WO2007136824A1

WO2007136824A1 - High-yield bacitracin-producing microorganism

Info

Publication number: WO2007136824A1
Application number: PCT/US2007/012057
Authority: WO
Inventors: Albert Fliss; Cherry Wu; Arthur Bailey; Avraham Yacobi
Original assignee: Taro Pharmaceuticals U.S.A., Inc.
Priority date: 2006-05-19
Filing date: 2007-05-21
Publication date: 2007-11-29

Abstract

The invention relates to bacteria which produces large amounts of bacitracin. The original wild-type bacterium (ATCC 10716) was genetically altered using nitrosoguanidine to produce the high-yield bacitracin-producing microbial colony no. 1335 (NRRL B-30918), which was screened for its ability to produce bacitracin. The high-yield bacitracin strain has improved capacity to produce bacitracin.

Description

HIGH-YIELD BACITRACIN-PRODUCING MICROORGANISM

FIELD OF THE INVENTION

The present invention relates to a process of preparing a high-yield bacitracin- producing mutant by chemically mutagenizing a wild-type bacitracin-producing bacterial strain. More specifically, the present invention is directed to a fermentation process for preparing bacitracin at an improved yield by employing the bacitracin-producing Bacillus licheniformis mutant.

BACKGROUND OF THE INVENTION

Bacitracin is an antibiotic polypeptide produced by Bacillus subtilis and Bacillus licheniformis. Bacitracin is active against many Gram (+) and a few Gram (-) bacteria species. There are several bacitracins identified (namely, bacitracins A, B, C₅ D, E₃ F and G), of which bacitracin A is of primary importance and is highly active. (Isolation: Johnson et al., Science 102, 376 (1945); Anker et al, J. Bacteriol 55, 249 (1948)).

Bacitracin B and C are active, but less so than bacitracin A, while the biological activity of the other bacitracin forms is relatively low. The zinc salt of bacitracin is especially valuable because it is stable to the effects of heat and storage over long periods of time.

Bacitracin preparation by organic chemical synthesis is not presently feasible.

The complex structure of bacitracin precludes the multi-step synthetic process, let alone the potential low yield accompanied with high cost and time that is required for its synthesis and purification after synthesis.

Fermentation is currently used to prepare bacitracins. Two bacteria strains are commonly used in the fermentation process, namely, Bacillus licheniformis and Bacillus subtilis. Using the Bacillus subtilis strain, Johnson et al. were the first to describe the fermentation production of bacitracin. (Science, vol. 102, pages 376-377). The highest yield reported in the Johnson's article was 10 units per milliliter. U.S. Pat. Nos. 2,457,887, 2,498,165 and 2,627,494 describe the effects of different nutrients (e.g., soybean meal and starch etc.) on bacitracin production. Bacitracin yield ranges from 10 to 92 units per milliliter of fermented medium. U.S. Pat Nos. 2,789,941 and 2,813,061 describe the yield improvement (about 80-90 to about 200-300 units/milliliter) with the use of dextrin and calcium carbonate as well as ammonium sulfate and a vegetable protein respectively. U.S. Pat. No. 2,567,698 discloses cultivating Bacillus subtilis in a suitable liquid nutrient fermentation medium and the yield is small, i.e., on the order of about 30-60 units per milliliter. U.S. Pat No. 2,828,246 describes the use of proteinaceous materials to improve the yield to about 325 units per milliliter, in 24 hours.

Using the Bacillus licheniformis strain, Haavik et al. describe an inducer role for amino acids (i.e. L-histidine and L-phenylalanine) in bacitracin production (FEMS Microbiology Letters. 10 (1981), 111-114). Supsk at al. describe enhancement of bacitracin biosynthesis by branched-chain amino acids (i.e., DL-4-azaleucine) in a Bacillus licheniformis mutant (Folia Microbio. 30, 342-348, 1985). Wild-type Bacillus licheniformis was first exposed to a chemical mutagen and subsequently cultured in the presence of DL-4-azaleucine (an inhibitor of L-leucine) to further select a DL-4- azaleucine resistant mutant. DL-4-azaleucine was utilized to enrich for leucine biosynthesis pathway mutants. The authors hypothesized that selected chemically- induced mutants that thrive in the presence of DL-4-azaleucine can produce higher amounts of bacitracin. Supek et al. reported the isolation of a DL-4-azaleucine-resistant mutant of Bacillus licheniformis after chemical mutagenesis using N-methyl-N'-nitro-N- nitrosoguanidine. However, the further selected DL-4-azaleucine-resistant mutant still produces a sub-optimal level of bacitracin (i.e. 4 times higher than the wild type).

