Asperpyridone A represents an unusual class of pyridone alkaloids with demonstrated potential for hypoglycemic activity, primarily by promoting glucose consumption in HepG2 cells. Trichodin A, initially isolated from the marine fungus Trichoderma sp. strain MF106, exhibits notable antibiotic activities against Staphylococcus epidermidis. Despite their pharmacological significance, the regulatory mechanisms governing their biosynthesis have remained elusive. In this investigation, we initiated the activation of a latent gene cluster, denoted as \"top\", through the overexpression of the Zn2Cys6 transcription factor TopC in Tolypocladium ophioglossoides. The activation of the top cluster led to the biosynthesis of asperpyridone A, pyridoxatin, and trichodin A. Our study also elucidated that the regulator TopC exerts precise control over the biosynthesis of asperpyridone A and trichodin A through the detection of protein-nucleic acid interactions. Moreover, by complementing these findings with gene deletions involving topA and topH, we proposed a comprehensive biosynthesis pathway for asperpyridone A and trichodin A in T. ophioglossoides.

The emergence of drug resistance highlights the importance of new drug discovery. Microbial secondary metabolites encoded in biosynthetic gene clusters (BGCs) are a prolific source of drugs, whereas most of these BGCs are cryptic. Thus, taking strategies to activate these cryptic BGCs is of great importance for potential drug discovery. In this work, three novel pentangular polyphenols lanthomicin A-C were identified by activating a cryptic aromatic polyketide BGC through promoter engineering combined with optimization of fermentation conditions. We further confirmed the involvement of lanthomicin (ltm) BGC in biosynthesis by CRISPR-Cpf1-assisted gene editing. Based on functional analysis of homologous genes, a putative biosynthetic pathway was proposed for the three lanthomicins. Particularly, lanthomicin A showed antiproliferative activity with IC50 0.17 μM for lung cancer cell line A-549. The discovery of lanthomicins brings new members to the pentangular polyphenol subclade of aromatic polyketide and demonstrates the potential of Streptomyces as a source for drug discovery.

Phytopathogenic fungi infect crops, presenting a worldwide threat to agriculture. Polyene macrolides are one of the most effective antifungal agents applied in human therapy and crop protection. In this study, we found a cryptic polyene biosynthetic gene cluster in Actinokineospora spheciospongiae by genome mining. Then, this gene cluster was activated via varying fermentation conditions, leading to the discovery of new polyene actinospene (1), which was subsequently isolated and its structure determined through spectroscopic techniques including UV, HR-MS, and NMR. The absolute configuration was confirmed by comparing the calculated and experimental electronic circular dichroism (ECD) spectra. Unlike known polyene macrolides, actinospene (1) demonstrated more versatile post-assembling decorations including two epoxide groups and an unusual isobutenyl side chain. In bioassays, actinospene (1) showed a broad spectrum of antifungal activity against several plant fungal pathogens as well as pathogenic yeasts with minimum inhibitory concentrations ranging between 2 and 10 μg/mL.

With the increase of drug resistance caused by the improper use and abuse of antibiotics, human beings are facing a global health crisis. Sequencing of Streptomyces genomes revealed the presence of an important reservoir of secondary metabolic gene clusters for previously unsuspected products with potentially valuable bioactivity. It has therefore become necessary to activate these cryptic pathways through various strategies. Here, we used RNA-seq data to perform a comparative transcriptome analysis of Streptomyces ansochromogenes (wild-type, WT) and its global regulatory gene disruption mutant ΔwblA, in which some differentially expressed genes are associated with the abolished nikkomycin biosynthesis and activated tylosin analogue compounds (TACs) production, and also with the oviedomycin production that is induced by the genetic manipulation of two differentially expressed genes (san7324 and san7324L) encoding RsbR. These results provide a significant clue for the discovery of new drug candidates and the activation of cryptic biosynthetic gene clusters.

Genome sequencing has revealed that each Streptomyces contains a wide range of biosynthetic gene clusters (BGCs) and has the capability to produce more novel natural products than what is expected. However, most gene clusters for secondary metabolite biosynthesis are cryptic under normal growth conditions. In Streptomyces tsukubaensis, combining overexpression of the putative SARPs (Streptomyces antibiotic regulatory proteins) and bioactivity-guided screening, the silent gene cluster (tsu) was successfully activated and a novel bioactive anthracycline tsukubarubicin was further isolated and identified. Biological activity assays demonstrated that tsukubarubicin possessed much better antitumor bioactivities against various human cancer cell lines (especially the breast cancer cell lines) than clinically used doxorubicin. Moreover, the previously unreported gene cluster (tsu) for biosynthesis of tsukubarubicin was first characterized and detailed annotations of this gene cluster were also conducted. Our strategy presented in this work is broadly applicable in other Streptomyces and will assist in enriching the natural products for potential drug leads. KEY POINTS: • Generally scalable strategy to activate silent gene clusters by manipulating SARPs. • The novel anthracycline tsukubarubicin with potent antitumor bioactivities. • Identification and annotation of the previously uncharacterized tsu gene cluster.

