Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides

Abstract

Commercial interest in microbial lipids is increasing due to their potential use as feedstock for biodiesel production. The supply of NADPH generated by malic enzyme (ME; NADP+-dependent; EC 1.1.1.40) has been postulated as being the rate-limiting step for fatty acid biosynthesis in oleaginous fungi, based mainly on data from the zygomycete Mucor circinelloides studies. This fungus contains five genes that code for six different ME isoforms. One of these genes, malA, codes for the isoforms III and IV, which have previously been associated with lipid accumulation. Following a strategy of targeted integration of an engineered malA gene, a stable strain overexpressing malA and showing high ME activity has been obtained, demonstrating the feasibility of this strategy to overexpress genes of biotechnological interest in M. circinelloides. This is the first report showing the integration and overexpression of a gene in Zygomycetes. Unexpectedly, the genetically modified strain showed a lipid content similar to that of a prototrophic non-overexpressing control strain, suggesting that another limiting step in the fatty acid synthesis pathway may have been revealed as a consequence of the elimination of malic enzyme-based bottleneck. Otherwise, the fact that prototrophic strains showed at least a 2.5-fold increase in lipid accumulation in comparison with leucine auxotrophic strains suggests that a wild-type leucine biosynthetic pathway is required for lipid accumulation. Moreover, increasing concentrations of leucine in culture medium increased growth of auxotrophs but failed to increase lipid content, suggesting that the leucine synthesized by the fungus is the only leucine available for lipid biosynthesis. These results support previous data postulating leucine metabolism as one of the pathways involved in the generation of the acetyl-CoA required for fatty acid biosynthesis.

Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

“…Recently, leucine metabolism was found to be another bottleneck in lipid accumulation in the oleaginous fungus M. circinelloides (37). In that work, the overexpression of the LEU2 gene (a gene encoding ␤-isopropylmalate dehydrogenase, involved in leucine biosynthesis) significantly increased the lipid content, in accordance with previous observations in Saccharomyces cerevisiae (74).…”

“…However, the molecular mechanism of efficient lipid biosynthesis is still not well understood in oleaginous fungi in general and in M. alpina in particular. PAH and its cofactor BH 4 have been suggested to be essential for lipid metabolism in higher organisms (31)(32)(33)(34)(35)(36), and some amino acid metabolism pathways have been postulated to be involved in fatty acid biosynthesis in oleaginous fungi (37,38). However, the functional significance of the phenylalanine-hydroxylating system in the biosynthesis of lipids and closely related compounds has yet to be fully elucidated.…”

Methylcrotonyl-CoA Carboxylase Regulates Triacylglycerol Accumulation in the Model Diatom Phaeodactylum tricornutum

“…It has been reported that Leu metabolism is involved in the generation of the acetyl-CoA required for fatty acid biosynthesis in the oleaginous fungus Mucor circinelloides (Vorapreeda et al, 2012;Rodríguez-Frómeta et al, 2013). In addition, catabolism of amino acids such as Ala and Gln could provide carbon skeletons directed to fatty acid elongation (Schwender et al, 2006;Junker et al, 2007;Hockin et al, 2012).…”

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

“…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).…”