Potent non-benzoquinone ansamycin heat shock protein 90 inhibitors from genetic engineering of Streptomyces hygroscopicus.

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain of Streptomyces hygroscopicus, shows high accumulation and long residence time in tumor tissue, is well-tolerated upon intravenous dosing, and is highly efficacious in the COLO205 mouse tumor xenograft model.

Cytotoxic hypothemycin analogues from Hypomyces subiculosus.

Three new hypothemycin analogues were isolated from the fungal strains Hypomyces subiculosus DSM 11931 and DSM 11932. The structures of these compounds were elucidated by spectroscopic methods, chemical conversion, and X-ray crystallographic analysis. One of the analogues, 4-O-demethylhypothemycin, exhibited potent and selective cytotoxic activity against cell lines with a BRAF mutation.

Characterization of product capture resin during microbial cultivations.

Various bioactive small molecules produced by microbial cultivation are degraded in the culture broth or may repress the formation of additional product. The inclusion of hydrophobic adsorber resin beads to capture these products in situ and remove them from the culture broth can reduce or prevent this degradation and repression. These product capture beads are often subjected to a dynamic and stressful microenvironment for a long cultivation time, affecting their physical structure and performance.

Production of 8-demethylgeldanamycin and 4,5-epoxy-8-demethylgeldanamycin from a recombinant strain of Streptomyces hygroscopicus.

Two new geldanamycin derivatives produced by genetic engineering of Streptomyces hygroscopicus strain K309-27-1 were isolated and characterized. Removal of the 8-methyl group of geldanamycin was achieved by replacing the AT4 domain of the polyketide synthase with a malonyl AT domain. The resulting strain produced 8-demethyl geldanamycin (2) and 4,5-epoxy-8-demethylgeldanamycin (3).

Improved bioconversion of 15-fluoro-6-deoxyerythronolide B to 15-fluoro-erythromycin A by overexpression of the eryK Gene in Saccharopolyspora erythraea.

The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution. This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter. Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S.

Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli.

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l(-1) (OD600 approximately 60) and the production of 700 mg l(-1) of 6-dEB.

A sensitive and robust method for quantification of intracellular short-chain coenzyme A esters.

A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites.

Precursor-directed biosynthesis of novel triketide lactones.

Precursor-directed biosynthesis was used to produce different triketide lactones (R-TKLs) in a fermentation process. Plasmids expressing engineered versions of the first subunit of 6-deoxyerythronolide B synthase (DEBS1) fused to the terminal DEBS thioesterase (TE) were introduced into three different Streptomyces strains. The DEBS1 protein fused to TE had either an inactivated ketosynthase domain (KS1 degrees ) or a partial DEBS1 lacking module 1 but containing module 2 (M2+TE).

Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues.

A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed.