Study On Metabolic Analysis And Regulation Of EPA Biosynthesis By Pythium

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with a wide range of functions in biological systems. EPA is important in the treatment of cardiovascular disease, such as atherosclerosis and cerebral thrombosis, EPA and its metabolites are used as biomedical products nutraceuticals, fortified foods and health supplements. Currently, there is a great demand for EPA worldwide, but the purified EPA was failed to meet the increasing demand. EPA produced by microorganisms is currently the main focus of studies. In this article, the metabolism of EPA biosynthesis was studied, and the rate-limiting steps in EPA biosynthesis pathway and its key enzymes have been further confirmed, moreover, to enhance the gene expression of key enzymes, the engineered Pythium strains were constructed, the study lay the foundation for the industrial application. The study will be of great importance in increasing the yield of EPA. The creative results are as follows:(1) The study on fatty acid metabolism genomics during Pythium EPA fermentation process revealed the synthesis pathway and the possible rate-limiting step of Pythium EPA lipid.There are two EPA synthesis pathways, n-3and n-6, in Pythium fermentation according to previous studies, however, the contributions of the two pathways are not clear. According to fatty acid composition analysis in Pythium EPA fermentation process, it was found that a-linolenic acid content (<0.23%) in n-3pathway is significantly lower than y-linolenic content (>1.11%) in n-6pathway, and no intermediate (6,9,12,15-parinaric acid) was detected from a-linolenic acid to EPA. Moreover, a positive correlation of EPA synthesis and ARA product in n-6pathway was found. For these reasons, it is concluded that Pythium EPA is mainly came from n-6pathway, produced by ARA conversion. In addition, oleic acid, linoleic acid, and ARA intermediates had more accumulation in EPA fats, indicating invertases (D12, D6, and D17dehydrogenases) catalyzed above intermediates in EPA fat synthesis pathway could be key enzymes of EPA synthesis. By comparative studies on fatty acid composition and gene expression changes of key enzymes in n-6pathway by high-ARA yield strains under different fermentation conditions, it was found high expression of D9, D12and D6dehydrogenase genes can conspicuously promote the conversion of stearic, oleic and linoleic acids and ARA synthesis. In addition, D9, D12and D6dehydrogenases are key enzymes in EPA synthesis.Based on EPA fatty acid composition analysis under different temperature and DO conditions, it was detected that the conversions of oleic and linoleic acids and the improvement of EPA yield could be promoted by either reducing temperature or increasing DO, which indicated that corresponding invertases (D12and D6) could be regulated by temperature and DO.(2) The analysis of transcriptome based on the optimization fermentation of EPA in Pythium revealed the molecular mechanism of EPA lipid synthesis and the key enzyme of EPA synthesis in Pythium.The transcriptome of Pythium was sequenced by RNA-seq technique.23,796Unigenes were obtained by genome sequencing assembly, and9,398Unigenes were annotated by nr. The1,945unique sequences obtained by GO classification were classified into functional groups of three categories. Six terms of the GO classification were (catalytic activity, binding, metabolic process, cellular process, cell, cell part) dominantly represented,7,300Unigenes were annotated by KEGG analysis and distributed into158pathways, among which metabolic pathways contained the most of Unigenes, followed by purine metabolism, spliceosome pathway, ammonia acyl-tRNA synthesis, fatty acid synthesis, etc. These results reflect the gene distribution of expression and function in Pythium, which can provide molecular basis for in further research of EPA synthesis mechanism.Gene expression of Pythium at different fermentation stages was analyzed by Digital Gene Expression Profiling and then the molecular mechanism of EPA lipid biosynthesis. The analysis of pathway significant enrichment,48h vs.100h, the differentially expressed gene was focused on leucine, isoleucine, and valine biosynthesis, Aminoacyl-tRNA biosynthesis, steroid biosynthesis, Pantothenate and CoA biosynthesis.100h vs.148h, the differentially expressed gene was focused on biosynthesis of secondary metabolites, glycolysis/Gluconeogenesis, fatty acid metabolism, it was found that enhancing the pathway of leucine, isoleucine, and valine biosynthesis, glycolysis/Gluconeogenesis, Pantothenate and CoA biosynthesis, and weaken the pathway of Aminoacyl-tRNA biosynthesis, which can regulate the EPA biosynthesis in Pythium. Futher, we found the31Unigene marked as transcription factors, among them, NF-Y transcription factor was reported relating to fatty acid synthesis, the express quantity of NF-Y transcription factor was highest in100h, which can promote biosynthesis of EPA by regulating the gene expression of fatty acid synthetase.Further comparative analysis of EPA synthesis gene under different fermentation stages, there are48EPA biosynthesis related genes were found, among them, Gene expression level of D6, acetyl-COA carboxylase, Elongase, diacylglycerol acyltransferase (DGAT) and fatty acid synthase the were large difference under different fermentation stages, providing D6, acetyl-COA carboxylase, Elongase, diacylglycerol acyltransferase (DGAT) and fatty acid synthasethe are key enzymes in EPA synthesis(3) We constructed engineering Pythium which included D6and vgb to improve the yield of EPA.We constructed engineering Pythium which included D6, two positive strains were selected, the content of EPA were improved16.18%and15.63%, respectively. We also constructed engineering Pythium which included vgb, one positive strain Pythium was selected. The optimized fermentation process showed that the content of EPA were improved20.70%with the low speed (120r/min).This study provide the theoretical and experimental basis for building many gene to regulate EPA biosynthesis.