WO2004111240A2

WO2004111240A2 - Sequence for the bacillomycin d synthesis in bacillus amyloliquefaciens fzb42

Info

Publication number: WO2004111240A2
Application number: PCT/DE2004/001249
Authority: WO
Inventors: Rainer Borriss; Alexandra Koumoutsi; Joachim Vater; Helmut Junge; Birgit Krebs
Original assignee: Rainer Borriss
Priority date: 2003-06-12
Filing date: 2004-06-14
Publication date: 2004-12-23
Also published as: WO2004111240A3; DE112004001054B4; DE112004001054D2

Abstract

The invention relates to the DNA and amino acid sequences for the Bacillomycin D-Operons and the use of the sequences for the biosynthesis of antibiotically effective lipopeptides and methods for the production thereof. The above is of application in medicine, biotechnology, agriculture and the pharmaceutical industry.

Description

Sequence for the BacillomycinD synthesis in Bacillus amyloliquefaciens FZB42

The invention relates to the DNA and amino acid sequence of the bacillomycin D operon 5 and the use of the sequences for the biosynthesis of antibiotic lipopeptides and methods for their production. Fields of application of the invention are medicine, biotechnology, agriculture and the pharmaceutical industry.

0 Antifungal and hemolytically active peptides have been isolated from Bacillus subtilis and related species and have been described in the literature. Particular attention is paid to the iturin-like compounds, which are cyclic heptapeptides that are linked via a ß-amino acid and modified with ß-fatty acid. The following arrangement of the 5 stereospecificity of the amino acids is characteristic of these compounds: LDDLLDL. Known representatives of this class of substances are: IturinA, D and E, bacillomycin L, D and F, mycosubtilin and bacillopeptin. Similar to eg surfactin and fengycin, these lipopeptides are synthesized non-tribosomally by multienzyme complexes directly according to a thiotemplate mechanism. The for the biosynthesis of mycosubtilin (Duitman et al. 0 1999. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an aminotransferase, and a fatty acid synthase. PNAS 96: 13294-13299) and Iturin A ( Tsuge et al. 2001. Cloning, sequencing, and characterization of the Iturin A Operon. J. Bacteriol. 183: 6265-6273) in B. subtilis responsible gene clusters have been described. ^' 5

The increased occurrence of antibiotic-resistant pathogenic microorganisms has led to an intensive search for new effective antibacterial and antifungal compounds. The search for antiviral compounds is at least as important. The discovery of new, suitable natural substances is becoming increasingly difficult. In addition to the screening of natural producer strains, combinatorial biosynthesis promises to be an efficient alternative for the development of new, effective drugs. Combinatorial biosynthesis is based on the genetic manipulation of biosynthetic pathways of "classic" antibiotics (e.g. lipopeptides), which are caused by polyketide synthases (PKS) or non-tribosomal peptide synthases (NRPSs) can be synthesized. Polyketides and non-tribosomal peptides are two large families of natural products that contain numerous clinically important compounds, e.g. antimicrobial compounds (penicillin, bacitracin, erythromycin, oleandomycin, tylosin and vancomycin), immunosuppressants (cyclosporin, FK506 and rapamycin) and antitumor compounds (blexor) epothilones). Most of the developments in combinatorial biosynthesis are based on compounds that are synthesized by modular PKSs. In addition, NRPSs and PKSs for aromatic polyketides are used for combinatorial biosynthesis.

Non-tribosomal peptide synthetases, NRPSs have a modular structure that represents the necessary functional units. According to the multiple “thiotemplate” mechanism of the non-tribosomal peptide synthesis, the respective substrate is recognized in the 1st step by the adenylation (A) domain and activated by ATP hydrolysis to the corresponding adenylate. Subsequently, the activated substrate is covalently activated with the 4′- Phosphopantetheinyl (4'-PPan) cofactor linked to a conserved serine residue of the peptidyl carrier protein (PCP) domain, which is located downstream of the A domain, and post-translational modification of the PCP domain through a 4'-PPan transferase (PPtase). PPtases are associated with most NRPS biosynthetic gene clusters. During the elongation reaction, the PCP-linked precursors are linked to the nascent peptide chain through the condensation (C) domain, which is between each consecutive Pair of activated units localized In addition to A, PCP and C domains, other modifications are domains in the modules that incorporate modified residues. The release of the complete peptide by cyclization or hydrolysis is catalyzed by a thioesterase-like (Te) domain at the COOH terminus of the last NRPS. As a consequence of this linear assembly mechanism, the primary sequence and the extent of modification of the final NRPSs product is determined by the sequence of the linear sequence of the catalytic domains and modules (Eppelmann et al. 2001. J.Biol. Chem. 276, 34824-34381).

Modular PKSs and NRPSs are polyfunctional "megasynthases" that are organized in repetitive units or "modules". Each module is for a discrete step responsible for the polyketide or polypeptide chain extension. Because of this modularity and because each module encodes a catalytic domain that determines the choice of the next amino acid (including stereospecificity), it is possible to synthesize new compounds by manipulating the domains or modules. Since the structures of polyketides are very complex and rich in stereocenters, this technique allows the manipulation of compounds that are difficult to obtain using conventional chemical methods (E. Rodriguez and R. Daniel. 2001. Combinatorial biosynthesis of antimicrobials and other natural products.Current Opinion in Microbiology 4: 526-534). A prerequisite for the use of combinatorial resynthesis is knowledge of the gene sequences which code for the required PKSs and NRPSs and a host strain which allows stable expression of large heterologous DNA regions.

For example, the srfA deletion strain B.subtilis KE10, a derivative of the surfactin-producing strain B.subtilis ATCC21322, proved to be suitable for the heterologous expression of a 49 kb bacitracin gene cluster from B.licheniformis (Eppelmann et al. 2001. Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis. J. Biol. Chem. 276, 34824-34831). Further convincing examples of the possibility of combinatorial lipopeptide synthesis, in particular of iturin compounds in genetically accessible, i.e. Host strains that can be transformed by DNA transformation, primarily of the genus Bacillus, are not known from the literature.

Iturins, ie non-tribosomally synthesized lipopeptides with a β-amino fatty acid modification, have tyrosine in the D configuration at the 2nd position of the ring-shaped heptapeptide. Two additional D-amino acids are located at positions 3 and 6. They have a strong anti-fungal and hemolytic as well as a limited antibacterial activity. The structure of the Iturine Mycosubtilin, Iturin A and Bacillomycin D is shown in Fig. 1. The responsible gene clusters (operons) are described for the synthesis of the very similar Iturine IturinA and Mycosubtilin. So far, the sequence information for the NRPS synthesis modules was still pending for bacillomycin D, which has deviations from iturin A in 4 out of 7 and mycosubtilin in 3 out of 7 positions of the peptide chain. Mass spectroscopic investigations had shown that FZB42 Bacillomycin D is synthesized in the late growth phase in an ammonium citrate medium (cf. Example 1).

The object of the invention was to provide gene sequences of polyketide synthases (PKS) or non-tribosomal peptide synthases (NRPSs) which can be used for combinatorial lipopeptide synthesis.

The invention is implemented according to the claims. An essential part of the invention is represented by the sequence in claims 1 and 2.

