DD246316A1 - METHOD FOR SYNTHESIS OF NOURSEOTHRICIN ACETYL TRANSFERASE IN HETEROLOGIC STREPTOMYCES HOSTES - Google Patents

Abstract

The invention relates to a process for the cloning of a gene for the enzyme nourseothricin acetyltransferase (NAT) and the use of heterologous Streptomyces producers for the bacterial production of the gene product by submerged fermentation. This object was achieved according to the invention in such a way that by shut-gun cloning the gene for the NAT enzyme from a donor strain of the species Streptomyces noursei was isolated by nourseothricin-resistant clones were selected in the transformation mixture. The DNA of the recombinant plasmid pNAT1 of one of these primary clones was then analyzed with restriction enzymes and part of the target DNA insert was subcloned into the plasmid vector pMG220. With the resulting NAT recombinant plasmids, including plasmids pMG2215 and pMG2220, u. a. Staemme heterologous Streptomyces species, preferably S. chrysomallus and S. lividans, transformed and the bacteria thus obtained finally used for NAT production by submerged fermentation.

Description

For this 2 pages drawings

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Field of application of the invention

The invention relates to a process for the cloning of a gene for nourseothricin acetyltransferase (NAT) and the use of heterologous producers of the genus Streptomyces for the bacterial production of the gene product by submerged fermentation. The field of application of the invention is thus in the microbiological pharmaceutical research and industry, especially in the genetic engineering of industrially important Streptomyces strains.

NAT is a protein synthesized by Streptomyces noursei, the creator of the streptothricin antibiotic nourseothricin, which confers resistance on this strain to its own antibiotic. According to the invention, heterologous we call such NAT-producing bacteria, which normally produce neither nourseothricin nor NAT, but have acquired this ability through targeted genetic manipulation.

NAT modifies nourseothricin to an inactive form by specific acetylation. This inactivating reaction is based on the potential use of this enzyme as a specific detection and test agent of the antibiotic.

Characteristic of the known technical solutions

Streptothricins of the Nourseothricin A and B type were detected by G.Bradler and H.Thrum (Z. Gen. Mikrobiol. 24 [1963], p.105ff.) For the strain Streptomyces noursei JA 3890b.

It was shown by I. Haupt and H.Thrum (J. Basic Microbiol. 25 [1985], p. 355 ff.) That various microorganisms, including the production strain S. noursei JA 3890 b, can acquire resistance by acetylation of the antibiotic. These authors also found the high specificity of the acetylation reaction by NAT.

For some years, methods for in vitro recombination of DNA developed by basic research have been increasingly exploited to isolate genes encoding industrially and medically important proteins. These genes can be spliced into vector DNA molecules in vitro and, in association with them, converted into suitable homologous or heterologous hosts. There they can replicate in association with the vector and, under certain conditions, also reach expression, this heterologous expression allowing the possibility of overproduction due to gene amplification with copy number vectors. Synthesis of the enzyme NAT has not yet been possible in heterologous hosts or using gene amplification.

Object of the invention

The aim of the invention is to isolate a nourseothricin acetyltransferase fNATJ gene in the synthesis of this enzyme in heterologous Streptomyces hosts.

Explanation of the essence of the invention

The invention has for its object to develop a genetic engineering method for the isolation and cloning of the NAT gene and provide heterologous Streptomyces strains for microbial production of the gene product. According to the invention, this object is achieved in a series of experimental steps as follows:

1. Selection of the gene donor

As a donor for DNA fragments harboring the intact gene for the enzyme NAT, a nourseothricin-producing microorganism of the species Streptomyces noursei, preferably the differentiation mutant MK84G2 of the plasmid-free production strain S. noursei JA3890b NG 13, is selected. This strain is related to an earlier patent application in the Microorganism Depository of the Central Institute for Microbiology and Experimental Therapy of AdW, GDR - 6900 Jena, Beutenbergstr. 11, filed under the registration number ZIMET 43760.

2. Isolation of the total DNA of the gene donor

The cellular DNA of the selected S. noursei donor, preferably of MK84G2, is isolated by lysis of the cell wall with lysozyme, by cleaning with detergent solution and with phenol and by precipitation in ethanol in the highest possible molecular weight (~ 100 kb). · "

3. Shut-gun cloning (primary cloning)

The purified total DNA obtained in the above process step is digested with the restriction enzyme BamHI in a manner known per se, and the resulting fragments are linked to the BglII-digested plasmid plJ702 (5.7 kb) in the usual way by the action of ligase. '. ;

With the ligation mixture obtained is a non-melanin-forming, nourseothricin-sensitive Streptomyces recipient strain, preferably the known per se strain S. lividans TK24 (sensitive to 30 / xg / ml Nourseothricin), I transformed.