It would be desirable to develop, using a chemical mutagenesis approach, a high- yield bacitracin-producing bacterial mutant. The present invention provides a bacitracin- producing mutant having a surprising capability of high-yield production of bacitracin.

SUMMARY OF THE INVENTION

The present invention provides a novel approach for the preparation and isolation of high-yield bacitracin-producing microorganisms. In particular, the present invention provides a unique fermentation process for the bacitracin-producing microorganisms to sufficiently produce bacitracin at an improved amount. It is an object of the present invention to provide a high-yield bacitracin- producing bacterial strain. Preferably, the high-yield bacitracin-producing bacterial strain is obtained by chemical-induced mutagenesis and is designated as microbial colony no. 1335 (NRRL B-30918).

It is another object of the present invention to provide a process of chemical mutagenizing a wild-type bacitracin-producing bacterial strain (Bacillus licheniformis, ATCC 10716) to provide a high-yield bacitracin-producing bacterial strain.

It is another object of the present invention to provide an isolating process for the high-yield bacitracin-producing strains.

It is another object of the present invention to provide a new and improved mutant of Bacillus licheniformis with increased bacitracin-producing ability.

It is yet another object of the present invention to provide a fermentation process using the high-yield bacitracin mutant bacterial strain to prepare bacitracin.

Other features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description of the invention. These advantages of the invention will be realized and attained by the methods particularly pointed out in the written description and claims hereof.

BRIEF DESCRIPTION QF THE DIAGRAMS

Figure 1 depicts a representative experiment of the fermentation production of bacitracin using a wild-type bacitracin strain (ATCC 10716)

Figure 2 depicts another representative experiment of the fermentation production of bacitracin using a wild-type bacitracin strain (ATCC 10716) Figure 3 depicts a representative experiment of the fermentation production of bacitracin using a low-yield bacitracin-producing control mutant (microbial colony no. 1006) Figure 4 depicts another representative experiment of the fermentation production of bacitracin using a low-yield bacitracin-producing control mutant (microbial colony no.

735)

Figure 5 depicts a representative experiment of the fermentation production of bacitracin using a high-yield bacitracin-producing mutant (microbial colony no. 1335, NRRL B-

30918)

Figure 6 depicts another representative experiment of the fermentation production of bacitracin using a high-yield bacitracin-producing mutant (microbial colony no. 1335,

NRRL B-30918)

DETAILED DESCRIPTION OF THE INVENTION

Definitions: As used herein, the term "isolated" refers to recovering the bacitracin from a fermentation broth; ''^■Bacillus licheniformis" refers to a species in the

Bacillus genus and is classified as a gram-negative rod; "wild-type" Bacillus licheniformis refers to the bacterial strain of ATCC 10716; "mutagenesis" refers to a process of forming or developing a mutation in a cell; "chemical-induced mutagenesis" refers to a mutagenesis process by exposing a cell to a chemical mutagen such as N- methyl-N'-nitro-N-nitrosoguanidine to form or develop a mutation in the cell;

"fermentation" broadly refers broadly to the bulk growth of microorganisms in a growth medium. No distinction is made between aerobic and anaerobic metabolism; "zone of inhibition" refers to a clear ring appearing around a paper disc containing an antibiotic.

If bacitracin is produced, a "zone of inhibition" appeared in the growth inhibition assay.

The larger the "zone of inhibition", the higher the amount of bacitracin produced;

"microbial colony" refers to the progeny of a single microbial cell in the original inoculum; and "passage" refers to a process of passing or maintaining a group of microorganisms or cells through a series of cultures.

For purposes of the present invention, the term "high-yield" refers to fermentation production of bacitracin in excess of about 3 mg/mL; the term "low-yield" refers to fermentation production of bacitracin of less than 1 mg/mL. For purposes of the present invention, the term "about" encompasses +/- 10% of a value.

For purposes of the present invention, the term "bacitracin" is intended to encompass all known bacitracins, such as bacitracin A, B, C, D, E, F, and G.

The present invention provides a chemically induced mutant of a Bacillus strain that is capable of producing bacitracin at a high yield in a fermentation.

