Process for producing indolmycin

Details Process for producing indolmycin Process for producing indolmycin

2001-01-23

2002-05-14

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S108000, C435S121000, C435S244000, C514S292000

Reexamination Certificate

active

06387687

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a method for producing an antibacterial agent, especially, indolmycin useful as an anti-H.pylori (Helicobacter pylori), by a fermentation.
BACKGROUND ART
Indolmycin is a compound represented by the following formula and useful as a pharmaceutical (for example, an antibacterial agent, especially anti-H.pylori agent), a veterinary agent and a herbicide.
A known method for producing indolmycin by a chemical synthesis is a method described in Journal of Organic Chemistry, Vol.51, p. 4920 (1986). However, this synthesis gives only a racemate since indolmycin contains 2 asymmetric carbon atoms. While another method is described in Chemistry Letters, p.163 (1980), it involves a complicated process for obtaining an optically active indolmycin and has a low yield.
Still another known method for producing indolmycin is a fermentation using Streptomyces griseus ATCC 12648 as a producer microorganism (The Journal of Antibiotics, Vol.27, p.49 (1974), The Journal of Antibiotics, Vol.34, p.551 (1981)), but its low yield is also problematic from an industrial point of view.
Since a conventional method for producing indolmycin cannot satisfactorily be applied to industrial production, a large scale and convenient method for producing indolmycin is desired.
DISCLOSURE OF INVENTION
We made an effort under the circumstance described above to establish a method for producing indolmycin by fermentation, and finally discovered that by obtaining a tryptophan analogue-resistant mutant of Streptomyces griseus and by using it, an accumulation of a large amount of indolmycin in a culture broth can be obtained. We also discovered that by supplementing the medium with L-tryptophan a further higher accumulation can be achieved. We also established a method for obtaining indolmycin conveniently from the culture broth at a higher yield by means of chromatography using an adsorption resin, and a weakly-basic anion exchange resin, a weakly acidic cation exchange resin as a support together with an aqueous organic solvent.
Thus, the present invention relates to:
(1) A microorganism having indolmycin-producing ability and tryptophan analogue resistance;
(2) A microorganism according to the above (1) wherein said microorganism is a microorganism belonging to the Genus Streptomyces;
(3) A microorganism according to the above (1) wherein said microorganism is a microorganism belonging to the species
Streptomyces griseus;
(4) A microorganism according to the above (1) wherein said tryptophan analogue is fluoro-DL-tryptophan;
(5) A method for producing indolmycin or its salt comprising cultivating a microorganism according to the above (1) in a medium to allow indolmycin or its salt to be accumulated in the culture broth, and then recovering a product;
(6) A method according to the above (5) wherein L-tryptophan is added to the medium;
(7) A method according to the above (5) wherein 0.2 to 5 g of L-tryptophan is added per 1 L of the medium;
(8) A method according to the above (5) wherein 0.2 to 10 g of anthranilic acid is added per 1 L of the medium;
(9) A method according to the above (5) wherein 0.2 to 3 g of L-tryptophan and 0.2 to 2 g of anthranilic acid are added per 1 L of the medium;
(10) A method according to the above (5) wherein a purification is performed using an adsorption resin, a basic anion exchange resin and/or an acidic cation exchange resin as a support and an aqueous organic solvent as an eluent.
A tryptophan analogue employed in the invention may for example be a halogenated tryptophan such as 5-fluoro-DL-tryptophan (hereinafter sometimes referred to as 5-FT), 6-fluoro-DL-tryptophan (hereinafter sometimes referred to as 6-FT) and the like. One preferable example is 5-fluoro-DL-tryptophan or 6-fluoro-DL-tryptophan.
A microorganism having tryptophan analogue resistance can be obtained by subjecting a microorganism having an indolmycin-producing ability to a mutation-inducing treatment ordinarily employed in manipulating microorganisms followed by screening for a strain capable of growing in a tryptophan analogue-containing medium described above. Such mutation-inducing treatment may for example be (1) a UV irradiation [spores of a microorganism having an indolmycin-producing ability as a parent strain are suspended for example in a 100 mM phosphate buffer (pH7.0) and irradiated for 90 seconds under a 15 W UV lamp placed at a distance of 30 cm] and (2) a chemical treatment for example with N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter sometimes referred to as NTG) [NTG treatment: spores of a microorganism having an indolmycin-producing ability as a parent strain are suspended for example in a 50 mM Tris.HCl buffer (pH8.0) supplemented with 1 mg/ml of NTG and allowed to stand at 30° C. for 1 hour].
Instead of a mutation-inducing treatment, a spontaneous mutation may also be employed to allow a strain to acquire ability of growing in a tryptophan analogue-containing medium, and such strain is encompassed in a microorganism having a tryptophan analogue resistance according to the invention.
A microorganism having an indolmycin-producing ability employed in this invention may be any microorganism having an indolmycin-producing ability, such as a microorganism of Streptomyces (for example,
Streptomyces griseus
), typically,
Streptomyces griseus
ATCC 12648 and
Streptomyces sp.
HC-21(IFO 15984, FERM BP-5571). The microbiological characteristics of
Streptomyces sp.
HC-21 are found in WO97/49703.
A microorganism having an indolmycin-producing ability and a tryptophan analogue resistance can be obtained as a tryptophan analogue-resistant mutant for example by subjecting an indolmycin-producing microorganism such as
Streptomyces griseus
ATCC 12648 as a parent strain to a mutation-inducing treatment such as a UV irradiation and an NTG treatment followed by incubating the strain in a medium (such as agar plate) containing a tryptophan analogue described above at a concentration at which the parent strain can not grow followed by screening the resultant colonies. The degree of the resistance of a mutant thus obtained against a tryptophan analogue can be determined for example as follows. Thus, an agar plate, for example as shown in Table 1, is supplemented with a certain amount of a tryptophan analogue (such as 5-fluoro-DL-tryptophan) and inoculated with a loopful of spores containing about 1×10
6
to about 1×10
8
spores/ml and then incubated under a condition suitable for the growth of the parent strain (for example at 24° C. for 8 days), whereby determining the concentration of tryptophan analog at which the parent strain can grow. “A microorganism having a tryptophan analogue resistance (a tryptophan analogue-resistant strain)” according to the invention is a mutant which became to be able to grow in a medium (such as agar medium) containing a tryptophan analogue at a concentration at which the parent strain can not grow.
A microorganism having a tryptophan analogue resistance according to the invention may be a microorganism which can grow in a medium (such as agar plate) containing a tryptophan analogue described above at a high concentration. Against 5-fluoro-DL-tryptophan, for example, a microorganism capable of growing at a concentration of 0.1 to 10000 &mgr;g/ml, preferably 1 to 5000 &mgr;g/ml, more preferably 10 to 2000 &mgr;g/ml is employed. Against 6-fluoro-DL-tryptophan, a microorganism capable of growing at a concentration of 50 to 1000 &mgr;g/ml, preferably 100 to 800 &mgr;g/ml is employed.
By cultivating a microorganism having a tryptophan analogue resistance obtained as described above followed by quantifying indolmycin in the culture broth, a strain giving an increased indolmycin accumulation can be selected.
Examples of a microorganism having an indolmycin-producing ability and a tryptophan analogue resistance are those described in the following Examples including
Streptomyces griseus
5FW-1-226-9 capable of growing in the presence of 5-FT at 10 &mgr;g/ml,
Streptomyces griseus
5FW-2-8-7 c

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