DD269166B5 - Process for producing recombinant gene products - Google Patents

Description

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Field of application of the invention

The invention relates to a microbial process for the production of recombinant gene products, in particular of enzymes, immunomodulators, immunodeterminators, hormones and of other biologically active peptide active ingredients, in genetically modified producer strains.

With the help of genetic engineering, the genes for numerous active substances from o.g. Product groups isolated, characterized and partially sequenced. Most of the products encoded by these genes are medicinally-applied substances, e.g. Streptokinase, interferon, interleukin, insulin; others play an important role in the food and beverage industry, eg. A-amylase, β-glucanase, chymosin.

The field of application of the invention is therefore in biotechnological productions in the pharmaceutical-chemical as well as in the food and beverage industry. Another field of application is microbiological and genetic engineering research.

Characteristic of the known state of the art

As producers (cloning hosts) of homologous and heterologous recombinant gene products, only bacterial, yeast and fungal strains whose cells have an intact cell envelope have been used (Genetics of Industrial Microorganisms - 1978, 1962 (Sebek OK and Laskin, AI, Edts Genetic Engineering I-VII [Setlow, JK and Hollaender, A.], Plenum Press, New York, 1979-1985, Biotechnology and Genetic Engineering Reviews, Vol. GE, Edt.), Intercept, Ponteland, Newcastle upon Tyne, 1984-1986; Biology of Industrial Microorganisms [A.L.Demaein and N.A. Solomon], The Benjamin / Cumminge Publishing Comp. Inc. London, 1985; Malke, H., Gerlach, D., Koehler, W. and Ferrettl, J.J., 1984, MoI. Gene. Genet. 196,360 to 363; Kleßen, C. and Malke, H. "1986, J. Basic Microbiol. 26.75 to 81; Patent Specifications DD 159782, DD 203071, DD 202307, DD 203330, DD 203331, DE-OS 31 52001, DD 245444, EP 0089692, EP 01 51337, WO 8S / 00382, WO 85/03945 and many others).

The insects are Escherichia coli, Bacillus subtilis, Actinomycetes, streptococci, pseudomonads and yeasts.

The use of these microorganisms for the production of practice-relevant Genprodukiu is; to varying degrees associated with the following disadvantages:

Predominantly intracellular localization of the gene products associated with aggregation into inclusion bodies and more or less less activity losses (eg IFN, prochymosin),

Destruction of the cells in the isolation of intracellular gene products and loading of the product fraction with intracellular proteins and tariff wall components,

High content of the product fraction of endotoxins, immunoreactive substances and other pollutants originating from the cytoplasm and in particular from the cell wall,

Destruction or inactivation of the gene products formed in the periplasmic space or in the culture medium by periplasmic or extracellular enzymes, in particular proteases.

Stable bacterial protoplast type L-forms, d. H. Bacterial cells that are viable without a cell wall have been the subject of basic microbiological research for about 30 years. They are experimentally derived from normal parent bacterium species organisms that can permanently proliferate without cell wall. The most important differences to the parent bacteria are:

- missing cell wall structures,

- spherical or pleomorphic cell forms of different sizes and without morphogenetic determination,

- altered colony morphology,

- formation of membranous granules,

Greater osmotic sensitivity,

- inability to reversion,

Resistance to β-lactams and other antibiotics which inhibit cell wall biosynthesis,

- Resistance to bacteriophages as well

- Existence of chemical and structural alterations of the cytoplasmic membrane (J. Gumpert & U. Taubeneck, Experientia Suppl., Vol. 46, 1983, 227).

In stable L-forms, cell wall biosynthesis is irreversibly blocked, and the organisms are considered to be extremely stable as well as pleiotropic mutants that share many phenotypic properties with the mycoplasma. By DNA-DNA hybridization (BH Hoyer & JR King, J. Bacteriol., Vol.97, 1969, 1516) and comparisons (agarose gel banding pattern of the chromosomal DNA after digestion with restrictases, differences in the genome of the L-forms and proven parents.

The L-forms grow on agar media and in liquid nutrient solutions with additions of 5% to 10% serum, of osmotic stabilizers (5% to 10% sucrose, 2% to 5% NaCl), of divalent cations (Mg ⁺ *, Ca ⁺⁺ ) and complex components such as yeast extract and peptones (J.Gumpert & U. Taubeneck, loc. Cit.) In recent years, the transformability of protoplast-type L-forms with naturally-formed resistance plasmids has been reported (see the related references in J Gumpert & U. Taubeneck, supra). These are experiments in which the L-forms were cultivated only on agar media and in which the detection of expression of antibiotic resistance could only be achieved with colony growth.

In any case, none of these findings has ever been used to develop a technical teaching which makes it possible to produce any desired heterologous gene products by providing in vitro recombinant expressor plasmids under conditions such as subrunner fermentation by means of L-forms.

Object of the invention

The aim of the invention is the production of recombinant gene products in an economical way.

Explanation of the essence of the invention

It is an object of the present invention to provide a genetic-microbiological process by means of which selected, biologically active recombinant gene products can be produced in high yields using expression plasmids and novel microorganism strains.

According to the invention, the object is in its basic feature achieved by prokaryotic or eukaryotic microorganism strains that are reproducible without or with greatly reduced cell wall (short name: L-forms), with a for expression of a recombinant gene product, in particular an enzyme, a Immunomodulator, an immunodeterminator, a hormone or other biologically active peptide drug, capable of replicable

recombined vector plasmid (abbreviated expression: Expressicnsplasmid), then stimulated in a fermentation medium for expression and isolated the gene product formed.

Stable bacterial or fungal L-form strains, in particular protoplast-type L-form strains, are preferably used in the present method.

