NtcA: a negative regulator of secondary metabolite biosynthesis in Sorangium cellulosum.

Microorganisms continue to be a source of novel, bioactive natural products for the treatment of human diseases. Notable among them are the myxobacteria, with some 50% of metabolites isolated from strains of a single species, Sorangium cellulosum. As native production in myxobacteria is often low, however, research has begun to address the regulatory systems governing the pathways, with the aim of increasing fermentation titers. These efforts are significantly enabled by whole genome sequencing data. We previously identified ChiR as a positive regulator of chivosazol biosynthesis in the genome sequencing strain S. cellulosum So ce56, only the second regulatory function known from myxobacterial secondary metabolism. As So ce56 is known to produce two additional compounds, the mixed polyketide etnangien (Irschik et al., 2007; Menche et al., 2008), and the siderophore myxochelin (Schneiker et al., 2007), we set out to further exploit the genome data to discover additional regulators of secondary metabolite biosynthesis. Here we report a novel function for a member of the NtcA family of nitrogen-responsive transcriptional regulators, as a negative transcriptional regulator of chivosazol biosynthesis. NtcA is a promoter binding protein (PBP), which recognizes a conserved sequence within the chivosazol promoter. Inactivation of ntcA enhanced the production of chivosazol by 4-fold, but also increased the yield of etnangien by 3.5-fold. The ammonia-induced repression of biosynthesis observed in wild type So ce56 was significantly attenuated in a ntcA mutant. Taken together, these data suggest that inhibition of chivosazol biosynthesis by environmental nitrogen is mediated, at least in part, by the NtcA protein. Our results also reinforce the idea that genomics-guided engineering of regulatory pathways is a viable strategy for improving metabolite yields through fermentation.

New natural epothilones from Sorangium cellulosum, strains So ce90/B2 and So ce90/D13: isolation, structure elucidation, and SAR studies.

In addition to epothilones A (1) and B (2), 37 natural epothilone variants and epothilone-related compounds were isolated from the culture broth of a 700 L fermentation of Sorangium cellulosum, strain So ce90/B2. Of these, only the 12,13-desoxyepothilones, epothilone C (14) and D (15), were produced in significant amounts (3-6 mg/L); the 21-hydroxy derivatives and epothilones E (3) and F (4), in low and variable amounts due to further degradation by the producing organism. Most of the other epothilone variants were produced only in 1-100 microg/L amounts. The new compounds are very similar in structure to the parent compounds 1, 2 and 14, 15 and are presumably the result of the imperfect selectivity of the biosynthetic enzymes for acetate and propionate. Further, epothilones containing an oxazole moiety (10-13) in the side chain instead of a thiazole as well as ring-expanded 18-membered macrolides, epothilones I (30-35), and a ring contracted 14-membered macrolide, epothilone K (36), were found as very minor metabolites. The mutant strain, So ce90/D13, instead of macrolactones, produced short-chain carboxylic acids 40, 41, and 42 bearing the characteristic thiazole side chain. The structures of the new epothilones were elucidated on the basis of comprehensive NMR and MS data. The new epothilone variants were tested in a cytotoxicity assay with mouse fibroblasts (cell line L929), and structure-activity relationships were established. Several new natural epothilones showed activity comparable to 1 and 2, but in no case exceeded that of 2.

Studies on the biosynthesis of epothilones: the PKS and Epothilone C/D monooxygenase.

Nonproducer mutants support the assumption that epothilones A and B are synthesized by the same polyketide synthase (PKS). The endproducts of the PKS, epothilones C and D, compete for the active site of a constitutively synthesized monooxygenase which is regulated by product inhibition. The postulated C-13 hydroxy-epothilones as direct precursors of epothilones C and D were not detected.

Identification of an L-dopa decarboxylase gene from Sorangium cellulosum So ce90.

During a screening program intended to identify genes encoding enzymes typical for secondary metabolism in Sorangium cellulosum So ce90, an aromatic amino acid decarboxylase gene (ddc) was detected. Expression of ddc in Escherichia coli and subsequent enzyme assays with cell-free extracts confirmed the proposed function derived from amino acid sequence comparisons. In contrast to other aromatic amino acid decarboxylases of eukaryotic origin, the S. cellulosum Ddc converted only L-dihydroxy phenylalanine. This is the first report of a gene encoding an L-dihydroxy phenylalanine decarboxylase in bacteria.

