The Role of Glyceraldehyde-3-Phosphate Dehydrogenases in NADPH Supply in the Oleaginous Filamentous Fungus Mortierella alpina.

Shunxian Wang,Hao Zhang,Guangfei Hao,Wei Chen,Xin Tang,Yong Q Chen,Haiqin Chen

doi:10.3389/fmicb.2020.00818

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme within the glycolytic pathway. GAPDH catalyzes the transformation of glyceraldehyde 3-phosphate to glycerate-1, 3-biphosphate, a process accompanied by the production of NADH. Its role in the NADPH production system of the oleaginous filamentous fungus Mortierella alpina was explored. Two copies of genes encoding GAPDH were characterized, then endogenously overexpressed and silenced through Agrobacterium tumefaciens-mediated transformation methods. The results showed that the lipid content of the overexpression strain, MA-GAPDH1, increased by around 13%. RNA interference of GAPDH1 and GAPDH2 (MA-RGAPDH1 and MA-RGAPDH2) greatly reduced the biomass of the fungus. The lipid content of MA-RGAPDH2 was found to be about 23% higher than that of the control. Both of the lipid-increasing transformants showed a higher NADPH/NADP ratio. Analysis of metabolite and enzyme expression levels revealed that the increased lipid content of MA-GAPDH1 was due to enhanced flux of glyceraldehyde-3-phosphate to glycerate-1, 3-biphosphate. MA-RGAPDH2 was found to strengthen the metabolic flux of dihydroxyacetone phosphate to glycerol-3-phosphate. Thus, GAPDH1 contributes to NADPH supply and lipid accumulation in M. alpina, and has a distinct role from GAPDH2.

Highlights

Mortierella alpina is an oleaginous filamentous fungus with a strong ability to accumulate polyunsaturated fatty acids (PUFAs) and has been used in industrial production of arachidonic acids (ARA) (Tsunehiro et al, 2001; Higashiyama et al, 2002; Dyal and Narine, 2005)
The aim of this study is to explore the role of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in NADPH production system in oleaginous filamentous fungus M. alpina
GAPDH1 can contribute to NADPH production, it does not make as much effect on fatty acid synthesis as either malic enzyme or the phosphate pentose pathway, which increase lipid content by

Summary

Introduction

Mortierella alpina is an oleaginous filamentous fungus with a strong ability to accumulate polyunsaturated fatty acids (PUFAs) and has been used in industrial production of arachidonic acids (ARA) (Tsunehiro et al, 2001; Higashiyama et al, 2002; Dyal and Narine, 2005). Great efforts have been made to improve its lipid yield, production and productivity Sakuradani et al, 2009; Hao et al, 2016). A traditional and effective way to achieve this is increasing the supply of NADPH, which is indispensable during lipid biosynthesis. Known enzymes involved in this process include malic enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconic dehydrogenase from the phosphate pentose pathway and isocitrate dehydrogenase (Hao et al, 2014a,b, 2016). A clear understanding of all NADPH sources for lipid biosynthesis is important for expounding the lipid biosynthesis process. Studies aimed to identify enzymes that contribute to NADPH production for lipid biosynthesis are necessary

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