ° C. in a dark place for 2 days.

10 ml of the medium 1 was dispensed into another 21 mmφ test tube and sterilized in the same manner as described above. To this, 0.5 ml of the above culture solution was subcultured, and incubated at 32 ° C. in a dark place for 18 minutes.
The cells were cultured with shaking for a time.

[0036]

[Table 1]

This culture solution was washed at 3000 g for 5 minutes.
After centrifugation for 1 minute, the supernatant was discarded and suspended in the same volume of Tris-maleate buffer (pH 6.0) as before centrifugation.
This washing operation was repeated twice more. After this, 30
After centrifugation at 00 g for 5 minutes, the supernatant was discarded.
Tris-maleic acid (pH 6.
0) and cultivated at room temperature for 80 minutes. The thus-mutated bacteria were washed three times in the same manner as described above, and then transferred to a test tube in which sterilized medium 1 was dispensed, and cultured with shaking at 32 ° C in a dark place for 2 days. The culture solution was diluted and applied to an agar plate prepared by adding 15 g / L of agar to Medium 1 and sterilizing at 121 ° C. for 15 minutes, followed by culturing at 32 ° C. for 4 days in the dark. As a result, about 15
2,000 colonies were obtained.

Next, 0.2 ml of a sterilized medium 1 per well was dispensed into a sterilized 96-well microplate.
About 15,000 mutant strains described above were inoculated respectively. This was shake-cultured in a dark place at 30 ° C. using a microplate shaker, and after 48 hours, glycine and levulinic acid were added to each well so that the final concentrations were 30 mM each. Shaking culture was performed for another 24 hours under the same conditions as above.

5-Aminolevulinic acid in each well was detected as follows (Erich reaction). (1) The culture solution was collected from each well into another microtiter plate, 0.1 ml of a 1 M acetate buffer containing 1% acetylacetone was added, and the plate was covered with a seal.
After incubation at 15 ° C. for 15 minutes, the microtiter plate was quenched in ice. (2) Next, 0.1 ml of an acetic acid solution containing 2% p-dimethylaminobenzaldehyde and 16% perchloric acid was added, and after 15 minutes at room temperature, reddish purple color due to the Erich reaction was observed, (3) At this time, a part of the reaction solution was taken, and by TLC analysis, a strain showing a purple-red coloration not derived from 5-aminolevulinic acid was excluded, and the mutant 6 having a high production of 5-aminolevulinic acid 6
Strains were selected.

The above 6 strains were cultured in a medium 1 test tube in a dark place at 30 ° C.
For 48 hours, and spread on an agar plate having the composition of Medium 1 to form a colony. Next, the sterilized 96
0.2 ml of sterilized medium 1 per well was dispensed into a well microplate, and 60 colonies derived from each of the above colonies were arbitrarily selected and inoculated. this,
After shaking culture using a microplate shaker in a dark place at 32 ° C., 48 hours later, glycine was added to each well to a final concentration of 30 mM, and levulinic acid was added to each of 20 colonies of the above 60 colonies. 30, 15,
1 mM was added. Further, the cells were shake-cultured under the same conditions as described above for 24 hours, and a culture solution was collected from each well, and the productivity of 5-aminolevulinic acid and the optimal concentration of levulinic acid were examined by Erich reaction. Repeat this operation three times, using the high productivity and the variation in productivity between colonies as indices,
One mutant strain with higher productivity was isolated more stably than the CR-002 strain. This was named CR-150 strain. The optimal concentration of levulinic acid was 30 mM. When the amount of accumulated 5-aminolevulinic acid was determined using a microplate reader to determine the amount of accumulated 5-aminolevulinic acid, the absorbance at 553 nm of the Erich reaction when 30 mM levulinic acid was added.
The CR-150 strain was 0.1 mM compared to 1 mM.

