<i>De Novo</i> Synthesis of Tyrosol and Hydroxytyrosol through Temperature-Inducible Systems and Metabolic Engineering

Hydroxytyrosol (HT) has various biological and pharmacological activities, including potent antioxidant activity. The efficient synthesis of HT and tyrosol has been achieved by microbial synthesis. However, more strategies are needed to enhance its yield and meet the demands of industrialization. In this study, <i>SceARO10</i> and <i>EcoyahK</i> were used for the <i>de novo</i> synthesis of tyrosol in <i>Escherichia coli</i> using a temperature-inducible system. Different sources of phenolic acid decarboxylase and alcohol reductase were investigated, with <i>YliARO10</i> and <i>YliPAR4</i> from <i>Yarrowia lipolytica</i> showing the best catalytic performance, yielding 4.05 g/L of tyrosol at 60 h in shake flasks, the highest yield reported. Next, <i>EblHpaBC</i> from <i>E. coli</i> BL21 (DE3) was introduced for HT biosynthesis, and the HT-related degradation gene <i>mhpB</i> was functionally characterized in <i>E. coli</i>. Subsequently, by enhancing precursor supply, eliminating competing metabolic pathways, and knocking out <i>mhpB</i>, the HT yield reached 1.28 g/L after 60 h. Finally, in a 5 L bioreactor, titers of 6.18 and 4.97 g/L of tyrosol and HT were achieved for the first time using a temperature-induced strategy. This study presents a method for the modification of microbial chassis for the efficient synthesis of tyrosol and HT.