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

ABSTRACT 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.