Example 2 The same operation as in Example 1 was carried out except that the strain used was CR-150, and about 15,000 mutant strains were induced. A selection test was performed in the same manner as in Example 1 to obtain a mutant strain CR-268 having improved productivity as compared to the CR-150 strain. 553 nm of Erich reaction using microplate reader
The amount of accumulated 5-aminolevulinic acid was determined by the absorbance of the strain CR-268 to be 0.3 mM.

Example 3 The same operation as in Example 1 was carried out except that the strain to be used was CR-268, and about 15,000 mutants were induced. A selection test was performed in the same manner as in Example 1 to obtain a mutant CR-368 strain whose productivity was improved as compared with the CR-268 strain. 553 nm of Erich reaction using microplate reader
The amount of accumulated 5-aminolevulinic acid was determined by absorbance of the strain CR-368 to be 0.5 mM.

Example 4 The same operation as in Example 1 was carried out except that the strain to be used was CR-368, and about 15,000 mutants were induced. A selection test was performed in the same manner as in Example 1 to obtain a mutant CR-405 strain having improved productivity as compared with the CR-368 strain. 553 nm of Erich reaction using microplate reader
The amount of accumulated 5-aminolevulinic acid was determined by the absorbance of the strain CR-405 to be 1.0 mM.

Example 5 The same operation as in Example 1 was carried out except that the strain to be used was CR-405 strain, and about 15,000 mutant strains were induced. A selection test was performed in the same manner as in Example 1 except that the concentration of levulinic acid to be added was 15 mM, to obtain a mutant strain CR-502 having improved productivity as compared to the CR-405 strain. When the amount of accumulated 5-aminolevulinic acid was determined by the absorbance at 553 nm of the Erich reaction using a microplate reader,
The CR-405 strain was 1.2 mM, whereas the CR-405 strain was CR-405.
502 strains were 2.1 mM.

Example 6 The same operation as in Example 1 was carried out except that the strain to be used was CR-502, and about 15,000 mutant strains were induced. A selection test was performed in the same manner as in Example 1 except that the concentration of levulinic acid to be added was 1 mM, to obtain a mutant strain CR-660 having improved productivity as compared with the strain CR-502. Using a microplate reader, the amount of accumulated 5-aminolevulinic acid was determined by the absorbance at 553 nm of the Erich reaction.
Whereas the R-502 strain was 2.4 mM, CR-6 was used.
60 strains were 3.3 mM.

Example 7 The same operation as in Example 1 was carried out except that the strain to be used was CR-660, and about 15,000 mutants were induced. A selection test was performed in the same manner as in Example 1 except that the concentration of levulinic acid to be added was changed to 1 mM, and mutants CR-0072001 and CR-007200 improved in productivity as compared with the CR-660 strain.
2. CR-0072009 strain was obtained. When the amount of accumulated 5-aminolevulinic acid was determined by the absorbance at 553 nm of the Erich reaction using a microplate reader,
CR-0072001, CR-0072002, CR-
0072009 strains are 5.9, 5.6 and 4.6 mM, respectively.
Met.

Example 8 Mutants CR-0072001 and CR-0 obtained in Example 7
072002, CR-0072009 strain,
The inoculated flask was inoculated into a 500 ml permeation flask prepared in 00 ml and shake-cultured at 32 ° C for 48 hours in a dark place. After culture, 5
The cells were collected by centrifugation at 000 g for 10 minutes, suspended in the following medium 2 so that the wet cell weight was 0.5 g / 10 ml, and 3 ml was dispensed into a 21 mm test tube. The test tube thus obtained was subjected to reciprocal shaking culture at 32 ° C. in the dark, and the 5-aminolevulinic acid in the culture solution after 20 hours was purified by the method of Okayama et al. (CLINICAL CHEMISTRY,
Vol. 36, no. 8, p-1494, 1990). As a result, CR-0072001, CR-0
072002 and CR-0072009 strains are 1
9.0, 21.5, and 31.0 mM.

