Identification of the genes a¡ecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Abstract

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondii Dvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondii Dfra1-45 mutant accumulated 1.8–2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Dvma1-17 and Dfes1-77 knockout strains could not grow at 37 1C in contrast to the wild-type strain and the Dfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 1C. Although the Dfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Dvma1-17 and Dfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.