| Eicosapentaenoic acid(EPA)and Docosahexaenoic acid(DHA)belong to the family of omega-3 polyunsaturated fatty acids,which play an important role in cell structure and signal transduction,and nerves system protection,vision enhancement and owning significant anti-tumor activity.Schizochytrium sp.is a microalgae with high lipid production,the main commercial source of polyunsaturated fatty acids.The main synthesis pathways of polyunsaturated fatty acids in Schizochytrium sp.,fatty acid synthase(FAS)and polyketide synthase pathway(PKS),are regulated by multiple genes and the mechanism is not resolved completely.There are few studies on the βoxidation pathway,an important part of fatty acid synthesis and metabolism.Previous research in our laboratory had identified the phospholipid metabolism pathway glycerophosphocholine phosphodiestera(GDPD),PKS pathway polyketoacyl reductase(KAR)and β oxidation pathway enoyl-CoA hydratase or isomerase(IS)three enzymes play a key role in the metabolic pathway of polyunsaturated fatty acids in Schizochytrium sp.In this study,Schizochytrium SR21 was used as the original strain,to explore the effects of three key genes on the synthesis of omega-3 polyunsaturated fatty acids by Schizochytrium sp..Meanwhile,enzymatic experiments in vitro,combined with traditional biological spectroscopy and computational simulation methods,are used to explore the interaction mechanism between protease and substrates.The main research contents and results are as follows:Gdpd was overexpressed in Schizochytrium SR21,and the biomass reached 34.91 g/L at 72 h,which was an increase of 3.22%compared to the original strain.Kar overexpression contributed to a decrease in the proportion of polyunsaturated fatty acids in the total lipid of Schizochytrium sp.,but the proportion of EPA reached 0.66%,which was 32%higher than that of the original strain.This might be related to the strengthening of the β-oxidation pathway,which led to an increase of intracellular oxidation level.Is overexpression resulted in the simultaneous decrease of biomass and total lipid production in Schizochytrium sp..while Is knockout resulted in the total lipid production of 23.63 g/L,which was 2.21%higher than the wild-type strain.The proportion of polyunsaturated fatty acids in the total lipid reached 40.75%,an increase of 7.04%,and the proportion of EPA and DHA reached to 0.54%and 33.27%,an increase of 8%and 11.87%compared with the wild-type strains respectively.Using RTqPCR to monitor the genes expression level of related metabolic pathways,the results showed that the FAS pathway,β-oxidation pathway,acetyl-CoA synthesis pathway and NADPH supply pathway of Is knockout strains were weakened,while the PKS pathway was strengthened,which resulted in an increase of total lipid production and the proportion of polyunsaturated fatty acids.To investigate the function of IS.Spectroscopy results showed that IS catalyzed the isomerization and hydration reactions of two short-chain fatty acid β oxidation intermediates,trans-3-hexenoic acid coenzyme A and trans-2-hexenoic acid coenzyme A respectively,verifying the isomerase and hydratase activity of IS.The results of molecular docking simulation experiments showed that it was mainly the binding of Arg134 at the catalytically active region with substrates to form a hydrogen bond when IS catalyzed the isomerization reaction,while the catalytic hydration reaction was mainly determined by the binding of Lys60 with substrates.In addition,the longer the chain length of the reaction substrates,the stronger the binding capacity,indicating the specificity of IS for the selection of substrates with different chain lengths.As a key enzyme in the β-oxidation pathway,IS participated in the oxidative decomposition and conversion of polyunsaturated fatty acids,leading to the accumulation of specific polyunsaturated fatty acids,which clarified the significance of IS in the metabolism and transformation of polyunsaturated fatty acids in Schizochytrium sp.. |
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