Kapustiak K. Mutation rib1-86 as instrument for find out a genesinvolved in regulaton of riboflavin biosynthesis and iron homeostasis in the yeasts Pichia guilliermondii

The thesis is devoted to investigation of the regulaton of riboflavin biosynthesis and ron homoeostasis in the yeasts Pichia guilliermondii. It is known, that Pichia guilliermondii can overproduces riboflavin (vitamin B2) in response to iron limitation. Molecular mechanisms of such regulation are still unknown. To study this phenomenon, it is necessary to select putative mutations leading to altered regulation of riboflavin biosynthesis and identify genes involved. To select mutants defective in regulation of riboflavin biosynthesis, we used P. guilliermondii RF deficient strain rib1-86. This strain lacks activity of GTP cyclohydrolase II (encoded by RIB1 gene) that catalyses hydrolysis of guanosine-5-triphosphate to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate - the first committed stage of riboflavin biosynthesis. In contrast to other P. guilliermondii rib1 mutants, strain rib1-86 is able to grow without riboflavin under condition of iron starvation. Also, this strain spontaneouslyreverted to riboflavin prototrophy at a frequency approximately 10-7. Rate of reversion is increased in conditions of RF deprivation. We hypothesize that riboflavin prototrophy in this strain can be restored by hyperexpression of RIB1 gene that can be caused by iron starvation, or mutations affecting regulation of riboflavin biosynthesis. To check this hypothesis, we studied the mutant allele rib1-86 of RIB1 gene and characterized spontaneous revertants (riboflavin prototrophs) of P.guilliermondii rib1-86 strain. 2.1 kbp DNA fragment encomprizing GTP cyclohydrolase II structural gene was amplified by PCR using chromosomal DNA of P.guilliermondii rib1-86 strain as a template. Obtained DNA fragment was purified, digested with Xba1 and BamH1 endonucleases and cloned into corresponding sites of pUC19 vector. Sequencing of the mutant allele rib1-86 and subsequent alignment of RIB1 and rib1-86 nucleotide sequences revealed a single point mutation: substitution of G620 to A, that converts a cysteine codone to tyrosine. Alignment of amino acid sequences of GTP cyclohydrolase II from different microorganisms revealed that in most cases this position (207) is occupied by valine. Earlier it was shown that three cysteine residues located near this position are necessary to release formate from the imidazole ring of GTP. It is possible that C207 is needed also for the catalytic hydrolysis of GTP to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate in P. guilliermondii. The presence of cysteine in the same position of GTP cyclohydrolase II of yeast Candida albicans which also overproduce RF in response to iron limitation can be a suggestion in favor of this speculation. Most likely, C207Y substitution inactivates P. guilliermondii GTP cyclohydrolase II not completely or makes it unstable. Subsequently, in the case of hyperexpression of the gene total level of GTP cyclohydrolase II activity is sufficient to provide riboflavin synthesis according to the cell needs. Results of Northern blotting demonstrated that an increase in RF production by P. guilliermondii cells caused by iron starvation or regulatory mutation correlates with elevated level of mRNAs of key enzymes involved in this biosynthetic pathway. We conclude that regulation of RF biosynthesis by iron in P. guilliermondii occurs at the transcriptional level. Obtained spontaneous revertants were crossed with the wild type strain, and segregants were analyzed. A collection of riboflavin producing haploid strains (without rib1-86 mutation) was obtained. Colonies of segregants were red on medium supplemented with threephenyltetrazoliumchloride (TTC). The genetic analysis of these segregants revealed six novel loci RED1-RED6 (reduction), as well as previously identified genes RIB81, RIB80 and HIT1. Newly identified mutations are not linked with the RIB1 locus, are recessive, monogenic and possessed nuclear localization. Compare to the wild-type strain, all red mutants possessed increased activity of GTP cyclohydrolase and elevated levels of RF production. All of them possessed increased ferric/cupric reductase activity and higher non-hemin iron content. In addition, they were more sensitive to transitional metals. The metal hypersensitivity can be prevented by increased extracellular iron. Study of one corresponding mutant, red6, showed derepression of RIB1 mRNA synthesis in iron-sufficient medium.