# Trepenoid Biosynthesis Dominates among Secondary Metabolite Clusters in _Mucoromycotina_ Genomes
## 3 Results and Discussion
### Heterogeneity of NRPS/NRPS-Like Homologues  
Functional NRPS clusters harbour an adenylation (A) domain, a condensation (C) domain and a thioesterase or peptidyl carrier protein (T/PCP) [56]. Due to their similarity with PKS, as well as the frequent presence of PKS/NRPS hybrid core genes, there are often SMCs bearing the features of both NRPS and PKS clusters. In [PERSON] et al. (2020) [39], the abundance of NRPS/NRPS-like clusters among _Mucorales_ and _Mortierellales_ was shown, while PKS dominated the landscape of _Zopagales_ genomes. In our study, the abundance of aryl-forming reductase (NPS) was detected in the genomes of _Mucorales_, _Endogonales_ and _Diversisporales_. In the setting of NRPS and NRPS-like clusters, MCL-based clustering revealed the limitations of rule-based SMC detection: the NRPS/NRPS-like labels could easily overlap, as the sequences are frequently fragmented (due to, for example, incomplete gene models). The largest grouping of NRPS-like sequences (MCL0003, \(n\) = 116, with an additional 29 SMCs labelled as NRPS; see Supplementary Table S3) concerns Class I adenylate reductases, most likely orthologous to _Schizosaccharomyces pombe_ LYS2, which is an essential aminoadipate reductase involved in the primary metabolic pathway of lysine biosynthesis across fungi [57]. This observation is further corroborated by the AdenylPred results, which classified most of the MCL0003 sequences as \"bulky mainly phenyl derivatives aa\", a category which encompasses lysine in the predictor output. The moderate overlap in predictions between NRPS and NRPS-like predicted types resulted from the detection of a condensation domain in the candidate SMCs (e.g., due to matching of the conserved orthologoue group signature SMCOG11207 or the fatty acid ligase similarity mentioned earlier). An extended analysis of the putative LYS2 orthologologues confirms that these candidate SMCs occur predominantly in a single copy per genome (\(n\) = 130; see Figure 4), with apparent multiple copies present in the assemblies of seven isolates representing the _Rhizopus_, _Mucor_ and _Apophysomyces_ genera. Since out of a limited number of isolates lacking the LYS2 signature (\(n\) = 11), the majority concern the well-characterised species of _Rhizopus oryzae_ (\(n\) = 6) and _R. delemar_ (\(n\) = 2); these are more likely to arise from the incompleteness of the assemblies for these particular isolates. Taken together, the ubiquity, uniqueness and readily detectable sequence signature of candidate LYS2 clusters enabled us to conclude that the conserved involvement of Type I reductases in lysine biosynthesis is likely an ancestral conserved trait across _Mucoromycota_. Of the other subtypes of NRPS-like sequences, Type II/III adenylate-forming reductases [57] (MCL0001) were present across 127 assemblies, with the highest numbers of SMCs (total \(n\) = 304) in _Rhizopus microsprous_ (\(n\) = 14), _Gigaspora margaria_ (\(n\) = 10) and _Gigaspora rose_ (\(n\) = 8). The densest representation of NRPS-like SMCs was confined to members of _Mucorales_ (\(n\) = 251) and two representatives of _Diversisporales_. Since no examples of Type IV adenylate reductases were noted (no ferredoxin-NADP+ reductase FNR domain in the C-terminal position [57]), one can conclude that most likely the terminal domain swap leading to a division between Type IV (L-serine reductases) and Type III (aryl acid reductases) was most likely confined to _Dikarya_. Further characterisation, including phylogenetic reconstruction on improved gene models, is necessary to unambiguously resolve whether the analysed representatives belong to Clade II (L-tyrosine reductases; indicative of a _Dikarya_ specific domain swap) or III (indicative of domain swapped Type IV ancestral genes being lost in _Mucornycota_). However, a cursory BLASTP search and an approximate maximum likelihood reconstruction of the phylogenetic tree of adenylate-forming reductases with FastTree [34] strongly suggested the latter possibility (a monophyletic clade of Type III reductases).