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Antimicrobial Agents and Chemotherapy, June 2006, p. 2113-2121, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.00007-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biosynthetic Gene Cluster for the Polyenoyltetramic Acid -Lipomycin

C. Bihlmaier,¹ E. Welle,¹ C. Hofmann,¹ K. Welzel,² A. Vente,² E. Breitling,³ M. Müller,³ S. Glaser,⁴ and A. Bechthold^1*

Albert-Ludwigs-Universität Freiburg, Institut für Pharmazeutische Wissenschaften, Pharmazeutische Biologie und Biotechnologie, Stefan-Meier-Straße 19, D-79194 Freiburg, Germany,¹ Combinature Biopharm AG, Robert-Rössle-Straße 10, 13125 Berlin, Germany,² Albert-Ludwigs-Universität Freiburg, Institut für Pharmazeutische Wissenschaften, Pharmazeutische und Medizinische Chemie, Stefan-Meier-Straße 19, D-79194 Freiburg, Germany,³ Technische Universität München, Institut für Organische Chemie und Biochemie II, Lichtenbergstraße 4, 85747 München, Germany⁴

Received 4 January 2006/ Returned for modification 2 March 2006/ Accepted 28 March 2006

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic -lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that -lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of D-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The tetramic acid (2,4-pyrrolidinedione) ring system has been known since the early 20th century, when the first simple derivatives were prepared. Natural products containing the tetramic acid moiety continue to receive interest due to the range of biological activities they display, including antibiotic and antiviral activity, mycotoxicity, cytotoxicity, and phospholipase A2 inhibition (38). Other members of this class are responsible for the pigmentation of certain molds and sponges. Examples of natural compounds containing a tetramic acid moiety are aurantosid B, militarinon B, erythroskyrin, fuligurubin, oleficin, and -lipomycin. Most of these compounds are produced by marine sponges or by fungi, and just a few, like oleficin and -lipomycin, are produced by microorganisms. Surprisingly, given the interesting pharmaceutical activities of some of these drugs, to date there has been no publication of a gene or gene cluster involved in the biosynthesis of a tetramic acid-containing molecule. Therefore we decided to focus our research on this interesting subject.

As a model substance we chose -lipomycin, an acyclic polyene antibiotic isolated from Streptomyces aureofaciens Tü117 (32). The orange-red compound was named lipomycin due to the fact that the antibiotic activity is antagonized by several lipids, including lecithin and some sterols. The structure of -lipomycin is similar to the mycotoxin erythroskyrin (5). A carbonyl group links a pentaene chain with a derivative of N-methyl-tetramic acid composing the aglycone. An interesting L-digitoxose molecule is attached to the polyketide part of the aglycone. While polyene antibiotics like nystatin and amphotericin interact with membrane steroles and therefore have activity against fungi but not bacteria (8), the acyclic polyene -lipomycin is active against gram-positive bacteria but has no effect upon the growth of fungi, yeasts, and gram-negative bacteria.

In this report, the cloning of the -lipomycin biosynthetic gene cluster as well as the generation of mutants with deletions in genes for different biosynthetic enzymes is described.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains, growth conditions, media, and vectors. For standard purposes, S. aureofaciens Tü117 (32) and mutant strains S. aureofaciens MT (MT), S. aureofaciens NRPS (NRPS), S. aureofaciens IntGTF (IntGTF), S. aureofaciens IntPKS (IntPKS), S. aureofaciens NRPSpPKS (NRPSpPKS), and S. aureofaciens MTpPKS (MTpPKS) were grown on HA medium (1% malt extract, 0.4% yeast extract, 0.4% glucose, and 1 mM CaCl₂), pH adjusted to 7.3, prepared as solid or liquid medium, at 28°C. For maintenance of mutant strains IntGTF, IntPKS, NRPSpPKS, and MTpPKS, apramycin was added to a final concentration of 25 µg/ml. For lipomycin production, HA liquid medium (100 ml in a double-baffled 300-ml Erlenmeyer flask) was used. DNA manipulation was carried out in Escherichia coli XL1-Blue (Stratagene, La Jolla, CA). An S. aureofaciens Tü117 genomic cosmid library was constructed in E. coli DH5 (Invitrogen) using pOJ436 (7) as the cosmid vector. Before transforming, S. aureofaciens Tü117 plasmids were propagated in E. coli ET 12567 (dam dcm hsdS Cm^r) to obtain unmethylated DNA. E. coli strains were grown on Luria-Bertani (LB) agar or liquid medium containing the appropriate antibiotic for selection. Vector pBluescript SK(–) (pBSK–) was from Stratagene; Litmus28 was from New England Biolabs (Frankfurt, Germany); and pKC1132, carrying the apramycin resistance gene, used for gene disruption, was from Eli Lilly and Company (Indianapolis, IN) (7). pSET-1cerm (21) was used for the generation of plasmids to complement the mutant strains.

