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Carbazomycins (1–8) are a subgroup of carbazole derivatives that contain oxygen at the C3 and C4 positions and an unusual asymmetric substitution pattern. Several of these compounds exhibit antifungal and antioxidant activities. To date, no systematic biosynthetic studies have been conducted on carbazomycins. In this study, carbazomycins A and B (1 and 2) were isolated from Streptomyces luteosporeus NRRL 2401 using a one-strain-many-compound (OSMAC)-guided natural product mining screen. A biosynthetic gene cluster (BGC) was identified, and possible biosynthetic pathways for 1 and 2 were proposed. The in vivo genetic manipulation of the O-methyltransferase-encoding gene cbzMT proved indispensable for 1 and 2 biosynthesis. Size exclusion chromatography indicated that CbzMT was active as a dimer. In vitro biochemical assays confirmed that CbzMT could repeatedly act on the hydroxyl groups at C3 and C4, producing monomethylated 2 and dimethylated 1. Monomethylated carbazomycin B (2) is not easily methylated; however, CbzMT seemingly prefers the dimethylation of the dihydroxyl substrate (12) to 1, even with a low conversion efficiency. These findings not only improve the understanding of carbazomycin biosynthesis but also expand the inventory of OMT-catalyzing iterative methylations on different acceptor sites, paving the way for engineering biocatalysts to synthesize new active carbazomycin derivatives.

Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives.

Abstract Fucosterol, a sterol isolated from brown algae, has been demonstrated to have anti-cancer properties. However, the effects and underlying molecular mechanism of fucosterol on non-small cell lung cancer remain to be elucidated. In this study, the corresponding targets of fucosterol were obtained from PharmMapper, and NSCLC related targets were gathered from the GeneCards database, and the candidate targets of fucosterol-treated NSCLC were predicted. The mechanism of fucosterol against NSCLC was identified in DAVID6.8 by enrichment analysis of GO and KEGG, and protein–protein interaction data were collected from STRING database. The hub gene GRB2 was further screened out and verified by molecular docking. Moreover, the relationship of GRB2 expression and immune infiltrates were analyzed by the TIMER database. The results of network pharmacology suggest that fucosterol acts against candidate targets, such as MAPK1, EGFR, GRB2, IGF2, MAPK8, and SRC, which regulate biological processes including negative regulation of the apoptotic process, peptidyl-tyrosine phosphorylation, positive regulation of cell proliferation. The Raf/MEK/ERK signaling pathway initiated by GRB2 showed to be significant in treating NSCLC. In conclusion, our study indicates that fucosterol may suppress NSCLC progression by targeting GRB2 activated the Raf/MEK/ERK signaling pathway, which laying a theoretical foundation for further research and providing scientific support for the development of new drugs.

Two new cyclodipeptide (CDP) derivatives (1–2) and another seven known cyclodipeptides (3–9) were isolated from Streptomyces 26D9-414 by the genome mining approach combined with genetic dereplication and the “one strain many compounds” (OSMAC) strategy. The structures of the new CDPs were established on the basis of 1D- and 2D-NMR and comparative electronic circular dichroism (ECD) spectra analysis. The biosynthetic gene clusters (BGCs) for these CDPs were identified through antiSMASH analysis. The relevance between this cdp cluster and the identified nine CDPs was established by genetic interruption manipulation. The newly discovered natural compound 2 displayed comparable cytotoxicity against MDA-MB-231 and SW480 with that of cisplatin, a widely used chemotherapeutic agent for the treatment of various cancers.