*2.3. Origin Verification of Compound* **2** *by Biotransformation Assay*

Unexpected accumulation of compound **2** as a hydroxylated derivative of **1** raises the question on the origin of the hydroxylation activity, due to no corresponding enzymes for the related hydroxylation reaction. Inspired by a previous work by Li et al. [23], we postulated that endogenous enzymes from *A. nidulans* were also in charge of the conversion of **1** to **2**. Intrigued by this hypothesis, we conducted a feeding experiment of compound **1** in *A. nidulans* A1145. After four days feeding with compound **1**, compound **2** was clearly present in the culture extract after LC-MS analysis (Figure 3). This proved that *A. nidulans* can modify the initial polyketide product **1** by hydroxylation at the methyl (Figure 4). Unfortunately, no *A. nidulans* candidate enzymes could be anticipated for the process.

**Figure 4.** Biosynthetic pathway of **1** and **2** in *A. nidulans* A1145.

#### *2.4. Bioactivities of Compounds* **1** *and* **2**

In previous reports, compound **1** was tested for cytotoxic activities against the human cancer cell lines Hela, A549, HepG2 and the mouse lymphoma cell line L5178Y, antimicrobial activity against Gram positive (*Staphylococcus aureus* ATCC 25923 and *Bacillus subtilis* ATCC 6633) and Gram negative (*Escherichia coli* ATCC 25922 and *Pseudomonas aeruginosa* ATCC 27853) bacteria, *Candida albicans* ATCC 10231, and multidrug-resistant isolates from the environment [17,18,24], while compound **2** was evaluated for the cytotoxicity activities against a panel of cancer cell lines (A549, HL-60, K562 and L5178Y) [17,25]. All the above investigations proved to be inactive. In our research, the antimicrobial activities against MR-CNS (Methicillin-Resistant Coagulase-Negative *Staphylococci*), MRSA (Methicillin-Resistant *Staphylococcus Aureus*), *S. aureus*, *Acinetobacter baumannii*, *B. cereus*, *B. subtilis*, *P. aeruginosa*, and *C. albicans* were evaluated, but none of them presented an antimicrobial effect under the concentration of 30 μM. Meanwhile, the radical scavenging assay based on 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) was used to test the radical scavenging activity of compounds **1** and **2**, and they showed similar effects with ED50 values of 67.4 μM and 104.2 μM (the value of ascorbic acid was 12.6 μM as positive control).

#### **3. Discussion**

Coumarins and isocoumarins are a large family of lactonic natural products, abundant in various organisms including bacteria, fungi, lichens, liverworts, sponges, plants and insects [22,26]. The widespread distribution and possession of a broad spectrum of pharmacological activities, including antifungal, anti-inflammatory, cytotoxic, and antimicrobial properties, have led to the continuous discovery of novel isocoumarin compounds [22,27,28]. Structurally, there are six chemical active positions (C3 to C8) of isocoumarin, which make a substantial contribution to the formation of diverse chemical derivatives containing alkyl, halogen, heterocyclic, aryl, etc. Isocoumarins have garnered significant attention in total synthesis due to their utility as key intermediates in the production of valuable compounds, such as isoquinolines and isochromenes [27,29,30]. These diverse chemical substitution patterns significantly augment the structural complexity and broaden the range of biological and pharmacological activities of isocoumarins [31–33].

Several biosynthetic gene and gene clusters responsible for coumarins and isocoumarins have been discovered [23,32–35]. In fungi, isocoumarin derivatives are primarily derived from the polyketide pathways, which are typically catalyzed by nonreducing polyketide synthase (nrPKS), containing domains such as starter unit ACP transacylase (SAT), βketoacyl synthase (KS), acyl transferase (AT), product template (PT), acyl carrier protein (ACP), methyltransferase (MT), and thioesterase (TE) [22,23,32]. However, despite recent significant advancements in the chemical synthesis of isocoumarins, there are relatively few PKSs responsible for their biosynthesis that have been documented, compared to the abundance of isocoumarin derivatives. In fungi, the elucidated biosynthetic PKSs could be divided into two categories. The first class, represented by Pcr9304 and cla3, have no MT domain, which corresponds with the absence of methyls on C3,5,7, while only one report of the second class exists with C3,5-dimethyl directed by AcreC. To date, there is no biosynthetic enzyme report about C7-methyl isocoumarin derivatives.

Inspired by the uncommon carbon substituent at C7-methyl of **1** and **2** with raising reported activities [22,26], we performed a comparison towards reported PKSs synthesizing isocoumarin derivatives by phylogenetic analysis (Figure 5). The results demonstrated that SlPKS4 is close to AcreC, which also contains an MT domain compared to others. NCBI BLASTP at the amino acid level between SlPKS4 and AcreC shows a sequence identity of 36.89%. However, the reason why the MT domain directed SAM on different sites remains unknown.

Despite being located in a cluster, the *SlPKS4* gene is sufficient for compound **1** production. The reasons why the tailoring enzymes were ineffective are still unknown. We proposed that it is possibly because of the inactivation of the genes encoding tailoring enzymes during the rearrangements and breakages, or as a result of being flanked by potential transposons [36–38]. Another hypothesis was that the post-translational modification rules may differ among different host strains, lead to wrong enzyme structures, and ultimately became inactive. Moreover, we did comparative analysis between the gene cluster containing SlPKS4 and other biosynthetic gene clusters producing isocoumarin using clinker (Figure S7). The results suggested that our gene cluster was low and/or similar as a whole, and the tailoring enzymes were least conversed.

**Figure 5.** Phylogenetic tree analysis of SlPKS4 and reported PKSs synthesizing isocoumarin derivatives.