In its further embodiment, the invention is in terms of their microorganisms aspect characterized in that from the field of bacterial L-form Stemmo preferably those are used, the

- of Gram-negative parent bacterium π from the genera Pseudomonas, Agrobacterium, Proteus and Escherichia

- of Gram positive parent bacteria of the genera Staphylococcus, Streptococcus (serological groups D, H and N), Bacillus, Lactobacillus, Streptomyces and Thermoactinomyces

originate. In particular, in this connection L-form strains

- On the one hand from the Baktenen species Pseudomonas aeruginosa, Pseudomonas putida, Agrobacterium tumefaciens, Proteus mirabilis

Proteus vulgaris and Escherichia coli

- on the other hand from the bacterial species Staphylococcus aureus, Streptococcus faecalis (Gro D) Streptococcus pyogenes (Gro D) Streptococcus sanguis (Gro H) Streptococcus cremoris (Gro. N) Streptococcus lactis (Gro. N) Bacillus subtilis

Bacillus licheniformis

Lactobacillus bulgaricus

Lactobacillus lactis

Streptomyces hygroscopicus

Streptomyces griseus

Streptomyces levoris

Streptomyces noursei as well

Thermoactinomyces vulgaris

used. In each variant of the further embodiment of the present method stable bacterial L-form strains are used, which

Isolated by treatment with inhibitors of cell wall biosynthesis,

- adapted to growth in liquid media and

- selectively cultivated over a period of several years

have been. In part, stable L-form strains have been used that have lost the capacity for cell wall resynthesis and reversion to parent forms for over 10 years. This is associated with profound feature changes that give these microorganisms novel properties, such as round and pleomorphic cell shape, loss of flagellar, fimbrial and mesosome formation, biochemical and structural changes of the cytoplasmic membrane, altered enzyme activities, eg. Lack of exoproteases, as well as altered antigenicity properties. In addition, the cell populations of stable bacterial L-form strains used in the variants of the further embodiment of the present method are plasmid-free. Cell material of such L-forms is transformed in each case with DNA of an expression plasmid, which in addition to at least one Replikationsoriyin

- Genes for at least one antibiotic resistance or auxotrophic marker (short word: marker genes) and

Genes for at least one polypeptide or for another protein (shorthand: structural gene [e]) in a reading frame compatible with an expression secretion unit (short name: ESE) or with an expression unit (short word: EE)

contains.

In various variants of the present invention, preference is given to so-called shuttle expression plasmids with at least one replication origin for Gram-negative as well as Gram-positive bacteria or with at least one replication origin for bacteria as well as for yeasts, in particular for Gram-negative bacteria as well as for yeasts of the family Saccharomycetaceae used.

With regard to the selection of a phenotype marker, preference is given in the various variants of the present invention to an expression plasmid having at least one marker gene for antibiotic resistance from the substance group (aminoglycosides, macrolides, tetracyclines, chloramphenicol, lincomycin),

especially for resistance from the antibiotic group

(Streptomycin, kanamycin, neomycin, erythromycin, tetracycline, oxytetracycline), or an expression plasmid with at least one marker gene for a leucine, a histidine, a uracil, a threonine or for a Isozitratlyase auxotrophy used.

From the point of view of selecting the expression unit (EE) or the expression secretion unit (ESE), in the various variants of the further embodiment of the present invention, preference is given to an expression plasmid with an ESE or EE of a gene for a yeast enzyme or a yeast proenzyme activator or with an ESE or EE

Either a gene for an enzyme, a coagulation factor or a proenzyme activator of bacterial origin

Or a gene located on a bacteriophage for an enzyme, for a clotting factor or for another protein

used. In this case, in particular, several times such ESE or EE is used, which after its primary insulation in a

polylinkerkenden shuttle vector, optionally in a bacterial yeast shuUle plasmid vector, has been recloned and made portable in this way.

In earlier inventions from the Central Institute, expression vectors or plasmids were provided which

The ESD of the Streptokinase gene of strain Streptococcus equisimilis H46A (skc-ESE),

- the EbB of bacteriophage 42 D staphylokinase gene of Staphylococcus aureus (SAK-ESE) or

- contain the portable ESE of the gene for the exotoxin A of the bacteriophage T12 of Streptococcus pyogenes (spA-ESE). Thus, those variants are preferably also provided for the present method in which expression plasmids are used which contain in one way or another the skc-ESE, the SAK-ESE or the speA-ESE.

Other variants of the described invention are designed to use an expression plasmid with a synthetic consensus ESE or an expression plasmid with an ESA inducible by a medium supplement. From the point of view of the choice of the particular structural gene, certain variants of the embodiment of the present invention are characterized in that an expression plasmid is used which carries a structural gene for an enzyme, for a proenzyme activator or for a coagulation factor in reading frame-compatible linkage with its native ESE , In particular, the present invention encompasses variants in which an expression plasmid is used which comprises the structural gene

for an alpha-amylase,

for H46A streptokinase,

for the 042 D-staphylokinase or

for the isocitrate lyase from Yarrowia lipholytica H 222-27, each in read-frame-compatible linkage with its native ESE.

However, the present invention predominantly contains those process variants in which an expressin plasmid which contains a heterologous structural gene is used in each case

for an enzyme of both pro- and eukaryotic origin,

for an immunomodulator,

for an immunodeterminator,

for another biologically active peptide drug

both pro- and eukaryotic origin

in frame-compatible association with the skc-ESE, with the SAK-ESE, with the speA-ESE, with a synthetic consensus ESE or with an e ^; NEN by an addition of medium inducible ESE has.

Furthermore, in its further embodiment, the present invention also encompasses Verfahrensvariantsn, in each of which an expression plasmid is used, which is obtained by insertion of an available from an available recombinant Trägorplasmid as a compact fragment (ESE structures) -DNS block in a cloning vector with repeat sequences , Such a cloning vector with repeat sequences is available, for example, with the streptococcal plasmid pDB 101 (molecular size 17.8 kb).

The transformation of the respectively selected L-form strain with one of the expression plasmids provided is carried out in the presence of polyethylene glycol (MW 4000 to 6000) and under osmotic stabilization, to which 0.3 M to 0.4 M sucrose is used. Following transformation with an expression plasmid, an agar medium containing an addition of 0.3 μg to 3 μg of selective labeling of the above labeling per ml of medium is selected for positive transformant clones (screening).

Thus obtained positive L-shape transformant colonies are then propagated through multiple passages on said agar medium until growth as a colony is reached. Subsequently, the plasmid DNA contained in H ° n thus obtained L-form transformant is analyzed, and thereby recombinant L-form clones with high gene expression and high extracellular product formation rate are selected.

