*2.4. Pathogenic Cryptococcal Species Have the Highest CYP Diversity*

CYP diversity analysis revealed that pathogenic cryptococcal species, along with non-pathogenic *C. wieringae* and the fungal parasite *N. encephela* UCDFST 68-887.2, have 100% CYP diversity in their genomes (Figure 5 and Supplementary Dataset 2, sheet 3). *Tremellomycetes* such as *C. curvatus*, *C. amylolentus* CBS 6273 and *T. asahii* var. *asahii* strains had the lowest CYP diversity percentage. This is due to the blooming of CYP5139 members in their genome (Supplementary Dataset 2, sheet 1). The highest CYP diversity observed in pathogenic cryptococcal species is perfectly matched with species belonging to the fungal subphylum *Saccharomycotina* [38]. One commonality can be found between the species belonging to *Tremellomycetes* and *Saccharomycotina*: It can be assumed that some species of *Tremellomycetes* lost CYPs, compared to their counterparts, which may be due to the adaptation to use simple carbon sources present in the host, as observed for species of *Saccharomycotina*, where the loss of CYPs in response to the adaptation to use simpler carbon sources was observed [38].

**Figure 5.** CYP diversity percentage analysis in *Tremellomycetes*.

#### *2.5. Most of the CYPs from the Species of Tremellomycetes Are Orphans with No Known Function*

Among CYPs from the species of *Tremellomycetes*, CYP51F1 of *C. neoformans* has been shown to be involved in 14α-demethylation of lanosterol [30] and the *CYP51F1* gene was cloned from *T. asahii* ATCC MYA-1296 = OMU239 = TIMM4014 [27]. Apart from CYP51F1, some of the CYPs' functions can be predicted based on characterized homolog CYPs. CYP61 family members are involved in membrane ergosterol biosynthesis where they catalyze C-22 sterol desaturase activity [43]. CYP505 family members are involved in oxidation of fatty acids [44]. CYP504 family members are involved in conversion of phenyl acetate to 2-hydroxyphenylacetate [45]. CYP53 family members are involved in detoxification of toxic molecules, including benzoate and its derived compounds [46–48]. The primary function of CYP53 is the conversion of benzoate to *para*-hydroxy-benzoate. A study reported that CYP53 could be an alternative antifungal drug target in view of its critical role in fungal organisms [49]. It is interesting to note that this CYP family is only present in three species belonging to the genus *Trichosporon* (Figure 4 and Supplementary Dataset 2, sheet 1). CYP55 family members are involved in

the reduction of nitric oxide (NO) to nitrous oxide (N2O) [50,51]. It is interesting to note that in addition to CYP53, CYP55 family members are also found in two species belonging to the genus *Trichosporon* (Figure 4 and Supplementary Dataset 2, sheet 1). Apart from the CYP families listed above, the rest of the CYPs found in species of *Tremellomycetes* are orphans.
