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.

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.