Penicillenols belong to the family of tetramic acids with anticancer and antibacterial activities. Here, we report the discovery of the biosynthetic gene cluster ( pnc) for penicillenol A 1 and E in Penicillium citrinum ATCC9849 by genome mining. We discover the pnc cluster based on the results of gene deletions in P. citrinum and gene cluster heterologous expression in Aspergillus nidulans . We also propose the assembly line of the PKS module in PncA with the reduction by PncB provides a highly reduce polyketide chain to be further linked with an L-threonine molecule and released from PncA to produce penicillenol E. Further formation of penicillenol A 1 requires the N -methylation of tetramic acid group by PncC. Our work deepens the understanding of the biosynthetic logic for N -methylated tetramic acids and contributes to the discovery of new penicillenols by genome mining.