HU184125B - New genetic process for preparing antibiotic producing micromono spora strains - Google Patents

Abstract

A process for the preparation of antibiotic-producer Micromonospora strains having modified genetic material, wherein following cultivation in a glycine medium, a protoplast suspension is prepared with lysozyme, under osmotically buffered conditions ensured by sucrose, from each culture of two mutants of the antibiotic- producer Micromonospora, at least one of which is genetically labelled, the two suspensions obtained are combined in the present of polyethylene glycol, and incubated at room temperature. The resulting fused protoplasts are suspended in soft agar, then plated on an agar plate containing prolin and inorganic salts, incubated for 20 to 30 days, and finally all mutants are selected from the regenerated colonies which are sure to have genetic material different from that of the parent strains.

Description

FIELD OF THE INVENTION The present invention relates to a method for producing genetically modified individuals of antibiotic-producing Micromonospora strains.

It is known that antibiotic production is genetically determined, and thus changes in the genetic stock of the producing strain also affect the ability to produce antibiotics. These stable genetic changes (mutations) also occur spontaneously with an extremely low frequency. The frequency of mutations can be increased by known methods using UV, near UV, gamma and X-rays as well as treatment with various mutagenic substances. These methods have been successfully used to increase antibiotic production capacity in different strains. The disadvantage of the method is that the induced mutation is not controllable, i.e. a random process, and that not all survivors of the mutation treatment are mutants. Thus, a very large number of survivors need to be screened while receiving a more productive mutant.

It is known that the levels of many enzymes increase with the increase of the gene dose, that is, if a gene doubles within a given chromosome, the level of the protein encoded by it is also approx. doubles. It is also known that raising the level of enzymes responsible for the synthesis of antibiotics enhances the synthesis of that antibiotic. The mechanism of gene duplication is unclear. It is also not known which mutagenic substance primarily causes gene duplication.

From the foregoing, it is understood that a method that significantly increases the number of gene duplications is of great importance in increasing the production of antibiotic-producing strains.

It may be industrially useful to produce a new species from two Micromonospora species producing similar antibiotics that have more favorable breeding or production properties than the parent strains, or to produce new valuable antibiotics that are related to the antibiotics produced by the parent strains.

The protoplast fusion method solves the above problems.

The object of the present invention is to enable the genetic modification of the Micromonospora strains to be advantageously altered by recombination.

With the exception of a single publication [Beretta et al. al. J. of. Bact. 107, 415 (1971)], no genetic information transfer method is known. Contrary to the 10 ' ⁶ recombination frequency for Streptomycetes, no classical methods were used to obtain recombinants for the Micromonospora strains, whereas the protolast fusion method for the same strains was capable of transmitting genetic information at 10' ^{3 to} 10 ' ⁴ frequencies.

Fusion of protoplasts combines the complete cytoplasm of two or more protoplasts, and thus the entire genetic stock therein. Selection yields either stable merodiploids or recombinants. In merodiploids, the number of genes responsible for antibiotic synthesis within the cell may increase, even if not directly selected for antibiotic production. Gene duplication may also occur during recombination.

Another advantage of the method is that, during protoplast fusion, large chromosome fragments from the donor to the recipient are much more frequently fixed in the survivors of selection than any other method used to date in bacterial genetics.

The protoplast fusion method has long been widely used to study the genetic properties of mammalian and plant cells and fungi.

The first reports of bacterial protoplast fusion due to difficulties in cell wall regeneration were published in 1976 by K. Fodor and L. Alföldi, Proc. Natl. Acad. Sci. USA 73, 2147 (1976); P. Schaeffer et al., Proc. Natl. Acad. Sci. USA 73, 2151 (1976); Hopwood D. A. et al., Natura 268, 171 (1977); R. H. Baltz: J. of Gén. Microb. 107: 93 (1978); 0. Godfrey et al., Can. J. Microbiol. 24, 994 (1978).

Successful protoplast fusion within the Micromonospora family has not been reported in the literature.