The resulting transformant cell mixture, preferably the resulting ZK24 cell mixture, in which, inter alia, recombinant clones which have in each case taken up an intact plJ702 derivative with inserted NAT gene can be expected, is now being screened, which is based on the following conditions:.

The use of the literature-known Streptomyces plasmid pIJ702 (E.Katz et al., J. Gen. Microbiol., 129 [1983] 2703) with a unique BglII cleavage site in its tyrosinase gene (tyrosinase enzymatically converts tyrosine into melanin and stains the corresponding Colony black) allows selection of recombinant clones due to the lack of pigment formation. Selection with the antibiotic nourseothricin leads to a further specification, since only the clones obtained according to the method can grow in the presence of nourseothricin, which are capable of inactivating this antibiotic.

The clone TK24 (7A) found as a result of this selection is then propagated and the DNA of the primary recombinant plasmid contained therein was given the name pNAT1-isolated in large quantity. This plasmid DNA is digested with the restriction enzymes SaIi, Sau3A and KpnI, characterized in this way and the map obtained in Fig. 1.

4. Subcloning of the NAT gene into the Streptomyces pMG220 vector

Subsequent to the characterization of the primary plasmid pNAT1 (6.9 kb), the NAT gene-carrying fragments of this plasmid were, for subcloning in a conventional manner, namely

a pNAT1 Bcll fragment (3.0kb)

a pNAT1 Sall fragment (1.3kb).

as well as partial digestion

a ρΝΑΤΊ Sau3A fragment (1.2kb),

provided.

The insertion of these fragments into the partially BamHI and Sail-digested Streptomyces vector pMG220 (5.3kb), obtained by the method of a prior patent application, yielded a variety of active-NAT plasmid derivatives upon transformation of S. Hvidans TK24, respectively -Gene. In particular, as a result of this process step, pMG220 derivatives pMG2215 (6.6kb) and pMG2220 (5.5kb) were obtained. Both neoplasms are Sall derivatives of pMG220, which have incorporated the NAT1 SalI fragment, but differ by the in vitro deletion of the SaIB and SalE fragments of pMG220 in pMG 2220 (Figure 2).

Both pNAT1 and pMG2215 and pMG 2220 are then used to transform S. lividans strains, preferably S.

lividans TK24, from S. chrysomallus strains, preferably S. chrysomallus 2, and nourseothricin-sensitive S. noursei strains, preferably S. noursei JA 3890 b NG 13/33. These heterologous host strains are cultured to form NAT in a manner known per se (shake flask scale). The further processing, characterization and biochemical application of NAT takes place in the cell-free system.

5. Identification of NAT activity in heterologous Streptomyces hosts

Phenotypically, the detection succeeds by recourse to known process steps on the basis of the fact that the said strains only in possession of one of the NAT gene carrying new plasmids pNAT1, pMG 2215 or pMG 2220 even at concentrations of more than 50 / ig / ml Nourseothricin to grow.

Biochemically, the detection is performed by incubation of the S 100 supernatant with specific substrates (see Section 3 of the embodiment). It could be shown that in the cell-free system only in the presence of coenzyme A and acetate an inactivation of nourseothricin takes place, whereby the identity of the nourseothricin acetyl transferase (NAT) is verified.

Advantages of the invention ·

The process according to the invention makes it possible to obtain the enzyme nourseothricin acetyltransferase (NAT) by submerged fermentation of bacteria which do not belong to the species Streptomyces noursei and, unlike them, do not form the antibiotic nourseothricin. Thus, the basic substance is provided for a highly specific detection reaction for nourseothricin.

In addition, expression of this resistance determinant by replicons of increased copy number allows for gene amplification. The presence of an isolated clone with the gene for NAT continues to open up diverse possibilities for in vitro construction. • The heterologous producers obtained according to the method finally permit further genetic manipulation with the aim of being able to provide NAT-producing performance strains.

embodiment

The following embodiment comprises the four parts:

1. Primary cloning of the NAT (shut-gun cloning)

2. Subcloning of the NAT gene

3. Expression of the NAT gene in heterologous hosts

4. Detection of a nourseothricin acetyltransferase in methodically obtained heterologous Streptomyces hosts. 1. Primary cloning of the NAT (shut-gun cloning)

1.1. Extraction of total DNA from S. noursei JA 3890b NG 13 MK84G2 The donor strain is incubated for 72 hours in Medium M 79:

1% glucose

1% peptone

0.2% yeast extract

0.6% NaCl

1% casein hydrolyzate

cultured at 28 ⁰ C in a reciprocating shaker.

