Microbial synthesis of the type I polyketide 6-methylsalicylate with Corynebacterium glutamicum.

Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4'-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.

Genomics-inspired discovery of massiliachelin, an agrochelin epimer from Massilia sp. NR 4-1.

A putative siderophore locus was detected in the genome of the violacein-producing bacterium Massilia sp. NR 4-1 and predicted to direct the biosynthesis of a molecule that is structurally related to the thiazoline-containing siderophore micacocidin. In order to track this compound, we analyzed the metabolic profiles of Massilia cultures grown under different iron concentrations. A compound which was found to be predominantly produced under iron deficiency was subsequently isolated. Its structural characterization by spectroscopic and bioinformatic analyses revealed a previously not known diastereomer of the cytotoxic alkaloid agrochelin. The structure of this natural product, which was named massiliachelin, corresponds to the assembly line encoded by the identified siderophore locus.

Herpetopanone, a diterpene from Herpetosiphon aurantiacus discovered by isotope labeling.

The genome of the predatory bacterium Herpetosiphon aurantiacus 114-95T harbors a number of biosynthesis genes, including four terpene cyclase genes. To identify the terpenes biosynthesized from H. aurantiacus 114-95T, we fed the strain with 13C-labeled glucose and, subsequently, searched for characteristic mass shifts in its metabolome. This approach led to the discovery of a new natural product, of which the isotope pattern is indicative for a diterpene originating from the methylerythritol phosphate pathway. After large-scale fermentation of H. aurantiacus 114-95T, the putative diterpene was isolated in sufficient quantity to enable NMR-based structure elucidation. The compound, for which the name herpetopanone is proposed, features a rare octahydro-1H-indenyl skeleton. Herpetopanone bears resemblance to cadinane-type sesquiterpenes from plants, but is structurally entirely unprecedented in bacteria. Based on its molecular architecture, a possible biosynthetic pathway is postulated.

Structure of Ralsolamycin, the Interkingdom Morphogen from the Crop Plant Pathogen Ralstonia solanacearum.

Ralsolamycin, an inducer of chlamydospore formation in fungi, was recently reported from the plant pathogenic bacterium Ralstonia solanacearum. Although interpretation of tandem mass data and bioinformatics enabled a preliminary chemical characterization, the full structure of ralsolamycin was not resolved. We now report the recovery of this secondary metabolite from an engineered R. solanacearum strain. The structure of ralsolamycin was elucidated by extensive spectroscopic analyses. Chemical derivatization as well as bioinformatics were used to assign the absolute stereochemistry. Our results identified an erroneous genome sequence, thereby emphasizing the value of chemical methods to complement bioinformatics-based procedures in natural product research.

Herpetosiphon gulosus sp. nov., a filamentous predatory bacterium isolated from sandy soil and Herpetosiphon giganteus sp. nov., nom. rev.

Three filamentous gliding bacteria from the German Collection of Microorganisms and Cell Cultures, Hp g11, Hp g471 and Hp g472, were subjected to a phylogenetic analysis. These organisms had previously been classified as members of the genus Herpetosiphon based on their growth physiology and morphology. However, a taxonomic assignment at the species level had not been carried out. Analysis of 16S rRNA sequences now confirmed the close relationship of strain Hp g472 to Herpetosiphon aurantiacus DSM 785T (98.6 % nucleotide identity) and Herpetosiphon geysericola DSM 7119T (97.7 %). The results of DNA-DNA hybridization experiments further implied that strain Hp g472 should be classified as a distinct species. The DNA G+C content of strain Hp g472 was 49.9 mol%. The major quinone was MK-10 and the predominant cellular fatty acids were C18 : 1, C16 : 1 and C16 : 0. Based on phenotypic, chemotaxonomic and phylogenetic data it was concluded that strain Hp g472 represents a novel species of the genus Herpetosiphon, for which the name Herpetosiphon gulosus sp. nov. is proposed. The type strain is Hp g472T (=DSM 52871T=NBRC 112829T). In contrast to Hp g472T, the strains Hp g11 and Hp g471 exhibited closest 16S rRNA gene sequence similarity (>99 %) with 'Herpetosiphon giganteus' Hp a2. The distinctive genotypic and phenotypic properties of the latter supported the revival of the name as Herpetosiphon giganteus (ex Reichenbach & Golecki, 1975) sp. nov., nom. rev. We propose the previously deposited reference strain DSM 589T=NBRC 112828T as the type strain.

Siderophores as molecular tools in medical and environmental applications.

Almost all life forms depend on iron as an essential micronutrient that is needed for electron transport and metabolic processes. Siderophores are low-molecular-weight iron chelators that safeguard the supply of this important metal to bacteria, fungi and graminaceous plants. Although animals and the majority of plants do not utilise siderophores and have alternative means of iron acquisition, siderophores have found important clinical and agricultural applications. In this review, we will highlight the different uses of these iron-chelating molecules.

Zincophorin - biosynthesis in Streptomyces griseus and antibiotic properties.

Zincophorin is a polyketide antibiotic that possesses potent activity against Gram-positive bacteria, including human pathogens. While a number of total syntheses of this highly functionalized natural product were reported since its initial discovery, the genetic basis for the biosynthesis of zincophorin has remained unclear. In this study, the co-linearity inherent to polyketide pathways was used to identify the zincophorin biosynthesis gene cluster in the genome of the natural producer Streptomyces griseus HKI 0741. Interestingly, the same locus is fully conserved in the streptomycin-producing actinomycete S. griseus IFO 13350, suggesting that the latter bacterium is also capable of zincophorin biosynthesis. Biological profiling of zincophorin revealed a dose-dependent inhibition of the Gram-positive bacterium Streptococcus pneumoniae. The antibacterial effect, however, is accompanied by cytotoxicity. Antibiotic and cytotoxic activities were completely abolished upon esterification of the carboxylic acid group in zincophorin.

