Regulation Of MTHFD On Fatty Acid Synthesis In Mortierella Alpina

Polyunsaturated fatty acids（PUFAs）have important physiological functions and can be used to prevent and treat various chronic diseases.Mortierella alpina（M.alpina）can de novo synthesis a variety of PUFAs,but the yield of fatty acids reached the bottleneck,so it is urgent to analyze the mechanism of fatty acid synthesis and carry out targeted molecular modification of M.alpina.NADPH can provide reducing power for fatty acid synthesis,traditional sources are 6-phosphoglucose dehydrogenase,glucose-6-phosphate dehydrogenase,malic enzyme and isocitrate dehydrogenase.Recently,cell and animal studies have found that the methylenetetrahydrofolate dehydrogenase（MTHFD）in folate metabolic pathway can convert5,10-CH₂-THF into 5,10-CH-THF with the generation of NAD（P）H,it’s an important component of the cell NADPH pool.Therefore,MTHFD may play an important role in fatty acid synthesis.Genome analysis showed that there are two MTHFDs in M.alpina,MTHFD1and MTHFDL.Here we proved that MTHFD1 and MTHFDL play key roles in fatty acid synthesis in M.alpina at the molecular level.We preliminary study regulatory mechanism of MTHFD1 and MTHFDL on reducing power of fatty acid synthesis.Hence our study lays a foundation for uncovering the mechanism of lipogenesis.The main results were as follows:Firstly,MTHFD1 overexpression strain（MA-MTHFD1）was constructed successfully.Total fatty acid increased by 40.1%and the relative content of arachidonic acid increased by91.1%in MTHFD1 overexpression strain（MA-MTHFD1）compared with M.alpina,which indicated that the overexpression of MTHFD1 not only increased fatty acid contents but also fatty acid desaturation.Secondly,MTHFDL overexpression strain（MA-MTHFDL）and MTHFDL RNA interference（MA-MTHFDLSilent）were constructed successfully.Total fatty acid increased by35.6%and the relative content of AA increased by 80.8%in MTHFDL overexpression strain（MA-MTHFDL）,which indicated that the overexpression of MTHFDL not only increased fatty acid contents but also fatty acid desaturation;the RNA interference of MTHFDL reduced total fatty acid by 45.0%,which indicated that RNA interference of MTHFDL significantly reduced the content of total fatty acid content in M.alpina.Finally,after analysis of NADPH content and transcript levels of NADPH-generating genes,the results showed that the contents of NADPH in MA-MTHFD1 and MA-MTHFDL increased by 26.4%and 32.4%respectively,and decreased by 19.0%in MA-MTHFDLSilent.Further analysis showed that after overexpressing MTHFD1 and MTHFDL,the transcript levels of other genes which also can produce NADPH were increased at the same time,which indicated overexpressing MTHFD1 and MTHFDL enhanced the sources of NADPH required for fatty acid synthesis.After RNA interference with MTHFDL gene,the transcript levels of other genes which also can produce NADPH were decreased at the same time,which indicated RNA interference MTHFDL reduced the sources of NADPH required for fatty acid synthesis.In conclusion,the MTHFD1 gene and MTHFDL gene in folate metabolic pathway play important roles in the synthesis and regulation of NADPH in fatty acid synthesis in M.alpina.