Enzyme promiscuity-driven co-production of flavonoid 7-O-glycosides in engineered Saccharomyces cerevisiae

Flavonoid 7-<i>O</i>-glycosides are a key class of bioactive flavonoid derivatives with broad application prospects in functional foods and nutraceuticals. Currently, their production is mainly dependent on unsustainable plant extraction or inefficient chemical synthesis. Microbial synthesis provides a promising green alternative for producing such functional ingredients; however, enzyme promiscuity hinders their ability to form specific products exclusively. To address this issue, we propose a strategy to harness enzyme promiscuity by engineering the metabolic context. This approach purposefully utilizes enzyme's catalytic flexibility to co-produce flavonoid 7-<i>O</i>-glycoside mixtures. We first optimized the (2<i>S</i>)-naringenin (NAR) biosynthesis module by overexpressing key enzymes, relieving feedback inhibition, and enhancing acetyl-CoA supply, achieving a yield of 318.08 mg/L NAR. Subsequently, by harnessing the promiscuity of 7-<i>O</i>-glucosyltransferase and implementing strategies including glycosidase elimination, <i>S</i>-adenosyl-l-methionine balancing, and uridine diphosphate-glucose supply optimization, we achieved efficient co-production of (2<i>S</i>)-isosakuranetin (ISOEIN) 7-<i>O</i>-glycoside and NAR 7-<i>O</i>-glycoside. This study establishes a sustainable and efficient biosynthesis platform for the production of complex flavonoid mixtures as potential functional food ingredients, demonstrates a green biosynthesis route for food-grade natural products, and exemplifies a novel paradigm of exploiting enzyme promiscuity through metabolic context engineering in microbial systems. This strategy is expected to be extendable to the synthesis of other structurally similar bioactive compounds for food and health applications.