General genetic manipulation and PCR. Standard molecular biology procedures were performed as described previously (40). Isolation of plasmid DNA from E. coli and DNA restriction/ligation were performed following the protocols of the manufacturers of the kits, enzymes, and reagents: Macherey & Nagel (Dueren, Germany), QIAGEN (Hilden, Germany), and Promega (Mannheim, Germany). PCRs were performed on a PerkinElmer GeneAmp 2400 thermal cycler. Primers were purchased from Operon Biotechnologies Inc. (Cologne, Germany). Oligonucleotide primers used are listed in Table 1. Reaction conditions involved 35 cycles of denaturing at 95°C for 1 min, annealing at 65°C for 40 s, and extending at 72°C for 2.5 min in the case of lipNrps primers NrpsF and NrpsR, 4 min in the case of complementation constructs for lipNrps and lipMt, and 1 min in the case of oligonucleotide primers Ap1 and Ap2. To amplify parts of lipMt and lipGtf used to generate the inactivation constructs, the elongation time was 1.75 min. A cosmid library was prepared using cosmid pOJ436 (7). For preparation of DNA, the mycelium was embedded in agarose. Cell disruption, partial digestion of the genomic DNA, and separation of DNA fragments were performed as described previously (36). Robotically produced high-density colony arrays (Hybond N+; Amersham Pharmacia, Freiburg, Germany) were utilized for the screening of a total of 2,300 cosmid clones with a strain-specific type I polyketide synthase probe (PKSI-52) obtained by PCR using primers KSIFOR und ATIREV (Table 1) and a nonspecific type I polyketide synthase probe (cosmid 2P6) from Streptomyces antibioticus Tü6040 (45), following standard nonradioactive hybridization procedures with digoxigenin (DIG)-labeled DNA probes. The detection was performed by incubation with anti-DIG antibodies conjugated with alkaline phosphatase and by incubation with Attophos following the protocols given by the manufacturer (Boehringer, Mannheim, Germany).

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TABLE 1. Sequences of oligonucleotide primers

DNA sequencing and computer-assisted sequence analysis. Nucleotide sequences were determined on an ABI sequencer at 4-Base Lab GmbH (Reutlingen, Germany) and at GATC Biotech AG (Konstanz, Germany) using standard primers (M13 universal and reverse, T3, and T7) or customized, internal primers. Computer-assisted analysis was done with the DNASIS software package (version 2.1, 1995; Hitachi Software Engineering) and the FramePlot software at http://www.nih.go.jp/jun/cgi-bin/frameplot.pl (25). Database comparison was performed with the BLAST search tools on the server of the National Center for Biotechnology Information, Bethesda, MD (2). To analyze polyketide synthase domains, the SEARCHPKS program (at http://www.nii.res.in/searchpks.html) offered by the National Institute of Immunology, New Delhi, India, was used (53).

Construction of gene inactivation plasmids. For the generation of chromosomal mutants of the -lipomycin producer S. aureofaciens Tü117 by homologous recombination, four gene disruption plasmids, pNRPS, pMT, pPKS, and pGT, were constructed as described below. The sequences of all gene disruption plasmids were confirmed by DNA sequencing.

pNRPS was generated by ligation of a 1.6-kb EcoRI-XbaI digested PCR fragment (Fig. 1, fragment D amplified with primers NrpsF and NrpsR) into pBSK– and successive BamHI restriction, treatment with T4 DNA polymerase, and religation. The mutated 1.6-kb fragment was cloned (EcoRI and XbaI) into pKC1132 to generate pNRPS.

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FIG. 1. Organization of the lipomycin biosynthetic gene cluster from S. aureofaciens Tü117. Cosmid clones isolated are shown above the map. Fragments F, G, and H were used as probes for hybridization. The solid line indicates the region sequenced during this study. Open reading frames are shown as arrows indicating the size and the direction of transcription. Fragments A, B, C, D, and E were used for insertional inactivation.

