Depsides and depsidones have attracted attention for biosynthetic studies due to their broad biological activities and structural diversity. Previous structure‒activity relationships indicated that triple halogenated depsidones display the best anti-pathogenic activity. However, the gene cluster and the tailoring steps responsible for halogenated depsidone nornidulin (3) remain enigmatic. In this study, we disclosed the complete biosynthetic pathway of the halogenated depsidone through in vivo gene disruption, heterologous expression and in vitro biochemical experiments. We demonstrated an unusual depside skeleton biosynthesis process mediated by both highly-reducing polyketide synthase and non-reducing polyketide synthase, which is distinct from the common depside skeleton biosynthesis. This skeleton was subsequently modified by two in-cluster enzymes DepG and DepF for the ether bond formation and decarboxylation, respectively. In addition, the decarboxylase DepF exhibited substrate promiscuity for different scaffold substrates. Finally, and interestingly, we discovered a halogenase encoded remotely from the biosynthetic gene cluster, which catalyzes triple-halogenation to produce the active end product nornidulin (3). These discoveries provide new insights for further understanding the biosynthesis of depsidones and their derivatives.

Marine-derived fungi are emerging as attractive producers of structurally novel secondary metabolites with diverse bioactivities. However, the lack of efficient genetic tools limits the discovery of novel compounds and the elucidation of biosynthesis mechanisms. Here, we firstly established an effective PEG-mediated chemical transformation system for protoplasts in two marine-derived fungi, Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7. Next, we developed a simple and versatile CRISPR-Cas9-based gene disruption strategy by transforming a target fungus with a single plasmid. We found that the transformation with a circular plasmid encoding cas9, a single-guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted and insertional gene mutations in both marine-derived fungal strains. In addition, the histone deacetylase gene rpd3 was mutated using the established CRISPR-Cas9 system, thereby activating novel secondary metabolites that were not produced in the wild-type strain. Taken together, a versatile CRISPR-Cas9-based gene disruption method was established, which will promote the discovery of novel natural products and further biological studies.

The rapid emergence of antimicrobial-resistant pathogenic microbes has accelerated the search for novel therapeutic agents. Here we report the discovery of antarmycin A (1), an antibiotic containing a symmetric 16-membered macrodiolide core with two pendant vancosamine moieties, one of which is glucosylated, from deep-sea-derived Pseudonocardia antarctica SCSIO 07407. The biosynthetic gene cluster of 1 was identified on a giant plasmid featuring transferable elements. In-depth biosynthetic investigation enabled us to (i) identify a set of seven genes associated with the product of the vancosamine moiety; (ii) discover two glycosyltransferases dedicated to the transfer of pendant sugars; and (iii) isolate rhamnose-modified antarmycin B (2) and a deglucosylated derivative antarmycin C (3) from genetically engineered mutant strains. Antibacterial assays revealed that 1 displays superior antibacterial properties with potent in vitro activities against the critical priority pathogens, multidrug-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus, fast bacterial killing, insusceptibility to antimicrobial resistance, and high in vivo efficiency in infection models. Mechanistic investigations revealed that 1 disrupts the bacterial cell membrane through a mechanism involving interactions between the vancosamine moieties and membrane-embedded phosphatidylglycerol/phosphatidylethanolamine. The results provide insights into the biological generation of vancosamine in natural products and demonstrate the potential of 1 as an effective lead to address the growing antimicrobial resistance threats.

Marine-derived fungi are emerging as prolific workhorses of structurally novel natural products (NPs) with diverse bioactivities. However, the limitation of available selection markers hampers the exploration of cryptic NPs. Recyclable markers are therefore valuable assets in genetic engineering programs for awaking silent SM clusters. Here, both pyrG and amdS-based recyclable marker cassettes were established and successfully applied in marine-derived fungi Aspergillus sp. SCSIO SX7S7 and Spiromastix sp. SCSIO F190, respectively. Using pyrG recyclable marker, a markerless 7S7-∆depH strain with a simplified HPLC background was built by inactivating a polyketide synthase (PKS) gene depH and looping out the pyrG recyclable marker after depH deletion. Meanwhile, an amdS recyclable marker system was also developed to help strains that are difficult to use pyrG marker. By employing the amdS marker, a backbone gene spm11 responsible for one major product of Spiromastix sp. SCSIO F190 was inactivated, and the amdS marker was excised after using, generating a relatively clean F190-∆spm11 strain for further activation of novel NPs. The collection of two different recycle markers will guarantee flexible application in marine-derived fungi with different genetic backgrounds, enabling the exploitation of novel structures in various fungi species with different genome mining strategies.