In one embodiment, the present invention utilizes a chemically-induced mutagenesis approach to obtain a Bacillus licheniformis mutant. The chemically- induced mutagenesis protocol of Supek et. al. is followed (Supek et. al, 1984). Other suitable protocols for chemically-induced mutagenesis may be used and are understood to be encompassed by the present invention.

Suitable chemical mutagens include, but are not limited to, N-methyl-N'-nitro-N- nitrosoguanidine, hydroxy 1 amine, ethyl methane sulphonate and the like. Preferably, the chemical mutagen is N-methyl-N'-nitro-N-nitrosoguanidine. The exposure time and concentration of mutagen may be modified and optimized by one skilled in the art to achieve an optimal chemically-induced mutagenesis.

In another embodiment, the present invention provides a mutant strain of Bacillus licheniformis strain (NRRL B-30918). The prepared mutant of the present invention is shown to produce bacitracin in a much improved amount. The prepared mutant strain is distinct from the wild-type bacterial strain of Bacillus licheniformis (ATCC 10716), when compared with their bacitracin-producing abilities. The prepared mutant of the present invention is morphologically indistinguishable from its wild-type counterpart; nor is there any observable difference in their growth patterns.

After chemically-induced mutagenesis, mutated strains derived from the wild- type bacterial strain Bacillus licheniformis were screened for improved bacitracin production potential. In accordance with the present invention, a microorganism mutant strain (NRRL B-30918) is shown to be a high-yield bacitracin producer when cultivated in a fermentation medium for a period of time.

It is to be understood that a high-yield bacitracin producing mutant (prepared in accordance with the present invention) may or may not have the same identifying biological characteristics of the parent or progenitor strain, as long as the mutant produces bacitracin at an improved amount.

Preferably, the mutated bacterial cell strain produces at least about 3 mg/mL of bacitracin in a fermentation process. More preferably, the mutated bacterial cell strain produces at least about 4 mg/mL of bacitracin in a fermentation process. More preferably, the mutated bacterial cell strain produces at least about 5 mg/mL of bacitracin in a fermentation process.

Medium for use in preparing inoculum may contain additional components as appropriate, such as peptone, N-Z Amine, enzymatic soy hydrolysate., additional yeast extract, malt extract, vegamine, supplemental carbon sources and various vitamins.

In accordance with the present invention, the high-yield bacitracin-producing mutants are cultured in a culture or fermentation medium that comprises a carbon source and a nitrogen source. One skilled in the art may determine the carbon source and the nitrogen source. Illustrative examples of suitable supplemental carbon sources include, but are not limited to, other carbohydrates, such as soybean flour, wheat flour, corn flour, glucose, fructose, mannitol, starch or starch hydrolysate, cellulose hydrolysate and molasses; organic acids, such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols, such as glycerol, inositol, mannitol and sorbitol.

Illustrative examples of suitable nitrogen sources include, but are not limited to, ammonia, including ammonia gas and aqueous ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea; nitrate or nitrite salts, and other nitrogen-containing materials, including amino acids as either pure or crude preparations, meat extract, peptone, fish meal, fish hydrolysate, corn steep liquor, casein hydrolysate, soybean cake hydrolysate, yeast extract, dried yeast, ethanol-yeast distillate, soybean flour, wheat flour, corn flour, cottonseed meal, and the like.

Besides the carbon and nitrogen sources, the culture medium contains suitable inorganic salts, and, as appropriate, various trace nutrients, growth factors and the like suitable for cultivation of the microorganism strain. Illustrative examples of suitable inorganic salts include, but are not limited to, salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper, molybdenum, tungsten and other trace elements, and phosphoric acid.

Illustrative examples of appropriate trace nutrients, growth factors, and the like include, but are not limited to, coenzyme A, pantothenic acid, pyridoxine-HCl, biotin, thiamine, riboflavin, flavine mononucleotide, flavine adenine dinucleotide, DL-6,8- thioctic acid, folic acid, Vitamin B12, other vitamins, amino acids such as cysteine and hydroxyproline, bases such as adenine, uracil, guanine, thymine and cytosine, sodium thiosulfate, sodium sulfate, calcium carbonate, dipotassium phosphate, p- or r- aminobenzoic acid, niacinamide, nitriloacetate, and the like, either as pure or partially purified chemical compounds or as present in natural materials.

The amount of each of these ingredients to be employed is preferably selected to maximize the bacitracin production. Such amounts may be determined empirically by one skilled in the art according to the various methods and techniques known in the art.