According to the technical solution described here, recombinant L-form strains were used for a number of species of Bakieria in this stage of their further embodiment, including, inter alia, for

Pseudomonas aeruginosa

Pseudomonas putida

Agrobacterium tumefaciens

Proteus mirabilis

Proteus vulgaris

Escherichia coli

Staphylococcus aureus

Streptococcus faecalis

Streptococcus pyogenes

Streptococcus sanguis

Streptococcus cremoris

Streptococcus lactis

Bacillus subtilis

Bacillus licheniformis

Lactobacillus bulgaricus

Lactobacillus lactis

Streptomyces hygroscopicus

Streptomyces griseus

Streptomyces levoris

Streptomyces noursei

Thermoactinomyces vulgaris, which have acquired the ability to produce heterologous gene products, including but not limited to streptokinase (abbreviated to SK),

Staphylokinase (short name: SAK), human alpha interferon (short name: hlFN-alpha) · or (hlFN-alpha) fusion proteins, prochymosin (short name: PChy) or Isozitratlyase (short name: ICL) to synthesize. The recombinant L-form strains obtained as a result of this selection and testing step are, after their adaptation to growth in liquid nutrient media, stimulated to express in a fermentation culture under technical culture conditions. Preferably, these strains are stimulated to express in technical submicronformation. In its further embodiment, the present invention is finally gekonnzeichnet that so-called for the adaptation of many of the recombinant L-form strains obtained in growth in liquid media. Liquid LFS medium (L-form standard medium) consisting of

Meatbouillon (Egg made from beef)

Yeast extract 0.3% to 1.0%

Sucrose 5.0% to 10%

Horse serum 2.0% to 10%,

optionally in microgram amounts siipplementiert with the respective selective antibiotic of the associated host-vector construction is used.

The same nutrient medium is used when, for the production of the respective heterologous gene product, a corresponding recombinant L-form strain obtained according to the method is fermented as a submerged shake culture (in quantities of 20 ml to 100 ml). This fermentation is carried out under aerobic, sterile conditions at temperatures of 32 ⁰ C to 37 ⁰ C for a period of 12 hours to 48 hours.

In further variants of the invention described, the technical cultivation is carried out in stirred fermentors or in fermentors of greater useful volume, with complex nutrient media on a meat extract or tissue extract basis with additions of peptone (0.5% to 1.0%), yeast extract (0.5%). up to 1.0%), sucrose (2.0% to 10%) and horse serum (2% to 10%). Here, the cultivation under sterile conditions is also carried out at temperatures of 32 to 37 ° C and at acidities of pH 6.0 to pH 8.0, wherein in the case of Rührfermentors the stirring speeds to values in the range between 100U / min and 400U / min as well as aeration rates of 1 to 2 volumes of air per 1 volume of medium.

For preferred embodiments of the method described are in particular the genotypically stable bacterial protoplast type L-form strains

Pr. MirabilisLVI

Pr. MirabilisLD52,

E.coliLW1655F (plus)

E.coliLW1655F (minus)

E.coliLCB,

Staph. aureusL66

Staph. aureus L 61,

Bac.subtilisL170

Bac. 1997, subtilis L

Strep, hygroscopius L 33-354

Strep, hygroscopius L. OB56

Strep. griseusL86

Strep, levoris L EK1331 as well

Th.vulgarisLK481

provided. Some of these strains are in the depository for microorganisms of the GDR, Central institute for microbiology and experimental therapy of the AdW, DDR - Jena, 6900 Beutenbergstr. 11, under the following registration numbers

Pr. MirabilisLVI ZIMET11199

Bac. subtilis L 1997 ZIMET 11 198

Bac. subtilis L 170 ZIMET 11 201

E.coliLW1655F (plus) ZIMET 11 200

. =. coliLW1655F (minus) ZIMET11 207

· hinterleo. Service.

For those variants of the present invention in which an expression plasmid containing a structural gene for an enzyme or for a proenzyme activator (including "its" respective terminator sequence for the translation stop) in unmodified association with its is used to transform the selected L-form strains In particular, in their preferred embodiments, native ESE carries

The shuttle plasmid pSM752 (molecule size 13.3 kb; erythromycin marker) coding for H46A streptokinase, or

- The plasmid pDB 15 (molecular size 8,9kb, erythromycin marker), coding for 042D-staphylokinase, used. Both expression plasmids have been prepared by methods which are the subject of prior patent applications by the Central Institute.

For those variants of the present invention in which an expression plasmid containing a heterologous structural gene for an enzyme of eukaryotic origin or for another biologically active peptide active (including "its" respective terminator sequence) is used to transform the selected L-form strains for Translationssiop) in read-frame-compatible linkage with an ESE from the following group

(skc-ESE, SAK-ESE, speA-ESE)

In particular, in their preferred embodiments

The shuttle expression plasmid p8B 203 (molecular size 10.8 kb; erythromycin markers; SAK-ESE) coding for a human alpha 1 interferon fusion protein,

The shuttle expression plasmid pJS 127 (molecular size 9.8 kb, erythromycin markers, spoA-ESE), codiorer.d fi'.r oin woitorc's human alpha 1 -Iiterferon fusion protein, or

The shuttle expression plasmid pS636 (Molokiil size 10.5 kb, erythromycin markers: spoA-ESE), coding for prochymosin,

used. The two first-mentioned expression plasmids are again prepared according to Verfahron, which are the subject of earlier patent applications of the Central Institute. By contrast, plasmid pS 636 has been constructed by a process which is the subject of a simultaneous patent application by the central institute.

After the stable protoplast-type L-form stems provided for the preferred embodiments of the invention were selected on agar medium following transformation with the above-mentioned expression plasmids and pre-grown to colonial growth, respectively plasmid DNAs containing the recovered L-form transformants - the recombinant L-form strains

Pr.mirabilis LVI (pSM752) Pr.mirabilis LD52 (pSM752) E.coli LW1655F (plus) (pSM752) E. Coli L W1655F (minus) (pSV.752) Bac. Subtilis L170 (pSM752) Bac. subtilis L 1997 (pSM 752) Strep, hygroscopicus L 33-354 (pSM 752) Strep, hygroscopicus L OB56 (pSM 752)

Pr.mirabilis LVI (pDB15) Pr.mirabilis LD52 (pDB15) E.coli LV1655F (plus) (pDB15) E.coli LV1655F (minus) (pDB15) E. coli LCB (pDB15) Staph. aureus L 66 (pDB 15) Staph. aureus L 61 (pDB 15) Bac. subtilis L 170 (pDB15) Bac.subtilis L1997 (pDB15) Strep, levoris L EK1331 (pDB15) Th.vulgaris LK481 (pDB15)

Pr.mirabilis LVI (pBB203) Pr.mirabilis LD52 (pBB203) E.coli LW1655F (plus) (pBB203) E.coli LW1655F (minus) (pBB203) Bac. Subtilis L170 (pBB203) Bac. subtilis L1997 (pBB 203) Strep, hygroscopicus L 33-354 (pBB203) Strep, hygroscopicus L OB 56 (pBB 203)

- Pr.mirabilis LVI (pJS127) Pr.mirabilis LD52 (pJS127) E.coliLW1655F (plus) (pJS127) E.coli LW1655F (minus) 'PJS127) E. coli LCB (pJS127) Staph. aureus L66 (pJS127) Staph. aureus L61 (pJS127) Bac.subtilis L170 (pJS127) Bac.subtilis L1997 (pJS127) Strep, griseus L86 (pJS127) Th.vulgaris LK481 (pJS127)

Pr.mirabilis LVI (pS636) Pr. Mirabilis L D 52 (pS636) Bac. subtilis L170 (pS636) Bac. subtilis L1997 (pS636)

be isolated as clones with high gene expression and high extracellular product formation rate.

