Physiological consequence of disruption of the VMA1 gene in the riboflavinoverproducer Ashbya gossypii

The vacuolar ATPase subunit A structural gene VMA1 of the biotechnologicall y important riboflavin overproducer Ashbya gossypii was cloned and disrupte d to prevent riboflavin retention in the vacuolar compartment and to redire ct the riboflavin flux into the medium, Cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequenc es of the Vma1 proteins from yeast and filamentous fungi, The deduced polyp eptide comprises 617 amino acids with a calculated molecular mass of 67.8 k Da. The deduced amino acid sequence is highly similar to that of the cataly tic subunits of Saccharomyces cerevisiae (67 kDa), Candida tropicalis (67 k Da), and Neurospora crassa (67 kDa) with 89, 87, and 60% identity, respecti vely, and shows about 25% identity to the beta-subunit of the FoF1-ATPase o f 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 t ype.