Hydroxytyrosol (HT) is a potent polyphenolic antioxidant widely utilized in the biomedical and food industries. However, its high-level microbial biosynthesis is primarily hindered by the metabolic flux imbalances and severe cellular toxicity. In this study, an artificial synthetic pathway from 4-hydroxyphenylpyruvate was constructed in an engineered l -phenylalanine producing E . coli chassis. Building on this, the endogenous precursor supply was strengthened via targeted promoter engineering of aroK , aroC , and tyrA , and the heterologous HT biosynthetic pathway was enhanced by overexpressing ARO10. To mitigate intermediate l -DOPA accumulation, co-expression of l -DOPA decarboxylase (DODC) and tyramine oxidase (TYO) reduced l -DOPA by 63.7%, while expression of l -amino acid deaminase (LAAD) reduced l -DOPA by 76.1%. Additionally, precise cofactor engineering was implemented; overexpressing the riboflavin metabolic genes ribH , ribC , and ribF , alongside introducing pntAB , increased HT production by 30.9% and 12.7%, respectively. Furthermore, transcriptomic analysis under HT stress revealed significant upregulation of genes related to transport and stress responses. Among these targets, overexpressing marR substantially improved cellular tolerance and HT production. Finally, during a 5-L bioreactor fermentation supplemented with Fe 2+ and ascorbic acid, the engineered strain achieved an HT titer of 9.25 g/L, a yield of 0.102 g/g glucose, and a productivity of 0.193 g/L/h. This study reports the highest HT titer to date in E. coli using glucose as the carbon source, providing a robust biomanufacturing platform.
Zuo et al. (2026) studied this question.