CA1309674C

CA1309674C - Signal peptide for the excretion of peptides in streptomycetes

Info

Publication number: CA1309674C
Application number: CA000481600A
Authority: CA
Inventors: Klaus-Peter Koller
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1984-05-17
Filing date: 1985-05-15
Publication date: 1992-11-03
Anticipated expiration: 2009-11-03
Also published as: ES543120A0; IL75214A0; JPH0797993B2; NO173452B; FI81113B; FI851929A0; ES8704540A1; FI81113C; EP0161629A1; DK217285A; DE3564118D1; AU580062B2; PT80483B; FI851929L; JPS60260598A; DE3418274A1; HUT38670A; IL75214A; ZA853672B; HU197351B

Abstract

Abstract of the Disclosure:

A prepeptide comprising 20 to 35 amino acids, of the formula Met-Arg-Val-Arg-Ala-Leu-Arg-X-Ala-Ser-Ala (I) in which X represents a hydrophobic region of 10 to 25 amino acids, acts in Streptomycetes as a signal peptide which removes from the cell a peptide which is linked to it via its amino terminal end, and excretes it into the culture medium. A corresponding gene structure can be incorporated upstream of a structural gene, and then on expression brings about the excretion of the desired peptide into the culture medium. For this purpose, suit-able plasmids are modified by incorporation of genes of this type, and host organisms, in particular Strepto-mycetes, are transformed with them.

Description

1 30967~

~OE R4/F 109 The invention relates to a signal peptide whic is a constituent of a propeptide which, in a Streptomyces cell ~hich contains a signal peptidase, is cleaved into the signal peptide and a polypeptide, the latter being removed from the cell and excreted into the culture medium.
The invention further relates to DNA sequences which code for this signal peptide, gene structures which contain this DNA sequence in the reading frame with a structural gene, plasmids which contain a gene structure of this type, and host organisms containing plasmids of this type. Further aspects of the invention and its preferred embodiments are illustrated in detail below.
A process for the preparatîon of tendamistat by fermentation of Str ~ o~ces ten~ae~ha~Oa ~e3~d~ n pro-~15 posed, in ~erman ~ , ~hichprocess comprises using S~ tendae strains which produce tendamistat and have been treated ~i~h sublethal doses of acriflavine. A DNA fragment containing the gene ~or ten-damistat was isolated from strains thus obtained, namely a 2.3 kb Pst I fragment. It was possible, by incorporation of this fragment in pBR 322 which had been cut ~ith Pst I to amplify this DNA in E~ coli and to re-isolate this DNA in pure form.
It has now been found that the signal peptide (prepeptide) of the formula I
Ar~-Val-Ar~-Ala~Leu-Arg-Y.-Ala-S~r-Ala (I) ;n wh;ch X represents a hydrophob;c region compr;s;ng 10 to 25, preferably 17 to ZO am;no acids (most likely 20 am;no ac;ds)~ ;s coded on this 2.3 kb fr3gment ;mmed;-ately upstream of the structural gene for tendam;stat.
It has also been found tha~ by use of th;s signalpept;de other peptides are excreted from host cells which conta;n an appropriate s;gnal pep~idase~ Thus the inven-~'ij9 '~

