Effects Of Gene PAH And SSADH On Lipogenesis In The Oleaginous Fungus Mortierella Alpina

Polyunsaturated fatty acids(PUFAs)are functional fatty acids that are significant in human's daily life.Mortierella alpina can synthesize a variety of omega-3 and omega-6 series of PUFAs and even can industrially produce arachidonic acid(AA).However,the lipid-producing ability of Mortierella alpina is limited and affected by many factors.Lipid synthesis and metabolism mechanism can be explored through the molecular modification of Mortierella alpina.Reduced nicotinamide adenine dinucleotide phosphate(NADPH)is the source of reducing power required for lipid synthesis.The phenylalanine hydroxylase(PAH)gene is the key enzyme in the phenylalanine hydroxylation system,which can eventually produce tartrahydric acid and enter malic acid circulation metabolism to produce NADPH.In the ?-aminobutyric acid(GABA)shunt pathway,semialdehyde succinate dehydrogenase(SSADH)catalyzes the production of succinic acid into the tricarboxylic acid cycle while producing NADPH.In order to improve the lipid synthesis ability of Mortierella alpina and explore its synthesis mechanism,the relationship between PAH or SSADH and fatty acid synthesis was analyzed.PAH was overexpressed and RNA interferencd in this study,and this approach was used to investigate how PAH regulate lipid metabolism in Mortierella alpina.Then RNA interference SSADH gene was constructed of recombinant Mortierella alpina and the relationship between lipid synthesis and SSADH was proved.The control mechanism of PAH and SSADH on lipid synthesis were analysed.The following are main results:1)PAH overexpression strain(M.alpina-PAH)was constructed successfully.TFA levels reached 37.6% of the cell dry weight and increased by 8.9% in M.alpina-PAH compared to the wild type,which indicates that the overexpression of PAH rised the content of fatty acid.Then by constructing RNA interference gene PAH recombinant M.alpina(M.alpina-iPAH),we found that the the content of total fatty acid accounted for 30.4% of the dry weight of cells and rised by 11.9% compared with the wild type which indicates that interfering with the key enzyme PAH gene in phenylalanine metabolism would affect the lipid synthesis of Mortierella alpina.Therefore,PAH plays an important role in lipid synthesis in Mortierella alpina.2)Analysis of the GABA pathway in Mortierella alpina strains demonstrated the presence of?-aminobutyric acid.The interfering strain(M.alpina-iSSADH)of the semialdehyde succinate dehydrogenase(SSADH)gene was successfully constructed.The transcription level of the SSADH gene in the recombinant strain was downregulated by 55.4% and lipid accumulation reached 27.3% of the dry weight of the cells,which decreased by 26.4% compared to the wild type.This indicates that the SSADH affects the lipid accumulation in Mortierella alpina.(3)After overexpressing of PAH gene,the content of NADPH in M.alpina-PAH was increased by 24.3% compared with wild-type Mortierella alpina,and the transcription level of NADPH gene in glycolysis,pentose phosphate pathway,tricarboxylic acid cycle and folic acid metabolism pathway were upregulated significantly which indicates that the increase of phenylalanine metabolism raised the source of NADPH.In addition,the relative content of NADPH was reduced by around 11.9% in M.alpina-iPAH,and the transcriptional level of PAH gene was significantly down-regulated.These indicate that the phenylalanine metabolic pathway regulates the synthesis of NADPH,which could be the regulatory mechanism of PAH on lipogenesis in Mortierella alpina.