Studies on the biosynthesis of epothilones: the biosynthetic origin of the carbon skeleton.

The biosynthetic origin of the epothilone skeleton was studied by the incorporation of 13C and radioactively labeled precursors by Sorangium cellulosum So ce90. The carbon atoms are derived from acetate, propionate, the methyl group of S-adenosyl-methionine, and cysteine which also introduces the sulfur and nitrogen atoms. Epothilone biosynthesis starts with the formation of the thiazole part from acetate and cysteine. The incorporation of acetate or propionate units results in the formation of epothilones A and B, respectively. To introduce the epoxide function of epothilones A and B molecular oxygen is used.

Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties.

An antifungal activity against Mucor hiemalis was detected in the culture broth of Sorangium cellulosum (Myxococcales) strain So ce90. The activity was excreted into the supernatant during the log and early stationary phase. When the adsorber resin XAD-16 was added to the culture, the active metabolites were quantitatively bound to the resin. The epothilons showed a high cytotoxicity for animal cells and mimic the biological effects of taxol (BOLLAG et al., Cancer Res. 55: 2325 approximately 2333, 1995).

The jerangolids: A family of new antifungal compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties of jerangolid A.

An antifungal activity was detected in the culture broth of the myxobacterium, Sorangium cellulosum strain So ce 307. The activity was excreted into the supernatant during the log and early stationary phase. When the organism was fermented in the presence of the adsorber resin XAD-16, the metabolite was quantitatively bound to the resin. The main component, jerangolid A, has structural similarities to ambruticin, which is also produced by strains of Sorangium cellulosum.

Chivosazol A, a new inhibitor of eukaryotic organisms isolated from myxobacteria.

A new antibiotic, chivosazol, was isolated from the culture broth of the myxobacterium Sorangium cellulosum strain So ce12. It is a macrocyclic ring with one oxazol ring and a glycosidically bound 6-deoxyglucose (quinovose) at C-11. The antibiotic shows antimicrobial activity against yeasts and filamentous fungi, and is especially potent against mammalian cells. It was not active against bacteria.

Ratjadon: a new antifungal compound from Sorangium cellulosum (myxobacteria) production, physio-chemical and biological properties.

An antifungal activity, ratjadon, was detected in the culture broth of Sorangium cellulosum (Myxococcales) strain So ce360. The metabolite was quantitatively bound to the adsorber resin XAD-16, which was added to the medium at the beginning of the fermentation. The antibiotic spectrum was narrow, but some important phytopathogenic fungi, especially species of Oomycetes, were inhibited at very low concentrations.

The ripostatins, novel inhibitors of eubacterial RNA polymerase isolated from myxobacteria.

A new antibiotic, ripostatin, was isolated from the culture supernatant of the myxobacterium, sorangium cellulosum strain So ce377. It is a macrocyclic lactone carbonic acid containing an unsubstituted phenyl ring in a side chain. The antibiotic acts especially on Staphylococcus aureus, but seems not to penetrate most bacteria. The MIC values are in the range of 1 microgram/ml. Ripostatin is an inhibitor of eubacterial RNA polymerase. It interferes with the initiation of RNA synthesis.

Disorazol A, an efficient inhibitor of eukaryotic organisms isolated from myxobacteria.

A new antibiotic, disorazol, was isolated from the culture broth of the myxobacterium, Sorangium cellulosum strain So ce 12. It is a macrocyclic compound containing two oxazole rings. The antibiotic acted against many fungi and mammalian cell cultures. The latter responded to extremely low doses (MIC 3-30 pg/ml). None of the tested bacteria and yeasts were inhibited.

The tartrolons, new boron-containing antibiotics from a myxobacterium, Sorangium cellulosum.

New antibiotics were isolated from the culture broth of the myxobacterium, Sorangium cellulosum, strain So ce 678. The antibiotics were active against Gram-positive bacteria and mammalian cells. They were named tartrolon A and B. Tartrolon B contains a boron atom. The boron binding region of tartrolon is identical with that of boromycin and aplasmomycin.

