Systematically Engineering Escherichia coli for Efficient and Complete Hydroxytyrosol Biosynthesis

Hydroxytyrosol (HT), a potent natural antioxidant in olive oil, has broad food and nutraceutical applications, but sustainable, scalable production remains challenging. Here, we achieved efficient de novo HT biosynthesis in Escherichia coli using glucose-glycerol dual carbon sources, combining metabolic engineering and rational enzyme design. We eliminated feedback inhibition, deleted competing pathways, and rationally mutated the rate-limiting enzyme HpaBC (S462A/M293Y), which enhanced catalytic efficiency by improving substrate entry, oxygen availability, and shortening reaction distance, as revealed by molecular dynamics simulations. Global cofactor optimization boosted HT titer to 4.90 g/L in shake flasks. Under optimized dissolved oxygen in a 5 L bioreactor, HT production reached 13.25 g/L, the highest reported titer in E. coli to date, providing a robust strategy for sustainable HT biomanufacturing.