The reversion of Pichia guilliermondii transformants to the wild-type phenotype

Pichia guilliermondii strain with blocked GTP cyclohydrolase II was transformed using replicative plasmids and their fragments containing the structural gene RIB7 of this enzyme. Experiments showed that the presence of an ARS element and the promoter region of the gene in the genome of transformants reduces the probability of their reversion to the wild-type phenotype. Different types of recombination in the yeast P. guilliermondii are discussed.     

The pleiotropic nature of <Emphasis Type="Italic">rib80, hit1</Emphasis>, and <Emphasis Type="Italic">red6</Emphasis> mutations affecting riboflavin biosynthesis in the yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis>

The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1, and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5–1.8-fold decrease in the activity of such Fe-S cluster proteins as aconitase and flavocytochrome b ₂, whereas the hit1 mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.     

Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the non-flavinogenic yeast Saccharomyces cerevisiae.     

Restoration of the Wild-Type Phenotype in Pichia guilliermondii Transformants

Pichia guilliermondii strain with blocked GTP cyclohydrolase II was transformed using replicative plasmids and their fragments containing the structural gene RIB1 of this enzyme. Experiments showed that the presence of the ARS element and the promoter region of this gene in the genome of transformants reduces the probability of their reversion to the wild-type phenotype. Different types of recombination in the yeast P. guilliermondiiare discussed.     

The pleiotropic nature of rib80, hit1, and red6 mutations affecting riboflavin biosynthesis in the yeast Pichia guilliermondii

The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1, and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5-1.8-fold decrease in the activity of such Fe-S cluster proteins as aconitase and flavocytochrome b2, whereas the hitl mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.