Selected recombinant L-form strains of this collection are cultured in the media indicated above (including LFS medium) under shake flask conditions as under fermentor conditions. As demonstrated by the exemplary embodiments, the recombinant L-form populations were able to grow with generation times of 60 minutes to 180 minutes, reaching cell counts above 10 ⁹ / ml and high extracellular concentrations of the respective heterologous gene product. In summary, the recombinant producer organisms used according to the method have the following advantages in comparison with conventional recombinant bacterial and yeast strains:

Easy transformability with recombinant expression plasmids in a broad host range;

Good secretion with high levels of extracellular gene product levels;

Lack of endotoxins, immunoreactive components and pollutants located in the cell wall and thus more favorable work-up procedures;

- Inability of genetically manipulated L-forms to survive outside laboratory conditions;

Increased or absolute resistance to bacterial highs due to lack of wall-bound receptor substances;

High stability of the formed and secreted gene products in the culture solution due to lack of extracellular protease activity;

Optionally easy workability of the L-form cells after application of an osmotic shock (by distilled water or by low addition of detergent).

Ausführunysboisploie

Example 1: Streptokinaso Formation by L-Forms of Protons mirabilis LVI

- Isolation of the L-formone;

After bivouac of Floischbuuillon-Agar-Plutton with Zuittzon of 10% Piotdosorum, 1% NaCI and 400E Penicillin G / ml with 0.1 ml of a culture of Proteus mirabilis Vl from the oxponmitiellon growth phase and Bobrutunghoi 37 T emerge after 3 to 5 days A- and B-type L-form colony (Gumport, J. and Tauboneck, U., Z. AiIg. Microbiol. 1b, 1975, 399). By transfer of A-type collo- rium to Ponicillin-free Moilium, stable L-till could be observed after> 10 to 10 Passayon.

- Adapt.ition to growth in liquid nutrient modification:

The adaptation of the L-form cultures to growth in flüsskjon Niihrmodion crfolyto by üinhrinuon dense asarlisnnnr agar blocks in 10 to 20ml LFS-Mcdium and incubation under Sdiutiolbedinyunyon (Rundscl wiuytisdi at 2D0U / min) at 37 ⁰ C. After 5 to 10 passages may be good Growth with Zoikliohton / on 10 * to 10 "colony-forming units / ml can be obtained.

Transformation with Streptokinase Recombinant Plasmid Vectors

The zw 1 ransformation the L-Formcn bonutzto Streptokinaso recombinant plasmid pSM 752 is that of Mr'ko (Malko, H. et al., Mol. Gon. Genet. 196.1984, 360), KlelSon, C. and Malko , H. (J. Basic Microbiol., 26, 1986, 70). pSM 75? has a molecular size of 13.3kb, is a fusion plasmid from E. coli pBR322 and dom streptococcal vector pSf.l 7, determines as a selective marker tetracycline and MLS rosistone and carries it as a 25G8 bplsort in the Pstl-place of the bla-Gons of pBR322.

The standard transformation procedure consists of the following steps: 18 to 24 hours old Scbiittol culture from LV! in LFS medium; Centrifuge at 5000g for 10 minutes; rosacea cluster in LFS medium at a concentration of vc (hi 'Zullon / nil, to 0.1 ml of this L-form suspension, 10 to 30 μg of plasmid DNA and 0.15 ml of polyothylone glycol solution (30%, MG 6000, b shake to 10 minutes boi 37 ⁰ C;; give in 0.4M sucrose); cool for 10 minutes in the ice bath, 0.9 ml of osmotically stabilized admit LFS medium;? 2 to 4 hours at 37 ⁰ C shaking, 0.1 ml of which on LFS-Aynr-Platton with and without 10py erythromycin, buffeting the Platton boi 37 ⁰ C. Isolation of the transformant clones grown on the erythromycin-onthaltone Ayarplniton is carried out according to the Überschichton with cinom ovorlay (TRIS-HCl-Ayar, 1: iig. with addition of 20% skimmed milk and 10pg plasminogen / ml) and seioation of those colonies, which after 2 to 18 hours huhrütunij at 37 ⁰ C Aufhellungszoi formed in the overlay hobon.

- Detection of P'asmid-'iNS in ClcnL-Form Cell 'and Characterization of the Banding Patterns:

The individual Transfo nantenkolonien be transferred using the agar-block method several times on frischos medium until abundant wax is present with the formation of colonies. From these cells obtained after flushing with 0.4M sucrose or from those grown in LFS medium, the plasmid DNA was isolated according to the method of Birnboim & DoIy (Nucleic Acid Research 7, 1979, 1513) and mixed with the agarose Goleloktrophoreso characterized (agarose Sigma 0.7%, TRIS buffer 36mM, NaH ₂ PO ₄ 3mMM, EDTA 1OmM, staining with ethidium bromide, Horizontaleloktrophorese at 8V / nm voltage).

- Selection of transformant strains and proof of SK formation:

Transformant culture with ample plasmid DNA content and banding patterns identical to the original plasmid are adapted to growth in liquid media, as will be done. To determine the SK-Bilduno inoculation of 50 ml LFS medium with the addition of lOpgErythromycin / ml with 5 ml of a 10 to 29 hours old shake culture. At intervals of 3 to 4 hours, 2 ml samples were taken and centrifuged at 5000 rpm in a laboratory centrifuge. Thirty to fifty μΙ of the undiluted and diluted supernatants were then placed in Stan / Iöclier agar plate. The agar consists of TRIS-HCl (50 mM) -NaCl (150 mM) buffer (pH8.1), 1% Difco agar, 10% to 20% skimmed milk and 1000 pg human plasminogen / ml. Figure 1 shows such a plate with the Proteus mirabilis LVI (L VIT1 to T6) proband next to that of Bacillus subtilis L 170 (L 170, 2, 3, 5, 6, 1), the dilutions of a streptokinase standard (20O). 5000 U / ml) and the controls (untransformed L-Formcn L Vl K and L 170 K without pSM 752 and AgarmeJium without plasminogen).

