This study reports the de novo biosynthesis of xanthohumol, a bioactive flavonoid from hops, in yeast. By balancing three parallel biosynthetic pathways—aromatic, malonyl-CoA, and mevalonate (MVA)—and engineering prenyltransferase (PTase), precursor supply, and peroxisomal pathways, the researchers achieved an 83-fold increase in the production of the key precursor demethylxanthohumol (DMX). They identified prenylation as the limiting step in DMX biosynthesis and optimized DMAPP availability and prenylation efficiency. The study also revealed that peroxisomal compartmentalization of the DMX biosynthetic pathway improved DMX production by enhancing PTase expression and precursor supply. Finally, the O-methyltransferase gene HlOMT1 was expressed to complete the de novo biosynthesis of xanthohumol in yeast, achieving a production level of 142 µg/L. The work provides a systematic approach for engineering yeast cell factories to produce complex natural products, offering a sustainable alternative to traditional methods of xanthohumol extraction from hops. The findings highlight the importance of balancing precursor supply, optimizing enzyme activity, and compartmentalizing metabolic pathways to enhance the efficiency of biosynthetic processes in yeast.This study reports the de novo biosynthesis of xanthohumol, a bioactive flavonoid from hops, in yeast. By balancing three parallel biosynthetic pathways—aromatic, malonyl-CoA, and mevalonate (MVA)—and engineering prenyltransferase (PTase), precursor supply, and peroxisomal pathways, the researchers achieved an 83-fold increase in the production of the key precursor demethylxanthohumol (DMX). They identified prenylation as the limiting step in DMX biosynthesis and optimized DMAPP availability and prenylation efficiency. The study also revealed that peroxisomal compartmentalization of the DMX biosynthetic pathway improved DMX production by enhancing PTase expression and precursor supply. Finally, the O-methyltransferase gene HlOMT1 was expressed to complete the de novo biosynthesis of xanthohumol in yeast, achieving a production level of 142 µg/L. The work provides a systematic approach for engineering yeast cell factories to produce complex natural products, offering a sustainable alternative to traditional methods of xanthohumol extraction from hops. The findings highlight the importance of balancing precursor supply, optimizing enzyme activity, and compartmentalizing metabolic pathways to enhance the efficiency of biosynthetic processes in yeast.