Rational metabolic reprogramming of yeast across multiple pathway nodes for de novo liquiritigenin biosynthesis from glucose

Liquiritigenin (LIG) is a natural flavonoid with diverse bioactivities, rendering it attractive for applications in food, pharmaceuticals, nutraceuticals, and cosmetics. However, its de novo biosynthesis in microbial hosts faces several major challenges, including the lack of efficient enzymes compatible with heterologous systems, insufficient supply of precursor amino acids, suboptimal cofactor availability and electron transfer efficiency. To evaluate these constraints, Saccharomyces cerevisiae was engineered through systematic stepwise comparison of p -coumaric acid-forming routes, downstream enzyme sources, aromatic amino acids precursor supply, NADPH availability, and fermentation conditions. The best-performing strain QL14 produced 4.09 mg/L LIG in shake-flask fermentation and 8.42 mg/L LIG in 5-L fed-batch fermentation. However, naringenin (NAR) remained the predominant downstream product. These results indicate that increased pathway input improved overall flavanone output but was insufficient to redirect flux toward the CHR-dependent LIG-forming branch. Therefore, downstream branch selectivity, rather than precursor or NADPH supply alone, represents the major constraint in the current yeast system. This work provides a basis for future CHR-focused engineering and controllable LIG/NAR co-production.