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

Sequence for the bacillomycin d synthesis in bacillus amyloliquefaciens fzb42 Download PDF

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WO2004111240A2
WO2004111240A2 PCT/DE2004/001249 DE2004001249W WO2004111240A2 WO 2004111240 A2 WO2004111240 A2 WO 2004111240A2 DE 2004001249 W DE2004001249 W DE 2004001249W WO 2004111240 A2 WO2004111240 A2 WO 2004111240A2
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biosynthesis
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fzb42
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WO2004111240A3 (en
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Rainer Borriss
Alexandra Koumoutsi
Joachim Vater
Helmut Junge
Birgit Krebs
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Rainer Borriss
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

Definitions

  • 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.
  • 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.
  • 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.
  • classic antibiotics e.g. lipopeptides
  • PKS polyketide synthases
  • NRPSs non-tribosomal peptide synthases
  • 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).
  • antimicrobial compounds penicillin, bacitracin, erythromycin, oleandomycin, tylosin and vancomycin
  • immunosuppressants cyclosporin, FK506 and rapamycin
  • antitumor compounds epothilones
  • Most of the developments in combinatorial biosynthesis are based on compounds that are synthesized by modular PKSs.
  • NRPSs and PKSs for aromatic polyketides are used for combinatorial biosynthesis.
  • Non-tribosomal peptide synthetases 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.
  • A adenylation
  • 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).
  • PCP peptidyl carrier protein
  • PPtases 4'-PPan transferase
  • PPtases are associated with most NRPS biosynthetic gene clusters.
  • 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
  • C condensation
  • other modifications are domains in the modules that incorporate modified residues.
  • Te thioesterase-like domain at the COOH terminus of the last NRPS.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • PKS polyketide synthases
  • NRPSs non-tribosomal peptide synthases
  • Bacillus amyloliquefaciens FZB42 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).
  • flanking genes as well as the bmyD and the bmyA gene 98% sequence agreement (at the protein level) with the corresponding genes of
  • 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.
  • 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).
  • 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.
  • 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.
  • NRPS individual enzymes
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 DH5 ⁇ . 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.
  • a 1.2 kb fragment of the bmyA gene was amplified with chromosomal DNA from FZB42 and ligated into pGEM-T vector.
  • 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
  • Competent FZB 42 cells were obtained using a modification of the "One Step Transformation Method" by Kunststoff 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
  • the cell preparation is used immediately for the DNA transformation.
  • 0.1 ml is plated on LB plates + 5 ⁇ g / ml erythromycin and 25 ⁇ g / ml lincomycin. Transformants are visible after 2 days and incubation at 37 ° C.
  • the lipopeptide spectrum of the gene disruption mutant AKl (AbmyA :: Em ⁇ ) 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).
  • 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.
  • 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 AK2.2 ⁇ l 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.
  • 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).
  • FIG. 1 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).
  • 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 / ⁇ g 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 / ⁇ g DNA) can be detected (dashed vertical arrows). No transformants were detectable after 3.5 and 4 h.
  • FIG. 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 lyophilizedGoodf ⁇ ltraten.
  • 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 ⁇ )
  • 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 11 Inhibition of Alternaria solani growth by FZB42 and mutants AKl, AK2, AK3, CHI, CH2

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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

Sequenz für die BacillomycinD Synthese in Bacillus amyloliquefaciens FZB42 Sequence for the BacillomycinD synthesis in Bacillus amyloliquefaciens FZB42
Die Erfindung betrifft die DNA- und Aminosäuresequenz des Bacillomycin D-Operons 5 und die Verwendung der Sequenzen für die Biosynthese von antibiotisch wirksamen Lipopeptiden bzw. Verfahren zu deren Herstellung. Anwendungsgebiete der Erfindung sind die Medizin, die Biotechnologie, die Landwirtschaft und die pharmazeutische Industrie.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 Aus Bacillus subtilis und verwandten Arten sind antifungal und hämolytisch wirksame Peptide isoliert und in der Literatur beschrieben worden. Besondere Beachtung finden dabei die iturinartigen Verbindungen, bei denen es sich um zyklische Heptapeptide handelt, die über eine ß-Aminosäure verknüpft sind und mit ß-Fettsäure modifiziert sind. Charakteristisch für diese Verbindungen ist die folgende Anordnung der 5 Stereospezifität der Aminosäuren: LDDLLDL. Bekannte Vertreter dieser Substanzklasse sind: IturinA, D und E, Bacillomycin L, D und F, Mycosubtilin und Bacillopeptin. Ähnlich wie z.B. Surfactin und Fengycin werden diese Lipopeptide nichtribosomal durch Multienzymkomplexe direkt nach einem Thiotemplate- Mechanismus synthetisiert. Die für die Biosynthese von 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) und Iturin A (Tsuge et al. 2001. Cloning, sequencing, and characterization of the Iturin A Operon. J. Bacteriol. 183: 6265-6273) in B. subtilis verantwortlichen Gencluster wurden beschrieben. '50 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
Das verstärkte Auftreten von Antibiotika-resistenten pathogenen Mikroorganismen hat zu einer intensiven Suche nach neuen wirksamen antibakteriellen und antifungalen Verbindungen geführt. Von mindestens ebenso großer Bedeutung ist die Suche nach antiviralen Verbindungen. Dabei wird die Entdeckung neuer, geeigneter Naturstoffe 0 immer schwieriger. Neben dem Screening von natürlichen Produzentenstämmen verspricht die kombinatorische Biosynthese eine effiziente Alternative für die Entwicklung neuer wirksamer Medikamente zu werden. Kombinatorische Biosynthese beruht auf der genetischen Manipulation von Biosynthesewegen „klassischer" Antibiotika (z.B. von Lipopeptiden), die durch Polyketidsynthasen (PKS) bzw. nichtribosomalen Peptidsynthasen (NRPSs) synthetisiert werden. Polyketide und nichtribosomale Peptide sind zwei große Naturstoff-Familien, die zahlreiche klinisch bedeutsame Verbindungen beinhalten, z.B. antimikrobielle Verbindungen ( Penicillin, Bacitracin, Erythromycin, Oleandomycin, Tylosin und Vancomycin), Immunsuppressiva (Cyclosporin, FK506 und Rapamycin) und Antitumorverbindungen (Doxorubicin, Bleomycin und Epothilone). Die meisten Entwicklungen der kombinatorischen Biosynthese basieren auf Verbindungen, die durch modulare PKSs synthetisiert werden. Daneben werden auch NRPSs und PKSs für die aromatische Polyketide für die kombinatorische Biosynthese eingesetzt.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.
Nichtribosomale Peptidsynthetasen, NRPSs weisen eine modulare Struktur auf, die die notwendigen funktionellen Einheiten repräsentieren. Entsprechend dem multiplen „thiotemplate" Mechanismus der nichtribosomalen Peptidsynthese wird im 1. Schritt durch die Adenylierungs (A-) Domäne, das jeweilige Substrat erkannt, und durch ATP Hydrolyse zu dem korrespondierenden Adenylat aktiviert. Nachfolgend wird das aktivierte Substrat kovalent mit dem 4'- Phosphopantetheinyl (4'-PPan) Kofaktor gebunden, der seinerseits an einen konservierten Serin-Rest der Peptidyl-Carrierprotein (PCP) Domäne geknüpft ist. Die PCP-Domäne ist stromabwärts von der A-Domäne lokalisiert. Die postranslationale Modifikation der PCP-Domäne erfolgt durch eine 4'- PPan Transferase (PPtase). PPtasen sind mit den meisten NRPS Biosynthesegen- Clustern assoziiert. Während der Elongationsreaktion werden die PCP gekoppelten Präkursoren mit der nascenten Peptidkette durch die Kondensations- (C-) Domäne verbunden. Diese ist zwischen jedem konsekutivem Paar der aktivierten Einheiten lokalisiert. Zusätzlich zu A-, PCP- und C-Domäne sind weitere modifizierende Domänen in den Modulen, die modifizierte Reste inkorporieren, vorhanden. Die Freisetzung des kompletten Peptids durch Cyclisierung oder Hydrolyse wird durch eine Thioesteraseartige- (Te-) Domäne am COOH Terminus der letzten NRPS katalysiert. Als Konsequenz dieses linearen Assemblierungsmechanismus, wird die Primärsequenz und das Ausmaß der Modifikation des finalen NRPSs Produktes durch die Aufeinanderfolge der linearen Sequenz der katalytischen Domänen und Module bestimmt (Eppelmann et al. 2001. J.Biol. Chem. 276, 34824-34381).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).