Mureidomycins (MRDs), a group of unique uridyl-peptide antibiotics, exhibit antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa. Our previous study showed that the cryptic MRD biosynthetic gene cluster (BGC) mrd in Streptomyces roseosporus NRRL 15998 could not be activated by its endogenous regulator 02995 but activated by an exogenous activator SsaA from sansanmycin\'s BGC ssa of Streptomyces sp. strain SS. Here we report the molecular mechanism for this inexplicable regulation. EMSAs and footprinting experiments revealed that SsaA could directly bind to a 14-nt palindrome sequence of 5\'-CTGRCNNNNGTCAG-3\' within six promoter regions of mrd. Disruption of three representative target genes (SSGG-02981, SSGG-02987 and SSGG-02994) showed that the target genes directly controlled by SsaA were essential for MRD production. The regulatory function was further investigated by replacing six regions of SSGG-02995 with those of ssaA. Surprisingly, only the replacement of 343-450 nt fragment encoding the 115-150 amino acids (AA) of SsaA could activate MRD biosynthesis. Further bioinformatics analysis showed that the 115-150 AA situated between two conserved domains of SsaA. Our findings significantly demonstrate that constitutive expression of a homologous exogenous regulatory gene is an effective strategy to awaken cryptic biosynthetic pathways in Streptomyces.

Fungal genomes carry many gene clusters seemingly capable of natural products biosynthesis, yet most clusters remain cryptic or down-regulated. Genome mining revealed an unconventional paraherquonin-like meroterpenoid biosynthetic gene cluster in the chromosome of Neosartorya glabra. The cryptic or down-regulated pathway was activated by constitutive expression of pathway-specific regulator gene berA encoded within ber biosynthetic gene cluster. Chemical analysis of mutant Ng-OE: berA extracts enabled the isolation of four berkeleyacetal congeners, in which two of them are new. On the basis of careful bioinformatic analysis of the coding enzymes in the ber gene cluster, the biosynthetic pathway of berkeleyacetals was proposed. These results indicate that this approach would be valuable for discovery of novel natural products and will accelerate the exploitation of prodigious natural products in filamentous fungi.

BACKGROUND: Actinomycete genome sequencing has disclosed a large number of cryptic secondary metabolite biosynthetic gene clusters. However, their unavailable or limited expression severely hampered the discovery of bioactive compounds. The whiB-like (wbl) regulatory genes play important roles in morphological differentiation as well as secondary metabolism; and hence the wblA so gene was probed and set as the target to activate cryptic gene clusters in deepsea-derived Streptomyces somaliensis SCSIO ZH66.
RESULTS: wblA so from deepsea-derived S. somaliensis SCSIO ZH66 was inactivated, leading to significant changes of secondary metabolites production in the ΔwblA so mutant, from which α-pyrone compound violapyrone B (VLP B) was isolated. Subsequently, the VLP biosynthetic gene cluster was identified and characterized, which consists of a type III polyketide synthase (PKS) gene vioA and a regulatory gene vioB; delightedly, inactivation of vioB led to isolation of another four VLPs analogues, among which one was new and two exhibited improved anti-MRSA (methicillin-resistant Staphylococcus aureus, MRSA) activity than VLP B. Moreover, transcriptional analysis revealed that the expression levels of whi genes (whiD, whiG, whiH and whiI) and wbl genes (wblC, wblE, wblH, wblI and wblK) were repressed by different degrees, suggesting an intertwined regulation mechanism of wblA so in morphological differentiation and secondary metabolism of S. somaliensis SCSIO ZH66.
CONCLUSIONS: wblA orthologues would be effective targets for activation of cryptic gene clusters in marine-derived Streptomyces strains, notwithstanding the regulation mechanisms might be varied in different strains. Moreover, the availability of the vio gene cluster has enriched the diversity of type III PKSs, providing new opportunities to expand the chemical space of polyketides through biosynthetic engineering.