Development of fluorescent reporter tagged RIB gene cassettes for replicative transformation, early expression, and enhanced riboflavin production in Eremothecium ashbyi.

Eremothecium ashbyi is a riboflavin overproducing filamentous fungus in which the metabolic pathways have not been genetically characterized. Two genes of the riboflavin biosynthetic (RIB) pathway, RIB1 and RIB3, which encode GTP-cyclohydrolase II (GCH II) and 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase respectively, were selected for the present study. The two RIB genes under their native promoters were obtained from Ashbya gossypii genomic library. Yeast enhanced green fluorescent protein (yEGFP) and mCherry genes were tagged to the C-terminal ends of RIB1 and RIB3 genes to analyse the functionality of the RIB transgenes in E. ashbyi. Shuttle vectors with the reporter tagged RIB genes contained the Escherichia coli kan(R) gene and Saccharomyces cerevisiae ARS element. On transformation with these plasmids, the ARS element was found to be functional in E. ashbyi. The E. ashbyi transcription factors could recognize the Ashbya RIB gene promoters and express the reporter tagged RIB genes as cytoplasmic proteins, in early cell development. Replicative transformants carrying RIB1-mCherry plasmids showed 2.95 times more GCH II activity and 2.44 times more riboflavin production when compared to untransformed. This is the first report of genetic transformation of E. ashbyi and is of significance as the first step towards genetic engineering of this genus.