Wang Hongchao, Zhang Chen, Feng Jinghan, Liu Yuan, Yang Qin, Chen Haiqin, Gu Zhennan, Zhang Hao, Chen Wei, Chen Yong Q
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA.
Microbiology (Reading). 2016 Sep;162(9):1544-1553. doi: 10.1099/mic.0.000345. Epub 2016 Aug 2.
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.
高山被孢霉是一种著名的能产生多不饱和脂肪酸的产油真菌。对高山被孢霉基因组的分析表明，存在一个推定的二氢叶酸还原酶（DHFR）基因，该基因在四氢生物蝶呤（BH4）的补救途径中发挥作用，此前在真菌中从未有过相关研究。DHFR是四氢叶酸的唯一来源，在维持BH4水平方面起着关键作用。转录组数据分析显示，当高山被孢霉开始积累脂质时，DHFR会因氮耗尽而上调。与在不含抑制剂的培养基上生长的高山被孢霉相比，在含有DHFR抑制剂的培养基上生长的高山被孢霉的脂肪酸谱发生了显著变化。为了探究DHFR在叶酸/BH4代谢中的作用及其与脂质生物合成的关系，我们在大肠杆菌中异源表达了高山被孢霉编码DHFR的基因，并将重组酶纯化至同质。通过液相色谱、质谱和可见-紫外光谱对酶活性进行了研究。还研究了动力学参数以及温度、pH、金属离子和抑制剂对DHFR活性的影响。参与叶酸代谢的胞质产生NADPH的基因的转录水平受到DHFR抑制剂的下调，这突出了DHFR在脂质生物合成中的功能重要性。因此，DHFR与脂质代谢之间的关系至关重要，叶酸代谢可能是脂肪酸合成中替代NADPH的来源。据我们所知，本研究首次报道了真菌中BH4补救途径的全面表征。
