*2.1. Bioinformatic Analysis of the Target nrPKS and the Gene Cluster in S. lamellicola HDN13-430*

To probe the metabolic potential of *S. lamellicola* HDN13-430, the whole-genome sequencing and analysis were performed. The prediction of secondary metabolites using antiSMASH indicated 12 PKSs, 16 NRPSs, 3 terpenes, 6 hybrids and 2 other types biosynthetic gene clusters (Figure S2). During analyzing the PKSs of strain *S. lamellicola* HDN13-430, an nrPKS which we termed as *SlPKS4* (Figure 2), exhibits low identities with known nrPKSs, while the highest similarities of 41.46% and 41.20% at the amino acid level were donated by pkbA [15] and andM [16], which are responsible for the biosynthesis of compounds 3-methylorsellinic and 3,5-dimethylorsellinic acid, respectively. The low-similarity PKS *SlPKS4*, located in a cluster with seven tailoring enzymes termed Sl4001-Sl4007, with proposed functionsas quinone oxidoreductase, dienelactone hydrolase, γ-glutamyl phosphate reductase, hypothetical protein, sulfide quinone reductase, threonine dehydratase, ketol-acid reductoisomerase, respectively (details in Table S2). The proposed functions of enzymes located in the cluster are all uncommon with a rare report in secondary metabolites biosynthesis. However, further analysis of gene transcription status by RT-PCR of six media based on OSMAC (one strain many compounds) and epigenetic regulation strategies, shows that the cluster including *SlPKS4* is totally silent under regular laboratory culture conditions (Figure S1). Also, there are no reports about the construction of a genetic operating system on the specie *S. lamellicola*, which prompts us to investigate the function by heterologous biosynthesis in *A. nidulans*.

**Figure 2.** Organization and proposed function of *SlPKS4* and tailoring genes.

#### *2.2. Heterologous Expression of the Gene Cluster and Elucidation of Compounds* **1** *and* **2**

To demonstrate the function of SlPKS4, a 9547 bp fragment containing the whole genomic sequence of SlPKS4, plus a downstream region of 526 bp containing the native terminator, were amplified via PCR and cloned into the expression vector pANU-*SlPKS4* by homologous recombination in *Saccharomyces cerevisiae*. The obtained construct, pANU-*SlPKS4*, was introduced into *A. nidulans* A1145 by polyethylene glycol (PEG)-mediated protoplast transformation. Integration transformants, including pANU-*SlPKS4*, were grown on liquid CD-Starch medium following selection by uridine and uracil autotrophy and subsequent confirmation by PCR amplification [17]. The cultures were extracted with ethyl acetate and analyzed by LC-MS for secondary metabolites. As shown in Figure 3, two additional peaks of compounds **1** and **2** were detected in the extract compared to the control strain containing the empty vector. The two compounds share similar UV spectra with absorption maxima at 240, 280 and 330 nm, together with [M + H]+ ions at *m/z* = 207.1 and 223.2, respectively, indicating similar structures and differences coming from hydroxylation (Figure S2). Following large-scale fermentation, isolation and structural elucidation by 1D NMR analysis (Tables S3 and S4 and Figures S8–S11) confirmed compounds **1** and **2** to be similanpyrone B and pestapyrone A, respectively [17,18]. The literature review concludes that both compounds belong to the group of isocoumarins.

**Figure 3.** HPLC analysis of the secondary metabolites in *A. nidulans* strains. UV absorptions at 280 nm are illustrated. HPLC full chromatogram of the original *A. nidulans* and the strain harboring SlPKS4 were provided to prevent the presence of compound **2** in original *A.nidulans* (Figure S5). HPLC analysis method: 5:95 to100:0 MeOH-H2O (with 0.1% trifluoroacetic acid), 40 min, 1 mL/min.

Similarly, the tailoring enzymes Sl4001-Sl4007 were separately cloned, constructed on expression plasmids and introduced into *A. nidulans* A1145 (Figure S4). Unexpectedly, after 4 days of culturing followed by extraction with ethyl acetate, no new compound, except **1** and **2**, was detected by LC-MS analysis (Figure 3). Double checking the gene transcription status by RT-PCR was performed, confirming that all seven tailoring enzymes, together with SlPKS4, were expressed properly (Figure S6), which exclude the possibility of unexpression.

Compound **1** has undergone investigation through chemical synthesis [19] and isotope labeling [20,21] since the 1980s, however, the enzyme responsible for the biosynthesis has not been reported until now. Our report about SlPKS4 is the first discovery of PKS responsible for compound **1**. Meanwhile, isocoumarins derivatives were generally discovered from the fungal genera *Penicillium*, *Ceratocystis*, *Fusarium*, *Artemisia*, *Aspergillus*, *Cladosporium*, *Oospora*, and *Hydrangea* [22]. To our best knowledge, there is no coumarins or isocoumarins reported from the fungal genus *Simplicillium,* so this is also the first time to prove that the fungal genus *Simplicillium* has the ability to produce isocoumarin derivatives.