1 3~961~
-- 3 --tion also relates to propeptides of the formuLa II
Sig-R (II) in ~hich Sig denotes the amino acid sequence of the formula I, and R represents the residue of a genetically c~dable peptide linked by its amino terminal end, in ~hich an acidic amino ac;d, ;n part;cular aspart;c acid, is prefer-ably located at the am;no terminal end.
Thus, one aspect of the invention relates to pep-tides of the formula II in which R denotes hydrogen or a peptide residue, for example the tendamistat residue~
Another aspect of the invention relates to the correspon-ding DNA sequence which can be obtained from Streptomyces tendae strains which produce tendamistat and which have preferably been treated with sublethal doses of acri-flavine, by isolation of the total DNA, digestion ~ithPst I, Southern hybridization ~ith the DNA sequence A, 5'-(32P-)CCT TCh GmG TCG T~T T~G r~ 3' !A) isolation of the 2.3 kb Pst I fragment, cutting ~ith BamHI, Southern hybridi~ation with the sequence A, isolation of the 0.94 kb PstI BamHI subfragment, cutting with Sau 3a, Southern hybridization with the sequence A, isolation of the 0.~25 kb BamHI Sau 3a subfragment and sequencing of the DNA, and which has the following features:
a) it is located immediately upstream of the tendamistat structural gene, b) it codes at the amino terminal end ~or Me~-Arg-V~l-Arg-~la-Leu-Ar~, c) it codes at the carboxy terminal end for Ala-Ser-~la 3~ and d) it codes in the middle for a hydrophobic region which comprises 10 to 25, preferably 17 to 20 amino acids.
This DNA sequence is called sequence 8 or signal peptide in the following textu The kb figures wh;ch were determined by compari-son with standard markers have the customary accuracy.
In place of the sequence A, it is possible to select for the Southern hybridization any desired sequence ~hich is complementary to the tendamistat gene or the 1 30967~

counterpart strand.
For the various steps for the characterization of the DNA sequence B, in each case in practice the DNA is introduced into a suitable vector, the latter is trans-formed into a host cell, amplified there, the transfor-mants determined by colony hybridi~ation with the sequence A, and the DNA reisolated. These steps are known per se.
The DNA sequence C, whose coding strand is repre-sented in the attachment, has the nucleotide sequence of 10 the tendamistat structural gene from S. tendae.
Thus, the gene structures mentioned contain the DNA sequence B in the reading frame with a structural gene which codes, for example, for tendamistat, preferably the DNA sequence C. ~
The invention also relates to plasmids which com-prise the DNA sequence 9 in the reading frame with a structural gene which codes, for example~ for tendamistat, in particular for the DNA sequence C. These plasmids can contain a replicon which is effective in E. coli, and are 20 then able to amplify and, possibly, also to express the DNA in E. coli.
Preferred plasmids additionally contain a replicon which is effective in Streptomycetes. If a Streptomyces is transformed with a plasmid of this type~ it becomes 25 able to express the peptide, which is determined by the structural gene~ in the form of the propeptide of the formula II, which is then cleaved by a signal peptidase during the processing, and the desired peptide is excreted into the culture medium.
So-called shuttle plasmids which contain both a replicon which is effective in E. coli and one which is effective in Streptomycetes are also advantageous. These shuttle vectors can be amplified in E. coli and, after re-isolation~ transformed into Streptomycetes, where 35 production of the desired polypeptide then takes place.
The invention also relates to host organisms which have been trar,sformed with the plasmids mentioned, in particular host organisms of the genus Streptomyces, especially of the species S. tendae or, in particular, 1 30967~

S. l;vidans~
Furthermore, the invention relates to a process for the preparation of a polypeptide of the general for-mula III
H2N-R (III) in which R has the meaning indicated for formula II, in which is used one of the transformed host organisms men-tioned, which contains a signal peptidase which splits off the propeptide of the formula II and secretes the desired 10 polypeptide.
While a plethora of vectors are available for Gram-negative bacteria, only a few vectors have been described for Gram-positive bacteria, in particular for Strepto-mycetes. Vectors for bacteria of the species S. tendae 15 have not hitherto been disclosed~ Thus, an approach for the utilization of S. tendae as host organisms is made possible by the invention.
A particular advantage of the inven~ion is that transformed Streptomyces strains, in particular S. lividans 20 strains, sporulate optimally, that is to say the content of recombinant plasmid does not adversely affect these strains in their generative phase. Thus, the transformed organisms are also suitable for further strain improve-ments, for example for the production and selection of 25 metabolic mutants involving the use of spores.
Compared with untransformed strains of S. tendae, the transformed strains, in particular S. lividans, do not form melanin. Thus, there is no necessity to remove it, and this makes the isolation of the desired peptide, for 30 example tendamistat, considerably easier and it prevents losses in yield.
Another advantage oF the invention is that foreign genes are also expressed in S. lividans, and the corres-ponding polypeptides are excreted, which likewise offers 35 a variety of possibilities for strain improvement and for modification of the polypeptide thus produced.
~ lowever, it is also possible according to the invention to transform other species of Streptomyces, for example S. ghanaensis or aureofaciens. When strains l 3ns67~