FIELD OF THE INVENTION The present invention relates to a method for producing modified genetically modified antibiotic-producing Micromonospora strains, wherein the two mutants of the antibiotic-producing Micromonospora strains, at least one of which is genetically labeled, are preferably protected with osmotic lysozyme after culturing in glycine medium. a suspension is prepared, the two suspensions thus obtained are mixed in the presence of polyethylene glycol and incubated at room temperature. The resulting fused protoplasts, preferably suspended in soft agar, are layered on agar plates containing sucrose, proline and inorganic salts, incubated for 20-30 days and selected from regenerated colonies whose genetic stock is distinctly different from that of the parents.

Preferably, 0.2-0.5% glycine is added to the culture medium to facilitate lysozyme lysis of the cell wall. Due to the inhibitory effect of glycine on cell wall biosynthesis, a common feature of glycine-sensitized cultures is that mycelial filaments are shorter and thicker than non-glycine-containing cultures and exhibit protrusions. Only cultures with such a microscopic image can be made into a sufficiently effective protoplast suspension.

Sensitized cultures are treated with osmotic protection (preferably 0.2-0.35 M sucrose) for 5 to 10 mg / ml lysozyme for 30-90 minutes.

Protoplast suspensions from different mutants of the strains are fused together in a 1: 1 ratio and treated with 6,000 molar polyethylene glycol. On a regeneration agar containing a suspension of fused protoplasts, containing sucrose for the osmotic protection, proline for the regeneration and inorganic salts, preferably calcium (II) chloride, niagnesium (II) chloride, potassium dihydrogen phosphate, incubate between temperatures. Protoplasts regenerate within 20-30 days.

The genetic characteristics of the selected parental pairs must be such that they are

-2ιο + tza treated recombinants could be selected by selection. Antibiotic resistance, which has already been used successfully in classical microbial genetics, and various auxotrophic indications can be used for this purpose.

In a preferred embodiment of the method of the invention, the strain Micisonospora inyoensis ATCC 27600, which produces sisomycin, requires rifampicin for growth but not rifampicin for growth (0.5 µg / ml rifampicin). already sensitive) parent pair. After fusion, we look for offspring that do not require casino acid for their growth but are resistant to rifampicin, i.e., form minimal colonies in the presence of 10 µg / ml rifampicin. Regeneration is carried out on a non-selective medium providing optimal conditions for regeneration. For selection of progeny of recombinant phenotype, regenerated colonies are washed and the suspension is plated on selective minimum agar medium containing 10 µg / ml rifampicin. Colonies grown here are derived from individuals with recombinant phenotypes resulting from fusion.

In another preferred embodiment of the method of the invention, a novel selection method is used wherein one parent is heat-labeled so that it completely loses its viability but its genetic stock remains intact. In this case, therefore, the entire genetic stock of one partner is introduced into the other fusion partner in heat-inactivated cytoplasm. The advantage of the method is that it allows post-fusion selection among parents, only one of which is genetically labeled.

This principle is used by gentamicin-producing Micromonospora purpurea var. nigrescent strain (MNG 00122) was used for a streptomycin-sensitive-streptomycin-resistant parent pair. To select for recombinants, the protoplasts of the resistant parent are inactivated by gentle heat treatment (55 ° C) prior to fusion. Because streptomycin susceptibility to streptomycin resistance is dominant, the selection of recombinants should allow 8 to 10 days (wait for phenotypic lag). After 8-10 days, 10,000 µg / ml streptomycin is plated on the regeneration plates. Colonies appearing within 20 to 30 days differ in genetic composition from parent pairs.

Applying the heat treatment inactivation technique, Micromonospora purpurea var. nigrescent strain was two orders of magnitude more resistant to streptomycin than Micromonospora inyoensis, we created an interspecific hybrid between the two strains by protoplast fusion. The essence of the method is that streptomycin-resistant Micromonospora purpurea var. a protoplast suspension of a nigrescent strain is heat-inactivated and then fused with a protoplast suspension of a streptomycin-sensitive Micromonospora inyoensis strain. After 8-10 days, 1000 µg / ml streptomycin agar is layered on the fused protoplasts to be regenerated. Colonies appearing after 20-30 days are interspecific hybrids.

