DE102004027335A1 - Sequences for the synthesis of antibacterial polyketides - Google Patents

Abstract

The invention relates to DNA sequences encoding polypeptides, which in turn are involved in the enzymatic biosynthesis of polyketide compounds, as well as the amino acid sequences of the polypeptides. DOLLAR A Furthermore, the invention encompasses the use of the DNA and amino acid sequences according to the invention for the combinatorial re-synthesis of polyketides which have improved stability and / or action against phyto-, animal- and / or human-pathogenic bacteria. DOLLAR A The fields of application of the invention are biology, medicine and pharmacy.

Description

The This invention relates to DNA sequences encoding polypeptides which turn to the enzymatic biosynthesis of Polyketidverbindungen involved as well as the amino acid sequences the polypeptides.

Farther 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 one Action against bacteria, preferably Gram-positive bacteria, however, has no inhibitory activity against eukaryotic organisms.

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

The spore-forming Gram-positive bacterium Bacillus amyloliquefaciens FZB42 promotes plant growth and suppressed the growth of phytopathogenic microorganisms in the area of 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 BacillomycinD and fengycin causes. The sequence for the Bacillomycin D operon indicates the presence of three modular peptide synthetases BmyA, B and C in FZB42 (Borriss et al. and Trademark Office C12N 15 / 52- Acc. 10326394.2).

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

The Elimination of fengycin and surfactin synthesis had no effect on the antibacterial effect of FZB42.

A antibacterial effect has already been conjugated for various Polyene antibiotics of the genus Bacillus (e.g., Zimmermann et al. Antibiotics 40: 1677-1681, Patel et al. 1995. J. Antibiotics 48, 997-1003).

gene sequences, the synthesis of these antibacterial agents in Bacillus Determine biocontrol strains but are not known.

The increased Occurrence of antibiotic-resistant pathogenic microorganisms has become an intense search for new effective antibacterial and antifungal compounds. At least as well greater Meaning is the search for antiviral compounds.

there the discovery of new natural products is becoming increasingly difficult. In addition to the screening of natural producer strains Combinatorial biosynthesis promises an efficient alternative for development to become a new effective medication.

polyketides and nonribosomal peptides are two major natural product families that many clinically important compounds, e.g. antimicrobial Compounds (penicillin, bacitracin, erythromycin, oleandomycin, Tylosin and vancomycin), immunosuppressants (cyclosporine, FK506 and Rapamycin) and antitumor compounds (doxorubicin, bleomycin and Epothilones).

polyketides are a class of secondary metabolites with remarkable diversity in structure and function, both antimicrobial as well as antiviral and antiparasitic properties can have.

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

The polyketide Bacillaen, produced in unknown ways 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 C35H4807. 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 Application of the "suppression Subtractive Hybridization "(SSH) Method, three gene clusters were identified in FZB42, homology to polyketide synthases (Koumoutsi et al., 2004. J.Bacteriol., 186). A determination of the complete DNA sequences as well as the translated However, proteins did not occur. A proof of the synthesis of polyketides in Bacillus amyloliquefaciens or an associated antibiotic activity was not accomplished. There was no assignment of the polyketide synthase gene cluster to Synthesis of a polyketide.

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

Combinatorial Biosynthesis is based on the genetic manipulation of biosynthetic pathways of "classical" antibiotics (e.g. of lipopeptides) produced by polyketide synthases (PKSs) and nonribosomal ones, respectively Peptide synthases (NRPSs) are synthesized.

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

There Each module contains the functional domains for recognition, activation and condensation of a substrate amino acid including stereospecificity determined, it is possible synthesize new connections by manipulating the domains or modules.

There the structures of polyketides are very complex and rich in stereocenters are, this technique allows the manipulation of compounds that difficult to obtain using conventional chemical methods are (E. Rodriguez and R. Daniel 2001. Combinatorial biosynthesis of antimicrobials and other natural products. Current Opinion in Microbiology 4: 526-534).

requirement for the Application of combinatorial resynthesis is the knowledge of Gene sequences for encode the required PKSs and NRPSs and a host strain, the stable expression of large heterologous DNA regions allowed.

