DE102004027335B4 - Sequences for the synthesis of antibacterial polyketides - Google Patents

Abstract

Recombinant expression vector, characterized in that it contains the DNA sequence SEQ ID NO: 2 of the strain Bacillus amyloliquefaciens FZB42.

Description

The invention relates to DNA sequences which encode polypeptides which in turn are involved in the enzymatic biosynthesis of polyketide compounds and the amino acid sequences of the polypeptides.

Furthermore, the invention relates to the first-time description of synthetic routes for the production of polyketide antibiotics from Bacillus amyloliquefaciens FZB42. At least one polyketide antibiotic has a specific activity against bacteria, preferably Gram-positive bacteria, but has no inhibiting action against eukaryotic organisms.

The fields of application of the invention are biology, medicine and pharmacy.

State of the art

The spore-forming Gram-positive bacterium Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses the growth of phytopathogenic microorganisms in the plant root zone (Idriss et al., 2002. Microbiology 148: 2097-2109).

The suppression of the phytopathogenic fungus Fusarium oxysporum is caused by the combined action of the cyclic lipopeptides bacillomycin D and fengycin. The sequence for the bacillomycin D operon indicates the presence of three modular peptide synthetases BmyA, B and C in FZB42 (Borriss et al., Unpublished application German Patent and Trademark Office C12N 15/52 Acc. 10326394.2, this application was withdrawn prior to publication ).

By inactivating the BmyA-encoding gene, not only the bacillomycin D synthesis but also the antifungal activity of FZB42 on Fusarium oxysporum was eliminated. In contrast, the antibacterial effect of culture filtrates of the FZB42 mutants on Gram-negative and Gram-positive bacteria persisted. (Koumoutsi et al., 2004, J. Bacteriol., 186, 1084-1096).

The elimination of fengycin and surfactin synthesis did not affect the antibacterial activity of FZB42.

An antibacterial effect has already been described for various conjugated polyene antibiotics of the genus Bacillus (eg Zimmermann et al 1987. J. Antibiotics 40: 1677-1681, Patel et al., 1995. J. Antibiotics 48, 997-1003) ,

US Pat. No. 4,681,846 describes difficidin as an antibacterial agent and discloses a process for producing difficidin and antibacterial compounds derived therefrom from Bacillus subtilis.

However, gene sequences that determine the synthesis of these antibacterial agents in Bacillus biocontrol strains are not known.

The increased occurrence of antibiotic-resistant pathogenic microorganisms has led to an intensive search for new effective antibacterial and antifungal compounds. At least as important is the search for antiviral compounds.

The discovery of new natural products 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.

Polyketides and nonribosomal peptides are two major natural product families that include numerous clinically important compounds, e.g. As antimicrobial compounds (penicillin, bacitracin, erythromycin, oleandomycin, tylosin and vancomycin), immunosuppressants (cyclosporin, FK506 and rapamycin) and antitumor compounds (doxorubicin, bleomycin and epothilones).

Polyketides are a class of secondary metabolites with remarkable diversity in structure and function that can exhibit both antimicrobial and antiviral and antiparasitic properties.

Their broad spectrum of activity makes them one of the most sought-after molecular groups in biological screens. For example, the polyketide mupirocin is responsible for the biocontrol function of the Gram-negative bacterium Pseudomonas fluorescens (El-Sayed et al., 2004. Chemistry & Biology 1: 419-430).

The polyketide bacillaen, which is produced in a hitherto unknown manner in some Bacillus subtilis strains, is known as an inhibitor of prokaryotic protein synthesis and has a molecular weight of 580 and an empirically determined empirical formula of C35H48O7. In the agar diffusion assay, bacillaen is active against a variety of bacteria but not against eukaryotic organisms (Patel et al., 1995. Journal of Antibiotics 48, 997-1003).

By applying the "suppression subtractive hybridization" (SSH) method, FZB42 identified three gene clusters homologous to polyketide synthases (Koumoutsi et al., 2004, J. Bacteriol, 186). A determination of the complete DNA sequences and the translated proteins did not occur. Evidence of the synthesis of polyketides in Bacillus amyloliquefaciens or an associated antibiotic activity was not performed. There was no assignment of the polyketide synthase gene cluster for the synthesis of a polyketide.

