Klaus Gerth

Complete genome sequence of the myxobacterium Sorangium cellulosum.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strains complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase–like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

Novel expression hosts for complex secondary metabolite megasynthetases: Production of myxochromide in the thermopilic isolate Corallococcus macrosporus GT-2

Although many secondary metabolites with diverse biological activities have been isolated from myxobacteria, most strains of these biotechnologically important gliding prokaryotes remain difficult to handle genetically. In this study we describe the new fast growing myxobacterial thermophilic isolate GT-2 as a heterologous host for the expression of natural product biosynthetic pathways isolated from other myxobacteria. According to the results of sequence analysis of the 16S rDNA, this moderately thermophilic isolate is closely related to Corallococcus macrosporus and was therefore named C. macrosporus GT-2. Fast growth of moderately thermophilic strains results in shorter fermentation and generation times, aspects which are of significant interest for molecular biological work as well as production of secondary metabolites. Development of a genetic manipulation system allowed the introduction of the complete myxochromide biosynthetic gene cluster, located on a transposable fragment, into the chromosome of GT-2. Genetic engineering of the biosynthetic gene cluster by promoter exchange leads to much higher production of myxochromides in the heterologous host C. macrosporus GT-2 in comparison to the original producer Stigmatella aurantiaca and to the previously described heterologous host Pseudomonas putida (600 mg/L versus 8 mg/L and 40 mg/L, respectively).

Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads

Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be “silent” under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.

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.

Marinoquinolines A-F, pyrroloquinolines from Ohtaekwangia kribbensis (Bacteroidetes).

Marinoquinoline A (1) was isolated from the gliding bacterium Ohtaekwangia kribbensis together with the novel marinoquinolines B-F (2-6). Their structures were elucidated from NMR and HRESIMS data. The pyrroloquinolines showed weak antibacterial and antifungal activities and moderate cytotoxicity against four growing mammalian cell lines with IC(50) values ranging from 0.3 to 8.0 μg/mL. In a screening against tropical parasites marinoquinolines A-F (1-6) showed activity against Plasmodium falciparum K1 with IC(50) values between 1.7 and 15 μM.

Molecular Basis of Elansolid Biosynthesis: Evidence for an Unprecedented Quinone Methide Initiated Intramolecular Diels–Alder Cycloaddition/Macrolactonization

Elansolids A1/A2 (1) and B1–B3 (2–4) and the structurally unusual and highly reactive elansolid A3 (5) are new metabolites from the gliding bacterium Chitinophaga sancti (formerly Flexibacter spec. ; Scheme 1). While elansolid A2 (1*) shows antibiotic activity against Gram-positive bacteria in the range of 0.2 to 64 mgmL 1 and cytotoxicity against L929 mouse fibroblast cells with an IC50 value of 12 mgmL , the atropisomer elansolid A1 (1) is significantly less active. 3] The elansolids feature a bicyclo[4.3.0]nonane core which in the case of elansolids A1/A2 is part of a 19-membered macrolactone. Elansolid B1 is the corresponding seco acid of elansolids A1/A2, while the elansolids B2 and B3 are workup artifacts that result from nucleophilic addition of methanol and NH3, respectively, to

