Development and Characterization of Expression Vectors for the Riboflavin Overproducing Fungus Eremothecium ashbyii using Ashbya gossypii Genes

Abstract

Eremothecium ashbyii is a filamentous hemi-ascomycete fungus and a natural overproducer of riboflavin. The present study was undertaken to characterize the molecular tools constructed for the genetic manipulation of this organism based on plasmids constructed for the related organisms A. gossypii and S. cerevisiae using two candidate genes. The candidate gene, SPR3 homolog of S. cerevisiae, is known to play a role in cytokinesis in S. cerevisiae. This gene was chosen to aid in future studies on the regulation of septation and its role in the excretion of riboflavin in E. ashbyii, as yeast cytokinesis is analogous to the septation of filamentous fungi. The second candidate gene was the S. cerevisiae RAD14 homolog, which is known to play a key role in the nucleotide excision repair pathway. Reporter plasmids, constructed previously in a preliminary study with the AgSPR3-like gene and the AgRAD14-like gene fused to the LacZ reporter gene, were used in this study. These plasmids were characterized by sequencing followed by homology searches. While the former revealed homology to the S. cerevisiae septin protein family, SPR3 gene, and the Neurospora crassa CDC12 gene involved in cell cycle regulation, the latter showed homology to the S. cerevisiae HOGI gene involved in the osmotic stress response.