Modulare PKSs und NRPSs sind polyfunktionelle „Megasynthasen", die in repetitiven Einheiten oder „Modulen" organisiert sind. Jedes Modul ist für einen diskreten Schritt der Polyketid- oder Polypeptidkettenverlängerung verantwortlich. Aufgrund dieser Modularität und weil jedes Modul eine katalytische Domäne kodiert, die die Wahl der nächsten Aminosäure bestimmt (einschließlich der Stereospezifität), ist es möglich, neue Verbindungen durch Manipulation der Domänen oder Module zu synthetisieren. Da die Strukturen von Polyketiden sehr komplex und reich an Stereozentren sind, erlaubt diese Technik die Manipulation von Verbindungen, die durch Anwendung konventioneller, chemischer Methoden schwer zu erhalten sind (E. Rodriguez and R. Daniel. 2001. Combinatorial biosynthesis of antimicrobials and other natural products. Current Opinion in Microbiology 4: 526-534). Voraussetzung für die Anwendung der kombinatorischen Neusynthese ist die Kenntnis der Gensequenzen, die für die erforderlichen PKSs und NRPSs kodieren und eines Wirtsstammes, der die stabile Expression großer heterologer DNA-Bereiche erlaubt.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.
Zum Beispiel erwies sich der srfA Deletion Stamm B.subtilis KElO, ein Derivat des Surfactin produzierenden Stammes B.subtilis ATCC21322, für die heterologe Expression eines 49 kb grossen Bacitracin Genclusters aus B.licheniformis, als geeignet (Eppelmann et al. 2001. Engineered biosynthesis of the peptide antibiotic Bacitracin in the Surrogate host Bacillus subtilis. J. Biol. Chem. 276, 34824-34831). Weitere überzeugende Beispiele für die Möglichkeit kombinatorischer Lipopeptidsynthesen, insbesondere von Iturinverbindungen in genetisch zugänglichen, d.h. durch DNA Transformation transformierbaren Wirtsstämmen, vornehmlich des Genus Bacillus, sind aus der Literatur nicht bekannt.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.
Iturine, d.h. nichtribosomal synthetisierte Lipopeptide mit einer ß- Aminofettsäuremodifikation, weisen Tyrosin in der D-Konfiguration an der 2. Position des ringförmigen Heptapeptids auf. Zwei zusätzliche D-Aminosäuren befinden sich an den Positionen 3 und 6. Sie besitzen eine starke antifüngale und hämolytische, sowie eine begrenzte antibakterielle Aktivität. Die Struktur der Iturine Mycosubtilin, Iturin A und Bacillomycin D wird aus der Abb. 1 ersichtlich. Für die Synthese der sehr ähnlichen Iturine IturinA und Mycosubtilin sind die verantwortlichen Gencluster (Operons) beschrieben. Für das Bacillomycin D, das gegenüber Iturin A in 4 von 7 und gegenüber Mycosubtilin in 3 von 7 Positionen der Peptidkette Abweichungen aufweist stand bisher die Sequenzinformation für die NRPS Synthesemodule noch aus. Massenspektroskopische Untersuchungen hatten gezeigt, dass durch FZB42 Bacillomycin D in der späten Wachstumsphase in einem Ammonium-Citrat-Medium synthetisiert wird (vgl. Beispiel 1).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).
Der Erfindung lag die Aufgabe zugrunde Gensequenzen von Polyketidsynthasen (PKS) bzw. nichtribosomalen Peptidsynthasen (NRPSs) bereitzustellen, die für kombinatorische Lipopeptidsynthesen eingesetzt werden können.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.
Die Erfindung wird gemäß den Ansprüchen realisiert. Ein wesentlicher Teil der Erfindung wird durch die Sequenz in den Ansprüchen 1 und 2 repräsentiert.The invention is implemented according to the claims. An essential part of the invention is represented by the sequence in claims 1 and 2.
Im Verlauf der Genomanalyse des phytostimulatorischen Rhizobakteriums 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) konnte überraschend das Genomcluster, das für die Synthese des Iturins Bacillomycin D kodiert, identifiziert werden. Eine Mutante mit spezifischer Insertion einer Antibiotikaresistenzkassette im bmyA Gen, war unfähig zur Bacillomycin D Biosynthese (Beispiel 6).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).
Während die flankierenden Gene sowie das bmyD und das bmyA Gen 98% Sequenzübereinstimmung (auf Proteinebene) mit den korrespondierenden Genen desWhile 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 Biosynthese von B.subtilis RB 14 aufweisen sind der 3 'Bereich des bmyB Gens und das gesamte bmyC Gen variabel. Entsprechend der ColinearitätshypotheseIturinA 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
(Übereinstimmung der Anordnung der Gen-Module mit der Peptidsequenzfolge) befinden sich hier die für die Verknüpfung des Peptids mit den abweichenden Aminosäuren L-Pro (4), L-GIu (5), D-Ser (6), und L-Thr (7) verantwortlichen Module.(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.
Die Genorganisation des Bacillomycin D Biosynthesecluster von FZB42 ist schematisch in der Abbildung 1 dargestellt.The gene organization of the bacillomycin D biosynthetic cluster of FZB42 is shown schematically in Figure 1.
Mit dem Stamm FZB42 wurde ein Stamm identifiziert, der sich nicht nur durch eine erstmalig identifizierte Sequenz für die nichtribosomale Bacillomycinbiosynthese auszeichnet, sondern gleichzeitig die Fähigkeit zur DNA-Aufnahme durch genetische Kompetenz besitzt. Diese Fähigkeit ist bisher nur bei wenigen Bacillusstämmen der Species Bacillus subtilis, vornehmlich in dem Modellorganismus B.subtilis 168, nachgewiesen worden. Überraschend konnte gezeigt werden, dass der Stamm nach einer Methode, die auf der Ausbildung natürlicher Kompetenz in einer bestimmten Phase des Wachstums beruht, die Fähigkeit zur Aufnahme und Rekombination von linearer und circulärer DNA (Plasmid- und chromosomaler DNA) ausbildet (Abb.4). In dem DNA-kompetenten Stamm B.subtilis 168 ist das erfindungsgemäße Gencluster für die Bacillomycin D Biosynthese nicht vorhanden. Es wird angenommen, dass dieses Gencluster ähnlich wie bei den Stämmen ATCC 6633 und RB 14 durch horizontalen Gentransfer in das Genom integriert wurde (Abb. 4). Allerdings sind weder ATCC6633 noch RB 14 genetisch kompetent und können somit nicht für die kombinatorische Biosynthese neuartiger Lipopeptide, vorzugsweise von Iturinderivaten manipuliert werden. Diese Möglichkeit eröffnet der Stamm FZB 42 in dem beliebige Module für die Iturinbiosynthese via homologer Rekombination austauschbar sind. Die modularen Anordnung von NRPSs erlaubt die gezielte Manipulation der nichtribosomalen Proteinmatrizen und befähigt somit zum rationalen Design neuer Peptidantibiotika. Funktionelle Hybrid-Matrizen können in vitro und in vivo hergestellt werden. Für die kombinatorische Biosynthese neuer iturinartiger Wirkstoffe ergeben sich durch die im bmyB und bmyC Gen anwesenden Module neuartige Verknüpfungsmöglichkeiten. Wahlweise können die Aminosäuren 4, 5, 6, und/oder 7 mit der L-Asn(l)-D-Tyr(2)-D-Asn(3) „core-Sequenz verknüpft werden und verschiedene Iturin-Hybrid- Varianten erhalten werden (Tabelle 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).
Weitere Kombinationsmöglichkeiten betreffen mögliche Positionsaustausche der colinear synthetisierten Aminosäuren durch „Domänen"shuffling"; Deletion (<7) oder Addition (>7) von Aminosäuren in dem Standard-Heptapeptid.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.
Tabelle 1 : Kombinatorische Biosynthese neuartiger Iturine durch ModulaustauschTable 1: Combinatorial biosynthesis of novel Iturins through module exchange
Figure imgf000006_0001
L-Asn D-Tyr D-Asn L-Pro L-GIu D-Ser L-Thr
Figure imgf000006_0001
L-Asn D-Tyr D-Asn L-Pro L-GIu D-Ser L-Thr
Mycosubtilir LMycosubtilir L
L-Asn D-Tyr D-Asn L-GIn L-Pro D-Ser L-AsnL-Asn D-Tyr D-Asn L-GIn L-Pro D-Ser L-Asn
Durch neuartige Verknüpfung der Epimerisierungsdomänen mit den AS 1, AS 4, AS 5 und AS 7 ergeben sich zahlreiche weitere Varianten für neuartige Iturinverbindungen. Der Stamm FZB42 ist kompetent für DNA-Transformation. „Gene targeting" Strategien durch homologe Rekombination sind daher gut anwendbar. Dadurch sind die dargestellten Manipulationen direkt in FZB 42 durchführbar. FZB42 enthält weitere Gencluster für die nichtribosomale Synthese von Fengycin und Surfactin (Koumoutsi et al. 2004. J. Bacteriol. 186, 1084-1092). Ein rekombinativer Austausch dieser DNA Bereiche durch weitere Syntheseoperons für variable Iturine ist möglich und gestattet die simultane Synthese von verschiedenen Iturinen mit synergistischem Effekt. So wird der antagonistische Effekt gegenüber phytopathogenen Pilzen durch den simultanen Effekt von Bacillomycin D und Fengycin verursacht (Beispiel 7, Tabelle 4). Es ist auch möglich, mehrere Kopien eines Iturin-Biosyntheseoperons in das FZB42 Chromosom zu rekombinieren und einen Gendosiseffekt mit erhöhter Syntheserate des jeweiligen Iturins zu erhalten.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.
