A Fungal DMAT-Type Prenyltransferase Catalyzes Regiospecific <i>O</i> -Farnesylation of Free <scp>l</scp> -Threonine and <scp>l</scp> -Serine

DMAT-type prenyltransferases (PTases) are essential for the biosynthesis of structurally diverse hybrid terpenoids. Here, we report the discovery and functional characterization of DiaB, a previously uncharacterized fungal PTase identified through genome mining. Heterologous expression of the <i>dia</i> gene cluster in <i>Aspergillus oryzae</i> NSAR1 led to the production of five new meroterpenoids. <i>In vivo</i> and <i>in vitro</i> studies revealed that DiaB is a PTase that catalyzes the regiospecific <i>O</i>-farnesylation of free l-threonine and l-serine, a previously unknown activity for this enzyme class in fungi. The functions of all enzymes in the cluster were elucidated, revealing a novel pathway to prenylated amino acids. This work expands the substrate scope of DMAT-type PTases and provides a new biocatalytic tool for meroterpenoid diversification.