ABSTRACT Kaitocephalin (KCP, 1 ) 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 ( 2 - 5 ). In vitro enzymatic assays with 2( S )-dechlorokaito lactate ( 4 ) 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. Table of Contents graphical abstract