RESUMO
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). The structures of both molecules were elucidated through interpretation of 1D and 2D NMR data as well as comparison with authentic geldanamycin derivatives. Compounds 2 and 3 displayed moderate cytotoxicity against the human breast cancer cell line SK-BR-3.
Assuntos
Antibióticos Antineoplásicos/isolamento & purificação , Engenharia Genética , Quinonas/síntese química , Quinonas/isolamento & purificação , Streptomyces , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Benzoquinonas , Linhagem Celular Tumoral , Humanos , Lactamas Macrocíclicas , Estrutura Molecular , Quinonas/química , Quinonas/farmacologia , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismo , Relação Estrutura-AtividadeRESUMO
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. erythraea K301-105B, compared to its parent. In shake flasks, K301-105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA). In bioreactor studies, K301-105B produced 1.3 g/L of 15F-EryA with 75-80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50-55% molar yield on fed precursor by the parent strain. At higher precursor feed rates, K301-105B produced 3.5 g/L of 15F-EryA while maintaining 75-80% molar yield on fed precursor.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Eritromicina/análogos & derivados , Eritromicina/biossíntese , Eritromicina/metabolismo , Melhoramento Genético/métodos , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Biotransformação , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas Recombinantes/metabolismo , Saccharopolyspora/classificação , Especificidade da EspécieRESUMO
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. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.