Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce non-methylated flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of these O-methylated flavonoids is currently still highly inefficient.In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperitin production from fed cinnamic acid precursors. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase from Hordeum vulgare in three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance in Escherichia coli towards producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than previously reported. Our findings will facilitate the further engineering of this enzyme towards higher production of methylated flavonoids.Competing Interest StatementThe authors have declared no competing interest.