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Registration Number

0224U032864, (0124U004341) , R & D reports

Title

Study of the mechanisms of action of new genes involved in the regulation of riboflavin synthesis in the flavinogenic yeast Candida famata

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Дослідження механізмів зв’язування активатора транскрипції Sef1 з промоторами структурних генів біосинтезу рибофлавіну використовуючи дріжджову моногібридну систему

Head

Fedorovych Dariya V., д.б.н.

Registration Date

09-12-2024

Organization

Institute of Cell Biology of the National Academy of Sciences of Ukraine

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To investigate the interaction of the previously identified transcription factor Sef1 with the promoters of the structural genes of riboflavin synthesis. To identify new genes involved in the regulation of riboflavin biosynthesis in the flavinogenic yeast Candida famata and to study their mechanisms of action by cloning and modifying them. To study the phenotypes of the obtained strains, namely, the accumulation of riboflavin, the activity of enzymes and the expression of the corresponding structural genes, as well as the content of iron in the cells.

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Bioinformatic work was carried out to search for and analyze the promoters of structural genes of riboflavin biosynthesis in the yeast Candida famata, namely RIB1, RIB2, RIB3, RIB5, RIB6 and RIB7. Integrative vectors containing the promoters of structural genes of the riboflavin biosynthesis pathway RIB1, RIB2, RIB3, RIB5, RIB6 and RIB7 in front of the LAC4 reporter gene were constructed. The Sef1 binding site was identified within the RIB1 gene promoter sequence. Plasmids containing the RIB1 gene promoter with a modified or deleted potential Sef1 interaction site were constructed. Cassettes containing the LAC4 reporter gene of Kluyveromyces lactis, which encodes β-galactosidase, under the control of different promoters of structural genes of riboflavin biosynthesis (RIB promoters) and the SEF1 gene of C. famata under the control of the GAL1 promoter, which is activated by galactose, were introduced into the genome of the yeast Saccharomyces cerevisiae. Transformants of S. cerevisiae containing all components of the monohybrid system were obtained, namely integrative plasmids with RIB1 promoters and the LAC4 reporter gene, and β-galactosidase activity was determined when cultivating these transformants in a medium with galactose (under induction conditions (GAL1 promoter)) and with glucose (control). The results obtained indicate that the transcription factor Sef1 directly binds and activates the RIB1 gene promoter, as well as the promoters of the RIB3, RIB6, RIB7 genes, as evidenced by the high β-galactosidase activity in the corresponding transformants. The transcription factor Sef1 does not directly interact with the promoters of the RIB2 and RIB5 genes. It was established that Sef1 binds to the sequence TAAAAATCCGAACCCCGG, as evidenced by the loss of β-galactosidase activity upon alteration or deletion of this site.

Product Description

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Dzanaieva Liubov S.
Romanov Serhii O.
Faiura Liubov R.
Tsyrulnyk Andrii O.

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2024-12-09