Modular pathway engineering enables gram-scale de novo biosynthesis of narirutin in yeast

Narirutin, a bioactive flavanone-7-O-disaccharide with antioxidant and lipid-lowering properties, is conventionally obtained via plant extraction, which suffers from low yields and seasonal dependence. In this study, we engineered Saccharomyces cerevisiae for de novo narirutin biosynthesis by partitioning the biosynthetic pathway into discrete functional modules for a stepwise optimization. First, a robust glycosylation chassis was developed by deleting the endogenous hydrolase EXG1 to prevent deglycosylation. Subsequently, an efficient two‑step glycosylation module was assembled through comprehensive enzyme screening to convert naringenin to narirutin. To achieve de novo production from glucose, a glycosylation module was genomically integrated into a high-yield naringenin-producing strain. Furthermore, enhancing the UDP-glucose (UDPG) supply through a "push-block" metabolic engineering strategy increased the titer to 216.8 mg/L. Finally, fed-batch fermentation with optimized stepwise feeding, dissolved oxygen (DO), and pH stabilization further boosted production to 1153.3 mg/L, representing the highest reported to date. This first demonstration of gram-scale narirutin biosynthesis establishes a sustainable microbial platform and offers a blueprint for producing other flavonoid glycosides.