Efficient production of hydroxytyrosol by directed evolution of HpaB in Escherichia coli

Hydroxytyrosol (HT) is an olive-derived phenolic phytochemical that has gained increasing commercial interest due to its natural antioxidant properties. It is widely used in the field of food supplement and medicine. It is reported that 4-hydroxyphenylacetate 3-hydroxylase (EcHpaB) and flavin reductase (EcHpaC) from E. coli BL21(DE3) can successfully express and catalyze the production of HT from tyrosol. In this study, the tyrosol production strain YMG5∗R as chassis cells, and a random mutant library of EcHpaB was established using error-prone PCR to improve the ability of EcHpaB to convert tyrosol to HT. Finally, a highly efficient HT synthetic mutant strainYMG5∗R-HpaB^TLEHC with high transformation efficiency was screened by directed evolution. The YMG5∗R-HpaB^TLEHC strain efficiently converted 50 mM tyrosol, with a yield of hydroxytyrosol reaching 48.2 mM (7.43 g/L) and a space-time yield reached 0.62 g/L·h. Overall, our study demonstrates the successful development of a highly efficient synthetic enzyme mutant for the production of HT, which has the potential to significantly improve the commercial viability of this natural antioxidant.