Fungal cytochrome P450 enzymes have been shown to catalyze regio- and stereoselective oxidative intermolecular phenol coupling. However, an enzyme capable of catalyzing undirected para-para (C4-4') coupling has not been reported. Here, we revealed the biosynthetic gene cluster (BGC) of phomoxanthone A from the marine fungus Diaporthe sp. SYSU-MS4722. We heterologously expressed 14 biosynthetic genes in Aspergillus oryzae NSAR1 and found that PhoCDEFGHK is involved in the early stage of phomoxanthone A biosynthesis to give chrysophanol and that chrysophanol is then processed by PhoBJKLMNP to yield penexanthone B. A feeding experiment suggested that PhoO, a cytochrome P450 enzyme, catalyzed the regioselective oxidative para-para coupling of penexanthone B to give phomoxanthone A. The mechanism of PhoO represents a novel enzymatic 4,4'-linkage dimerization method for tetrahydroxanthone formations, which would facilitate biosynthetic engineering of structurally diverse 4,4'-linked dimeric tetrahydroxanthones.