Most developments in combinatorial biosynthesis are based on compounds synthesized by modular polyketide synthases (PKSs). In addition, non-ribosomal peptide synthases (NRPSs) and PKSs are also used for the synthesis of aromatic polyketides and for combinatorial biosynthesis.

Combinatorial biosynthesis relies on the genetic manipulation of biosynthetic pathways of "classical" antibiotics (eg, lipopeptides) synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs), respectively.

Modular PKSs and NRPSs are polyfunctional "megasynthases" organized in repetitive units or "modules". Each module is responsible for a discrete step of polyketide or polypeptide chain extension.

Since each module determines the functional domains for recognition, activation, and condensation of a substrate amino acid, including stereospecificity, it is possible to synthesize new compounds through manipulation of 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 (Rodriguez and R. Daniel 2001. Combinatorial biosynthesis of antimicrobials and other natural Products, Current Opinion in Microbiology 4: 526-534).

The prerequisite for the application of combinatorial resynthesis is the knowledge of the gene sequences which code for the required PKSs and NRPSs and of a host strain which allows the stable expression of large heterologous DNA regions.

The object of the present invention was the identification and characterization of gene clusters in the genome of the bacterium Bacillus amyloliquefaciens FZB42, which are responsible for the synthesis of polyketides, as well as the identification of synthetic pathways for polyketide antibiotics. Another object of the invention was the identification of polyketide antibiotics produced by these synthetic routes.

The invention is realized according to the claims.

The present invention relates to DNA sequences which code for polypeptides, which in turn are involved in the enzymatic biosynthesis of Polyketidverbindungen and the amino acid sequences of the polypeptides.

Furthermore, the invention relates to the description of synthetic routes for the production of polyketide antibiotics from Bacillus amyloliquefaciens FZB42. At least one polyketide antibiotic has a specific activity against bacteria, preferably Gram-positive bacteria, but has no inhibiting action against eukaryotic organisms.

In strain FZB42, three gene clusters for the synthesis of polyketides have been identified and fully characterized by sequence analysis.

Surprisingly, in the genome of the bacterium Bacillus amyloliquefaciens (strain FZB42), the DNA sequences with SEQ ID NO: 1, 2 and 3 could be identified and characterized. The DNA sequences encode polypeptides, which in turn are involved in the enzymatic biosynthesis of polyketide compounds having antibacterial activity.

Extensive investigations have shown that the expression products of the sequence SEQ ID NO: 1 cause the enzymatic biosynthesis of the antibacterial polyketide Bacillaen. The expression products of the sequence SEQ ID NO: 2 cause the enzymatic biosynthesis of the antibacterial polyketides difficidin and / or oxydifficidin and the expression products of the sequence SEQ ID NO: 3 are responsible for the synthesis of a previously unknown antibacterial polyketide.

The invention further comprises a recombinant expression vector, characterized in that it contains the DNA sequence SEQ ID NO: 2 of the strain Bacillus amyloliquefaciens FZB42. The invention also relates to a method, characterized in that cells are transformed or transfected with a recombinant expression vector containing the DNA sequence SEQ ID NO: 2 of the strain Bacillus amyloliquefaciens FZB42, the cells are cultured in a suitable culture medium and the polyketide compound isolated from the culture supernatant.

With this method, the polyketides difficidin and / or oxydifficidin can be produced with antibacterial activity.

Furthermore, the invention encompasses the use of the expression vector according to the invention, which contains the DNA sequence SEQ ID NO: 2 of the strain Bacillus amyloliquefaciens FZB42 for the combinatorial re-synthesis of difficidin and / or oxydifficidin, which has an improved stability and / or action against phyto- animal - and / or human pathogenic bacteria.

The amino acid sequences of polypeptides involved in the enzymatic biosynthesis of polyketide compounds comprising the sequences of SEQ ID NOs: 4 to 42 and the use of the amino acid sequences for combinatorial resynthesizing of polyketides, which have improved stability and / or Have activity against phyto-, animal and / or human pathogenic bacteria are also the subject of the invention.

The modular arrangement of type 1 polyketide synthases allows the targeted manipulation of non-ribosomal protein matrices and thus enables the rational design of new polyketide antibiotics according to the invention.

Functional hybrid templates can be made in vitro and in vivo. Numerous modifying enzymes (eg reductases, dehydratases, methyltransferases) from one of the three PKS clusters present in FZB42 can also be used for the combinatorial biosynthesis of new polyketide agents.