Preferably, the culture medium (for fermentation) used in bacitracin production contains soy flour (80 grams), corn flour (40 grams), sodium sulfate (5 grams), calcium carbonate (5 grams), vegamine (3 grams) and dipotassium phosphate (0.3 gram).

The fermentation culture conditions employed, including temperature, pH, ' aeration rate, agitation rate, culture duration, and the like, may be determined empirically by one of skill in the art to maximize bacitracin production. The selection of specific culture conditions depends upon factors such as the particular inventive microorganism strain employed, medium composition and type, culture technique, and similar considerations,

In a preferred embodiment of the present invention, when employing the mutant bacterial strain (e.g., microbial colony no. 1335, NRRL B-30918), cultivation takes place at a temperature in the range of about 37°C (i.e., about 35°C — about 39°C, and at a pH in the range of about 7 to about 9, preferably in the range of about 8 to about 8.5. The culture conditions employed can, of course, be varied by known methods at different time-points during cultivation, as appropriate, to maximize bacitracin production. Preferably, the time of fermentation is about 19 to about 22 hours.

Cultivation of the high-yield bacitracin-producing microorganism strain may be accomplished using any of the submerged fermentation techniques known to those skilled in the art, such as airlift, traditional sparged-agitated designs, or in shaking culture.

After cultivation for a sufficient period of time, such as, for example, from 19 to 22 hours, the bacitracin that has accumulated in the cells and/or culture broth is isolated according to suitable known methods including ion exchange chromatography, gel filtration, solvent extraction, affinity chromatography, or any combination thereof. For example, solvent extraction using a lower alkyl alcohol is suitable. Preferably, solvent extraction using N-butanol is suitable. Other methods that are suitable with the conditions employed for cultivation may also be used; illustrative examples of suitable methods for recovering bacitracin are described in U.S. Pat. Nos. 2,498,165, 2,609,324, 2,776,240, 2,915,432, and 3,795,663; illustrative examples of suitable methods for preparation of Zn bacitracin are described in U.S. Pat. No. 3,937,694.

Microorganism Deposit A subculture of the high-yield bacitracin-producing Bacillus licheniformis mutant

(i.e., microbial colony no. 1335, NRRL B-30918) has been deposited in connection with the present invention. The present microbial strain, prepared and used for carrying out the examples, has been deposited at the Agricultural Research Service Culture Collection CNRRL), located at 1815 North University Street, Peoria, Illinois 61604 U.S.A., pursuant to the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. AH restrictions on the availability of the materials deposited will be irrevocably removed upon the issuance of a patent thereon.

EXAMPLES

MATERIALS AND METHODS Strain and Cultivation

A strain of wild-type Bacillus licheniformis (ATCC 10716) was cultivated and maintained in 2X YT medium at 30⁰C. 2X YT medium contains 16 grams/L of tryptone, 10 grams/L of yeast extract, and 5 grams/L of NaCl.

Analytical Methods

Quantification of Bacitracin: Micrococcus luteus Growth Inhibition Test Bacitracin activity was determined by an agar diffusion method (see US Pharmacopoeia method) using Micrococcus luteus (ATCC 10240) as a test microorganism. In this method, 5 mL of enriched nutrient broth, containing 5.4 grams/L of nutrient broth, 2.5 grams/L of yeast extract, and 12.5 grams/L of heart infusion broth, were inoculated with a loop of M. luteus and incubated at 37°C for 18 hours. Culture plates were prepared with base agar containing 800 ml, of Antibiotic Medium I (DF263- 17) and seed agar containing 200 mL of Antibiotic Medium I and 5 mL of M. luteus culture. 20 ml, of base agar at 50⁰C was poured onto sterile polyethylene culture plates with a diameter of 10 cm. After 30 minutes, 5 mL of seed agar at 45°C was poured onto the base agar. Poured plates could be kept for 2 weeks at 4°C.

In order to assay for bacitracin activity, one (1) mL aliquot of culture media was centrifuged at 13,000 rpm for 5 minutes to remove the cells. Ten (10) μl of each of the supernatants were pipetted onto a sterile filter paper disc. The sterile filter paper discs were dried in air for approximately 5 minutes. The sterile filter paper discs were carefully applied onto the surface of the Micrococcus luteiis plates (described above). Negative controls included liquid culture media. In order to quantify bacitracin production, standards were utilized (ranging from 0.5 mg/mL to 5 mg/mL).

