The order Mucorales is a group of ancient fungi with limited tools for gene manipulation. The main consequence of this manipulation unwillingness is the limited knowledge about its biology compared to other fungal groups. However, the emerging of mucormycosis, a fungal infection caused by Mucorales, is attracting the medical spotlight in recent years because the treatments available are not efficient in reducing the high mortality associated with this disease. The result of this renewed interest in Mucorales and mucormycosis is an extraordinarily productive effort to unveil their secrets during the last decade. In this review, we describe the most compelling advances related to the genetic study of virulence factors, pathways, and molecular mechanisms developed in these years. The use of a few genetic study models has allowed the characterization of virulence factors in Mucorales that were previously described in other pathogens, such as the uptake iron systems, the mechanisms of dimorphism, and azole resistances. More importantly, recent studies are identifying new genes and mechanisms controlling the pathogenic potential of Mucorales and their interactions with the host, offering new alternatives to develop specific strategies against mucormycosis.Genes 2020, 11, 317 2 of 17 Mucorales are a neglected phylogenetic group compared to others such as Ascomycetes and Basidiomycetes. The limited knowledge about the genetics of Mucorales is a consequence of the restricted tools for gene manipulation, as most of them cannot be transformed. However, DNA can be introduced in Mucor circinelloides, Rhizopus delemar, and Rhizopus oryzae [6,7]. These genetic models and the alarm raised for the emerging cases of mucormycosis are attracting the interest of the scientific community. Thus, the last decade has produced several studies related to genes, pathways, and mechanisms showing a direct connection with virulence in Mucorales [8,9]. One of the most studied mechanisms has been the process of gene silencing or RNA interfering (RNAi) in M. circinelloides [10]. After the dissection of the gene silencing machinery, the knowledge of this mechanism allowed the unveiling of a new and particular type of antifungal resistance mediated by temporal epigenetic changes [11]. In addition, the applied use of gene silencing enabled the development of functional genomics techniques, which have been used for the identification of several new virulence factors [12]. Along with silencing, gene disruption driven by homologous recombination has also allowed the study of the particular role in M. circinelloides of virulence factors identified in other fungi, such as the role of a high-affinity iron uptake mechanism, the protein family of CotH, and the calcineurin pathway. Moreover, the implementation of the new omics technologies has produced a long list of candidate genes not previously related to virulence, becoming promising targets for the development of new treatments against mucormycosis. Finally, the diversity of molecular and cell methodolo...

Fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, NADPH, and the precursor of fatty acids, acetyl-CoA, which is generated in the cytosol being produced by ATP: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. This transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (Fig. 1). The role of malate transporter in lipid accumulation in oleaginous fungi is not fully understood, however. Therefore, the expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70 % (from 13 to 22 % dry cell weight, CDW), whereas the lipid content in mt knockout mutant decreased about 27 % (from 13 to 9.5 % CDW) compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain and increased in the mt knockout strain compared with the wild-type strain. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.

BackgroundCarotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. They also have important industrial applications as food colourants, animal feed additives and nutraceuticals. Some of them, such as β-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. The mucoral Blakeslea trispora is used by the industry to produce β-carotene, although optimisation of production by molecular genetic engineering is unfeasible. However, the phylogenetically closely related Mucor circinelloides, which is also able to accumulate β-carotene, possesses a vast collection of genetic tools with which to manipulate its genome.ResultsThis work combines classical forward and modern reverse genetic techniques to deepen the regulation of carotenoid synthesis and generate candidate strains for biotechnological production of β-carotene. Mutagenesis followed by screening for mutants with altered colour in the dark and/or in light led to the isolation of 26 mutants that, together with eight previously isolated mutants, have been analysed in this work. Although most of the mutants harboured mutations in known structural and regulatory carotenogenic genes, eight of them lacked mutations in those genes. Whole-genome sequencing of six of these strains revealed the presence of many mutations throughout their genomes, which makes identification of the mutation that produced the phenotype difficult. However, deletion of the crgA gene, a well-known repressor of carotenoid biosynthesis in M. circinelloides, in two mutants (MU206 and MU218) with high levels of β-carotene resulted in a further increase in β-carotene content to differing extents with respect to the crgA single-null strain; in particular, one strain derived from MU218 was able to accumulate up to 4 mg/g of β-carotene. The additive effect of crgA deletion and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA.ConclusionsThe use of a mucoral model such as M. circinelloides can allow the identification of the regulatory mechanisms that control carotenoid biosynthesis, which can then be manipulated to generate tailored strains of biotechnological interest. Mutants in the repressor crgA and in the newly identified regulatory mechanism generated in this work accumulate high levels of β-carotene and are candidates for further improvements in biotechnological β-carotene production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0493-8) contains supplementary material, which is available to authorized users.

RNA interference (RNAi) was discovered at the end of last millennium, changing the way scientists understood regulation of gene expression. Within the following two decades, a variety of different RNAi mechanisms were found in eukaryotes, reflecting the evolutive diversity that RNAi entails. The essential silencing mechanism consists of an RNase III enzyme called Dicer that cleaves double-stranded RNA (dsRNA) generating small interfering RNAs (siRNAs), a hallmark of RNAi. These siRNAs are loaded into the RNA-induced silencing complex (RISC) triggering the cleavage of complementary messenger RNAs by the Argonaute protein, the main component of the complex. Consequently, the expression of target genes is silenced. This mechanism has been thoroughly studied in fungi due to their proximity to the animal phylum and the conservation of the RNAi mechanism from lower to higher eukaryotes. However, the role and even the presence of RNAi differ across the fungal kingdom, as it has evolved adapting to the particularities and needs of each species. Fungi have exploited RNAi to regulate a variety of cell activities as different as defense against exogenous and potentially harmful DNA, genome integrity, development, drug tolerance, or virulence. This pathway has offered versatility to fungi through evolution, favoring the enormous diversity this kingdom comprises.

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 "2oxoglutarate: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.