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

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme within the glycolytic pathway. GAPDH catalyzes the transformation of glyceraldehyde 3phosphate 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-3phosphate. Thus, GAPDH1 contributes to NADPH supply and lipid accumulation in M. alpina, and has a distinct role from GAPDH2.

Validated Growth Rate-Dependent Regulation of Lipid Metabolism in Yarrowia lipolytica

“…Moreover, conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P) by upregulated triose phosphate isomerase could possibly enhance lipid accumulation. This could promote the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the NADH product of which can be transformed into NADPH in the cytoplasm through the pyruvate–oxaloacetate–malate cycle [ 12 ].…”

Autophagy Improves ARA-Rich TAG Accumulation in Mortierella alpina by Regulating Resource Allocation

“… E. coli TOP10 and A. tumefaciens AGL-1 were cultured in LB and yeast extract peptone (YEP; 10 g/L yeast extract, 10 g/L tryptone, 5 g/L NaCl) media, respectively. The preparation methods of the media used for A. tumefaciens - mediated transformation, including antibiotic-free minimal medium (MM), induction medium (IM), and uracil-free synthetic-complete (SC) agar plates, have been described previously ( 43 ). M. alpina activation in GY medium and flask fermentation in nitrogen-limited Kendrick medium were performed as described previously ( 44 ).…”

“… A. tumefaciens -mediated transformation of M. alpina MAU1 was performed as described previously ( 43 ). A single bacterial colony of A. tumefaciens harboring the vector was successively activated in YEP liquid medium and MM.…”

Production, Biosynthesis, and Commercial Applications of Fatty Acids From Oleaginous Fungi

“…Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an enzyme highly conserved in the glycolytic pathway. The lipid-producing filamentous fungus M. alpina was used to characterize two copies of the gene encoding GAPDH, and the overexpression strain MA-GAPDH1 increases the lipid content by about 13% ( 143 ). First, the total lipid accumulation was increased by overexpressing a malic enzyme from Crypthecodinium cohnii to elevate NADPH supply.…”