LAUSR

Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics that inhibit bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years ago, 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 revealed 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 sulfur insertion and C−S bond formation chemistry but rather in the construction of the five‐membered thiolactone ring as, upon its deletion, we observed the alternative production of the six‐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.