**5. Conclusions**

In summary, guided by the bioinformatic analysis of *S. lamellicola* HDN13-430, we successfully activated the silent nrPKS gene *SlPKS4* in *A. nidulans*, and proved its function as an isocoumarin synthase. Furthermore, we demonstrated by feeding experiment that **2** are modification products of **1** by uncharacterized endogenous host enzymes. Based on the DPPH scavenging assay, Compounds **1** and **2** showed moderate radical scavenging activities with ED50 values of 67.4 μM and 104.2 μM, respectively, which was the first time for this to be measured. Our results provide one additional example that the products of a heterologous expressed gene can be further converted by host enzymes. Furthermore, this is the first report of nrPKS responsible for the biosynthesis of compound **1**. This is also the first report that the fungal specie *S. lamellicola* could produce isocoumarin derivatives, which expands the production methods of isocoumarins compounds.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/md21090490/s1, Table S1: The primers used in this study; Table S2: Analysis of gene cluster; Table S3: NMR data of **1** in DMSO-*d*6; Table S4: NMR data of **2** in CD3OD; Figure S1: RT-PCR analysis for gene transcription status. The results showed that all seven tailoring enzymes together with SlPKS4 were totally silent under 6 regular laboratory conditions; Figure S2: AntiSMASH analysis results of the genome of the strain *S. lamellicola* HDN13-430; Figure S3: UV absorptions of compounds **1** and **2**; Figure S4: Maps of the vectors pANU-*SlPKS4*, pANU-*SlPKS4*+*Sl4004*, pANR-*Sl4001+5+7* and pANP-*Sl4002+3+6*; Figure S5: HPLC full chromatogram of the original *A. nidulans* and the strain harboring SlPKS4. The results prevent the presence of compound **2** in original *A.nidulans*; Figure S6: RT-PCR analysis for gene transcription status. The results showed that all seven tailoring enzymes together with SlPKS4 were expressed properly; Figure S7: Comparative analysis between the gene cluster and other isocoumarin BGCs by clinker; Figure S8: 1H NMR (500 MHz, DMSO-*d*6) spectrum of compound **1**; Figure S9: 13C NMR (125 MHz, DMSO-*d*6) spectrum of compound **1**; Figure S10: 1H NMR (500 MHz, CD3OD) spectrum of compound **2**; Figure S11: 13C NMR (125 MHz, CD3OD) spectrum of compound **2**. Gene Sequence of *SlPKS4.*

**Author Contributions:** The contributions of the respective authors are as follows: J.Y. drafted the work, constructed the plasmids and performed the fermentation, extraction, as well as the isolation. X.L., C.M. and Y.Z. were involved in the fermentation, extraction, elucidation of the constituents and bioinformatic analysis. J.Y., X.L. and C.L. performed the biological evaluations. Q.C. and G.Z. contributed to checking and confirming all the procedures of the isolation and identification. T.Z. and D.L. designed the study, supervised the laboratory work, and contributed to the critical reading of the manuscript, and was also involved in the structural determination and bioactivity elucidation. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (U1906212, 81991522), Qingdao Marine Science and Technology Center (2022QNLM030003), the Fundamental Research Funds for the Central Universities (202172002 and 202262015), Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City (2021CXLH0012), Taishan Scholar Youth Expert Program in Shandong Province (tsqn 201812021, tsqn 202103153), Major Basic Research Programs of Natural Science Foundation of Shandong Province (ZR2021ZD28).

**Data Availability Statement:** The gene sequence of *SlPKS4* was uploaded to GeneBank (Accession Number: OR519897).

**Acknowledgments:** The authors recognize material contributions from Yi Tang (University of California, Los Angeles) for providing plasmids.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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