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

The Invention is realized according to the claims.

object of the present invention are DNA sequences encoding polypeptides which, in turn, participate in the enzymatic biosynthesis of polyketide compounds involved as well as the amino acid sequences the polypeptides.

Farther the invention relates to the description of synthesis routes for the preparation of polyketide antibiotics from Bacillus amyloliquefaciens FZB42. there At least one polyketide antibiotic has a specific effect against bacteria, preferably Gram-positive bacteria but no inhibition against eukaryotic organisms.

In the tribe FZB42 were three gene clusters for the synthesis of polyketides identified and fully characterized by sequence analysis.

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

extensive Investigations revealed that the expression products of the sequence with the SEQ ID NO: 1 the enzymatic biosynthesis of the antibacterial Polyketides cause bacilli. The expression products of the sequence with the SEQ ID NO: 2 cause the enzymatic biosynthesis of antibacterial Polyketides difficidin and / or oxydifficidin and the expression products the sequence of SEQ ID NO: 3 are for the synthesis of a hitherto unknown antibacterial polyketide responsible.

• a) DNA-Sequenzen gemäß den SEQ-ID NO: 1, 2 oder 3 oder deren komplementären Strängen;
• b) DNA-Sequenzen, die unter stringenten Bedingungen an die unter a) genannten Sequenzen oder an Fragmente davon hybridisieren;
• c) alle Variationen und durch Substitution, Insertion oder Deletion entstandenen Mutanten der unter a) und b) definierten DNA-Sequenzen, die isofunktionelle Expressionsprodukte ergeben,

sowie rekombinante Expressionsvektoren umfassend mindestens eine unter a) bis c) definierte DNA-Sequenz.The invention further includes DNA sequences selected from:

• a) DNA sequences according to SEQ ID NO: 1, 2 or 3 or their complementary strands;
• b) DNA sequences which hybridize under stringent conditions to the sequences mentioned under a) or to fragments thereof;
• c) all variations and mutants resulting from substitution, insertion or deletion of the DNA sequences defined under a) and b) which give isofunctional expression products,

as well as recombinant expression vectors comprising at least one DNA sequence defined under a) to c).

object the invention are also prokaryotic or eukaryotic cells, those having at least one DNA sequence defined under a) to c) or transformed with at least one recombinant expression vector or transfected as well as methods for enzymatic biosynthesis of polyketide compounds having antibacterial activity.

The Methods are characterized in that cells with at least a defined under a) to c) DNA sequence or at least be transformed or transfected into a recombinant expression vector, the cells are cultured in a suitable culture medium and the polyketide compound is isolated from the culture supernatant.

With this method can the polyketides bacillaen, difficidin and / or oxydifficidin as well a previously uncharacterized polyketide with antibacterial Effect can be produced.

Farther comprises the invention the use of the DNA sequences defined under a) to c) and the expression vectors of the invention on the combinatorial re-synthesis of polyketides, which has improved stability and / or effect phyto, animal and / or human pathogenic bacteria.

The Amino acid sequences of polypeptides involved in the enzymatic biosynthesis of polyketide compounds comprising the sequences having SEQ ID NO: 4 to 42 as well as the use of the amino acid sequences for combinatorial Synthesis of polyketides that provide improved stability and / or Effect over 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 nonribosomal protein matrices and thus empowers According to the invention for rational Design of new polyketide antibiotics.

functional Hybrid matrices can be prepared in vitro and in vivo. Numerous modifying enzymes (e.g., reductases, dehydratases, methyltransferases) from any of three PKS clusters in FZB42 can also be used for combinatorial Biosynthesis of new polyketide agents are used.

Farther allows the natural Competence for DNA uptake and recombination and knowledge of the DNA sequences at FZB42 the targeted use of stronger and regulatable promoters to increase the product yield.

