JPH0367677B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention provides two compounds that have herbicidal activity and are useful as herbicides.
-amino-4-(hydroxymethylphosphinyl)
-butyryl-L-alanyl-L-alanine (hereinafter
The present invention relates to an improvement in the culture production method for SF-1293 (substance referred to as SF-1293). (Prior art) SF-1293 substance is Streptomyces hygroscopicus
SF-1293 strain (see Special Publication No. 51-639; FERM
-BP-130, ATCC-21705), and was subsequently discovered to have strong herbicidal activity (see Japanese Patent Publication No. 59-23282), and its development as a herbicide has progressed. . In order to use it as a herbicide, it is essential to conduct research and development on its safety and efficacy, as well as to improve the manufacturing method to produce it at a low cost. As a report on the improvement of the manufacturing method, a method of adding 2-amino-4-(hydroxymethylphosphinyl)-butyric acid (hereinafter referred to as AMPB), which is a component of the SF-1293 substance, was reported (Japanese Patent Application Laid-Open No. 55-21754). (see official bulletin), as well as
Method of adding biosynthetic intermediates of SF-1293 substance (see JP-A-58-219191 and JP-A-58-146591)
etc. can be mentioned. (Problems to be Solved by the Invention) Conventional herbicides are widely used artificially synthesized compounds through organic synthesis, contributing to environmental pollution, but the SF-1293 substance is produced by microorganisms.
It is attracting attention as an ideal herbicide because it is rapidly decomposed. To date, no herbicides produced by microorganisms have been put into practical use or widely used, and one reason for this is that it is difficult to produce large amounts of secondary metabolites produced by microorganisms in good yields. It is. The present invention focuses on the above-mentioned problems and provides an SF-
We aim to solve this problem by establishing an improved method that enables high-yield production of 1293 substances. (Means and effects for solving the problems) The present inventors have developed an SF-1293 substance-producing bacterium using SF-1293 substance-producing bacteria for the purpose of improving the production method of SF-1293 substance.
-1293 substance production When various phosphorus-containing compounds were added to the culture medium and the relationship with SF-1293 substance production was investigated, it was found that 4 The addition of -(hydroxymethylphosphinyl)-2-oxobutyric acid (hereinafter referred to as OMPB) significantly increases SF-
It was discovered for the first time that production of 1293 substances could be improved. OMPB is a known substance, and its manufacturing method and physicochemical properties are described in, for example, Japanese Patent Application Laid-Open No. 56-92897. AMPB has stereoisomerism, and only the L-isomer is used in the previously proposed method, whereas OMPB has no stereoisomerism and can be completely utilized in the method of the present invention, and the number of synthesis steps is The present invention is an extremely advantageous method from the viewpoint of synthesis yield and cost. Therefore, the present invention provides SF-1293 substance-producing bacteria belonging to the genus Streptomyces by the following formula: 4-(hydroxymethylphosphinyl) represented by
- Phosphorus-containing compound SF characterized by being cultured in the presence of 2-oxobutyric acid, its salt, its monoester, its diester, or its amide -
It provides a high-yield production method for 1293 substances. The present invention will be explained in more detail below. As the Streptomyces strain used in the culture method of the present invention in which OMPB is added to increase the yield of SF-1293 substance, any Streptomyces strain that produces SF-1293 substance can be used. For example, the aforementioned bacterial strain SF-1293 (FERM-BP-130; isolated from soil and named Streptomyces hygroscopicus );
ATCC-21705). The mycological properties of Streptomyces hygroscopicus SF-1293 strain are specified in Japanese Patent Publication No. 1983-639. Streptomyces hygroscopicus SF−
Strain 1293 is susceptible to changes in its properties, as seen in the case of other strains of the genus Streptomyces, and can be easily mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, chemicals, etc. Even if it is a mutant strain, it has the ability to produce SF-1293 substance,
Or AMPB or SF, which is a component of SF-1293 substance
Any strain of the genus Streptomyces that exhibits the ability to produce the SF-1293 substance by adding an intermediate for biosynthesis of the -1293 substance or by adding the OMPB of the present invention can be used in the present invention. In the production method of the present invention, strain SF-1293 is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing Streptomyces bacteria can be used. Examples of carbon sources include glucose, starch, glycerin, sucrose, starch syrup, and molasses. These may be used alone or in combination. Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, dry yeast, cornstarch liquor, ammonium sulfate, ammonium nitrate, etc. are used alone or in combination. Calcium carbonate, salt, etc. as necessary.
Inorganic salts such as potassium chloride and phosphates can be added. According to the present invention, the yield of SF-1293 substance can be increased by adding OMPB during the production culture of SF-1293 substance as described above.
It is more effective to add OMPB in portions during the culture rather than all at once at the beginning of the production culture. The amount of OMPB added is not critical, but it can usually be added at a rate of about 0.1 to 20 mg/ml, preferably about 0.5 to 5 mg/ml, to the production medium. In addition, when adding OMPB, normal SF-
This method is extremely advantageous because it allows the use of a diluted medium than in the production culture of 1293 substances, contributing to a reduction in manufacturing costs. Furthermore, SF−1293
It is stated in the specification of Japanese Patent Application No. 60-197753 that the production of SF-1293 substance is significantly improved by adding alanine at the same time to the known method of adding AMPB during the production culture of the substance. However, in the case of OMPB addition according to the present invention,
The production of SF-1293 substance can be further enhanced by simultaneously adding inexpensive dl-alanine. The most suitable culture method is liquid culture, especially deep culture. Cultivation is performed under aerobic conditions, and the suitable temperature for cultivation is 25 to 35°C, but in most cases it is cultured at around 28 to 32°C. The number of culture days can be shorter than that of normal SF-1293 substance production culture, and the appropriate number of culture days is usually 4 to 7 days. For other details of the culture method itself, please refer to the description in the above-mentioned Japanese Patent Publication No. 51-639, which describes the method for producing SF-1293 substance. Quantification of SF-1293 substances in the culture solution is performed using an amino acid analyzer. In other words, after adjusting the culture solution to pH 2.0, the supernatant was obtained by centrifugation (3000 rpm, 15 minutes) or filtration, and then analyzed using an amino acid analyzer (manufactured by ATTO).
Separation and quantification are performed using MLC-703 (retention time: 14 minutes). SF-1293 from the culture solution obtained according to the present invention
The method for purifying the substance is the same as the usual purification method from SF-1293 culture solution, and for details, please refer to the description in the above-mentioned Japanese Patent Publication No. 51-639. (Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 Streptomyces hygroscopicus SF-
One mutant strain of 1293 strains was cultured in preculture medium (soluble starch 2.0
%, polypeptone 1.0%, meat extract 0.3%, dipotassium hydrogen phosphate 0.05%, PH 7.0).
This was cultured with shaking at 28℃ for 24 hours, and the same medium was further incubated at 80℃.
ml and cultured with shaking at 28°C for 24 hours, with a ratio of 3%: glucose 7.0%, wheat germ 3.9%, sungrain 2.5%, monopotassium phosphate.
The cells were inoculated into a production medium consisting of 0.3% cobalt chloride and 0.0001% cobalt chloride, and cultured with shaking at 28°C. OMPB
and alanine were added according to the concentrations and methods shown in Table 1 below. After making the culture solution acidic, it was centrifuged (3000 rpm, 15 minutes) to obtain a supernatant, which was then analyzed using an amino acid analyzer (MLC-703 model manufactured by ATTO, retention time 14 minutes).
The production amount of SF-1293 substance was measured. The results are shown in Table 1.