In the course of genome analysis of the phytostimulatory rhizobacterium Bacillus amyloliquefaciens FZB42 (Idriss et al. 2002. Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant growth promoting effect. Microbiology 148, 2097-2109), the genome cluster that was responsible for the synthesis of Bacillomycin D encoded, can be identified. A mutant with a specific insertion of an antibiotic resistance cassette in the bmyA gene was unable to bacillomycin D biosynthesis (Example 6).

While the flanking genes as well as the bmyD and the bmyA gene 98% sequence agreement (at the protein level) with the corresponding genes of

IturinA biosynthesis of B.subtilis RB 14, the 3 'region of the bmyB gene and the entire bmyC gene are variable. According to the colinearity hypothesis

(Correspondence of the arrangement of the gene modules with the peptide sequence sequence) are the ones for linking the peptide with the different amino acids L-Pro (4), L-Glu (5), D-Ser (6), and L-Thr (7) responsible modules.

The gene organization of the bacillomycin D biosynthetic cluster of FZB42 is shown schematically in Figure 1.

The strain FZB42 was identified as a strain that is not only characterized by a sequence identified for the first time for non-tribosomal bacillomycin biosynthesis, but also has the ability to take up DNA through genetic competence. This ability has so far only been found in a few Bacillus strains of the species Bacillus subtilis, primarily in the model organism B.subtilis 168, has been proven. Surprisingly, it was shown that the strain develops the ability to absorb and recombine linear and circular DNA (plasmid and chromosomal DNA) using a method based on the development of natural competence in a certain phase of growth (Fig. 4). , The gene cluster according to the invention for bacillomycin D biosynthesis is not present in the DNA-competent strain B.subtilis 168. It is assumed that this gene cluster was integrated into the genome by horizontal gene transfer similar to the ATCC 6633 and RB 14 strains (Fig. 4). However, neither ATCC6633 nor RB 14 are genetically competent and therefore cannot be manipulated for the combinatorial biosynthesis of novel lipopeptides, preferably iturin derivatives. The strain FZB 42 opens this possibility in which any modules for iturin biosynthesis can be exchanged via homologous recombination. The modular arrangement of NRPSs allows the targeted manipulation of the non-tribosomal protein matrices and thus enables the rational design of new peptide antibiotics. Functional hybrid matrices can be produced in vitro and in vivo. For the combinatorial biosynthesis of new iturin-like active ingredients, the modules present in the bmyB and bmyC genes result in new types of linkage. The amino acids 4, 5, 6 and / or 7 can optionally be linked to the L-Asn (1) -D-Tyr (2) -D-Asn (3) "core sequence and obtain different Iturin hybrid variants be (Table 1).

Other possible combinations relate to possible position changes of the colinearly synthesized amino acids by "domains" shuffling "; Deletion (<7) or addition (> 7) of amino acids in the standard heptapeptide.

Table 1: Combinatorial biosynthesis of novel Iturins through module exchange

L-Asn D-Tyr D-Asn L-Pro L-GIu D-Ser L-Thr

Mycosubtilir L

L-Asn D-Tyr D-Asn L-GIn L-Pro D-Ser L-Asn

The novel linking of the epimerization domains with the AS 1, AS 4, AS 5 and AS 7 results in numerous further variants for novel iturin compounds. The strain FZB42 is competent for DNA transformation. "Gene targeting" strategies by homologous recombination can therefore be used well. As a result, the manipulations shown can be carried out directly in FZB 42. FZB42 contains further gene clusters for the non-tribosomal synthesis of fengycin and surfactin (Koumoutsi et al. 2004. J. Bacteriol. 186, 1084 A recombinant exchange of these DNA areas by additional synthetic operons for variable iturins is possible and allows the simultaneous synthesis of different iturins with a synergistic effect. The antagonistic effect against phytopathogenic fungi is caused by the simultaneous effect of bacillomycin D and fengycin (example 7, Table 4. It is also possible to recombine several copies of an iturin biosynthesis operon into the FZB42 chromosome and to obtain a gene dose effect with an increased synthesis rate of the respective iturin.

Using the techniques mentioned, the respective iturin biosynthesis operons can be placed under the control of a strong, inducible promoter in order to obtain an increased biosynthesis rate in FZB42. It is also possible to produce individual enzymes (NRPS) of non-tribosomal peptide synthesis for targeted synthesis in "in vitro" systems.

The strain FZB42 can also be used for the simultaneous production of bacillomycin D or an iturin-like combinatorial product together with the surfactant and fengycin, which also have an antifungal effect, because of their accessibility for genetic manipulations and their genetically determined biosynthetic potential.

Genetic organization of the Bacillomycin D biosynthesis cluster of FZB42: The peptide synthetases responsible for the biosynthesis of the heptapeptide BacillomycinD are encoded by the genes bmyA (Al), bmyB (Bl, B2, B3, B4), and bmyC (Cl and C2). These genes are part of an approximately 38 kb gene cluster that is inserted between the conserved genes yxjF and xynD and also the bmyD gene that presumably encoded an S-malonyl synthetase. This gene organization is largely similar to the structure of the Iturin A operon described in Bacillus subtilis RB 14 (K. Tsuge, T. Akiyama, M.Shoda, 2001 J.Bacteriol. 183, 6265-6273). In addition, the bacillomycin D operon is similar to the mycosubtilin operon of B. subtilis ATCC 6633 (Duitman et al. 1999, PNAS 96, 13294-13299) (Fig. 2). Three of the 7 adenylation domains, which determine the specificity of the amino acid sequence of the heptapeptide, are highly conserved and determine the sequence L-Asn-D-Tyr-D-Asn, which is identical in the iturins Iturin A, Mycosubtilin and Bacillomycin D. The following four adenylation domains differ significantly from the corresponding domains in the mycosubtilin and iturin A operon and determine a sequence L-Pro-L-Glu-D-Ser-L-Thr that is different from the other two iturins (Fig. 2). Epimerization domains on the modules B1, B2 and Cl determine the D configuration of D-Tyr (2), D-Asn (3) and D-Ser (6).

A Clustal W analysis of the sequences of conserved and non-conserved adenylation domains of all three known Iturin biosynthesis operons revealed that the adenylation domains of bmy_B3 (Pro), bmy_B4 (GIu), bmy_Cl (Ser) and bmy_C2 (Thr) clearly differ from the corresponding domains in the alternative Iturin biosynthesis clusters differ (Fig. 3). The amino acid to be activated is of greater influence than the respective position in the Iturin gene cluster. Thus bmy_B3 (Pro) is more similar to itu_B4 (Pro) and mycB4 (Pro) than to itu_B3 (GIn) and myc_B3 (GIn). The situation is similar for bmy_B4 (GIu), which has the greatest homology to itu_B3 (GIn) and myc_B3 (GIn). Bmy_Cl (Ser) is relatively similar to myc__Cl (Ser) and itu_C2 (Ser). Here too, the degree of homology corresponds to the position of the amino acid in the iturin derivative (Ser6 for mycosubtilin; Ser7 for IturinA). Bmy_C2 (Thr7) takes a separate position, for which - similar to Glu5 - there is no equivalent amino acid in the other two Iturines (Fig.3).

With the specific adenylation domains bmy_B4 (GIu) and bmy_C2 (Thr) according to the invention, there are particularly interesting combination effects for novel iturins, since the amino acids GIu and Thr are new in mycosubtilin and iturin A- Derivatives can be introduced. The best homology (% identical residues) of the constant and variable adenylation domains is listed in Table 2.

