An antibiotic R106 represented by the general formula (I): ##STR1## wherein: R is methyl or ethyl; X.sub.1 is MePhe, .beta.-HOMePhe or Phe; X.sub.2 is allo-Ile, Val or Leu; X.sub.3 is MeVal or Val; X.sub.4 is .beta.-HOMeVal, .gamma.HOMeVal, MeVal, Val, N,.beta.-MeAsp, .beta.-HOMePhe, MePhe, MeDH.sub.2,3 Val or MeDH.sub.3,4 Val is produced by a process which comprises culturing a strain of the genus Aureobasidium that is capable of producing the said antibiotic R106 and collecting the said antibiotic from the fermentation broth. The antibiotic R106 compounds are useful in the treatment of fungal infection.
This application is a division of application Ser. No. 379,629, filed Jul. 13, 1989 now U.S. Pat. No. 5,057,493.
The present invention relates to novel antibiotics R106 which are useful as therapeutic agents for the treatment of fungal infection, and a process for producing the same as well as for the use thereof.
As therapeutic agents for the treatment of fungal infection, there are known approximately 20 antibiotics including amphotericin B, nystatin, trichomycin, griseofulvin, pyrrolnitrin, clotrimazole, miconazole nitrate, etc. However, these antibiotics are questionable in activity and toxicity.
An object of the present invention is to provide novel antibiotics which have a high activity but low toxicity as therapeutic agents for treating fungal infection.
The present inventors isolated a number of microorganisms from the surface of plant leaves or from the soil, for purposes to discover novel antibiotics, purified antibiotics produced by these microorganisms and examined the biological properties of these antibiotics. As a result, it has been found that a series of antibiotics showing an antimicrobial activity against pathogenic fungi such as Candida albicans, Cryptococcus neoformans, etc. can be produced in fermentation broth of microorganisms belonging to the genus Aureobasidium.
These antibiotics have been isolated from the fermentation broth; and as the result of examination of physicochemical properties of the antibiotics, it has been confirmed that these antibiotics are novel compounds that are not found in the literature. These antibiotics have been named R106. Thus, the present invention provides antibiotics R106 having the following structural formula and a process for production thereof as well as application thereof: ##STR2## wherein: R is methyl or ethyl;
X.sub.1 is MePhe, .beta.-HOMePhe or Phe;
X.sub.2 is allo-Ile, Val or Leu;
X.sub.3 is MeVal or Val;
X.sub.4 is .beta.-HOMeVal, .gamma.-HOMeVal, MeVal, Val, N,.beta.-MeAsp, .beta.-HOMePhe, MePhe, MeDH.sub.2,3 Val or MeDH.sub.3,4 Val.
The abbreviations for amino acids used in the above formula (I) are given in Table 9 later shown.
First, microorganisms used for the present invention can be a strain of the genus Aureobasidium which is capable of producing antibiotics R106 of the present invention represented by the general formula (I). An example of the microorganisms is Aureobasidium No. R106 (hereinafter this strain is referred to as strain No. R106), which the present inventors newly isolated from a soil sample collected at Kamitushima-cho, Kamiagata-gun, Nagasaki-ken, Japan. This strain is a new strain that has the characteristics described above and can produce advantageously novel antibiotics R106 of the present invention. Thus, this strain is one of the microorganisms which can be effectively used for the method of the present invention. Further, not only mutants of strain No. R106 which are prepared by natural or artificial mutation, but all microorganisms of the genus Aureobasidium which are capable of producing the antibiotics R106 of the present invention can be used for the method of the present invention.
Strain No. R106 has the following mycological characteristics.
(1) Growth on various nutrient media.
The following table shows cultural characteristics of strain No. R106 on various nutrient agar media after incubation for 4, 7, and 14 days at 25.degree. C.
