Role of Malic Enzyme during Fatty Acid Synthesis in the Oleaginous Fungus Mortierella alpina

Abstract

The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi. Oleaginous fungi, such as the commercial production species Mortierella alpina and Mucor circinelloides, can accumulate fatty acids to more than 20% of their cell dry weight (1). However, the mechanism of fatty acid biosynthesis in these organisms is still not fully understood. The carbon flux pathway and provision of NADPH are two major events during fatty acid synthesis. NADPH is particularly important as the sole source of reducing power during fatty acid synthesis in oleaginous fungi (2-5).The NADPH-generating enzyme malic enzyme (ME) (NADP ϩ dependent; EC 1.1.1.40), catalyzing the decarboxylation of malate to pyruvate (malate ϩ NADP ϩ ϭ pyruvate ϩ CO 2 ϩ NADPH), was speculated to play a pivotal role during fatty acid synthesis in oleaginous fungi (3, 5). When ME activity was inhibited, the cell fatty acid content of M. circinelloides decreased from 24% to 2% (6). Moreover, in the M. circinelloides strain R7B, overexpression of the ME genes from M. alpina and M. circinelloides produced a 2.5-fold increase in fatty acid content (7). These results led to the conclusion that a ratelimiting step in fatty acid biosynthesis is the generation of NADPH by ME (7). However, a recent study revealed that leucine auxotrophy caused a 2.5-fold decrease in cell fatty acid content and that leuA gene expression restored its level in M. circinelloides strain R7B. ME overexpression, however, did not alter the fatty acid content despite a significant increase in ME activity (8). It was thus proposed that the leucine metabolic pathway, by participating in acetyl coenzyme A (acetyl-CoA) generation, may be critical during fatty acid synthesis in M. circinelloides (8,9).M. circinelloides R7B, generated by random mutagenesis (10), may contain other, unknown mutations in addition to that in the leuA gene, which can complicate the interpretation of the abovementioned results. Therefore, a recipient strain with a known genetic mutation(s) and stable fatty acid synthesis characteristics will be advantageous for future research. To this end, a uracil auxotroph of M. alpina ATCC 32222 was generated via homologous recombination. The M. alpina ATCC 32222 cytosolic ME (isoform E)-encoding gene (11), named malE1, was cloned and homologously overexpressed in order to evaluate the role of ME in fatty acid synthesis. MATERIALS AND METHODSStrains and gr...

Metabolic Engineering of Mortierella alpina for Enhanced Arachidonic Acid Production through the NADPH-Supplying Strategy

“…Many enzymes can generate NADPH; however, their relative contributions to lipogenesis are still unclear. We have previously shown that ME1 overexpression increases total fatty acid production whereas ME2 facilitates fatty acid desaturation (5,8). This finding suggests that various sources of NADPH may play different roles during fatty acid synthesis and that the simultaneous overexpression of cytosol-and membrane-type NADPH-producing enzymes would augment the levels of total fatty acids as well as their desaturation.…”

“…The genes g6pd1, g6pd2, g6pd3, pgd, and idh1 from M. alpina were overexpressed in the same organism. Two pairs of previously described specific primers (HisproF1/TrpCR1 and HisproF2/ TrpCR2) ( Table 1) were applied to confirm the presence of the integrated transfer DNA (T-DNA) in the genomic DNA of transformants (1,5,8). Expression levels, enzymatic activities, NA-DPH, and fatty acid levels were analyzed in three replicates of each transformed strain, including ME1 (malE1)-and ME2 (malE2)-overexpressing strains that were previously constructed (5).…”

“…Agrobacterium tumefaciens-mediated transformation (ATMT) was performed essentially as described previously (5,8). The spores of M. alpina uracil-auxotrophic strain CCFM 501 were harvested from 2-week cultures grown on GY agar medium containing 5-FOA and uracil, followed by centrifugation at 12,000 ϫ g for 20 min and dilution with fresh liquid IM to 10 8 spores/ml.…”

“…Malic enzyme (ME; EC 1.1.1.40) was considered the sole NADPH provider for fatty acid synthesis in oleaginous microbes (3). Some evidence, however, indicated that ME is only one of several NADPH sources (4,5). We and others have recently demonstrated that the pentose phosphate pathway (PPP), specifically the glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and the 6-phosphogluconate dehydrogenase (PGD; EC 1.1.1.44) genes, plays a major role during microbial fatty acid synthesis (1,2,6).…”

“…An expression system using the uracil auxotroph strain CCFM 501 was recently established and reliably applied for gene manipulation (1,5,8,12, 13). In this study, the M. alpina genes encoding G6PD, PGD, and IDH were overexpressed in the same organism, and their effects on fatty acid synthesis were evaluated and compared with the effect of ME (5, 8).…”