Durch die genannten Techniken können die jeweiligen Iturinbiosyntheseoperons unter die Kontrolle eines starken, induzierbaren Promotors gestellt werden, um eine erhöhte Biosyntheserate in FZB42 zu erhalten. Es ist auch möglich, einzelne Enzyme (NRPS) der nichtribosomalen Peptidsynthese für gezielte Synthese in „in vitro" Systemen zu produzieren.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.
Der Stamm FZB42 kann aufgrund seiner Zugänglichkeit für genetische Manipulationen und seines genetisch determinierten Biosynthesepotentials auch für die simultane Produktion von Bacillomycin D bzw. eines iturinartigen kombinatorischen Neuprodukts zusammen mit den ebenfalls antifungal wirkenden Surfactin und Fengycin, herangezogen werden.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.
Genorganisation des Bacillomycin D Biosyntheseclusters von FZB42: Die für die Biosynthese des Heptapeptids BacillomycinD verantwortlichen Peptidsynthetasen werden durch die Gene bmyA (Al), bmyB (Bl, B2, B3, B4), und bmyC (Cl und C2) kodiert. Diese Gene sind Bestandteil eines ca. 38 kb großen Genclusters, das zwischen die konservierten Gene yxjF und xynD inseriert ist und dem auch das bmyD Gen, das vermutlich eine S-Malonyl-Synthetase kodiert, angehört. Diese Genorganisation ähnelt weitgehend der bei Bacillus subtilis RB 14 (K. Tsuge, T. Akiyama, M.Shoda, 2001 J.Bacteriol. 183, 6265-6273) beschriebenen Struktur des Iturin A Operons. Ausserdem weist das Bacillomycin D-Operon Ähnlichkeit zum Mycosubtilin-Operon von B. subtilis ATCC 6633 (Duitman et al. 1999, PNAS 96, 13294-13299) auf (Abb. 2). Drei der insgesamt 7 Adenylierungsdomänen, die die Spezifität der Aminosäuresequenz des Heptapeptids bestimmen, sind hochkonserviert und determinieren die Sequenz L-Asn- D-Tyr-D-Asn, die in den Iturinen Iturin A, Mycosubtilin und Bacillomycin D identisch ist. Die folgenden vier Adenylierungsdomänen sind im Vergleich zu den entsprechenden Domänen im Mycosubtilin und Iturin A-Operon deutlich abweichend und determinieren eine im Vergleich zu den anderen zwei Iturinen unterschiedliche Sequenz L-Pro-L-Glu-D-Ser-L-Thr (Abb.2). Epimerisierungsdomänen an den Modulen Bl, B2 und Cl determinieren die D-Konfiguration von D-Tyr (2), D-Asn (3) und D-Ser (6).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).
Eine Clustal W Analyse der Sequenzen von konservierten und nichtkonservierten Adenylierungsdomänen aller drei bekannten Iturin- Biosyntheseoperons ergab, dass die Adenylierungsdomänen von bmy_B3 (Pro), bmy_B4 (GIu), bmy_Cl (Ser) und bmy_C2 (Thr) deutlich von den entsprechenden Domänen in den alternativen Iturin- Biosyntheseclustern abweichen (Abb. 3). Dabei ist die zu aktivierende Aminosäure von größerem Einfluß als die jeweilige Position im Iturin-Gencluster. So weist bmy_B3 (Pro) eine höhere Ähnlichkeit zu itu_B4 (Pro) und mycB4 (Pro) auf als zu itu_B3 (GIn) und myc_B3 (GIn). Ähnlich ist die Situation bei bmy_B4 (GIu), das die größte Homologie zu itu_B3 (GIn) und myc_B3 (GIn) aufweist. Eine relativ hohe Ähnlichkeit weist bmy_Cl (Ser) zu myc__Cl (Ser) bzw. itu_C2 (Ser) auf. Auch hier entspricht der Grad der Homologie der Position der Aminosäure im Iturinderivat (Ser6 bei Mycosubtilin; Ser7 bei IturinA). Eine separate Stellung nimmt bmy_C2 (Thr7), für das - ähnlich Glu5 - keine äquivalente Aminosäure in den anderen beiden Iturinen existiert (Abb.3).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).
Mit den erfindungsgemäßen spezifischen Adenylierungsdomänen bmy_B4 (GIu) und bmy_C2 (Thr) ergeben sich besonders interessante Kombinationseffekte für neuartige Iturine, da durch sie die Aminosäuren GIu und Thr neu in Mycosubtilin- und Iturin A- Derivate eingeführt werden können. Die beste Homologie (% identische Reste) der konstanten und variablen Adenylierungs-Domänen ist in Tabelle 2 aufgeführt.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.
Tabelle 2: Beste Homologie (% identischer Reste) bei den variablen Domänen mby_B3, mby_B4, mby_C 1 , und mby_C2Table 2: Best homology (% identical residues) for the variable domains mby_B3, mby_B4, mby_C 1, and mby_C2
Figure imgf000009_0001
Figure imgf000009_0001
Die erfindungsgemäße DNA-Sequenz des BacillomycinD-Operons weist überraschend große Unterschiede in repräsentativen Bereichen zur Sequenz des Mycosubtilin und IturinA-Operons auf. Der besondere Vorteil der Erfindung gegenüber dem Stand der Technik ist die Möglichkeit, neuartige Stoffe mit antibiotischer, insbesondere antifungaler und antiviraler Wirkung produzieren zu können, die wegen dem gehäuften Auftreten von Resistenzen im medizinischen Bereich von großer Bedeutung sind.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.
Da das von Bacillus FZB42 produzierte Bacillomycin D einen antagonistischen Effekt auf phytopathogene Pilze, insbesondere in Kombination mit Fengycin, einem ebenfalls in FZB 42 produzierten Lipopeptid, ausübt (siehe Ausführungsbeispiel 7), ist der Einsatz im Pflanzenschutz besonders vorteilhaft.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.
Durch die Präsenz der erfindungsgemäßen DNA-Sequenz im Bacillus-Stamm Bacillus amyloliquefaciens FZB42 und dessen DNA-Kompetenz eröffnet sich für die Fachwelt nicht nur die Möglichkeit für genetische Experimente, sondern es werden gleichzeitig ideale Voraussetzungen für die kombinatorische Lipopeptidbiosynthese geschaffen. Ausführungsbeispiele: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
Beispiel 1: Massenspektroskopische Analyse von FZB 42: Nachweis von Bacillomycin D, Fengycin und Surfactin: Für den Nachweis von Lipopeptidprodukten in ganzen Zellen wurde B.amyloliquefadens FZB42 auf Landy-Agarmedium (Landy, M. et al. 1948. Proc. Doc. Exp. Biol. Med. 67, 539-541) bei 37°C 24 h kultiviert. Zur Analyse der Massenspektren durch MALDI MS wurde Zellmaterial von der Agarplatte entnommen und mit Matrix Medium (gesättigte Lösung von α-Cyanocinnaminsäure in 40% Acetonitril - 0.1% Trifluor-Essigsäure) bedeckt und luftgetrocknet wie vorher beschrieben (Leenders, F. et al. 1999. Rapid Commun. Mass Spectrom. 13, 943-949). Alternativ wurde eine kleine Probe des gefriergetrockneten Kulturfϊltrats mit 70% Acetonitril - 0,1% Trifluor- Essigsäure extrahiert. Der Extrakt wurde 1:1 (Vol./Vol.) mit Matrixmedium gemischt. Ein 1 μl-Aliquot wurde auf das Target aufgetragen und bis zur MSMessung luftgetrocknet (Vater,J. et al. 2002. Appl. Environ. Microbiol. 68, 6210-6219).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).
Die Massenspektren waren bei dem Einsatz von ganzen Zellen und Kulturfiltraten identisch. Dabei wurden drei Gruppen erhalten, die den Lipopeptiden Suractin, Bacillomycin D und Fengycin entsprechen. Ihre Massenzahlen sind in der Tabelle 3 zusammengefasst.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.