Furthermore, the natural competence for DNA uptake and recombination and the knowledge of the DNA sequences in FZB42 allows the targeted use of stronger and regulatable promoters to increase the product yield.

The invention is explained in more detail below and illustrated by embodiments, without these explanations would have a restrictive effect.

Antibacterial activity of FZB42

Culture filtrates of FZB42 after 24 h or 48 h culture have both antibacterial activity against most of the tested bacteria and antifungal activity (Arxula adeninovorans, Saccharomyces cerevisiae). The highest sensitivity was observed in Gram-positive bacteria (except Bacillus subtilis, Staphylococcus aureus). The verified representatives of the α-proteobacteria were also clearly inhibited by the culture filtrate of FZB42. In contrast, representatives of enterobacteria and other representatives of γ-proteobacteria were relatively insensitive (see Table 1). Table 1: Inhibition of microorganisms by culture filtrates of FZB42 (24 and 48 h growth). The diameter of the zones of inhibition is given in cm. tribe 24 hours 48 h taxon Bacillus subtilis - - 0.3 cloudy Gram + lowGC Bacillus megaterium 1.3 clear 1.1 clear Gram + lowGC Paenibacillus larvae larvae 1 ¹ clear 1,3 ² clear Gram + lowGC Streptococcus faecalis 0.8 clear 0.6 clear Gram + lowGC Micrococcus luteus - - 0.5 clear Gram + lowGC Staphylococcus aureus - - - - Gram + lowGC Arthrobacter spec. 0.7 clear 0.9 clear Gram + lowGC Streptomyces coelicolor 40/233 1.6 clear 1.2 clear Gram + highGC Streptomyces viridochromogenes 40110 1.6 clear 1.2 clear Gram + highGC Streptomyces F2 0.8 clear 0.8 clear Gram + highGC Agrobacterium tumefaciens 0.4 clear 0.9 clear α-proteobacteria Agrobacterium tumefaciens AGL1 0.5 clear 1 clear α-proteobacteria Flavobacterium spec. 0.3 cloudy 0.3 cloudy α-proteobacteria Acinetobacter spec. - - - - α-proteobacteria Klebsiella terrigena 0.3 clear 0.2 clear γ-proteobacteria Klebsiella pneumoniae 0.2 cloudy 0.2 clear γ-proteobacteria Enterobacter spec. 0.3 cloudy - - γ-proteobacteria Erwinia carotovora 0.3 cloudy 0.4 cloudy γ-proteobacteria Serratia marcescens 0.6 clear 0.5 clear γ-proteobacteria Proteus vulgaris 0.2 cloudy 0.2 cloudy γ-proteobacteria ¹ determination after 5 days growth on blood agar
² Determination after 8 days growth on blood agar Pseudomonas aeruginosa 0.2 cloudy 0.4 cloudy γ-proteobacteria Pseudomonas fluorescens 0.5 cloudy 0.4 cloudy γ-proteobacteria Pseudomonas fluorescens 15 0.2 cloudy - - γ-proteobacteria Pseudomonas aureofaciens PS49 - - - - γ-proteobacteria Pseudomonas putida 62 - - - - γ-proteobacteria Pseudomonas spec. 14a 0.3 cloudy 0.2 clear γ-proteobacteria Pseudomonas spec. 20 0.4 clear 0.3 clear γ-proteobacteria Pseudomonas spec. 79 - - - - γ-proteobacteria Pseudomonas spec. K19 - - - - γ-proteobacteria Arxula adeninovorans 0.2 clear 0.6 clear yeasts Saccharomyces cerevisiae 1.2 clear 1.1 clear yeasts

In the culture filtrate of FZB42 and in mutants in which the synthesis of the antifungal-acting lipopeptides bacillomycin D, fengycin and surfactin was eliminated, surprisingly, a specific antibacterial activity, in particular against Gram-positive bacteria (inter alia, Bacillus sp., Streptomyces sp., Streptococcus sp.).

By eliminating the pks3 gene sequence in the mutant CH8, the antibacterial effect against the bacteria mentioned was largely abolished. This finding demonstrates that the polyketide synthetases of the pks3 gene cluster are used to synthesize a polyketide with specific antibacterial activity. A chemical Characterization of the polyketide by mass spectroscopy was previously not possible. A similarity to the previously described from Bacillus polyketides (Bacillaen and Difficidin / Oxydifficidin) could be excluded.