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

Antibacterial activity of FZB42

Culture filtrates of FZB42 after 24 h or 48 h culture exhibit both antibacterial activity compared to the most checked bacteria, as well as antifungal activity (Arxula adeninovorans, Saccharomyces cerevisiae). The highest sensitivity was in Gram-positive bacteria (except Bacillus subtilis, Staphylococcus aureus). Also the verified representatives the α-proteobacteria were significantly inhibited by the culture filtrate of FZB42. On the other hand were representatives of enterobacteria and other representatives of γ-proteobacteria 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.

in the Culture filtrate of FZB42 and in mutants, in which the synthesis of the antifungal-acting lipopeptides Bacillomycin D, fengycin and surfactin was turned off, surprisingly a specific antibacterial effect, especially against Gram-positive Bacteria (among others Bacillus sp., Streptomyces sp., Streptococcus sp.) be detected.

By Removal of the pks3 gene sequence in the mutant CH8 became the antibacterial Effect against the bacteria mentioned largely canceled. This Findings prove that the polyketide synthetases of the pks3 gene cluster a polyketide synthesized with specific antibacterial activity becomes. A chemical characterization of the polyketide by mass spectroscopy was not possible until now. A similarity to the previously described from Bacillus polyketides (Bacillaen and difficidin / oxydifficidin) could be excluded.

in the Culture filtrate of the bacterium Bacillus amyloliquefaciens FZB42 and single mutants could surprisingly the polyketides bacillaen and difficidin / oxydifficidin detected become.

To Separation of the culture filtrate from FZB42 by HPLC could be performed in the after 18 min. Retention time eluted peak by electrospray mass spectrometry (ESM) a peak with m / z of 581.1, the bacillaene equivalent. The highest intensity this peak was observed after 48 h culture in Landy's medium.

One optimized drug based on the polyketide Bacillaen provides an alternative when used against pathogenic gram-positive bacteria of the genera Staphylococcus sp. and Streptococcus sp. whose, Medical treatment due to the rapidly spreading multi-resistance across from usual Antibiotics is becoming increasingly difficult.

in addition comes that the pks3 polyketide does not inhibit eukaryotic cells caused and thus potentially for use in the human area suitable is.

These particularly advantageous property could be occupied by that in the mutant CH8 no reduction of antifungal activity across from Fusarium oysporum or Saccharomyces cerevisiae was caused.

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

Genome analysis: pks gene cluster

At different positions in the genome of B. amyloliquefaciens FZB42 were three big ones Gene cluster with homology to the polyketide synthase operon (pksX) B.subtilis (Albertini et al., 1995. Microbiology 141, 299-309) demonstrated. Pks1 (SEQ ID NO: 1, 72612 bp, Tables 2 and 7), pks2 (SEQ ID NO: 2, 53724 bp, Table 3 and 8) and pks3 (SEQ ID NO: 3, 74700, Tab.4 and 9) have structural Similarities and are probably due to gene duplication originated 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. 1084-1096). pMX39 (Em ^R ) was digested with Spel. 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 pks1Up 5'-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 (KS 1, 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 'part 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 (KS 1, 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 cloned into the vector pGEM-T. 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 confirmed with the specific primers.

Table: Strains and plasmids. For the production of the desired knockout strains, the plasmids pCH6, pCH7 and pCH8 were used for the transformation into FZB42.

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, 1 ). 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 spectroscopy (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. megegate, the inhibitory effect of the culture filtrate was slightly reduced, compared 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 on St. aureus remained ( 2 ).

Although so far no 3rd polyketide by the one of us used in investigation methods in FZB 42, the significant decrease in the inhibitory activity towards the B.megaterium indicator strain in the pks-3 knock out mutant CH8 shows that this gene cluster produces a potent antibiotic with specific activity ( 1 , 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 causative agent of foul brood in Bee larvae, Paenibacillus larvae larvae and the human pathogenic Staphylococcus aureus) of interest.