Table 2: Best homology (% identical residues) for the variable domains mby_B3, mby_B4, mby_C 1, and mby_C2

The DNA sequence of the BacillomycinD operon according to the invention has surprisingly large differences in representative regions from the sequence of the Mycosubtilin and IturinA operon. The particular advantage of the invention over the prior art is the possibility of being able to produce novel substances with antibiotic, in particular antifungal and antiviral activity, which are of great importance in the medical field because of the frequent occurrence of resistance.

Since the bacillomycin D produced by Bacillus FZB42 has an antagonistic effect on phytopathogenic fungi, in particular in combination with fengycin, a lipopeptide also produced in FZB 42 (see exemplary embodiment 7), use in crop protection is particularly advantageous.

The presence of the DNA sequence according to the invention in the Bacillus strain Bacillus amyloliquefaciens FZB42 and its DNA competence not only opens up the possibility for experts to carry out genetic experiments, but also creates ideal conditions for combinatorial lipopeptide biosynthesis. EXAMPLES

Example 1: Mass Spectroscopic Analysis of FZB 42: Detection of Bacillomycin D, Fengycin and Surfactin: For the detection of lipopeptide products in whole cells, B.amyloliquefadens FZB42 was used on Landy agar medium (Landy, M. et al. 1948. Proc. Doc. Exp Biol. Med. 67, 539-541) at 37 ° C for 24 h. For analysis of the mass spectra by MALDI MS, cell material was removed from the agar plate and covered with matrix medium (saturated solution of α-cyanocinnamic acid in 40% acetonitrile - 0.1% trifluoroacetic acid) and air-dried as previously described (Leenders, F. et al. 1999 Rapid Commun. Mass Spectrom. 13, 943-949). Alternatively, a small sample of the freeze-dried culture filtrate was extracted with 70% acetonitrile - 0.1% trifluoroacetic acid. The extract was mixed 1: 1 (v / v) with matrix medium. A 1 μl aliquot was applied to the target and air-dried until the MS measurement (Vater, J. Et al. 2002. Appl. Environ. Microbiol. 68, 6210-6219).

The mass spectra were identical when using whole cells and culture filtrates. Three groups were obtained which correspond to the lipopeptides suractin, bacillomycin D and fengycin. Their mass numbers are summarized in Table 3.

Table 3: Lipopeptide products of B. amyloliquefaciens FZB42 detected by

MALDI-TOF-MS

Product and observed mass peaks (m / z) determined as

Surfactin (1)

1,0308, 1,046.8 C13 surfactin [M + Na, K] ⁺

1,044.8, 1060.8 C14 surfactin [M + Na, K] ⁺

1,058.8, 1,074.8 C 15 surfactin [M + Na, K] ⁺

Bacillomycin D (2)

1,031.7; 1,053.7, 1,069.7 C14 bacillomycin D [M + H, Na, K] ⁺

1,045.7, 1,067.7, 1,083.7 C15 bacillomycin D [M + H, Na, K] ⁺

1,059.7, 1,081.7, 1,097.7, 1,095.7, 1,111.7 C16 bacillomycin D [M + H, Na, K] ⁺

Fengycin (3)

1,449.9, 1,471.9, 1,487.9 Ala-6-C15 fengycin [M + H, Na, K] ⁺

1,463.9, 1,485.9, 1,501.9 Ala-6-C16 fengycin [M + H, Na, K] ⁺ 1,477.9, 1,499.9, 1,515.9 Ala-6-C17 fengycin [M + H, Na, K] ⁺

1,491.8, 1,513.9, 1,529.9 Val-6-C16 fengycin [M-H, Na, K] ⁺

1,505.8, 1,527.8, 1,543.8 Val-6-C17 fengycin [M-H, Na, K] ⁺

Ensemble 2's lipopeptide products were identified as bacillomycin D by their postsource decay (PSD) mass spectra. The PSD-MALDI-TOF-MS were carried out with the same samples. Monoisotopic mass numbers were recorded. The (rare) protonated species are advantageous for sequence analysis because they break down into fragments much faster than the alkali adducts. For example, the lipopeptide with the mass number m / z of 1,031.5 was identified as a protonated form of a bacillomycin D isoform with a fatty acid of 14 carbon atoms. Their sequence (Figure 6) was derived from a series of b "r, Y""(- H ₂ O) - and proline-derived b" ₂ fragment ions. The peptide ring of this bacillomycin D is at the peptide bond between its amino -Fatty acid residue and threonine at position 7 and at the N-termimis of proline-5 cleaved.

Example 2: Genome analysis of FZB42 by shot gun sequencing:

Chromosomal DNA was prepared by FZB42 using a standard method (CRHarwood & SM Cutting) and fragmented using partial restriction digestion (Sau IIIA in suboptimal concentration) or by hydrodynamic shear. The DNA inserts were ligated into a Smal-linearized, dephosphorylated pTZ19 vector plasmid. Selection of the white clones after transformation in E.coli DH5α. Sequence analysis in an automatic ABI396 sequencing system. In total, more than 39,000 sequence runs with an average reading range of 655 bp were carried out and assembled into approximately 800 "contigs". With an expected genome size of 3,800 kb, this corresponds to a 6.5-fold repetition ("coverage") of the non-redundant ones Sequence. Gaps between the "contigs" were closed by PCR reactions with sequence-specific primers. The bacillomycin D operon was identified as an approximately 38 kb DNA sequence on a total of 56 kb "contig" by comparing the homology of the deduced amino acid sequences with the database (Blast P ). The greatest similarity was found for the Iturin A biosynthesis operon from Bacillus subtilis RB 14 and the Mycosubtilin biosynthesis operon from Bacillus subtilis ATCC6633. Example 3: Identification of functional domains by sequence analysis of the bacillomycin D gene cluster

By comparative analysis of the Bacillomycin D gene sequences with different sequence homology programs (BLAST, Pfam) conserved domains were identified in the genes bmyA, bmyB and bmyC which are responsible for the non-tribosomal synthesis of the Bacillomycin D peptide. The colinear position of the identified, conserved or variable adenylation, epimerization and thioesterase domains is shown in Fig. 2.

Example 4: Preparation of an AbmyA :: Em ^κ mutant of FZB42

A 1.2 kb fragment of the bmyA gene was amplified with chromosomal DNA from FZB42 and ligated into pGEM-T vector. The erythromycin resistance gene from the

Vector pMX39 was inserted in the central region of the bmyA gene. The resulting plasmid with the Em resistance determinant in the bmyA gene was subsequently

Linearization by double cross over recombined in the bmyA wt gene of FZB 42. Competent FZB 42 cells were obtained using a modification of the "One Step Transformation Method" by Kunst and Rapoport (1993).

The cells showed maximum competence in a short period of time at the end of the logarithmic growth phase (Fig. 5). The method for the production of competent cells and the DNA transformation is shown in detail in Example 5. The correct insertion of the resistance gene into the FZB42 genome was carried out by PCR with chromosomal DNA and by Southern hybridization of wt and EmR

Transformants detected.

Example 5: Production of competent cells and DNA transformation of FZB42:

- 10 ml of LB were inoculated with a fresh colony of FZB42 and at 28 ⁰ C and 210 rpm overnight incubation.