which contain no plasmid and are able to synthesize a specific signal peptidase are transformed with ~he hybrid plasmids according to the invention, then stable trans-formants ~hich express and secrete the coded peptide are 5 obtained.
Particularly preferred embodiments of the inven-tion are illustrated in detail in the examples which follow. In these examples, percentage data relate to weight unless other~ise specified. The figures repre-10 senting the hybrid plasmids show the restriction sitestrue to scale.
The following vectors, known from the literature, were used in the examples: single-strand phages M 13 mp 8 and M 13 mp 9: Messing et al., 6ene 19 (1982) 269; pUC 8:
15 Vierra et al., Gene 19 (1982) 259; pAC 177 and 184:
Chang et al., J. Bacteriology 134 (1978) 1141; pIJ 102 and 350: Kieser et al., Mol. Gen. Genet. 185 (1982) Z23.
Maintenance of the S. tendae strain is described ;n U.S. Patent 4,226,764. In principle, the tendamistat 20 gene can be isolated from any strain which produces ten-damistat. However, the procedure of German ~sh 3 ~ ~6~'c~ ~ ~is particularly advantageous~ the iso-lation of the DNA being described ;n Example 3 therein.
This isolated complete DNA is the starting material for 25 Example 1 below.
Example 1 5 ~ug of DNA is completely digested with the res-triction enzyme Pst I and, after fractionation in a 0.8%
agarose gel, transferred to nitrocellulose filters 30 (Southern transfer). The filter with the bound, denatured DNA is prehybridized for 6 hours in 5 ml of prehybridiza-tion medium (0.6 M NaCl, 0.06 M Na EDTA, 0.1X sodium dodecyl sulfate solution, 100 ug/ml sonicated calf thymus DNA and 4-fold concentrated Denhardt's solution). It is 35 then again treated with 5 ml of the prehybridization medium to which, however, 500,000 cpm/ml of radiolabeled DNA have been added. This radiolabeled probe is obtained as follows:
The DNA sequence A is synthesized chemically by 1 30~67~

the phosph;te process. It contains 20 nucleotides (mole-cular ~eight about 13,000) and is complementary to the putative 9NA sequence for tendamistat~ deriYed from the amino acid sequence of tendamistat beQinniny from amino acid 37 of tendamistat, using the triplets preferred by E.
coli. This DNA sequence A is radiolabeled at the ~' end using r-32P-ATP and nucleotide kinase.
For the hybridization of this radioactive probe to the complementary DNA sequence in the complete DNA, the 10 mixture is allo~ed to stand at 37C for 24 hours. Then the non-bound radioactive DNA is removed, and the filter is washed at 37C with 5 x 200 ml of hybridization medium for 30 minutes each time, and then subjected to auto-radiography. After exposure for 24 hours, the hybridiza-15 tion signals show that the gene is located on the 2.3 kbPst I fragment. This fragment is obtained by electro-elution of a section corresponding to this fragment size, cut out of a preparative agarose gel on which the Pst I
digested total DNA had been fractionated. The eluted DNA
20 is cloned in the Pst I restriction site of the plasmid pUC 8.
These hybrid plasmids are transformed into E. coli JM 103 and are amplified. The clones which carry the insertion with the desired tendamistat gene are detected by colony hybridization using the radioactive DNA probe A. The 25 hybrid plasmids pKAI 1a and 1 b thus obtained are represen-ted in Figures 1 a and b.
The localization of the gene can be determined exactly by further Southern hybr;dization steps against the isolated 2.3 kb Pst I fragment and its subfragments 30 (Figures 2a to 2c).
Example 2 The 2.3 kb Pst I fragment from S. tendae is cloned in the unique Pst I restriction site in the plasmid pIJ 102.
The hybrid plasmid pAX 1 a and 1 b thus obtained, ~hich 35 differ in their orientation of the insertion, confer the ability to produce tendamistat on S. lividans strains after hav;ng been transformed into them. Figure 3 shows the plasmid pAX 1 a.

