Biosynthesis of Kaitocephalin: A Neuroprotective Natural Product Featuring a Peptide‐Like yet Nonpeptidic Scaffold

Kaitocephalin (KCP) is a neuroprotective natural product that acts as an antagonist of ionotropic glutamate receptors, making it a highly promising lead for drug discovery. It possesses a unique scaffold composed of three amino acids connected via C─C bonds, which appears peptide-like but is formed without peptide bonds. In this study, we identified the KCP biosynthetic gene cluster (kpb cluster) in the producing fungus Eupenicillium shearii through integrated genomic and transcriptomic analyses. LC-MS/MS profiling and chemical derivatization of E. shearii extracts led to the discovery of four novel pathway-related metabolites. In vitro enzymatic assays with 2(S)-dechlorokaito lactate, one of the four identified metabolites, as a substrate enabled functional characterization of KpbI, KpbM, and KpbB involved in KCP formation. Among them, the dioxygenase KpbI was found to catalyze an unprecedented two-step oxidation to form the d-serine moiety. In addition, isotope tracing experiments provided new insights into the origin of the l-proline moiety. These findings establish a foundation for future studies aimed at elucidating the complete biosynthetic mechanism of KCP.