Tabelle 3: Lipopeptidprodukte von B. amyloliquefaciens FZB42 detektiert durchTable 3: Lipopeptide products of B. amyloliquefaciens FZB42 detected by
MALDI-TOF-MSMALDI-TOF-MS
Produkt und beobachtete Massenpeaks (m/z) Bestimmung alsProduct and observed mass peaks (m / z) determined as
Surfactin (1)Surfactin (1)
1 0308, 1,046,8 C13-Surfactin [M + Na, K]+ 1,0308, 1,046.8 C13 surfactin [M + Na, K] +
1 044,8, 1060,8 C14-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] + 1,058.8, 1,074.8 C 15 surfactin [M + Na, K] +
Bacillomycin D (2)Bacillomycin D (2)
1 031,7; 1 053,7, 1 069,7 C14-BacillomycinD [M + H, Na, K] + 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-BacillomycinD [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, 1095,7, 1 111,7 C16-BacillomycinD [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)Fengycin (3)
1 449,9, 1,471,9, 1,487,9 Ala-6-C15 Fengycin [M + H, Na, K] + 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-C 17 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] + 1,491.8, 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] + 1,505.8, 1,527.8, 1,543.8 Val-6-C17 fengycin [M-H, Na, K] +
Die Lipopeptid Produkte von Ensemble 2 wurden als Bacillomycin D durch ihre postsource decay (PSD) Massenspektren identifiziert. Die PSD-MALDI-TOF-MS wurden mit den gleichen Proben durchgeführt. Es wurden die monoisotopischen Massenzahlen aufgezeichnet. Für die Sequenzanalyse sind die (seltenen) protonierten Species vorteilhaft, da sie viel schneller in Fragmente zerfallen als die Alkali-Addukte. Z.B. wurde das Lipopeptid mit der Massenzahl m/z von 1 031,5 als protonierte Form einer Bacillomycin D Isoform mit einer Fettsäure von 14 C- Atomen identifiziert. Ihre Sequenz (Abbildung 6) wurde von einer Serie von b„r, Y„"(-H2O)- und von Prolin- abgeleiteten b„2 Fragment Ionen abgeleitet. Der Peptidring von diesem Bacillomycin D ist an der Peptidbindung zwischen ihrem Amino-Fettsäurerest und Threonin an Position 7 sowie an N-Termimis von Prolin-5 gespalten.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.
Beispiel 2: Genomanalyse von FZB42 durch shot gun Sequenzierung:Example 2: Genome analysis of FZB42 by shot gun sequencing:
Chromosomale DNA wurde von FZB42 durch Anwendung einer Standardmethode (C.R.Harwood & S. M. Cutting) präpariert und durch Anwendung von partiellem Restriktionsverdau (Sau IIIA in suboptimaler Konzentration) bzw. durch hydrodynamische Scherung fragmentiert. Die Ligation der DNA-Inserts erfolgte in ein Smal-linearisiertes, dephosphoryliertes pTZ19 Vektorplasmid. Selektion der weißen Klone nach Transformation in E.coli DH5α. Sequenzanalyse in einem automatischen ABI396 Sequenziersystem. Insgesamt wurden mehr als 39 000 Sequenzläufe mit einer durchschnittlichen Leseweite von 655 bp durchgeführt und zu ca. 800 „Contigs" assembliert. Das entspricht bei einer voraussichtlichen Genomgröße von 3 800 kb einer ca. 6,5 fachen Wiederholung („coverage") der nichtredundanten Sequenz. Lücken zwischen den „Contigs" wurden durch PCR-Reaktionen mit sequenzspezifischen Primern geschlossen. Das Bacillomycin D-Operon wurde als ca 38 kb große DNA Sequenz auf einem insgesamt 56 kb großen „Contig" durch Homologievergleich der deduzierten Ammosäuresequenzen mit der Datenbank identifiziert (Blast P). Die größte Ähnlichkeit wurde zu dem Iturin A-Biosynthese-Operon von Bacillus subtilis RB 14 und dem Mycosubtilin-Biosyntheseoperon von Bacillus subtilis ATCC6633 ermittelt. Beispiel 3: Identifizierung funktioneller Domänen durch Sequenzanalyse des Bacillomycin D-GenclustersChromosomal 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
Durch vergleichende Analyse der Bacillomycin D Gensequenzen mit verschiedenen Sequenzhomologieprogrammen (BLAST, Pfam) wurden in den Genen bmyA, bmyB und bmyC konservierte Domänen identifiziert, die für die nichtribosomale Synthese des Bacillomycin D Peptids verantwortlich sind. Die colineare Position der identifizierten, konservierten bzw. variablen Adenylierungs-, sowie der Epimerisierungs- und Thioesterasedomänen geht aus der Abb. 2 hervor.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.
Beispiel 4: Herstellung einer AbmyA:: Emκ Mutante von FZB42Example 4: Preparation of an AbmyA :: Em κ mutant of FZB42
Ein 1.2 kb Fragment des bmyA Gens wurde mit chromosomaler DNA von FZB42 amplifiziert und in pGEM-T Vektor ligiert. Das Erythromycin-Resistenzgen aus demA 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
Vektor pMX39 wurde in den zentralen Bereich des bmyA-Gens eingefügt. Das resultierende Plasmid mit der Em-Resistenzdeterminante im im bmyA Gen wurde nachVector 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
Linearisierung durch doppelten cross over in das bmyA wt Gen von FZB 42 rekombiniert. Kompetente FZB 42 Zellen wurden durch Anwendung einer Modifikation der „Ein Schritt-Transformationsmethode" von Kunst und Rapoport (1993) erhalten.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).
Die Zellen wiesen maximale Kompetenz in einem kurzen Zeitabschnitt am Ende der logarithmischen Wachstumsphase auf (Abb.5). Die Methode für die Herstellung kompetenter Zellen und die DNA Transformation ist in Beispiel 5 ausfuhrlich dargestellt.Die korrekte Insertion des Resistenzgens in das FZB42 Genom wurde durch PCR mit chromosomaler DNA und durch Southern Hybridisierung von wt und EmRThe 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
Transformanten nachgewiesen.Transformants detected.
Beispiel 5: Herstellung kompetenter Zellen und DNA-Transformation von FZB42:Example 5: Production of competent cells and DNA transformation of FZB42:
- 10 ml LB werden mit einer frischen Kolonie von FZB42 inokuliert und bei 280C und 210 rpm über Nacht inkubiert.- 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 werden mit 2 ml der Vorkultur beimpft und bei 370C und 210 rpm inkubiert. Am Ende der logarithmischen Wachstumsphase, bei einer OD600 nm zwischen 2 - 4, wird die Kultur mit jeweils 20 ml MD Medium 1:1 verdünnt und weiter unter gleichen Bedingungen 1 h geschüttelt. Anschliessend wird die Kultur bei Raumtemperatur und 6 000 rpm zentrifugiert und das Zellsediment in 4 ml des Überstands der Zentrifugation aufgenommen (Anreicherung 1:10).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).
- Die Zellpräparation wird sofort für die DNA Transformation verwendet.- The cell preparation is used immediately for the DNA transformation.
- In einem 100 ml Erlenmeyer-Kolben werden 200 μl frische, kompetenter Zellen mit 500 - 1000 ng DNA in 200 μl frisch angesetztem Transformationspuffer für 20 min. bei 37°C unter gelegentlichem Schütteln inkubiert - Zugabe von 1 ml vorgewärmten (37°C) LB, das für die Induktion der Em Resistenz 0,1 μg Erythromycin (2 μl Em [50 μg/ml] in 1 ml LB) enthalten kann und Schütteln bei 370C für weitere 90 min.- 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 wird auf LB-Platten + 5 μg/ml Erythromycin und 25 μg/ml Lincomycin ausplattiert. Transformanten sind nach 2 Tagen und Bebrütung bei 37°C sichtbar.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.
Beispiel 6: Analyse des Lipopeptidspektrums der Mutante AKl (ΔbmyAr.En^)Example 6: Analysis of the lipopeptide spectrum of the mutant AKl (ΔbmyAr.En ^)
Das Lipopeptidspektrum der Gen-Disruption Mutante AKl (AbmyA::Emκ) wurde, wie in Beispiel 1 dargestellt, durch MALDI-TOF MS bestimmt. Während die Surfactin- Produktion unverändert blieb, waren im Massenspektrum keine Bacillomycin D Signale nachweisbar (Abbildung 7).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).
Beispiel 7: Antifungale Aktivität von FZB42 und MutantenstämmenExample 7: Antifungal activity of FZB42 and mutant strains
Die phytopathogenen Pilzstämme Fusarium oxysporum f. sp.cucumerinum DSMZ 62313 (Welkekrankheit u.a. bei Spargel und Erbse), Alternaria solani (Dürrfleckenkrankheit Tomate), Gaeumannomyces graminis (Schwarzbeinigkeit Kartoffel), Rhizoctonia solani (Fußkrankheiten, Fäulen) und Pythium aphanidermatum (Wurzelbrand Gurke) wurden auf Waksman-Medium kultiviert. Die Bäckerhefe Saccharomyces cerevisiae wurde auf Malz-Agar angezogen. Mit diesen Stämmen wurde die antifungale Aktivität von FZB42 und den Mutanten AKl (ΔbmyA::Emκ, defizient in Bacillomycin D-Produktion, Herstellung ist in Beispiel 4 beschrieben), AK2 (ΔfenA::CmR, defizient in Fengycin-Produktion), AK3 (ΔbmyA::Emκ ΔfenA::CmR , defizient in Bacillomycin D - und Fengycin-Produktion), CHI (ΔsrfA::EmR, defekt in Surfaktinproduktion), CH2 ((AsrfA::EmR AfenA::CmR , defekt in Surfaktin- und Fengycin-Produktion) untersucht. Die Herstellung der Mutanten erfolgte wie beschrieben (Koumoutsi, A. et al. 2004 J. Bacteriol. 186, 1084-1096). Die Doppel- Mutante CH2 wurde durch Transformation von CHI mit chromosomaler DNA von AK2 erhalten. 2μl einer 20 h Kultur in Landy-Medium von FZB 42 bzw. der Mutantenstämme wurden auf Waksman-Agar mit den regulär angeordneten, aktiv wachsenden, Pilzkulturen aufgetragen. Die Platten wurden 3 - 5 Tage bei 27°C inkubiert. Die Hemmwirkung der Bakterienkulturen ist durch die Ausbildung von reduzierten Wachstumszonen der Pilze sichtbar (Abb. 8 A, 9, 10 und 11). Die antibiotische Aktivität auf Saccharomyces cerevisiae und Bacillus megaterium wurde mit verdünntem Kulturfiltrat im Agar-Diffusionstest (beschrieben bei Koumoutsi et al. 2004 J. Bacteriol. 186, 1084-1096) bestimmt. Die hämolytische Wirkung wurde im Lochtest auf Blutagarplatten bestimmt (Vater, J. et al. 2002 Appl. Environ. Microbiol. 68, 6210-6219). Die Ergebnisse sind in der Tabelle 4 zusammengefasst.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.
