<i>De Novo</i> Synthesis of Tyramine in Engineered <i>Escherichia coli</i> Using Two-Stage Dissolved Oxygen-Controlled Fermentation

Metabolic regulation and fermentation strategies limit the synthesis of tyramine. Here, a <i>de novo</i> synthetic tyramine-producing strain, LAN 25, was constructed. First, tyramine-producing strain was obtained by modifying the key metabolic nodes of tyrosine synthesis and overexpressing the tyrosine decarboxylase gene from <i>Lactobacillus brevis</i>. Then, a two-stage dissolved oxygen (DO)-controlled fermentation process was established in a 5 L fed-batch bioreactor. In the first stage, sufficient DO was supplied to accumulate tyrosine precursors, while in the second stage, limited DO was provided to promote tyramine synthesis. Next, <i>ldhA</i> and <i>adhE</i> were deleted to improve the strain's robustness. To enhance the redox flux (ATP/ADP ratio) under limited DO conditions, the anaerobic promoter, <i>Pvgb</i>, was used to control the expression of a <i>ppk</i>-based ATP regeneration system in response to DO changes. Additionally, the tyrosine internal transport system was modified. Finally, a titer of 21.33 g/L of tyramine with a yield of 0.092 g/g glucose was obtained.