EcoRI and BamHI restriction sites were introduced upstream and inside of lipMt, respectively, using primers Mt1F and Mt1R. The 0.9-kb EcoRI-BamHI PCR fragment (Fig. 1, fragment B) was ligated into pBSK– to create plasmid pMT1. A second 0.9-kb PCR fragment (Fig. 1, fragment C) was amplified using primers Mt2R and Mt2F and cloned into pMT1 using the restriction sites BamHI and XbaI. The resulting plasmid pMT12 contained the gene lipMt with an in-frame deletion of 303 bp and an additional BamHI restriction site. After restriction with EcoRI and XbaI, the insert was transferred to pKC1132 to generate pMT.

To generate the plasmid pPKS, a 3.2-kb BamHI-PvuII fragment (Fig. 1, fragment E) from cosmid 6I03 encoding an internal part of the gene lipPks1 was cloned into the BamHI-EcoRV site of pKC1132. The insert of pPKS encodes the acyl carrier protein of the loading module as well as the ketosynthase domain and acyltransferase domain of the first extension module from LipPks1.

To generate pGT, a 1.0-kb internal fragment of the gene lipGtf (Fig. 1, fragment A, amplified with primers GtfF and GtfR) was ligated into pKC1132 using the restriction sites EcoRI and XbaI.

Generation of chromosomal mutants of S. aureofaciens Tü117. S. aureofaciens Tü117 was transformed with the four gene disruption plasmids (plasmid DNA was isolated from E. coli strain ET12567 and denatured by alkaline treatment) as described previously (29) using PEG2000 (Merck, Darmstadt, Germany). After a 16-h incubation at 28°C, cells with integrated plasmids were selected by covering the transformation plates with 3.5 ml soft agar containing 1 mg apramycin, and the integration of the plasmids into the chromosome was shown by PCR analysis using primers Lip-1 and Lip-2 (when using pNRP), MT-E and MT-X (when using pMT), and Ap1 and Ap2 (when using either pPKS or pGT).

For the generation of NRPS and MT, single-crossover mutants were screened for loss of resistance as a consequence of a double-crossover event. Deletions within both genes were confirmed by PCR. PCR fragments obtained from the double-crossover mutant NRPS using primers Lip-1 and Lip-2 could not be restricted by BamHI, whereas the PCR fragments obtained from S. aureofaciens Tü117 could be digested by the enzyme. The size of the PCR fragment from the double-crossover mutant MT obtained using primers MT-E and MT-X was 1.9 kb, whereas the size of the fragment from S. aureofaciens Tü117 was 2.2 kb.

To generate the double-mutant strains NRPSpPKS and MTpPKS, the plasmid pPKS was transferred into protoplasts of the mutant strains NRPS and MT as described previously. The integration of pPKS into the chromosomes was confirmed by PCR analysis as described above.

Construction of complementation plasmids. For the generation of plasmids used to complement the mutant strains NRPS and MT, lipNrps and lipMt were amplified by PCR using Pfu polymerase. Suitable restriction sites (for lipNrps, HindIII and XbaI; for lipMt, EcoRI and XbaI) were introduced upstream and downstream of each gene using primers CN-F/CN-R and Mt1F/Mt2R (Table 1), respectively. The 2.6-kb PCR product of lipNrps was restricted with HindIII and XbaI and ligated into Litmus28 to create plasmid pLit-Nrps. Plasmid pSET-1cerm was restricted with MunI and XbaI to remove urdGT1c, and the EcoRI-XbaI fragment from pLit-Nrps containing lipNrps was fused to ermE* to generate complementation plasmid pSET-Nrps. To generate the complementation plasmid pSET-Mt, a 2.2-kb fragment containing lipMt was amplified. After restriction with EcoRI and XbaI, the fragment was ligated into pSET-1cerm after removal of urdGT1c, in the same way described for pSET-Nrps.

Biological properties. The antimicrobial activity of -lipomycin and its derivatives was determined by agar plate diffusion assay using Bacillus subtilis as the test strain. Susceptibility of staphylococci, streptococci, and enterococci to -lipomycin was determined by microdilution according to CLSI (formerly NCCLS) guidelines (34).

Feeding of MT with N-methylated glutamic acid. The mutant MT was inoculated in 100 ml of HA liquid medium. After 24 h, 1 ml of this MT culture broth was subcultured into 100 ml HA liquid medium supplemented with 1.5 mg N-methylated glutamic acid and incubated. Fifty-five hours later, an additional 5 mg of N-methylated glutamic acid was added to the medium. N-Methyl-DL-glutamic acid was synthesized according to the method of J. C. Watkins (48) starting from -keto glutaric acid. The product was obtained as colorless crystals with a melting point of 152°C. Other analytical data (infrared, ¹H nuclear magnetic resonance [NMR], and ¹³C NMR) are in accordance with data from the literature (23).

