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J Biol Chem, Vol. 274, Issue 14, 9442-9448, April 2, 1999

Physiological Consequence of Disruption of the VMA1 Gene in the Riboflavin Overproducer Ashbya gossypii

Carola Förster, Maria A. Santos^§, Susanne Ruffert, Reinhard Krämer, and José L. Revuelta^§

From the  Institut für Biochemie der Universität zu Köln, Zülpicher Strasse 47, 50674 Köln, Germany and the ^§ Departamento de Microbiologia y Genetica, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain

The vacuolar ATPase subunit A structural gene VMA1 of the biotechnologically important riboflavin overproducer Ashbya gossypii was cloned and disrupted to prevent riboflavin retention in the vacuolar compartment and to redirect the riboflavin flux into the medium. Cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequences of the Vma1 proteins from yeast and filamentous fungi. The deduced polypeptide comprises 617 amino acids with a calculated molecular mass of 67.8 kDa. The deduced amino acid sequence is highly similar to that of the catalytic subunits of Saccharomyces cerevisiae (67 kDa), Candida tropicalis (67 kDa), and Neurospora crassa (67 kDa) with 89, 87, and 60% identity, respectively, and shows about 25% identity to the -subunit of the F_oF₁-ATPase of S. cerevisiae and Schizosaccharomyces pombe. In contrast to S. cerevisiae, however, where disruption of the VMA1 gene was conditionally lethal, and to N. crassa, where viable disruptants could not be isolated, disruption of the VMA1 gene in A. gossypii did not cause a lethal phenotype. Disruption of the AgVMA1 gene led to complete excretion of riboflavin into the medium instead of retention in the vacuolar compartment, as observed in the wild type.

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