1 30~67~

Example 3 The commercially available strain S. lividans TK 24 (John Innes Institute, Norwich, England) is converted ;nto protoplasts in known manner, and 108 protoplasts 5 are added to 1 ~9 of hybrid plasmid pAX 1 a in the pre-sence of 20% polyethylene glycol 6000. The transformed protoplasts are incubated on regeneration medium R2YE
(Thompson et al., Nature 286 (1980) 525) at 30C for 5 days.
The formation of an extracellular amylase inacti-vator can be demonstrated by a plate test:
5 ml of an aqueous solution containing 0.4 to 1.0 mgtml pancreatin is poured over the regenerated colo-nies, and the mixture is incubated at 37C for 1 hour.
15 The solution is then removed and replaced by 5 ml of a 2% starch agar. AFter incubation of the plates at 37C
for 2 hours, 5 ml of an iodine/potassium iodide solution is poured over them to develop them. Colonies with a blue halo indicate that the clones synthesi~e and excrete ten-20 dam;stat.
As a check, the plasmid DNA of strains which pro-duce tendamistat and sporulate well can be isolated and mapped. All strains which produce tendamistat carry pAX 1 plasmid DNA.
25 _ample 4 The process is carried out in accordance with Example 2, but the plasmid pIJ 350 which carries a thio-strepton-resistance gene as a selectable marker in Strepto-mycetes is used. The hybrid plasmids pAX 350 a and b are 30 thus obtained (which differ in the orientation of the insertion). Figure 4 shows the plasmid pAX 350 a.
After transformation in accordance with Example 3, resistant clones are selected on minimum medium (Hopwood, Bacteriological Reviews 31 (1967) 373 - 403) in the pre-35 sence of 50 ~g/ml thiostrepton, and are tested for pro-duction of tendamistat either directly on minimum medium or after being transferred to non-selective R2YE agar.

9 ~ 7 aT

xample 5 Hybrid plasmids ~hich contain the 2~3 kb Pst I
fragment and, in addition to the Streptomyces replicon~
contain an E. coli replicon have a number of advantages as shuttle vectors; because of thç E. coli replicon and re-sistance markers effective in E~ coli~ they are able to be well amplified in these organisms. After isolation and transformation into Streptomycetes, in particular into S.
lividans, they have high stability~ As a consequence of their selection markers uhich are effective in Strepto-mycetes, and of the Streptomyces replicon, they can also be well amplified in these organisms and can express and secrete tendamistat.
The plasmid pAC 184 is completely digested with the restriction enzyme Sal I, and the en2yme is removed by extraction with phenol/chloroform. The protruding 5' ends are filled using the enzyme DNA polymerase (Klenow frag-ment) in the presence of ATP, CTP, GTP and TTP. A Pst I
linker of the structure 5 ' TCG AGC TGC AGC TCG A 3 ' 3 ' AGC TCG AC~ TC5 AGC T 5 ' is ligated to the blunt ends in a ligase reaction at 22C
(2 ~9 of linker to 0.4 ~g o~ PNA). The DNA is extracted with phenol/chloroform and, after precipitation, is diges-ted with the enzyme PSt I to obtain Pst I ends which can be 25 ligated~ The Pst I ends of the vector are then dephos-phorylated, again extracted with phenol/chloroform, and l;gated with plasmid pAX 1 a which has been partially digested with Pst I. The ligation mixture is transformed in-to E. coli (HB 101 or MC 1061). Clones resistant to chlor-amphenicol are rinsed off the plate, and the plasmid DNA isisolated. S. lividans TK 24 is transformed with 1 to 2 ~g of plasmid DNA and tested for production of tendamistat~
Clones with a positive reaction in the tendamistat test are isolated, the plasmid DNA is isolated by rapid alkaline lysis and introduced into E. coli HB 101 or MC 1061 by back-transformation. Plasmids re-isolated after amplification do not differ from the plasmids isola-ted from S. lividans strains. The recombinant plasmids , `1 ~0967~

