Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina.

Mortierella alpina is a well-known polyunsaturated fatty acid-producing oleaginous fungus. Analysis of the Mort. alpina genome suggests that there is a putative dihydrofolate reductase (DHFR) gene playing a role in the salvage pathway of tetrahydrobiopterin (BH4), which has never been explored in fungi before. DHFR is the sole source of tetrahydrofolate and plays a key role in maintaining BH4 levels. Transcriptome data analysis revealed that DHFR was up-regulated by nitrogen exhaustion, when Mort. alpina starts to accumulate lipids. Significant changes were found in the fatty acid profile in Mort. alpina grown on medium containing DHFR inhibitors compared to Mort. alpina grown on medium without inhibitors. To explore the role of DHFR in folate/BH4 metabolism and its relationship to lipid biosynthesis, we expressed heterologously the gene encoding DHFR from Mort. alpina in Escherichia coli and we purified the recombinant enzyme to homogeneity. The enzymatic activity was investigated by liquid chromatography and MS and VIS-UV spectroscopy. The kinetic parameters and the effects of temperature, pH, metal ions and inhibitors on the activity of DHFR were also investigated. The transcript level of cytosolic NADPH-producing gene involved in folate metabolism is down-regulated by DHFR inhibitors, which highlights the functional significance of DHFR in lipid biosynthesis. The relationship between DHFR and lipid metabolism is thus of major importance, and folate metabolism may be an alternative NADPH source in fatty acid synthesis. To our knowledge, this study is the first to report the comprehensive characterization of a BH4salvage pathway in a fungus.