DE3331860A1 - Process for the preparation of tendamistat - Google Patents

Abstract

The treatment of tendamistat-producing strains of Streptomyces tendae with sublethal doses of acriflavine produces mutants which provide very much higher yields of tendamistat. In contrast to the initial strains, which harbour the tendamistat gene on a 4.7 kb Pst I fragment, in the mutants it is located on a 2.3 kb Pst I fragment. The gene is multiplied in the mutants by a factor of about 20.

Description

HOECHST AKTIENGESELLSCHAFT "HOE 83 / F 175 Dr.KL / mü Process for the production of tendamistat

Tendamistat is a ^ -amylase inactivator that works by fermentation from Streptomyces tendae. In the German Offenlegungsschrift 27 01 890 (or the US patents' 4,226,764, 4,282,318, and 4,339,436)

£ Φ 5 describes a fermentation process in which the strain

S. tendae ATCC 31 210 is used. According to the published European patent application (EP-A 2) 49 _c ^ this inactivator is a peptide of 74 amino acids (in a mixture with other, partially degraded peptides), which is particularly advantageous by fermentation with S. tendae DSM 1912 (ATCC 31 969) is obtained. In this publication, the inactivator is also referred to as HOE 467 or the components as HOE 467-A and HOE 467-B. Isolation and purification are carried out by absorption or chromatography. A crystallization process has also been proposed (German patent application P 33 09 059.9).

It has now been found that the yield of tendamistat is still considerably improved by improving the strain can be. While according to the known method with the strain DSM 1912 (ATCC 31 969) a tendamistat yield of 0.07 g / l is achieved, this is achieved by the trunk improvement according to the invention to at least 1, mostly even increased by 1.5 to 1.8 g / l. The inventive Strain improvement is preferably done by treating the tendamistat producing S. tendae strains from S. tendae DSM 2727, with the mutagen acriflavin. reached in sublethal doses. The strain DSM 2727 became isolated from strain DSM .1912 by passive selection. Tendamistat formation served as selection criterion, this strain provides approx. 0.0 9 g / l.

The invention further relates to the improved strains, a DNA segment characterizing these strains of approx. 37 kb (kilobase pairs) in size with a defined fragmentation pattern,

GOPY /

the gene for tendamistat, hybrid plasmids containing this gene and host organisms containing such hybrid plasmids. The various aspects of the invention and its preferred embodiments are set out in the claims Are defined. Advantageous embodiments of the invention are explained in more detail below:

A strain improvement in terms of overproduction of a protein such as Tendamistat can be through improvement the permeability of the protein through the cell membrane as well as by increasing the synthesis rate or by a combination can be achieved by both (J. Mandelstam et al., Biochemistry of Bacterial growth; Oxford, London, Edinburgh, Boston, Melbourne; Blackwell Scientific Publications, 1982). One The rate of synthesis can be increased, for example, in a physiological way by inducing gene activity by means of an inductor; genetically by mutative removal of a repressor blocking the reading of the gene, by structural changes in regulatory areas (promoters) in front of the structural gene, which result in an increased reading rate of the gene, or by increasing the number of copies of a gene including its regulatory Areas about genetic engineering. The combination of these possibilities is usually done with the help of genetic engineering trodden by plasmids (R.E. Glass, Gene Function. London; Croom Helm, 1982).

It has now been found that - in particular starting from the strain DSM 2727 - isolates significantly more efficient mutants can be according to a mutation technique not previously described for streptomycetes. The principle of mutation . is that acriflavin in a low dosage of about 5 to 50, preferably 10 to 30, ug per ml of culture solution acts over the entire period of cell growth in a shaking culture. It is advantageous to use the Fermentation medium as a shaking culture medium. The mutated cells become 12-24 hours after the stationary growth phase has been reached

Cell associations homogenized and those consisting of 5-10 cells Mycelial fragments plated out on an agar medium. The outgrown, mutated cell clones from this become propagated on agar slants and tested in the shaking culture. Overproducing strains are selected by measuring the (^ -amylase content in the fermentation medium. Mutants selected in this way, such as the strains 1-9353 »1-9362 and 1-9418, occur with exceptionally high mutation rates on. The increase in production compared to the state of the art '(DSM 1912) is often more than 2000%.

It was also found that the mutants, compared to the Staiim DSM 1912, have a further 60 % less formation of a red pigment (cf. EP-A 49 847). The increased formation of tendamistat as well as the significantly lower level of contamination of the culture solution consequently facilitate the processing of the inactivator and thus represent a significant technological advance.

The mutants obtained have been found to be genetically stable; H. their productivity remains over several Generations received. The strains and their descendants are thus suitable for the production of inactivator on a large scale Scale through fermentation.