are designated pSA 2 a or b (Figures 5a and b) depending on the orientation o~ the insertion which carries the tendamistat gene.
Example 6 The process ;s carried out in accordance with Example 5, but starting from plasmid pAX 350 a, selecting in E. coli for chloramphenicol resistance and in S~ livi-dans for thiostrepton resistance and production of ten-damistat, and the plasmids pSA 351 a and b ~Figures 6a/b) are obtained~
Example 7 The process is carried out in accordance w;th Example 5, but starting from the plasmid pAC 177 in place of pAC 184, and the plasmids pSA 3 a and b (Figures 7 a/b) are obtained.
For this purpose, the plasmid pAX 1 a is par-tially cut with Pst I, and the enzyme is heat-inactivated by heating at o8C for 15 minutes. The DNA is ligated into the plasmid pAC 177 which has been cut w;th Pst I, dephos-phorylated and deproteinized. After transformation ofE~ coli HB 101 or MC 1061~ clones resistant to kanamycin are rinsed off the plate, the plasmid DNA is isolated, and S. lividans TK Z4 is transformed ~ith 1 to 2 ~9 of this DNA. Clones which produce tendamistat are selected and the plasmids are characterized.
Example 8 The process is carried out in accordance with Example 7, but the plasmid pAX 350 a is used ;n place of the plasmid pAX 1, selection in S. lividans for thio-strepton resistance is carried out, and the plasmids pSA
352 a and b ~Figures 8 a/b) are obtained.
Example 9 As is evident from Figure 2, the gene which codesfor tendamistat and the signal sequence is ~v 0.3 kb long.
The 2.3 kb fragment used in the examples detailed above can thus be used in a shortened form for the construction of hybrid plasmids wh;ch br;ng about the product;on of tendamistat:
The plasmid pKAI 1 a is d;gested with Sal I and 'I 30q67~

rel;gated. In th;s manner, the plasmid pKAI 2 which has been shortened by about 750 base-pairs is obtained. It is cloned, isolated and cut with Pst I~ The DNA is dephos-phorylated using alkaline phosphatase from calf intestines, and is deproteinized with phenol/chloroform.
The plasmid pIJ 102 is completely cut ~ith Pst I
and, after heat-inactivation of the enzyme, the fragments are ligated in the Pst I restriction site of pKAI 2. The ligation mixture is transformed into E. coli H8 101 or MC
1061. The plasmid DNA from clones which are resistant to ampicillin is isolated by the rapid alkaline lysis process, and S. lividans TK 24 is transformed with 1 to 2 ~9 of th;s DNA. Clones wh;ch produce tendam;stat are seLected and the plasm;d DNA from them ;s isolated by rapid alkaline lysis.
After re-transformation into E. coli HB 101 or MC 1061 and after isolation of the plasmid DNA from the transformed E.
coli strains, the plasmid pSA 1 is obtained and charac-terized by restriction analysis (Figure 9). The plasmid shows no difference from the plasmids isolated from the S.
lividans strains, but the DNA work-up from E. coli is more productive and possible in a shorter time.