After 3 to 6 hours, a clearing of the cloudy agar begins as a result of proteolysis of the milk casein by the streptokinase around positive SK samples. Since no spread of the clarifying zones is longer detectable (18 to 24 hours incubation at 37 ⁰ C), the diameters are ausgemesseri and SK-concentration determined by comparison with don zone diameters of the standard samples

 Figure 1 documents for the embodiment 1 that

- the 6 investigated P. mirabilis form L-shape transformants SK,

- with 4 transformants without special adaptation maximum values of over 6000E / ml can be achieved

- The SK remains stable even after 48 hours cultivation under shaking conditions at 37 ⁰ C and without additions of Protcinaschcmmcrn in the culture solution.

Example 2: Streptokinase-Bilclung dutch L-formone of Bac. subtilis L 170

The isolation of the L-forms of Bac. subtilis 170 is made according to the gradient method by adding 1000E / ml penicillin G or 500pg lysozyme / ml into puncture holes of agar medium from meat broth (FB), 10% horse serum and 3% NaCl. The L-form colonies are formed in the lysis zone of the bespateln with an 8 to 24 hour old culture of bac. subtilis developing bacteria lawn. Stable L-forms are obtained by continuous transfer of the L-form Kohnien with LFS agar media without penicillin and Lysozymzusätze.

In the adaptation to growth in liquid nutrient media, the transformation with SK plasmids, the detection of the plasmid DNA in the L-form cells, the selection of the transformant strains with SK formation and the determination of the SK production is carried out as in the exemplary embodiment 1

 Figure 1 documents for embodiment 2,

- that the 6 transformants studied by Bac. form subtilis L 170 SK,

- that the maximum values without any gonotypic and phenotypic follow-up adjustments are between 200 and 500E / ml SK,

- That the SK remains stable even after 48 hours of culturing under shaking at 37 ⁰ C and despite partial lysis of the L-form cells and without the addition of proteinase inhibitors in the culture solution.

Example 3: Human interferon alphai formation by L-forms of Escherichia coli

- Isolation of L-forms:

The primary isolation of the L-forms of E. coli W1055 plus plus E. coli W1655F minus is achieved by inoculation of pre-dried agar plates (meat water, 1% bacteriological peptone, 0.5% NaCl, 10% horse serum, 6 to 8% sucrose, 0 , 8 to 1% Bacto agar, 500 to 20000E penicillin G / ml) with a 18 to 24 hour liquid culture and incubation under semiaerobic conditions at 37 ° C. After multiple transfer by agarblock technique, the L-form colonies also grow under normal aerobic conditions and reduced penicillin concentrations (1 000 U / ml). Genotypically stable L-forms are obtained by continuous transfer of L-form colonies to agar media without penicillin or other cell wall inhibiting substances.

The adaptation of the L-form strains to growth in liquid LFS medium, the transformation with the vector plasmid pBB203 and the plasmid detection are carried out according to the methodology described in Example 1.

The plasmid pBB 203 has a molecular size of 10.9 kb. It carries as 600-bp sequence the hlFN-alpha 1 gene from the E.

coli plasmid pHRW500, as a 420 bp Hinf I-TagI partial fragment, the native expression secretion unit of the staphylokinase gene from the temperate staph. aureus phage 42D and MLS resistance.

- Evidence of hlFN-Alphai Formation:

Isolated clones adapted to liquid LFS medium are fermented in 50 to 200 ml LFS medium supplemented with 10 μg erythromycin / ml submerged under shaking conditions at 37 ° C. In the samples taken at intervals of 2 to 4 hours, a separation of the L-form cells from the culture solution is carried out by centrifuging at 5000 g. The detection of the extracellular hlFN-alpha 1 takes place in the culture solution with and without the addition of phenylmethylsulfonyl fluoride (PMSF). To determine the intracellular hlFN-alpha 1, the sediment from 50 to 100ml in Tris (50mM) sucrose (20%) - HCI buffer with addition of DNAse and PMSF (50μΙΛηΙ) resuspended and then by treatment with ultrasound (3- to 6 times for 30 seconds) or lysed by adding TRITON X-100. The hlFN-alphai determination is done with the supernatant of the lysate after 20 minutes centrifugation at 15,000 g and 4 ⁰ C. The quantitative determination is carried out hlFN alpha 1 in the bioassay method according to the method of Tonev and happiness (J. Basic Microbiol. 26,198b, 173-189, based on Rubinstein (Rubinstein et al., J. Virology 2, 1981, 555-760) The basis for the evaluation is the 50% inhibition of the cytopathic effect of vesicular stomatitis viruses (VSV Indiana) on the bovine cell line MDBK (FIG. Machin Darby Bovine Kidney.) The reading is made microscopically and the titer determination is made on the basis of an entrained, internationally standardized laboratory standard for hlFN-Alpha 1. Documenting the results summarized in Table 1,

- That is formed by each of the respectively selected transformant clones of the strains E. coli L W1655 F plus and E. coli L W1655 F minus hlFN-alpha 1 and excreted in the culture medium. (Without phenotypic growth adjustment, extracellular concentrations are between 1.5χ10 ⁷ and 5χ10 ⁸ EHlFN-AlphaVI. The highest values were measured with E.coli LW1655FplusTE11507 and E.coliLW1655F minusTE 141507 after 20 hours of shaking culture at 37 ° C),

That the formation and secretion of the hlFN-alpha 1 starts with the onset of growth and reaches the maximum in the early stationary growth phase after 10 to 20 hours of incubation as a function of the growth rate,

- that the hlFN-alpha 1 amounts formed are maintained in the culture solution for up to 48 hours while culturing is continued (microscopically detectable lysing processes in the culture and longer storage of the samples without PMSF additions and freezing did not lead to a substantial reduction under the medium conditions used the hlFN alpha 1 concentrations.),

That the hlFN-alpha 1 formed by E. coli L forms is almost quantitatively excreted into the nutrient medium (the intracellular hlFN-alpha1 concentrations were 0.1% to 10% on the basis of the same sample volumes for E. coli L. W1655 F DlusTE 11507, 8% and for E. coli MLW1655F minus 141507 0.6% to 1.2% of the measured extracellular levels.).