The biomass thus produced is collected by centrifugation, washed twice with 0.9% NaCl solution and then 1 g of wet mycelium in 10 ml of lysis solution:

10 mM EDTA, pH 8.0 25 mM Tris-HCl 1 mg / ml lysozyme

treated with lysozyme. After 15 minutes incubation at room temperature, the EDTA concentration is increased to 5OmM to

The addition of 10ml detergent solution:

0.4% Na-desoxycholate

1% Brij35

5OmMTMs-HCl

5OmMEDTA

causes resolution of cell integrity and release of high-viscosity cell contents. A subsequent phenol treatment at 4 ^C C (10 ml phenol; pH 7.5) leads to centrifugation (7000 r / min) for the formation of 3 phases, the upper phase containing the dissolved nucleic acids. Addition of this solution with chloroform (chloroform: isoamyl alcohol, 24: 1) causes further purification, especially of lipids, phenol residues and residual protein. From the upper phase, the DNA is precipitated by addition of 2 volumes of ethanol (in fragments of ~ 100kb size).

1.2. Fragmentation of total DNA with BamHI

After resuspension in TE buffer, the total DNA is in the batch

100μΙ DNA solution (50Mg) 15μΙ TM buffer 32μΙΗ ₂ Ο

3μ \ BamHI (50E). , -

by digestion for one hour at 37 ° C with the restriction enzyme BamHI digested. The DNA fragment solution is deproteinized by addition of EDTA (10 mM final concentration), Na dodecyl sulfate (.1%) and phenol, purified with chloroform, precipitated with ethanol after addition of NaCl and in this form for step 1.5. this embodiment provided.

1.3. Extraction of the vector DNA

The vector used was the plasmid pIJ702 (E. Katz, supra). pIJ 702 is a derivative of pJ10147 (J.Pernodet, M.Guerineau, Mol. Genet., 182 [1981] pp. 53-59) with a broad host range and average copy number. This vector has two selectable markers (thiostrepton resistance determinant and gene for tyrosinase) with e.g. a unique cleavage site for the restriction enzyme BglII. Insertion of foreign DNA into this locus leads to the inactivation of tyrosinase. Transformants with such a recombinant vector are capable of growing on thiostrepton and colorless, whereas the transformants with the original vector form the pigment melanin, i. to turn black.

Strain S. lividans 3131 containing this plasmid is cultured for 72 hours at 28 ° C on a reciprocating shaker in medium M79, collected by centrifugation and washed twice with 0.9% NaCl solution. The pellet (about 20 g wet weight) is dissolved in 100 ml of solution I:

25mM Tris-HCl, pH 7.5 25mM EDTA, pH 8.0 0.9% Glucose 10% sucrose

and 20mg lysozyme resuspended, after 10 to 30 minutes exposure at room temperature with 250ml of freshly prepared solution II:

0.2N NaOH 2% SDS

and incubated on ice for 15 minutes. Addition of 1.80 ml of Na acetate (pH 6.0) leads to the precipitation of the proteins and the single-stranded DNA, while the plasmid DNA is able to renature. After centrifugation at 7 000 U / min. For 20 minutes, a clear supernatant is obtained, from which the plasmid DNA is precipitated by adding 0.6 volumes of isopropanol. For the purpose of separating residues of chromosomal DNA and RNA, the DNA dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) is admixed with 1 g / ml CsCl and, after addition of 0.4 ml ethidium bromide solution (10 mg / ml ) Centrifuge each 5 ml of CsCl solution at 45,000 rpm for 36 hours and eliminate the resulting plasmid band. After removal of the ethidium bromide by shaking with n-butanol (TE saturated), the DNS-CsCl solution is dialyzed against 2x11 TE buffer overnight. The DNA thus obtained is then digested with the enzyme BglII.

