Dihomo-gamma linolenic acid (DGLA, 20:3, n-6) is the elongated product of Gamma linolenic acid (GLA, 18:3, n-6) catalyzed by the enzyme delta-6 elongase (D6E) or gamma linolenic acid elongase (GLELO). Construction of engineered oleaginous microbes have been attracting significant interest to produce DGLA because of its nutritional value and medicinal applications. Mucor circinelloides is a GLA producing filamentous fungus which can be a useful tool to produce DGLA. We have, therefore, overexpressed the D6E (GLELO) gene in this fungus to construct DGLA producing cell factory. To produce DGLA in M. circinelloides, homologous overexpression of D6E (GLELO) gene was analyzed. When the gene was overexpressed in M. circinelloides CBS277.49, up to 5.72% DGLA was produced in this strain. To our knowledge, this is the first report describing the overexpression of D6E (GLELO) gene in M. circinelloides to construct DGLA producing cell factory. A new scope for further research has been established by this work for improved production of DGLA in this fungus, specifically in its high lipid-producing strain, WJ11.

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.

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 β 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.

Mucorales is the largest and most well-studied order of the phylum Mucormycota and is known for its rapid growth rate and various industrial applications. The Mucorales fungi are a fascinating group of filamentous organisms with many uses in research and the industrial and medical fields. They are widely used biotechnological producers of various secondary metabolites and other value-added products. Certain members of Mucorales are extensively used as model organisms for genetic and molecular investigation and have extended our understanding of the metabolisms of other members of this order as well. Compared with other fungal species, our understanding of Mucoralean fungi is still in its infancy, which could be linked to their lack of effective genetic tools. However, recent advancements in molecular tools and approaches, such as the construction of recyclable markers, silencing vectors, and the CRISPR-Cas9-based gene-editing system, have helped us to modify the genomes of these model organisms. Multiple genetic modifications have been shown to generate valuable products on a large scale and helped us to understand the morphogenesis, basic biology, pathogenesis, and host–pathogen interactions of Mucoralean fungi. In this review, we discuss various conventional and modern genetic tools and approaches used for efficient gene modification in industrially important members of Mucorales.

The fungus,Mucor lusitanicus, is of great interest for microbial lipids, because of its ability to accumulate intracellular lipid using various carbon sources. The biosynthesis of fatty acid requires the reducing power NADPH, and acetyl-CoA, which is produced by the cleavage of citrate in cytosol. In this study, we employed different strategies to increase lipid accumulation in the low lipid-producing fungi via metabolic engineering technology. Hence, we constructed the engineered strain ofM. lusitanicusCBS 277.49 by using malate transporter (mt) and 2-oxoglutarate: malate antiporter (sodit)fromM. circinelloidesWJ11. In comparison with the control strain, the lipid content of the overexpressed strains ofmtandsoditgenes were increased by 24.6 and 33.8%, respectively. These results showed thatmtandsoditcan affect the distribution of malate in mitochondria and cytosol, provide the substrates for the synthesis of citrate in the mitochondria, and accelerate the transfer of citrate from mitochondria to cytosol, which could play a significant regulatory role in fatty acid synthesis leading to lipids over accumulation.

Eicosatetraenoic acid (ETA, 20:4, ω-3) is the desaturation product of dihomo-gamma linolenic acid (DGLA, 20:3, ω-6) catalyzed by delta-17 desaturase, which is considered as a healthy product that helps to lower risk of heart diseases. The oleaginous filamentous fungus, Mucor circinelloides, has been used for a long time as a model micro-organism for GLA production at industrial scales. However, M. circinelloides lacks the key enzymes to synthesize C20 polyunsaturated fatty acids (PUFAs). M. circinelloides could produce DGLA by overexpressing the D6E(GLELO) gene, which could be a useful tool to produce ETA due to the availability of established genetic manipulation tools. Therefore, in this study, delta-17 desaturase (PpD17 and PaD17) genes from Phytophthora parasitica and Pythium aphanidermatum, respectively, were introduced into M. circinelloides to construct an ETA-producing cell factory. Our results showed that the PaD17 and PpD17 overexpression strains’ biomass increased by 25.98 and 23.34 g/L (39.98 and 25.75%), respectively, compared with the control strain. Meanwhile, the lipid contents of the recombinant strains also increased and reached up to 28.88% in Mc-PaD17 and 30.95% in Mc-PpD17, respectively, compared with the control strain (23.38% in Mc-2076). The RT-qPCR results showed that overexpression of delta-17 desaturase genes promoted the expression of cme2, fas2, and D6E, thereby contributing to lipid biosynthesis in M. circinelloides. Meanwhile, the content of ETA reached up to 1.95%, and the yield of ETA was up to 114.69 mg/L in PpD17 overexpression mutants at 96 h. This study provided the first report on the construction of an ETA-producing cell factory by heterologous overexpression of the PpD17 gene in M. circinelloides, which established a new scope for further research in the production of ETA in oleaginous fungi.