The results define the enzymatic logic and temporal organization of persiathiacin biosynthesis, providing a conceptual framework for rational diversification of complex thiopeptide natural products.
Abstract
Thiopeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that form complex bioactive scaffolds through extensive enzymatic tailoring. The polyglycosylated thiopeptides persiathiacins, exhibit potent activity against multidrug-resistant Mycobacterium tuberculosis (Mtb) and methicillin-resistant Staphylococcus aureus (MRSA). The persiathiacin biosynthetic gene cluster encodes six cytochrome P450 (CYP) enzymes, but the logic of their oxidative modifications was unknown. Here, we establish a protoplast-based genetic system for Actinokineospora and systematically assign functions to all P450s. We demonstrate that PerX hydroxylates the central thiazole, PerV installs the third indole–core crosslink required for macrocyclization, and PerT, not PerU, catalyses indole N-hydroxylation. Combined gene inactivation and metabolite profiling reveal a hierarchical enzymatic sequence leading to the mature scaffold prior to sugar installation. Notably, the intermediate accumulating in the ΩperX mutant exhibits enhanced anti-M. tuberculosis potency compared to persiathiacin A (IC50 = 0.07 vs 1.5 µg mL−1). These results define the enzymatic logic and temporal organization of persiathiacin biosynthesis, providing a conceptual framework for rational diversification of complex thiopeptide natural products.
The global rise of antibiotic resistance poses a public health challenge, emphasizing the urgent need for novel antibiotics featuring unique chemical scaffolds and mechanisms of action. Here, we report the discovery and characterization of darumycins, guanidine-containing pentacyclic sesterterpene antibiotics that exhibit potent activity against high-priority Gram-positive pathogens and mycobacteria. The darumycin biosynthetic gene cluster was identified through genome mining in the Actinobacterium Micromonospora rubida and heterologously expressed in Streptomyces chassis strains. Gene cluster engineering facilitated the discovery of novel darumycin derivatives, thereby expanding the chemical diversity within the sesterterpene class of natural products and revealing nuanced variations in their bioactivity. Targeted gene deletions, along with LC–MS and NMR analyses, enabled us to propose a darumycin biosynthetic pathway, further complemented by in vitro biochemical characterization of two O-methyltransferase tailoring enzymes, DarM and DarG. The high-resolution crystal structure of DarM in complex with SAH provided valuable insights into the enzymatic mechanism and revealed a distinct architecture compared to other methyltransferases acting on terpene scaffolds.
An alkaliphilic strain of Streptomyces S9 was isolated and taxonomically characterised through multilocus sequence analysis (MLSA) using Streptomyces-specific primers, indicating its genetic potential to biosynthesise aromatic polyketides.
This study expands the enzymatic repertoire of licorice OMTs, provides new insights into the structure-function relationships underling multiple methylations, and establishes a foundation or sustainable biocatalytic production of highly bioactive methylated flavonoids in G. inflata and related species.
Xiaoman Yang, Yun Huang, Xiaoju Liang et al.· International Journal of Bio...· 0 citations
Two new 4-hydroxy-2-pyridone alkaloids, prebassianins F (5) and G (6), featuring a rare 1,4-cyclohexanediol moiety, were isolated from rice cultures of Akanthomyces araneicola GY29011. Genome mining and heterologous expression of the corresponding biosynthetic gene cluster (pre) in Aspergillus nidulans afforded six additional new analogues (3−4 and 7−10), along with two known compounds (1−2). Combinatorial expression and substrate feeding experiments enabled the elucidation of the biosynthetic pathway and functional assignment of the tailoring enzymes. Notably, PreC was identified as a rare bifunctional fusion enzyme comprising old yellow enzyme (OYE) and short-chain dehydrogenase (SDR) domains. Truncation of PreC led to the accumulation of a key intermediate (10), supporting its role in sequential reductions. cDNA analysis confirmed that PreC is encoded as a single open reading frame containing four introns, including a rare GC−AG intron. Compounds 3 and 4 exhibited antibacterial activity against Bacillus subtilis (MIC = 16 μg/mL). These findings expand the structural diversity of 4-hydroxy-2-pyridone alkaloids and provide new insights into the enzymatic logic and evolution of fungal biosynthetic pathways.
Haoyu Yu, Wei Lin, Jianbin Xiao et al.· Journal of Natural Products· 0 citations
Gram-negative bacteria present a major clinical challenge but also remain an underexplored source of antibacterial natural products. Resistance-guided genome mining of the entomopathogenic symbiont Xenorhabdus identified the rdb biosynthetic gene cluster, which encodes a putative prodrug antibiotic, pre-rhabdobranin. However, the inability to isolate the proposed active metabolite, rhabdobranin, has prevented direct functional evaluation. Here we report a convergent total synthesis of the proposed structure of pre-rhabdobranin B, which revealed a stereochemical misassignment at the N-terminal arginine residue. Synthesis of both rhabdobranin epimers showed that, although they are nearly indistinguishable by standard analytical methods, inversion at this single stereocenter has a pronounced effect on antibacterial activity. Biological evaluation of the revised rhabdobranin structure revealed potent antibacterial activity against Gram-negative pathogens, including WHO critical-priority carbapenem-resistant Klebsiella pneumoniae. Cellular and biochemical profiling implicated inhibition of protein biosynthesis as its principal antibacterial mechanism. We further show that the GNAT-family acetyltransferase RdbK N-acetylates rhabdobranin, attenuating its activity and establishing a secondary self-resistance mechanism. These findings validate resistance-gene-guided discovery in Gram-negative symbionts as a strategy for uncovering cryptic antibiotics and identify rhabdobranin as a promising scaffold for Gram-negative antibiotic development.
Woonkee S. Jo, Zaynoun Attieh, Jan J. Crames et al.· Journal of the American Chem...· 0 citations
The symbiotic bacterium Photorhabdus is a rich source of bioactive secondary metabolites that mediate tripartite interactions with nematodes and insect hosts. However, natural products of ribosomal origin remain largely underexplored within this ecological niche. Here, we report the identification of aphotorhaptin A, a darobactin-like peptide (daropeptide) natural product from Photorhabdus asymbiotica, which structurally features an ether crosslink and an N-terminal acetyl unit. Biosynthetic investigation uncovers aphotorhaptin A is matured via an unexpected leader cleavage step, and the subsequent N-terminal acetylation confers metabolic stability that maintains the hexapeptide scaffold integrity. Biochemical and structural studies demonstrate the acetyltransferase PasC exhibits remarkable substrate promiscuity, facilitated by an expansive active-site cavity that accommodates diverse acyl-CoA donors and peptide substrates. Unlike the antimicrobial darobactin, aphotorhaptin A appears to lack antibacterial activity but modulates nematode development, and this activity requires the ether crosslink and the N-terminal acetyl group in the hexapeptide scaffold. These findings expand the chemical and biosynthetic space of ribosomal peptide family and establish its link with nematode development and reproduction.
Suze Ma, Ru Li, Xiang-Yang Gao et al.· Proceedings of the National...· 0 citations
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