# Trepenoid Biosynthesis Dominates among Secondary Metabolite Clusters in _Mucoromycotina_ Genomes
## 3 Results and Discussion
### Predominance of Terpene Biosynthesis in Non-Dikaryu Genomes  
Terpenes are synthesised from isoprenyl diphosphates by terpene synthases. In fungi, terpenoids are predominantly produced from mevalonate. Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are condensed into products of diverse size by an isoprenyl diphosphate synthase (IDS) and further modified by terpene synthase (TPS) [44]. This condensation of two geranylgeranyl pyrophosphate (GGPP) units results in the formation of _cis_-phytoene, which is further isomerized to a _trans_-phytoene linear polyene structure that is the basic element for further formation of all Carotenes (or carotenoids in general). The subsequent activity of cyclases leads to the formation of rings on one or two ends of this chain, and the introduction of conjugated double bonds leads to the formation of the characteristic carotenoid structures (tetraterpenoids). The presence of conjugated double bonds is responsible for providing the characteristic yellow, orange or reddish colours of these hydrocarbons [45]. Apart from plants and bacteria, carotenoids are often present in some fungal lineages. Neurosporaxanthin produced by an ascomycetus fungus (_Neurospora crassa_) or astaxanthin synthesised by the basidiomycete yeast _Xanthophyllomyces dendorhous_ are good examples [46; 47]. However, \(\beta\)-carotene production seems to be the most widespread in the fungal kingdom and is especially consistent within _Mucoronycotina_. Its synthesis has been studied, among others, in _Phycomyces blackseleaanus_, _Blakeslea trispora_ and _Mucor cireincloides_[47]. All these different carotenoids in fungi can act as protecting agents against oxidative stress [48] or play a photoprotective role [49]. In _Mucoronycotina_, they are additionally involved in sexual reproduction, as carotenoids are precursors of trisprooric acid (C\({}_{18}\)), which are basic building blocks of sex pheromones in the sexual reproduction of _Mucorales_[50]. Therefore, the predominance of terpene biosynthesis in this group of fungi seems to manifest. [PERSON] et al. (2020) [39] described the dominance of terpene cyclases unique for this group of fungi. Our results support the importance of terpene biosynthesis in _Mucoronycotina_. Particularly, the presence of squalene- and geranylogenanyl phosphate synthase-coding genes (SQS, SQS_NDUF6 and GGPS) was observed throughout the phylogenetic tree of _Mucoronycotina_, except for the majority of _Rhizopus_ genomes, pointing out the probable loss of this character in this lineage or missing predictions due to a lack of dedicated gene annotation. SQS indicates the canonical squalene synthase found in fungal SMCs, whereas SQS_NDUF6 has similarity to NDUF46 (NDUF6, mitochondrial complex assembly factor 6), which is known to contain a nonfunctional squalene/phytoene synthase domain. Interestingly, the terpene biosynthesis-related genes were detected mainly within plant-related _Diversipandes_ and _Gomerales_, while carotenoid synthesis-related genes were observed mainly within saprotrophic _Mucorales_ (Figure 4). Although carotenoids belong to the tetraterpenoids, they form a well-characterised subgroup that, in several aspects, may be delimited and discussed separately. Indeed, the retention of a conserved genomic cluster can best be seen among terpenoid SMCs, where carotenoid biosynthesis likely selects for tight linkage of carB and carRA orthologous. The crucial enzymes responsible for the condensation (geranyl diphosphate synthase, GPPS; polyprenyl_synt, PF003480), phytoene and/or squalene synthase activity (SQS_PSY, PF00494), desaturation (phytoene desaturase, PDS; amino-oxidase, PF01593)) and cyclisation (lycopene cyclase activity of LCPS; no Pfam, IPR017825, TIGR03462) stepsare all accounted for the detected SMCs (see also Figure 4). Regarding the canonical terpene synthases, these occur in two major candidate families: homologues of lanosterol synthase (yeast ERG7, \(n\) = 33) and terpene synthase Type II homologues (\(n\) = 4).