Domain shuffling within the three pks gene clusters in FZB 42 allows the synthesis of "tailor-made" polyketides, which have improved shelf-life and efficacy against the target pathogenic microorganisms, since all three polyketides have no activity against the eukaryotic model organisms S. cerevisiae and Fusarium oxysporum can also be used in the medical field Table 6: Antibacterial, antifungal and hemolytic activity of gene knock-out mutants Production of the mutants CH6, CH7 and CH8 Other mutants as described (Koumoutsi et al. 2004, J. Bacteriol., 186, 1084-1096)

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 (bp 1..71249)

Genes of Bacillus amyloliquefaciens pks1 operon (pks1B, C, D, E, F, G, H, I, J, L, M, N, R) and the pks1S gene

Table 8: The pks2 genometer of Bacillus amyloliquefaciens FZB42

• Source: SEQ ID NO: 2, GenBank Accession Number: AJ634061, Length: 53724 bp, contains the pks2 operon (bp 1 .. 52130)

Genes of Bacillus amyloliquefaciens pks2 operon (pks2A, B, C, D, E, F, G, H)

Table 9: The pks3 genometer 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 the pks3 operon (pks3A, B, C, D, E, F, G, H, I, J, K, L, M gene) from Bacillus amyloliquefaciens

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (20)

DNA sequences encoding polypeptides which turn to the enzymatic biosynthesis of Polyketidverbindungen comprising the sequences having SEQ ID NO: 1, 2 and 3. DNA sequences according to claim 1, characterized in that that the sequences are from a bacterium of the family Bacillus. DNA sequences according to one of the preceding claims, characterized in that the sequences consist of a bacterium of the genus Bacillus amyloliquefaciens. DNA sequences according to one of the preceding claims, characterized characterized in that the sequences from the strain FZB42 of the bacterium Bacillus amyloliquefaciens. DNA sequences according to one of the preceding claims, whose Expression products for the enzymatic biosynthesis of polyketides with antibacterial Effect, comprising the sequence with the SEQ ID NO: 1, 2 and 3. DNA sequences according to one of the preceding claims, whose Expression products the enzymatic biosynthesis of antibacterial Polyketides cause or are involved in Bacillaen the sequence having SEQ ID NO: 1. DNA sequences according to any one of claims 1-5, whose Expression products the enzymatic biosynthesis of antibacterial Polyketides Difficidin and / or Oxydifficidin cause or because comprising the sequence having SEQ ID NO: 2. DNA sequences according to any one of claims 1-5, whose Expression products the enzymatic biosynthesis of one not yet cause or involved in known antibacterial polyketide comprising the sequence of SEQ ID NO: 3. DNA sequences according to one of the preceding claims comprising DNA sequences selected from: a) DNA sequences according to the SEQ ID NO: 1, 2 or 3 or their complementary strands; b) DNA sequences, under stringent conditions to the sequences mentioned under a) or hybridize to fragments thereof; c) all variations and by substitution, insertion or deletion mutants the DNA sequences defined under a) and b), the isofunctional Expression products result. Recombinant expression vector comprising at least a sequence according to any one of claims 1 to 9. Prokaryotic or eukaryotic cell with at least one DNA according to any one of claims 1 to 9 or at least a recombinant expression vector according to claim 8 transformed or transfected. Process for the enzymatic biosynthesis of polyketide compounds, characterized in that a) cells with at least one DNA according to any one of claims 1 to 9 or with at least one recombinant expression vector transformed or transfected according to claim 8; b) the Cells are cultured in a suitable culture medium; and c) the polyketide compound is isolated from the culture supernatant. Process according to claim 12 for the preparation of polyketides with antibacterial effect. Process according to claim 13 for the preparation of the polyketide Bacillaene. Process according to claim 13 for the preparation of the polyketides Difficidin and / or oxydifficidin. Process according to claim 13 for the production of a previously uncharacterized polyketide with antibacterial activity. Polyketide with antibacterial action preferably against gram-positive bacteria, made from strain FZB42 of the bacterium Bacillus amyloliquefaciens, wherein the DNA sequence with the SEQ ID NO: 2 for the Polyketidsynthetasen encoded. Use of DNA sequences according to one of claims 1 to 9 or recombinant expression vectors according to claim 10 Combinatorial re-synthesis of polyketides for improved stability and / or Effect over phyto, animal and / or human pathogenic bacteria. Amino acid sequences of polypeptides involved in the enzymatic biosynthesis of polyketide compounds comprising the sequences having SEQ ID NO: 4 to 42nd Use of amino acid sequences according to claim 16 for the combinatorial re-synthesis of polyketides, which has an improved stability and / or effect phyto, animal and / or human pathogenic bacteria.