At the end of the 18-hour incubation, plates were visualized and checked for the presence of any zones of inhibition exhibited by the supernatants from respective microbial colonies. In order to determine bacitracin concentration, diameters of the zone of inhibition for the test samples were compared to that of the known bacitracin standards.

The following non-limiting examples below serve for further explanation of the invention.

Example 1

Chemical Mutagenesis of Bacillus licheniformis A high-yield bacitracin-producing mutant strain was generated by chemicalinduced mutagenesis. The protocol for chemical-induced mutagenesis was adopted from Supek et al. (1984), using N-methyl-N'-nitro-N-nitrosoguanidine with minor modifications.

In brief, an exponentially growing culture of Bacillus licheniformis was washed twice in normal saline by centrifugation. Pellets were resuspended in 50 mM Tris- maleic acid buffer. The mutagen (N-methyl-N'-nitro-N-nitrosoguanidine) was dissolved in cold (4°C) 50 mM Tris-maleic buffer at a concentration of 2 mg/mL. The mutagen was added to the Bacillus licheniformis culture to induce mutagenesis and incubated for 1, 2 or 3 hours at a final concentration of 1 mg/mL of mutagen at room temperature. The mutagen was removed by washing the cells twice with 2X YT. Cells were subsequently resuspended in 2X YT medium. One-tenth mL of different dilutions was placed on 2X YT agar plates, which were further incubated at 30⁰C for 1 day.

Example 2 Preliminary studies were initially performed using DL-4-azaleucine in an attempt to isolate a high-producing bacitracin mutant. Results suggest that this approach failed, because the mutagenesis protocol (Supek et. al.) allowed only for the isolation of DL- 4azaleucine enriched mutants having minimal increases in bacitracin production, (data not shown). Subsequently, we researched a novel screening approach. This novel approach does not utilize any branch-chain amino acids (i.e. DL-4-azaleucine).

Unexpectedly, we discovered that the omission of the DL-4-azaleucine in the screening protocol resulted in the isolation of higher yield bacitracin producing mutants. Accordingly, we adopted this direct screening approach (i.e. immediately after chemical mutagenesis the isolated clones were screened for bacitracin producing capability).

Direct Screening Approach for High-Yield Bacitracin-Producing Mutants

After the chemical-induced mutagenesis, high-yield bacitracin-producing mutant strains were obtained by screening for improved ability to produce bacitracin. Screening was performed by using the Micrococcus ϊuteus Growth Inhibition Test (described above) and compared to that of the wild-type Bacillus licheniformis.

Approximately twenty-five (25) rounds of chemical-induced mutagenesis were performed. Out of an approximately one hundred thousand (~100,000) mutant clones, only one stable mutant clone exhibited a high-yield production for bacitracin as compared to wild-type Bacillus licheniformis. While some mutants appeared to exhibit high-yield bacitracin production in the first passage, the ability of the high-yield bacitracin production was lost during further passages. Without wishing to be bound by a theory, it is believed that the rare occurrence of a stable high-yield bacitracin-producing mutant strain is in agreement with the rare occurrence of a proper mutation at the single gene level. This particular mutant (i.e., microbial colony no. 1335, NRRL B-30918) is shown to consistently produce bacitracin at a concentration about 8-10 folds more than that of the wild-type Bacillus licheniformis. The microbial colony no. 1335 (NRRL fi¬

ll 30918) was found to be stable after several passages. More specifically, the microbial colony no. 1335 (NRRL B-30918) produces bacitracin in a fermentation broth in the amount of at least 3 mg/mL.

Example 3

Fermentation Production of Bacitracin Using Wild-type Bacillus licheniformis

Wild-type Bacillus licheniformis were cultured under the following fermentation condition in a 2 L ferm enter for optimal production of bacitracin.

Preliminary Culture of Wild-type Bacillus licheniformis

Seed culture was prepared by inoculating 3 ml of 2X YT medium with a loop of wild-type Bacillus licheniformis cells in a 10-mL sterile culture tube. The seed culture was incubated with rotary shaking (250 rpm) at 37°C for 15 to 18 hours. 1 mL of subculture was then transferred to a 100 mL of 2X YT medium, under identical conditions describe above, for 6 to 8 hours.

