Comprehensive Structural and Biochemical Analysis of the Terminal Myxalamid Reductase Domain for the Engineered Production of Primary Alcohols.

The terminal reductase (R) domain from the non-ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce the myxalamide family of secondary metabolites. Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Here we report crystal structures for MxaA R, both in the absence and, for the first time, in the presence of the NADPH cofactor. Molecular dynamics simulations were employed to provide a deeper understanding of this domain and further identify residues critical for structural integrity, substrate binding, and catalysis. Aggregate computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity toward highly reduced substrates.

A highly unusual polyketide synthase directs dawenol polyene biosynthesis in Stigmatella aurantiaca.

Enormous progress in the field of polyketide biosynthesis has led to the establishment of rules for general text book biosynthetic logic and consequently to the assumption that biosynthetic genes can be easily correlated with the corresponding natural products. However, non-textbook examples of polyketide assembly continue to be discovered suggesting the gene to product and product to gene predictions need improvement, especially as they are increasingly used in the post-genomic era. Here, we analyzed the genomic blueprint of a myxobacterial multi-producer of secondary metabolites, Stigmatella aurantiaca DW4/3-1, for its biosynthetic potential by genome-mining. In addition to the five polyketide synthase and/or nonribosomal peptide synthetase gene clusters of known function we identified a further 13 genomic regions exemplifying the enormous genetic potential for the production of additional chemical diversity by this strain. We show by gene inactivation and heterologous expression of the newly identified biosynthetic pathway for dawenol that the biosynthesis of this known polyene does not follow text book biosynthetic logic. Intriguingly, a genomic locus encoding an unusual polyketide synthase exhibiting similarity to gene loci involved in the formation of polyunsaturated fatty acids and secondary lipids was identified.

A multifunctional enzyme is involved in bacterial ether lipid biosynthesis.

Fatty acid-derived ether lipids are present not only in most vertebrates but also in some bacteria. Here we describe what is to our knowledge the first gene cluster involved in the biosynthesis of such lipids in myxobacteria that encodes the multifunctional enzyme ElbD, which shows similarity to polyketide synthases. Initial characterization of elbD mutants in Myxococcus xanthus and Stigmatella aurantiaca showed the importance of these ether lipids for fruiting body formation and sporulation.

Evolutionary divergence of sedoheptulose 7-phosphate cyclases leads to several distinct cyclic products.

Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules.

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases.

AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg(2+). Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.

Discovery of the rhizopodin biosynthetic gene cluster in Stigmatella aurantiaca Sg a15 by genome mining.

The field of bacterial natural product research is currently undergoing a paradigm change concerning the discovery of natural products. Previously most efforts were based on isolation of the most abundant compound in an extract, or on tracking bioactivity. However, traditional activity-guided approaches are limited by the available test panels and frequently lead to the rediscovery of already known compounds. The constantly increasing availability of bacterial genome sequences provides the potential for the discovery of a huge number of new natural compounds by in silico identification of biosynthetic gene clusters. Examination of the information on the biosynthetic machinery can further prevent rediscovery of known compounds, and can help identify so far unknown biosynthetic pathways of known compounds. By in silico screening of the genome of the myxobacterium Stigmatella aurantiaca Sg a15, a trans-AT polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene cluster was identified that could not be correlated to any secondary metabolite known to be produced by this strain. Targeted gene inactivation and analysis of extracts from the resulting mutants by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-HRMS), in combination with the use of statistical tools resulted in the identification of a compound that was absent in the mutants extracts. By matching with our in-house database of myxobacterial secondary metabolites, this compound was identified as rhizopodin. A detailed analysis of the rhizopodin biosynthetic machinery is presented in this manuscript.

Completing the puzzle of aurachin biosynthesis in Stigmatella aurantiaca Sg a15.

The aurachins are a family of secondary metabolites, with the main members aurachin A, B, C, and D, produced by the myxobacterium Stigmatella aurantiaca Sg a15. These isoprenoid quinoline alkaloids are classified as A-type or C-type aurachins according to the position of the farnesyl residue either at C4 or C3 of the quinoline core, respectively. Previous feeding studies revealed that the C-type aurachins are converted to A-type aurachins by late stage tailoring reactions. While the core gene cluster coding for the functionalities required for the biosynthesis of the basic structure aurachin D is known, neither of the genes encoding for the successively acting tailoring enzymes was known up to date, which was assumed to be due to a split cluster organisation. Here we describe the identification of a total of five genes, located upstream of the aurachin core cluster and at additional two loci elsewhere in the genome, encoding for the aforementioned functionalities. The generation and evaluation of respective inactivation mutants of S. aurantiaca Sg a15 allowed for the first time to propose an exhaustive model for aurachin biosynthesis. One of the deduced biosynthetic transformations corresponds to a pinacol rearrangement, an unprecedented tailoring reaction in secondary metabolite biosynthesis.

Unprecedented anthranilate priming involving two enzymes of the acyl adenylating superfamily in aurachin biosynthesis.