Strain No. R106 shows good growth on potato-dextrose agar, Czapek agar, and malt extract agar media. Colonies of the strain are usually mucoid, pasty, or rarely velvety, and then become leathery as time goes by. The colonies are white to creamy or light pink in color, then changing olive green to light brown or brown, sometimes finally to black with production of dark brown pigments, which is insoluble, as time goes by. Rhizoidlike structures are often formed around the colonies. Hyphae with a size of 2 to 15 .mu.m in diameter elongate into the agar medium without forming aerial mycelia. Blastic conidia with a size of 1-5.times.2-10 .mu.m, are often formed intercalary or terminal on hyphae like finger tips and sometimes form ball-like clusters. Vegetative cells in early stage of growth are yeast-like, and ellipsoidal or lemon-like in shape with a size of 3-5.times.8-15 .mu.m and multiply by polyblastic budding. The strain forms arthrospores with a size of 4-10.times.8-20 .mu.m and chlamydospores with a size of 5-25.times.10-25 .mu.m. No ascospores are observed.
(2) Physiological characteristics.
1) Temperature range permitting growth
Temperature permitting growth : 12.5.degree.-29.0.degree. C.
Optimum temperature for growth : 23.0.degree.-29.0.degree. C.
2) Vitamin requirement
Growth in vitamin-free medium : Good growth.
3) Pigment formation
Insoluble dark brown pigments are produced. From the foregoing mycological characteristics, strain No. R106 is considered to belong to the genus Aureobasidium. Among the known species of Aureobasidium listed in "W. B. Cooke: Mycopathlogia et Mycologia Applicata, 17 , 1-43 (1962)", "J. A. von Arx, The Genera of Fungi Sporulating in Pure Culture, J. Cramer, Lehre (1970)", G. S. de Hoog & E. J. Hermanides-Nijhoff, "Studies in Mycology" No. 15, p. 141-166, CBS, Baarn (1977) and other references, strain No. R106 is considered to be a member of A. pullulans from its characteristics. But known A. pullulans did not produce an antibiotic R106. So we designated the strain as Aureobasidium pullulans No. R106, and the strain was deposited on Jul. 8, 1988 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, under deposit number FERM BP-1938.
The antibiotics R106 of the present invention can be produced by inoculating and culturing the strain described above in a nutrient medium.
In culturing the R106-producing organisms, there may be appropriately used as carbon sources, for example, glucose, fructose, saccharose, starch, dextrin, glycerin, molasses, thick malt syrup, oils and fats, organic acids, etc.; as nitrogen sources, organic nitrogen compounds or inorganic nitrogen compounds such as soybean powder, cotton seed powder, corn steep liquor, casein, peptone, Casamino acids, yeast extract, meat extract, germ, urea, amino acids, ammonium salts, etc.; as salts, inorganic salts, for example, sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, etc.; singly or in appropriate combination. If necessary and desired, heavy metals salts, e.g., iron salts, copper salts, zinc salts, cobalt salts, etc.; vitamins such as biotin, vitamin B1, etc.; other organic or inorganic compounds which can assist growth of the producing organisms and accelerate production of R106, may be suitably added. Furthermore, defoaming agents or surface active agents such as silicone oil, polyalkylene glycol ethers, etc. may also added to the medium.
For culture, conventional techniques used for production of antibiotics by fermentation of microorganisms may be adopted, and liquid culture methods especially by shaking or tank fermentation with aeration and agitation are most suitable. Further by appropriately supplementing carbon sources, nitrogen sources, trace salts, etc. during the course of incubation, the amount of antibiotics R106 production can be increased. A preferred temperature for the culture is generally in a range of 15 to 30.degree. C. A pH value for the culture is preferably in a range of 2 to 8. The number of days for fermentation may vary depending upon culture conditions but is sufficient generally 1 to 14 days.
The R106 thus accumulated in the fermentation broth can be advantageously collected therefrom by utilizing the physicochemical properties of the antibiotics.