Chemical chain termination resolves the timing of ketoreduction in a partially reducing iterative type I polyketide synthase.

Synthetic chain terminators were used to capture the biosynthetic intermediates from a partially reducing iterative type I polyketide synthase, which is integrated into a multimodular biosynthesis enzyme. The off-loaded metabolites clarified the timing of ketoreduction and aromatization in the assembly of the antibiotic micacocidin.

Precursor-directed biosynthesis of micacocidin derivatives with activity against Mycoplasma pneumoniae.

Micacocidin is a promising natural product for the treatment of Mycoplasma pneumoniae infections. In the biosynthesis of this antibiotic, a fatty acid-AMP ligase (FAAL) activates the starter unit hexanoic acid as acyl-adenylate and forwards it to an iteratively acting polyketide synthase. Biochemical analysis of the FAAL revealed an extended substrate tolerance, thereby opening the door for the modification of a micacocidin residue that is barely accessible via semisynthesis. A total of six new analogues were generated by precursor-directed biosynthesis in this study and profiled against M. pneumoniae.

An iterative type I polyketide synthase initiates the biosynthesis of the antimycoplasma agent micacocidin.

Micacocidin is a thiazoline-containing natural product from the bacterium Ralstonia solanacearum that shows significant activity against Mycoplasma pneumoniae. The presence of a pentylphenol moiety distinguishes micacocidin from the structurally related siderophore yersiniabactin, and this residue also contributes to the potent antimycoplasma effects. The biosynthesis of the pentylphenol moiety, as deduced from bioinformatic analysis and stable isotope feeding experiments, involves an iterative type I polyketide synthase (iPKS), which generates a linear tetraketide intermediate from acyl carrier protein-tethered hexanoic acid by three consecutive, decarboxylative Claisen condensations with malonyl-coenzyme A. The final conversion into 6-pentylsalicylic acid depends on a ketoreductase domain within the iPKS, as demonstrated by heterologous expression in E. coli and subsequent site-directed mutagenesis experiments. Our results unveil the early steps in micacocidin biosynthesis and illuminate a bacterial enzyme that functionally resembles fungal polyketide synthases.

Structure and biosynthetic assembly of cupriachelin, a photoreactive siderophore from the bioplastic producer Cupriavidus necator H16.

The bacterium Cupriavidus necator H16 produces a family of linear lipopeptides when grown under low iron conditions. The structural composition of these molecules, exemplified by the main metabolite cupriachelin, is reminiscent of siderophores that are excreted by marine bacteria. Comparable to marine siderophores, the ferric form of cupriachelin exhibits photoreactive properties. Exposure to UV light induces an oxidation of its peptidic backbone and a concomitant reduction of the coordinated Fe(III). Here, we report the genomics-inspired isolation and structural characterization of cupriachelin as well as its encoding gene cluster, which was identified by insertional mutagenesis. Based upon the functional characterization of adenylation domain specificity, a model for cupriachelin biosynthesis is proposed.

Biosynthesis of a complex yersiniabactin-like natural product via the mic locus in phytopathogen Ralstonia solanacearum.

A genome mining study in the plant pathogenic bacterium Ralstonia solanacearum GMI1000 unveiled a polyketide synthase/nonribosomal peptide synthetase gene cluster putatively involved in siderophore biosynthesis. Insertional mutagenesis confirmed the respective locus to be operational under iron-deficient conditions and spurred the isolation of the associated natural product. Bioinformatic analyses of the gene cluster facilitated the structural characterization of this compound, which was subsequently identified as the antimycoplasma agent micacocidin. The metal-chelating properties of micacocidin were evaluated in competition experiments, and the cellular uptake of gallium-micacocidin complexes was demonstrated in R. solanacearum GMI1000, indicating a possible siderophore role. Comparative genomics revealed a conservation of the micacocidin gene cluster in defined, but globally dispersed phylotypes of R. solanacearum.

Coordinated regulation of expression of Salmonella pathogenicity island 1 and flagellar type III secretion systems by ATP-dependent ClpXP protease.

Salmonella enterica serovar Typhimurium delivers a variety of proteins via the Salmonella pathogenicity island 1 (SPI1)-encoded type III secretion system into host cells, where they elicit several physiological changes, including bacterial invasion, macrophage apoptosis, and enteropathogenesis. Once Salmonella has established a systemic infection, excess macrophage apoptosis would be detrimental to the pathogen, as it utilizes macrophages as vectors for systemic dissemination throughout the host. Therefore, SPI1 expression must be restricted to one or a few specific locations in the host. In the present study, we have demonstrated that the expression of this complex of genes is repressed by the ATP-dependent ClpXP protease, which therefore suppresses macrophage apoptosis. Depletion of ClpXP caused significant increases in the amounts of two SPI1-encoded transcriptional regulators, HilC and HilD, leading to the stimulation of hilA induction and therefore activation of SPI1 expression. Our evidence shows that ClpXP regulates cellular levels of HilC and HilD via the control of flagellar gene expression. Subsequent experiments demonstrated that the flagellum-related gene product FliZ controls HilD posttranscriptionally, and this in turn activates HilC. These findings suggest that the ClpXP protease coregulates SPI1-related virulence phenotypes and motility. ClpXP is a member of the stress protein family induced in bacteria exposed to hostile environments such as macrophages.