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

Abstract

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 (BH 4 ), which has never been explored in fungi before. DHFR is the sole source of tetrahydrofolate and plays a key role in maintaining BH 4 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/BH 4 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 BH 4 salvage pathway in a fungus.

Screening chemical modulators of benzoic acid derivatives to improve lipid accumulation in Schizochytrium limacinum SR21 with metabolomics analysis

“…The main functional component of FA is p -ABA [21]. Chen et al suggested that FA could promote lipid synthesis in microorganisms [12]. This may be the direct effect of p -ABA on lipid synthesis.…”

“…S4B). The increase in the two enzymes accelerated the metabolism of the PPP, and the enhancement of tetrahydrofolate dehydrogenase and the PPP in the process was also observed, and more NADPH substrates could be provided [12]. In general, NADPH is the key substrate for lipid synthesis.…”

“…HX-308by 16.16% and 30.44%, respectively, by adding 9 g/L ascorbic acid [11]. Wang et al found that folic acid could promote the synthesis of lipids by Mortierella alpine by increasing NADPH [12]. The synthesis of PUFAs in Schizochytrium also requires the involvement of NADPH [13, 14].…”

The role of phenylalanine hydroxylase in lipogenesis in the oleaginous fungus Mortierella alpina

“…Because the genes involved in the phenylalanine hydroxylation system are absent in most fungal genomes [19], investigations of phenylalanine hydroxylation in the fungal kingdom have been lacking. Our laboratory is the first, to our knowledge, to sequence the whole genome of M. alpina [20] and comprehensively characterize the phenylalanine hydroxylation system in a fungus [3,19,21]. When M. alpina were cultured on a PAH inhibitor-containing medium, significant changes were observed in the lipid composition.…”

The role of MTHFDL in mediating intracellular lipogenesis in oleaginous Mortierella alpina

“…After sequencing the whole genome of M. alpina , we explored the presence of a putative MTHFDL gene in this fungus, which was first characterized in fungi in our laboratory [16, 17]. The inhibition of folate metabolism led to a decrease in the content of total fatty acid (TFA) and AA in M. alpina [18]. In addition, the differentiation of OP9 bone marrow stromal cells into fat cells was inhibited by the inhibition of folate metabolism [19].…”