Fermentation Culture of Wild-type Bacillus licheniformis

Sixty (60) mL of seed culture was inoculated into a 3.3 L BioFlo III fermentor from New Brunswick Scientific Instruments (New Jersey, USA) containing 2L of fermentation medium (80 grams of soy flour, 40 grams of wheat flour, 5 grams of sodium sulfate, 5 grams of calcium carbonate, 3 grams of wheat vegamine and 0.3 gram of dipotassium phosphate).

During the fermentation, a temperature of 37°C was maintained. The culture was sparged with sterile air at 0.5 vol/vol/min aeration and stirred at a mechanical stirrer speed of 500 rpm.

The fermentation time was 19-22 hours. At the end of the fermentation period, the pH of the fermentation broth was usually pH 8-8.5. After growth in the fermentation culture, cells were centrifuged for 5 minutes at 13,000 rpm and the supernatant was collected. Bacitracin activity of the fermentation supernatant was measured (see Micrococcus luteus Growth Inhibition Test above). In brief, sterile filter papers (0.5 cm) were gently placed onto each culture plate, and 10 μL of supernatant or bacitracin standards was pipetted onto the circular filter discs. Plates were incubated at 37°C for 18 hours. After incubation, the circular zones of inhibition were compared between the test samples (wild-type strain and mutant strain fermentation supernatant) and the bacitracin standards. Purified bacitracin (0.5 mg/mL to 5 mg/mL) was used as a standard. Concentrations of bacitracin produced were calculated by comparing the diameters of the zone of inhibition of the tested samples to those of the bacitracin standards.

Figures 1 and 2 show two separate representative experiments of the fermentation production of bacitracin using the wild-type Bacillus licheniformis. The yield of bacitracin was consistently below 1 mg/mL. For example, the yield was 0.5 mg/mL in one experiment (see Figure 1), and the yield was also 0.5 mg/mL in another experiment (see Figure 2).

Example 4

Fermentation Production of Bacitracin Using Bacillus licheniformis Mutants

The high-yield Bacillus licheniformis mutant (i.e., microbial colony no. 1335, NRRL B-3091S) was similarly prepared as described in Example 1 and cultured to produce bacitracin using the fermentation process as described in Example 3.

It was observed that the growth pattern of the high-yield Bacillus licheniformis mutant (i.e., microbial colony no. 1.335, NRRL B-30918) was identical to that of the wild-type Bacillus licheniformis. Both the colony appearance and the colony morphology of the high-yield Bacillus licheniformis mutant (i.e., microbial colony no. 1335, NRRL B-30918) appeared to be also identical to that of wild-type Bacillus licheniformis. The production of bacitracin by the high-yield Bacillus licheniformis mutant (i.e., microbial colony no. 1335, NRRL B-30918) remained the same even after several passages (i.e., 5-10 passages). Figures 5 and 6 show two separate representative experiments of the fermentation production of bacitracin using the high-yield bacitracin-producing mutant strain (i.e., microbial colony no. 1335, NRRL B-30918). The yield was 4.0 mg/mL for microbial colony no. 1335 (NRRL B-30918) (see Figure 5). The yield was 5.0 mg/mL for microbial colony no. 1335 (NRRL B-30918) (see Figure 6).

Example 5

Comparative Studies:

Fermentation Production of Bacitracin Using Bacillus licheniformis Mutants that Have Low-Yield of Bacitracin Production

In a comparative control study, fermentation production of bacitracin was evaluated using mutants that have undergone the chemical-induced mutagenesis process. Illustrated in two separate representative experiments were two low-yield bacitracin- producing mutants (i.e., microbial colony nos. 735 and 1006). These two low-yield Bacillus licheniformis mutants were prepared as described in Example 1 and cultured to produce bacitracin using the fermentation process as described in Example 3.

Figures 3 and 4 show two separate representative experiments of the fermentation production of bacitracin using the control low-yield bacitracin-producing mutant strains (i.e., microbial colony nos. 735 and 1006). The yield was 0.5 mg/mL for microbial colony no. 735 (see Figure 3) and the yield was 0.5 mg/mL for microbial colony no.1006 (see Figure 4). Accordingly, these comparative studies indicate the specificity for the high-yield bacitracin-producing strain and not the result of chemical-induced mutagenesis in general.

