Recently, a research team led by Professor Li Shengying from the State Key Laboratory of Microbial Technology at Shandong University (SDU), in collaboration with Professor Wang Binju and Professor Zhang Yandong’s teams from Xiamen University, published aresearch articletitled "Deciphering the Thiolactonization Mechanism in Thiolactomycin Biosynthesis" in Journal of the American Chemical Society. This work successfully unravels the biosynthetic pathway of the sulfur-containing antibiotic thiolactomycin, revealing a unique mechanism for constructing the γ-thiolactone scaffold in nature.

Thiolactomycin, a natural product derived from actinomycetes, demonstrates strong antibacterial activity against both Gram-positive and Gram-negative bacteria. Its distinctive γ-thiolactone ring selectively targets bacterial type II fatty acid synthase while sparing human and mammalian fatty acid synthesis pathways, rendering it a highly promising antibiotic candidate. Over the past four decades, research has systematically elucidated its pharmacological properties, mechanisms of action, and chemical synthesis. Nevertheless, the biosynthetic mechanism of its pharmacophore—the γ-thiolactone ring—remained elusive.

This study isolated thiolactomycin-type compounds from Streptomyces sp. S6043a, an actinobacterium strain previously isolated from an Antarctic deep-sea sponge. Through bioinformatics analysis and in vivo gene inactivation experiments, the researchers identified key enzymes responsible for γ-thiolactone ring formation: the Cy domain of nonribosomal peptide synthetase (NRPS) TlnC, the A-PCP domain of TlnD, and the cytochrome P450 enzyme TlnA.To elucidate the catalytic roles of these enzymes, the authors adopted an NRPS domain-splitting strategy to obtain all the soluble recombinant proteins. Using chemically synthesized polyketide intermediate as substrate, they successfully reconstitutedtheγ-thiolactone ring formation process in a one-pot in vitro reaction. Their findings revealed that the Cy domain of TlnC mediates a rare sulfur-transfer reaction, incorporating a cysteine-derived sulfur atom into the polyketide backbone to generate an unstable thiocarboxylic intermediate. Subsequently, the P450 enzyme TlnA catalyzes a novel C–S bond cyclization, converting this intermediate into thiolactomycin.

To further elucidate the catalytic mechanisms of the Cy domain and P450 enzyme, the authors employed an integrated approach combining in vitro biochemical assays, isotopic labeling experiments, protein structural analysis, site-directed mutagenesis, quantum mechanics/molecular mechanics (QM/MM) simulations, density functional theory (DFT) calculations, primary kinetic isotope effect (KIE) measurements, and time-course NMR experiments. The results demonstrated that the Cy domain facilitates sulfur transfer through a sequential mechanism involving: amide bond formation, intramolecular cyclization, ring-opening rearrangement, and C–S bond cleavage. Notably, while characterized Cy domains typically catalyze thiazole/oxazole ring formation via a "condensation-cyclization-dehydration" pathway, this study reveals a Cy domain with a novel catalytic function. Mechanistic investigations established that the P450 TlnA mediates stereoselective intramolecular C–S cyclization through a "distal radical" mechanism, ultimately generating the γ-thiolactone ring. These findings revised the previously proposed epoxide intermediate hypothesis.

In summary, this study comprehensively elucidates the thiolactonization mechanism in thiolactomycin biosynthesis, reporting the first enzymatic γ-thiolactone ring formation identified in nature. The work establishes crucial genetic, enzymatic, and mechanistic foundations for engineering this promising antibiotic's biosynthetic pathway. Furthermore, these findings significantly advance our understanding of sulfur-containing natural product biosynthesis and provide new enzymatic tools for synthetic biology applications.

Additionally, the research team recently deciphered the biosynthetic mechanism of the thiocarboxylic acid moiety in the sulfur-containing natural product thioquinolobactin. Through an innovative "element engineering" approach, they developed a chemo-enzymatic platform for producing selenocarboxylic acid analogs. These findings were published in the Journal of Natural Products under the title "Carboxylic Acid Tailoring in Thioquinolobactin Biosynthesis."

These research projects were supported by the the National Key Research and Development Program of China, the National Natural Science Foundation of China, and the Shandong Provincial Natural Science Foundation. The State Key Laboratory of Microbial Technology serves as the primary and corresponding author affiliation.