20 ml MDCH medium with 2 ml of the preculture and grown at 37 ⁰ C and incubated rpm 210th At the end of the logarithmic growth phase, at one OD600 nm between 2 - 4, the culture is diluted 1: 1 with 20 ml MD medium and shaken for 1 h under the same conditions. The culture is then centrifuged at room temperature and 6,000 rpm and the cell sediment is taken up in 4 ml of the supernatant from the centrifugation (enrichment 1:10).

- The cell preparation is used immediately for the DNA transformation.

- In a 100 ml Erlenmeyer flask, 200 ul fresh, competent cells with 500-1000 ng DNA in 200 ul freshly prepared transformation buffer for 20 min. Incubated at 37 ° C with occasional shaking - Add 1 ml preheated (37 ° C) LB, which may contain 0.1 μg erythromycin (2 μl Em [50 μg / ml] in 1 ml LB) to induce Em resistance and shaking at 37 ⁰ C for a further 90 min.

0.1 ml is plated on LB plates + 5 μg / ml erythromycin and 25 μg / ml lincomycin. Transformants are visible after 2 days and incubation at 37 ° C.

Example 6: Analysis of the lipopeptide spectrum of the mutant AKl (ΔbmyAr.En ^)

The lipopeptide spectrum of the gene disruption mutant AKl (AbmyA :: Em ^κ ) was, as shown in Example 1, determined by MALDI-TOF MS. While the surfactin production remained unchanged, no bacillomycin D signals were detectable in the mass spectrum (Figure 7).

Example 7: Antifungal activity of FZB42 and mutant strains

The phytopathogenic fungal strains Fusarium oxysporum f. sp.cucumerinum DSMZ 62313 (wilting disease including asparagus and peas), Alternaria solani (drought-stained disease tomato), Gaeumannomyces graminis (black-legged potato), Rhizoctonia solani (foot diseases, rot) and Pythium aphanidermatum (root-burned cucumber). The baker's yeast Saccharomyces cerevisiae was grown on malt agar. With these strains, the antifungal activity of FZB42 and the mutants AKl (ΔbmyA :: Em ^κ , deficient in bacillomycin D production, production is described in Example 4), AK2 (ΔfenA :: Cm ^R , deficient in fengycin production), AK3 (ΔbmyA :: Em ^κ ΔfenA :: Cm ^R , deficient in bacillomycin D and fengycin production), CHI (ΔsrfA :: Em ^R , defective in Surfactin production), CH2 ((AsrfA :: Em ^R AfenA :: Cm ^R , defective in surfactant and fengycin production) was investigated. The mutants were produced as described (Koumoutsi, A. et al. 2004 J. Bacteriol. 186, 1084-1096) The double mutant CH2 was obtained by transforming CHI with chromosomal DNA from AK2.2μl of a 20 h culture in Landy medium from FZB 42 or the mutant strains were on Waksman agar with the regularly arranged, actively growing The plates were incubated for 3-5 days at 27 ° C. The inhibitory effect of the bacterial cultures is visible through the formation of reduced growth zones of the fungi (Fig. 8 A, 9, 10 and 11). The antibiotic activity on Saccharomyces cerevisiae and Bacillus megaterium was determined with dilute culture filtrate in the agar diffusion test (described in Koumoutsi et al. 2004 J. Bacteriol. 186, 1084-1096). The hemolytic effect was determined in the hole test on blood agar plates (Vater, J. et al. 20 02 appl. Environ. Microbiol. 68, 6210-6219). The results are summarized in Table 4.

Table 4: Antifungal, antibacterial and hemolytic activity of FZB42 and mutant strains with defective lipopeptide synthesis

Legends for the illustrations

Figure 1: Structure of Iturins with an antifungal effect. The gene organization of the BacillomycinD biosynthetic cluster of FZB42 is shown schematically below. The numbers correspond to the order of the amino acids in the iturin molecule. Highly preserved areas are marked in black, variable areas in red. E indicates the presence of an epimerization domain (responsible for D configuration).

Figure 2: Gene organization of the Bacillomycin D cluster.

Figure 3: ClustalW comparison of the ACL domains Al, Bl, B2, B3, B4, Cl and C2 of NRPS of iturin biosynthesis in FZB42 (bmy, BacillomycinD), RB 14 (itu, Iturin A) and ATCC 6633 (myc , Mycosubtilin).

Figure 4: The Bacillomycin D gene cluster is an insertion in the region of the 1 944/1 955 kb region (green) of the B. subtilis genome. With the bacillomycin D gene cluster, smaller genome fragments from the regions 4,000 - 4,003 kb (yellow) and 3088 - 3094 (blue) were also integrated into this recombination site.

Figure 5: Growth of FZB42 and AKl in MDCH medium. Maximum competence (> 1000 TF / μg DNA) is achieved after 2.33 h at an O.D. 600 nm of 1.4 (red arrow). After 2 h (O.D. 1.1) and 2.67 h (O.D. 2), only a few transformants (<50 TF / μg DNA) can be detected (dashed vertical arrows). No transformants were detectable after 3.5 and 4 h.

Figure 6: Structural analysis of the lipopeptide product with m / z 1 031.5 in situ by PSD-Maldi TOF-MS of whole cells of B. amyloliquefaciens FZB42. The structure was obtained from a series of C- and N-terminal fragments [b "and Y" (- H ₂ O) ions as well as from proline-derived P "fragments.

Figure 7: MALDI TOF MS analysis of bacillomycin D and surfactin lipopeptides from B. amyloliquefaciens FZB42 and mutant strains. A: Detection of surfactin and bacillomycin D mass peaks in extracts from lyophilized Kulturfϊltraten. B: Detection of surfactin and bacillomycin D mass peaks in FZB 42 cells. C: Detection of surfactin but not of bacillomycin D in the mutant AKl (AbmyA :: Em ^κ )

Figure 8: Inhibition of the growth of Fusarium oxysporum DSMZ 62313 (A, B) and Bacillus megaterium by FZB42 and mutants AKl, AK2, CHI

Figure 9: Inhibition of the growth of Gaeumannomyces graminis by FZB42 and mutants AKl, AK2, AK3, CHI and CH2

Figure 10: Inhibition of Rhizoctonia solani growth by FZB42 and mutants AKl, AK2, AK3, CHI and CH2

Figure 11: Inhibition of Alternaria solani growth by FZB42 and mutants AKl, AK2, AK3, CHI, CH2

Claims

claims

1. DNA sequence of the non-tribosomal peptide synthases for bacillomycinD characterized by the following sequence (SEQ-ID-NO: 1):

LOCUS bmy_feng_c 80841 bp DNA 8-MAY-2003

DEFINITION Complementary copy of gbalvlδr.