[0048]

[Table 2]

Example 9 The mutant strain CR-0072009 was inoculated into a 300 ml Erlenmeyer flask equipped with baffles prepared by preparing 50 ml of Medium 3 and cultivated with shaking in a dark place at 32 ° C. for 48 hours. This is 3L
When 1.8 L of the medium 3 was prepared in a fermenter having a capacity of 0.5 ml, the whole amount was inoculated and cultured with stirring at 32 ° C., aeration rate of 0.36 L / min, and 400 rpm. The culture was continued while controlling the pH to 6.5 to 6.6 using sulfuric acid and sodium hydroxide, and after 40 hours of culture, 0.210 g of levulinic acid, 8.1 g of glycine, yeast extract (manufactured by Nippon Pharmaceutical Co., Ltd., D -3) 18 g was added, the culturing was continued at a stirring rotation speed of 325 rpm, and the pH was maintained at 6.3 to 6.4 using sulfuric acid and sodium hydroxide. After 12, 26 and 38 hours, 8.1 g of glycine was added. The culture was stopped 50 hours after the addition of levulinic acid. 5-Aminolevulinic acid in this culture solution was analyzed by the method of Okayama et al. (CLINICAL CHEMISTRY,
Vol. 36, no. 8, p-1494, 1990) and found to be 60 mM. At this time, the average value of the dissolved oxygen concentration in the culture solution after the addition of levulinic acid was 0.01
ppm. Further, the oxidation-reduction potential in the culture solution after the addition of levulinic acid was in the range of −180 mV to −50 mV.

[0050]

[Table 3]

Example 10 The mutant CR-0072009 strain was inoculated into a 300 ml Erlenmeyer flask with a baffle prepared by preparing 50 ml of Medium 3 and cultivated with shaking in a dark place at 32 ° C. for 48 hours. This is 3L
When 1.8 L of the medium 3 was prepared in a fermenter having a capacity of 0.5 ml, the whole amount was inoculated and cultured with stirring at 32 ° C., aeration rate of 0.36 L / min, and 400 rpm. The culture was continued while controlling the pH to 6.5 to 6.6 using sulfuric acid and sodium hydroxide, and after 40 hours of culture, 0.210 g of levulinic acid, 8.1 g of glycine, and 18 g of yeast extract were added. The culture was continued while maintaining the pH at 6.3 to 6.4 using sulfuric acid and sodium hydroxide at 0.72 L / min and a stirring rotation speed of 600 rpm.
Glycine was added after 12, 24, and 38 hours.
1 g each was added. The culture was stopped 50 hours after the addition of levulinic acid. 5-Aminolevulinic acid in this culture solution was analyzed by the method of Okayama et al. (CLINICAL CHEMISTRY, Vo).
l. 36, no. 8, p-1494, 1990) to be 13 mM. At this time, the average value of the dissolved oxygen concentration in the culture solution after the addition of levulinic acid was 2.3 ppm. Further, the oxidation-reduction potential in the culture solution after the addition of levulinic acid was in the range of 50 mV to 70 mV.