Example 6

Recovery of Bacitracin from the Fermentation Broth

Bacitracin was recovered from the fermentation broth using an extraction method. Specifically, two (2) L of fermentation broth were mixed with two (2) L of water-saturated N-butanol (1 :1 vol/vol). The mixture was shaken in a separatory funnel (about 2 minutes) and allowed to settle for about 30 minutes at room temperature. Two phases resulted. The upper phase (i.e., organic phase) contains the bacitracin. The lower phase (i.e., aqueous phase) was extracted again. The extraction was similar to that described before; that is, an equal volume of the lower phase was mixed with an equal volume of water-saturated N-butanol (1:1 vol/vol). The two (2) organic phases were combined and concentrated by rotary evaporation to remove the N-butanol. The concentration of bacitracin recovered from the fermentation was determined using the Micrococcus luteus Growth Inhibition Test as detailed above.

Example 7

Preparation of Zinc Bacitracin Zinc bacitracin may be conveniently prepared using a standard available protocol

(See, for example, U.S. Pat. No.3,937,694).

In brief, a fermentation broth is first acidified to a pH of 2-3. The acidified fermentation broth containing bacitracin is then increased to a pH above 7.0 (by adding, for example, NaOH), in the presence of an optimal amount of soluble zinc salt and ammonium sulfate. This process is performed under constant mechanical stirring to ensure optimal mixing of the reactants (i.e., bacitracin, zinc salt and ammonium sulfate). During this process, the Zn bacitracin is precipitated and a majority of the zinc hydroxide (i.e., Zn(OH)Z) is maintained in a soluble form. The resulting zinc bacitracin precipitate is filtered and dried to obtain zinc bacitracin.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. The disclosures of the cited publications in the present application are incorporated by reference herein in their entireties by reference. It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims. Bacteria sp. MASF-1335; Accession No. B-30918

AUSTRALIA

The applicant hereby gives notice that the furnishing of a sample of a microorganism shall only be effected prior to the grant of a patent, or to the lapsing, refusal or withdrawal of the application, to a person who is a skilled addressee without an interest in the invention (Regulation 3.25(3) of the Australian Patents Regulations).

CANADA

The applicant hereby requests that, until either a Canadian patent has been issued on the basis of the application or the application has been refused, or is abandoned and no longer subject to reinstatement, or is withdrawn, the furnishing of a sample of deposited biological material referred to in the application only be effected to an independent expert nominated by the Commissioner of Patents.

DENMARK

The applicant hereby requests that, until the application has been laid open to public inspection (by the Danish Patent and Trademark Office), or has been finally decided upon by the Danish Patent and Trademark Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Danish Patent and Trademark Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Danish Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Danish Patent and Trademark Office or any person approved by the applicant in the individual case.

FINLAND

The applicant hereby requests that, until the publication of the mention of the grant of a patent by the National Board of Patents and Registration of Finland, or for 20 years from the date of filing if the application has been finally decided upon without resulting in the grant of a patent by the National Board of Patents and Registration of Finland, the furnishing of a^' sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the National Board of Patents and Registration of Finland or any person approved by the applicant in the individual case.

GERMANY

The applicant hereby request that, until the grant of a patent or for 20 years from the date of filing if the application is refused or withdrawn, a sample shall only be issued to an independent expert nominated by the applicant. The request shall be filed with the German Patent and Trade Mark Office before technical preparations for publication of the international application are considered to be completed. Bacteria sp. MASF-1335; Accession No. B-30918

ICELAND

The applicant hereby requests that, until a patent has been granted or a final decision taken by the Icelandic Patent Office concerning an application which has not resulted in a patent, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Icelandic Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Icelandic Patent Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Icelandic Patent Office or ariy^?person -approved by the applicant in the individual case. "' " ^?

NORWAY

The applicant hereby requests that, until the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant' with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Norwegian Patent office or any person approved by the applicant in the individual case.

SINGAPORE

The applicant hereby requests that the furnishing of a sample of a microorganism shall only be made available to an expert. The request to this effect must be filed by the applicant with the International Bureau before the completion of the technical preparations for international publication of the application.

SPAIN

The applicant hereby request that until the publication of the mention of the grant of a Spanish patent or for 20 years from the date of filing if the application is refused or withdrawn, the biological material shall be made available as provided in Article 45 SPL only by the issue of a sample to an independent expert, the applicant must, by a written statement, inform the International Bureau accordingly before completion of technical preparations for publication of international application. Such statement must be separate from the description and claims of the international application and must preferably be made on Form PCT/RO/134, referred to in Section 209 of the Administrative Instructions under the PCT and reproduced in Annex Z of Volume I of the PCT Applicant 's Guide.