The process is carried out in accordance with Example 9, but the plasmid pIJ 350 is used in place of the plasmid pIJ 102~ it then being possible additionally to select for thiostrepton resistance in S. lividans, an d the plasmids pSA 350 a and b are obtained. Figure 10 shows the plasmid pSA 350 a. In shake cultures, this plasm;d leads to higher yields of tendamistat than does the plasmid pSA 350 b which contains the insertion with the tendamistat gene in the reverse orientation. In con-trast, the reduction in size of the insertion to 1.5 kb has no significant effect on the formation of product.
Example 11 To determine the structure and nucleotide sequence of the tendamistat gene, the 0.94 kb Pst I/Bam HI sub-fragment tFigures 2a and b) and 295 bp Sau 3a/8am HI sub-fragment (Figure 2c) were cloned in the single-strand phages M 13 mp 8 and M 13 mp 9. The primer used for the `I 30967~

dideoxy sequencing reaction was the 20 nucleotide DNA
sequence A and a commercially available 15 bp primer (Bethesda Research Laboratories GmbH, Neu-Isenburg). The DNA sequence C ~as found.
Example 12 Upstream of the structural gene of tendamistat~
there is on ~he DNA an open read1ng frame up ~o the start codon ATG (Met) for a protein ~hich is located immediately upstream of the amino terminal end of tendamistat. This s;gnal peptide corresponds to DNA sequence B.

Appendix:
DNA sequence C ~coding strand) 5'-GAC ACG ACC GTC TCC GAG CCC GCA CCC TCC TGC GTG
NH2-Asp Thr Tilr ~al Ser Glu Pro Ala Prc Ser Cys Val ACG CTC TAC CAG AGC TGG CGG TAC TCA CAG GCC GAC
Thr Leu Tyr Gln Ser Trp Arg Tyr Ser Gln Ala ~sp 3s AAC GGC TGT GCC GAG ACG GTG ACC GTG AAG GTC GTC
Asn Gly Cys Ala Glu Thr Val Thr ~Tal Lys Val Val 4~
TAC GAG GAC GAC ACC GAA GGC CTG TGC TAC GCC GTC
Tyr Glu Asp Asp Thr Glu Gly Leu Cys Tyr Ala '~al sO 55 ~o GCh CCG GGC CAG ATC ACC ACC GTC GGC 5AC GGC TAC
Ala Pro Gly Gln Ile Thr Thr Val Gly Asp Gly Tyr ATC GG5 TCG ChC GGC CAC GCG CGC TAC CTG GCT CGC
Ile Gly Ser His Gly His Ala Ar~ Tyr Leu Ala Ar~

