Minimization of the Thiolactomycin Biosynthetic Pathway Reveals that the Cytochrome P450 Enzyme TlmF Is Required for Five‐Membered Thiolactone Ring Formation

Abstract

Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics inhibiting bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years, the study of TLM biosynthesis remains in its infancy. We recently discovered the biosynthetic gene cluster (BGC) for TLM from the marine bacterium Salinispora pacifica CNS-863. Here, we report the investigation of TLM biosynthetic logic through mutagenesis and comparative metabolic analyses. Our results reveal that only four genes (tlmF, tlmG, tlmH, and tlmI) are required for the construction of the characteristic γ-thiolactone skeleton of this class of antibiotics. We further showed that the cytochrome P450 TlmF does not directly participate in the sulfur insertion and C-S bond formation chemistry but rather in the construction of the 5-membered thiolactone ring, as upon its deletion, we observed the alternative production of the 6-membered δ-thiolactomycin. Our findings pave the way for future biochemical investigation of the biosynthesis of this structurally unique group of thiotetronic acid natural products.

The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research

“…Thiotetrnate antibiotics are potent fatty acid synthase inhibitors bearing a thiolactone core structure. The isolation and structure identification of several thiotetronate antibiotics have been published 170,171. In comparison with the truncated 1 H NMR spectra (0–9.0 ppm selected) in the ESI,† 170,171 shown in Fig.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…188,189 This ring, found at the C-terminus of the molecule, contains a sulfur atom that originates from the side chain of cysteine that is selected and activated by the nal module of the PKS/NRPS. Following this, the exact mechanism by which the sulfur is inserted into the molecule is unclear, although the role of the P450 is believed to centre on the epoxidation of the C4-C5 double bond (Fig.…”

Biosynthesis of antibiotic chuangxinmycin from Actinoplanes tsinanensis

“…A cytochrome P450-mediated C–S bond formation has been elucidated in several microbial secondary metabolite biosynthesis, such as SgvP in griseoviridin biosynthesis 47 and TlmF in five-membered thiolactone ring formation 48 . In thn cluster, a P450 (ThnC) was speculated to catalyze the second C–S bond formation in a similar manner in indolethiophene scaffold of thienodolin 39 .…”