Purification of -lipomycin and new metabolites. Wild-type and mutant strains were cultured in production medium for 5 days at 28°C in a rotary shaker (180 rpm). Mycelia were collected by centrifugation and extracted with acetone at room temperature. After removal of the mycelia by filtration, the extract was evaporated to reduce the amount of acetone. Finally this mycelium extract, combined with the supernatant, was extracted twice with an equal volume of ethyl acetate. The solvent of the organic phase was removed, and the residue was dissolved in acetonitrile. This solution was used for high-pressure liquid chromatography (HPLC)-UV/visible light and HPLC-mass spectrometry (MS) analysis. For further purification of -lipomycin, this crude extract was washed with water-chloroform and fats were removed with petroleum ether as described previously (45). Finally, pure -lipomycin for the microdilution assay was obtained by using a preparative reversed-phase HPLC Waters type 600 controller (Waters Associates, Eschborn, Germany) with an XTerra Prep C₁₈ column (5 µm, 7.8 by 150 mm) and an XTerra UP1 precolumn. As the solvent, 50% acetonitrile in 0.5% acetic acid was used at a flow rate of 3.5 ml/min.

Analysis of -lipomycin and its intermediates. Detection of -lipomycin and its intermediates by HPLC-UV/visible light was performed on a reversed-phase column (XTerra MS C₁₈; 3.5 µm, 4.6 by 100 mm) and precolumn (XTerra VA-1) from Waters Associates (Eschborn, Germany), with acetonitrile and 0.5% acetic acid in H₂O as the solvent (nonlinear gradient from 25% to 100% acetonitrile in 50 min, at a flow rate of 0.5 ml/min). Detection and spectral characterization of peaks were accomplished with a photodiode array detector and Millennium software (Waters Associates). HPLC-MS was performed on an Agilent 1100 system (Agilent Technologies, Waldbronn, Germany) with an electrospray chamber and a quadrupole detector. HPLC analysis was carried out on a Zorbax XDB-C₈ column (5 µm, 4.6 by 150 mm) with a Zorbax SB-C₁₈ precolumn (5 µm, 4.6 by 12.5 mm) from Agilent Technologies. A nonlinear gradient from 20% to 95% acetonitrile in 0.5% acetic acid over 30 min at a flow rate of 0.7 ml/min was used. The column temperature was 23°C, and the UV detection wavelengths were 254 nm, 270 nm, 310 nm, and 460 nm. The mass selective detector chamber settings were as follows: drying gas flow rate, 12 liters/min; nebulizing pressure, 50 psi/g; drying gas temperature, 350°C. The samples were analyzed in positive- and negative-scan modes with a mass range of 100 to 1,100 Da.

Nucleotide sequence accession number. The sequence reported here has been deposited into the GenBank database under the accession number DQ176871.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
In order to verify the antibiotic activity described by Kunze et al. (32), -lipomycin was tested to determine its activity against several gram-positive strains. As shown in Table 2, its MIC is in the range of 8 µg/ml to 32 µg/ml. There were no appreciable differences in the MICs of nonresistant and antibiotic-resistant strains.

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TABLE 2. Susceptibilities of staphylococci, streptococci, and enterococci to -lipomycin

Cloning and identification of the -lipomycin cluster. Due to the chemical structure of -lipomycin, it was likely that a type I polyketide synthase was involved in the biosynthesis of lipomycin. Therefore, a mix of three different DNA probes derived from (i) the simocyclinone biosynthesis gene cluster containing the PKSI genes simC1A, simC1B, and simC1C (45) and the putative thioesterase gene simC2, (ii) parts of the gene simC1A, and (iii) a PCR fragment amplified from S. aureofaciens Tü117 with primers directed against conserved gene sequences of the acyltransferase domain of PKSI were used for screening of the genomic cosmid library of the -lipomycin producer S. aureofaciens Tü117. From the 20 cosmids identified by this initial screening, 6 cosmids containing overlapping DNA inserts were selected by restriction mapping. Two cosmids of this group, 6I03 and 5A16, were chosen for random sequencing of subcloned BamHI fragments. The sequence analysis revealed that both cosmids contained overlapping DNA inserts with significant homology to the expected type I polyketide synthase (PKS). As revealed by additional hybridization experiments with probes specific for the detection of NDP-glucose-4,6-dehydratase and NDP-4-keto-6-deoxyhexose-2,3-dehydratase genes, cosmid 6I03 also carries deoxysugar biosynthetic genes. Cosmids 6I03 and 5A16 were chosen for sequencing. To complete the sequence of the lipomycin cluster (Fig. 1), BamHI fragments from the borders of cosmids 6I03 and 5A16 were used as additional probes to screen the cosmid library. Based on restriction mapping of the cosmids identified by this hybridization cosmids 2L13 and 1F15 were selected for further sequencing. Sequence alignment of all four cosmid inserts revealed a 74.5-kb DNA fragment with an average G+C content of 71.6%, well within the range of the reference value for Streptomyces sp. DNA (52). The deduced open reading frames (ORFs) were functionally designated on the basis of database searches (Table 3). The genetic organization of the lipomycin biosynthetic gene cluster (lip gene cluster) is shown in Fig. 1.