TGC CTT TAG-3' Cys Le~ Stp ,,,,~,~ o~nle~ s~lri~
V ~ ~ 1 and 3 of German Pa~ pp~sc~4=--Example 1 Mutat;on procedure: About 0.25 cm2 of sporulated 1 309~74 mycelium of the strain DSM 2727, which has been cultured on oatmeal agar tEuropean Patent Application A 49,847), is transferred into 100 ml conical flasks which contain 25 ml of the following culture med;um:
Soybean flour Z %
Glucose 3 Corn starch 0.2 X
Urea 0.1 %
Ammonium citrate 0.1 %
Malt extract 0.5 %
KH2Po4 0.2 %
The medium is autoclaved at 121C and 1 bar for 30 minutes, and the pH is about 7Ø The inoculated flasks are incubated at 30C on a shaking machine for 2 days. Then 2 ml of the precul~ure which has grown are transferred into 20 ml of main culture medium in 100 ml conical flasks (European Patent Application A 49,847):
Soluble starch 2-~ %
Soybean flour 0.4 %
Cornsteep liquor 0.4 %
Skim milk powder 0.7 X
Glucose 1.0 %
(NH4) ~PO4 1.2 X
After autoclaving, the pH of the medium is 7.6.
Acriflavine is added at a concentration of 10-25 ~gt ml to this medium as the mutagenic agent. The culture is shaken at 220 rpm for 5 days at 28C. The culture solution is then centrifuged (1,300 x g, 5 minutes) and the cell pellet is washed in main culture medium~ The washed cells are fra~mented using a glass homogenizer and are plated out on agar plates containing the following medium:
Sucrose 0.3 %
Dextrin 1.5 X
NaCl 0.05 %
K2HP04 0.05 %
FeS04 x 7 ~l2 0.001%
Tryptone soya broth 0.5 X
(Oxoid) 1 3()967~1 ~ 14 -Meat extract 0.1 (Difco) Yeast extract 0.2 YO
(Difco) Agar (Merck) 1.5 X
The pH of the solution is 7~1 (autoclaving con-ditions as above).
The plates are ;ncubated at 2~C for 5 days, and individual colonies are transferred into slant tubes con~
tain;ng oatmeal agar. These are incubated as described until the mycelium has sporulated. The sporulated mycelium is propagated as described above in the preculture and main culture, and the culture filtrate after a 5-day main culture is tested for its content of tendamistat tcF.
European Patent Application A 49,847). It ~as possible in this manner to select the strains I-9353, I-9362, I-9417 and I-9418 which produce, during their period of growth under the conditions described, about 90,000-100,000 IU
tendamistat/l culture solution~ corresponding to 1.5-1~ 9 inactivator/l.
Example 3 Isolation of the deoxyribonucleic acid from S.
tendae and determination of its molecular biological characteristics.
The cells are cultured in accordance with Example 1. After growth for 3 days in the main culture medium, the cells in a conical flask are harvested by centrifuga-tion at 3~000 x 9 and 4C for 5 minutes and are washed twice with 50 ml of a solution of 50 mM Tris/HCl buffer, 30 100 mM NaCl and 25 mM EDTA. In a typical preparation, 3 9 of cells which have been compacted by centrifugation are instantaneously deep-frozen in liquid nitrogen at -198C and then homo~enized to a fine powder in the pre-sence of N2 using a blade-homogenizer tWarring Commercial ~lender). This powder is taken up in 10 ml of the above-mentioned bufFer, dissolved on ice and incubated with 0.8 ml of lysozyme solution ~30 mg/Ml) at 2~C for 20 min-utes, shaking gently~ 0.~ ml of a proteinase K soluticn 1 3 n~67/1, tl5 mg/ml) and 0.8 ml of a 2~% solution of the sodium salt of N-lauroylsarcos;ne are then added to the suspension.
After careful mixing, the mixture is ;ncubated on ice for 30 minutes and at room temperature for a further 5 15 minutes. After addition and dissolut;on of 22 g of solid cesium chloride, the volume is made up to 22 mL
using the abovementioned buffer, and the suspension is centrifuged at 12,000 x 9 and 4C for 25 minutes. Ethidium bromide is added to the clear centrifugate and the refrac-10 tive index is adjusted with CsCl to n = 1.3920 using arefractometer. After preparative ultracentrifugation (120,000 x 9, 36 hours, 15C), the band of chromosomal DNA is removed from the centrifuge tubes and is recen-trifuged under the same conditions. The ethidium bromide 15 is removed from the isolated band by extraction with n-butanol, and the band ;s dialyzed to remove all the CsCl. The DNA thus obtained can be cut with, for example, the enzymes Pst I, Bam H I, Sau 3A, Cla I, Bgl II and ~ho I, and cannot be cut with, for example, the endo-nucleases 2a Eco R I and Hind III.
Compared with the DNA from ATCC 31210 and DSM 2727 the DNA obtained from, for example, the mutants I-9353 and I-9362 has an amplified genetic element, the individual fragments of which, after staining with the fluorescent 25 dyestuff ethidium bromide, are clearly visible and stand out from the background of fragments. The size of the amplified element and the characteristic individual fragments after digestion with restriction endonucleases are shown below:

1 30q674 ~ 16 -FragmentFragment si2e (kbp) No. Pst IXho I _ Bam_H I
1 9.0 13.5 7.2 2 7.0 11.5 6.1 3 6.8 9.3 S.3 4 4.6 2.8 2,9 3.4 0.7 2~6 6 3.1 2.5 7 2.3 2.15 8 1.1 2.1 ~ 1 a 5 1 O 1 ~ 2 1 1 1 . 1 12 1aO
13 0.8 14 0.5 0.48 Total 37.3 3708 37.43

Claims

1. A peptide of the formula Met-Arg-Val-Arg-Ala-Leu-Arg-X-Ala-Ser-Ala-R

in which R denotes hydroxy or the residue of a genetically codable peptide, and X represents a hydrophobic region which comprises 10 to 25 amino acids.

2. DNA sequence B obtainable from Streptomyces tendae strains, which produce tendamistat and have been treated with sublethal doses of acriflavine, by isolation of the complete DNA, digestion with Pst I, Southern hybridization with the DNA sequence A, 5'-(32P-)CCT TCA CTG TCG TCT TCG TA-3' (A) isolation of the 2.3 kb Pst I fragment, cutting with BamHI, Southern hybridization with the sequence A, isolation of the 0.94 kb PstI BamHI subfragment, cutting with Sau 3a, Southern hybridization with the sequence A, isolation of the 0.525 kb BamHI Sau 3a subfragment and sequencing of the DNA, and which has the following features:
a) it is located immediately upstream of the tendamistat structural gene, b) it codes at the amino terminal end for Met-Arg-Val-Arg-Ala-Leu-Arg, c) it codes at the carboxy terminal end for Ala-Ser-Ala and d) it codes in the middle for a hydrophobic region which comprises 10 to 25 amino acids.

3. A gene structure containing the DNA sequence B.

4. The gene structure as claimed in claim 3, contain-ing the DNA sequence B in the reading frame with a struc-tural gene.

5. The gene structure as claimed in claim 3 wherein the structural gene codes for tendamistat.

6. The gene structure as claimed in claim 4 wherein the structural gene codes for tendamistat.

7. The gene structure as claimed in claim 5 wherein the tendamistat gene is DNA sequence C.

8. A plasmid having a DNA sequence as claimed in claim 2.

9. A plasmid having a DNA sequence as claimed in claim 3, 4 or 5.

10. The plasmid as claimed in claim 8, having a replicon effective in streptomycetes.

11. The plasmid as claimed in claim 8, having a replicon effective in E. coli.

12. The plasmid as claimed in claim 10, having a replicon effective in E. coli.

13. A host microorganism containing a plasmid as claimed in claim 8.

14. A host microorganism containing a plasmid as claimed in claim 10.

15. A host microorganism containing a plasmid as claimed in claim 11.

16. The host organism as claimed in claim 13, belonging to the genus Streptomyces.

17. The host organism as claimed in claim 14, belonging to the genus Streptomyces.

18. The host organism as claimed in claim 15 belonging to the genus Streptomyces.

19. The host organism as claimed in claim 16 belonging to the species S. tendae or S. lividans.

20. The host organism as claimed in claim 17 belonging to the species S. tendae or S. lividans.

21. The host organism as claimed in claim 18 belonging to the species S. tendae or S. lividans.

22. A process for the preparation of a polypeptide which comprises using a host microorganism as claimed in claim 13, 16 or 19, which contains a signal peptidase which detaches the prepeptide corresponding to the DNA
sequence B.

23. A process for the preparation of a polypeptide which comprises using a host microorganism as claimed in claim 13, 16 or 19, which contains a signal peptidase which detaches the prepeptide corresponding to the DNA
sequence B, and wherein the host organism used has a plasmid which contains a gene structure containing the DNA sequence B, wherein the structural gene codes for tendamistat.