Surprisingly, the polyketides bacillaen and difficidin / oxydifficidin could be detected in the culture filtrate of the bacterium Bacillus amyloliquefaciens FZB42 and individual mutants.

After separation of the culture filtrate of FZB42 by HPLC could in the after 18 min. Retention time eluted peak detected by electrospray mass spectrometry (ESM) a peak with m / z of 581.1, which corresponds to the bacillaen. The highest intensity of this peak was observed after 48 h of culture in Landy's medium.

An optimized drug based on the polyketide Bacillaen represents an alternative in the use against pathogenic gram-positive bacteria of the genera Staphylococcus sp. and Streptococcus sp. whose medical treatment is increasingly complicated by the rapidly spreading multidrug resistance compared to conventional antibiotics.

In addition, the pks3 polyketide does not cause any inhibition of eukaryotic cells and is therefore potentially suitable for use in the human field.

This particularly advantageous property could be demonstrated by the fact that in the mutant CH8 no reduction of the antifungal activity against Fusarium oysporum or Saccharomyces cerevisiae was caused.

To identify the gene cluster (pks1, pks2, pks3) possibly responsible for the antibacterial activity, all three gene clusters were completely sequenced (SEQ ID NO: 1 = pks1, SEQ ID NO: 2 = pks2, and SEQ ID NO: 3 = pks3) and further characterized by homology sequence comparison.

Genome analysis: pks gene cluster

At various positions in the genome of B. amyloliquefaciens FZB42, three large gene clusters with homology to the polyketide synthase operon (pksX) of B. subtilis (Albertini et al., 1995. Microbiology 141, 299-309) were detected. Pks1 (SEQ ID NO: 1, 72612 bp, Tab. 2 and 7), pks2 (SEQ ID NO: 2, 53724 bp, Tab. 3 and 8) and pks3 (SEQ ID NO: 3, 74700, Tab 4 and 9) have structural similarities and are probably the result of gene duplication from a common ancestor.

Construction of gene-knock-out mutants in FZB42

Gene expression was eliminated by cassette mutagenesis into the respective first keto-synthase module (KS) using erythromycin (Em ^R ) and chloramphenicol-resistance (Cm ^R ) -mediating genes (Koumoutsi et al., 2004. J. Bacteriol. 1084 to 1096). pMX39 (Em ^R ) was digested with SpeI. The resulting 2.9 kb fragment was digested with HindIII and a 1.8 kb emrAM fragment obtained. emrAM was inserted into the polylinker region of HindIII digested pUC18. The plasmid (pUCemR) was used for further gene insertion experiments to introduce erythromycin resistance. A 1.8 kb fragment with the CmR marker gene was obtained by SphI and HindIII digestion from pDG268 and cloned into pUC18. In the resulting plasmid (pUCcmR), the Cm ^R gene was flanked by the polylinker region of pUC18. Using the primers pks1Up5'-AAAGGAGCGGAGTGCAACATC and pks1Dw 5'-TGAGATGATGCCGTCCTCTTC, a 3.3 kb fragment was amplified by PCR and cloned into the vector pGEM-T. This was digested with EcoRI and HpaI and a 1.4 kb fragment with the first KS domain (KS1, Table 2) was removed. The Cm ^R gene from EcoRI / SmaI cut pUCcmR was inserted and the plasmid pCH6 was obtained. After linearization by ScaI, the plasmid was transformed into competent B. amyloliquefaciens FZB42 cells (Koumoutsi et al., 2004. J.Bacteriol., 186, 1084-1096). Gene substitution via homologous recombination yielded the Cm ^R pks1 mutant CH6. A 3.0 kp PCR fragment containing the 5 'portion of the pks2 operon was obtained by the primers pks2Up 5'-AGCTCATTGACAGCGATGCTGC and pks2Dw 5'-ATGATCGCCGCTTCCTCATCAG and cloned into the vector pGEMT. A 1.3 kb fragment with the first KS domain of pks2 (KS1, Tab. 3) was excised by EcoRI and KpnI and replaced by the 1.3 kb Cm ^R gene from pUCCmR. After linearization with ScaI, competent FZB42 cells were transformed and the Cm ^R pks2 knock out mutant CH7 was obtained. The pks3 gene was destroyed by insertion of an Em ^R cassette from pUCemR. A 2.2 kb pks3 gene fragment was amplified with the primers pks3Up 5'-ACATTGACCGCATCCTCAATCTG and pks3Dw 5'-TCAGCTGCTGTTCGGAATGTG and inserted into the vector pGEM-T cloned. Digestion with EcoRI and Eco72I removed a 376 bp fragment in the middle of the amplified region. The ermAM cassette (1.9 kb) was obtained from pUCemR by EcoRI / PvuII digestion and inserted. The resulting plasmid pCH8 was linearized with ApaI and transformed into competent FZB42 cells. The EmR pks3 mutant CH8 was obtained. The genetic configuration of the mutants was confirmed by PCR of the chromosomal DNA with the specific primers. Table 5: Strains and plasmids. For the production of the desired knockout strains, the plasmids pCH6, pCH7 and pCH8 were used for the transformation into FZB42. Description, resistance B. amyloliquefaciens strains Source / Production FZB42 Producer of surfactin, fengycin, bacillomycin FZB Biotech GmbH Berlin CH6 Δpks1 :: Cmr pCH6 → FZB42 CH7 Δpks2 :: Cmr pCH7 → FZB42 CH8 Δpks3 :: Emr pCH8 → FZB42 plasmids pMX39 Ap ^R , Emr, Tetr Derivative of pDB101 pDG364 amyE Ap ^R, Cm ^R Integrative vector pGEM-T Cloning Vector, Ap ^R Laboratory collection pUC18 Cloning Vector, Ap ^R Laboratory collection pUCemR pUC18 with erm_amp, Ap ^R pUCcmR pUC18 with cat, Amp ^R , Cm ^R pCH6 pGEM-T with a 1.9 kb fragment of pks1 :: Cmr, Ap ^R pCH7 pGEM-T with a 1.7 kb fragment from pks2 :: Cmr, Ap ^R pCH8 pGEM-T with a 1.8 kb fragment from pksf3 :: Emr, Ap ^R

