Multi-step metabolic engineering optimization of Escherichia coli BL21 Star (DE3) for high-efficient production of...

Hydroxytyrosol (HT), a key member of natural polyphenolic compounds, is enriched in lipid-bound forms in olive tissues, has remarkable antioxidant activity and great application potential in food, pharmaceutical and cosmetic industries. This study developed a genome integration strategy based on Escherichia coli BL21 Star (DE3) to achieve efficient de novo synthesis of HT through multidimensional metabolic engineering modifications. Firstly, key functional genes (HpaBC, ARO10^D331C, ADH6, tyrC, aroG^fbr) were introduced into the host to establish the basic de novo HT synthesis capacity. Optimizing the copy number ratio of key enzyme genes regulated carbon flux to enhance target metabolism. Additionally, knocking out competitive branch genes (tyrR, pheA) alleviated precursor competition, and strengthening the shikimate pathway increased HT precursor concentrations. For the tyrosine-to-HT conversion step, the LAAD gene was introduced to construct an efficient catalytic module; molecular docking was used to analyze substrate-binding characteristics of cytochrome P450 enzymes and HpaBC, verifying their catalytic potential for HT synthesis. Finally, the engineered strain achieved efficient synthesis in a 5 L bioreactor, reaching an HT titer of 7460 mg/L at 43 h of fermentation. Through the synergistic strategies of gene integration, metabolic flux optimization, and catalytic module reinforcement, this study established an efficient HT biosynthetic system, providing a novel technical pathway for the green biomanufacturing of natural polyphenolic compounds.