1.4. Digestion of the vector with BglII

200 / xl (20 g) plJ702 DNA solution. 60μΙ TM buffer 30μΙ H ₂ O 10μΙ BgIII (20E)

are incubated for 60 minutes at 37 ° C and then the enzyme is inactivated after addition of EDTA (1OmM), SDS (1%) and phenol and separated by centrifugation. The clear upper phase is further extracted with chloroform. Subsequently, the vector DNA precipitated with ethanol and for process step 1.5. provided.

1.5. Ligation of the fragments

ΙΟμ, Ι (4 / xg) BglII-digested vector DNA 50μ, Ι (25 ^ g) BamHI-digested donor DNA + 6μΙ NaCl

+ 165 μλ EtOH are precipitated together,

in 14μ.Ι H ₂ 0.2μΙ ligation buffer, 2μ, 1 ATP dissolved, incubated for 2 minutes at 70 ⁰ C, cooled to 14 ° C and 2 / xl T4 ligase was added. This mixture is filled after 2 hours to 100 / ul and incubated at 25 ⁰ C overnight.

1.6. Transformation of Streptomyces lividans TK24 with in vitro recombined DNA

The transformation is carried out in a manner known per se, ind.em 4 x 10 ⁹ protoplasts of S. lividans TK 24 in 0.3 ml medium P;

103 g sucrose 0.25 g K ₂ SO ₄ 2.03 g MgCl ₂ : 6H ₂ O 2 ml of trace element solution

after autoclaving

10ml KH ₂ PO ₄ (0.5%)

100ml CaCl ₂ -2H ₂ O (3.68%) >,

100 ml TES buffer (0.25 M, pH 7.2)

Trace elements per liter:

40 mg ZnCl ₂

200 mg of FeCl ₃ -6H ₂ O 10mg CNCI ₂ .2H ₂ O 10mg MnCl ₂ -4H ₂ O 10 mg Na ₂ B ₄ O ₇ · 10H ₂ O

10 mg (NH ₄ J ₆ Mo ₇ O ₂₄ -4H ₂ O

with 20 μl of the ligation mixture and 0.32 ml of PEG 1000 (40%). This mixture is taken up in 12ml R2-DS agar

103g sucrose 0.2OgK ₂ SO ₄ 10.12g MgCl ₂ -6H ₂ O 2 ml trace elements 10g glucose

0.1 g DifcoCasaminoacids

22g Difco Bacto Agar. '-

after autoclaving,

10 ml KH ₂ PO ₄ (0.5%) 80 ml CaCl ₂ -2H ₂ O (3.68%) 15 ml L-proline (20%) 10 OmITES (0.25M, pH 7.2) ''

plated on 4 plates with R2 agar. 3 plates are overlaid with 30 μg / ml nourseothricin and one plate with 50 μg of thiostrepton. After 10 days 2 colonies could be isolated on nourseothricin-containing medium, one of which was designated clone TK24 (7A) containing the plasmid pNATI.

On the thio-strepton-containing control plate, 370 colonies resulted, of which 30 showed the black coloration typical of the reconstituted vector plasmid.

DNA prepared from the colony containing the primary plasmid pNATI gave in a retransformation batch in S. lividans TK24 10 ⁶ transformants when selected with 30 //. G / ml nourseothricin.

1.7. Mapping and sizing of the insert in plasmid pNATI

Using standard methods of genetic engineering, the DNA of pNAT 1 is isolated and digested with restriction enzymes. No cleavage sites were found for the enzymes BamHI, BglII, EcoRI, EcoRV, Bell, Pvull, PstI, Hindill, Sphl, Xbal, Xhol. With the enzymes Kpnl, Sail, Sau3 A the map was determined according to Fig. 1.

This map suggests that the NAT gene, i. the nourseothricin resistance determinant (ntr) can be recloned as: *

1st Bcl fragment after complete digestion with Bell

2. Sau3 Α fragment after partial digestion with Sau3 A,

3. Sall fragment after complete digestion with Sail.

However, the latter possibility will only be successful if the small Sall-Sau3 Α fragment, which is lost, contains no information that is essential for NAT.

2. Subcloning of the NAT gene in heterologous hosts

As a vector for the subcloning of the plasmid pMG 220 is selected, which has a 7- to 8-fold higher copy number than plJ702. This vector has two BamHI sites suitable for cloning and 6 sites for Sail. Partial digestion of the vector with these enzymes and their ligation with corresponding fragments of the primary plasmid

1. pNAT1 (Bell) + pMG220 (BamHI part.)

2. pNAT1 (Sau3 A part.) + PMG220 (BamHI part.)