Elansolid A3, a Unique p‐Quinone Methide Antibiotic from Chitinophaga sancti

The genus Chitinophaga (Flexibacter) is well known as a producer of peptides with antibacterial activity. Only recently, elansolid A was isolated as the first macrolide antibiotic from Chitinophaga sancti (comb. nov.= new combination), strain GBF13. Remarkably, elansolid A occurred as two, stable, separable atropisomers, A1 (1) and A2 (1*). Detailed NMR studies combined with molecular modeling revealed that the atropisomerism is caused by the rigidity of two differently folded macrolide rings with either C7 or C6 “folded-in” the lactone ring. Importantly, the isomers differ in their biological activity. Whereas 1* showed antibiotic activity against Gram-positive bacteria in the range of 0.2– 64 mg mL 1 and cytotoxicity against L929 mouse fibroblast cells with a value for IC50 of 12 mg mL , the other atropisomer 1 was clearly less active. Our observation of a metabolite production depedent on the cultivation conditions of Chitinophaga sancti and the unaccounted occurrence of elansolids B1 (3) and B2 (4), which are assumed to be artifacts from treatment with water or methanol, required an in-depth analysis. We now report on the isolation and characterization of two further unique metabolites of which the exceptional, polyketide-based quinone methide antibiotic elansolid A3 (2) constitutes the major fraction (Scheme 1). For the production of 2, C. sancti was cultivated under slightly acidic conditions (pH 5.85) in the presence of Amberlite XAD 16 resin. After 150 h the neutral adsorber resin along with the adhering cell mass was harvested by sieving and then immediately frozen at 30 8C. Rapid HPLC-UV-HRMS analysis of a fresh acetone extract from the XAD resin additionally revealed a more polar peak of 2 at Rt 5.4 min. The elemental composition was determined to be C37H48O6, which is identical to 1 (Rt 8.7 min) and 1* (Rt 7.6 min). However, isomer 2 differed in the UV spectrum with a new band at 324 nm. During attempted workup under mild conditions, the isolation of this new metabolite (2) turned out to be impossible as it readily reacted with water or methanol, thus yielding elansolid variants 3 and 4. Because pH control did not stabilize the new metabolite 2 sufficiently, an unorthodox isolation process had to be developed. Unexpectedly, the final workup procedure had to be performed by carefully avoiding any contact of the fermentation products with water; this was achieved by using nitrogen as inert gas and all chromatographic steps were limited to the use of dry aprotic organic solvents. The freeze-dried Amberlite XAD 16 was extracted with dry acetone and 2 was isolated by silica-gel column chromatography, which finally yielded about 10 mg of 2 per plate by thick-layer chromatography. The NMR spectra were recorded in dry [D6]acetone (see the Supporting Information, Table S1) and clearly showed [a] Dr. R. Jansen, Dr. K. Gerth, Dipl.-Ing. H. Steinmetz, S. Reinecke, Dipl.-Ing. W. Kessler, Prof. Dr. R. M ller Helmholtz Centre for Infection Research Research Group Microbial Drugs Inhoffenstr. 7, 38124 Braunschweig (Germany) Fax: (+49) 531-61819499 E-mail : rolf.jansen@helmholtz-hzi.de [b] Prof. Dr. R. M ller Helmholtz Institute for Pharmaceutical Sciences Saarland (HIPS) Saarland University, P.O.Box 151150 66041 Saarbr cken (Germany) Fax: (+49) 681-30270202 E-mail : rom@mx.uni-saarland.de [c] Prof. Dr. A. Kirschning Institut f r Organische Chemie und Biomolekulares Wirkstoffzentrum (BMWZ) Leibniz Universit t Hannover Schneiderberg 1B, 30167 Hannover (Germany) Supporting information for this article, including tables of NMR data, biological activities, and full experimental data of 2 and 5, is available on the WWW under http://dx.doi.org/10.1002/chem.201100457. Scheme 1. Structures of elansolids 1, 1*, 2, 3, and 4.

Deciphering regulatory mechanisms for secondary metabolite production in the myxobacterium Sorangium cellulosum So ce56

Sorangium cellulosum strains produce approximately 50% of the biologically active secondary metabolites known from myxobacteria. These metabolites include several compounds of biotechnological importance such as the epothilones and chivosazols, which, respectively, stabilize the tubulin and actin skeletons of eukaryotic cells. S. cellulosum is characterized by its slow growth rate, and natural products are typically produced in low yield. In this study, biomagnetic bead separation of promoter‐binding proteins and subsequent inactivation experiments were employed to identify the chivosazol regulator, ChiR, as a positive regulator of chivosazol biosynthesis in the genome‐sequenced strain So ce56. Overexpression of chiR under the control of T7A1 promoter in a merodiploid mutant resulted in fivefold overproduction of chivosazol in a kinetic shake flask experiment, and 2.5‐fold overproduction by fermentation. Using quantitative reverse transcription PCR and gel shift experiments employing heterologously expressed ChiR, we have shown that transcription of the chivosazol biosynthetic genes (chiA–chiF) is directly controlled by this protein. In addition, we have demonstrated that ChiR serves as a pleiotropic regulator in S. cellulosum, because mutant strains lack the ability to develop into regular fruiting bodies.

Pellasoren: structure elucidation, biosynthesis, and total synthesis of a cytotoxic secondary metabolite from Sorangium cellulosum.