The soraphens: a family of novel antifungal compounds from Sorangium cellulosum (Myxobacteria). I. Soraphen A1 alpha: fermentation, isolation, biological properties.

An antifungal activity was detected in the culture broth of Sorangium cellulosum (Myxococcales), strain So ce26. The activity was excreted into the supernatant during the log and early stationary phase. The active substance was quantitatively bound to XAD absorber resin added to the medium at the beginning of the fermentation. The new secondary metabolite was called soraphen and is of special interest to plant disease control for its inhibitory activity against numerous phytopathogenic fungi.

Hollow fiber supported gas membrane for in situ removal of ammonium during an antibiotic fermentation.

To study the influence of ammonium on an antibiotic cultivation, mass transfer measurements of ammonium through microporous hydrophobic membranes using different stripping methods were carried out and compared. The higher overall mass transfer coefficients for ammonium were obtained with an acid stripping solution compared to water, vacuum, or sweeping air. A hollow fiber module for in situ removal of ammonium during cultivation was designed and operated in an external bypass to a 10-L fermentor. Compared to a control fermentation, the cell dry mass could be increased 2.6 times and the antibiotic concentration 8 times, if the in situ ammonium removal was in operation.

Saframycin Mx1, a new natural saframycin isolated from a myxobacterium.

A new natural saframycin was discovered in the culture broth of the myxobacterium, Myxococcus xanthus strain Mx x48. The fermentation and isolation of the antibiotic are described. The name, saframycin Mx1, is proposed. The compound appears to interact with cellular DNA.

Vortex chamber for in situ recovery of the antibiotic Myxovirescin A in continuous cultivation.

A vortex chamber for continuous adsorption of the antibiotic Myxovirescin A on XAD resins was developed. In this paper the design and use of the vortex chamber in an external bypass of a continuous process is described. Compared with the normal continuous process, the specific production rate of the antibiotic is four to five times higher when the antibiotic is continuously adsorbed. A semicontinuous process could be performed by using two chambers for adsorption and regeneration alternatively.

The sorangicins, novel and powerful inhibitors of eubacterial RNA polymerase isolated from myxobacteria.

A new antibiotic, sorangicin, was isolated from the culture supernatant of the myxobacterium, Sorangium (Polyangium) cellulosum strain So cel2. It is a macrocyclic lactone carbonic acid and is produced in two structural variants, sorangicins A and B. In addition small quantities of the respective glycosides, sorangiosids A and B, may be found. The antibiotic acts mainly against Gram-positive bacteria, including myocobacteria, with MIC values between 0.01 and 0.1 microgram/ml, but at higher concentrations (MIC 3 approximately 30 micrograms/ml) Gram-negatives are also inhibited. Yeasts and molds are completely resistant. The new antibiotic is a specific inhibitor of eubacterial RNA polymerase which it blocks, however, only if added before RNA polymerization has started.

Determination of bacterial ammonia pools using Myxococcus virescens as an example.

Samples (150 microliters) from liquid cultures of known cell density of Myxococcus virescens (Myxobacterales) were used for the determination of the intracellular NH3/NH+4 concentrations (= total ammonia). The cells were separated from the culture broth within 30 s by centrifugation through a silicone layer and were lysed immediately with 20 microliters of a disintegration liquid at the bottom of the centrifugation tube. The ammonia concentrations of the lysates were determined with a Dohrmann nitrogen analyzer. The intracellular ammonia concentrations were calculated after corrections for trapped supernatant had been made by adding radioactive glucose, which cannot be taken up by the organism. Control experiments with permeabilized cells and radioactive methylamine corroborated the reliability of the method.

The corallopyronins, new inhibitors of bacterial RNA synthesis from Myxobacteria.

From the culture broth of the myxobacterium, Corallococcus (Myxococcus) coralloides, three new antibiotics have been isolated: corallopyronin A, B and C. The compounds, which are chemically related to the recently discovered myxopyronins, act mainly on Gram-positive bacteria, with MIC values between 0.1 and 10 micrograms/ml, and only exceptionally or at much higher concentrations (MIC values; 100 and more micrograms/ml) on Gram-negatives. They do not inhibit eukaryotic organisms and show no toxicity for mice (sc). The corallopyronins appear to block specifically eubacterial RNA polymerase.