Tabelle 4: Antifungale, antibakterielle und hämolytische Aktivität von FZB42 und Mutantenstämmen mit defekter LipopeptidsyntheseTable 4: Antifungal, antibacterial and hemolytic activity of FZB42 and mutant strains with defective lipopeptide synthesis
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000014_0001
Figure imgf000015_0001
Legenden zu den AbbildungenLegends for the illustrations
Abbildung 1: Struktur von Iturinen mit antifungaler Wirkung. Die Genorganisation des BacillomycinD Biosynthesecluster von FZB42 ist schematisch unten dargestellt. Die Zahlen entsprechen der Reihenfolge der Aminosäuren im Iturinmolekül. Hochkonservierte Bereiche sind schwarz, variable Bereiche rot gekennzeichnet. E weist auf das Vorhandensein einer Epimerisierungsdomäne (verantwortlich für D- Konfiguration) hin.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).
Abbildung 2: Genorganisation des Bacillomycin D-Clusters.Figure 2: Gene organization of the Bacillomycin D cluster.
Abbildung 3: ClustalW- Vergleich der ACL-Domänen Al, Bl, B2, B3, B4, Cl und C2 von NRPS der Iturin-Biosynthese in FZB42 (bmy, BacillomycinD), RB 14 (itu, Iturin A) und ATCC 6633 (myc, Mycosubtilin).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).
Abbildung 4: Das Bacillomycin D-Gencluster ist eine Insertion im Bereich der 1 944/1 955 kb Region (grün) des B.subtilis Genoms. Mit dem Bacillomycin D-Gencluster wurden auch kleinere Genomfragmente aus den Regionen 4 000 - 4 003 kb (gelb) und 3088 - 3094 (blau) in diesen Rekombinationsort integriert.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.
Abbildung 5: Wachstum von FZB42 und AKl in MDCH Medium. Maximale Kompetenz (> 1000 TF/μg DNA) wird nach 2, 33 h bei einer O.D.600 nm von 1,4 erreicht (roter Pfeil). Nach 2 h (O.D. 1,1) und 2,67 h (O.D. 2) sind nur wenige Transformanten (< 50 TF/μg DNA) nachweisbar (gestrichelte vertikale Pfeile). Nach 3,5 und 4 h sind keine Transformanten nachweisbar.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.
Abbildung 6: Strukturelle Analyse des Lipopeptidproduktes mit m/z 1 031,5 in situ durch PSD-Maldi TOF-MS von ganzen Zellen von B. amyloliquefaciens FZB42. Die Struktur wurde von einer Serie C- und N-terminaler Fragmente [b„ und Y„(-H2O) Ionen sowie von Prolin-abgeleiteten P„ Fragmenten erhalten.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.
Abbildung 7: MALDI TOF MS Analyse von Bacillomycin D und Surfactin Lipopeptiden von B. amyloliquefaciens FZB42 und Mutantenstämmen. A: Nachweis von Surfactin und Bacillomycin D Massen Peaks in Extrakten von lyophilisierten Kulturfϊltraten. B: Nachweis von Surfactin und Bacillomycin D Massen Peaks in FZB 42 Zellen. C: Nachweis von Surfactin aber nicht von Bacillomycin D in der Mutante AKl (AbmyA::Emκ)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 )
Abbildung 8: Hemmung des Wachstums von Fusarium oxysporum DSMZ 62313 (A,B) und Bacillus megaterium durch FZB42 und Mutanten AKl, AK2, CHIFigure 8: Inhibition of the growth of Fusarium oxysporum DSMZ 62313 (A, B) and Bacillus megaterium by FZB42 and mutants AKl, AK2, CHI
Abbildung 9: Hemmung des Wachstums von Gaeumannomyces graminis durch FZB42 und Mutanten AKl, AK2, AK3, CHI und CH2Figure 9: Inhibition of the growth of Gaeumannomyces graminis by FZB42 and mutants AKl, AK2, AK3, CHI and CH2
Abbildung 10: Hemmung des Wachstums von Rhizoctonia solani durch FZB42 und Mutanten AKl, AK2, AK3, CHI und CH2Figure 10: Inhibition of Rhizoctonia solani growth by FZB42 and mutants AKl, AK2, AK3, CHI and CH2
Abbildung 11 : Hemmung des Wachstums von Alternaria solani durch FZB42 und Mutanten AKl, AK2, AK3, CHI, CH2 Figure 11: Inhibition of Alternaria solani growth by FZB42 and mutants AKl, AK2, AK3, CHI, CH2

Claims

Patentansprüche claims
1. DNA-Sequenz der nichtribosomalen Peptidsynthasen für BacillomycinD gekennzeichnet durch folgende Sequenz (SEQ-ID-NO: 1):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-2003LOCUS bmy_feng_c 80841 bp DNA 8-MAY-2003
DEFINITION Complementary copy of gbalvlδr.DEFINITION Complementary copy of gbalvlδr.
SOURCESOURCE
ORGANISM Bacillus amyloliquefaciens FZB42ORGANISM Bacillus amyloliquefaciens FZB42
COMMENT containing bacillomycinD and fengycin gene ClusterCOMMENT containing bacillomycinD and fengycin gene clusters
COMMENT This file is created by Vector NTI http : //www. informaxinc . com/COMMENT This file is created by Vector NTI http: // www. informaxinc. com /
FEATURES Location/Qualifiers CDS compleraent (69..1604)FEATURES Location / Qualifiers CDS compleraent (69..1604)
/label=xynD/ Label = xynD
CDS complement (1932..9788)CDS complement (1932..9788)
/label=bτnyC CDS complement (9876 . .25964)/ label = bτnyC CDS complement (9876. .25964)
/label=bmyB CDS complement (26012..37957)/ label = bmyB CDS complement (26012..37957)
/label=bmyA CDS complement (37980..39179)/ label = bmyA CDS complement (37980..39179)
/label=bmyD CDS complement (39742..40524)/ label = bmyD CDS complement (39742..40524)
/label=yxjF CDS complement (40537..41220)/ label = yxjF CDS complement (40537..41220)
/label=scoB CDS complement (41217..41918)/ label = scoB CDS complement (41217..41918)
/label=scoA CDS complement (41946..43379)/ label = scoA CDS complement (41946..43379)
/label=yxj C/ label = yxj C
CDS complement (43687..44880)CDS complement (43687..44880)
/label=biol/ Label = biol
CDS complement (44885..45901)CDS complement (44885..45901)
/label=bioB/ Label = bioB
CDS complement (46605..47762)CDS complement (46605..47762)
/label=bioF/ Label = bioF
CDS complement (47755..49194)CDS complement (47755..49194)
/label=bioA CDS complement (49095..49835)/ Label = bioA CDS complement (49095..49835)
/label=bioW/ Label = BIOW
CDS 50091..50546CDS 50091..50546
/label=yngA CDS 50556..51445/ label = yngA CDS 50556..51445
/label=yngB/ Label = yngB
CDS 51514..52113CDS 51514..52113
/label=yngC CDS complement (52171..53697)/ label = yngC CDS complement (52171..53697)
/label=yngE CDS complement (53715..54494)/ label = yngE CDS complement (53715..54494)
/label=yngF/ Label = yngF
CDS complement (54511. .55407)CDS complement (54511. .55407)