Antibacterial and antifungal activity of gene knock-out mutants

By eliminating the pks3 gene by cassette mutagenesis, almost all antibacterial activity in the mutant strain CH8 was eliminated (indicator strain B. megaterium, ). In contrast, the pks2 mutant CH7 exhibited unchanged antibacterial activity against the indicator strains Bacillus megaterium and Arthrobacter spec. on. Both mutants were not altered in their antifungal activity (Saccharomyces cerevisiae, Fusarium oxysporum) compared to the wild type. Conversely, none of the lipopeptide synthesis mutants (AK1, AK2, AK3, CH1, CH2, CH3) were effective against Bacillus megaterium and Arthrobacter spec. affected. The elimination of the sfp gene, which codes for a phosphopantetheinyl transferase, results in the loss of the synthesis of biologically active lipopeptides and polyketides (Table 5). Of particular interest is the effect of the yczE mutation (CH4), which inhibits both the synthesis of lipopeptides of the iturin family and the polyketides. The failure of the synthesis of bacillomycin D in CH4 (yczE), and of bacillomycin D, fengycin and surfactin in the sfp, yczE double mutant CH5 could be detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS). The failure of the synthesis of bacillaen and difficidin / oxydifficidin in CH4 (yczE), and of bacillomycin D, fengycin and surfactin in the sfp, yczE double mutant CH5 could be detected by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI TOF MS). The corresponding analysis of the culture filtrates of the pks mutants (CH6-CH8) revealed that the pks1 gene cluster causes the synthesis of bacillaen and the pks2 gene cluster the synthesis of difficidin / oxydifficidin. Surprisingly, the elimination of the bacillaen biosynthesis had no drastic effect on the inhibitory effect against the indicator strains used in the agar diffusion test. Compared to B. megaterium, the inhibitory effect of the culture filtrate was slightly reduced, in contrast to St. aureus - as in the CH8 pks3 knock out mutant - in the first case detectable. The cause could be the increased formation of difficidin and oxydifficidin in both mutants compared to the wild type. After elimination of the pks2 gene cluster, no difficidin / oxydifficidin was detected in the culture filtrate of CH7. The antibiotic activity towards B. megaterium remained unchanged while no inhibitory activity against St. aureus remained ( ). Although no 3rd polyketide has been used by us in any of our investigation methods FZB 42 was detected, the significant decrease in the inhibitory activity towards the indicator strain B. megaterium in the pks-3 knock out mutant CH8 shows that this gene cluster produces a potent antibiotic with specific activity ( , Table 6). In particular, the gene clusters pks2 (difficidin / oxydifficidin biosynthesis) and pks3 (unknown polyketide) are for the synthesis of drugs with specific activity against phyto-, animal- and human pathogenic bacteria (eg the causative agent of potato scab, Streptomyces scabies, the pathogen the foul brood in bee larvae, Paenibacillus larvae larvae and the human pathogenic Staphylococcus aureus) of interest.

