De Novo Artificial Biosynthesis of 3-Hydroxyphloretin in <i>Escherichia coli</i>

3-Hydroxyphloretin (3-OH phloretin), a dihydrochalcone compound containing a catechol moiety, is naturally present in apples and exhibits potent anti-adipogenic, anti-obesity, and anticancer activities. In this study, we developed a modular co-culture platform enabling the de novo biosynthesis of 3-OH phloretin from glucose in <i>Escherichia coli</i>. We demonstrated that 4-coumarate 3-hydroxylase (Sam5), derived from <i>Saccharothrix espanaensis</i>, efficiently catalyzes the hydroxylation of phloretin to 3-OH phloretin. The engineered co-culture system comprised two functional modules: an upstream module that converts l-tyrosine to phloretic acid through the expression of tyrosine ammonia-lyase and enoate reductase genes, and a downstream module that converts phloretic acid to 3-OH phloretin via the sequential action of 4-coumarate-CoA ligase, a mutated chalcone synthase, and Sam5. Using this system, we successfully achieved the de novo production of 3-OH phloretin at a titer of 4.69 mg/L from glucose. In parallel, the artificial biosynthetic pathway also yielded phloretic acid and 3-hydroxyphloretic acid (3-OH phloretic acid) at titers of 161.7 and 176.2 mg/L, respectively, in an engineered l-tyrosine-overproducing <i>E. coli</i> strain. To the best of our knowledge, this study represents the first successful establishment of an artificial biosynthetic route for the production of both 3-OH phloretic acid and 3-OH phloretin directly from glucose in <i>E. coli</i>. This platform lays the groundwork for the microbial production of valuable dihydrochalcone compounds and holds promise for further optimization toward industrial-scale applications.