The Regulation Of Mechanism Of Tetrahydrobiopterin To Lipogenesis In Mortierella Alpina

Polyunsaturated fatty acids?PUFAs?are functional and essential fatty acids and necessary for human health.M.alpina is a one of the highest?PUFAs?-producing well-known oleaginous fungus,can produce arachidonic acid?AA?,Eicosapentaenoic acid?EPA?,etc.But the content of fatty acids of oleaginous microorganisms achieves the bottleneck,and the molecular mechanism of fatty acid synthesis and desaturation is still not well understood in oleaginous fungi in general,particularly in M.alpina.Tetrahydrobiopterin?BH₄?is a required cofactor for phenylalanine hydroxylation by phenylalanine hydroxylase?PAH?and nitric oxide synthesis by nitric oxide synthase?NOS?.BH₄ is essential for lipid metabolism in higher organisms,but there is no direct biochemical evidence that BH₄ is functionally significant in lipid metabolism.GTP cyclohydrolase I?GTPCH?is the first and rate-limiting enzyme for biosynthesis of tetrahydrobiopterin?BH₄?,whose gene was overexpressed in this study,and this approach was used to preliminarily investigate how intracellular BH₄ levels regulate lipid metabolism in M.alpina.Our study lays a foundation for uncovering the mechanism of efficient lipogenesis.The main results were described as follows:1.Our transcriptome data analysis revealed that GTPCH was up-regulated 2.8 fold by nitrogen exhaustion,when M.alpina starts to accumulate lipids.Significant changes were found in the fatty acid profile in M.alpina grown on medium containing BH₄ synthesis inhibitor?DAHP?,consequently,total fatty acid?TFA?and AA accumulation levels decreased by about 36% and 33%,respectively;GTPCH overexpression strain?MA-GTPCH?was constructed successfully,TFA levels were approximately 37% higher and AA levels were 27% higher in MA-GTPCH,which indicates that the overexpression of GTPCH not only increases cell fatty acid contents but also fatty acid desaturation.Hence,BH₄ has a metabolic correlation with lipogenesis in M.alpina.2.The overexpression of GTPCH led to total biopterin levels were 24-fold higher,BH2 levels were 36-fold higher,BH₄ levels were 7-fold higher than in the wild type strain,but folate content decreased by 25%,which indicates GTPCH overexpression is highly effective in augmenting BH₄ levels in M.alpina,and can be a valid strategy to research the function of BH₄ in M.alpina.3.The overexpression of GTPCH led to a 56% up-regulation in the expression of PAH,and enhanced tyrosine production by about 45%,which indicates that the function of phenylalanine hydroxylation is improved by the increased BH₄ levels;The overexpression of GTPCH led to a 48% up-regulation in the expression of NOS,enhanced NO level by 32% and decreased ROS level by above 90%,which indicates BH₄ can regulate the role of nitric oxide synthesis,and then influence NO/ROS dynamic equilibrium;Overexpression of GTPCH promoted an up-regulation of NADPH generating genes involved in glycolysis,pentose phosphate pathway,TCA cycle,and folate metabolism,and increased the reducing power,NADPH levels,for lipid synthesis.Hence,BH₄ could regulate the reducing power for lipid synthesis by enhancing the function of phenylalanine catabolism and nitric oxide synthesis pathway,which could be the regulatory mechanism of BH₄ on lipogenesis in M.alpina.