# Trepenoid Biosynthesis Dominates among Secondary Metabolite Clusters in _Mucoromycotina_ Genomes
## 3 Results and Discussion
### Heterogeneity of NRPS/NRPS-Like Homologues
Prediction of substrate specificity on adenylate forming reductases has shown 'cinnamate and succinylbenzoate derivatives' to be the most frequent predicted class (\(n\) = 133 candidate SMCs). This AdenylPred class of adenylate-forming reductases encompasses enzymes involved in the production of coumarate, cinnamate, vanillate, caffeine and ferulate. The next most abundant groups of compounds were 'luciferin' (\(n\) = 57), fatty acids (\(n\) = 32; 'c13 through c17'), and salicate, 2,3-dihydroxybenzoate, anthranite, benzoate, naphthoate xanthenurate and quinolate (\(n\) = 30; 'aryl and biaryl derivatives'). Furthermore, 10 SMCs had adenylate-forming reductases predicted to utilise short organic acids ('c2 through c5') and aromatic amino acids and lysine ('bulky mainly phenyl derivatives'). Taken together, the AdenylPred results suggest the substantial diversity of reductase substrates across different aromatic organic compounds of importance. Among adenylation enzymes, we found that the _Gloomeromycotina_ are unique in coding for candidate SMCs containing fatty acid acyl-CoA AMP ligase (FAAL). It has been documented previously that their symbiotic relationship with plants includes the exchange not only of carbohydrates but also of lipids [58]. Pertinently, the _Gloomeromycotina_ are unable to produce fatty acids independently of the host plant. Apparently, they have lost Type I fatty acid synthase and retain only a mitochondrial Type II FAS synthetase of unknown molecular function. In stark contrast to NRPS-like adenylating enzymes, canonical nonribosomal peptide synthases were densely represented only in _Mortierella alipina_ (a number ranging from 19 to 25 across three representatives) and _Gigaspora_ (_G. margaria_, \(n\) = 13; _G.rosea_, \(n\) = 12). On basis of sequence similarity (core peptide synthase), we were able to unambiguously assign putative SMCs (GCA_001021685_13_glim, GCA_000240685_19_glim, GCA_000507065_21_glim) for the recently described malpicyclin biosynthesis [12], which was present across all three _Mortierella alipina_ isolates (ATCC 32222, B6842, CCTCC M207067) (Figure 5). Interestingly, this core SMC from _M. alipina_ ATCC 32222 and B6842 encompasses three putative accessory genes: a saccharide deacetylase (similar to known chitin deacetylase from SwissProt), a major facilitator superfamily transporter (MFS, sugar transporter homologue) and alpha-methylacyl-CoA racemase/epimerase (also matching carnitine dehydratase SMCOG in AntiSmash predictions). While, at first glance, only the racemase accessory gene is present in the _M. alipina_ CCTCC M207067 isolate, the 5'-terminal location of the SMC (relative to contig ends) suggests full biosynthetic capacity and ties with biosynthesis/transformation/efflux of glycoconjugates across _M. alipina_. AdenylPred prediction of small hydrophobic amino acid adenylation specificity fits well with the main substrate of leucine/valine residues incorporated into the cyclic peptides. The SMC is often accompanied by a zinc finger transcription factor-coding gene from the GATA zinc finger family (PF00320). The SMC is accompanied by two P-type ATPase genes and a putative toxin comprising a methylase and PIN-fold nuclease DUF1308 [59]. Proteins with such an architecture are numerous in _Gloomeromycotina_ (e.g., _Rhizophagus irregularis_ A0A21 IGJI1_9 GLOM) and _Zoopagomycota_ (_Thamucephaluspharospora_ A0A4P9 XIN5_9 FUNG) genomes and have not been characterised so far. An additional candidate mulpibaldin cluster (GCA_000507065_29_glim, _M. alpina_ B6842) was selected based on sequence similarity to the MalB malpibaldin synthetase protein sequence. However, the number of gaps suggested assembly or detection issues in this case, and no putative accessory genes were annotated. The neighbouring genes are rich in protein repeat domains (Ankirin, leucine-rich repeats) and disordered regions. The remaining NRPS SMCs identified in _Mortierellomycotina_ genomes likely produce distinct cyclic peptides not characterised so far.