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TABLE 3. Deduced functions of ORFs shown in Fig. 1

The lipomycin polyketide synthases. Four large ORFs in the -lipomycin cluster (lipPks1 to -4) encode proteins with the highest homology to several type I polyketide synthases. They all have the same modular organization as that first described for the erythromycin PKS genes, with each module catalyzing a single condensation and reduction cycle (16). In the lip cluster each gene encodes an enzyme with two modules. lipPks1 encodes a loading module and an extension module, and lipPks2, lipPks3, and lipPks4 encode polyketide synthases with two extension modules (Fig. 2). The loading module (module L) of LipPks1 contains only an acyltransferase (AT) domain and an acyl carrier protein (ACP), whereas several other PKS loading modules from polyene biosynthesis clusters (9, 11) and simocyclinone (45) contain an additional ß-ketoacyl synthase (KS) domain. Module I might catalyze the first extension reaction, as it consists of KS, AT, ketoreductase (KR), and ACP domains and lacks the dehydratase (DH) domain which is necessary to remove the hydroxyl group from the ß-carbon (Fig. 2). The next five extension modules, including module II and module III of LipPks2, module IV and module V of LipPks3, and module VI of LipPks4, all feature the same domain pattern, with KS, AT, DH, KR, and ACP domains that account for the five double bonds in the final lipomycin molecule. The last extension module (module VII) of LipPks4 consists of KS, AT, KR, and ACP domains. KS domains of type I PKSs are described as the most highly conserved of all the constituent domains in these multifunctional enzymes (4, 6). Likewise, the identities among LipPks KSs are high (67 to 96%), and these KSs contain the described signature active-site sequence DTACSS with an invariant cysteine residue and the two invariant histidine residues located 135 and 175 amino acids towards the C terminus from the active-site cysteine (4). Comparison with other type I KS domains from the database revealed the highest homology (69 to 74% identity) to avermectin (24) and amphotericin (11) KSs. All LipPks AT domains contain the consensus sequence GxSxG, with the catalytic serine residue common in all these enzymes. The substrate specificity of such AT domains for malonyl-coenzyme A (CoA) or methylmalonyl-CoA is determined by a stretch of 20 amino acids N terminal to the catalytic serine. As defined by this sequence stretch, AT domains of module I and module II convert methylmalonyl-CoA (20), and the other five AT domains of the extension modules III to VII will incorporate acetate extender units from malonyl-CoA (20). Short branched-chain carboxylic acids derived from the amino acids valine, leucine, and isoleucine commonly serve as fatty acid synthase starter units in bacteria (27). In a similar fashion, several modular PKSs utilize amino acid-derived short branched-chain carbonyl-CoAs such as isobutyryl-CoA and 2-methylbutyryl-CoA as starter units, as was demonstrated by labeling studies in the case of avermectin (12). Based on the structure of -lipomycin we would expect isobutyryl-CoA as a starter unit. The AT domain of the LipPks1 loading module shows 50% identity to the AT domain of the avermectin loading module, making it very likely that isobutyryl-CoA is incorporated. All modules contain one ACP domain which includes the 4'-phosphopantetheine binding site. The ACPs from extension modules II to VI all show the signature LGFDS, which fits well to the active-site sequence (LGxDS) of prokaryotic ACPs (46). The ACPs of the loading module (LGITS) and from module I (VGFDS) and module VII (LGFAS) deviate from this sequence. Modules II, III, IV, and V each contain a DH domain with the conserved active-site motif LxxHxxxGxxxxP (6). In contrast, the most probable active DH domain in module VI contains a serine instead of the glycine. In general, KR domains show a potential motif (GxGxxGxxxA) for NADPH binding at the N-terminal end of the domain (43). Only the KR domain of module I contains a sequence that fits perfectly to this motif. Most KRs connected with the lip cluster (modules II to VI) contain an alanine (A) at the first position (AxGxxGxxxA), which was also described for modules of the pimaricin (3) and rapamycin (4) polyketide synthases. The region GxGxxG constitutes a tight turn at the end of the first strand of a ß-sheet and marks the beginning of the succeeding -helix in the ß//ß fold (Rossmann fold) for cofactor binding (49). In most NADP-binding domains, the same fingerprint region can be identified, although in some cases the first or the third glycine is replaced by alanine. However, the KR domain from LipPks4 module VII shows two nonconservative replacements, displaying the sequence GxDxxRxxxA. This alteration should preclude the formation of the turn and hence make such a KR inactive. Also, a characteristic Lx(S,G)RxG motif with an invariant arginine (43) can be found in all KRs, except the one of module VII. The catalytic triad consisting of a conserved tyrosine (Y), a serine (S), and a lysine (K) located in the active site (37) can be found in KRs from modules II to VI. KRs carry out stereospecific ketoreduction of the ß-carbonyl functionalities, and each domain naturally generates only one of the two possible epimers at the resulting hydroxyl centers. Sequence analysis of natural KR domains discovered motifs that accurately predict the direction of ketoreduction (10). The KRs from modules II to VI are all showing the conserved LDD motif, which is characteristic for the B-type KRs. This motif is absent in the KR of module I, which contains a tryptophan (W) at position 141 and belongs to the A-type KRs.