SOURCE

ORGANISM Bacillus amyloliquefaciens FZB42

COMMENT containing bacillomycinD and fengycin gene clusters

COMMENT This file is created by Vector NTI http: // www. informaxinc. com /

FEATURES Location / Qualifiers CDS compleraent (69..1604)

/ Label = xynD

CDS complement (1932..9788)

/ label = bτnyC CDS complement (9876. .25964)

/ label = bmyB CDS complement (26012..37957)

/ label = bmyA CDS complement (37980..39179)

/ label = bmyD CDS complement (39742..40524)

/ label = yxjF CDS complement (40537..41220)

/ label = scoB CDS complement (41217..41918)

/ label = scoA CDS complement (41946..43379)

/ label = yxj C

CDS complement (43687..44880)

/ Label = biol

CDS complement (44885..45901)

/ Label = bioB

CDS complement (46605..47762)

/ Label = bioF

CDS complement (47755..49194)

/ Label = bioA CDS complement (49095..49835)

/ Label = BIOW

CDS 50091..50546

/ label = yngA CDS 50556..51445

/ Label = yngB

CDS 51514..52113

/ label = yngC CDS complement (52171..53697)

/ label = yngE CDS complement (53715..54494)

/ Label = yngF

CDS complement (54511. .55407)

/ label = yngG CDS complement (55635..56891)

/ label = yngH CDS complement (57006..58643)

/ label = yngl CDS complement (58692..59831)

/ label = yngJ CDS complement (59974..61509)

/ label = yngK CDS complement (61415..62002)

/ label = yngL CDS complement (62087..65887)

/ label = ppsE CDS complement (65909..76681)

/ label = ppsD CDS complement (76710..80828)

/ Label = PPSC

BASE COUNT 18956 a 20424 c 18453 g 23008 t

2. Amino acid sequences of the non-tribosomal peptide synthases for BacillomycinD characterized by the following sequences:

SEQ ID NO: 2

> CDS 37980 ... 39179 (complementary) Translation of bmyD_FZB42 (laa - 400aa) mnnlaflfpgqgsqfvgmgkqfwndfvlakrlfeeasdaisldvkklcfngdmneltktmnaqpailtvsviafq vymqeigvkprflaghslgeysalvcagalsfrdavtlvrergilmqnadpqqqgamaavthlslqtlqeicski stedfpagvacmnseqqhvisghrqavervikmaeekgaaytylnvsapfhssmirsaseqfqtvlhqysfrdaa wpiisnvtarpyssgnsisehlkqhmtmpvrwtesmhylllhgvteviemgpnnvlagllrkttnhivpyplgqt sdΛφplsnsaerkkhivhlrkkqlnklmiqsviarnynkdsaaysnmttplftqiqelkqqkrhilvcksekermkrelevs

SEQ ID NO: 3

> CDS 26017 ... 37957 (complementary) Translation of bmyA_FZB42 (laa - 3982aa) mytsqfqtlvdvirersnisdrgirfiesdknetwsyrqlfeeaqgylgylqhlgikpkqeivfqiqenksfw afwacilggmipvpvsigedddhklkvwriwnilnhpfliasekvldkikkyaaehdlqdfhhqlneksdviqdq iydypasfyepdadelafiqfssgstgdpkgvmlthhnlihntcaignalhvhskdsflswmplthdmgliachl vpfitginqnlmptelfirrpilwmkkahehkasilsspnfgynyflkflknepdwdlshikviangaepilpel cdeflkrcaafnlkrsailnvyglaeasvgaafskigkefvpvylhrdylnlgeravkvskedqncasfvevgqp idycqlrisdetnervedgiighihikgdnvtqgyynnpestekaltsdgwvktgdlgfisesgnlwtgrekdi ifvngkniyphdiervaiemeevdlgrvaacgvydqktqsgeivlfwykkspekfaplvkeikkhlykrggwsi kevlpirklpkttsgkvkryelarqyeagnfstesaaineclesspetsgqtpiheietellsifsevlngkkvh ladsyfdmganslqlsqiaerieqkfgcelavsdlftypsitdlaaylsesraeikqdaaakpshvtpkdiaiig mslnvpgastksdfwnllekgehsireypesrlkdaadylksiqsefnesqfvkggyldeidrfdysffglapka aqfmdpnqrlflqsawhaiedagyaggsmngsrvgvyagyskvgydyerllsanypeelhqyivgnlpsvlasri ayflnlkgpavtvdtacssslaavhmackslisgdcemalaggirtsllpicigldmessdgytktfskdsdgtg tgegaaavllkpl qdavrdgdhiygvikgsamnqdgttagitapnpaaqtevietawkdagiapetlsfieahgt gtklgdpvefnglckafekytakkqfcaigsvksnighlfeaagivgliksvlmlnhkknpplahfnepnplihf hsspfyvnqeaaeftsgdeplrggvssfgfsgtnahwleeyisqseyapedehgphlfvlsahtekslyelaqq yrqyvsddsqaslksicytastgrahldhgiamivsgkqelsdkltrliqgdrnlpgvyigyknmkemlpahkee Inqqaaalikqrlrtqderitwlnraaelfvqgavidwralysgetvqktplplypfersrcwaeadqlrlnege kkggaalninqskahiesflktvisnasgiraeeldlnahfiglgmdsimlsqvkkaiadefgvdipmdrffdtm nnlqsvidylaetvpssfasappqenvpaqeiqviseaqsesdrreghqehmlekiiasqnqliqdtlqaqlnsf nllrnsghhsdekeyakaqeksipsvkqghptvtaekkaaqeakpyvpfqpqnlndqghytarqkqyledfikky adktkgskqytdntrfahannrnlssfrsywkeivypiiaersdgskmwdidgneyiditmgfgvnlfghhpsfi tqviddstrsslpplgpmsdvagevadrirtctgvervafynsgteavmvalrlaraatgrkkwafsgsyhgtf dgvlgvagtkggaasanplapgilqsfmddliilhynnpdsldvirslgdelaavlvepvqsrrpdlqpqaflke lraitqqsgtalimdeiitgfriglggaqewfgiqadlvtygkiigggqplgwagkaefmnaidggtwqygdds ypqdeakrtfvagtfnthpltmrmslavlrhlqtegehlyeqlnqktaylvdelnrcfeqaqvpirmvrfgslfr fvssldndlffyhlnykgvyvwegrncf lsaahtaddieniiqavkdtvedlrrggfipegpdspdgggrkksgt relspeqkqlvmashygneasaalnqsimlkvkgelqytslkqavrhivdrhealrtvihpddevqqvlermnid ipvidftghphehrepeiqkwltedakrpfhfheqkplfrihvltsahnehlivltfhhiiadgwsiavfvqele snyaaivqgkpispkeadvsfrqyldwqqaqidsghyeegvrywrrhfsepiqqpilpstasvrypngyegdrct vrlgrplsealrslsiqmknsvfvtmlgafhlflhrltklsglvigvpaagqshmkqhdligncvnraipvkntss sestlsgylgsmkesvnlamrhqavpmtlvarelphdqvpdmriifnldrpfrklhfgkaeaepvaypvkctlyd lflnitdahqeyvldfdfntnvispeimkkwgtgftkllqkmvegdsiplnammmfsdeeqhdlqalyaehqkri ssigsntanfteayeapvneterqlariweelfglervgrsdrflalggnslqatlmlskvqktfhqkvsigqff nhqtvkelagfiqnetkwhlpmkaaekkayyptspaqqrvyflhqlepdqlaqnmfghisitgkydeqalissl qqvtnqrheafrtyfdiidgdivqklenevdfnvhvrtmsrdefdaysdrfvkpfrldqaplvraelikieneqae llidrahhiisdgysvniltnellalyhqkplpdiefeykdfaewqnqrlnddamkrqetywleqfqdeipildlp tdgskaaerssegqrvtcslqpdvirslqdlaqkaettlytvllaaynvllhkytgqediwgtpasgrnhpdie kiigifiqtigirtkphanrtftdyleevkrqtldafenqdypfdrlveklnvqrettgkslfntmfvfqniefh eirhnectfkvkernpgvslydlmltiedagqqiemhfd ykpgqftkdtieqitrhytailnslveqpemtlssv pmlseterhqlltecngtktpyphketvyrwfemqaeqspdheavifgnerytyrqlneranrlartlrtkgvqa dqfvaiicphrielivgilavlkaggayvpidpeypedriqymlrdsraewltqrslldqlpydgdwlldeen syheehsnlesdsdahdlaymiytsgstgnpkgvliehqgladyiwwakevyvrgektnfplyssisfdltvtsi ftplvtgntiivfdgedksavlseimrdsridmikltpahlhvikemniaggtairkmivggenlstrlaksvse qfkgrldifneygpteawgcmiyqfdaerdkrefvpigtpaantdiyvadasrnlvpigvigeiyisgpgvarg ywnrpdltaekfvenpyvpgakmyksgdlakrlkdgnlvyigrvdeqvkirghrielgeieaamhnaeavqkaav tvkeeedglkqlcayyvsdkpiaaaqlreqlssglpdymvpsyfvrlehmpltsngkinrkalpapeaslqqtae yvppgneteskltdlwkevlgishagikhnffdlggnsiraaalaarihkeldvnlslkdifkfptieqladkal hmdknryvpipvakkmpyypvssaqrrmyllshteggeltynmtgamnvegtidperlnaafrkliarhealrts fdlyegepaqrihqnvdftieriqaseeeaedrvldfikafdlakpplmraglieieparhvlwdmhhiisdgv svnilmkdlsriyegnepdplsiqykdfavwqqsdiqkrniknqeaywldqfhgdipvldmpadyerpairdyeg esfeflipehlkqrlsqmeedtgatlyraillasytillskysgqediivgtpsagrthldvepwgmfvntlvir nhpagrktfdaylnevkenmlnayknqdypleeliqhlhlpkdssrnplfdtmfv lqnldhaeltfdslqlkpyp fhhsvakfdltlsiqadrdnyyglfeyskklfkksrievlsndylhilsaileqpsiliehiglsgsneeeenal dsiqlnf SEQ ID NO: 4