Example 4: Prochymosin Formation in L-forms of Bacillus subtilis L 1997

Isolation of the L-form of Bac. subtilis 1997 is carried out according to the same procedure as described in Example 2. The methods of adaptation to growth in liquid nutrient media, transformation with the plasmid pS636, detection of the plasmid DNA in the L-form cells and selection of the clones with prochymosin formation are essentially the same as set forth in Example 1.

The vector pS636 is a chymosin expression plasmid with a molecular size of 10.5 kb. It contains the chymosin gene under control of the speA expression secretion unit and MLS resistance. For the selection and cultivation of the transformants, 5 to 5 μg erythromycin / ml are used.

- Fermentation:

Round-bottomed flasks containing 20 to 100 ml of LFS medium to which 5 μg erythromycin / ml are added are inoculated with a preculture of 12 to 24 hours and incubated under shaking conditions at 37 ^° C. for 20 to 30 hours. After separation of the cells by centrifugation, the detection of the formed prochymosin takes place directly in the culture solution.

Immunological detection of chymosin: 1 ml each of the culture solutions are adjusted to pH8.0 with crystalline Tris and brought to a final concentration of 1% with SDS. This solution is heated for 5 minutes while boiling in a water bath. Subsequently, each 80μΙ in 10% polyacrylamide SDS flat gels are separated by electrophoresis according to the method of Laemmli (Laemmli, M.K., Nature 227.1970,680-685). The transfer of the separated protein mixture to nitrocellulose film (Schleicher & Schult) is carried out by the method of Kyhse-Andersen (Kyhse-Andersen J., J. Biochem., 3iophys., Methods 10, 194, 203-209). The immunological detection of prochymosin on the nitrocellulose film is carried out by the method of Towbin et al. (Towbin et al., Proc. Natl. Acad., U.S.A. 76, 1979, 7350-4343). The primary antibody used is rabbit anti-calf chymosin serum and secondary antibody for detection

an immunological reaction, an anti-rabbit IgG peroxidase conjugate from the goat. Diaminobenzidine is used as the color reagent.

The overexpressed prochymosin (molecular weight of 37.5KD) can be completely converted into active chymosin (molecular weight 35.5KD) by the method of Foltmann (method in Enzymology 19, 1970, 421-436) in the culture filtrate after an activation time of 20 hours. The activated enzyme is detected after 10-fold concentration by ethanol filling in Ouchterlony double diffusion test; it shows an identity reaction with Kalchchosine.

- proof of milk clotting activity:

For detection of milk clotting activity, 1% skimmed milk agar in 0.1 M sodium acetate buffer, 0.01 M CaCl ₂ , pH 6.2, is used. Skimmed milk powder is suspended to 12% in the above buffer at 37 ⁰ C under stirring and it was added and 0.25 ml to 10 ml temperature-controlled at 50 ° C agar mixed. The homogeneous mixture is poured to 1 mm layer thickness on a glass plate. In the solidified agar layer holes are punched in the diameter of 3 to 4 mm. The culture filtrates of the strains to be tested are activated 2 to 3 hours at pH 2.5 according to the method of Foltmann (supra). After the pH has been adjusted to 6.2, 7 μΙ of the test solution and its dilutions are applied to the punched holes. As a standard, SDS-electrophoretically unitary vesicular myosin purified from rumenylab to hislidyl sepharose by the method of Amouracho and Vijayalakshmif Amourache & Vijayalakshmi, J. Chromatography 303, 1984, 285-290) is used. lOpg / ml of the standard chymosin used brings within 100 seconds 10 ml skimmed milk (prepared from dried milk) at 3O ⁰ C for coagulation (Foltmann, supra).

Figure 2 shows the milk clotting activity in culture solutions of three different Bac. subtilis L 1997 (pS636) clones neat and in a 1: 2 ratio compared to different levels of calf chymosin. The results of the fermentation tests show

- that different clones of 3ac. subtilis L 1997 (pS636) during submerged fermentation to form prochymosin and secrete into the nutrient medium,

- that education takes place predominantly in the active growth phase,

That the formed prochymosin is almost completely activatable and the resulting chymosin is identical to the calf-chymosin,

That with the obtained clones between 5pg and 15μg chymosin / ml of culture solution are obtained,

- That the secreted prochymosin remains stable with prolonged fermentation time up to 40 hours.

Example 5: Formation of Prochymosin by L-Formation of Bacillus subtilis L 1997 in the Laboratory Fermentor For fermentation on a 2-l scale, chymosin clones of Bac. subtilis L 1997, as described in Example 4. A glass laboratory fermenter with stirrer and aeration device and automatic pH and pO _{2 control} is filled with 1.91 LFS medium supplemented with 5 μg erythromycin / ml and after heating to 36 ° C. with 200 ml of an 18-hour bac. inoculated subtilis L 1997 (pS636) culture. The fermentation takes place with stirring speeds between 300 to 400 rpm, aeration rates of 0.5 to 1 volume of air per 1 volume of medium, pH adjustment to pH 6.5 to pH 7.2 and temperature control to 35 ° C to 37 ° C. The samples taken at 2 hour intervals are centrifuged for 10 minutes at 6000g. The determination of the prochyinosine formation takes place in the supernatant according to the methods described in Example 4. The results show

- that Bac. subtilis L 1997 (pS636) clones grow under the described fermentation conditions with generation times between 100 minutes and 130 minutes and cell numbers above 10 ⁹ cells / ml are achieved,

That the formation and secretion of activatable prochymosin begins with the onset of growth,

- That after 10 hours of fermentation prochymosin concentrations between 5 to 10 μg / ml can be achieved.

