Biosynthesis of Chlorinated Lactylates in Sphaerospermopsis sp. LEGE 00249

[Image: see text] Lactylates are an important group of molecules in the food and cosmetic industries. A series of natural halogenated 1-lactylates, chlorosphaerolactylates (1–4), were recently reported from Sphaerospermopsis sp. LEGE 00249. Here, we identify the cly biosynthetic gene cluster, containing all the necessary functionalities for the biosynthesis of the natural lactylates, based on in silico analyses. Using a combination of stable isotope incorporation experiments and bioinformatic analysis, we propose that dodecanoic acid and pyruvate are the key building blocks in the biosynthesis of 1–4. We additionally report minor analogues of these molecules with varying alkyl chains. This work paves the way to accessing industrially relevant lactylates through pathway engineering.