A novel Hansenula polymorpha transcriptional factor HpHAP4-B, able to functionally replace the S. cerevisiae HAP4 gene, contains an additional bZip motif.

The transcriptional regulator HAP4, whose expression is induced on respiratory substrates, has been shown to be involved in the balance between fermentation and respiration in Saccharomyces cerevisiae. We have previously identified a HAP4 orthologue in the yeast Hansenula polymorpha, called HpHAP4-A, which, despite its very limited sequence conservation (a 16 amino acid N-terminal motif), is fully functional in S. cerevisiae. Based on the same N-terminal motif, a second gene has now been identified in the same organism. It was shown to contain an additional cis-binding motif of the bZip type. We report on the cloning, heterologous expression and analysis in S. cerevisiae of this novel ScHAP4 orthologue. From these experiments we could conclude that, as with HpHAP4-A, the novel orthologue, designated HpHAP4-B, could functionally replace the S. cerevisiae gene but to a lesser extent. The relationship between the presence of the additional cis-binding motif and the weaker potential as a HAP4 functional homologue is discussed.

A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the HAP transcriptional complex is involved in the fermentation-respiration shift. This complex is composed of four subunits. Three subunits are necessary for DNA-binding, whereas the Hap4p subunit, glucose-repressed, contains the transcriptional activation domain. Hap4p is the key regulator of the complex activity in response to carbon sources in S. cerevisiae. To date, no HAP4 homologue has been identified, except in Kluyveromyces lactis. Examination of these two HAP4 sequences led to the identification of two very short conserved peptides also identified in other yeasts. In the yeast Hansenula polymorpha, two possible HAP4 homologues have been found. Their deduced amino acid sequences are similar to the ScHap4p and KlHap4p proteins only in the N-terminal 16-amino-acid basic motif. Since molecular genetic tools exist and complete genome sequence is known for this yeast, we expressed one of these putative HpHap4 proteins in S. cerevisiae and showed that this protein is able to restore the growth defect of the S. cerevisiae hap4-deleted strain. A set of experiments was performed to confirm the functional homology of this new gene with ScHAP4. The discovery of a Hap4-regulatory protein in H. polymorpha with only the N-terminal conserved domain of the S. cerevisiae protein indicates that this domain may play a crucial role during evolution.

[Cloning of structural genes involved in riboflavin synthesis of the yeast Candida famata].

The riboflavin overproducing mutants of the flavinogenic yeast Candida famata isolated by conventional selection methods are used for the industrial production of vitamin B2. Recently, a transformation system was developed for C. famata using the leu2 mutant as a recipient strain and Saccharomyces cerevislae LEU2 gene as a selective marker. In this paper the cloning of C. famata genes for riboflavin synthesis on the basis of developed transformation system for this yeast species is described. Riboflavin autotrophic mutants were isolated from a previously selected C. famata leu2 strain. C. famata genomic DNA library was constructed and used for cloning of the corresponding structural genes for riboflavin synthesis by complementation of the growth defects on a medium without leucine and riboflavin. As a result, the DNA fragments harboring genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding GTP cyclohydrolase, reductase, dimethylribityllumazine synthase, dihydroxybutanone phosphate synthase and riboflavin synthase, were isolated and subsequently subcloned to the smallest possible fragments. The plasmids with these genes successfully complemented riboflavin auxotrophies of the corresponding mutants of another flavinogenic yeast Pichia guilliermondii. This suggested that C. famata structural genes for riboflavin synthesis and not some of the supressor genes were cloned.