[Table] As shown in Table 1, the effect of adding OMPB or its esters, etc. is clear. Example 2 The same production medium as in Example 1 and its 70% concentration medium were used as seeds for a jar fermenter (3-volume mini-jar made by Iwashiya) at a rate of 2%, which was cultured in the same preculture medium as in Example 1. (Preparation amount 2) was inoculated. In a jar fermenter, aeration stirring culture was carried out at 28°C, and the effects of adding OMPB and alanine were investigated in the same manner as in Example 1. however,
In this example, OMPB and alanine were added semi-continuously using a peristaltic pump and an interlapper. The results are shown in Table 2.

[Table] As shown in Table 2, SF− due to the addition of OMPB
The effect of improving the production of 1293 substances is obvious. (Effect of the invention) As shown in the examples, it is possible to increase the production amount of SF-1293 substance by adding OMPB or its salt or ester form during production culture of SF-1293 substance. It became clear. It was also revealed that the effect of adding OMPB was further enhanced when alanine was added. In the case of OMPB supplemented culture, regular SF-
This method is extremely advantageous because it allows the use of a medium with a lower concentration than when producing and culturing 1293 substances, and it also contributes to reducing raw material costs. Also, normal SF-
The production lap is faster than the production culture of 1293 substances, so the culture time can be shortened, and utility costs such as electricity and steam required for tank operation can be reduced, and the annual production per culture tank can be reduced. It is possible to increase the amount. This shows that the present invention is an extremely effective method for producing substances such as herbicides that need to be produced at low cost and in large quantities.

Claims (1)

[Scope of Claims] 1. SF-1293 substance producing bacteria belonging to the genus Streptomyces is prepared by the following formula: 4-(hydroxymethylphosphinyl) represented by
- Phosphorus-containing compound SF characterized by being cultured in the presence of 2-oxobutyric acid, its salt, its monoester, its diester, or its amide -
High-yield production method for 1293 substances.