**1. Introduction**

Fungi have proven to be a tremendous source of new bioactive lead compounds with thousands of bioactive compounds isolated [1,2]. Meanwhile, whole-genome sequencing data revealed that the number of biosynthetic gene clusters encoded in fungi is much larger than the types of natural products isolated, which indicates that a major portion of biosynthetic gene clusters are still silent or poorly expressed [3,4]. To activate these silent gene clusters and increase the silent metabolic potential, a variety of techniques have been developed, including epigenetics regulation, co-culture, precursor feeding, heterologous expression, changing fermentation parameters and ribosome engineering, etc. [5,6].

Among these, heterologous expression has unique advantages, especially to achieve the de novo biosynthesis of compounds in a heterologous host, which benefits from genetic tractability, short life-cycle, and high bio-safety [7]. As for the activation of silent gene clusters in fungi, heterologous expression shows special superiority, such as: (1) it is more controllable compared to other activation methods, especially, the activation is orientated instead of randomly; (2) the ideal chassis cells with simple metabolite backgrounds make it easy to perform the isolation of targeted compounds; (3) it is still effective without regulators, selective markers, or strain genetic operating system, which is frequently the major obstacle in non-model fungi [8–10].

The fungal specie, *Simplicillium lamellicola*, has great ecological and commercial importance due to the exceptional bioactivities, particularly in microbial biopesticide [11–14].

**Citation:** Yu, J.; Liu, X.; Ma, C.; Li, C.; Zhang, Y.; Che, Q.; Zhang, G.; Zhu, T.; Li, D. Activation of a Silent Polyketide Synthase SlPKS4 Encoding the C7-Methylated Isocoumarin in a Marine-Derived Fungus *Simplicillium lamellicola* HDN13-430. *Mar. Drugs* **2023**, *21*, 490. https://doi.org/10.3390/ md21090490

Academic Editor: Georg Pohnert

Received: 24 August 2023 Revised: 6 September 2023 Accepted: 12 September 2023 Published: 13 September 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

However, to our best knowledge, four new compounds, reported in the specie (Figure 1), indicate a great potential for bioactive secondary compounds from *S. lamellicola*. Isolated from a marine sediment collected in Pritz Bay, *S. lamellicola* HDN13-430 was the first strain of this specie isolated from Antactica, which made the genome mining worthwhile. During our ongoing genome mining work on the fungal strain *S. lamellicola* HDN13-430, a nonreducing polyketide synthase (nrPKS), termed SlPKS4, attracts our attention. SIPKS4 showed a low sequence identity to other PKSs. With the expression test of cDNA, the nrPKS, together with the gene cluster, were proven to be completely silent under regular laboratory conditions (Figure S1). Due to the lack of regulators in the native strain, the following heterologous expression of SlPKS4 in *Aspergillus nidulans* lead to the yield of two isocoumarin derivatives: similanpyrone B (**1**) and pestapyrone A (**2**). Compounds **1** and **2** showed radical scavenging activities, while no activity in the antibacterial bioassays was observed. Although compounds **1** and **2** were first described more than 50 years ago, the biosynthetic process is still not entirely understood. We proposed the biosynthetic pathway of compounds **1** and **2**, and conducted phylogenetic analysis with other PKSs responsible for the synthesis of isocoumarin derivatives.

**Figure 1.** Bioactive compounds reported from *S. lamellicola*.

#### **2. Results**
