RESUMEN
The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus Delta relA strains.
Asunto(s)
Glicerol/metabolismo , Redes y Vías Metabólicas/genética , Familia de Multigenes , Streptomyces/genética , Secuencia de Bases , Ácido Clavulánico/biosíntesis , ADN Bacteriano/química , ADN Bacteriano/genética , Eliminación de Gen , Dosificación de Gen , Perfilación de la Expresión Génica , Orden Génico , Genes Bacterianos , Glucosa/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Streptomyces/crecimiento & desarrolloRESUMEN
A protein binding to the autoregulatory element (ARE) upstream of the regulatory ccaR gene of Streptomyces clavuligerus was isolated previously by DNA affinity binding. The areB gene, encoding this protein, is located upstream and in opposite orientation to the leuCD operon of S. clavuligerus; it encodes a 239-amino-acid protein of the IclR family with a helix-turn-helix motif at the N-terminal region. An areB-deleted mutant, S. clavuligerusDeltaareB, has been constructed by gene replacement. This strain requires leucine for optimal growth in defined media. Expression of the leuCD operon is retarded in S. clavuligerusDeltaareB, because AreB binds the areB-leuCD intergenic region acting as a positive modulator. Clavulanic acid and cephamycin C production are improved in the DeltaareB mutant although no drastic difference in ccaR expression was observed. Pure recombinant AreB protein does not bind the ARE(ccaR) sequence (as shown by EMSA) unless filtered extracts from S. clavuligerus ATCC 27064-containing molecules of Mr lower than 10 kDa are added to the binding reaction. Restoration of binding to the ARE(ccaR) sequence is not observed when filtered extracts are obtained from the DeltaareB mutant, suggesting that biosynthesis of the small-molecular-weight effector is also controlled by AreB.
