Armeniaspirols (1–3) are potent antibiotics against Gram-positive pathogens. Through a biosynthetic investigation, we identified four enzymes involved in the structural modification of 1–3. Manipulation of their activity led to the generation of 4–6 and nine novel analogues, 7–15. Bioactivity assessments revealed that the pyrrole chloro group and the methyl group are important for the antimicrobial activities of armeniaspirols, which lays the foundation for future structure optimization and mechanism of action studies of armeniaspirols.
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.
DepR, a LysR-type transcriptional regulator encoded by the last gene of the putative min operon (orf21-20-19-depR) located at the downstream region of the anticancer agent FK228 biosynthetic gene cluster in Chromobacterium violaceum No. 968, positively regulates the biosynthesis of FK228. In this work, the mechanism underlining this positive regulation was probed by multiple approaches. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay (DIFA) identified a conserved 35-nt DNA segment in the orf21-orf22 intergenic region where the purified recombinant DepR binds to. Quantitative reverse transcription PCR (RT-qPCR) and green fluorescent protein (GFP) promoter probe assays established that transcription of phasin gene orf22 increases in the depR deletion mutant of C. violaceum (CvΔdepR) compared to the wild-type strain. FK228 production in the orf22-overexpressed strain C. violaceum was reduced compared with the wild-type strain. DepR has two conserved cysteine residues C199 and C208 presumed to form a disulfide bridge upon sensing oxidative stress. C199X point mutations that locked DepR in a reduced conformation decreased the DNA-binding affinity of DepR; T232A or R278A mutation also had a negative impact on DNA binding of DepR. Complementation of CvΔdepR with any of those versions of depR carrying a single codon mutation was not able to restore FK228 production to the level of wild-type strain. All evidences collectively suggested that DepR positively regulates the biosynthesis of FK228 through indirect metabolic networking.
Anisomycin (1), a pyrrolidine antibiotic, exhibits diverse biological and pharmacologic activities. The biosynthetic gene cluster of 1 has been identified previously and the multistep assembly of the core benzylpyrrolidine scaffold was characterized. However, enzymatic modifications, such as acylation involved in 1 biosynthesis are unknown. In this study, the genetic manipulation of aniI proved that it encoded indispensable acetyltransferase for 1 biosynthesis. Bioinformatics analysis suggested AniI as a member of LbH-MAT-GAT sugar O-acetyltransferase, but the biochemical assay identified that its target site was the hydroxyl group of the pyrrolidine ring. AniI was found to be tolerant of acyl donors with different chain length for the biosynthesis of 1 and derivatives 12 and 13 with butyryl and isovaleryl groups, respectively. Meanwhile, it showed comparable activity towards biosynthetic intermediates and synthesized analogues, suggesting promiscuity to the pyrrolidine ring structure of 1. These data may inspire new viable synthetic routes for the construction of more complex pyrrolidine ring scaffolds in 1. Finally, the overexpression of aniI under the control of strong promoters contributed to the higher productivities of 1 and its analogues. These findings reported here not only improved the understanding of anisomycin biosynthesis but also expand the substrate scope of O-acetyltransferase working on the pyrrolidine ring and pave the way for future metabolic engineering construction of high-yield strain. IMPORTANCE Acylation is an important tailoring reaction during natural products biosynthesis. Acylation could increase the structural diversity, affect the chemical stability, volatility, biological activity and even the cellular localization of specialized compounds. Many acetyltransferases have been reported in natural product biosynthesis. The typical example of LbH-MAT-GAT sugar O-acetyltransferase subfamily was reported to catalyze the CoA-dependent acetylation of the 6-hydroxyl group of sugars. However, no protein of this family has been characterized to acetylate non-sugar secondary metabolic product. Here, AniI was found to catalyze the acylation of the hydroxyl group of the pyrrolidine ring and be tolerant of diverse acyl donors and acceptors, which made the biosynthesis more efficient and exclusive for 1 and its derivatives biosynthesis. Moreover, the overexpression of aniI serves as a successful example of genetic manipulation of a modification gene for the high production of final products and might set the stage for future metabolic engineering.
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