Tabelle 7: Das pks1-Genkluster von Bacillus amyloliquefaciens FZB42 Quelle: SEQ-ID-NO: 1, GenBank Accession Number: AJ634060, Länge: 72612 bp, beinhaltet das pks1-Operon (bp1...71249) Gene des Bacillus amyloliquefaciens pks1 Operons (pks1B, C, D, E, F, G, H, I, J, L, M, N, R) und das pks1S Gen Gen CDS Produkt Protein-ID SEQ-ID-NO: pks1B 1...690 hydroxyacylglutathione hydrolase CAG23949.1 4 pks1C 1009...1878 malonyl-CoA-[acyl-carrier-prote] transacylase CAG23950.1 5 pks1D 1850...2989 malonyl-CoA-[acyl-carrier-protein] transacylase CAG23951.1 6 pks1E 2991...5231 malonyl-CoA-[acyl-carrier protein] transacylase CAG23952.1 7 pks1F 5297...5545 acyl carrier protein CAG23953.1 8 pks1G 5597...6859 3-hydroxy-3-methylglutaryl CoA synthase CAG23954.1 9 pks1H 6847...7629 enoyl-CoA hydratase CAG23955.1 10 pks1I 7639...8388 enoyl-CoA-hydratase CAG23956.1 11 pks1J 8428...23376 putative polyketide synthase CAG23957.1 12 pks1L 23360...36805 putative polyketide synthase CAG23958.1 13 pks1M 36802...47358 putative polyketide synthase CAG23959.1 14 pks1N 47348...63649 putative polyketide synthase CAG23960.1 15 pks1R 63753...70886 putative polyketide synthase CAG23961.1 16 pks1S 71247...72494 putative cytochrome P450 107K1 CAG23962.1 17 Tabelle 8: Das pks2-Genkluster von Bacillus amyloliquefaciens FZB42 Quelle: SEQ-ID-NO: 2, GenBank Accession Number: AJ634061, Länge: 53724 bp, beinhaltet das pks2-Operon (bp1...52130) Gene des Bacillus amyloliquefaciens pks2 Operons (pks2A, B, C, D, E, F, G, H) Gen CDS Produkt Protein-ID SEQ-ID-NO: pks2A 1...2307 malonyl CoA [acyl-carrier-protein] transacylase CAG23963.1 18 pks2B 2329...14589 putative polyketide synthase typeI CAG23964.1 19 pks2C 14589...19361 putative polyketide synthase CAG23965.1 20 pks2D 19379...28117 putative polyketide synthase typeI CAG23966.1 21 pks2E 28110...35114 putative polyketide synthase typeI CAG23967.1 22 pks2F 35129...40849 putative polyketide synthase typeI CAG23968.1 23 pks2G 40846...48228 putative polyketide synthase typeI CAG23969.1 24 pks2H 48279...52130 putative polyketide synthase CAG23970.1 25 Tabelle 9: Das pks3-Genkluster von Bacillus amyloliquefaciens FZB42 Quelle: SEQ-ID-NO: 3, GenBank Accession Number: AJ634062, Länge: 74700 bp, beinhaltet das pks3-Operon (bp 3469...73761) rbam01165 Gen, rbam01163 Gen, nusG Gen, proI Gen und das pks3 operon (pks3A, B, C, D, E, F, G, H, I, J, K, L, M Gen) von Bacillus amyloliquefaciens Gen CDS Produkt Protein-ID SEQ-ID-NO: rbam 01165 99...1115 