Table 1: Extracellular and Intracellular hlFN-alpha1 Concentrations in Transformant Cultures of the Stable Protoplast-Type L Forms of E. coli LW1655 F "and E. coli LW1655 F ~

Transformant strain extracellular hlFN E / L nach18Std. nach40Std. intracellular hlFN E / L " percentage ownership shake shake after 18 or % 1.5-5x10 " 1.5-5 x 10 ⁸ 40 hours 0.1-0.8 E. coli LWF "TE 1 1507 3-5 χ 10 ⁷ 3-5x10 ' 1-13 χ 10 ^s 0.1-0.5 E. coli LWF ^ TE 21507 1,5-5x10 ⁸ 1.5-5 x 10 ⁸ 4-15 x 10 ⁴ 0.2-0.3 E. coli LWF ^ TE 12002 1,5-5 χ 10 ⁷ 1.5-5 x 10 ⁷ 1-15 x 10 ^s 0.1-0.2 E. coli LWF "TE 11507 1.5-5 x 10 ⁷ 1-3x10 ⁷ 1,5-9x10 ⁴ E. coli LWF "TE 31507 1.5-5 x 10 ⁸ 1,5-4,5x10 ⁸ 0.6-1.2 E. coli LWF "TE 141507 9-50 x 10 ⁵

converted in the ratio of the intracellular IFN of cells from 1 ml of culture to extracellular IFN in 1 ml of culture solution.

Claims (35)