It has been proven by molecular biological analysis that the overproduction of the tendamistat is due to the multiplication of the corresponding gene. However, this genetic manipulation was not carried out in the usual way by means of introduction of a multicopy plasmid containing the gene. The type of mutation described here resulted in much more causes a profound reorientation and reorganization of the DNA of the tribe, within the framework of which a genetic,

apparently chromosomal DNA element of approx. 37 kb Size has been redesigned and selectively increased. The gene for tendamistat was

as part of this genome rearrangement from its original Location on a 4.1 kb Pst I fragment on the chromosomal DNA of the parent strains on the

37 kb element of the mutants displaced (transposed). Simultaneously thus, with the multiplication of the 37 kb element, amplification of the tendamistat gene occurred. Spontaneously occurring Amplifications of DNA are described for streptomycetes in the most recent literature (H. Schrempf, Mol. Gen. Genet. 189 (1983) 501), but there is no comparable case of amplification of a defined gene and the associated overproduction of a protein have so far been found.

The amplified DNA element from the overexpressing strains becomes visible after cleavage of the isolated chromosomal DNA with various restriction endonucleases and after electrophoretic separation in 0.5-1.5 % agarose gels by staining with ethidium bromide. Molecular hybridizations using generally described methods (Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, 1982) with cloned sequences of the amplified DNA show that only parts of the

Elements present in the original roots

are, but are not in abundance.

It has been found that the chromosomal DNA of Streptomyces tendae and the mutants can isolate after centrifugation in the CsCl density gradient if the cells are in the main culture medium cultured, washed, treated with lysozyme in buffer containing Tris-EDTA-NaCl and treated with a detergent (Sodium salt of N-lauroyl sarcosine) can be lysed. The number of copies of the inactivator gene can be estimated if a sample homologous to the gene is radioactively labeled and used in a Southern hybridization experiment (Southern, 1975) against equivalent amounts of chromosomal DNA from the parent strain

(e.g. DSM 2727) and isolated mutants hybridized. The amount of bound radioactivity is then proportional to the frequency of the gene in the compared DNA species. (Kafatos FC et al., Nucleic Acids Res. 7 (1979) 1541). The homologous sample represents either the isolated gene itself or a chemically synthetic part of the gene, the DNA sequence of which was derived from the primary sequence of the protein. The sample then hybridizes to a 2.3kb Pst I fragment of the amplified DKA, eg the overproducing mutant 1-9362, but to a 4.7kb Pst I fragment of the _{DNA of the} starting strains. The hybridization rate is approx. 20 times higher to 1-9362 DNA, so that a gene verification can be concluded by a factor of> 20. This corresponds to the increase in product formation due to the new mutants.

The DNA containing the tendamistat gene can be used in a known manner Way (Maniatis et al., Op. Cit.) Incorporated into plasmids such as pBR 322 and cloned in host organisms such as E. coil and be increased. The replicated DNA can be used for further strain improvements.

Tendamistat is of great therapeutic interest in the treatment of widespread metabolic diseases such as Diabetes mellitus, obesity or hyperlipoproteinemia: when taken orally, the inactivator inhibits the intestinal tract occurring human ts <amylase. Depending on the dose, it can go through the inactivator can now influence the amount of glucose accumulating in the intestine, which is removed by the * C-amylase from the Starch is formed, and thus also directly on the glucose content in the blood. By applying the inactivator, the dreaded, in the case of the above Diseases uncontrolled, regulated rise in blood sugar levels after meals and opened up a new route to the therapy of metabolic diseases (Meyer et al., The Lancet, April 1983, 934).

In the following examples, percentages relate to weight, unless stated otherwise.

Characterization of the starting strain DSM 2727 Streptomyces tendae:

Morphology of the spore chain * - retxnaculum apertum

Spore surface - smooth Spore shape - spherical Color aerial mycelium - gray (e) Substrate mycelium - olive-red-brown

physiolog. Test: negative melanin formation

Recovery of

D-glucose +

D-arabinose +

D-xylose +

Fructose +

Rhamnose +

Sucrose +

Raffinose +

Mannitol +

Inositol -

Lactose +

example 1

Mutation method: Mycelium of strain DMS 2727, which was grown on oat flake agar, was transported about 0.25 cm (EP-A 49 847) are inoculated into 100 ml Erlenmeyer flasks, which are filled with 25 ml of the following culture medium are:

Soy flour 2 %

Glucose 3 %

Corn starch 0.2 %

Urea 0.1 %

Ammonium citrate 0.1 %

Malt extract 0.5 %

KH ₂ PO ₄ 0.2 %

τ - "

The medium is autoclaved for 30 minutes at 121 ^° C. and 1 bar, the pH is 7.0. The seed flasks are incubated for 2 days at 30 ⁰ C on a shaker. Then 2 ml of the grown preculture are inoculated into 20 ml main culture medium, which is in 100 ml Erlenmeyer flasks (EP-A 49 847):

soluble starch 2 - 4 %

Soy flour 0.4 %

Q Cornsteep liquid 0.4 %

Skimmed milk powder 0.7 %

Glucose 1.0 %

^ pn 1? <£

The pH of the medium after autoclaving is 7.6.

