Brigitte Kunze

New lessons for combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene cluster of Stigmatella aurantiaca DW4/3-1.

The biosynthetic mta gene cluster responsible for myxothiazol formation from the fruiting body forming myxobacterium Stigmatella aurantiaca DW4/3-1 was sequenced and analyzed. Myxothiazol, an inhibitor of the electron transport via the bc 1-complex of the respiratory chain, is biosynthesized by a unique combination of several polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS), which are activated by the 4′-phosphopantetheinyl transferase MtaA. Genomic replacement of a fragment of mtaB and insertion of a kanamycin resistance gene into mtaA both impaired myxothiazol synthesis. Genes mtaC and mtaDencode the enzymes for bis-thiazol(ine) formation and chain extension on one pure NRPS (MtaC) and on a unique combination of PKS and NRPS (MtaD). The genes mtaE and mtaF encode PKSs including peptide fragments with homology to methyltransferases. These methyltransferase modules are assumed to be necessary for the formation of the proposed methoxy- and β-methoxy-acrylate intermediates of myxothiazol biosynthesis. The last gene of the cluster,mtaG, again resembles a NRPS and provides insight into the mechanism of the formation of the terminal amide of myxothiazol. The carbon backbone of an amino acid added to the myxothiazol-acid is assumed to be removed via an unprecedented module with homology to monooxygenases within MtaG.

The Biosynthesis of the Aromatic Myxobacterial Electron Transport Inhibitor Stigmatellin Is Directed by a Novel Type of Modular Polyketide Synthase

Deductions from the molecular analysis of the 65,000-bp stigmatellin biosynthetic gene cluster are reported. The biosynthetic genes (stiA–J) encode an unusual bacterial modular type I polyketide synthase (PKS) responsible for the formation of this aromatic electron transport inhibitor produced by the myxobacterium Stigmatella aurantiaca. Involvement of the PKS gene cluster in stigmatellin biosynthesis is shown using site-directed mutagenesis. One module of the PKS is assumed to be used iteratively during the biosynthetic process, which seems to involve an unusual transacylation of the biosynthetic intermediate from an acyl carrier protein domain back to the preceding ketosynthase domain. Finally, the polyketide chain which is presumably catalyzed by a novel C-terminal domain in StiJ that does not resemble thioesterases, is cyclized and aromatized. The presented results of feeding experiments are in good agreement with the proposed biosynthetic scheme. In contrast to all other PKS type I systems reported to date, each module of StiA–J is encoded on a separate gene. The gene cluster contains a “stand alone” O-methyltransferase and two unusualO-methyltransferase domains embedded in the PKS. In addition, inactivation of a cytochrome P450 monooxygenase-encoding gene involved in post-PKS hydroxylation of the aromatic ring leads to the formation of two novel stigmatellin derivatives.

Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga15

BACKGROUND Myxobacteria have been well established as a potent source for natural products with biological activity. They produce a considerable variety of compounds which represent typical polyketide structures with incorporated amino acids (e.g. the epothilons, the myxothiazols and the myxalamids). Several of these secondary metabolites are effective inhibitors of the electron transport via the respiratory chain and have been widely used. Molecular cloning and characterization of the genes governing the biosynthesis of these structures is of considerable interest, because such information adds to the limited knowledge as to how polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) interact and how they might be manipulated in order to form novel antibiotics. RESULTS A DNA region of approximately 50000 base pairs from Stigmatella aurantiaca Sga15 was sequenced and shown by gene disruption to be involved in myxalamid biosynthesis. Sequence analysis reveals that the myxalamids are formed by a combined PKS/NRPS system. The terminal NRPS MxaA extends the assembled polyketide chain of the myxalamids with alanine. MxaA contains an N-terminal domain with homology to NAD binding proteins, which is responsible during the biogenesis for a novel type of reductive chain release giving rise to the 2-amino-propanol moiety of the myxalamids. The last module of the PKS reveals an unprecedented genetic organization; it is encoded on two genes (mxaB1 and mxaB2), subdividing the domains of one module from each other. A sequence comparison of myxobacterial acyl-transferase domains with known systems from streptomycetes and bacilli reveals that consensus sequences proposed to be specific for methylmalonyl-CoA and malonyl-CoA are not always reliable. CONCLUSIONS The complete biosynthetic gene cluster of the myxalamid-type electron transport inhibitor from S. aurantiaca Sga15 has been cloned and analyzed. It represents one of the few examples of combined PKS/NRPS systems, the analysis and manipulation of which has the potential to generate novel hybrid structures via combinatorial biosynthesis (e.g. via module-swapping techniques). Additionally, a new type of reductive release from PKS/NRPS systems is described.