1. A process for the preparation of recombinant gene products, in particular enzymes, immunomodulators, immunodeterminators, hormones and other biologically active peptide active ingredients, on a microbial route, characterized in that prokaryotic or eukaryotic microorganism strains that are reproducible without or with greatly reduced cell wall (Abbreviation : L-forms), transformed with a replicable recombinant vector plasmid capable of expressing the respective gene product (short term: expression plasmid), stimulated for expression in a fermentation medium and the gene product isolated. 2. The method according to claim 1, characterized in that one uses stable bacterial or fungal L-form strains. 3. Process according to Claims 1 and 2, characterized in that protoplast-type L-form strains are used. 4. The method according to claim 1 to 3, characterized in that one uses L-form strains derived from Gram-negative parent bacteria. 5. The method according to claim 4, characterized in that one L-form strains of the bacterial genera Pseudomonas, Agrobacterium, Proteus and Escherichia. 6. The method according to claim 5, characterized in that L-form strains of the bacterial species Pseudomonas aeruginosa, Pseudomonas putida, Agrobacterium tumefaciens, Proteus mirabilis, Proteus vulgaris and Escherichia coli. 7. The method according to claim 1,2, C and 6, characterized in that for the transformation, the genotypically stable protoplast-type L-form strains Pr. Mirabilis LV Pr. Mirabilis L D52, E. coli L W1655 (minus) and E. coli L CB. 8. The method according to claim 1 to 3, characterized in that one uses L-form strains derived from Gram positive parent bacteria. 9. A method according to claim 8, characterized in that L-form strains from the bacterial genera Staphylococcus, Streptococcus (serologic groups D, H and N), Bacillus, Lactobacillus, Streptomyces as well Thermoactinomyces used. Ίθ. Process according to claim 9, characterized in that L-form strains of the bacterial species Staphylococcus aureus, Streptococcus faecalis, Streptococcus pyogenes, Streptococcus sanguis, Streptococcus cremoris, Streptococcus lactis, Bacillus subtilis, Bacillus licheniformis, Lactobacillus bulgaricus, Lactobacillus lactis, Streptomyceo hygroscopicus, Streptomyces griseus, Streptomyces levoris, Streptomyces noursei and Thermoactinomyces vulgaris. 11. The method according to claim 1, 2, 3 and 10, characterized in that, for the transformation, the genotypically stable protoplast-type L-form strains Staph. aureus L 66, Staph. aureus L61, Bac. subtilis L 170, Bac. subtilis L 1997, Strep, hygroscopic L 33-354, Strep, hygroscopicus L OB56, Strep, griseus L 86, St ^r ep. levoris L EK 1331 and Th. vulgaris LK 481. 12. The method according to claim 1 to 3, 6 and 7 and 10 and 11, characterized in that one uses stable bacterial L-form-stemmines, the Isolated by treatment with inhibitors of cell wall biosynthesis, - adapted to growth in liquid media and - have been selectively cultivated over a period of several years. 13. A method according to claim 1, characterized in that in each case an expression plasmid is used to transform the selected L-form strain, which in addition to at least one replication origin - Genes for at least one antibiotic resistance or auxotrophic marker as well Genes for at least one polypeptide or for another protein in read-frame-compatible linkage with an expression secretion unit (short name: ESE) contains. 14. The method according to claim 13, characterized in that one uses a shuttle expression plasmid with at least one replication origin for both Gram-negative and Gram-positive bacteria. 15. The method according to claim 13, characterized in that one uses a shuttle expression plasmid with at least one Replikationsorigin for bacteria and for yeasts. 16. The method according to claim 15, characterized in that one uses a shuttle expression plasmid with at least one replication origin for Gram-negative bacteria and for yeasts of the family Saccharomycetaceae. 17. The method according to claim 1 and 13 to 16, characterized in that one uses an expression plasmid with at least one marker gene for antibiotic resistance from the following substance group (aminoglycosides, macrolides, tetracyclines, chloramphenicol, lincomycin). 18. Method according to claim 17, characterized in that an express onsplasmid with at least one N'arkergen for a resistance from the following antibiotics group (streptomycin, kanamycin, neomycin, erythromycin, tetracycline, oxytetracycline) is used. 19. The method according to claim 1 and 13 to 16, characterized in that one uses an expression plasmid with at least one marker gene for a leucine, a histidine, a uracil, a threonine or a Isozitratlyase auxotrophy. 20. The method according to claim 1 and 13 to 19, characterized in that an expression plasmid with a, possibly after their primary isolation by recloning in a polylinkertragenden shuttle vector (bacterial yeast shuttle) made portable ESE of a gene for a yeast enzyme or a yeast proenzyme activator. 21. The method according to claim 1 and 13 to 18, characterized in that an expression plasmid with a, possibly after their primary isolation by recloning in a polylinkertragenden plasmid vector portable made ESE Either a gene for an enzyme, a coagulation factor or a proenzyme activator of bacterial origin Or a gene located on a bacteriophage for an enzyme, for a clotting factor or for another protein used. 22. The method according to claim 21, characterized in that one The ESE of the streptokinase gene of strain Streptococcus equisimilis H46A (skc-ESE), - the portable ESE of bacteriophage 42 D staphylokinase gene of Staphylococcus aureus (SAK-ESE) or The portable ESE of the gene for the exotoxin A of the bacteriophage T12 of Streptococcus pyogenes (speA-ESE) used. 23. The method according to claim 1 and 13 to 16, characterized in that one uses an expression plasmid with a synthetic consensus ESE. 24. The method according to claim 1 and 13 to 18, characterized in that one uses an expression plasmid with an inducible by a medium addition ESE. 25. The method according to claim 1 and 13 to 22, characterized in that one uses an expression plasmid, which is a structural gene for an enzyme, for a proenzyme activator or
for a coagulation factor
in a frame-compatible way with its native ESE. 26. The method according to claim 25, characterized in that one uses an expression plasmid carrying a structural gene for an alpha-amylase, for the H46A streptokinase or for the 42 D-staphylokinase each in read-frame-compatible linkage with its native ESE. 27. The method according to claim 13,14,18 and 26, characterized in that one The shuttle expression plasmid pSM 752 (erythromycin marker) coding for H 46A streptokinase, or - the expression plasmid pDB 15 (erythromycin marker), coding for 42 D-staphylokinase used. 28. The method according to claim 1 and T3 to 24, characterized in that in each case one uses an expression plasmid , which is a structural gene for an enzyme of both pro- and eukaryotic origin, for a / mmunmodu / ator, for an immunodeterminator, \\ i \ a hormone or \\\ x e \ nen \ Ne \ lerenb \ o \ ujj \ active PepV \ dwuV.stotf as well \ pro \ \ s also eukaryotic origin in frame-compatible association with the skc-ESE, with the SAK-ESE, with the speA-ESE, with a synthetic consensus ESE or with a medium-addition-inducible ESE. 29. The method according to claim 13,14,18 and 28, characterized in that one The shuttle expression plasmid pBB203 (erythromycin M? SAK-ESE) encoding a human alpha 1-interferon fusion protein, The shuttle expression plasmid pJS 127 (erythromycin marker, speA-ESE), encoding another human alpha interferon fusion protein, or The shuttle expression plasmid pS63ö (erythromycin marker, speA-ESE), encoding prochymosin, used. 30. The method according to claim 13.18 and 25 to 29, characterized in that one uses an expression plasmid, which is obtained by insertion of a removed from a carrier plasmid as a compact fragment (ESE structural gene DNA block in a cloning vector with repeat sequences. 31. The method according to claim 30, characterized in that the cloning vector with repeat sequences, the streptococcal plasmid pDB 101 used. 32. The method according to claim 1,7,11 and 13 to 31, characterized in that the transformation of the respective L-form strain with an expression plasmid in the presence of polyethylene glycol (MW 4000 to 6000) and under osmotic stabilization by means of 0.3 M to 0.4M sucrose. 33. The method according to claim 1,7,11 and 13 to 32, characterized in that following the transformation with one of the Expressirnsplasmide provided - Selected positive transformant clones on agar medium with addition of 0.3Mg to 30pg selective antibiotic / ml. - This L-form transformant colonies multiplied by multiple passages on this agar medium increased until a growth is achieved as colonies and - After analysis of the plasmid DNA contained in these transformants each one of the following strains Pr. Mirabilis LVI (pSM752) Pr.mirabilisLD52 (pSM752) E. coli L W1655 F (plus) (pSM 752) E.coli L W1655 F (minus) (pSM 752) Bac.subtilis L170 (pSM752) Bac.subtilis L1997 (pSM752) Strep, hygroscopicus L 33-354 (pSM 752) Strep, hygroscopicus L OB 56 (pSM 752) - Pr. Mirabilis LVKpDB 15) Pr. Mirabilis L D 52 (pDB 15) E. coli LW1655 F (plus) (pDB 1B ) E. coli L W1655 F (minus) (pDB 15) E. coli LCB (pDB 15) Staph. aureus L66 (pDB15) Staph.aureus L61 (pDB15) Bac.subtilis L170 (pDB15) Bac. subtilis L 1997 (pDB 15) Strep, levoris L EK 1331 (pDB 15) Th.vulgaris LK481 (pDB15) - Pr. Mirabilis L VI (pBB 203) Pr. Mirabilis L D 52 (pBB 203) E. coli L W1655 F (plus) (pBB 203) E. coli L W1655 F (minus) (pBB 203) Bac. subtilis L170 (pBB 203) Bac. subtilis L1997 (pBB 203) Strep, hygroscopicus L 33-354 (pBB 203) Strep, hygroscopicus L OB 56 (pBE 203) Pr. mirabilis LVKpJS 127) Pr. mirabilis L D 52 (pJS 127) E. coli L W1655 Γ (plus) (pJS 127) E. coli LW1655 F (minus) (pJS 127) F. Coli LCB (DJS 127) Staph. aurous L 66 (pJS 127) Staph.aureusL61 (pJS127) Bac.subtilisL170 (pJS127) Bac. subtilis L1997 (pJS 127) Strep, griseus L 86 (pJS 127) Th.vulgarisLK481 (pJS127) Pr. MirabilisLVI (pS636) Pr. MirabilisLD52 (pS636) Bac. subtilis L170 (pS 636) Bac. subtilis L1997 (p. 636) as a recombinant L-form clone with high gene expression and high extracellular product formation rate. 34. The method according to claim 1 and 33, characterized in that the resulting recombinant L-form clones after their adaptation to growth in liquid Nührmedien in a fermentation medium under technical cultivation conditions for expression stimulates. 35. The method according to claim 34, characterized in that one stimulates the recombinant L-form clones obtained in technical Submersfermentation for expression. 36. The method according to claim 33 to 35, characterized in that one - For the adaptation of the obtained recombinant L-form clones liquid LFS medium used, consisting of meat broth Yeast Extract 0.3% to 1.0% Sucrose 5.0% to 10% Horse serum 2.0% to 10%, and optionally supplemented in microgram quantities with selective antibiotic, - The L-form clones as submerged shake cultures in amounts of 20rrl to 100 ml in this medium at temperatures from 32 ⁰ C to 37 ° C for a period of 12 hours to 48 hours sterile and aerobically fermented and - Determines the extracellular and optionally also the intracellular product concentration. 37. The method according to claim 33 to 36, characterized in that one For cultivating the recombinant L-form clones obtained in the stirred fermentor complex nutrient media on meat extract or tissue extract base with additions of peptone (0.5% to 1.0%), yeast extract (0.5% to 1.0%), sucrose (2, 0% to 10%) and horse serum (2% to 10%) urd In media of this composition, the fermentation of the recombinant L-form strains under sterile conditions at temperatures of 32 ° C to 37 ° C at acidities of pH 6.0 to pH 8.0 at stirring speeds between 100U / min and 400U / min and at aeration rates of 1 to 2 volumes of air per 1 volume of medium.