Example 11 The mutant strain CR-0072009 was inoculated into a 300 ml Erlenmeyer flask with a baffle prepared by preparing 50 ml of Medium 3 and cultivated with shaking in a dark place at 32 ° C. for 48 hours. This is 3L
When 1.8 L of the medium 3 was prepared in a fermenter having a capacity of 0.5 ml, the whole amount was inoculated and cultured with stirring at 32 ° C., aeration rate of 0.36 L / min, and 400 rpm. The culture was continued while controlling the pH to 6.5 to 6.6 using sulfuric acid and sodium hydroxide, and after 40 hours of culture, 0.210 g of levulinic acid, 8.1 g of glycine, and 18 g of yeast extract were added. The culture was continued while maintaining the pH at 6.0 to 6.5 with sulfuric acid and sodium hydroxide at 0.18 L / min and a stirring rotation speed of 200 rpm. After 12 hours and 24 hours, 8.1 g of glycine was added. The culture was stopped 50 hours after the addition of levulinic acid. 5-Aminolevulinic acid in this culture solution was analyzed by the method of Okayama et al. (CLINICAL CHEMISTRY, Vol.
36, no. 8, p-1494, 1990) and was 15 mM. At this time, the dissolved oxygen concentration in the culture solution after the addition of levulinic acid was below the detection limit. The oxidation-reduction potential in the culture solution after the addition of levulinic acid was −
The range was from 220 mV to -180 mV. Rhodobacter spha as shown in Example 7.
The CR-0072009 strain obtained by repeatedly mutating the eroides strain CR-002 as a source bacterium was used at an extremely high concentration of 5% by using inexpensive raw materials such as glucose and glycine.
-Aminolevulinic acid is a strain that can produce amino-levulinic acid, and it is possible to industrially produce 5-aminolevulinic acid using this strain. Further, as can be seen from Examples 10 and 11, in order to enhance the productivity, the dissolved oxygen concentration in the culture solution after adding levulinic acid was 2.0 ppm or less, or the oxidation-reduction potential in the culture solution was from -220 mV to 50 mV. Is desirably controlled within the range described above, and this can be easily performed by lowering the stirring rotation speed.

Example 12 Three 300 ml Erlenmeyer flasks with baffles prepared by preparing 50 ml of the culture medium 3 were prepared, and each of them was a mutant strain CR-0072.
The 009 strain was inoculated and cultivated with rotation and shaking at 32 ° C. for 48 hours in a dark place. The whole was inoculated into 1.8 L of medium 3 prepared in three 3 L fermenters and cultured at 32 ° C. The pH was adjusted to 6.5 using sulfuric acid and sodium hydroxide.
While controlling the pressure at 6.6, the aeration rate was set to 0.36 L / min, the stirring speed was set to 400 rpm, and the stirring speed was set to 250 rpm after 25 hours of culturing for one of them, and the culturing was continued for 90 hours each. During the culture, the dissolved oxygen concentration and 5-aminolevulinic acid synthase activity were measured. 5-Aminolevulinic acid synthase activity was measured as follows.

About 30 ml of the culture was sampled, and the cells were collected by centrifugation. The collected cells were added to a phosphate buffer (5
After washing with 0 mM, pH 7.2), the cells were resuspended in 5 ml of a buffer having the same composition, and the cells were disrupted by a French press using a conventional method and centrifuged at 10,000 g for 30 minutes. -An aminolevulinic acid synthase crude enzyme solution was used.

1 ml of a phosphate buffer (50 mM, pH 7.2)
Glycine 50 mM and pyridoxal phosphate 0.1 mM
And 1 mM EDTA, and the above crude enzyme solution was further added so as to be 1.0 mg / ml in terms of protein amount to prepare an enzyme reaction solution. To this was added succinyl-CoA at a concentration of 0.2 mM and incubated at 37 ° C. After 15 minutes, 1 ml of 10 vol% trichloroacetic acid was added to stop the reaction. This was centrifuged at 3500 rpm for 10 minutes, 1 ml of the supernatant was taken, 2 ml of 1M acetic acid buffer (pH 4.7) containing 1% acetylacetone was added, and the mixture was reacted at 100 ° C. for 15 minutes and immediately cooled with ice. 3.5 ml of Erich reagent was added, and the amount of pyrrole compound produced was measured from the absorbance at 553 nm after 15 minutes, and the amount of 5-aminolevulinic acid produced by the enzyme reaction (a) was calculated. The 5-aminolevulinic acid production rate was determined by the following equation (1), and this was defined as 5-aminolevulinic acid synthase activity. FIG. 1 shows the relationship between the dissolved oxygen concentration in the culture solution and the activity of 5-aminolevulinic acid synthase at that time.
And Table 4 below. The dissolved oxygen concentration of 10% in FIG.
At 7 ppm, a dissolved oxygen concentration of 90% corresponds to 6.6 ppm.

