Three important strains of Mucor circinelloides grown in complete and minimal media for specified period (72 h, 120 h and 168 h) under submerged fermentation conditions were investigated for their potential antioxidants/secondary metabolite production. All mycelial extracts demonstrated effective antioxidant activities in terms of β-carotene/linoleic acid bleaching, radical scavenging, reduction of metal ions and chelating abilities against ferrous ions. Different extraction methods and solvent systems affected the recovery yield and antioxidant activities of the extracts significantly (p ≤ 0.05). Ethanolic extracts were found to be rich source of antioxidant components and subsequently more effective in antioxidant properties. Fermentation period and media used also significantly affected (p ≤ 0.05) the antioxidant production and the resulting antioxidant properties. The (ethanolic) extracts of all the strains from late exponential growth phase (120 h) showed highest antioxidant production with topmost reducing, chelating and radical scavenging capabilities. Strain MC277.49 was found to be the highest producer of antioxidants followed by MC108.16 and WJ11. Phenolic compounds were detected significantly in higher (p ≤ 0.05) amount succeeded by the condensed tannins and flavonoids. Total phenol content of each extract was attributed to overall antioxidant capacity. Submerged fermentation with nutritional stress conditions were found to be excellent way of producing surplus amount of natural antioxidants/secondary metabolites with their vast potential commercial application in food and pharmaceutical industries.

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.

Stearidonic acid (SDA; 18:4, n-3) is the delta 15-desaturase product of gamma linolenic acid (GLA; 18:3, n-6) and delta 6-desaturase product of alpha linolenic acid (ALA; 18:3, n-3). Construction of engineered oleaginous microbes have been attracting significant interest in producing SDA because of its nutritional value and pharmaceutical applications. Mucor circinelloides is a GLA producing filamentous fungus, which can be a useful tool to produce SDA. This study has, therefore, overexpressed the delta-15 desaturase (D15D) gene from Mortierella alpina in this fungus to construct a SDA-producing cell factory. To produce SDA in M. circinelloides, the homologous overexpression of D15D gene was analyzed. When the gene was overexpressed in M. circinelloides CBS 277.49, up to 5.0% SDA was accumulated in this strain. According to current knowledge, this is the first study describing the construction of a SDA-producing cell factory by overexpression of D15D gene in oleaginous fungus M. circinelloides. A new scope for further research has been established by this work to improve SDA production in this fungus, specifically in its high lipid-producing strain, WJ11.