Endophytic fungi are emerging as attractive producers of natural products with diverse bioactivities and novel structures. However, difficulties in the genetic manipulation of endophytic fungi limit the search of novel secondary metabolites. In this study, we improved the polyethylene glycol (PEG)-mediated protoplast transformation method by introducing the CRISPR/Cas9 system into endophytic fungus Pestalotiopsis fici. Using this approach, we performed genome editing such as site-specific gene insertion, dual-locus mutations, and long DNA fragment deletions in P. fici efficiently. The average efficiency for site-specific gene insertion and two-site gene editing was up to 48.0% and 44.4%, respectively. In addition, the genetic manipulation time with long DNA fragment (5–10 kb) deletion was greatly shortened to one week in comparison with traditional methods such as Agrobacterium tumefaciens-mediated transformation (ATMT). Taken together, the development of the CRISPR/Cas9 system in the endophytic fungus will accelerate the discovery of novel natural products and further biological study.

Fungal secondary metabolites (SMs) represent an invaluable source of therapeutic drugs. Genomics-based approaches to SM discovery have revealed a vast and largely untapped biosynthetic potential within fungal genomes. Here, we used the publicly available fungal genome sequences from the NCBI public database, as well as tools such as antiSMASH, BIG-SLiCE, etc., to analyze a total of 11,598 fungal genomes, identifying 293,926 biosynthetic gene clusters (BGCs), which were subsequently categorized into 26,825 gene cluster families (GCFs). It was discovered that only a tiny fraction, less than 1%, of these GCFs could be mapped to known natural products (NPs). Some GCFs that only contain a single BGC internally are crucial for the biodiversity of fungal biosynthesis. Evident patterns emerged from our analysis, revealing popular taxa as prominent sources of both actual and potential biosynthetic diversity. Our study also suggests that the genus rank distribution of GCF is generally consistent with NP diversity. It is noteworthy that genera Xylaria, Hypoxylon, Colletotrichum, Diaporthe, Nemania, and Calonectria appear to possess a higher potential for SM synthesis. In addition, 7213 BGCs match possible known compound structures, and homologous gene clusters of well-known drugs can be located in different genera, facilitating the development of derivatives that share structural similarity to these drugs and may potentially possess similar biological activity. Our study demonstrated the various types of fungi with mining potential, assisting researchers in prioritizing their research efforts and avoiding duplicate mining of known resources to further explore fungal NP producers.

Microbial genetic circuits are vital for regulating gene expression and synthesizing bioactive compounds. However, assessing their strength and timing, especially in multicellular fungi, remains challenging. Here, an advanced microfluidic platform is combined with a mathematical model enabling precise characterization of fungal gene regulatory circuits (GRCs) at the single‐cell level. Utilizing this platform, the expression intensity and timing of 30 transcription factor‐promoter combinations derived from two representative fungal GRCs, using the model fungus Aspergillus nidulans are determined. As a proof of concept, the selected GRC combination is utilized to successfully refactor the biosynthetic pathways of bioactive molecules, precisely control their production, and activate the expression of the silenced biosynthetic gene clusters (BGCs). This study provides insights into microbial gene regulation and highlights the potential of platform in fungal synthetic biology applications and the discovery of novel natural products.

Fungal secondary metabolism (SM) is highly correlated with physiological processes that are typically regulated by pleiotropic regulators. In this study, we purposefully altered PratfA, a crucial regulator associated with oxidative stress in Penicillium raistrickii CGMCC 3.1066. After the knockout of PratfA, a novel polyketide (PK) raistrilide A (1) and the known nonribosomal peptide (NRP) tunicoidine (2) subsequently disappeared. Notably, compound 1 is a rare octaketone derivative and contains two unsubstituted cis-double bonds, demonstrating its unique biosynthetic mechanism. The knockout of PratfA resulted in the disappearance of 1–2 and greatly increased the susceptibility of ΔPratfA mutant strain to oxidative stress, rendering it nearly impossible to survive in such environments. At present, the OE⸬PratfA strain showed no phenotypic or oxidative stress sensitivity differences compared to the wild-type strain. Our findings highlight that the oxidative-stress-related transcription factor (TF) PratfA influences SM pathways in P. raistrickii. The manipulation of regulatory factors can guide the discovery of novel natural products (NPs).

Fungal bioleaching, as a green and sustainable technology for metal recovery, demonstrates unique advantages in addressing growing global metal demand and utilizing secondary resources. This review systematically summarizes recent progress and technological advances in fungal bioleaching for metal recovery. Fungi solubilize metals through three core mechanisms, acidolysis, complexolysis, and redoxolysis, mediated by organic acids (citric, oxalic, gluconic), while cell wall functional groups enable biosorption. Most frequently used genera in modern bioleaching (Aspergillus, Penicillium, Trichoderma) have achieved remarkable recoveries from e-waste (Cu, Li, Co up to 100%), rare earth elements (consortia up to 76%), tailings, and spent catalysts. Optimization of key parameters (pH, temperature, pulp density) and application of intelligent strategies (response surface methodology, machine learning) have further enhanced leaching efficiency. Strain engineering and synthetic biology offer new avenues for constructing hyper-efficient strains. Despite remaining bottlenecks such as slow kinetics and scale-up difficulties, fungal bioleaching holds broad industrial prospects for advancing the circular metal economy and the green metallurgical transition.