putative NADH dependent flavin oxidoreductase CAG23971.1 26 rbam 01163 1818...2594 RBAM01163 protein CAG23972.1 27 nusG 2938...3468 putative NusG CAG23973.1 28 pks3A 4085...6460 malonyl CoA-acyl carrier protein transacylase CAG23974.1 29 pks3B 7775...9139 putative long-chain fatty acid CoA ligase CAG23975.1 30 pks3C 9154...9891 putative 3-oxoacyl-[acyl-carrier protein] reductase CAG23976.1 31 pks3D 9931...22521 putative polyketide synthase typeI CAG23977.1 32 pks3E 22540...28836 putative polyketide synthase typeI CAG23978.1 33 pks3F 28861...34602 putative polyketide synthase typeI CAG23979.1 34 pks3G 34720...50268 putative polyketide synthase typeI CAG23980.1 35 pks3H 50378...57991 putative polyketide synthase typeI CAG23981.1 36 pks3I 58014...64166 putative polyketide synthase typeI CAG23982.1 37 pks3J 64163...70378 putative polyketide synthase CAG23983.1 38 pks3K 70454...71614 cytochrome P450 109 CAG23984.1 39 pks3L 71672...72919 putative hydroxymethyl glutaryl-CoA synthase CAG23985.1 40 pks3M 72979...73725 enoyl CoA hydratase CAG23986.1 41 proI 73762...74601 pyrroline-5-carboxylate reductase 2 CAG23987.1 42 Domain shuffling within the three pks gene clusters in FZB 42 provides the opportunity to synthesize "tailored" polyketides that have improved shelf life and efficacy against the target pathogenic microorganisms. Since all three polyketides had no effect on the eukaryotic model organisms S. cerevisiae and Fusarium oxysporum used, the use of the active ingredients is also possible in the medical field. Tab. 6: Antibacterial, antifungal and hemolytic activity of gene knock-out mutants. Preparation of mutants CH6, CH7 and CH8 as well as other mutants as described (Koumoutsi et al., 2004, J. Bacteriol., 186, 1084-1096) tribe mutation Polyketides ³ S. cerevisiae Fusarium oxysporum Bacillus megaterium St. aureus hemolysis AK1 bmyD ^- Bacilli, difficidin +++ + +++ +++ - A k2 fen ^- Bacilli, difficidin +++ +++ +++ +++ ++ AK3 bmyD ^- & fen ^- Bacilli, difficidin +++ - +++ +++ - CH1 srfA ^- Bacilli, difficidin +++ +++ +++ +++ +++ CH2 fen ^- & srfA ^- Bacilli, difficidin +++ +++ +++ +++ +++ CH5 sfp ^- & yczE ^- - - - - - - CH6 pks1 ^- Difficidin +++ +++ +++ +++ ++ CH7 pks2 ^- bacillaene +++ +++ +++ + ++ CH8 pks3 ^- Bacilli, difficidin +++ +++ + +++ +++ CH10 pks3_TE nb nb ⁴ nb + (+) +++ FZB42 wild type Bacilli, difficidin +++ +++ +++ (+) +++ ³ Detection in the culture filtrate by HPLC chromatography and mass spectroscopy
⁴ not determined

