Biotransformation of Kaempferol to Icaritin in Engineered <i>Saccharomyces cerevisiae</i>

Flavonoids are bioactive natural products known for their pharmaceutical properties and health-promoting applications. Microbial transformation offers a promising and sustainable approach for the biosynthesis of flavonoids. However, challenges such as the lack of well-established synthesis pathways and inefficient heterologous expression of key enzymes limit the flavonoid production such as icaritin. Here, a <i>Saccharomyces cerevisiae</i> strain was engineered to produce icaritin from kaempferol through a metabolic engineering strategy. Enzyme screening strategies identified the functional prenyltransferases, enabling the construction of a bioconversion pathway. The engineered isopentenol and mevalonate pathway boosted the supply of dimethylallyl pyrophosphate, producing 10.4 mg/L 8-prenylkaempferol. Redesigning the N-terminal of prenyltransferase resulted in a 7.5-fold increase in the titer of 8-prenylkaempferol. Cofactor engineering strategies of S-adenosyl-l-methionine recycling resulted in a substantial 139.8% increase in icaritin production. Additionally, the rational design of rate-limiting enzymes significantly improved catalytic performance, enhancing overall icaritin production. Ultimately, engineered <i>S. cerevisiae</i> transformed kaempferol to icaritin successfully through engineered enzymatic modifications with a titer of 14.4 mg/L. This study offers valuable insights into the enzyme design and sustainable natural products production.