Regulation of AreA on lipid biosynthesis under different nitrogen sources and C/N ratios in the model oleaginous fungus Mucor circinelloides.

Mucor circinelloides has been exploited as model filamentous fungi for studies of genetic manipulation of lipogenesis. It is widely recognized that lipid accumulation is increased when there is a lack of nitrogen source in oleaginous microorganism. Nitrogen metabolism in filamentous fungi is a complex process that can be regulated by the global nitrogen regulator AreA. In this study, we cultivated the areA-knockout and -overexpression strains obtained in our previous study, using 20 different nitrogen sources. It emerged that the disruption of AreA in M. circinelloides reduced its sensitivity to nitrogen availability, resulting in increased lipid synthesis. Specially, the areA-knockout strain was unable to fully utilize many nitrogen sources but the ammonium and glutamate. We continued to investigate lipid production at different molar C/N ratios using glucose as sole carbon source and ammonium sulfate as sole nitrogen source, of which the high C/N ratios activate high lipid accumulation. By comparing the experimental results with transcriptional analysis, we were able to identify the optimal process conditions suitable for lipid accumulation and potential targets for future metabolic engineering.

Identification and Functional Characterization of Adenosine Deaminase in Mucor circinelloides: A Novel Potential Regulator of Nitrogen Utilization and Lipid Biosynthesis.

Adenosine deaminase (ADA) is an enzyme distributed in a wide variety of organisms that cleaves adenosine into inosine. Since inosine plays an important role in nitrogen metabolism, ADA may have a critical function in the regulation of fatty acid synthesis. However, the role of ADA in oleaginous fungi has not been reported so far. Therefore, in this study, we identified one ada gene encoding ADA (with ID scaffold0027.9) in the high lipid-producing fungus, Mucor circinelloides WJ11, and investigated its role in cell growth, lipid production, and nitrogen metabolism by overexpressing and knockout of this gene. The results showed that knockout of the ada altered the efficiency of nitrogen consumption, which led to a 20% increment in the lipid content (25% of cell dry weight) of the engineered strain, while overexpression of the ada showed no significant differences compared with the control strain at the final growth stage; however, interestingly, it increased lipid accumulation at the early growth stage. Additionally, transcriptional analysis was conducted by RT-qPCR and our findings indicated that the deletion of ada activated the committed steps of lipid biosynthesis involved in acetyl-CoA carboxylase (acc1 gene), cytosolic malic acid enzyme (cme1 gene), and fatty acid synthases (fas1 gene), while it suppressed the expression of AMP-activated protein kinase (ampk α1 and ampk ß genes), which plays a role in lipolysis, whereas the ada-overexpressed strain displayed reverse trends. Conclusively, this work unraveled a novel role of ADA in governing lipid biosynthesis and nitrogen metabolism in the oleaginous fungus, M. circinelloides.

Improved protective and controlled releasing effect of fish oil microcapsules with rice bran protein fibrils and xanthan gum as wall materials.

This study aimed to prepare fish oil microcapsules by freeze-drying an emulsion co-stabilized by rice bran protein fibrils (RBPFs) and xanthan gum (XG) to improve the oxidation stability and controlled release effect. Emulsions stabilized either solely by RBPFs or unfibrillated rice bran protein (RBP) or by a combination of RBP and XG were also fabricated as microcapsule templates for comparison. The rheological properties, particle size, and zeta potential of the emulsions were examined. In addition, the characteristics of the fish oil microcapsules such as surface oil content, encapsulation efficiency, water activity, moisture content, morphological structure, oxidation stability, and digestive performance were also assessed. The rheological properties revealed that the addition of XG increased the storage modulus of the emulsion and reduced the loss modulus and apparent viscosity. At shear rates of 0-100 s-1, the fish oil emulsion did not exhibit any gel properties or shear thinning. Fibrillation increased the particle size of the fish oil emulsion, whereas adding XG reduced the droplet size. The combination of RBP fibrillation and XG addition provided the highest encapsulation efficiency for fish oil. Fibrillation reduced the water activity and moisture content of the fish oil microcapsules. The anisotropy of the fibrils and the high viscosity of XG produced a layer of wrapping on the continuous heterogeneous surface of the freeze-dried powder particles. RBPF/XG microcapsules stored at 45 °C for 1 month had the lowest peroxide value and thiobarbituric acid value, the lowest surface oil content, and the lightest yellowness. These results suggest that the combination of RBPFs and XG provides better encapsulation and protective effects for fish oil microcapsules. Upon simulated digestion, the microcapsules containing XG and RBPFs exhibited a more favorable controlled release of free fatty acids. These findings indicate that microcapsules formed from emulsions co-stabilized by XG and RBPFs are suitable for encapsulating fish oil in functional foods.

Simultaneous overexpression of ∆6-, ∆12- and ∆9-desaturases enhanced the production of γ-linolenic acid in Mucor circinelloides WJ11.

Mucor circinelloides WJ11, an oleaginous filamentous fungus, produces 36% lipid of its cell dry weight when cultured in a high C/N ratio medium, however, the yield of γ-linolenic acid (GLA) is insufficient to make it competitive with other plant sources. To increase the GLA content in M. circinelloides WJ11, this fungus was engineered by overexpression of its key genes such as Δ6-, Δ12-, and Δ9-desaturases involved in GLA production. Firstly, we tried to overexpress two Δ6-desaturase isozymes to determine which one played important role in GLA synthesis. Secondly, Δ6-and Δ12-desaturase were co-overexpressed to check whether linoleic acid (LA), the precursor for GLA synthesis, is a limiting factor or not. Moreover, we tried to explore the effects of simultaneous overexpression of Δ6-, Δ12-, and Δ9-desaturases on GLA production. Our results showed that overexpression (1 gene) of DES61 promoted higher GLA content (21% of total fatty acids) while co-overexpressing (2 genes) DES61 and DES12 and simultaneous overexpressing (3 genes) DES61, DES12, and DES91 increased the GLA production of engineered strains by 1.5 folds and 1.9 folds compared to the control strain, respectively. This study provided more insights into GLA biosynthesis in oleaginous fungi and laid a foundation for further increase in GLA production into fungus such as M. circinelloides.