Structure and Biosynthesis of Myxochromides S1–3 in Stigmatella aurantiaca: Evidence for an Iterative Bacterial Type I Polyketide Synthase and for Module Skipping in Nonribosomal Peptide Biosynthesis†

The myxobacterium Stigmatella aurantiaca DW4/3–1 harbours an astonishing variety of secondary metabolic gene clusters, at least two of which were found by gene inactivation experiments to be connected to the biosynthesis of previously unknown metabolites. In this study, we elucidate the structures of myxochromides S1–3, novel cyclic pentapeptide natural products possessing unsaturated polyketide side chains, and identify the corresponding biosynthetic gene locus, made up of six nonribosomal peptide synthetase modules. By analyzing the deduced substrate specificities of the adenylation domains, it is shown that module 4 is most probably skipped during the biosynthetic process. The polyketide synthase MchA harbours only one module and is presumably responsible for the formation of the variable complete polyketide side chains. These data indicate that MchA is responsible for an unusual iterative polyketide chain assembly.

Archazolid and apicularen: Novel specific V-ATPase inhibitors

BackgroundV-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research.ResultsArchazolid A and B, two recently discovered cytotoxic macrolactones produced by the myxobacterium Archangium gephyra, and apicularen A and B, two novel benzolactone enamides produced by different species of the myxobacterium Chondromyces, exerted a similar inhibitory efficacy on a wide range of mammalian cell lines as the well established plecomacrolidic type V-ATPase inhibitors concanamycin and bafilomycin. Like the plecomacrolides both new macrolides also prevented the lysosomal acidification in cells and inhibited the V-ATPase purified from the midgut of the tobacco hornworm, Manduca sexta, with IC50 values of 20–60 nM. However, they did not influence the activity of mitochondrial F-ATPase or that of the Na+/K+-ATPase. To define the binding sites of these new inhibitors we used a semi-synthetic radioactively labelled derivative of concanamycin which exclusively binds to the membrane Vo subunit c. Whereas archazolid A prevented, like the plecomacrolides concanamycin A, bafilomycin A1 and B1, labelling of subunit c by the radioactive I-concanolide A, the benzolactone enamide apicularen A did not compete with the plecomacrolide derivative.ConclusionThe myxobacterial antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. While archazolid at least partly shares a common binding site with the plecomacrolides bafilomycin and concanamycin, apicularen adheres to an independent binding site.

In vitro reconstitution of the myxochelin biosynthetic machinery of Stigmatella aurantiaca Sg a15: Biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases

Microorganisms produce iron-chelating compounds to sequester the iron essential for growth from the environment. Many of these compounds are biosynthesized by nonribosomal peptide synthetases, some in cooperation with polyketide synthases. Myxochelins are produced by the myxobacterium Stigmatella aurantiaca Sg a15, and the corresponding gene cluster was cloned recently. We have undertaken to express heterologously the myxochelin biosynthetic machinery in Escherichia coli. To activate the involved proteins posttranslationally, they were coexpressed with the phosphopantetheinyltransferase MtaA from the myxothiazol biosynthetic gene cluster. Phosphopantetheinylation of the carrier proteins could be verified by protein mass analysis. Six active domains in proteins MxcE, MxcF, and MxcG are capable of assembling myxochelin from ATP, NAD(P)H, lysine, and 2,3-dihydroxybenzoic acid in vitro. This fact demonstrates that the condensation domain of MxcG performs two condensation reactions, creating the aryl-capped α-amide and the aryl-capped γ-amide of the molecule. A previously unknown type of reductive release is performed by the reduction domain of MxcG, which alternatively uses NADPH and NADH to set free the peptidyl-carrier protein-bound thioester as an aldehyde and further reduces it to the alcohol structure that can be found in myxochelin A. This type of reductive release seems to be a general mechanism in polyketide and nonribosomal peptide biosynthesis, because several systems with C-terminal similarity to the reductase domain of MxcG can be found in the databases. Alternatively, the aldehyde can be transaminated, giving rise to a terminal amine.

