Cloning of Genes Sef1 and Tup1 Encoding Transcriptional Activator and Global Repressor in the Flavinogenic Yeast Meyeroz
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ing of Genes Sef1 and Tup1 Encoding Transcriptional Activator and Global Repressor in the Flavinogenic Yeast Meyerozyma (Candida, Pichia) guilliermondii D. Fedorovycha, *, V. Boretskya, Y. Pynyahaa, I. Bohovychb, Y. Boretskya, c, and A. Sibirnya, d aInstitute
of Cell Biology National Academy of Sciences of Ukraine, Lviv, 79005 Ukraine b University of Nebraska-Lincoln, Lincoln, NE 68588 United States c Lviv State University of Physical Culture, Lviv, 79000 Ukraine d University of Rzeszow, Rzeszow, 35-601 Poland *e-mail: [email protected]
Received March 10, 2020; revised March 24, 2020; accepted September 18, 2020
Abstract—Two Meyerozyma (Candida, Pichia) guilliermondii genes coding for homologs of transcriptional factor Sef1p of Candida famata and Tup1p of Candida albicans were identified, cloned, and deleted. Deletion of a homologue of Sef1p transcriptional factor in M.(P.) guilliermondii completely blocked over-synthesis of riboflavin under conditions of iron deficiency. The results of genetic complementation analysis suggest that previously reported rib83 mutants and newly constructed knock-out strains belong to the same complementation group and are defective in the same SEF1 gene. Inactivation of the identified homolog of the TUP1 gene in M.(P.) guilliermondii wild-type strain led to 1.5-fold increase in cellular iron content and 1.5–1.7-fold increase in riboflavin production. Introduction of a plasmid-borne copy of the TUP1 gene did not restore metabolic defects of the riboflavin overproduction and iron accumulation in mutant strain M. (P.) guilliermondii m3, bearing the hit1 mutation. The obtained results suggest that both transcription factors Sef1p and Tup1p are involved in the regulation of iron acquisition and riboflavin biosynthesis by yeast belonging to the CUG-clade. The molecular mechanism of action Tup1p on riboflavin biosynthesis in M.(P.) guilliermondii required further elucidation. Keywords: yeast, riboflavin, iron assimilation, transcriptional regulation DOI: 10.3103/S0095452720050072
INTRODUCTION Candida guilliermondii (teleomorph Pichia guilliermondii, Meyerozyma guilliermondii since 2010) is an ascomycete yeast widespread in the environment and is also part of the human saprophytic microflora [1]. M.(P.) guilliermondii belongs to the Crabtree-negative yeast, which cannot grow under strictly anaerobic conditions and has all three phosphorylation points in the respiratory chain [2]. M.(P.) guilliermondii and Candida albicans, like a majority of representatives of the so-called CUG clade, are capable of overproducing riboflavin (vitamin B2) under insufficient iron supply conditions [1, 3]. Mutants of M.(P.) guilliermondii with impaired regulation of riboflavin biosynthesis were selected and it was shown that they also have impairments of iron supply regulation and changes in response to oxidative stress [4]. For example, the mutant M.(P.) guilliermondii hit1-1 (high iron transport), which constitutively overproduces riboflavin, is characterized by increased cell reductase activity, high ir
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