Biosynthesis of many polyketide-derived secondary metabolites is initiated by incorporating starter units other than acetate. Thus, understanding their priming mechanism is of importance for metabolic engineering. Insight into the loading process of anthranilate into the biosynthetic pathway for the quinoline alkaloids aurachins has been provided by the sequencing of a partial biosynthetic gene cluster in the myxobacterium Stigmatella aurantiaca. The cluster encodes a predicted aryl:CoA ligase AuaE that was hypothesized to activate and transfer anthranilate to the acyl carrier protein AuaB. However, gene inactivation and in vitro experiments described here contradicted this model. Aided by the genome sequence of S. aurantiaca, we identified an additional aryl:CoA ligase homologue, AuaEII, encoded in a different gene operon, which is additionally required for anthranilate priming. We report the characterization of both enzymes and the elucidation of a novel non-acetate priming strategy in thio-templated biosynthetic machineries.

AuaA, a membrane-bound farnesyltransferase from Stigmatella aurantiaca, catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the biosynthesis of aurachins.

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA-auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. (1) H NMR and HR-EI-MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. K(M) values of 43 and 270 µM were determined for FPP and 2-methyl-4-hydroxyquinoline, respectively. Like other known membrane-bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg(2+) , Mn(2+) and Co(2+) , although no typical (N/D)DXXD binding motif was found in the sequence.

The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

Steroid biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)-oxidosqualene cyclase from the myxobacterium Stigmatella aurantiaca.

Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene-hopene cyclase in bacteria and (S)-2,3-oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid-synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol-producing myxobacteria. Stigmatella aurantiaca is shown to produce cycloartenol, the well-known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site-directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.

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.

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 alpha-amide and the aryl-capped gamma-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.

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.

The mtaA gene of the myxothiazol biosynthetic gene cluster from Stigmatella aurantiaca DW4/3-1 encodes a phosphopantetheinyl transferase that activates polyketide synthases and polypeptide synthetases.

Myxothiazol is synthesized by the myxobacterium Stigmatella aurantiaca DW4/3-1 via a combined polyketide synthase/polypeptide synthetase. The biosynthesis of this secondary metabolite is also dependent on the gene product of mtaA. The deduced amino acid sequence of mtaA shows similarity to 4'-phosphopantetheinyl transferases (4'-PP transferase). This points to an enzyme activity that converts inactive forms of the acyl carrier protein domains of polyketide synthetases (PKSs) and/or the peptidyl carrier protein domains of nonribosomal polypeptide synthetases (NRPSs) of the myxothiazol synthetase complex to their corresponding holo-forms. Heterologous co-expression of MtaA with an acyl carrier protein domain of the myxothiazol synthetase was performed in Escherichia coli. The proposed function as a 4'-PP transferase was confirmed and emphasizes the significance of MtaA for the formation of a catalytically active myxothiazol synthetase complex. Additionally, it is shown that MtaA has a relaxed substrate specificity: it processes an aryl carrier protein domain derived from the enterobactin synthetase of E. coli (ArCP) as well as a peptidyl carrier protein domain from a polypeptide synthetase of yet unknown function from Sorangium cellulosum. Therefore, MtaA should be a useful tool for activating heterologously expressed PKS and NRPS systems.

The myxochelin iron transport regulon of the myxobacterium Stigmatella aurantiaca Sg a15.

The biosynthetic gene cluster of the myxochelin-type iron chelator was cloned from Stigmatella aurantiaca Sg a15 and characterized. This catecholate siderophore was only known from two other myxobacteria. The biosynthetic genes of 2,3-dihydroxybenzoic acid are located in the cluster (mxcC-mxcF). Two molecules of 2, 3-dihydroxybenzoic acid are activated and condensed with lysine in a unique way by a protein homologous to nonribosomal peptide synthetases (MxcG). Inactivation of mxcG, which encodes an adenylation domain for lysine, results in a myxochelin negative mutant unable to grow under iron-limiting conditions. Growth could be restored by adding Fe3+, myxochelin A or B to the medium. Inactivation of mxcD leads to the same phenotype. A new type of reductive release from nonribosomal peptide synthetases of the 2, 3-dihydroxybenzoic acid bis-amide of lysine from MxcG, catalyzed by a protein domain with homology to NAD(P) binding sites, is discussed. The product of a gene, encoding a protein similar to glutamate-1-semialdehyde 2,1-aminomutases (mxcL), is assumed to transaminate the aldehyde that is proposed as an intermediate. Further genes encoding proteins homologous to typical iron utilization and iron uptake polypeptides are reported.

Stigmatella aurantiaca Sg a15 carries genes encoding type I and type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases: involvement of a type II synthase in aurachin biosynthesis.

3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases catalyse the first step of the shikimate pathway. Two unrelated DAHP synthase types have been described in plants and bacteria. Two type II (aroA(A2) and aroA(A5)) and one type I DAHP synthase gene (aroA001) were identified from the myxobacterium Stigmatella aurantiaca Sg a15. Inactivation of aroA(A5) leads to a mutant that is impaired in the biosynthesis of aurachins, which are electron transport inhibitors and contain an anthranilate moiety. Feeding of anthranilic acid to the mutant culture restores production of aurachins. Inactivation of aroA(A2) and aroA001 does not impair production of aurachins or other known secondary metabolites of S. aurantiaca Sg a15.