SWEDEN

The applicant hereby requests that, until the patent has been granted by the by the Swedish Patent and Registration Office, or has been finally decided upon without resulting in the grant of the patent, the furnishing of a sample shall only be effected to an expert in the art. The same is applied to rejected or withdrawn application within a period of 20 years from the Bacteria sp. MASF-1335; Accession No. B-30918

filing date. The request to restrict the furnishing of a sample to an expert in the art shall be filed by the applicant with the Swedish Patent and Registration Office, at the latest, by the day upon which technical preparations for publication of the application are considered to be completed.

UNITED KINGDOM

The applicant hereby requests that the furnishing of a sample of a microorganism shall only be made available to an expert. The request to this effect must be filed by the applicant in writing to the International Bureau before technical preparations for publication of the international application are completed.

EUROPEAN PATENT ORGANIZATION

The applicant hereby request that, until the publication of the mention of the grant of a European patent or for 20 years from the date of filing if the application is refused or withdrawn or deemed to be withdrawn, the biological material shall be made available as provided in Rule 28(3) EPC only by the issue of a sample to expert nominated by the requestor (Rule 28(4) EPC), the applicant must, by a written statement, inform the International Bureau accordingly before completion of technical preparations for publication of the international application. Such statement must be separate from the description and the claims of the international application and must preferably be made on Form PCT/RO/134, referred to in Section 209 of the Administrative Instructions under the PCT and reproduced in Annex Z of Volume 1 of the PCT Applicant 's Guide.

Claims

We claim:

I. A mutant oi Bacillus licheniformis, said mutant is designated as microbial colony no. 1335 (NRRL B-30918). 2. The mutant according to claim 1, wherein the mutant is obtained by chemically induced mutagenesis.

3. A biological pure culture of ^'a Bacillus licheniformis mutant, said mutant is designated as microbial colony no. 1335 (NRRL B-30918) and said culture being capable of producing bacitracin. 4. A process of producing bacitracin, comprising the steps of a) culturing a mutant of Bacillus licheniformis designated as microbial colony no. 1335 (NRRL B-30918) in a fermentation medium to produce a bacitracin; and b) isolating the bacitracin from the fermentation medium. 5. The process according to claim 4, wherein the mutant is obtained by chemically induced mutagenesis.

6. The process according to claim 4, wherein the culturing step is performed at a temperature of about 35°C to about 39°C.

7. The process according to claim 4, wherein the culturing step is performed at a temperature of about 37°C.

8. The process according to claim 4, wherein the culturing step is performed at a pH of about 7 to about 9.

9. The process according to claim 4, wherein the culturing step is performed at a pH of about 8 to about 8.5. 10. The process according to claim 4, wherein the culturing step is performed for about 19 to about 22 hours.

I 1. The process according to claim 4, wherein the culturing step is performed for about 19 hours.

12. The process according to claim 4, wherein the isolating step is performed by solvent extraction using N-butanol.

13. The process according to claim 4, wherein the isolated bacitracin is in excess of about 3 mg/mL.

14. The process according to claim 4, wherein the isolated bacitracin is in excess of about 4 mg/mL.

15. The process according to claim 4, wherein the isolated bacitracin is in excess of about 5 mg/mL. 16. A process of preparing a high-yield bacitracin-producing bacterial strain, comprising the steps of a) exposing a wild-type Bacillus licheniformis ATCC 10716 to a chem ical mutagenizing agent selected from the group consisting of hydroxylamine, N-methyl-N'- nitro-N-nitrosoguanidine, and ethyl methane sulphonate to prepare a mutant of Bacillus licheniformis; and b) isolating the mutant of Bacillus licheniformis that produces at least about 8 to about 10 fold more bacitracin as compared to that of the wild-type Bacillus licheniformis in a fermentation process.

17. The process according to claim 16, wherein the mutagenizing agent is N- methyl -N ' -nitro-N -nitrosoguan idine.

18. The process according to claim 16, wherein the mutant of Bacillus licheniformis is a subculture of microbial colony no. 1335 (NRRL B-30918).

19. The process according to claim 16, wherein the mutant of Bacillus licheniformis produces at least about 3 mg/mL of bacitracin in a fermentation process. 20. The process according to claim 16, wherein the mutant of Bacillus licheniformis produces at least about 4 mg/mL of bacitracin in a fermentation process.

21. The process according to claim 16, wherein the mutant of Bacillus licheniformis produces at least about 5 mg/mL of bacitracin in a fermentation process.