/label=yngG CDS complement (55635..56891)/ label = yngG CDS complement (55635..56891)
/label=yngH CDS complement (57006..58643)/ label = yngH CDS complement (57006..58643)
/label=yngl CDS complement (58692..59831)/ label = yngl CDS complement (58692..59831)
/label=yngJ CDS complement (59974..61509)/ label = yngJ CDS complement (59974..61509)
/label=yngK CDS complement (61415..62002)/ label = yngK CDS complement (61415..62002)
/label=yngL CDS complement (62087..65887)/ label = yngL CDS complement (62087..65887)
/label=ppsE CDS complement (65909..76681)/ label = ppsE CDS complement (65909..76681)
/label=ppsD CDS complement (76710..80828)/ label = ppsD CDS complement (76710..80828)
/label=ppsC/ Label = PPSC
BASE COUNT 18956 a 20424 c 18453 g 23008 tBASE COUNT 18956 a 20424 c 18453 g 23008 t
2. Aminosäuresequenzen der nichtribosomalen Peptidsynthasen für BacillomycinD gekennzeichnet durch folgende Sequenzen:2. Amino acid sequences of the non-tribosomal peptide synthases for BacillomycinD characterized by the following sequences:
SEQ-ID-NO: 2SEQ ID NO: 2
> CDS 37980 ... 39179 (complementary) Translation of bmyD_FZB42 (laa - 400aa) mnnlaflfpgqgsqfvgmgkqfwndfvlakrlfeeasdaisldvkklcfngdmneltktmnaqpailtvsviafq vymqeigvkprflaghslgeysalvcagalsfrdavtlvrergilmqnadpqqqgamaavthlslqtlqeicski stedfpagvacmnseqqhvisghrqavervikmaeekgaaytylnvsapfhssmirsaseqfqtvlhqysfrdaa wpiisnvtarpyssgnsisehlkqhmtmpvrwtesmhylllhgvteviemgpnnvlagllrkttnhivpyplgqt sdΛφplsnsaerkkhivhlrkkqlnklmiqsviarnynkdsaaysnmttplftqiqelkermkrhedvlseqele hsihlckliceakqlpaweelrilk> CDS 37980 ... 39179 (complementary) Translation of bmyD_FZB42 (laa - 400aa) mnnlaflfpgqgsqfvgmgkqfwndfvlakrlfeeasdaisldvkklcfngdmneltktmnaqpailtvsviafq vymqeigvkprflaghslgeysalvcagalsfrdavtlvrergilmqnadpqqqgamaavthlslqtlqeicski stedfpagvacmnseqqhvisghrqavervikmaeekgaaytylnvsapfhssmirsaseqfqtvlhqysfrdaa wpiisnvtarpyssgnsisehlkqhmtmpvrwtesmhylllhgvteviemgpnnvlagllrkttnhivpyplgqt sdΛφplsnsaerkkhivhlrkkqlnklmiqsviarnynkdsaaysnmttplftqiqelkqqkrhilvcksekermkrelevs
SEQ-ID-NO: 3SEQ 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 tgegaaavllkplqdavrdgdhiygvikgsamnqdgttagitapnpaaqtevietawkdagiapetlsfieahgt gtklgdpvefnglckafekytakkqfcaigsvksnighlfeaagivgliksvlmlnhkknpplahfnepnplihf hsspfyvnqeaaeftsgdeplrggvssfgfsgtnahwleeyisqseyapedehgphlfvlsahtekslyelaqq yrqyvsddsqaslksicytastgrahldhgiamivsgkqelsdkltrliqgdrnlpgvyigyknmkemlpahkee Inqqaaalikqrlrtqderitwlnraaelfvqgavidwralysgetvqktplplypfersrcwaeadqlrlnege kkggaalninqskahiesflktvisnasgiraeeldlnahfiglgmdsimlsqvkkaiadefgvdipmdrffdtm nnlqsvidylaetvpssfasappqenvpaqeiqviseaqsesdrreghqehmlekiiasqnqliqdtlqaqlnsf nllrnsghhsdekeyakaqeksipsvkqghptvtaekkaaqeakpyvpfqpqnlndqghytarqkqyledfikky adktkgskqytdntrfahannrnlssfrsywkeivypiiaersdgskmwdidgneyiditmgfgvnlfghhpsfi tqviddstrsslpplgpmsdvagevadrirtctgvervafynsgteavmvalrlaraatgrkkwafsgsyhgtf dgvlgvagtkggaasanplapgilqsfmddliilhynnpdsldvirslgdelaavlvepvqsrrpdlqpqaflke lraitqqsgtalimdeiitgfriglggaqewfgiqadlvtygkiigggqplgwagkaefmnaidggtwqygdds ypqdeakrtfvagtfnthpltmrmslavlrhlqtegehlyeqlnqktaylvdelnrcfeqaqvpirmvrfgslfr fvssldndlffyhlnykgvyvwegrncflsaahtaddieniiqavkdtvedlrrggfipegpdspdgggrkksgt relspeqkqlvmashygneasaalnqsimlkvkgelqytslkqavrhivdrhealrtvihpddevqqvlermnid ipvidftghphehrepeiqkwltedakrpfhfheqkplfrihvltsahnehlivltfhhiiadgwsiavfvqele snyaaivqgkpispkeadvsfrqyldwqqaqidsghyeegvrywrrhfsepiqqpilpstasvrypngyegdrct vrlgrplsealrslsiqmknsvfvtmlgafhlflhrltklsglvigvpaagqshmkqhdligncvnraipvkntss sestlsgylgsmkesvnlamrhqavpmtlvarelphdqvpdmriifnldrpfrklhfgkaeaepvaypvkctlyd lflnitdahqeyvldfdfntnvispeimkkwgtgftkllqkmvegdsiplnammmfsdeeqhdlqalyaehqkri ssigsntanfteayeapvneterqlariweelfglervgrsdrflalggnslqatlmlskvqktfhqkvsigqff nhqtvkelagfiqnetkwhlpmkaaekkayyptspaqqrvyflhqlepdqlaqnmfghisitgkydeqalissl qqvtnqrheafrtyfdiidgdivqklenevdfnvhvrtmsrdefdaysdrfvkpfrldqaplvraelikieneqae llidrahhiisdgysvniltnellalyhqkplpdiefeykdfaewqnqrlnddamkrqetywleqfqdeipildlp tdgskaaerssegqrvtcslqpdvirslqdlaqkaettlytvllaaynvllhkytgqediwgtpasgrnhpdie kiigifiqtigirtkphanrtftdyleevkrqtldafenqdypfdrlveklnvqrettgkslfntmfvfqniefh eirhnectfkvkernpgvslydlmltiedagqqiemhfdykpgqftkdtieqitrhytailnslveqpemtlssv pmlseterhqlltecngtktpyphketvyrwfemqaeqspdheavifgnerytyrqlneranrlartlrtkgvqa dqfvaiicphrielivgilavlkaggayvpidpeypedriqymlrdsraewltqrslldqlpydgdwlldeen syheehsnlesdsdahdlaymiytsgstgnpkgvliehqgladyiwwakevyvrgektnfplyssisfdltvtsi ftplvtgntiivfdgedksavlseimrdsridmikltpahlhvikemniaggtairkmivggenlstrlaksvse qfkgrldifneygpteawgcmiyqfdaerdkrefvpigtpaantdiyvadasrnlvpigvigeiyisgpgvarg ywnrpdltaekfvenpyvpgakmyksgdlakrlkdgnlvyigrvdeqvkirghrielgeieaamhnaeavqkaav tvkeeedglkqlcayyvsdkpiaaaqlreqlssglpdymvpsyfvrlehmpltsngkinrkalpapeaslqqtae yvppgneteskltdlwkevlgishagikhnffdlggnsiraaalaarihkeldvnlslkdifkfptieqladkal hmdknryvpipvakkmpyypvssaqrrmyllshteggeltynmtgamnvegtidperlnaafrkliarhealrts fdlyegepaqrihqnvdftieriqaseeeaedrvldfikafdlakpplmraglieieparhvlwdmhhiisdgv svnilmkdlsriyegnepdplsiqykdfavwqqsdiqkrniknqeaywldqfhgdipvldmpadyerpairdyeg esfeflipehlkqrlsqmeedtgatlyraillasytillskysgqediivgtpsagrthldvepwgmfvntlvir nhpagrktfdaylnevkenmlnayknqdypleeliqhlhlpkdssrnplfdtmfvlqnldhaeltfdslqlkpyp fhhsvakfdltlsiqadrdnyyglfeyskklfkksrievlsndylhilsaileqpsiliehiglsgsneeeenal dsiqlnf SEQ-ID-NO : 4> 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 lrmifektpdgyapritgtdeselfhlevmnykgetdpaqaiadkaneiqssmvldkgplmklglfqcpdgdhll iaihhllidgvswrilledfasgyeqaergqtiqlpqktdsfpswadqlskyaaetdmeeeiaywtelssikpqp lpkdtisegsllrdseevtiqwtkeeteqllkqanrayntdindllltslglavhkwtgtediwnleghgrepi ipdadisrtigwftsqypwlrmeagknlsqrikivkeglrripdkgmnysiikyisghseadslqlnpeisfny lgqfdqdlkhqalrispfstglsmnenqertavldlngraiaegtlsltlsysskqyerstmaqfarglkeslqev iahcvsrqqtsltpsdillkdisideleqlleqtrelgeaeniypltpmqkgmlfhslfdpnsgayfqqtmfdlh gdleidsfsksldglsqkydifrtnfyrgwkdqplqiifktkkigfqfndlremkesqkeamiqqyaredktnrgf dlekgalmrlfilrtdektyrfiwsfhhilmdgwclplitkeifenyfallqqkqpeqssitpysqyiewlgrqd akeataywdqylegyeeqtglpkdhhaaedgryvpekvtcdissdltskmkrtagkhhvtlntllqtawavllqk ynrsrdwfgswsgrpagipnvetmiglfintipvrfrceagttfaelmkeaqeravasqqfethplydiqart tqkqdlithlmifenypvdqymesigrqngtsitisnvqmeeqtnydfnltvipgdemnisfeynanvydrasie