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Lipid biosynthesis is a significant metabolic response to nitrogen starvation in oleaginous fungi. The oleaginous fungus Mucor circinelloides copes with nitrogen stress by degrading AMP through AMP deaminase (AMPD). However, the mechanism of AMPD in regulating lipogenesis remains largely unclear. To elucidate the mechanism of AMPD in lipid synthesis in this M. circinelloides, we identified two genes (ampd1 and ampd2) encoding AMPD and constructed an ampd double knockout mutant. The engineered M. circinelloides strain elevated cell growth and lipid accumulation, as well as the content of oleic acid (OA) and gamma-linolenic acid (GLA). In addition to the expected increase in transcription levels of genes associated with lipid and TAG synthesis, we observed suppression of lipid degradation and reduced amino acid biosynthesis. This suggested that the deletion of AMPD genes induces the redirection of carbon towards lipid synthesis pathways. Moreover, the pathways related to nitrogen metabolism, including nitrogen assimilation and purine metabolism (especially energy level), were also affected in order to maintain homeostasis. Further analysis discovered that the transcription factors (TFs) related to lipid accumulation were also regulated. This study provides new insights into lipid biosynthesis in M. circinelloides, indicating that the trigger for lipid accumulation is not entirely AMPD-dependent and suggest that there may be additional mechanisms involved in the initiation of lipogenesis., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In the oleaginous fungus Mucor circinelloides, lipid accumulation is regulated by nitrogen metabolism, which is regulated by the areA gene, a member of the GATA zinc finger transporter family and a major regulator for nitrogen metabolism. However, the role of areA in lipid accumulation in this fungus has not been reported. In order to explore the regulatory effect of areA gene on nitrogen metabolism and lipid accumulation in M. circinelloides, we constructed areA gene knockout and overexpression strains. Then, the recombinant strains were cultured and their biochemical indexes were measured. Simultaneously, transcriptomic studies on the recombinant strains were conducted to infer the regulatory mechanism of areA. The results showed that the areA knockout strain accumulated more lipid, which is 42 % higher than the control. While the areA overexpressing strain obtained the higher biomass accumulation (23 g/L) and used up the nitrogen source in the medium earlier than the control strain and knockout strain. Transcriptome data analysis showed that nr and nit-6 genes related to nitrogen metabolism were up-regulated. And the expression levels of key genes acc and aclY were higher in the areA knockout strain than others, which was positively correlated with the increased lipid accumulation. In addition, in knockout strains, protein catabolism tended to provide substrates for the lipid production, and the expression levels of the related genes were also higher than others. These results indicated that the areA gene not only controls the transcription level of genes related to nitrogen metabolism but also affects lipid accumulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Linolenic acid (LA) is gaining more interest within the scientific community. This is because it has a potential medical role in reducing the risk of inflammation, carcinogenesis, atherosclerosis and diabetes and is a valuable nutraceutical for human health. The oleaginous fungus Mucor circinelloides produces a high lipid content (36%), including valuable polyunsaturated fatty acids (PUFAs). However, the critical step in which oleic acid (OA) is converted into LA is not efficient at supplying enough substrates for PUFA synthesis. Hence, we propose a method to increase LA production based on genetic engineering. The overexpression of the Δ12-desaturase gene from M. circinelloides and Mortierella alpina increased the LA content and improved the lipid accumulation (from 14.9% to 21.6% in the Δ12-desaturase gene of the M. circinelloides overexpressing strain (Mc-D12MC) and from 14.9% to 18.7% in the Δ12-desaturase gene of M. alpina overexpressing strain (Mc-D12MA)). Additionally, the up-regulated expression levels of these genes targeted the genes involved in NADPH production, implying that the elevated Δ12-desaturase gene may function as a critical regulator of NADPH and lipid synthesis in M. circinelloides . This study provides the first evidence to support the design of metabolic engineering related to LA and PUFA production in M. circinelloides for potential industrial applications.

Malate as an important intermediate metabolite, its subcellular location, and concentration have a significant impact on fungal lipid metabolism. Previous studies showed that the mitochondrial malate transporter plays an important role in lipid accumulation in Mucor circinelloides by manipulating intracellular malate concentration. However, the role of plasma membrane malate transporters in oleaginous fungi remains unexplored. Therefore, in this work, two plasma membrane malate transporters "2-oxoglutarate:malate antiporters" (named SoDIT-a and SoDIT-b) of M. circinelloides WJ11 were deleted, and the consequences in growth capacity, lipid accumulation, and metabolism were analyzed. The results showed that deletion of sodit-a or/and sodit-b reduced the extracellular malate, confirming that the products of both genes participate in malate transportation. In parallel, the lipid contents in mutants increased approximately 10-40% higher than that in the control strain, suggesting that the defect in plasma membrane malate transport results in an increase of malate available for lipid biosynthesis. Furthermore, transcriptional analysis showed that the expression levels of multiple key genes involved in the lipid biosynthesis were also increased in the knockout mutants. To the best of our knowledge, this is the first report that demonstrated the association between plasma membrane malate transporters and lipid accumulation in M. circinelloides .

The carotene producer Mucor circinelloides is the fungus within the Mucoromycota phylum with the widest repertoire of molecular tools to manipulate its genome. The initial development of an effective procedure for genetic transformation and later improvements have resulted in an expansion of available tools, which include gene replacement, inactivation of gene expression by RNA silencing, gene overexpression, and functional genomics. Moreover, sequencing of its genome has given a definitive boost to these techniques making attainable the study of genes involved in many physiological or developmental processes, including carotenoid biosynthesis. Here, we describe in detail the latest molecular techniques currently used in M. circinelloides that have made it a valuable model for studying gene function within its phylum.

Objectives: To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation., Results: Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains., Conclusions: A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The carotene producer fungus Mucor circinelloides is the zygomycete more amenable to genetic manipulations by using molecular tools. Since the initial development of an effective procedure of genetic transformation, more than two decades ago, the availability of new molecular approaches such as gene replacement techniques and gene expression inactivation by RNA silencing, in addition to the sequencing of its genome, has made Mucor a valuable organism for the study of a number of processes. Here we describe in detail the main techniques and methods currently used to manipulate M. circinelloides, including transformation, gene replacement, gene silencing, RNAi, and immunoprecipitation.

Molecular approaches to study the biology of the zygomycete Mucor circinelloides depend mainly on the existence of a polyethylene glycol-based transformation method, which is one of the most efficient in zygomycete fungi. However, the poor reliability and low transformation rates of this method are major obstacles in the molecular study of a number of biological processes. This paper describes an easy and reliable method to transform M. circinelloides protoplasts by electroporation. A high-voltage pulse of 25μF capacitance, 400Ω resistance, and 4kV/cm field strength were seen to be the optimal electrical conditions for delivering DNA into M. circinelloides protoplasts. Under these electrical conditions, successful transformations were carried out with several self-replicative plasmid and strain combinations, producing up to more than 500 transformants per μg DNA. Targeted DNA integration of a transgene (atfA gene of Acinetobacter baylyi) in a particular locus (carRP) was also achieved. This transformation method will considerably facilitate in-depth molecular genetic studies of the biology of this fungus., (Copyright © 2011 Elsevier B.V. All rights reserved.)