The above principles were also applied to 4 other pairs of genetically tagged parents. One parent requires casino acid to grow and the other does not. To select for recombinants, protoplasts of the parent requiring casino acid for growth are inactivated by gentle heat treatment prior to fusion. Fused protoplasts are layered on minimal agar. Colonies appearing after 20-30 days have recombinant properties.

Suitably, the methods described in the literature for biosynthesized antibiotics may be used to test the ability of the strains of the genetically modified strains obtained by the method of the invention. so e.g. Micromonospora purpurea var., a gentamicin complex. nigrescens (MNG 00122), the strain was cultivated under fermentation conditions found to be optimal for antibiotic production.

I. et al., U.S. Pat. No. 168,778. Hungarian Patent (1973)], and then the total biological activity of the culture was measured using the agar diffusion method using the test organism Staphylococcus epidermidis and the composition of the biosynthesized gentamicin complex was determined by the Code of Federal Register (April 21, 1977) § 444.20 a / b 433-435. page).

The protoplast fusion method has been found to be capable of transmitting genetic information to other aminoglycoside-producing Micromonospora strains at relatively high frequencies. The following strains were also tested:

Micromonospora olivoasterospora

Micromonospora echinospora

Micromonospora purpurea (holotype)

The following examples illustrate the process of the present invention:

Example 1

The following two mutants of the micomonospora inyoensis strain ATCC 27600 producing schizomycin

1. Casaminic acid-demanding, rifampicin-resistant Cas'Rif ^R

2. In a 500 ml Erlenmeyer flask, 100 ml of sterile inoculum medium containing% soy flour% potato starch% sucrose was inoculated under frozen conditions with rifampicin-free vegetative mycelium of rifampicin-sensitive Cás ⁺ RifS free from casino acid.

0.4% calcium carbonate suspended in tap water.

The culture of the Cas'Rif ^R strain inoculated into the inoculum medium is incubated for 40 hours at 37 ° C on a shaker and sterile inoculated in a 500 ml Erlenmeyer flask.

100 ml sterile medium containing the composition

0.1% potassium nitrate

0.05% dipotassium hydrogen phosphate

0.05% magnesium sulfate water (1/7)

0.05% sodium chloride% glucose

0.1% yeast extract

0.3% casino acid

0.2% glycine in distilled water.

After inoculation, the culture is incubated on this medium for 48 hours on a shaker at 37 ° C,

The culture of the Cas ⁺ RifS strain inoculated into the inoculum medium is incubated for 65 hours at 37 ° C on a shaker and sterilized in 100 ml sterile medium in a 500 ml Erlenmeyer flask.

0.1% potassium nitrate

0.05% dipotassium hydrogen phosphate

0.05% magnesium sulfate water (1/7)

0.05% sodium chloride% glucose

0.1% yeast extract

0.5% glycine dissolved in distilled water.

After inoculation, the culture is incubated on this medium for 23 hours on a shaker at 37 ° C.

The cultures of both mutants are then centrifuged (6000 min- ¹ ) and the pellet is suspended in 20 ml of Pj medium, which is dissolved in the composition. The lysozyme cultures were slowly shaken in a 25 ml Erlenmeyer flask at 37 ° C. Cultures are quantitatively converted to protoplast after 30-60 minutes under microscopic observation. The resulting protoplast suspensions were sterilized by centrifugation and resuspended in 2 ml of P and repeated once more.

To carry out the protoplast fusion, the two different mutant protoplast suspensions were mixed at a 1: 1 ratio and allowed to stand for 0.1 min with 0.1 mL of the suspension with 0.9 mL of 50% aqueous polyethylene glycol (Ms 6000) containing 15% dimethyl sulfoxide. 0.05 ml of the polyethylene glycol-treated suspension is dropped into soft Rj medium containing 0.5% agar. The resulting mixture was layered on solid Rj medium containing 2.2% agar. Composition of R ₍ culture medium)

0.2 M saccharose 0.25 g / 1 potassium (I) sulfate 1 % glucose 0.3 % proline 0.01 % casamino 2 ml / 1 trace element solution 0,025 M TRIS-HCl buffer pfl 7.5 50 mM magnesium um (11) -chloride d 10 mM Calcium (II) chloride 0.2 mM potassium dihydrogen phosphate 0.1 % yeast extract

in distilled water.