rvrehfmqilhqwtdadirvdqaelltegerrtllqtlndtaapfpqtpvhqlfeeqsqrtpdqaavidkdrql tygelnkranrlartlrakgvqtdqpvaiitrnsieswgilavlksggayvpidpeypqdrirymlddsqagiv lmqrdvrkqlayegvtvllddegsyhqdgsdlapindashlayviytsgstgrpkgvliehrgltnyiwwakevy vkgekanfplyssisfdltvtsiftplvtgnaiivydgedktallesivrdprvdiikltpahlqvlkemniadq tavrrmivggenlstrlarsiheqfegrieicneygptetwgcmiyrydaakdrresvpigtaaantsiyvlde nmkpapigvpgeiyisgagvargylnrpeltaekfvddpfepgakmyktgdlakwladgnieyagrideqvkirg yrielgeieaallqeeaikeawtaredvhgfkqlcayyvsggqttaarlrkqlshslagymvpayfieldempl tsngkinrkglpapdfelqdraeykaprskaeeilvsawesvlgaenvsildnffdlggdsiksiqvssrlnqqg ykmeikdlfqyatiaelsphitqnlriadqgevkgkvsltpiqhwffdhittdqhyynqavmlhasegfqetqlr qtlqklaehhdalrmtfrttengceaqneeiaqsglyhlevmnlkedpdpgrtieakadeiqssmrlsdgplmka glfqcangdhlliaihhllidgvswrilledfasgyeqaergqtiqfpqktdsfqlwakrlseyaqsetikqeqe ywtkieqtevkplpkdfhethttakdsetaavewtkeetelllkqanrayhteindllltslslsishwsgleqi pihleghgreqiiqdidisrtvgwftslypwlhaqpgkeisdyikttkeglrqiphkgigygiarylsggmpsk lnpeisfnylgqfdqdlqqhgvqlssyscgsdssgnqerpyvlningmitegrlkltisysskqyaketimrlse tiqsrlrtiithcvhkeqseltpsdillkgisideldqlliqlphagevenvypltpmqkgmlfhslldeasssy feqasfdlqgelkidwfkaslerlfetyavlrtrfysgwndtplqivyktqtpqihfadlrdreehlredaiaay qredkakgfdlardplmriaifrmedrkyhliwsfhhivmdgwclslitkevfdhysaleegrepeppsaapysd yiewldrqdqgaakrywsgylegykgettllhkiaqheqkeyayanvicrfdheqtkqlqqianqhqvtlntliq tlwgvllqkysgsadwfgswsgrpaeipdveqmiglfintipvrircdedstfadtmqmvqqnalasqsydty plyeiqaqteqkqnlidhimifenypigqqyekghdaadlnivnfhieehthydfnwvipgeqltihfdfnqne ygqseaerirghfeqlihqilqqpsvkieemelltqqekeqlltplsggtekpeyetihtmferqaaqtpqeiai qyegaeisykelnetanklarilrkrgvkhqepvaviagrspslaiavlgilkaggaivpidpshpaeriryiie nsscthwtekdrsvpeaatqivtfieeaetepdgsnvqtintaedllyviytsgttgkpkgvllehrnmanlly dqftnsgidfktnvlqyaspafdvcyqelfsallsggtlhivpesikrdaaqlfsfinkhqtdivffptafakml fneesyaysfprcvkhlitageqltvsrlfqqvlrthglhlhnhygpsethwstytiqpgddipeyppigkpic hnnmyvinknkqlqpfgiagelyisgantgrgyvnnpaltgekflpdpfregavmyrtgdlarlredgqieyigr iddqakirgyriepkevevilanhpavreaavliqknalgenelcaycsvskatdpsalrkdlaknlpdymipvk wafvesipltangkvdrkalpepeggvqtgieyvaprtaaeaqlahiwqevlglprigvkdnffdigghslratt ltaklhkemgvslplrevfrsptieemaetitgmkhtaytsiptveakeyyavssaqkrlyilnqlkggelsynm psvmradgaldrklvekairkliqrhetlrtgfelidgepvqriyddvpfaveftqakeeqaealvhgfvrafdl ekapllrvglielakdrhlllfdmhhiisdgvsiknlieefvslyegkelppl-rvqykdyaawqlsdmqsermkk qeaywldvfsgevpvldmptdygrpaarsfeggqiefvigpelteqlkglavksestlymvllaayttmlakysg qediwgspiagrthsdlesligmfvgtlairtnpvgektfreyvqevkehtlkayenqeypfdelvdklnvsrd fsrhplfdtmfiwqntergelslddvrftpypdnhamakfdltfqaaenedgitgmisyaaalykqetaermakh fiqlieaiandpqtplsslemitakekeqiverwgqpaidcprdktihqlfeeqaertpehtaavyeksrftyre lneranrlarilrsegvqpdqpvgilaerslemivgimailkaggayvpmdpdspqeriryiledsgakvllaqp hlqdirvsfageilllndermnsgdgsnlvtaagpdhlayviytsgttgkpkgtliehrqvlhlmeglrgqvygay dsglrvsllapyyfdasvkqifaallgghalyivpkasvsdgyalsnyyrthridvtdgtpshlqlliaadslhg vtirhmliggealpqatvaqllelfasngssmplitnvygptetcvdasvfhivpetlasaddggyvpigkplgn nrvyivdshdrmlpigvkgelciagdgvgrgylnlpeltgvkfvadpfvigermyktgdlarylpdgnieyagrk dhqvkirgyrielgeveaallniehvqeavilarenaegqsdlyayftgekslpinqlkeklsdqipgymvpsyl mqleqmpltsngkvnrsalplpeaglqtgidyvaprtrpeeqlvhiwkevlkveqvgvkdnffdlgghslrgmtl vtkihkqfdksislrevfqyptieemarviagaetsgpdeipvaeakdiypvssvqkmvylstqieggelsynmp giltlegrmdmnrlqsafqsliqrheslrtgfemmrgelvqvikpqadfsierykaadeeveelfrnfvrpfdls qapllraglieleqdrhvfmfdmhhivsdgasranifveeliqlydgkeltplriqykdytvwqqqaeqrerikrq enywlnvfheelppfelpkdfarprirsfegkqynfaldenwqgikqmeeltgstaymillsaynillakysgq ediwgtpiagrmhgdlqhiigmfvntlairtapagektfmdyvtetketmlkayenqeypfeelveklgvkrdl srnplfdtmfvlqnteqtdieldslavrpyeqtntaakfdlqltfvmnpheiqgsfeyctklfkqktiatlskdy amilsaiikdpsiplkeiqlsekvnksehfaseielnfaggtairkmivggenlstrlaksvseqfkgrldifne ygpteawgcmiyqfdaerdkrefvpigtpaantdiyvadasrnlvpigvigeiyisgpgvargywnrpdltaek fvenpyvpgakmyksgdlakrlkdgnlvyigrvdeqvkirghrielgeieaamhnaeavqkaavtvkeeedglkq lcayyvsdkpiaaaqlreqlssglpdymvpsyfvrlehmpltsngkinrkalpapeaslqqtaeyvppgnetesk ltdlwkevlgishagikhnffdlggnsiraaalaarihkeldvnlslkdifkfptieqladkalhmdknryvpip vakkmpyypvssaqrrmyllshteggeltynmtgamnvegtidperlnaafrkliarhealrtsfdlyegepaqr ihqnvdftieriqaseeeaedrvldfikafdlakpplmraglieieparhvlwdmhhiisdgvsvnilmkdlsr iyegnepdplsiqykdfavwqqsdiqkrniknqeaywldqfhgdipvldmpadyerpairdyegesfeflipehl kqrlsqmeedtgatlymillasytillskysgqediivgtpsagrthldvepwgmfvntlvirnhpagrktfda ylnevkenmlnayknqdypleeliqhlhlpkdssrnplfdtmfvlqnldhaeltfdslqlkpypfhhsvakfdlt lsiqadrdnyyglfeyskklfkksrievlsndylhilsaileqpsiliehiglεsgsneeeenaldsiqlnf> 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: 5SEQ 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 mkkitehhdalrmvyvptedgyearnrgidegdlfslevislreeknvsqtietisneiqqsihlpegplmklgl frcqegdhlliavhhlvidgvswrillediaaayeqlqngeairlpkktdsyllwaeqlnryaesqefeaenqyw frqkhnpqltlpkdneqetglakdretvivqwtveeterllknahraystdmndllltglgtaihrwtgyedili dleghgresiipdldisrtvgwftslypvslqikadqdipqriktvkenlrqipqkgigyglikylsdhpkahew tghpeirfnylgqfdqdvrngkmevspyssgktasdnrpltytldingmisdgrlslaisycgkqyqretmeaca dllksslqqviahcdaqdqihltpsdislkgitigeldqfvqqtshlgdieniypltpmqkgmlfhslidsasea yfeqaafdlkgfldidafkmslahlaekydilrtlfytewkdqplqivfrqkpietavedirsmnshqrsefiad farrdkargfnltrdalmrvsilrteedqarliwsfhhilmdgwclplvtkevfetyyaileqrqpkrgavtpys ryiewldqqdhkqasaywrnylegyegqtvllqeqssdrakgyekgehefrlgkrltdeikraasrqqvtvntwi qtawglllqryngsqdwfgtwsgrpaeipgietmvglfintipvrihtqpemtaaqvlkmnqeralasqkydt fplydiqaqteqkqqlinhimvfenypvekqiehmkqddnaldildfhmeehthydftfivmpdgeidirfvynr dvydqasvermqthfmqimkqmaddqeirvqdldivtadersllidkfndtaaeypkektihqlfeeqaertpeq aaivfedkkmtyrivneranqlartlvakglqaeelvgimaerspemvigilailkaggvyvpidpdypkerihy mledsnvsilllqhhllegtdyqshtvflddpssygaeasnlklnvmpnqlayviytsgttgnpkgtliehknw rllfnnknvfdfnasdtwtlfhsfcfdfsvwemygallyggklviipkqiaknperylqllkseavtilnqtpsy fyqlmqeeradpesnlnirkiifggealnpsflkdwklkypltqlinraygitettvhvtykeitereidegrsni gqpiptlqayildeyqriqvmgipgelyvageglargylnrpeltgekfvehpfaagekmyktgdvarwlpdgni eylgridhqvkirgyrieigeveaallqlesvkeawiaieeegskqlcaylsgddslntaqlkhhllnklpaym ipayfvqmekmpitangkidrkalpapegnrltgteyeapgtliekqlaeiwknilalsdpgikdnffdvgghsl kvlqvihqindrmgikmhyqavydfptietmaraiqaavfesktdnvfvkmnqngsipvfcfppligyglvynem akrldgrctvyaadfleepsyekeivdryaesmigiqeqgpfvllgyssgsnlafevakalekrgrivsdimmld skravsvnyfseeeteeiihrnldiipdyyrelltipsikdkirsyltyhnklinsgavnanihhflcgeltdrg wkqstaqhyleyklkgdhvtifdphnieentdtirsiikrieerhhhglvleeqlsmgsfagdakfdkm > 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. Verwendung der Sequenzen gemäß den Ansprüchen 1 oder 2 für die Lipopeptidbiosynthese.3. Use of the sequences according to claims 1 or 2 for lipopeptide biosynthesis.
