Increased Lipid Accumulation in Mucor circinelloides by Overexpression of Mitochondrial Citrate Transporter Genes

Mucor circinelloides has been commonly used as the model microbe to investigate lipid production as an oleaginous fungus. Mitochondrial citrate transporter can catalyze the translocation of the citrate, accumulated from TCA cycle, across the mitochondrial inner membrane. The extra-mitochondrial citrate is then cleaved by ATP-citrate lyase to oxaloacetate (OAA) and acetyl-CoA. Acetyl-CoA together with NADPH generated in cytosol is used for fatty acid biosynthesis. Thus, citrate transporters provide a link between TCA cycle in mitochondria and fatty acid biosynthesis in cytosol. However, the role of citrate transporters for lipid accumulation in oleaginous fungi is not clear. Two genes coding for citrate transporters, named citrate transporter (ct) and tricarboxylate transporter (tct) respectively, were present in the genome of oleaginous fungus M. circinelloides WJ11, a high lipid producing strain (36%, lipid/cell dry weight). As the mutant of strain CBS 277.49 (15%, lipid/cell dry weight) has been constructed and its genetic engineering tools are available for gene manipulation, so in this work, we investigated the role of citrate transporters in regulating lipid biosynthesis by overexpressing the citrate transporters of M. circinelloides WJ11 in CBS 277.49. Results: Our results showed that overexpression of ct and tct led to increased lipid accumulation by 44% (from 13.0% to 18.8%, w/w, CDW) and 68% (from 13.0% to 21.8%, w/w, CDW), respectively. Moreover, extracellular citrate concentration in ct-overexpressing strains (4.91 mM) and tct-overexpressing (3.25 mM) were significantly decreased by 20% and 47% respectively compared to the control (6.09 mM). Furthermore, overexpression of the citrate transporter genes activated the downstream steps in lipid biosynthesis, such as ATP citrate lyase (acl gene) and fatty acid synthases (fas1 and fas2 genes), indicating a greater flux of carbon went into fatty acid biosynthesis. Conclusions: This is the first report showing that citrate transporters involved in lipid accumulation in M. circinelloides. Both citrate transporter and tricarboxylate transporter could transport mitochondrial citrate to cytoplasm, which could provide more citrate to be cleaved by increased ACL to provide more acetyl-CoA and NADPH for increased FAS to synthesize fatty acids, thus, play a vital role in lipid biosynthesis in oleaginous fungus M. circinelloides.