Myxobacteria are efficient producers of numerous secondary metabolites, and the genus Sorangium is frequently described as an proficient source for new, biologically active natural products. We report here the discovery and complete structure elucidation of pellasoren from the myxobacterium Sorangium cellulosum. Identification of the corresponding pel gene cluster from S. cellulosum So ce38 allowed us to establish a model for pellasoren biosynthesis, providing evidence for an unusual route to glycolate extender unit generation. Moreover, we present the first total synthesis of pellasoren and thereby confirm the absolute configuration of this natural product. Pellasoren (1; Scheme 1) was initially isolated from S. cellulosum So ce35 in the course of an activity-guided discovery program. Additionally, we recently identified 1 in relatively high amounts in extracts from the related strain S. cellulosum So ce38 by using LC-MS analysis. Here we determine its cytotoxicity against HCT-116 human colon cancer cells at a concentration of 155 nm (IC50). Full structural elucidation by NMR and ESI-MS analysis was performed and confirmed the identity of pellasoren from both sources (Figures S1–S3 and Table S1 in the Supporting Information). The pellasoren scaffold features an unusual enol ether moiety, also known from a small number of other natural products, which was corroborated by specific HMBC correlations between a methoxy signal and sp-hybridized carbon atoms (Figure S1 in the Supporting Information). The lactone moiety was identified through HMBC correlation between C1 and C5 and characteristic shifts for H-5 and C5 of d = 4.02 and 90.3 ppm, respectively. The position of the amide bond was assigned on the basis of indicative fragments in CID spectra. Efforts were made to establish the molecule s relative configuration by using NOE spectroscopy: ROESY interactions together with molecular modeling suggest an anti configuration of the substituents at C4 and C5, as well as a syn configuration of the methyl groups at C2 and C4 (Figure S2 in the Supporting Information). The stereocenter at C14 maintains the configuration derived from the incorporation of an l-alanine building block during biosynthesis. Stereochemical assignments that could initially not be validated by NOE analysis, such as the configuration at C6, were later established following total synthesis. Additionally, two isomeric pellasorens differing in the configuration of the C10– C11 double bond were isolated from S. cellulosum extracts. Pellasoren A (1 a) represents the all-E configuration while pellasoren B (1b) has a Z-configured double bond at C10– C11 which can be rationalized by photochemical isomerization. Following the structural elucidation of pellasoren, we set out to identify the underlying biosynthetic machinery using fragmentary whole-genome sequence information for strain S. cellulosum So ce38. Assuming that pellasoren is most likely the product of a hybrid PKS/NRPS biosynthetic pathway, the full complement of putative PKR/NRPS-related domains encoded in the So ce38 genome was annotated using bioinformatic tools. Using the presumed incorporation of alanine into pellasoren as a guide, we identified a genomic region roughly 57 500 bp in size, containing seven characteristic PKS modules organized as an apparent operon which also encodes one adenylation (A) domain exhibiting the Scheme 1. Structure of pellasoren A (1a) and B (1b).

Myxobacterium-Produced Antibiotic TA (Myxovirescin) Inhibits Type II Signal Peptidase

ABSTRACT Antibiotic TA is a macrocyclic secondary metabolite produced by myxobacteria that has broad-spectrum bactericidal activity. The structure of TA is unique, and its molecular target is unknown. Here, we sought to elucidate TAs mode of action (MOA) through two parallel genetic approaches. First, chromosomal Escherichia coli TA-resistant mutants were isolated. One mutant that showed specific resistance toward TA was mapped and resulted from an IS4 insertion in the lpp gene, which encodes an abundant outer membrane (Brauns) lipoprotein. In a second approach, the comprehensive E. coli ASKA plasmid library was screened for overexpressing clones that conferred TAr. This effort resulted in the isolation of the lspA gene, which encodes the type II signal peptidase that cleaves signal sequences from prolipoproteins. In whole cells, TA was shown to inhibit Lpp prolipoprotein processing, similar to the known LspA inhibitor globomycin. Based on genetic evidence and prior globomycin studies, a block in Lpp expression or prevention of Lpp covalent cell wall attachment confers TAr by alleviating a toxic buildup of mislocalized pro-Lpp. Taken together, these data argue that LspA is the molecular target of TA. Strikingly, the giant ta biosynthetic gene cluster encodes two lspA paralogs that we hypothesize play a role in producer strain resistance.