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FIG. 2. Biosynthetic scheme for -lipomycin. Domain organization and biosynthetic intermediates for LipPks1, LipPks2, LipPks3, LipPks4, and LipNrps are shown at the top. The KR domain is believed to be inactive. C, condensation domain.

The nonribosomal peptide synthetase of the lipomycin cluster. Computer-based sequence analysis revealed that LipNrps is similar to nonribosomal peptide synthetases (NRPS).

NRPS are multifunctional proteins organized into modules containing domains responsible for the adenylation, thioester formation, and condensation of an amino acid (30). Amino acid recognition and activation by formation of the corresponding adenylate are assignments of the adenylation domain (A domain) (13). This adenylate is subsequently transferred to the cysteamine group of an enzyme bound 4'-phosphopantetheinyl (4'-PP) cofactor located on the thiolation domain (T domain), also called peptidyl carrier protein (PCP). The stepwise amino-to-carboxy-terminal elongation of the thioesterified intermediates is catalyzed by the condensation domain (C domain). These domains form the three core domains of every elongation module (33). LipNrps consists of only one module with these three core domains (Fig. 2) in the order C-A-T.

The C domain of LipNrps contains an HHxxxDA motif with significant homology to the described conserved core motif HHxxxDG (28) of C domains. The PCP on the T domain of LipNrps contains the 4'-PP binding site motif LGGHSL. The A domain contains a binding pocket that determines the amino acid specificity (13). Based on the lipomycin structure, glutamic acid should be the substrate recognized by LipNrps. Most interestingly, the A domain of LipNrps is about 280 amino acids shorter than other previously described NRPS A domains, making it impossible to predict the constituents of the substrate binding pocket. However, lipNrps is functional and essential as shown by the LC-MS analysis of a lipNrps mutant strain, which did not produce lipomycins. LipNrps interacts with LipPks4 module VII to channel the polyketide from a PKS module to the NRPS module (Fig. 2). The interaction between PKSs and NRPS for the synthesis of hybrid peptide-polyketide metabolites has previously been characterized in the gene clusters of bleomycin (18) and leinamycin (14). NRPS and PKS cannot be functional unless their carrier proteins are posttranslationally modified by an enzyme known as phosphopantetheinyl transferase (PPTase) that catalyzes the transfer of the phosphopantetheine moiety from coenzyme A to the hydroxyl group of a conserved serine residue of the carrier protein (47). Such a PPTase could not be identified in the -lipomycin cluster. We expect that this enzyme is encoded somewhere else in the genome, maybe showing a broad carrier protein specificity as was described for the PPTase Svp from the bleomycin producer Streptomyces verticillus (41).

