Streptomyces Mediterranei Mutant Capable of Production of Rifamycin B

It has previously been reported that during fermentation in normal growth media, Streptomyces mediterranei synthesizes a family of antibiotics collectively referred to as the rifamycin complex (P. Sensi, et al., Antibiotics Annual 1959-1960, page 262). Subsequent work revealed that the addition of sodium diethyl barbiturate to the culture medium resulted essentially in the formation of a single fermentation product, rifamycin B (Margalith P. and Pagani H., Applied Microbiology, 9, 325, 1961).

The present invention relates to the isolation of a mutant strain of Streptomyces mediterranei and the elaboration by it of essentially rifamycin B only, irrespective of the presence or absence of sodium diethyl barbiturate in the fermentation medium, so that an improved process for making rifamycin B results. The rifamycin B is recovered from the fermentation medium in a conventional manner.

The following is a written description of the invention and of the manner and process of making and using it to enable the art skilled to make and use the same and sets forth the best mode contemplated by the inventors of carrying out their invention.

A suspension of spores of Streptomyces mediterranei ATCC 13685 was treated with N-methyl-N'-nitroso-N-nitroguanidine at 1 mg/ml in a pH 9.0 tris(hydroxymethyl)aminomethane buffer for 60 minutes at 28.degree. C. The mutagen-treated spores were then washed and plated onto Petri dishes containing Bennett agar. After 14 days incubation at 28.degree. C., the surviving colonies were picked off and examined for their ability to produce rifamycin B in liquid medium with and without sodium diethyl barbiturate. The mutant producing high quantities of rifamycin B in the absence of sodium diethyl barbiturate was further examined. Such mutant was called M 18 and deposited with the ATCC under the number 21789 and its characteristics will be described later in detail.

An industrial strain of Streptomyces mediterranei ATCC 13685 and strain M 18 were propagated on Bennett agar for 6 to 8 days at 28.degree. C. The vegatative medium was inoculated with a slant of Bennett agar (1/2 slant per flask) and the flasks were incubated on a rotary shaker at 28.degree. C. for 72 hours. The vegetative medium used contained:

The pH was adjusted to 7.3 with NaOH. An amount of 100 ml. vegetative medium was used in 500 ml. Erlenmeyer flasks. After 72 hours growth the vegetative medium was used to inoculate (5% v/v) the fermentation medium.

Two fermentation media were used:

RFB 744: a synthetic medium

RFB 2244: a complex medium

Fermentation were always carried out using 50 ml. of medium in a 500 ml. Erlenmeyer flask.

The composition of the two media was as follows:

The rifamycin B content of the filtered broths was assayed by a differential spectrophotometric technique described by Pasqualucci, et al., Journal of Pharmaceutical Sciences 59, 685, 1970.

Samples of fermentation medium were adjusted to pH 2.0 with hydrochloric acid and extracted with ethyl acetate. The organic extract was then extracted with the phosphate buffer, pH 7.38; under these conditions rifamycin B is extracted by the buffer whereas the rifamycin complex remains in the exhausted organic phase. The rifamycin complex in the exhausted ethyl acetate was estimated spectrophotometrically by measuring the optical density at 460 m.mu. of an aliquot suitably diluted in ethyl alcohol containing 1% ascorbic acid.

In industrial practice, the product rifamycin B is recovered from a fermented broth in usual ways.

For the investigation of growth characteristics, Str. mediterranei and its mutant strain M 18 were grown in a variety of standard media according to Gottlieb and Shirling, (Methods of Characterization of Streptomyces Species, Intern. J. Syst. Bact. 16, 313-340, 1966); and, in addition, some media recommended by Waksman were used (The Actinomycetes, Vol. II, Williams and Wilkins Co., 1961).

The following Tables report the growth characteristics and properties of the mutant strain M 18 ATCC 21789 and of Streptomyces mediterranei ATCC 13685.