Characterization of NAD+/NADP+-Specific Isocitrate Dehydrogenases From Oleaginous Fungus Mortierella alpina Involved in Lipid Accumulation.

Xin Tang,Jianxin Zhao,Xiaoqi Sun,Yong Q Chen,Haiqin Chen,Hao Zhang,Wei Chen,Xuxu Wang

doi:10.3389/fnut.2021.746342

Abstract

Mortierella alpina has a strong capacity for lipid accumulation. Isocitrate dehydrogenase (IDH) plays an important role in affecting the flow of intracellular carbon sources and reducing power NADPH for lipid biosynthesis. In this study, the effect of various IDHs (NAD+- and NADP+-specific) in M. alpina on the lipid accumulation was investigated through homologous overexpression. The results showed that the transcription level and enzyme activity of the IDHs from M. alpina (MaIDHs) in homologous overexpressing strains were higher than those of the control strain, but that their biomass was not significantly different. Among the various NAD+-specific MaIDH1/2/3 overexpression, NAD+-MaIDH3 reduced total lipid content by 12.5%, whereas overexpression NAD+-MaIDH1 and NAD+-MaIDH2 had no effect on fatty acid content. Intracellular metabolites analysis indicated that the overexpression NAD+-MaIDH3 strain had reduced the fatty acid accumulation, due to its greater carbon flux with the tricarboxylic acid cycle and less carbon flux with fatty acid biosynthesis. For the NADP+-MaIDH4/5/6 recombinant strains overexpressing only NADP+-MaIDH4 enhanced the total fatty acid content by 8.2%. NADPH analysis suggested that this increase in lipid accumulation may have been due to the great reducing power NADPH is produced in this recombinant strain. This study provides theoretical basis and guidance for the analysis of the mechanism of IDH function and the potential to improve lipid production in M. alpina.

Highlights

Mortierella alpina has strong fatty acid synthesis ability and can synthesize a variety of biologically active polyunsaturated fatty acids (PUFAs), including arachidonic acid (AA) and eicosapentaenoic acid (EPA) [1]
MaIDH1/2/3 were annotated as mitochondrial NAD+specific Isocitrate dehydrogenase (IDH), suggesting that its subcellular localization may occur in mitochondria
MaIDH4/5/6 were annotated as eukaryotic NADP-specific IDHs, which may be present in cytoplasm, mitochondria, and chloroplasts, but their amino terminus was very similar to that of the cytoplasmic form, indicating that they may be located in the cytoplasm

Summary

Introduction

Mortierella alpina has strong fatty acid synthesis ability and can synthesize a variety of biologically active polyunsaturated fatty acids (PUFAs), including arachidonic acid (AA) and eicosapentaenoic acid (EPA) [1]. Isocitrate dehydrogenase (IDH) is an enzyme that plays an important role in energy metabolism, amino acid synthesis and vitamin synthesis. It catalyzes the oxidative dehydrogenation of isocitrate to form the intermediate product, oxalosuccinic acid, followed by oxidative decarboxylation to produce 2-oxoglutarate [6]. NADP+-IDH is mainly localized in eukaryotic chloroplasts [12], mitochondria [13], peroxisomes [14], cytoplasm [15], and prokaryotic cells [16], and serves as one of the sources of NADPH It plays a crucial role in cell defense against oxidative damage [17,18,19]. Many enzymes are cofactor dependent and they frequently require NADH or NADPH in stoichiometric quantities for their catalytic function [20,21,22]

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