Myxobacteria: a source of new antibiotics

Abstract Myxobacteria form highly colored macroscopic fruiting bodies on rotting wood and other substrates. The organisms can move by gliding or creeping, for example, across glass and agar surfaces. They also produce a large number of unusual secondary metabolites some of which have considerable potential as antibiotics. The large-scale cultivation of myxobacteria has also, therefore, become of great interest.

The mode of action of stigmatellin, a new inhibitor of the cytochrome b-c1 segment of the respiratory chain☆

The new antibiotic stigmatellin, obtained from the myxobacterium Stigmatella aurantiaca, was found to block the electron flow in the respiratory chain of bovine heart submitochondrial particles at the site of the cytochrome b-c1 segment. Its inhibitory potency was identical with that of antimycin and myxothiazol, and like these antibiotics, stigmatellin caused a shift in the spectrum of reduced cytochrome b. Difference spectroscopic studies with the three inhibitors in various combinations indicated that the binding site of stigmatellin was different from that of antimycin, but more or less identical with that of myxothiazol. Experiments with 14 synthesized derivatives of stigmatellin showed that good inhibitory activity can be expected only if the side chain was kept relatively lipophilic, and the keto and the hydroxy groups of the chromone system were left intact.

New Structural Variants of Homoserine Lactones in Bacteria

N‐Acylhomoserine lactones (AHLs) are used by a wide variety of bacteria for cell–cell communication in “quorum‐sensing”. These compounds are derived from L‐homoserine lactone and a fatty acid, which varies in chain‐length, degree of saturation, and the presence or absence of an oxygen atom at C‐3. In this study we describe for the first time the occurrence of acyl chains carrying a methyl branch, and present a GC‐MS‐based method that can be used to distinguish these compounds from unbranched isomers. The bacterium Aeromonas culicicola produces several methyl branched AHLs. In Jannaschia helgolandensis—a marine bacterium of the Roseobacter clade—a doubly unsaturated AHL, (2E,9Z)‐N‐(2,9‐hexadecadienoyl)‐L‐homoserine lactone, occurs. The location and configuration of the double bonds was proven by spectrometric investigation and synthesis. Finally, a method was developed to establish the absolute configuration of 3‐hydroxyalkanoyl‐HSLs by mild cleavage and chiral gas chromatography. The AHLs synthesized during this study were tested in sensor systems specific for certain AHL types. The results show that these compounds display varying responses to the respective sensors; this underlines the importance of determining the whole bouquet of AHLs and its function to fully understand their importance for regulatory functions in bacteria.

Multiple hybrid polyketide synthase/non-ribosomal peptide synthetase gene clusters in the myxobacterium Stigmatella aurantiaca.

Many bacterial and fungal secondary metabolites are produced by polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). Recently, it has been discovered that these modular enzymatic systems can also closely cooperate to form natural products. The analysis of the corresponding biosynthetic machineries, in the form of hybrid systems, is of special interest for combinatorial biosynthesis, because the combination of PKS and NRPS can lead to an immense variety of structures that might be produced. During our screening for hybrid PKS/NRPS systems from myxobacteria, we scanned the genome of Stigmatella aurantiaca DW4/3-1 for the presence of gene loci that encode both the PKS and NRPS genes. In addition to the previously characterized myxothiazol system, we identified three further hybrid loci, three additional PKS and one further NRPS gene locus. These were analyzed by hybridization, physical mapping, PCR with degenerate oligonucleotides and sequencing of fragments of the gene clusters. The function of these genes was not known but it had already been speculated that one compound produced by the strain and detected via HPLC was a secondary metabolite. This was based on the observation that its production is dependent on an active copy of the phosphopantetheinyl transferase gene mtaA. We show here that one of the identified hybrid gene loci is responsible for the formation of this secondary metabolite. In agreement with the genetic data, the chemical structure resembles a cyclic polypeptide with a PKS sidechain. Our data show that S. aurantiaca has a broader genetic capacity to produce natural products than the number of compounds isolated from the strain so far suggests.