> CDS 9876 ... 25964 (coraplementary) Translation of bmyB_FZB42 (laa -5363aa) msvfknqetywenlfdeedglsafpyfkaadkaslartgyqekcicrslspevsqritntmanhsdmaaylillag iecllykytdrtslilgiptvskqkagqsavnnivllkntlsnestfktvfgqlkeavndslknqnlpfrkmvrh lsvqyndehmplihtwslneihslqckedtatdtlfhfdlenngihlklfyngnlyderyinqivshldqllsv ilfqpqaaihtaeilpeaekqkllfdfndtvrdfsgsrtvyqlfeeqaertpehtavkfkndhltyrelnekanr lartlrncgvqpdtlvailadrslemivsiiavwkaggayvpldpeypkerlqyllhdadadvllvqhhlknsla fdgpvidlndetsyhadcsllspvaghshlayviytsgttgkpkgvmvehggivnslqwkkaffkhspadrvlvl ypyvfdafilnffgplisgatlhllpneenketfaiqnaikqerithfstsprllktmieqmnredfihvqhvw ggeqleadtveklhslqpririnneygptenswstfhpvqsadeqitigspvanhqayilgahhqiqpigvpge lyvggagvargylnrpelteekfvehlhvpgqkmyktgdlarwlpdgrieylgridhqvkirgyrieigeveaam fnlenvreaawaredadgakqlyayyvgepsltaaqfreelsrelpnymipsrfipleripltsngkidlkalp aadentraeneyiaprntieellasiwqevlgaerigildnffdfggdsiksiqvssrlyqagykvdmkhlfkhp siaelsqfvapvsrvadqgevnggtkltpiqhwffeqkmphahhynqavmlysaegfkegplrrtraeriashhda lrmifektpdgy apritgtdeselfhlevmnykgetdpaqaiadkaneiqssmvldkgplmklglfqcpdgdhll iaihhllidgvswrilledfasgyeqaergqtiqlpqktdsfpswadqlskyaaetdmeeeiaywtelssikpqp lpkdtisegsllrdseevtiqwtkeeteqllkqanrayntdindllltslglavhkwtgtediwnleghgrepi ipdadisrtigwftsqypwlrmeagknlsqrikivkeglrripdkgmnysiikyisghseadslqlnpeisfny lgqfdqdlkhqalrispfstglsmnenqertavldlngraiaegtlsltlsysskqyerstmaqfarglkeslqev iahcvsrqqtsltpsdillkdisideleqlleqtrelgeaeniypltpmqkgmlfhslfdpnsgayfqqtmfdlh gdleidsfsksldglsqkydifrtnfyrgwkdqplqiifktkkigfqfndlremkesqkeamiqqyaredktnrgf dlekgalmrlfilrtdektyrfiwsfhhilmdgwclplitkeifenyfallqqkqpeqssitpysqyiewlgrqd akeataywdqylegyeeqtglpkdhhaaedgryvpekvtcdissdltskmkrtagkhhvtlntllqtawavllqk ynrsrdwfgswsgrpagipnvetmiglfintipvrfrceagttfaelmkeaqeravasqqfethplydiqart tqkqdlithlmifenypvdqymesigrqngtsitisnvqmeeqtnydfnltvipgdemnisfeynanvydrasie rvrehfmqilhqwtdadirvdqaelltegerrtllqtlndtaapfpqtpvhqlfeeqsqrtpdqaavidkdrql tygelnkranrlartlrakgvqtdqpvaiitrnsieswgilavlksggayvpidpeypqdrirymlddsqagiv lmqrdvrkqlayegvtvllddegsyhqd gsdlapindashlayviytsgstgrpkgvliehrgltnyiwwakevy vkgekanfplyssisfdltvtsiftplvtgnaiivydgedktallesivrdprvdiikltpahlqvlkemniadq tavrrmivggenlstrlarsiheqfegrieicneygptetwgcmiyrydaakdrresvpigtaaantsiyvlde nmkpapigvpgeiyisgagvargylnrpeltaekfvddpfepgakmyktgdlakwladgnieyagrideqvkirg yrielgeieaallqeeaikeawtaredvhgfkqlcayyvsggqttaarlrkqlshslagymvpayfieldempl tsngkinrkglpapdfelqdraeykaprskaeeilvsawesvlgaenvsildnffdlggdsiksiqvssrlnqqg ykmeikdlfqyatiaelsphitqnlriadqgevkgkvsltpiqhwffdhittdqhyynqavmlhasegfqetqlr qtlqklaehhdalrmtfrttengceaqneeiaqsglyhlevmnlkedpdpgrtieakadeiqssmrlsdgplmka glfqcangdhlliaihhllidgvswrilledfasgyeqaergqtiqfpqktdsfqlwakrlseyaqsetikqeqe ywtkieqtevkplpkdfhethttakdsetaavewtkeetelllkqanrayhteindllltslslsishwsgleqi pihleghgreqiiqdidisrtvgwftslypwlhaqpgkeisdyikttkeglrqiphkgigygiarylsggmpsk lnpeisfnylgqfdqdlqqhgvqlssyscgsdssgnqerpyvlningmitegrlkltisysskqyaketimrlse tiqsrlrtiithcvhkeqseltpsdillkgisideldqlliqlphagevenvypltpmqkgmlfhslldeasssy feqasfdlqgelkidwfkaslerlfetyavlrtrfysgwndtp lqivyktqtpqihfadlrdreehlredaiaay qredkakgfdlardplmriaifrmedrkyhliwsfhhivmdgwclslitkevfdhysaleegrepeppsaapysd