after day, colonies grown at 32 ° C are washed with physiological saline and plated on the following medium:

0.2 M saccharose 0.25 g g / lg potassium (I) sulfate 0,025 M TR1S-HC1 buffer pH 7.2 (TRIS - tris-hydroxymethyl) -aminome perhaps 50 mM magnesium (II) chloride 10 inyl Calcium (II) chloride 0.8 mM Dipotassium hydrogen phosphate 2 ml / 1 trace element solution

0.1 0.05 % % potassium nitrate dipotassium hydrogen phosphate 0.05 % magnesium sulfate water (1/7) 0.05 % NaCI 0,001 % iron sulfate water (1/7) 2 % water-soluble starch 0,001 % rifampicin 1.5 % agar dissolved in distilled water.

dissolved in distilled water.

Composition of the trace element solution

40 mg / 1 zinc (II) chloride 200 mg / 1 ferric chloride water (1/6) 10 mg / l copper (II) chloride \ flavor (1/2) 10 mg / 1 manganese (II) chloride water (1/4) 10 mg / 1 disodium tetraborate water (1/10) 10 mg / 1 ammonium molybdate (VI) water (1/4)

The resulting suspensions were sterile centrifuged (6000 min ¹ ) and resuspended in 2 ml of P medium. To each of the two suspensions is added 1 to 1 ml of a sterile lysozyme solution (20 mg / ml) at a final concentration of 10 mg / ml / l. Lysosin [Calbiochem (San Diego California) 17,000 U / mg] P1 medium —After 6 days of incubation, colonies grown on this medium have a changed genetic stock relative to the parents.

Example 2

Gentamicin-producing Micromonospora purpurea var. nigrescens (MNG 00122)

Streptomycin-resistant Sni 1

Seeds of Streptomycin-sensitive Sm 2 frozen in vegetative mycelium were inoculated under sterile conditions with 100 ml of sterile medium in a 500 ml Erlenmeyer loinbik

1 % glucose 0.1 % ammonium chloride 0.4 % peptone 0.03 % magnesium (II) chloride water (1/6) 0.4 % Yeast! train 0.0058% ' NaCI 0.3 % glycine 0.03 % sodium sulfate water (1/10) 0.2 % potassium dihydrogen phosphate 0.5 / G / ml iron (II) chloride 0.4 % dipotassium hydrogen phosphate 0.35 M saccharose 0.05 % magnesium sulfate water (1/7) 2 ml / 1 trace element solution dissolved in distilled water. The pH of the medium is reduced to 7.5

dissolved in distilled water. 10

The culture was incubated on a shaker (320 min ^-1 ) for 42-44 hours at 37 ° C. Then, each culture was aseptically szuzsperdáljuk centrifuged (6000 min ^-1, 0 ° C) and the pellet in 20 ml of P medium that contained 15

0.2 M sucrose

0.025 g / l potassium sulfate

0.025 M 1RIS-HCl Buffer pH 7.2 mM Potassium (II) chloride 20 mM Nagium (II) chloride

0.8 mM potassium dihydrogen phosph: ml ml / L trace element solution

The flask is incubated without shaking at 55 ° C for 3 hours, and the protoplasts are resuspended by gentle shaking. The streptomycin sensitive non-heat treated protoplast suspension and the streptomycin resistant heat inactivated protoplast suspension were mixed in a 1: 1 ratio and 0.1 mL of the resulting suspension was added to 0.9 mL of 50% aqueous polyethylene glycol solution (Ms 6000) and stand for 10 minutes. 0.05 ml of the suspension containing the fused protoplasts are dropped into 3 ml of R ₂ medium containing 0.4% agar. The resulting mixture was layered onto ₂ /2% agar on silane R ₂ and incubated at 37 ° C. At day 8, the plates were layered with 3 ml of TR1S medium containing 0.5% agar at 1000 µg / ml streptomycin. The winters that appeared after day 20 grew as a result of protoplast fusion and subsequent recombination.

in distilled water.

