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.

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.

Carotenoids 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. This 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. The 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.

The classification of Mucorales encompasses a collection of basal fungi that have traditionally demonstrated an aversion to modern genetic manipulation techniques. This aversion led to a scarcity of knowledge regarding their biology compared to other fungal groups. However, the emergence of mucormycosis, a fungal disease caused by Mucorales, has attracted the attention of the clinical field, mainly because available therapies are ineffective for decreasing the fatal outcome associated with the disease. This revitalized curiosity about Mucorales and mucormycosis, also encouraged by the recent COVID-19 pandemic, has spurred a significant and productive effort to uncover their mysteries in recent years. Here, we elaborate on the most remarkable breakthroughs related to the recently discovered genetic advances in Mucorales and mucormycosis. The utilization of a few genetic study models has enabled the identification of virulence factors in Mucorales that were previously described in other pathogens. More notably, recent investigations have identified novel genes and mechanisms controlling the pathogenic potential of Mucorales and their interactions with the host, providing fresh avenues to devise new strategies against mucormycosis. Finally, new study models are allowing virulence studies that were previously hampered in Mucorales, predicting a prolific future for the field.

The study of the Mucoralean fungi physiology is a neglected field that the lack of effective genetic tools has hampered in the past. However, the emerging fungal infection caused by these fungi, known as mucormycosis, has prompted many researchers to study the pathogenic potential of Mucorales. The main reasons for this current attraction to study mucormycosis are its high lethality, the lack of effective antifungal drugs, and its recent increased incidence. The most contemporary example of the emergence character of mucormycosis is the epidemics declared in several Asian countries as a direct consequence of the COVID-19 pandemic. Fortunately, this pressure to understand mucormycosis and develop new treatment strategies has encouraged the blossoming of new genetic techniques and methodologies. This review describes the history of genetic manipulation in Mucorales, highlighting the development of methods and how they allowed the main genetic studies in these fungi. Moreover, we have emphasized the recent development of new genetic models to study mucormycosis, a landmark in the field that will configure future research related to this disease.