yiewldrqdqgaakrywsgylegykgettllhkiaqheqkeyayanvicrfdheqtkqlqqianqhqvtlntliq tlwgvllqkysgsadwfgswsgrpaeipdveqmiglfintipvrircdedstfadtmqmvqqnalasqsydty plyeiqaqteqkqnlidhimifenypigqqyekghdaadlnivnfhieehthydfnwvipgeqltihfdfnqne ygqseaerirghfeqlihqilqqpsvkieemelltqqekeqlltplsggtekpeyetihtmferqaaqtpqeiai qyegaeisykelnetanklarilrkrgvkhqepvaviagrspslaiavlgilkaggaivpidpshpaeriryiie nsscthwtekdrsvpeaatqivtfieeaetepdgsnvqtintaedllyviytsgttgkpkgvllehrnmanlly dqftnsgidfktnvlqyaspafdvcyqelfsallsggtlhivpesikrdaaqlfsfinkhqtdivffptafakml fneesyaysfprcvkhlitageqltvsrlfqqvlrthglhlhnhygpsethwstytiqpgddipeyppigkpic hnnmyvinknkqlqpfgiagelyisgantgrgyvnnpaltgekflpdpfregavmyrtgdlarlredgqieyigr iddqakirgyriepkevevilanhpavreaavliqknalgenelcaycsvskatdpsalrkdlaknlpdymipvk wafvesipltangkvdrkalpepeggvqtgieyvaprtaaeaqlahiwqevlglprigvkdnffdigghslratt ltaklhkemgvslplrevfrsptieemaetitgmkhtaytsiptveakeyyavssaqkrl yilnqlkggelsynm psvmradgaldrklvekairkliqrhetlrtgfelidgepvqriyddvpfaveftqakeeqaealvhgfvrafdl ekapllrvglielakdrhlllfdmhhiisdgvsiknlieefvslyegkelppl-rvqykdyaawqlsdmqsermkk qeaywldvfsgevpvldmptdygrpaarsfeggqiefvigpelteqlkglavksestlymvllaayttmlakysg qediwgspiagrthsdlesligmfvgtlairtnpvgektfreyvqevkehtlkayenqeypfdelvdklnvsrd fsrhplfdtmfiwqntergelslddvrftpypdnhamakfdltfqaaenedgitgmisyaaalykqetaermakh fiqlieaiandpqtplsslemitakekeqiverwgqpaidcprdktihqlfeeqaertpehtaavyeksrftyre lneranrlarilrsegvqpdqpvgilaerslemivgimailkaggayvpmdpdspqeriryiledsgakvllaqp hlqdirvsfageilllndermnsgdgsnlvtaagpdhlayviytsgttgkpkgtliehrqvlhlmeglrgqvygay dsglrvsllapyyfdasvkqifaallgghalyivpkasvsdgyalsnyyrthridvtdgtpshlqlliaadslhg vtirhmliggealpqatvaqllelfasngssmplitnvygptetcvdasvfhivpetlasaddggyvpigkplgn nrvyivdshdrmlpigvkgelciagdgvgrgylnlpeltgvkfvadpfvigermyktgdlarylpdgnieyagrk dhqvkirgyrielgeveaallniehvqeavilarenaegqsdlyayftgekslpinqlkeklsdqipgymvpsyl mqleqmpltsngkvnrsalplpeaglqtgidyvaprtrpeeqlvhiwkevlkveqvgvkdnffdlgghslrgmtl vtkihkqfdksislrevfqyptieemarviagaetsgpdeipvaeakdiypvssvqkmvylstqieggelsynmp giltlegrmdmnrlqsafqsliqrheslrtgfemmrgelvqvikpqadfsierykaadeeveelfrnfvrpfdls qapllraglieleqdrhvfmfdmhhivsdgasranifveeliqlydgkeltplriqykdytvwqqqaeqrerikrq enywlnvfheelppfelpkdfarprirsfegkqynfaldenwqgikqmeeltgstaymillsaynillakysgq ediwgtpiagrmhgdlqhiigmfvntlairtapagektfmdyvtetketmlkayenqeypfeelveklgvkrdl srnplfdtmfvlqnteqtdieldslavrpyeqtntaakfdlqltfvmnpheiqgsfeyctklfkqktiatlskdy amilsaiikdpsiplkeiqlsekvnksehfaseielnfaggtairkmivggenlstrlaksvseqfkgrldifne ygpteawgcmiyqfdaerdkrefvpigtpaantdiyvadasrnlvpigvigeiyisgpgvargywnrpdltaek fvenpyvpgakmyksgdlakrlkdgnlvyigrvdeqvkirghrielgeieaamhnaeavqkaavtvkeeedglkq lcayyvsdkpiaaa qlreqlssglpdymvpsyfvrlehmpltsngkinrkalpapeaslqqtaeyvppgnetesk ltdlwkevlgishagikhnffdlggnsiraaalaarihkeldvnlslkdifkfptieqladkalhmdknryvpip vakkmpyypvssaqrrmyllshteggeltynmtgamnvegtidperlnaafrkliarhealrtsfdlyegepaqr ihqnvdftieriqaseeeaedrvldfikafdlakpplmraglieieparhvlwdmhhiisdgvsvnilmkdlsr iyegnepdplsiqykdfavwqqsdiqkrniknqeaywldqfhgdipvldmpadyerpairdyegesfeflipehl kqrlsqmeedtgatlymillasytillskysgqediivgtpsagrthldvepwgmfvntlvirnhpagrktfda ylnevkenmlnayknqdypleeliqhlhlpkdssrnplfdtmfvlqnldhaeltfdslqlkpypfhhsvakfdlt lsiqadrdnyyglfeyskklfkksrievlsndylhilsaileqpsiliehiglεsgsneeeenaldsiqlnf