lipTe encodes a type II thioesterase with 57% identity to RifR, a thioesterase from the rifamycin gene cluster. The conservative motif of the TE catalytic center and the conservative histidine located about 130 amino acids downstream can be located (GHSMG-128 aa-PGGHF) (31). These type II thioesterases serve as editing enzymes to regenerate nonproductive carrier proteins in PKS and NRPS biosynthesis (22, 42). The deduced amino acid sequence of the lipMt product is very similar to methyltransferases, especially to O-methyltransferases. The closest resemblance was found for GilMT, a putative O-methyltransferase of the gilvocarcin V biosynthetic gene cluster from Streptomyces griseoflavus, and DnrK, a carminomycin 4-O-methyltransferase from Streptomyces peuceticus. LipMt perfectly contains the conserved motif site for S-adenosylmethionine binding (VLDVGGxxG) (26).

Enzymes involved in digitoxose biosynthesis. Deoxysugars are not only structural components of many natural products, they are also responsible for drug target interactions (19). Several deoxysugar biosynthetic gene clusters have been isolated in the past. Based on this information, six genes presumably involved in the biosynthesis and attachment of the D-digitoxose moiety were found in the lipomycin cluster. LipDig1 shows considerable homology to NDP-D-glucose synthases, and LipDig2, to NDP-hexose-4,6-dehydratases. We suppose that these two enzymes catalyze the steps from glucose-1-phosphate to NDP-4-keto-6-deoxy-D-glucose (Fig. 2). The gene product of lipDig5 is similar to many NDP-4-keto-6-deoxy-D-glucose 2,3-dehydratases. The closest match (60% identity) was with UrdS. The deduced amino acid sequence of LipDig3 showed homology to several TDP-4-keto-6-deoxyhexose 2,3-reductases. It also showed 58% identity to TylCII, a putative NDP-hexose 2,3-enoyl reductase of Streptomyces fradiae, and 63% identity to EryBII, a putative 3-ketoreductase of Saccharopolyspora erythreae. Participation of such a 2,3-dehydratase and a ketoreductase acting in sequence would be consistent with the proposed biosynthetic pathway for other dideoxysugars (17). Thus, LipDig5 and LipDig3 might catalyze the steps from NDP-4-keto-6-deoxy-D-glucose to produce NDP-4-keto-2,6-dideoxy-allose as a key intermediate. LipDig4 resembled NDP-hexose-4-ketoreductases like TylD (47%) of the tylosin producer S. fradiae. This matches perfectly with the proposed last step leading to NDP-D-digitoxose (Fig. 2). One gene in the cluster (lipGtf) encodes a protein with similarity to OleI (35) and TylCV, both identified as glycosyltransferases, indicating that LipGtf attaches the digitoxose moiety to the aglycone.

Genes putatively involved in regulation and resistance. In the lip cluster, six genes which might be important for regulation and resistance could be identified. They are all located together at the left border of the lipomycin cluster. lipReg1 and lipReg2 seem to encode a two-component system showing homology to two-component system response regulators and two-component system sensor kinases, respectively. The closest match was with a putative two-component system from Streptomyces coelicolor A3(2). LipReg2 and LipReg1 are also similar to the described two-component system of S. coelicolor AbsA1 and AbsA2, with 31% and 44% identity, respectively (39). It has been demonstrated that the temporal regulation of expression of the antibiotic gene clusters results from growth- phase-regulated expression of a pathway-specific activator (44). The first pathway-specific regulatory proteins characterized in actinomycetes belonged to a protein family called SARPs (Streptomyces antibiotic regulatory proteins) (50). In recent years, a novel family of transcriptional regulators has been identified (15). The name given to this group is "large ATP-binding regulators of the LuxR family" (LAL). LipReg4 belongs to this LAL family of regulators and may therefore be responsible for turning on the expression of biosynthetic genes in the lip cluster. LipReg4 is closely related to PikD, the first LAL protein from streptomycetes characterized in detail (51), and also shows 26% identity to NysRI and 27% identity to NysRIII (9). The LipReg3 regulatory protein exhibits sequence similarity to regulatory proteins of the MarR family. These proteins control an assortment of biological functions, including the expression of resistance to multiple antibiotics, organic solvents, detergents, and oxidative stress agents (1). Most members act as repressors and only a few as activators.

One of the mechanisms of self-resistance in bacteria is the removal of the secondary metabolite from the cytoplasm by transporting it out of the cell. The export of the -lipomycin is likely to be catalyzed by LipEx1, which is similar to several efflux proteins like Pur8, a puromycin resistance protein from Streptomyces alboniger.

