RESUMEN
A DNA segment cloned from Streptomyces purpurascens ATCC 25489 close to a region that hybridized to a probe containing part of the actinorhodin polyketide synthase caused S. galilaeus ATCC 31615 to produce new anthracyclines. When transformed with certain sub-clones of this segment, the host produced glycosides of epsilon-rhodomycinone, beta-rhodomycinone, 10-demethoxycarbonylaklavinone and 11-deoxy-beta-rhodomycinone in addition to those of aklavinone, the natural anthracyclines of S. galilaeus. The first two compounds are S. purpurascens products and the other two are novel compounds that conceptually are structural hybrids between S. galilaeus and S. purpurascens products. Three glycosides of one of the novel aglycones, 11-deoxy-beta-rhodomycinone, were purified and found to possess cytotoxic activity against L1210 mouse leukaemia cells. Separate regions of the cloned S. purpurascens DNA are responsible for modification of the S. galilaeus host product at the 10- and 11-positions.
Asunto(s)
Aciltransferasas/genética , Aminoglicósidos/biosíntesis , Antraciclinas , Antibióticos Antineoplásicos/biosíntesis , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Genes Bacterianos , Streptomyces/genética , Aciltransferasas/metabolismo , Aminoglicósidos/química , Aminoglicósidos/farmacología , Animales , Antraquinonas/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Proteínas Bacterianas/metabolismo , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Leucemia L1210 , Ratones , Estructura Molecular , Naftacenos/metabolismo , Sintasas Poliquetidas , Recombinación Genética , Eliminación de Secuencia , Especificidad de la Especie , Streptomyces/clasificación , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.