SEQ ID NO: 5

> CDS 1932 ... 9788 (complementary) Translation of bmyC_FZB42 (laa - 2619) msefkqqelfwssmfdaedrpsaipsfqmsdstiehdassapnrihsslssdvslrimkmtnkspmavymvllvg iecllykytgeegvwgvptfedetdedlrldqvmlikqninadstfksifnefkhklngailhqhvpfdkmagp lnlnydsnhlpmihtivsldqlhpirfietaaadtlfqfsiendsihlkltyneqaydrqymmqviehvnrifsi llfqpdlmirqlnilsdsetnqliaynqtaaeyprektihqfleeqaertpdqtawyedsrltyrelneranql artlqsegvqpdqpvgimaersldraivgifgilkaggayvpidpgypeervryiledsdtklllvqnqsqervpf tgkvldmkdpqnfcedgsnvepaagpdhlayviytsgstgkpkgvmvehrsvinrlvwmqekyplderdailqkt aitfdvsvwelfwwtisgsrlvllpnggeknpelildtiaqkgvstmhfvpamlhaflesmdqkpsgmlkqklas lrhvfasgealkpvhvagfkriitsvsqaqiinlygpteatidvsyfdcqteetyasipigkpisniqlyilhad lehmqpigvagelciagdglargylnrpeltaekfvnhpiasgeriyrtgdlarwlpdgnieylgridhqvkirg yrieigevegaffqlpaikeaiiiareidgetslcayytaqhaltagelreelsrqlpsymipayfvqleemplt fngkidrkslpsprenltgmnyeaprtelekilaavweavlglervgisdhffelggdsiksiqvssrlyqagyk feikhlfkyptiselvpyvepvtriaeqgeikgralltpiqhwffdqkypelhhynqavmlywkeeldesklrdv mkkitehhdalrmv yvptedgyearnrgidegdlfslevislreeknvsqtietisneiqqsihlpegplmklgl frcqegdhlliavhhlvidgvswrillediaaayeqlqngeairlpkktdsyllwaeqlnryaesqefeaenqyw frqkhnpqltlpkdneqetglakdretvivqwtveeterllknahraystdmndllltglgtaihrwtgyedili dleghgresiipdldisrtvgwftslypvslqikadqdipqriktvkenlrqipqkgigyglikylsdhpkahew tghpeirfnylgqfdqdvrngkmevspyssgktasdnrpltytldingmisdgrlslaisycgkqyqretmeaca dllksslqqviahcdaqdqihltpsdislkgitigeldqfvqqtshlgdieniypltpmqkgmlfhslidsasea yfeqaafdlkgfldidafkmslahlaekydilrtlfytewkdqplqivfrqkpietavedirsmnshqrsefiad farrdkargfnltrdalmrvsilrteedqarliwsfhhilmdgwclplvtkevfetyyaileqrqpkrgavtpys ryiewldqqdhkqasaywrnylegyegqtvllqeqssdrakgyekgehefrlgkrltdeikraasrqqvtvntwi qtawglllqryngsqdwfgtwsgrpaeipgietmvglfintipvrihtqpemtaaqvlkmnqeralasqkydt fplydiqaqteqkqqlinhimvfenypvekqiehmkqddnaldildfhmeehthydftfivmpdgeidirfvynr dvydqasvermqthfmqimkqmaddqeirvqdldivtadersllidkfndtaaeypkektihqlfeeqaertpeq aaivfedkkmtyrivneranqlartlvakglqaeelvgimaerspemvigilailkaggvyvpidpdypkerihy mledsnvsilllqhhllegtdyqshtvf lddpssygaeasnlklnvmpnqlayviytsgttgnpkgtliehknw rllfnnknvfdfnasdtwtlfhsfcfdfsvwemygallyggklviipkqiaknperylqllkseavtilnqtpsy fyqlmqeeradpesnlnirkiifggealnpsflkdwklkypltqlinraygitettvhvtykeitereidegrsni gqpiptlqayildeyqriqvmgipgelyvageglargylnrpeltgekfvehpfaagekmyktgdvarwlpdgni eylgridhqvkirgyrieigeveaallqlesvkeawiaieeegskqlcaylsgddslntaqlkhhllnklpaym ipayfvqmekmpitangkidrkalpapegnrltgteyeapgtliekqlaeiwknilalsdpgikdnffdvgghsl kvlqvihqindrmgikmhyqavydfptietmaraiqaavfesktdnvfvkmnqngsipvfcfppligyglvynem akrldgrctvyaadfleepsyekeivdryaesmigiqeqgpfvllgyssgsnlafevakalekrgrivsdimmld skravsvnyfseeeteeiihrnldiipdyyrelltipsikdkirsyltyhnklinsgavnanihhflcgeltdrg wkqstaqhyleyklkgdhvtifdphnieentdtirsiikrieerhhhglvleeqlsmgsfagdakfdkm

3. Use of the sequences according to claims 1 or 2 for lipopeptide biosynthesis.

4. Use of the sequences according to claims 1 or 2 for the biosynthesis of antibiotic substances.

5. Use of the sequences according to claims 1 or 2 for the biosynthesis of antifungal substances.

6. Use of the sequences according to claims 1 or 2 for the biosynthesis of substances with antagonistic activity against phytopathogenic fungi.

7. Use of the sequences according to claims 1 or 2 for the biosynthesis of substances with antagonistic activity against phytopathogenic fungi of the genera Fusarium sp., Alternaria sp., Pythium sp., Rhizoctonia sp., Gaeumannomyces sp.

8. Use of the sequences according to claims 1 or 2 for the biosynthesis of substances with antagonistic activity against phytopathogenic fungi of the species Fusarium oxysporum, Alternaria solani, Pythium aphanidermatum, Rhizoctonia solani, Gaeumannomyces graminis.

9. Use of the sequences according to claims 1 or 2 for the biosynthesis of bacillomycin D.

10. Use of the sequences according to claims 1 or 2 for the biosynthesis of novel iturin compounds.

11. Use of the sequences according to claims 1 or 2 for the combinatorial biosynthesis of novel iturin compounds by exchanging amino acids 4, 5, 6 and / or 7 of the bacillomycin D sequence:

AS l AS 2 AS 3 AS 4 AS 5 AS 6 AS 7

L-Asn D-Tyr D-Asn L-Pro L-GIu D-Ser L-Thr

12. Use according to claim 7, characterized by the following amino acid exchanges:

AS 4 (L-Pro) is replaced by L-GIn,

AS 5 (L-GLU) is replaced by L-Pro,

AS 6 (D-Ser) is replaced by D-Asn, and / or

AS 7 (L-Thr) is replaced by L-Ser or L-Asn.

13. Use according to claims 1-12, characterized in that the antibiotic effect takes place through a single biosynthesis product.

14. Use according to claims 1-12, characterized in that the antibiotic effect takes place by combining the biosynthesis product with other lipopeptides.

15. Use according to claims 1-12, characterized in that the antibiotic effect is achieved by combining the biosynthesis product with other lipopeptides that are synthesized in Bacillus amyloliquefaciens.

16. Use according to claims 1-12, characterized in that the antibiotic effect is achieved by combining the biosynthesis product with fengycin.

17. Use according to claims 3-16, characterized in that the biosynthesis takes place in Bacillus amyloliquefaciens.

18. Use according to claims 3-9, characterized in that the biosynthesis takes place in the Bacillus strain Bacillus amyloliquefaciens FZB42.

19. A process for the preparation of lipopeptides, in particular of novel iturin derivatives, using the sequences according to claims 1 or 2.

20. The method according to claim 11, characterized in that the non-tribosomal protein matrices are manipulated in a targeted manner.

21. The method according to claim 11 or 12, characterized in that optionally the amino acids 4, 5, 6, and / or 7 linked to the sequence L-Asn (l) -D-Tyr (2) -D-Asn (3) different Iturin hybrid variants are obtained.

22. The method according to claim 11 - 13 characterized by position exchanges of the colinearly synthesized amino acids by "domains" shuffling "; Deletion (<7) or addition (> 7) of amino acids in the standard heptapeptide.