[0056]

(Equation 1)

Comparative Example 1 The procedure was as in Example 10, except that the strain used was CR-002. The results are shown in FIG. As can be seen from FIG. 1, the CR-0072009 strain inhibits 5-aminolevulinic acid synthase activity as seen in a wild-type photosynthetic bacterium even under conditions where the dissolved oxygen concentration in the culture solution exceeds 2 ppm. Is less likely to occur.

[0058]

[Table 4]

Example 13 The medium 3 (Table 3) used in Example 9 contains 5.0 g / L of yeast extract B (manufactured by Oriental Yeast Co., Ltd., B-2) instead of the yeast extract contained in Medium 3. The following experiment was performed using the medium. As in Example 9, mutant CR-007
2009 strain in a 300 ml Erlenmeyer flask with baffle
The culture solution cultured for 8 hours was subjected to 1.8 L fermentation in a 3 L fermenter.
L. Yeast extract B, inoculate the whole amount into medium 3 prepared
C., aeration rate 0.36 L / min, stirring culture at 400 rpm,
0.210 g of levulinic acid, 8.1 g of glycine, and 18 g of yeast extract B were added, and the stirring speed was reduced to 325 rpm to continue the culture. After adding levulinic acid, the dissolved oxygen remained at 0.5 ppm for about 4 hours, but after 5 hours the dissolved oxygen began to rise. Six hours later this rose to 4 ppm,
5-Aminolevulinic acid production stopped at 10 mM. To this, when ferric chloride was added to 20 μM, the dissolved oxygen became 0.5 ppm or less again within 2 hours after the addition, and the production of 5-aminolevulinic acid resumed.
Glycine was added after an additional 12, 26, and 38 hours.
1 g each was added. The culture was stopped 50 hours after the addition of levulinic acid. 5-Aminolevulinic acid in this culture solution was analyzed by the method of Okayama et al. (Clinical Chemistry, Vo).
l. 36, no. 8, p-1494, 1990) and found to be 40 mM.

As is clear from Example 13, the addition of iron has the effect of restoring respiration.

Reference Example Yeast extract used in Example 9, Yeast extract B used in Example 13, and yeast extract C of another manufacturer (Industrial yeast extract manufactured by Oriental Yeast Co., Ltd.) used in the following Examples
The iron content of the yeast extract was determined by the ICPmass method to be 73 μg, 25 μg, and 15 μg / g of yeast extract, respectively.
It was 5 μg. From the iron content, it can be seen that the iron content contained in the medium 3 prepared using each yeast extract was 6.64, 2.27, and 14.1 μM, respectively. Thus, in Example 9, the culture solution contained 20 μM iron at the time of producing 5-aminolevulinic acid, and the medium before iron addition in Example 13 had 6.8 μM,
It can be seen that after the addition of iron, 26.8 μM iron was contained.

Example 14 The above yeast extract C was replaced with 3 g / L of the yeast extract in the medium 3.
In a 300 ml Erlenmeyer flask with a baffle prepared by adding 50 ml of a medium obtained by adding ferric chloride to the medium prepared as described in Table 5 to a concentration shown in Table 5, the mutant strain CR-0072009 [48 ml of yeast extract C in a test tube of 21 mmφ with 48 ml) was added. 1 ml of a culture solution (0.2 at an optical density of 660 nm)], which had been cultured for an hour, was inoculated, and cultured with shaking at 32 ° C. in a dark place. When combined with the iron content contained in the yeast extract, the amount of iron content shown in Table 5 is contained. Table 5 shows the results obtained by measuring the cell concentration after culturing for 30 hours at an optical density of 660 nm.

[0063]

[Table 5]

As is clear from Table 5, it can be seen that the addition of an appropriate iron content has the effect of increasing the growth rate.

