Study on molecular mechanisms of uptake, conversion, and excretion of lipids in yeast.

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To develop yeast systems for production of useful chemicals from alkanes and fatty acids, it is necessary to study how these natural resources are utilized by yeasts. In this project, we analyzed two yeast species that can utilize n-alkanes and fatty acids as carbon sources, Yarrowia lipolytica and Candida maltosa. Y.lipolytica has a haploid genome of which sequence analysis was completed recently. C.maltosa has a diploid genome and has been used for production of long chain dicarboxylic acids (DCA). We obtained following new results.1.Y.lipolytica takes up n-hexadecane in an energy dependent manner. This uptake is induced by alkane and repressed by glycerol and by mutations in peroxin genes.2.The production of long chain DCA by C.maltosa was improved by the increased expression of CmCDR1 gene that encodes an ABC transporter.3.We identified an alkane-responsive element ARE1 on the promoter of alkane-inducible genes. ARE1 dependent lacZ reporter expression system was constructed and integrated into the Y.lipolytica genome. By mutagenizing the resultant recombinant strain, we obtained one mutant defective in YAS1 gene. The yas1 mutant and Δyas1 did not grow on n-decane but grow on oleic acid. Yas1p is localized in nucleus and has a basic helix loop helix (bHLH) motif. CHIP assay proved that Yas1p-HA bound the ALK1 promoter region. From these results, we concluded that Yas1p is a transcription factor for n-alkane inducible gene expression. Since Yas1p bHLH region has homology to Ino4p bHLH of S.cerevisiae and Ino4p forms heterodimer with Ino2p, another protein with bHLH motif, we identified a gene YAS2 by searching Y.lipolytica genome sequence data and proved that Yas2p binds to Yas1p and is responsible for the alkane-responsible gene expression.