*3.4. Secondary Metabolite Biosynthesis Clusters*

Fungi produce numerous secondary metabolites, which have a multitude of roles in cellular processes, such as transcription and development [41]. Many of these compounds have significant applications in the medical field (e.g., antibiotics and antitumor drugs), as well as in the agriculture sector (e.g., insecticides). Based on recent genome sequencing results, the ability of fungi to produce secondary metabolites has been largely underestimated, as the gene clusters associated with secondary metabolite biosynthesis are not expressed under laboratory growth conditions [10].

The biosynthesis gene clusters (BGCs) identified using antiSMASH v6.0 were classified according to their types, and BGCs assigned to the production of a certain product are described in Figure 3. Only 14 BGCs within five classifications were found in the genome of *A*. *resinae* KUC3009. The number of identified BGCs was significantly lower than that of other common ascomycetes. For reference, the number of predicted BGCs for *Penicillium* and *Aspergillus* ranges between 29 and 85 [36,42,43]. Here, only five clusters were associated with the production of certain metabolites: 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), neurosporin A, brefeldin, phomopsins, and asperphenamate. One particular BGC in Type 3 polyketide synthase (T3-PKS) is predicted to produce phomopsins, and the gene exhibited a 100% similarity with that of *Phomopsis leptostromiformis*. Notably, phomopsins are a group of hexapeptide mycotoxins with potent antimitotic activity and, therefore, represent promising antitumor agents [44]. Beyond computational analysis, additional studies to prove the gene's function are needed for using the strain as a source of antitumor production.

**Figure 3.** All identified biosynthetic gene clusters (BGCs) in the genome of *A. resinae* KUC3009 and their predicted assigned product. Values in parentheses indicate the similarity with a known cluster.

Our antiSMASH v6.0 analyses indicated that the genome of *A. resinae* retains T1-PKS BGC to produce 1,3,6,8-THN (Figure 3), which is the intermediate metabolite of the dihydroxy naphthalene (DHN)–melanin synthesis pathway (Figure 4A). This T1-PKS BGC of *A. resinae* exhibited a 100% similarity with that of *Bipolaris oryzae* and *Nodulisporium* sp. ATCC74245 (Figure 4B). These modules typically contain conserved domains that are comprised of acyl-carrier protein transacylase (SAT), β-ketoacyl synthase (KS), acyltransferase (AT), two acyl-carrier proteins (ACPs), and thioesterase (TE) (Figure 4C) [45,46].

**Figure 4.** (**A**) Putative scytalone synthesis pathway of *A*. *resinae* KUC3009 (**B**) 1,3,6,8-THN BGC alignment between *A. resinae* and previously analyzed species. (**C**) Domain structure of the T1-PKS gene, consisting of an acyl-carrier protein transacylase (SAT) domain in gray, a β-ketoacyl synthase (KS) domain shown in green, an acyltransferase (AT) domain in pink, two acyl carrier protein (ACP) domains in blue, and a thioesterase (TE) domain in purple. (**D**) Comparison of flaviolin content in *A. resinae* mycelia cultured on PDA and tricyclazole-supplemented PDA (100 μg/mL). (**E**) Comparison of morphological properties of *A. resinae* cultured on PDA and tricyclazole-supplemented PDA (100 μg/mL).

> The T1-PKS gene cluster associated with 1,3,6,8-THN synthesis is predicted to be involved in the scytalone synthesis but not with DHN-melanin synthesis (Figure 3). Through genome annotation analysis, we confirmed that complete putative tetra-hydroxynaphthalene reductase (4-HNR) coding genes were present in the genome, whereas scytalone dehydratase and tri-hydroxynaphthalene reductase (3-HNR) coding genes were not identified. For reference, 1,3,6,8-THN molecule goes through a series of catalytic reactions to form the final product (1,8-DHN) aided by 4-HNR, scytalone dehydratase, and 3-HNR, serially. Finally, the DHN molecules are polymerized to form DHN-melanin using oxidation enzymes, such as laccase or other phenol oxidases [47,48].

> A series of biochemical evidence supports the existence of the T1-PKS gene associated with 1,3,6,8-THN and 4-HNR genes. Given that flaviolin is the auto-oxidative product of 1,3,6,8-THN, its detection in mycelia suggests that T1-PKS genes produce 1,3,6,8-THN [49]. Additionally, the amounts of flaviolin increased when the 4-HNR enzyme is inhibited [50]. We then sought to detect flaviolin and monitor its concentration after tricyclazole treatment to confirm whether our results were consistent with previous literature. GC–MS analyses confirmed the existence of flaviolin in mycelia cultured on PDA. Moreover, the flaviolin concentration in mycelia exhibited a 1.2-fold increase when cultured on PDA supplemented with 100 μg/mL of tricyclazole (Figure 4D).

> The study with tricyclazole inhibition assays supported that 1,3,6,8-THN BGC from *A*. *resinae* is not involved in the DHN melanin formation. As tricyclazole inhibits DHNmelanin formation by repressing activity of 3-HNR as well as 4-HNR, fungal pigmentation with DHN-melanin was also inhibited when cultured with tricyclazole [50]. When cultured on tricyclazole-supplemented media, the effect of tricyclazole on pigment formation of

*A*. *resinae* was poor, suggesting the pigmentation is not related to DHN-melanin synthesis. However, the gray-brown color of *A*. *resinae* cultures slightly changed to reddish-brown, which was likely due to an accumulation of shunt products (Figure 4E).
