Effect Of Isocitrate Dehydrogenase On Lipid Biosynthesis In Mortierella Alpina

Motierella alpina has strong fatty acid synthesis ability and can synthesize various polyunsaturated fatty acids(PUFAs)including arachidonic acid(AA)and eicosapentaenoic acid(EPA).It has broad development prospects in the food industry.As a basic metabolic enzyme,isocitrate dehydrogenase(IDH)can exert diversified functions in organisms.It can affect the distribution of intracellular carbon sources and produce NADPH,which has a regulatory effect on lipid biosynthesis and is indispensable in metabolism.In this paper,MaIDH was homologously overexpressed in M.alpina.The transcription level,enzyme activity,cell growth,lipid accumulation and related metabolites of MaIDHs at different fermentation time points were analyzed under nitrogen-limited culture conditions to study the effects of MaIDH isoenzymes on the lipid accumulation process in M.alpina.In addition,by heterologously expressed in E.coli,the enzymatic properties of MaIDHs were studied,which provided a reference for understanding the properties of MaIDH.This research provided a theoretical basis and guidance for the industrial production of polyunsaturated fatty acids by M.alpina.The main results in this study are listed as below:1.Bioinformatics analysis revealed that there are six MaIDH isozymes in M.alpina.The analysis of the conserved domain of MaIDHs revealed that MaIDHs belong to the isocitrate dehydrogenase superfamily,among which MaIDH1/2/3 are NAD~+-dependent MaIDHs and MaIDH4/5/6 are NADP~+-dependent MaIDHs.None of the six MaIDHs has a transmembrane structure and is not secreted proteins.The transcription level of the overexpressed recombinant strains of M.alpina was found to be higher than that of the control strain,and the transcription level increased by 1.3~34.4 times,respectively.This indicated that overexpression of MaIDH in M.alpina can effectively increase the transcription level of MaIDH.2.Under nitrogen-limited culture condition,overexpression of NAD~+-MaIDH1/2/3resulted in a decrease of total lipid of 3.7%,5.3%and 12.5%in the recombinant strains of M.alpina.While overexpressing NADP~+-MaIDH4/5/6 increased the total lipid of the recombinant strains by 8.2%,1.6%and 3.4%,respectively.Among them,NAD~+-MaIDH3 and NADP~+-MaIDH4 had the most significant effect on the total lipid of M.alpina.The above results indicated that the isozymes played different roles in the lipid accumulation process in M.alpina.3.The enzyme activity of MaIDH homologous overexpressing strains under nitrogen source restriction showed that the activities of MaIDH in the overexpressed MaIDH recombinant strains were higher than that of the control strain.The enzyme activity of all recombinant strains decreased with the prolongation of fermentation time.The activity of MaIDH3 in NAD~+-dependent MaIDHs was 37.4%higher than that in the control group,while the activity of MaIDH5 in NADP~+-dependent MaIDHs was 25.5%higher than than that in the control group.This suggested that the activity of MaIDH can be further influenced at the protein level by interfering with the transcriptional level of M.alpina.4.The metabolite analysis of MaIDH homologous overexpressing strains under nitrogen source restriction showed that overexpressed NAD~+-MaIDH1/2/3 significantly reduced the content of intracellular citric acid in the lipid accumulation phase of M.alpina,which decreased by 52.9%,24.2%and 23.9%,respectively.And overexpression of NADP~+-MaIDH4/5/6 can significantly increase the content of NADPH in M.alpina,increasing by 37.6%,46.2%and30.1%,respectively.This suggested that overexpression of MaIDH in M.alpina can affect intracellular carbon flux by affecting TCA cycle or affect cytosolic NADPH content.5.In order to explore the enzymatic properties of MaIDH,NAD~+-MaIDH3 and NADP~+-MaIDH4,which have relatively significant effects on the lipid biosynthesis of M.alpina,were selected to construct E.coli heterologous expression strains.A large amount of target protein was obtained by inducing expression in E.coli and MaIDH4 was detected the activity in vivo.The optimum temperature and optimum pH of MaIDH4 were 35°C and pH 8.0,respectively.The MaIDH4 had decent thermal stability below 40°C and high enzymatic activity in the range of pH 6.5~8.0.The analysis of enzymatic properties was beneficial to optimize the industrial fermentation conditions of M.alpina,and provided theoretical basis for regulating lipid biosynthesis of M.alpina.