Genes of unknown function. The gene product of lipX1 shows similarity to several guanyl-specific ribonucleases, with the closest match to a putative one from Streptomyces avermitilis MA-4680. lipX2 is located directly upstream of lipNrps. It encodes a protein with similarity to parts of a putative nonribosomal peptide synthetase from S. avermitilis MA4680 and to SyrE2 from Xanthomonas axonopodis pv. citri strain 306. SyrE2 belongs to the superfamily of adenylate-forming enzymes, and therefore LipX2 might be involved in the adenylation of glutamic acid before it is bound to the PCP of LipNrps. LipX3 resembles Rhs proteins from different sources. It is most similar to RhsA, a putative Rhs protein from S. avermitilis MA-4680. Homologues of its gene are present in the genomes of many different bacterial strains, e.g., E. coli, B. subtilis, and streptomycetes. The amino acid sequence of the lipX4 product is similar to the dipeptidil carboxypeptidase DdcA from S. fradiae.

Genes putatively located outside the cluster. Six ORFs were found close to the margins of the sequenced DNA. They probably do not belong to the lipomycin cluster. Five are located on one side of the cluster (ORF1 to ORF5) and the sixth is near the opposite margin (ORF6). The ORF1 product is similar to a putative gentisate 1,2-dioxygenase from Bacillus halodurans C-125. The deduced amino acid sequence of the ORF2 product resembles parts of a transcriptional regulator from the GntR family. ORF3 encodes a protein with similarity to Ocd1, an ornithine cyclodeaminase from Haloarcula marismortui ATCC 43049. ORF4 and ORF5 show homology to genes encoding 4,5-dihydroxyphthalate decarboxylases and 3,4-dihydroxyphthalate 2-decarboxylases, respectively. The ORF6 product is most similar to a Glu-tRNA amidotransferase subunit A from Nostoc sp. strain PCC 7120.

Mutational analysis of the lip cluster. Plasmid pNRPS was constructed to disrupt the putative NRPS by insertional mutagenesis. The resulting mutant, NRPS, did not produce any lipomycin derivative, indicating that lipNrps encodes an enzyme required for lipomycin production. pMT was constructed to disrupt the putative methyltransferase gene lipMt. The resulting mutant (MT) also did not produce any lipomycin derivative, indicating that the formation of N-methylated glutamic acid is essential for ß-lipomycin production (Fig. 2).

The production of -lipomycin was restored after introducing lipNrps and lipMt into NRPS and pMT, respectively. Feeding experiments of MT with N-methylated glutamic acid could not restore the production of -lipomycin. This makes it more likely that glutamic acid is bound as a substrate to the NRPS and that the methylation is taking place after this binding.

A mixture of compounds (around 50) was produced by each mutant strain (NRPS and MT) as indicated by HPLC-UV-MS analysis. When we tried to isolate at least one of these compounds for NMR analysis, we noticed that none of these compounds was produced in a sufficient amount. The same mixture of compounds was produced by the double mutants MTpPKS and NRPSpPKS, both in addition containing a disrupted loading module and extension module in LipPks1, indicating that the accumulation of these compounds did not result from the activity of the PKS of the lipomycin cluster.

Plasmid pGT was introduced into S. aureofaciens, allowing the inactivation of the putative glycosyltransferase LipGtf by integration. Culture extracts from the resulting mutant, IntGTF, and from the wild-type strain were analyzed by HPLC-MS. -Lipomycin production was clearly detected in the wild-type strain but was not detectable in the mutant strains. Instead, IntGTF produced ß-lipomycin lacking the digitoxose moiety.

All these results clearly showed that we indeed had cloned the lipomycin biosynthetic gene cluster. This sets the basis for future studies on the engineering of more-potent lipomycin analogs through genetic engineering.

 
We thank Annette Wittmer and Klaus Pelz (University of Freiburg) for antimicrobial activity testing.

This work was supported by a BMBF (GenoMik) grant to A.B.

 
* Corresponding author. Mailing address: Albert-Ludwigs-Universität Freiburg, Institut für Pharmazeutische Wissenschaften, Pharmazeutische Biologie und Biotechnologie, Stefan-Meier-Straße 19, D-79194 Freiburg, Germany. Phone: 49-761-203-8371. Fax: 49-761-203-8383. E-mail: andreas.bechthold{at}pharmazie.uni-freiburg.de.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Antimicrobial Agents and Chemotherapy, June 2006, p. 2113-2121, Vol. 50, No. 6
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