Example 15 The above yeast extract C was replaced with 3 g / L of the yeast extract C in the medium 3.
The mutant strain CR-0072009 [a culture obtained by culturing for 48 hours with 10 ml of yeast extract C in a test tube of 21 mmφ] in a 300 ml Erlenmeyer flask with a baffle prepared by adding 50 ml of medium to which 20 μM ferric chloride was further added to the medium prepared as described above. 1 ml (0.2 at an optical density of 660 nm)], and cultivated with shaking in the dark at 32 ° C. for 48 hours. This is 3L
1.8 L of the above ferric chloride-supplemented medium was prepared in a fermenter with a volume of 32 ° C., and inoculated at 32 ° C. with an aeration rate of 0.36 L /
The mixture was stirred and cultured at 400 rpm for a minute. After 24 hours of culture, 0.210 g of levulinic acid, 8.1 g of glycine, yeast extract C
9 g was added, the cultivation was continued at a stirring rotation speed of 325 rpm, and the pH was maintained at 6.3 to 6.4 using sulfuric acid and sodium hydroxide. After 12, 26, and 38 hours, 8.1 g of glycine was added. The culture was stopped 50 hours after the addition of levulinic acid. 5-Aminolevulinic acid in this culture solution was analyzed by the method of Okayama et al. (CLINICAL CHE).
MISTRY, Vol. 36, no. 8, p-149
4, 1990) was 55 mM. At this time, the average value of the dissolved oxygen concentration in the culture solution after the addition of levulinic acid was 0.01 ppm. The oxidation-reduction potential in the culture solution after the addition of levulinic acid was from -180 mV to -50.
It was in the range of mV.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between dissolved oxygen and 5-aminolevulinic acid synthase activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:01) (72) Inventor Tomonori Kanaga 1134-2 Gongendo, Satte City, Saitama Prefecture Cosmo Research Institute R & D Center Co., Ltd. (72) Inventor Kikuo Watanabe 1134-2 Gongendo, Satte City, Saitama Prefecture Inside Cosmo Research Institute R & D Center (72) Inventor Shinya Miyachi 1134-2 Gongendo, Sate City, Saitama Prefecture R & D Cosmo Research Institute Inc. Within the center (72) Inventor Keitaro Watanabe 1134-2, Gongendo, Satte City, Saitama Prefecture Inside the R & D Center, Cosmo Research Institute, Inc. (72) Koji Hotta 1134-2, Gongendo, Satte City, Saitama Cosmo Research Institute, Inc. research and development Center in (56) reference Patent flat 8-168391 (JP, a) (58 ) investigated the field (Int.Cl. ^7, B name) C12N 1/20 C12P 13/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (7)

(57) [Claims] [1] Rhodob spheroides (Rhodob)
acter sphaeroides) or a mutant thereof, which is a 5-aminolevulinic acid-producing microorganism having a dissolved oxygen concentration of 1.
Under aerobic culture conditions of 46-5.86 ppm, 3.5
A 5-aminolevulinic acid-producing microorganism, which exhibits a 5-aminolevulinic acid synthetase activity of from 5.6 to 5.6 (nmol / min / mg protein). 2. The method according to claim 1, wherein the bacterium is Rhodob.
The 5-aminolevulinic acid-producing microorganism according to claim 1, which is named acter sphaeroides) CR-0072009 and has been deposited as FERM BP-6320. 3. culturing the microorganism according to claim 1 or 2,
A method for producing 5-aminolevulinic acid, comprising collecting 5-aminolevulinic acid from the obtained culture. 4. The method for producing 5-aminolevulinic acid according to claim 3, wherein the culturing is performed in a medium to which levulinic acid or glycine is added. 5. The method for producing 5-aminolevulinic acid according to claim 4, wherein the amount of glycine added is 200 mM / time or less. 6. The cultivation, wherein the dissolved oxygen concentration in the culture solution is reduced to 0.
001 to 2 ppm or the oxidation-reduction potential is -220.
The method is carried out under the condition of ５０50 mV.
6. The method for producing 5-aminolevulinic acid according to any one of 5. 7. The method according to claim 3, wherein the culture is performed in a medium containing 5 to 500 μM of an iron component.
The method for producing 5-aminolevulinic acid according to the above item.