Acriflavine is added to this medium in a concentration of 10 25 / Ug / ml as a mutagenic agent. The culture will shaken for 5 days at 28 ° C and 220 rpm. Then the culture solution is centrifuged (1,300 g, 5 minutes) and that Cell pellet washed in main culture medium. The washed cells are fragmented and ground with a glass homogenizer plated out on agar plates with the following medium:

25th Cane sugar 0.3 Dextrin 1.5 NaCl 0.05 K ₂ HPO ₄ 0.05 FeSO ^ χ 7 H20 0.001 30th Tryptone soy
Broth (Oxoid) 0.5 Meat extract
(Difco) 0.1 • yeast extract
(Difco) 0.2 35 Agar (Merck) 1.5

The pH fourth of the solution is 7.1 (autoclaving level) conditions as above).

The plates are incubated for 5 days at 28 ° C. and individual colonies in slanted tubes are inoculated with oat flake agar. These are incubated as described until the mycelium is displaced. The transported mycelium is propagated in the preliminary and main culture as described above and the culture filtrate after 5 days Main culture checked for its content of tendamistat (cf. EP-A 49 847). This way the strains 1-9353, 1-9362, 1-9417, 1-9418 are selected in their Growth period under the conditions described approx. 90,000 - 100,000 IU Tendamistat / 1 culture solution, accordingly Produce 1.5 - 1.8 g inactivator / 1.

Example 2

The procedure is as in Example 1, but the main fermentation is carried out in a fermeter with a total volume of 10 1 with a 7 1 filling with main culture medium as described in Example 1. The strains 1-9353 and 1-9362 are fermented as strains. The main culture is inoculated with 100-300 ml of the preculture described in Example 1. The fermentation is carried out at 3O ⁰ C at an aeration rate of 300 l / h and a stirring of 70 0-900 rpm.

The fermentation process is monitored and tracked with regard to the inactivator activity, the substrate degradation and the biomass development by taking samples. The fermentation is stopped at the end of the exponential growth phase. The maximum inhibitory activity is reached after approx. 60 -SO hours only approx. 1.2 - ¹ > ⁵ S ^{/: L.} The fermentation content is then fed to processing.

Example 3

Isolation of deoxyribonucleic acid from S. tendae and its molecular biological characterization.

Culturing of the cells is carried out according to example 1. After 3 days of growth in the main culture medium, the cells are of an Erlenmeyer flask by centrifugation for 5 minutes at 3000 g at ¹ J ⁰ C harvested and washed twice with 50 ml of a solution of 50 mM Tris / HCl Buffer, 100 mM NaCl and 25 mM EDTA. In a typical preparation, 3 g of solid-centrifuged cells are shock-frozen in liquid nitrogen at -198 ° C and then with a cutting knife homogenizer (Warring Commercial Blender) in the presence of N _?

homogenized to a fine powder. This powder is taken up in 10 ml of said buffer, dissolved on ice and incubated with 0.8 ml lysozyme solution (30 mg / ml) for 20 minutes at 28 ° C. with gentle shaking. 0.8 ml of a proteinase K solution (15 mg / ml) and 0.8 ml of a 20 % solution of the sodium salt of N-lauroyl sarcosine are then added to the suspension. After gentle mixing, the mixture is incubated for 30 minutes on ice and an additional 15 minutes at room temperature. After addition and solution of 22 g of solid cesium chloride the volume with the above buffer to 22 ml and aie suspension at 12 000 g 4 ⁰ C centrifuged for 25 minutes. Ethidium bromide is added to the clear centrifugate and the refractive index is adjusted to n = 1.3920 by refractometry using CsCl. After preparative ultracentrifugation (120 000 g for 36 hrs., 15 ⁰ C) is taken from the band ehromosomaler DNA from the centrifuge tube and recentrifuged under the same conditions. The isolated band is freed from ethidium bromide by shaking with n-butanol and dialyzed to remove the CsCl completely. The DNA obtained in this way can be cut, for example, with the enzymes Pst I, Bam HI, Sau 3a, qj BgI II and Xho I, and cannot be cut, for example, with the endonucleases Eco RI and Hind III.