That is, antibiotics R106 are contained in the fermentation broth and mycelial cake and can thus be obtained by extracting the whole fermentation broth with a hydrophobic organic solvent, for example, an organic solvent such as ethyl acetate, butyl acetate, chloroform, butanol, methyl isobutyl ketone, etc. Furthermore, antibiotics R106 can also be obtained after separation of the fermentation broth into the broth filtrate and mycelial cake by filtration or centrifugation. For isolation of R106 from the broth filtrate, the broth filtrate may be extracted with the aforesaid hydrophobic organic solvents. Alternatively, the broth filtrate may be brought into contact with an appropriate supporting resin to adsorb R106 in the filtrate thereto followed by elution with an appropriate solvent. For the purpose, supporting resins such as, for example, activated charcoal, cellulose powder, adsorptive resin, etc., which separate compounds according to their differences in the adsorbability to the resin, can be advantageously used. In order to elute antibiotics R106 from these resins aqueous solution of hydrophilic organic solvents, for example, aqueous acetone, aqueous alcohol, etc. can be used in appropriate combination, though the combination varies depending upon kind and property of the resin. Antibiotics R106 can be obtained from the mycelial cake by extracting with a hydrophilic organic solvent such as acetone or the like.
Crude R106 thus obtained can be further purified by conventional purification methods used for lipophilic antibiotics. An example of the methods is column chromatography with a supporting resin such as silica gel, activated aluminum, activated charcoal and adsorbtive resin.
In the silica-gel column chromatography, antibiotics R106 can be eluted with chloroform, ethylacytated, methanol, acetone, water, etc. which are used singly or in appropriate combination.
Isolation and purification by high performance liquid chromatography can also be advantageously utilized. Examples of the supporting resins which can be used include silica gel, silica gel with chemical bonds such as octadecyl, octyl or amino groups, or polystyrene type porous polymer gel, etc.
As a mobile phase, there may be used a mixed solvent of hexane, isopropyl alcohol and chloroform, etc., aqueous methanol or aqueous acetonitrile, etc.
Countercurrent chromatography which is a purification method based on differences of compounds in partition between two liquid phases can also be advantageously utilized. As the partition solvent system, there may be used a solvent mixture of hexane-ethyl acetate-acetonitrile, chloroform-methanol-water or the like.
The physicochemical and biological properties of these new antibiotics R106 are as explained below by referring partly to the accompanying drawings wherein:
Priority Data
Jul 19, 1988 [JP] 63-180095 Feb 16, 1989 [JP] 63-36736 Jun 19, 1989 [JP] 63-158112
The invention provides isolated nucleic acid compounds encoding the multiple drug resistance protein of Aureobasidium pullulans. Vectors and transformed host cells comprising the multiple drug resistance-encoding DNA of Aureobasidium pullulans are also provided. The invention further provides assays which utilize these transformed host cells.
The invention provides an isolated gene coding for a protein regulating aureobasidin sensitivity; a process for cloning the gene with the use of the gene or a part of the same as a probe; a nucleic acid probe being hybridizable with the gene; an antisense DNA or RNA of the gene; a recombinant or transformant having the gene contained therein; an isolated protein regulating aureobasidin sensitivity and a process for producing the same by using the transformant; an antibody for the protein; a process for detecting the protein or the gene; and a process for screening an antimycotic by using the protein or the transformant. The invention is useful in the diagnosis and treatment of diseases including mycoses.
A gene associated with sensitivity to the antimycotic agent, aureobasidin A, has been isolated. The gene can be detected in a variety of cell types, and variant forms of the gene have been identified in mutant yeast strains. The proteins encoded by these genes are useful in diagnosing and treating mycoses.
The invention provides isolated nucleic acid compounds encoding a multiple drug resistance protein of Aspergillus flavus. Vectors and transformed host cells comprising the multiple drug resistance-encoding DNA of Aspergillus flavus MDR-1 are also provided. The invention further provides assays which utilize these transformed host cells.
The invention provides isolated nucleic acid compounds encoding a multiple drug resistance protein of Aspergillus fumigatus. Vectors and transformed host cells comprising the multiple drug resistance-encoding DNA of Aspergillus fumigatus MDR-1 are also provided. The invention further provides assays which utilize these transformed host cells.