The resulting sziszpenzi steriler were centrifuged (6000 min ^-1, 0 ° C) then re-suspended in 2 ml ndáljuk P2 medium. To 1 ml of kf t suspension is added 1 ml of a 10 mg / ml sterile lysozyme solution (lysozyme final concentration 5 mg / ml / lysozyme [Calbiochem (San Diego California) 17,000 U / mg)) The lysozyme cultures were shaken in a 25 ml Erlenmeyer-lorik beaker at 37 ° C (30 min ^-1 ) for 30-45 minutes by microscopic observation. The resulting protoplast suspensions were sterilized by centrifugation (6000 min ^-1 ) and the strain ptomycin-sensitive mutant protoplast was resuspended twice in 10 ml of R2 medium and repeated after centrifugation.

Composition of R ₂ ; 0.35 M saccharose 0.25 g ( potassium sulfate 1 % glucose 0.3 % proline 0.01 casein -hi d olizate um 50 mM calcium (chloride there Hi 50 nM magnesium (II) chloride 0.2 ΓιΜ potassium dihydrogen phosphate 2 nl / 1 trace element solution

example

The gentamicin tern, Micromonospora purpurea var. nigrescens strain below

1. It requires casino acid for growth

2. Growth without protoplast fusion requiring no azo amino acid; using the conditions described in Example 2. The non-casino acid mutant is heat-inactivated as described in Example 2 for the spheromycin-resistant mutant. The heat-inactivated protoplast suspension of prototrophic mesh and the non-heat-inactivated protoplast suspensions of casino acid were fused as in Example 2. The resulting mixture was layered on solid R ₂ medium without casino acid containing 2.2% agar and incubated at 37 ° C. Colonies appearing after 20-30 days grew as a result of protoplast fusion and subsequent recombination.

Example 4

To verify inter-species fusion, two bi-pairs were used:

distilled water; dissolved in ·

The streptomycin resistant mutant is also washed twice

P ₂ medium] In a 25 ml flask preheated to 1 ° C and then 55 ° C: 10 ml of TRI! is poured onto a medium with a composition

1. Micromonospora purpurea var. nigrescer

100 µg / ml streptomycin resistant

2. Micromonospora myoensis

0.5 µg / ml resistance to strept micin

1.2% TRIS

0.035% potassium-kk rid

Micromonospora purpurea var. nigrescen, strain 60 liters of heat-inactivated protoplast was prepared

-5184 125 suspensions. The heat-treated protoplasts are mixed in a 1: 1 ratio with the protoplast suspension prepared from Micromonospora inyoensis strain as in Example 1, the resulting suspension fused as in Example 1 and then regenerated. Regenerative protoplasts are inoculated on day 8 with 3 ml of 1000 µg / ml streptomycin in TR1S medium containing 0.5 '/ <' agar. Colonies appearing after day 20 grow as a result of protoplast fusion and recombination between the two species.

Claims (8)

Patent claims CLAIMS 1. A method for producing antibiotic-producing Micromonospora strains of non-genetic strains, wherein the two mutants of the antibiotic-producing Micromonospora strains, at least one of which is genetically labeled, are preferably provided with salucose after culturing in glycine medium. Prepare a protoplast suspension with lysosinr, mix the resulting two suspensions in the presence of polyethylene glycol and incubate at room temperature, then regenerate the resulting fused protoplasts with soft agar, preferably containing salarose, proline and inorganic salts. we select those whose genetic stock is different from their parents. 2. The method of claim 1, wherein the protoplasts are prepared from a culture at the beginning of the logarithmic phase. The method of claim 1 or 2, wherein the protoplasts of one parent are heat-inactivated. 4. The method of claim 1, 2 or 3, wherein the strain producing schizomycin is Micromonospora inyoensis. 5. A method according to claim 1, 2 or 3, wherein the gentamicin termini strain is Micromonospora purpurea var. nigres15 is used. 6. A method according to claim 1, 2 or 3 wherein the gentamicin producing strain is Micromonospora purpureate (holotype). 20 The method of claim 1, 2 or 3, wherein the gentamicin producing strain is Micromonospora eehinospora. 8. The method of claim 1, 2 or 3 wherein the fortimycin producing strain is Micromonospora olivoasterospora.