For example, those obtained from mutants 1-9353 and 1-9362 Compared to the DNA of ATCC 31210 or DSM 2727, DNA is characterized by an amplified genetic Element whose individual fragments are clearly visible after staining with the fluorescent dye ethidium bromide

emerge against the background of unselectively propagated fragments. The size of the amplified element as well as the characteristic individual fragments after digestion with Restriction endonucleases are shown below:

fragment Pst I Fragment size (kbp) Bam H I No. 9.0 Xho I 7.2 1 7.0 13.5 6.1 2 6.8 11.5 5.3 3 4.6 9.3 2.9 4th 3.4 2.8 2.6 5 3.1 0.7 2.5 6th 2.3 2.15 7th 'υ 2.1 8th 1.5 9 1.2 10 1.1 11 1.0 12th 0.8 13th 0.5 14th 0.48 15th 37.3 37.43 total 37.8

Example 4

Cloning of partial sequences of the amplified genetic Elements from mutant 1-9362.

100 µg of the DNA isolated according to Example 3 from 1-9362 are completely digested with the Pst I restriction endonuclease. The DNA fragments produced during digestion are separated by gel electrophoresis in a 0.8 # gel made of low melting point agarose and made visible under UV light after staining with the fluorescent dye ethidium bromide. Areas of the gel that

striking by their intense fluorescence, amplified DNA fragments contained are carefully excised and the DNA after melting of the gel at 68 ⁰ C (2 min) was isolated by extracting twice with phenol and wassergesattigtem followed by ether extraction. The DNA is concentrated from the aqueous phase thus obtained by ammonium acetate precipitation and reprecipitated for further purification with N, N'-bis- (3-aminopropyl) -1,4-butanediamine (spermine). The DNA obtained in this way is ligated in a 25 μl reaction mixture with the DNA of the purified, Pst I-digested plasmid, eg pBR 322, which has been treated with the enzyme alkaline phosphatase, using the enzyme T4 DNA ligase. The ligation mixture is transformed into cells of Escherichia coli HB 101 which have been made competent for uptake of DNA with CaCl3.

Transformed cells containing a recombinant plasmid with a DNA fragment inserted into the Pst I site are resistant to the antibiotic tetracycline, but sensitive to the antibiotic ampicillin. The selection of clones with the hybrid plasmid is accordingly carried out by replica-plating agar plates with tetracycline on agar plates with ampicillin.

To check in which E. coli clone a recombinant Plasmid with an insert from the amplified DNA is located, the plasmid DNA is from the individual E. coli clones isolated with the aid of the "rapid boiling" method (Holmes and Quigley, Analyt. Biochem. 114 (198I) 193) and with digested by the enzyme Pst I. Alternatively, the DNA is digested, for example with the endonucleases Bam H I and Sal I, since the interfaces of these enzymes are specific for the particular cloned fragment. The obtained fragment patterns are compared after separation in agarose gels with the fragment patterns of the correspondingly digested DNA from the low-melting agarose. In the case of matching The size and number of partial fragments becomes the plasmid DNA " then additionally radioactively marked by means of nick translation

333 * 880

and in a Southern hybridization experiment against the Restriction fragments of the total DNA from 1-9362 after digestion with the o. A. Enzymes hybridized. The identification a cloned fragment as part of the amplified genetic element is considered to be certain if a) the fragment patterns are identical to those of the DNA from 1-9362 after using various restriction enzymes and if b) the cloned fragment exclusively against the homologous fragment from the amplified DNA of 1-9362 TO hybridizes.

In this way, a 2.3 kb fragment of the amplified DNA cloned in E. coli which, according to Example 3, contains the complete gene for tendamistat. The 2.3 kb The Pst I fragment with the tendamistat gene is characterized by the sequence, location and size of each Partial fragments produced by the restriction enzymes Pst I, Bam H I and Sal I. The structure of the recombinant plasmid pKAI 1 is in Figure 1, the structure of the cloned 2.3 kb fragment shown in FIG. The individual molecular biological working methods used here are in Maniatis et al. (loc. cit.).

- blank page -

Claims (10)

Patent claims: 1. Process for the production of tendamistat by fermentation from Streptomyces tendae, characterized in that tendamistat-producing S. tendae strains be used with sublethal doses Acriflavine were treated. 2. The method according to claim 1, characterized in that 5 to 50 µg of acriflavin are used per ml of culture solution will. 3. The method according to claim 1 and 2, characterized in that 10 to 3.0 μg of acriflavin are used per ml of culture solution will. 4. The method according to one or more of the preceding claims, characterized in that the acriflavin acts over the entire period of cell growth. 5. The method according to one or more of the preceding claims, characterized in that the S. tendae strain DSM 2727 is used. 6. Tendamistat-producing strains of the species S. tendae,
who were treated with sublethal doses of acriflavin and whose tendamistat production was increased to at least 1 g / l. 7. Streptomyces tendae Starnm DSM 2727. 8. DNA fragment with the gene for tendamistat, available
from strains according to claim 6. 9. Hybrid plasmido containing the DNA according to claim 8. 10. Host organism containing a plasmid according to claim COPY J