3. ρΝΑΤΊ (sail) + pMG220 (sail part.)

led after introduction of these ligation mixtures in protoplasts of S. lividans TK24 in each case to Nourseothricinresistenten, d. H. to NAT-forming transformants.

Further studied were those clones recovered after recombination of the pNAT1 Sall fragment with the vector pMG 220 and transformation as above. The appearance of these clones proves that the information essential for NAT synthesis is limited to the region indicated in the map (Figure 1).

Mapping of the isolated clones revealed that all insertions were made in the small BamHI fragment of pMG 220, e.g. B.

Furthermore, in the case of pMG2220, the deletion of the SaIB and Ε fragments of the vector and the incorporation of the NAT gene occurred in their place (Figure 2).

3. Expression of the NAT gene in heterologous hosts

With the recovered recombinant plasmids, soz.-B. with pNAT1, pMG.2215 and pMG 2220, heterologous Streptomyces hosts, for example S. lividans TK24, S. chrysomallus 2 and S. noursei JA 3890b NG 13/33, are transformed and thus these strains have the ability to produce the nourseothricin acetyltransferase awarded.

4. Detection of Nourseothricin Acetyltransferase in Heterologous Streptomyces Hosts Obtained by the Method, for Example in Streptomyces lividans TK24 (7 A)

4.1. principle

The activity of nourseothricin acetyltransferase is determined in a cell-free extract as an enzyme source in the presence of ATP, coenzyme A and ¹⁴ C-acetate. The formed ¹⁴ C-acetylnourseothricin is detected by the phosphocellulose paper binding technique. The detection is based on the binding of the basic antibiotic to the cation exchange paper. After leaching of unused ¹⁴ C-acetate and ¹⁴ C-acetyl-coenzyme A, the bound ¹⁴ C-acetylnourseothricin is measured in the liquid scintillation spectrometer.

Parallel to the detection of ¹⁴ C-acetylnourseothricin, the acetylation of biologically active nourseothricin to inactive acetylnourseothricin is determined by measuring the biological activity in the perforated plate test with Bacillus subtilis 6633 as a test germ.

4.2. methodology

To obtain enzyme extracts, the centrifuged mycelium is disrupted with an ultrasonic homogenizer and subjected to cell disruption-a fractionated centrifugation. The cell-free postribosomal extract (S100 supernatant) is used after dialysis for the determination of acetyltransferase activity

 The standard reaction mixture for the detection of nourseothricin acetyltransferase contains in 0.1 ml:

45mMTris-HCl, pH7.5

1OmMMgCI ₂

5OmMNH ₄ CI. , ,

6mM mercaptoethanol

4mM ATP

0.2mM Coenzyme A

0.01 mM ¹⁴ C-acetate (94.9 mCi / mmol)

0.2mM Mg-acetate

5μg Nourseothricin 20μΙ Enzyme Extract (100 ^ g protein).

After 3 hours of incubation at 37 ° C δΟμΙ samples are applied to phosphocellulose paper (1.5 x 1.5 cm). The paper sheets are washed 3 times in H ₂ O, the first wash water having a temperature of 85 ° C. After drying the filters, the radioactivity in the toluene scintillator is measured in the liquid scintillation spectrometer. To determine the specificity of the acetyltransferase, in addition to nourseothricin, the aminoglycoside antibiotics streptomycin, neomycin and kanamycin have also been used as substrates.

4.3. Results

Detection and Substrate Specificity of Nourseothricin Acetyltransferase from Streptomyces lividans TK24 (7A)

1. Determination of radioactively labeled acetylnourseothricin

2. Measurement of the biological activity of nourseothricin after enzymatic acetylation (punch hole diameter: 9 mm).

1 ¹⁴ C-acetate units 2 S. livid. antibiotics transfer of Perforated plate test TC 24 (7 A) (Lpm) S. livid. Hemmhof 0 (mm) 9 S. livid. TC 24 (7 A) S. livid. 22 TC 24 1950 Tk 24 22 nourseothricin 22 0 20 23 streptomycin 0 80 22 neomycin 53 10 22 kanamycin 37 23

The detected acetyltransferase is nourseothricin specific. Aminoglycoside antibiotics such as streptomycin, neomycin and kanamycin are not acetylated or inactivated by this enzyme.

