Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora

► α-Oxygenated 4-hydroxyphenylpropanoids are substrates of a PKS III polyketide synthase. ► The sensitivity with which NMR detected 18O was enhanced by O-methylation with 13C-diazomethane. ► The biosynthetic sequence of oxygen incorporation into phenylphenalenones was elucidated. ► Lachnanthocarpone is involved in phenylphenalenone biosynthesis in Wachendorfia thyrsiflora.