4. Verwendung der Sequenzen gemäß den Ansprüchen 1 oder 2 für die Biosynthese antibiotisch wirksamer Substanzen.4. Use of the sequences according to claims 1 or 2 for the biosynthesis of antibiotic substances.
5. Verwendung der Sequenzen gemäss den Ansprüchen 1 oder 2 für die Biosynthese antifüngal wirkender Substanzen.5. Use of the sequences according to claims 1 or 2 for the biosynthesis of antifungal substances.
6. Verwendung der Sequenzen gemäss den Ansprüchen 1 oder 2 für die Biosynthese von Substanzen mit antagonistischer Wirkung gegen phytopathogene Pilze.6. Use of the sequences according to claims 1 or 2 for the biosynthesis of substances with antagonistic activity against phytopathogenic fungi.
7. Verwendung der Sequenzen gemäss den Ansprüchen 1 oder 2 für die Biosynthese von Substanzen mit antagonistischer Wirkung gegen phytopathogene Pilze der Gattungen Fusarium sp., Alternaria sp., Pythium sp., Rhizoctonia sp., Gaeumannomyces sp.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. Verwendung der Sequenzen gemäss den Ansprüchen 1 oder 2 für die Biosynthese von Substanzen mit antagonistischer Wirkung gegen phytopathogene Pilze der Arten Fusarium oxysporum, Alternaria solani, Pythium aphanidermatum, Rhizoctonia solani, Gaeumannomyces graminis.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. Verwendung der Sequenzen gemäß den Ansprüchen 1 oder 2 für die Biosynthese von Bacillomycin D.9. Use of the sequences according to claims 1 or 2 for the biosynthesis of bacillomycin D.
10. Verwendung der Sequenzen gemäß den Ansprüchen 1 oder 2 für die Biosynthese neuartiger Iturinverbindungen.10. Use of the sequences according to claims 1 or 2 for the biosynthesis of novel iturin compounds.
11. Verwendung der Sequenzen gemäß den Ansprüchen 1 oder 2 für die kombinatorische Biosynthese neuartiger Iturinverbindungen durch Austausch der Aminosäuren 4, 5, 6 und/oder 7 der Bacillomycin D Sequenz: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 7AS 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 L-Asn D-Tyr D-Asn L-Pro L-GIu D-Ser L-Thr
12. Verwendung gemäß Anspruch 7, gekennzeichnet durch folgende Aminosäureaustausche:12. Use according to claim 7, characterized by the following amino acid exchanges:
AS 4 (L-Pro) wird ausgetauscht durch L-GIn,AS 4 (L-Pro) is replaced by L-GIn,
AS 5 (L-GLU) wird ausgetauscht durch L-Pro,AS 5 (L-GLU) is replaced by L-Pro,
AS 6 (D-Ser) wird ausgetauscht durch D-Asn, und/oderAS 6 (D-Ser) is replaced by D-Asn, and / or
AS 7 (L-Thr) wird ausgetauscht durch L-Ser oder L-Asn.AS 7 (L-Thr) is replaced by L-Ser or L-Asn.
13. Verwendung gemäss den Ansprüchen 1 - 12, dadurch gekennzeichnet, dass die antibiotische Wirkung durch ein einzelnes Biosyntheseprodukt erfolgt.13. Use according to claims 1-12, characterized in that the antibiotic effect takes place through a single biosynthesis product.
14. Verwendung gemäss den Ansprüchen 1 - 12, dadurch gekennzeichnet, dass die antibiotische Wirkung durch Kombination des Biosyntheseproduktes mit weiteren Lipopeptiden erfolgt.14. Use according to claims 1-12, characterized in that the antibiotic effect takes place by combining the biosynthesis product with other lipopeptides.
15. Verwendung gemäss den Ansprüchen 1 - 12, dadurch gekennzeichnet, dass die antibiotische Wirkung durch Kombination des Biosyntheseproduktes mit weiteren Lipopeptiden, die in Bacillus amyloliquefaciens synthetisiert werden, erfolgt.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. Verwendung gemäss den Ansprüchen 1 - 12, dadurch gekennzeichnet, dass die antibiotische Wirkung durch Kombination des Biosyntheseproduktes mit Fengycin erfolgt.16. Use according to claims 1-12, characterized in that the antibiotic effect is achieved by combining the biosynthesis product with fengycin.
17. Verwendung gemäß den Ansprüchen 3 - 16, dadurch gekennzeichnet, dass die Biosynthese in Bacillus amyloliquefaciens erfolgt.17. Use according to claims 3-16, characterized in that the biosynthesis takes place in Bacillus amyloliquefaciens.
18. Verwendung gemäß den Ansprüchen 3 - 9, dadurch gekennzeichnet, dass die Biosynthese in dem Bacillus-Stamm Bacillus amyloliquefaciens FZB42 erfolgt.18. Use according to claims 3-9, characterized in that the biosynthesis takes place in the Bacillus strain Bacillus amyloliquefaciens FZB42.
19. Verfahren zur Herstellung von Lipopeptiden insbesondere von neuartigen Iturinderivaten unter Verwendung der Sequenzen gemäß den Ansprüchen 1 oder 2.19. A process for the preparation of lipopeptides, in particular of novel iturin derivatives, using the sequences according to claims 1 or 2.
20. Verfahren gemäß Anspruch 11, dadurch gekennzeichnet, dass die nichtribosomalen Proteinmatrizen gezielt manipuliert werden. 20. The method according to claim 11, characterized in that the non-tribosomal protein matrices are manipulated in a targeted manner.
21. Verfahren gemäß Anspruch 11 oder 12, dadurch gekennzeichnet, dass wahlweise die Aminosäuren 4, 5, 6, und/oder 7 mit der Sequenz L-Asn(l)-D-Tyr(2)-D-Asn(3) verknüpft werden und verschiedene Iturin-Hybrid- Varianten erhalten werden.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. Verfahren gemäß Anspruch 11 - 13 gekennzeichnet durch Positionsaustausche der colinear synthetisierten Aminosäuren durch „Domänen"shuffling"; Deletion (<7) oder Addition (>7) von Aminosäuren in dem Standard-Heptapeptid. 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.
PCT/DE2004/001249 2003-06-12 2004-06-14 Sequence for the bacillomycin d synthesis in bacillus amyloliquefaciens fzb42 WO2004111240A2 (en)

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