Claims (17)

Invention claim: A process for the synthesis of nourseothricin acetyltransferase in heterologous Streptomyces hosts using standard genetic engineering techniques of DNA recombination characterized by Fragments of the total DNA of a nourseothricin-producing microorganism of the species Streptomyces noursei, each harboring a gene for nourseothricin acetyltransferase (NAT gene) cloned on the Streptomyces vector plasmid pIJ702 and the resulting plJ702 derivatives a screening for NAT ⁺ subject recombinant primary plasmids (NAT ⁺ screening) (method step I), Extracts fragments with a functional NAT gene from each of one of the NAT ⁺ recombinant primary plasmids obtained therefrom and, in order to obtain NAT ⁺ recombinant secondary plasmids, subclone these in each case on a vector plasmid from the group of streptomyces vectors with increased copy number (method step II), - NAT ⁺ recombinant plasmid vectors obtained in steps I and II are transformed into heterologous Streptomyces strains (method step III) and - used such genetically engineered Streptomyces strains in Submerskultivierung for the production of nourseothricin acetyltransferase (step IV). 2. The method according to item 1, characterized in that one uses in step I as a donor for the NAT gene strain S. noursei JA 3890 b or one of the mutants of this strain. 3. The method according to Punct 1 and 2, characterized in that one uses in step I as a donor for the "NAT gene strain S. noursei JA 3890 b NG 13 or one of the mutants of this strain. 4. The method according to item 1 to 3, characterized in that one uses in step I as a donor for the NAT gene strain S. noursei JA 3890b NG 13 MK84G2 (strain ZIMET 43760). 5. The method according to item 1 and 4, characterized by subjecting a designated plasmid pNATI plJ702 derivative to a NAT ⁺ screening. 6. The method according to item 1, characterized in that the plasmid pMG220 is used in step II as streptomyces vector with increased copy number. 7. The method according to item 1.5 and 6, characterized in that one takes from the primary plasmid pNAT1 fragments with functional NAT gene and this subcloned to obtain NAT ⁺ recombinant secondary plasmids on pMG 220 (step II). 8. Method according to item 7, characterized in that one takes from the primary plasmid pNAT 1 either a BclI fragment (3.0 kb), a SalI fragment (1.3 kb) or a Sau3A fragment (1.2 kb), each subclones these functional NAT fragments onto plasmid pMG 220 and subjects each of the resulting recombinant secondary plasmids to NAT ⁺ screening. 9. Method according to item 8, characterized in that the Sall fragment of pNAT1 is inserted into plasmid pMG 220 and the resulting secondary plasmid pMG 2215 (6.6 kb) is subjected to NAT ⁺ screening. 10. The method according to item 8, characterized in that the Sall fragment of pNAT 1 is inserted into plasmid pMG 220 and the secondary plasmid pMG2220 (5.5 kb) obtained under in vitro deletion of the fragments pMG220-SalB and pMG220-SalE Subject to NAT ⁺ screening. 11. The method according to item 1, characterized in that one uses for the transformation according to method step III either strains of the Streptomyces genera S. lividans, S. chrysomallus and S. griseus or from the group of Nourseothricinsensitive S. noursei strains. 12. The method according to item 1, 5.9 to 11, characterized in that one uses for the transformation of the NAT ⁺ recombinant plasmids pNAT1, pMG 2215 and pMG 2220 strain S. lividans TK24. 13. The method according to item 1, 5, 9 to 11, characterized in that one uses for the transformation of the NAT ⁺ recombinant plasmids pNAT1, pMG 2215 and pMG 2220 strain S. chrysomallus 2. 14. The method according to item 1,5,9 to 11, characterized in that one uses for the transformation of the NAT ⁺ recombinant plasmids pNAT1, pMG2215 and pMG2220 strain S. griseus JA 3933. 15. The method according to item 1,5,9 to 11, characterized in that one uses for the transformation of the NAT ⁺ recombinant plasmids pNAT 1, pMG 2215 and pMG 2220 of the nourseothricin-sensitive strain S. noursei JA 3890 b NG 13/33 , 16. The method according to item 1,11 to 15, characterized in that one carries out the subsequent workup and characterization of the enzyme nourseothricin acetyltransferase in a cell-free system following a submersed cultivation of each now available heterologous NAT ⁺ Streptomyces strain. 17. Method according to items 1, 11 to 16, characterized in that the activity of nourseothricin acetyltransferase in the cell-free system is determined biochemically, i. by inactivation of nourseothricin in the presence of coenzyme A and acetate.