The Study Of The Transcriptional Regulation And The Source Of Reducing Power During Fatty Acid Synthesis In Mortierella Alpina

Mortierella alpina is an industrail oleaginous fungi that could accumulate lipid up to50%of its dry weight and have a record of complete safety. In this study, based on theanalysis of transcriptome data, the mechanism of transcriptional regulation in M. alpinaconverting from proliferative stage into lipid accumulation stage has been revealed. The roleof malic enzyme, glucose-6-phosphate dehydrogenase and6-phosphogluconatedehydrogenase during fatty acid synthesis was evaluated by a series of homologiousoverexpression an RNA interference experiment. The main results are described as follows:1．Based on the work of transcriptome analysis of M. alpina culturing from theproliferative stage into the lipid accumulation stage. The results indicated that thetranscriptional regulation of this stage transition mainly focused on the two aspects bellow:improving the supply of Acetyl-CoA and NADPH while reducing their consumption by othermetabolic pathway; accelerating the synthesis of triglycerides while reducing its catabolism.We also believe: the NADPH used for fatty acid synthesis in M. alpina are mainly frompentose phosphate pathway and malic enzyme.2. The M. alpina OPRTase coding gene (ura5gene) has been disrupted via homologousrecombination through Agrobacterium tumefaciens-mediate gene knock-out to generate M.alpina uracil auxtroph with clear genetic background.. The M. alpina genetic manuplationsystem including the vector pBIG2-ura5s-ITs and uracil auxtrophic strain CCFM501, will bea strongly support for the following theoretical study of fatty acid synthesis and geneticengineering of M. alpina.3. Based on the newly construted M. alpina transformation system, the malE1gene washomologously overexpressed or knocked down, which led to the fatty acid content a increasedfor30%or decreased for15%of cell dry weight compared with the control. ME1wasrevealed as a source of NADPH for fatty acid synthesis in M. alpina. Overexpression of ME2gene improved the AA content for60%compared with the control. It could be concluded thatthe fatty acid synthesis and desaturation in M. alpina are regulated by multiple steps.4. For further evaluating the main NADPH source during fatty acid synthesis in M.alpina, we have constructed RNAi expression vectors based on pBIG2-ura5s-ITs, followed bythe ATMT to knock down the genes encoding glucose-6-phosphate dehydrogenase and6-phosphogluconate dehydrogenase and the fatty acid content decreased for40%and15%respectively compared with the control. The NADPH during fatty acid synthesis in M. alpinaproved to be supplied by glucose-6-phosphate dehydrogenase,6-phosphogluconate dehydrogenase and malic enzyme. The glucose-6-phosphate dehydrogenase played a relativemore important role in supplying of NADPH.