Table 7: The pks1 gene cluster of Bacillus amyloliquefaciens FZB42 Source: SEQ ID NO: 1, GenBank Accession Number: AJ634060, length: 72612 bp, contains the pks1 operon (bp1 ... 71249) genes of Bacillus amyloliquefaciens pks1 operon (pks1B, C, D, E, F, G, H, I, J, L, M, N, R) and the pks1S gene gene CDS product Protein ID SEQ ID NO: pks1B 1 ... 690 hydroxyacylglutathione hydrolase CAG23949.1 4 pks1C 1009 ... 1878 malonyl-CoA [acyl-carrier-prote] transacylase CAG23950.1 5 pks1D 1850 ... 2989 malonyl-CoA [acyl carrier protein] transacylase CAG23951.1 6 pks1E 2991 ... 5231 malonyl-CoA [acyl-carrier protein] transacylase CAG23952.1 7 pks1F 5297 ... 5545 acyl carrier protein CAG23953.1 8th pks1G 5597 ... 6859 3-hydroxy-3-methylglutaryl CoA synthase CAG23954.1 9 pks1H 6847 ... 7629 enoyl-CoA hydratase CAG23955.1 10 pks1I 7639 ... 8388 enoyl-CoA hydratase CAG23956.1 11 pks1J 8428 ... 23376 putative polyketide synthase CAG23957.1 12 pks1L 23360 ... 36805 putative polyketide synthase CAG23958.1 13 pks1M 36802 ... 47358 putative polyketide synthase CAG23959.1 14 pks1N 47348 ... 63649 putative polyketide synthase CAG23960.1 15 pks1R 63753 ... 70886 putative polyketide synthase CAG23961.1 16 pks1S 71247 ... 72494 putative cytochrome P450 107K1 CAG23962.1 17 Table 8: The pks2 gene library of Bacillus amyloliquefaciens FZB42 Source: SEQ ID NO: 2, GenBank Accession Number: AJ634061, length: 53724 bp, contains the pks2 operon (bp1 ... 52130) genes of Bacillus amyloliquefaciens pks2 operons (pks2A, B, C, D, E, F, G, H) gene CDS product Protein ID SEQ ID NO: pks2A 1 ... 2307 malonyl CoA [acyl carrier protein] transacylase CAG23963.1 18 pks2B 2329 ... 14589 putative polyketide synthase typeI CAG23964.1 19 pks2C 14589 ... 19361 putative polyketide synthase CAG23965.1 20 pks2D 19379 ... 28117 putative polyketide synthase typeI CAG23966.1 21 pks2E 28110 ... 35114 putative polyketide synthase typeI CAG23967.1 22 pks2F 35129 ... 40849 putative polyketide synthase typeI CAG23968.1 23 pks2G 40846 ... 48228 putative polyketide synthase typeI CAG23969.1 24 pks2H 48279 ... 52130 putative polyketide synthase CAG23970.1 25 Table 9: The pks3 gene library of Bacillus amyloliquefaciens FZB42 Source: SEQ ID NO: 3, GenBank Accession Number: AJ634062, length: 74700 bp, contains the pks3 operon (bp 3469 ... 73761) rbam01165 gene, rbam01163 gene , nusG gene, proI gene and pks3 operon (pks3A, B, C, D, E, F, G, H, I, J, K, L, M gene) from Bacillus amyloliquefaciens gene CDS product Protein ID SEQ ID NO: rbam 01165 99 ... 1115 putative NADH dependent flavin oxidoreductase CAG23971.1 26 rbam 01163 1818 ... 2594 RBAM01163 protein CAG23972.1 27 nusG 2938 ... 3468 putative NusG CAG23973.1 28 pks3A 4085 ... 6460 malonyl CoA acyl carrier protein transacylase CAG23974.1 29 pks3B 7775 ... 9139 putative long-chain fatty acid CoA ligase CAG23975.1 30 pks3C 9154 ... 9891 putative 3-oxoacyl [acyl-carrier protein] reductase CAG23976.1 31 pks3D 9931 ... 22521 putative polyketide synthase typeI CAG23977.1 32 pks3E 22540 ... 28836 putative polyketide synthase typeI CAG23978.1 33 pks3F 28861 ... 34602 putative polyketide synthase typeI CAG23979.1 34 pks3G 34720 ... 50268 putative polyketide synthase typeI CAG23980.1 35 pks3H 50378 ... 57991 putative polyketide synthase typeI CAG23981.1 36 pks3I 58014 ... 64166 putative polyketide synthase typeI CAG23982.1 37 pks3J 64163 ... 70378 putative polyketide synthase CAG23983.1 38 pks3K 70454 ... 71614 cytochrome P450 109 CAG23984.1 39 pks3L 71672 ... 72919 putative hydroxymethyl glutaryl-CoA synthase CAG23985.1 40 pks3M 72979 ... 73725 enoyl CoA hydratase CAG23986.1 41 PROI 73762 ... 74601 pyrroline-5-carboxylate reductase 2 CAG23987.1 42

Legend to the pictures

: Antibiotic effect on Bacillus megaterium

: Antibiotic effects on Staphylococcus aureus

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

Claims (3)

Recombinant expression vector, characterized in that it contains the DNA sequence SEQ ID NO: 2 of the strain Bacillus amyloliquefaciens FZB42. Process for the enzymatic biosynthesis of the polyketides difficidin and / or oxydifficidin having an antibacterial activity, characterized in that a) cells are transformed or transfected with the recombinant expression vector according to claim 1; b) the cells are cultured in a suitable culture medium; and c) the polyketide compounds difficidin and / or oxydifficidin are isolated from the culture supernatant. Use of the recombinant expression vector according to claim 1 for the combinatorial re-synthesis of difficidin and / or oxydifficidin.

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